Chronic Liver Failure: Mechanisms and Management (Clinical

CLINICAL GASTROENTEROLOGY

Series Editor George Y. Wu University of Connecticut Health Center, Farmington, CT, USA

For further volumes: http://www.springer.com/series/7672

Chronic Liver Failure MECHANISMS AND MANAGEMENT

Edited by PERE GINÈS Liver Unit, Hospital Clinic University of Barcelona, Barcelona, Spain

PATRICK S. KAMATH Division of Gastroenterology and Hepatology Mayo Clinic, College of Medicine Rochester, MN, USA

VICENTE ARROYO Liver Unit, Hospital Clinic University of Barcelona, Barcelona, Spain Editors Pere Ginès, MD Patrick S. Kamath, MD Liver Unit Division of Gastroenterology and Hepatol Hospital Clinic College of Medicine University of Barcelona Mayo Clinic Villarroel 170 200 First St. S.W 08036 Barcelona, Spain Rochester, MN 55905, USA [email protected] [email protected]

Vicente Arroyo Liver Unit Hospital Clinic University of Barcelona Villarroel 170 08036 Barcelona, Spain [email protected]

ISBN 978-1-60761-865-2 e-ISBN 978-1-60761-866-9 DOI 10.1007/978-1-60761-866-9 Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 201093895

© Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identiﬁed as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

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Humana Press is part of Springer Science+Business Media (www.springer.com) Preface

Chronic liver failure is a frequent event in clinical practice. For people aged between 18 and 55 years, it ranks eighth as the cause of death and is the most common reason for hospital admission in gastroenterology or liver units and the main indication for liver transplantation. Chronic liver failure is probably the most complex clinical syndrome in human pathology. In addition to problems associated with the impairment of hepatic function, there are complications related to portal hypertension and to the strategic situation of the liver between the intestine and the systemic circulation. Encephalopathy, due to impaired hepatic metabolism of ammonium and other neurotransmission inhibitory substances produced in the intestines, is the most important event directly associated with hepatic failure. Patients may even progress to deep coma. Mortality associated with hepatic encephalopathy is however low, and most patients recover. Only when hepatic encephalopathy develops in association with other organ failure, particularly renal failure, the prognosis is poor. Coagulopathy, due to reduced hepatic synthesis of procoagulant factors, is also a remarkable feature of chronic liver failure, but the clinical relevance of this problem is also low due to the simultaneous decrease in the synthesis of anticoagulant factors by the liver. Finally, hypoalbuminemia, due to reduced hepatic synthesis of albumin, is another characteristic feature of chronic liver failure. Our concept of the relevance of hypoalbuminemia in liver failure has changed over time. At the beginning, it was considered essential in the pathogenesis of circulatory failure and ascites. Subsequently, these problems were related to splanchnic arterial vasodilatation rather than to low albumin synthesis, and our focus on the importance of albumin in liver failure has shifted. Finally, recent studies suggest that reduced serum albumin concentration and particularly impairment of albumin function could be relevant as a mechanism of chronic liver failure. Albumin is an essential transporter of hormones and other important endogenous substances for organ function. It is also a fundamental transporter of endogenous toxic substances from tissues to excretory organs such as the liver or the kidneys and of therapeutic agents to their target cells. Finally, albumin has specific biological functions, the most important being its antioxidant properties. The albumin binding and transport capacity in chronic v vi Preface liver failure is almost totally absent due to saturation of the molecular binding sites and also due to profound alterations in the molecular structure. Although the most characteristic complication of portal hypertension in chronic liver failure is gastrointestinal hemorrhage due to esophageal varices, the most relevant event associated with the increased portal pressure is without doubt the development of a cardiovascular dysfunction due to reduced splanchnic arterial vascular resistance and impaired cardiac inotropic and chronotropic functions and cardiac output. Splanchnic reduction in vascular resistance is due to both arterial vasodilation and increased angiogenesis. The mechanisms of the impaired cardiac function are still not well understood. Both disorders are progressive during the course of the disease, compromising arterial pressure and leading to homeostatic activation of the renin–angiotensin system, sympathetic nervous system, and vasopressin. These systems are powerful vasoconstrictors and impair the renal ability to excrete sodium and free water, leading to ascites, water retention and dilutional hyponatremia, and extrasplanchnic vasoconstriction. Vasoconstriction within the liver increases portal pressure and reduces hepatic blood flow. Vasoconstriction within the kidney is the mechanism of hepatorenal syndrome. Finally, there is vasoconstriction in other territories such as the muscles and brain. Recent studies indicate that reduction in cerebral blood flow and brain edema related to dilutional hyponatremia are important features in the predisposition of patients with advanced chronic liver failure to develop encephalopathy linking circulatory and cerebral dysfunction in chronic liver failure. The liver contains most of the cells of the reticuloendothelial system (Kupffer cells) and this particular allocation of the phagocytic activity is an essential mechanism for preventing the translocation of viable bacteria and bacterial products from the intestinal lumen to the systemic circulation. Intestinal motility is markedly reduced in advanced cirrhosis, probably as a consequence of the sympathetic nervous system overactivity, and this leads to intestinal bacterial overgrowth. Portal hypertension produces anatomic changes in the intestinal mucosa and increases intestinal permeability. Finally, the phagocytic activity of hepatic reticuloendothelial system is markedly reduced in patients with advanced cirrhosis. The combination of these three features is one of the most important pathological events in chronic liver failure. It makes the patients vulnerable to endogenous bacterial infections, mainly from intestinal origin. On the other hand, it also determines the continuous passage of bacterial products (endotoxin, bacterial DNA) into the systemic circulation, leading to a chronic inflammatory state with persistent activation of the innate immune system and cytokine synthesis. Preface vii

Malnutrition and cardiocirculatory dysfunction associated with chronic liver failure may be related to this feature. The development of an acute liver failure over a chronic liver failure (a condition known as acute-on-chronic liver failure) is another common complication in patients with advanced cirrhosis. It usually occurs in close chronological relationship to a precipitating event, commonly an infection. In addition to a deterioration of liver function, as manifested by increased bilirubin and INR, these patients present an acute and severe deterioration in the function of many other organs including the brain, kidneys, heart, peripheral circulation, lungs, and adrenal glands. Acute-on-chronic liver failure is one of the main causes of death of cirrhosis. Mortality relates to the number of organ failures, being greater than 90% in those with more than three organ failures. The incidence of acute-on-chronic liver failure is particularly high in patients with advanced chronic liver failure in the waiting list for liver transplantation. Prevention of bacterial infection, improvement in the intensive care management of multiorgan failure, and development of effective artificial liver support systems are essential features to improve survival in these patients. Chronic liver failure is, therefore, the consequence of not only a decreased hepatic function but also the impairment in the function of many other organs. It is a difficult field to study. Investigators in chronic liver failure should ideally be physicians expert in clinical hepatology and intensive care, with a profound knowledge of cardiovascular and renal pathophysiology and bacterial infections. This type of investigator is infrequent and probably explains why the percentage of papers deal- ing with chronic liver failure published in the main hepatology journals represents less than 5% of the total number of articles despite being the most frequent cause of hospital admission and the main cause of death in patients admitted to gastroenterology or hepatology units. The aim of this book is not only to review the current state of the art in the pathophysiology and treatment of chronic liver failure but also to stimulate young investigators to enter into this complex research area.

Vicente Arroyo Patrick Kamath Pere Ginès

Acknowledgements

The editors would like to dedicate this book to various members of their families: Vicente Arroyo to Paula, Max, Pau, and Isabel. Patrick Kamath to Janine, Amika, and Marielle. Pere Ginès to Núria, Anna, Núria, Marta, and Dolors. The authors would like to acknowledge the work of Mrs. Nicki van Berckel in the preparation of this book.

Contents

Preface ...... v Acknowledgements ...... ix Contributors ...... xv

Part I The Organ

Cells in the Liver—Functions in Health and Disease ...... 3 Fabio Marra and Maurizio Parola Liver Physiology ...... 33 Alexander Sendensky and Jean-François Dufour Assessment of Liver Function in Clinical Practice ...... 47 Hamed Khalili, Barham Abu Dayyeh, and Lawrence S. Friedman Physiology of the Splanchnic and Hepatic Circulations ...... 77 Gautam Mehta, Juan-Carlos García-Pagán, and Jaime Bosch Fibrosis as a Major Mechanism of Chronic Liver Disease ...... 91 Lars P. Bechmann and Scott L. Friedman Stem Cells and Chronic Liver Failure: Potential New Therapeutics . 109 Aiwu Ruth He, Arun Thenappan, Feras J. Abdul Khalek, and Lopa Mishra The Role of Inﬂammatory Mediators in Liver Failure ...... 131 Joan Clària, Marta López-Parra, Esther Titos, and Ana González-Périz Genomics of the Liver in Health and Disease ...... 155 Konstantinos N. Lazaridis

Part II Effects of Liver Failure on Organ Systems

Hepatic Encephalopathy and Alterations of Cerebral Function ... 171 Juan Córdoba and Rita García-Martinez

xi xii Contents

Bacterial Translocation and Alterations of the Digestive System ... 189 Reiner Wiest SIRS, Bacterial Infections, and Alterations of the Immune System .. 219 J. Macnaughtan, V. Stadlbauer, R.P. Mookerjee, and R. Jalan Regulation of the Extracellular Fluid Volume and Renal Function . 239 Jens H. Henriksen The Heart in Chronic Liver Failure ...... 269 Hongqun Liu, Soon Woo Nam, and Samuel S. Lee Haemostasis Abnormalities in Chronic Liver Failure ...... 289 Armando Tripodi The Systemic and Splanchnic Circulations ...... 305 Yasuko Iwakiri Hepatic Microcirculation ...... 323 Chittaranjan Routray and Vijay Shah Angiogenesis and Vascular Growth in Liver Diseases ...... 343 Manuel Morales-Ruiz, Sònia Tugues, and Wladimiro Jiménez Pulmonary Alterations in Chronic Liver Failure ...... 361 Michael J. Krowka and Aynur Okcay Adrenal Function in Chronic Liver Failure ...... 377 Javier Fernández and Juan Acevedo

Part III Management of Chronic Liver Failure

Antibiotic Prophylaxis and Management of Bacterial Infections ... 395 Joseph K. Lim, Puneeta Tandon, and Guadalupe Garcia-Tsao Management of Ascites and Hyponatremia ...... 411 Andrés Cárdenas and Pere Ginès Management of Renal Failure ...... 429 Vicente Arroyo and Mónica Guevara Correction of Abnormalities of Haemostasis in Chronic Liver Disease ...... 453 Marco Senzolo and Andrew Kenneth Burroughs The Treatment and Prevention of Variceal Bleeding ...... 477 Juan G. Abraldes, Jaime Bosch, and Juan Carlos García-Pagan Extracorporeal Artiﬁcial Liver Support Systems ...... 501 Rafael Bañares and María-Vega Catalina Contents xiii

Issues in Transplantation of Patients with Chronic Liver Failure .. 521 Michael D. Leise, W. Ray Kim, and Patrick S. Kamath Chronic Liver Disease in the Intensive Care ...... 541 Andrew Slack and Julia Wendon Subject Index ...... 561

Contributors

JUAN G. ABRALDES • Hepatic Hemodynamic Laboratory, Liver Unit, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain JUAN ACEVEDO • Liver Unit, IMDiM and IDIBAPS, Hospital Clínic, University of Barcelona and CIBERehd, Spain VICENTE ARROYO • Liver Unit, Hospital Clinic, University of Barcelona, Spain RAFAEL BAÑARES • Liver Unit, Hospital General Universitario Gregorio Marañón, Universidad Complutense de Madrid, CIBEREHD (Centro de Investigación Biomédica en Red). Instituto de Salud Carlos III. Spain, c/ Dr. Esquerdo 46 28007 Madrid, Spain LARS P. B ECHMANN • Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY, USA JAIME BOSCH • Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clínic, C.Villarroel 170, 08036 Barcelona, Spain ANDREW KENNETH BURROUGHS • The Royal Free Sheila Sherlock Liver Centre and University Department of Surgery, UCL and Royal Free Hospital, London, UK ANDRÉS CÁRDENAS • Gastrointestinal Unit, Hospital Clínic and University of Barcelona School of Medicine, Institut d’Investigacions Biomèdiques August Pi-Sunyer (IDIBAPS), Ciber de Enfermedades Hepaticas y Digestivas (CIBERHED), Barcelona, Spain MARÍA-VEGA CATALINA • Liver Unit, Hospital General Universitario Gregorio Marañón, Universidad Complutense de Madrid, CIBEREHD (Centro de Investigación Biomédica en Red). Instituto de Salud Carlos III. Spain, c/ Dr. Esquerdo 46 28007 Madrid, Spain JOAN CLÀRIA • Department of Biochemistry and Molecular Genetics, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain JUAN CÓRDOBA • Servei de Medicina Interna-Hepatologia, Hospital Universitari Vall d’Hebron, Pg. Vall d’Hebron 119, Barcelona 08035, Spain

xv xvi Contributors

BARHAM ABU DAYYEH • Harvard Medical School, Gastrointestinal Unit, Massachusetts General Hospital, Boston, MA, USA JEAN-FRANÇOIS DUFOUR • Universitätsklinik für Viszerale Chirurgie und Medizin, Inselspital, 3010, Bern, Switzerland JAV I E R FERNÁNDEZ • Liver Unit, IMDiM and IDIBAPS, Hospital Clínic, University of Barcelona and CIBERehd, Spain SCOTT L. FRIEDMAN • Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY 10029, USA LAWRENCE S. FRIEDMAN • Department of Medicine, Newton-Wellesley Hospital, 2014 Washington Street, Newton, MA, 02462, USA GUADALUPE GARCIA-TSAO • National HCV Resource Center, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA PERE GINÈS • Liver Unit, Hospital Clínic and University of Barcelona School of Medicine, Institut d’Investigacions Biomèdiques August Pi-Sunyer (IDIBAPS), Ciber de Enfermedades Hepaticas y Digestivas (CIBERHED), Barcelona, Spain ANA GONZÁLEZ-PÉRIZ • Department of Biochemistry and Molecular Genetics, Hospital Clínic, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain MÓNICA GUEVARA • Liver Unit, Institut of Digestive and Metabolic Disease, IDIBAPS, Ciberehd, Hospital Clinic, University of Barcelona, Barcelona, Spain AIWU RUTH HE • Cancer Genetics, Digestive Diseases, and Developmental Molecular Biology, Department of Surgery, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA JENS H. HENRIKSEN • Department of Clinical Physiology and Nuclear Medicine, 239, Faculty of Health Sciences, Hvidovre Hospital, University of Copenhagen, DK-2650, Hvidovre, Denmark YASUKO IWAKIRI • Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA R. JALAN • The UCL Institute of Hepatology, Royal Free Hospital, London, UK WLADIMIRO JIMÉNEZ • Service of Biochemistry and Molecular Genetics, Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Spain FERAS J. ABDUL KHALEK • Cancer Genetics, Digestive Diseases, and Developmental Molecular Biology, Department of Surgery, Contributors xvii

Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA HAMED KHALILI • Harvard Medical School, Gastrointestinal Unit, Massachusetts General Hospital, MA, USA PATRICK S. KAMATH • Gastroenterology and Hepatology Mayo Clinic Transplant Center, Mayo Clinic, Rochester, MN, USA WRAY KIM • Mayo Clinic Transplant Center, Mayo Clinic, Rochester, MN, USA MICHAEL J. KROWKA • Division of Pulmonary and Critical Care Medicine, Mayo Clinic, MN, Rochester, USA KONSTANTINOS N. LAZARIDIS • Division of Gastroenterology and Hepatology, Center for Basic Research in Digestive Diseases, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA SAMUEL S. LEE • Liver Unit, University of Calgary, Calgary, Canada MICHAEL D. LEISE • Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA JOSEPH KLIM • Yale Viral Hepatitis Program, Section of Digestive Diseases, Yale University School of Medicine, New Heaven, CT, USA HONGQUN LIU • Liver Unit, University of Calgary, Calgary, Canada AYNUR OKCAY • Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN, USA MARTA LÓPEZ-PARRA • Department of Biochemistry and Molecular Genetics, Hospital Clínic, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain J. MACNAUGHTAN • The UCL Institute of Hepatology, Royal Free Hospital, London, UK FABIO MARRA • Dipartimento di Medicina Interna, University of Florence, Viale Morgagni 85 I-50134 Florence, Italy RITA GARCÍA MARTINEZ • Servei de Medicina Interna-Hepatologia, Hospital Vall d’Hebron, Barcelona, Spain GAUTAM MEHTA • Institute of Hepatology, University College London, London, UK LOPA MISHRA • Department of Gastroenterology, Hepatology and Nutrition, The University of Texas MD Anderson Cancer Center, 1400 Pressler Street, FCT13.6038, Unit Number: 1466, Houston, TX 77030, USA R.P. MOOKERJEE • The UCL Institute of Hepatology, Royal Free Hospital, London, UK MANUEL MORALES-RUIZ • Service of Biochemistry and Molecular Genetics, Hospital Clínic, Villarroel 170, Barcelona, 08036, Spain xviii Contributors

SOON WOO NAM • Division of Gastroenterology/Hepatology, Catholic Medical College, Daejeon, South Korea JUAN-CARLOS GARCÍA-PAGÁN • Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clínic-IDIBAPS and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain MAURIZIO PAROLA • Dipartimento di Medicina e Oncologia Sperimentale, Centro Interuniversitario di Fisiopatologia Epatica, University of Torino, Torino, Italy CHITTARANJAN ROUTRAY • Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA ALEXANDER SENDENSKY • University Clinic for Visceral Surgery and Medicine, Inselspital, Bern, Switzerland MARCO SENZOLO • Department of Surgical and Gastroenterological Sciences, University-Hospital of Padua, Italy VIJAY SHAH • Gastroenterology and Hepatology Mayo Clinic Transplant Center, Mayo Clinic, Rochester, MN, USA ANDREW SLACK • Institute of Liver Studies, King’s College Hospital, Denmark Hill, London, UK V. S TADLBAUER • The UCL Institute of Hepatology, Royal Free Hospital, London, UK PUNEETA TANDON • Division of Gastroenterology, University of Alberta, Edmonton, Alberta, Canada ARUN THENAPPAN • Cancer Genetics, Digestive Diseases, and Developmental Molecular Biology, Department of Surgery, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA ESTHER TITOS • Department of Biochemistry and Molecular Genetics, Hospital Clínic, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain ARMANDO TRIPODI • Department of Internal Medicine, IRCCS Ospedale Maggiore/Mangiagalli/Regina Elena Foundation and Università degli Studi di Milano, Via Pace 9, 20122 Milano, Italy SÒNIA TUGUES • Service of Biochemistry and Molecular Genetics, Hospital Clínic-Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Spain JULIA WENDON • Institute of Liver Studies, King’s College Hospital, Denmark Hill, London REINER WIEST • Department of Internal Medicine, University of Regensberg, Regensberg, Germany Part I The Organ

Cells in the Liver—Functions in Health and Disease

Fabio Marra and Maurizio Parola

CONTENTS INTRODUCTORY REMARKS HEPATOCYTES KUPFFER CELLS HEPATIC STELLATE CELLS SINUSOIDAL ENDOTHELIAL CELLS CONCLUSIONS REFERENCES

Key Words: Acetaminophen, Alcoholic liver disease, Angiogenesis, Apoptosis, Microbial infections, Cancer, Cytokines, Epithelial-to- mesenchymal transition, Extracellular matrix, Fibrosis, Hepatocellular carcinoma, Hepatocytes, Hepatic stellate cells, Inﬂammation, Innate immunity, Ischemia–reperfusion injury, Liver regeneration, Lipopolysaccharide, Matrix metalloproteinases, Metabolism, Myoﬁbroblasts, Nonalcoholic fatty liver disease, Pericytes, Platelet-derived growth factor, Portal hypertension, Sinusoidal endothelial cells, Space of Disse, Toll-like receptors, Transforming growth factor-β, Tumor necrosis factor, Viral hepatitis

1. INTRODUCTORY REMARKS The liver lobule is formed by hepatocytes and cholangiocytes, constituting the two hepatic epithelial cell populations, as well as by cells that are collectively deﬁned as nonparenchymal cells (1–4). Morphometric and functional analyses indicate that hepatocytes occupy almost 80% of the total liver volume and perform the majority of liver functions.

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_1, C Springer Science+Business Media, LLC 2011

3 4 Marra and Parola

Nonparenchymal liver cells, which contribute only 6.5% to the liver volume, but represent approximately 40% of the total number of liver cells, are localized in the sinusoidal compartment of the organ. The walls of hepatic sinusoids are lined by at least three different cell types, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and hepatic stellate cells (HSCs, formerly known as fat- storing cells, Ito cells, lipocytes, perisinusoidal cells, or vitamin A-rich cells) (2, 3). Additionally, intrahepatic lymphocytes are often present in the sinusoidal lumen, including particularly the so-called pit cells, which have been recognized as liver-speciﬁc natural killer (NK) cells (3). Under both normal and pathophysiological conditions, many functions of the hepatocytes are regulated by mediators released from neighboring nonparenchymal cells. Because of space constraints, in this chapter only selected information related to the different hepatic cell populations in normal as well as in either acute or chronic liver injury will be recalled and the interested reader is referred to more exhaustive and specialized references. A schematic representation of the relations among cells in the hepatic sinusoid is shown in Fig. 1.

Sinusoidal endothelial cell Hepatic Hepatocyte stellate cell

Kupffer cell

Space of Disse

Fig. 1. Schematic representation of the space of Disse and of the relations among cells discussed in this chapter.

2. HEPATOCYTES Parenchymal liver cells (i.e., hepatocytes) are polygonal/polyhedral cells, sometimes binucleated, with a diameter ranging between 20 and 30 μm, accounting for approximately 80% of the liver mass and usually Cells in the Liver—Functions in Health and Disease 5

Fig. 2. Immunohistochemistry for hypoxia-inducible factor-2α (HIF2 α), identifying hypoxic hepatocytes in a biopsy of a cirrhotic human patient (chronic HCV infection). characterized by three major surface domains: (a) the basal or sinusoidal domain, showing short microvilli and involved in the exchange of molecules with sinusoidal blood; (b) the lateral domain, characterized by junctional complexes between adjacent hepatocytes; and (c) the bile canalicular domain (2). Hepatocytes represent the key hepatic effector cells since most of the liver functions can be attributed to their activity and are also selectively targeted by hepatotropic viruses as well as by many toxic compounds, including ethanol, which are specifically metabolized by the organ, leading to either acute or chronic liver injury (Fig. 2). Hepatocytes can also be injured as a consequence of autoimmunity, metabolic derangement, or genetic mutations. In this section, after brieﬂy recalling the major and well-established physiological roles of parenchymal cells, we will limit our analysis to two well-deﬁned and timely topics: (a) hepatocyte apoptosis as a driving force for either acute or chronic liver injury and (b) hepatocyte as a putative cell source for MFs through epithelial-to-mesenchymal transition.

2.1. Hepatocytes in Physiological Conditions As far as physiological functions are concerned, hepatocytes have a well-established role in (5):

– regulation of glucose blood levels by means of glycogen synthesis, glycogenolysis, and gluconeogenesis; 6 Marra and Parola

– lipid metabolism, either by synthesizing lipoproteins or via intracellular catabolism of exogenous or endogenous lipids, through mitochondrial β-oxidation and peroxisomal oxidation; – metabolism/inactivation of several exogenous (drugs, ethanol, toxins, environmental pollutants, carcinogens) and endogenous (steroid hormones, bilirubin) compounds by either phase I (oxidation and reduction) or phase II (conjugation and hydrolysis) reaction; – synthesis of plasma proteins, including albumin, acute-phase proteins, components of the complement and clotting systems, ﬁbrinogen, and others; – inactivation of ammonia by the urea cycle; – metabolism of retinoids and other vitamins; – homeostasis of iron, copper, and zinc; – a contribution to bile secretion.

From a metabolic point of view, a signiﬁcant “zonation” or heterogeneity of parenchymal cells has been described depending on their location. As an example, periportal hepatocytes (or hepatocytes of zone 1 of the Rappaport’s acinus), which receive a blood supply rich in oxygen and substrates, are predominantly gluconeogenic whereas perivenous hepatocytes (hepatocytes of zone 2 of Rappaport’s acinus), which receive blood already partially depleted of glucose, oxygen, and substrates, are predominantly glycolitic. A similar degree of zonation has been described for oxidative, amino acid, xenobiotic, and ammonia metabolisms (4).

2.2. Hepatocyte Apoptosis: A Driving Force for Acute and Chronic Liver Injury Liver injury is characterized by either necrotic or apoptotic cell death as well as, more often, by a combination of the two, and indeed it is well known that the same stimulus can induce both types of irreversible cell death (6, 7). As nicely pointed out by Malhi and Gores (8), on a cellular basis liver necrosis may even simply represent the result of an overwhelming or dysregulated apoptosis, as it may be the case for necrosis following exaggerated mitochondrial dysfunction by apoptotic signaling cascades. Indeed, current evidence is that apoptosis can be found in any type of liver injury and is often predominant (9–11), as the detection of serum levels of M30 (an epitope formed by proteolytic cleavage of the hepatocytic cytokeratin 18 by caspase 3) is starting to reveal (11–14). Hepatocyte apoptosis can be triggered by several extracellular as well as intracellular signals or conditions, with mitochondrial dysfunction Cells in the Liver—Functions in Health and Disease 7 playing a major role. Indeed, any event able to trigger apoptosis, including death receptor activated signaling cascades (i.e., by ligands such as TNF, TRAIL, and FasL), endoplasmic reticulum (ER) stress, activation of c-Jun N-terminal kinases (JNKs), and lysosomal permeabilization (see (8) for more details), can result in mitochondrial outer membrane permeabilization, either through Bax and Bak activation or through mitochondrial permeability transition. Moreover, hepatocyte apoptosis has been described in all major conditions of acute and chronic liver injury and the following are the best characterized examples. Acute liver failure (ALF). Acetaminophen overdose, as a model of ALF, has been shown to trigger apoptosis by activating JNK isoforms 1 and 2 and translocation of Bax to mitochondria (15), by an increased expression of TNF as well as by an increased expression of Fas and FasL (16, 17) with NK also playing a role (15). Nonalcoholic steatohepatitis. In the liver of NASH patients, apoptosis correlates with inflammation and fibrosis, and steatotic hepatocytes are sensitized to FasL- and TRAIL-mediated apoptosis (16–18). In these clinical settings, a proapoptotic role is also attributed to the so- called free fatty acid induced and JNK-mediated lipoapoptosis (19)as well as, likely, to the activation of ER stress (8). Alcoholic steatohepatitis (ASH). In the proinflammatory cytokine microenvironment of ASH, again apoptosis plays a major role, with a predominant role for TNF (as released by activated Kupffer cells) which mediates both apoptosis and inflammation (20), and again circulating levels of TNF, Fas, FasL, and TNFR1 are significantly increased (10, 21). Apoptosis in ASH is likely to be also triggered by increased generation of ROS as well as by induction of ER stress by either ROS or acetaldehyde (i.e., a major metabolite of ethanol) (8, 22, 23). Chronic viral hepatitis. Low-grade, Fas-mediated apoptosis has been found to correlate with the severity of inflammatory response (24) because of Fas-enhanced expression of HCV-infected hepatocytes and the action of FasL-expressing T lymphocytes (25, 26). Sensitization to TRAIL-mediated apoptosis, following interaction of the ligand with TRAIL receptors 1 and 2, has also been described in human chronic viral hepatitis as well as in NAFLD (26). In addition, hepatocytes can be sensitized to either TRAIL-, TNF-, or FasL-mediated apoptosis by HCV core or envelope protein ((8) and references therein). Almost homologous findings have been described for hepatocyte apoptosis in either acute or chronic injury by HBV infection (8). Cholestatic liver injury. Hepatocyte apoptosis is a prominent in vivo and in vitro feature of experimental cholestatic liver injury (27). Glycine-conjugated chenodeoxycholic acid (GCDCA) can induce 8 Marra and Parola apoptosis by either a FasL-dependent (28) or a FasL-independent mechanism, the latter operating through spontaneous oligomerization and phosphorylation of Fas on hepatocyte plasma membrane (29, 30). GCDCA can also upregulate expression of TRAIL-R2 and then sensitize hepatocytes to TRAIL-induced apoptosis (31, 32). Some more general messages should be emphasized. First, apoptosis in liver injury, irrespective of the etiology (excess accumulation of triglycerides, action of GCDCA, viral proteins, etc.), is likely to predominantly occur in damaged, “vulnerable,” or stressed hepatocytes, a condition which is also believed to sensitize cells to the action of death ligands such as TNF, TRAIL, or FasL. Moreover, hepatocyte apoptosis can be relevant for fibrogenesis progression because of at least two mechanisms: (a) apoptotic bodies, when avidly taken up by Kupffer cells, lead to KC activation and release of profibrogenic and proinflammatory cytokines, chemokines, ROS, and other mediators able to perpetuate inflammatory reaction and positively affect HSC (8); and (b) apoptotic bodies can be engulfed also by HSC and this can lead to the activation of HSC, as indicated by increased expression of α-SMA, procollagen type I, and TGF-β1(33). Apoptosis blockade has then been envisaged as a therapeutic strategy for either acute or chronic liver diseases, as the first promising studies employing TRAIL receptor antibodies or pan-caspase inhibitors (34, 35) suggest. Moreover, it has been suggested that part of the therapeutic efficacy of ursodeoxycholic acid in cholestatic disease may be related to inhibition of hepatocyte apoptosis (36, 37).

2.3. Hepatocytes as a Putative Source of Myofibroblasts via Epithelial-to-Mesenchymal Transition Epithelial-to-mesenchymal transition (EMT) is a process, paradigmatic of the concept of cell plasticity, that leads epithelial cells to lose their polarization and specialized junctional structures, to undergo cytoskeleton reorganization, and to acquire morphological and functional features of mesenchymal-like cells. Although EMT has been originally described in embryonic development, where cell migration and tissue remodeling have a primary role in regulating morphogenesis in multicellular organisms, recent literature has provided evidence suggesting that the EMT process is a more general biological process that is also involved in several pathophysiological conditions, including cancer progression as well as kidney and lung fibrosis through transition into (myo)fibroblast-like profibrogenic cells (38–42). Concerning the role of EMT in chronic liver diseases, typical EMT changes in vitro were first reported in cultured rat neonatal hepatocytes (43, 44), in cultured Cells in the Liver—Functions in Health and Disease 9 primary mouse hepatocytes (45, 46), or in different nontumorigenic hepatocytic cell lines (46, 47) following exposure to several growth factors and cytokines. In particular, TGF-β1, through Smad2/3 signaling, induced all canonical EMT-related changes (SNAI1 induction, E-cadherin and hepatocyte transcriptional factor HNF4 downregulation, upregulation of mesenchymal and invasiveness markers) (45–47). In one of these studies (46), progressive appearance in the injured livers of cells positive for fibroblast-specific protein 1 (FSP-1, a marker of EMT) was described, although less than 10% of FSP-1 positive cells were shown to coexpress the MF marker α-SMA. The involvement of EMT was also suggested by lineage-tracing experiments using AlbCre.R26RstoplacZ double transgenic mice, indicating that approximately 15% of hepatic cells were FSP-1 positive at the time of severe fibrosis and that approximately 5% of the hepatic cells were coexpressing either FSP-1 or albumin. Moreover, progression of fibrosis and generation of putative EMT-derived fibroblasts were significantly inhibited by BMP-7, which is known to antagonize TGF-β1 signaling. Similar results were also described by others by employing a transgenic mouse model of Smad7 overexpression in hepatocytes to counteract CCl4-induced fibrosis (48). However, at present, we still lack clinical studies properly designed in order to ascertain the real contribution of EMT in progressive fibrogenesis associated to the most common forms of human CLDs where the major role of HSC/MFs has extensively been described (see later in this chapter).

3. KUPFFER CELLS Kupffer cells (KCs) are named after the pathologist Carl von Kupffer, who identiﬁed them as resident liver macrophages (49). KCs constitute around 80% of all the tissue macrophages of the reticuloendothelial system and about 15% of all liver cells (50). KCs are mainly concentrated in the periportal area of the lobule and have the ability to migrate along the sinusoidal wall (51). KCs derive from circulating monocytes, but are also capable of self-renewal (52). After liver transplantation, donor KCs are replaced within 1 year in humans (53). KCs are the ﬁrst macrophage population to encounter gut-derived bacteria, endotoxin (LPS), and antigens and function as antigen- presenting cells (54), being a critical component of innate immunity, together with NK cells, dendritic cells, and soluble factors (55). In addition, they participate in clearance of senescent red blood cells and hemoglobin–aptoglobin complexes (56). This action has a relevance for oxidative damage and microcirculation, as the heme group 10 Marra and Parola of hemoglobin is degraded by heme-oxygenase-1, leading to production of antioxidant biliverdin and carbon monoxide, which protects the hepatic microcirculation (57). KCs may be isolated and studied in culture. The most effective factors inducing KC activation are complement factors C3a and C5a and microbial products, such as LPS. Once activated, KCs secrete several mediators that play a pivotal role in host defense and in tissue injury. Activation of NADPH oxidase results in production of superoxide anion, which helps to destroy phagocytosed organisms but may be harmful to surrounding cells (58). KCs also express constitutive and inducible cyclooxygenase isoforms, leading to prostaglandins and thromboxane synthesis. Prostaglandins modulate TNF production by KC (59), while thromboxane A2 induces HSC contraction, thus potentially contributing to portal hypertension (60). A diagram summarizing the major features of Kupffer cells is shown in Fig. 3.

Key role in innate immunity Clearance of senescent red blood cells Production of arachidonic acid metabolites Generation of reactive oxygen intermediates Expression of proinflammatory cytokines (TNF, IL-1, IL-6) Pathogenic role in acute and chronic liver injury Release of nitric oxide (may be protective in some conditions) Involvement in pathophysiologic states: Kupffer cells Defense from microbial infections Alcoholic liver disease Insulin resistance and nonalcoholic fatty liver disease Acetaminophen-mediated liver injury Ischemia–reperfusion injury Liver regeneration Portal hypertension Liver cancer

Fig. 3. Summary of the major physiological and pathophysiological characteristics of Kupffer cells.

3.1. Kupffer Cells in Acute and Chronic Liver Injury Like for other hepatic cells, evidence for the role of KCs in health and disease comes from studies in animal models more than from human studies. KCs have an important physiological role in the context of innate immunity (61). They contribute to eliminate microorganisms, Cells in the Liver—Functions in Health and Disease 11 dead cells, LPS, and, to a less extent, toxic agents, including ethanol. In particular, KCs contribute to liver injury in animals challenged with carbon tetrachloride (62), LPS (63), galactosamine, and acetaminophen (64, 65), with KC being a relevant source of proinflammatory cytokines (including TNF and IL-6) and high levels of oxidative stress-related products, proteolytic enzymes, and eicosanoids (64). The release of ROS by KCs can kill bacteria and other micro-organisms, but can also contribute to tissue damage. In addition, KCs produce nitric oxide (NO) that has a controversial role in hepatic pathophysiology. In some instances, NO has been shown to be protective toward liver injury, such as in the case of rodents intoxicated with carbon tetrachloride. However, in animals subjected to hepatic ischemia/reperfusion, NO may combine with oxidative stress-related products to form toxic peroxynitrites (66). KCs may be protective also in models of cholestasis, where expression of IL-6 has been shown to reduce damage and inflammation (67). More specifically, KCs play a major role in the following condition of liver injury: Endotoxin-mediated injury. Endotoxin-mediated KC activation is a major mechanism contributing to liver injury, and indeed increased concentration of endotoxin in the portal blood is a feature common to several conditions, including alcoholic liver disease (68). In rats challenged with CCl4, administration of endotoxin aggravated liver damage (69). Secretion of proteolytic enzymes is an additional mechanism leading to hepatic injury in several models. Hepatic fibrosis. Hepatic stellate cells (HSCs) are the pivotal players in the process of liver fibrosis (see Chapter 5). However, KCs are believed to play a role both in the process of HSC activation and in the maintenance of fibrogenesis. In particular, expression of TGF-β1 by KCs has been shown to contribute to the activation process (70). In addition, KC-conditioned medium leads to the upregulation of PDGF receptors, which are a hallmark of the activation process, increasing PDGF-mediated proliferation (71). Secretion of proinflammatory cytokines by activated KCs leads to secretion of chemotactic cytokines, such as monocyte chemoattractant protein-1, by HSC, amplifying the inflammatory process. KCs also produce gelatinases such as MMP-2, which degrade normal extracellular matrix and lead to HSC activation (72). Host defense. Rapid elimination of bacteria depends on a close interaction between KCs and neutrophils. KCs bind the bacteria, which are then internalized and killed by neutrophils (73). KCs also take part in neutrophil clearance, a critical event in the resolution of inflammation (74). KCs have been implicated in the response of the liver to sepsis, where LPS pretreatment has been linked to an increase in the number 12 Marra and Parola of KCs and an improvement of prognosis (75). In contrast, susceptibility to infection is increased in the presence of reduced KC function (76). In a model of listeriosis, inactivation of KCs was associated with reduced ability to clear infection (77). The role of KCs in infection clearance is linked to the secretion of proinflammatory cytokines and chemokines and the resulting recruitment of inflammatory cells (78). Interestingly, KCs are infected by some germs such as cytomegalovirus and Leishmania, before expanding to the rest of the liver (79). As a correlate, in patients with liver failure, susceptibility to infection may depend, at least in part, on the loss of the ability of KCs to clear bacteria that translocate from the intestine. Another clinical correlate of this mechanism is the presence of endotoxemia in patients with advanced liver disease. Finally, KCs may also be directly involved in the process of hepatocyte apoptosis via CD95, during viral infection or other conditions associated with liver injury, such as graft rejection (80). Alcoholic and nonalcoholic liver disease. In the context of alcohol- induced damage, release of cytokines, proinflammatory mediators, and ROS by KCs has a role in the pathogenesis of injury (81). Mediators expressed under the control of NF-κB play a major role, including TNF, IL-6, and inflammatory chemokines, such as IL-8. The relevance of TNF is also demonstrated by limitation of alcohol-induced damage in TNFR1-knockout mice (82). KCs have an activated phenotype and increase in number in conditions of chronic alcohol intake (83). Of note, the expression of CD14, the receptor for endotoxin, is increased on KCs during alcohol-induced damage (84), thus amplifying the effects of chronic endotoxemia associated with alcohol ingestion. Moreover, LPS acts synergistically with ethanol to increase NF-κB activation and cytokine expression (81). KC activation has been described in models of nonalcoholic steatohepatitis (85) and is mediated, at least in part, by proinflammatory mediators such as TNF (86). Recently, Kodama et al. (87) have reported that activation of JNK1 in KCs is a critical mediator of inflammation and fibrosis during experimental steatohepatitis, using a chimeric mouse model. In addition, chitotriosidase expression in KCs was found to be increased in NASH patients and to be related to activation of HSCs and fibrogenesis (88). KCs also modulate insulin resistance and lipid metabolism in hepatocytes, identifying an additional level of interaction that may be relevant for this condition (70). Release of endocannabinoids by KCs has also been shown to contribute to liver injury in experimental steatohepatitis (89). In both alcoholic and nonalcoholic steatohepatitis, toll-like receptor-4 activation by endotoxin contributes to the increase in proinflammatory cytokines and ROS, and genetic inactivation of TLR-4 Cells in the Liver—Functions in Health and Disease 13 protects from damage caused by both conditions (90). The pathways downstream of TLR-4 include activation of MyD88 and TRIF, but only TRIF deletion ameliorates alcohol-induced damage (91). Activation of NADPH oxidase is another pathway that contributes to TNF expression in response to LPS, as indicated by the protection afforded by the lack of p47phox to ethanol-induced injury (90). An interesting aspect is related to the role of adiponectin, a fat-derived cytokine that protects from both alcoholic and nonalcoholic steatohepatitis (92). Adiponectin has a number of metabolic and anti-inflammatory actions, which include inhibition of TNF release by activated KCs (92, 93). Similarly, intracellular generation of cyclic AMP decreases the expression of inflammatory cytokines (81). Acetaminophen-mediated injury. In this condition, depletion of intracellular glutathione and generation of reactive oxygen species are considered critical mechanisms. While some data show that acetaminophen toxicity can be reduced inactivating KCs (94), recent evidence indicates that KCs may actually be beneficial in this condition. In fact, elimination of KCs with liposomal clodronate results in more severe liver injury, due to reduced release of anti-inflammatory molecules (95). The protective action may be mediated, in part, by production of nitric oxide by KCs (96) and by chemokines of the ELR-CXC group that induce hepatocyte proliferation in this model (97). In addition, IL-10 and IL-18, secreted by KCs, may participate in the protection from acetaminophen-induced injury (98). Ischemia–reperfusion and liver transplantation. Ischemia–reperfusion injury is critical for the outcome of liver resection or liver transplantation, and KC activation plays a key role in mediating injury (99). Generation of reactive oxygen species via NADPH oxidase, and the resulting activation of NF-κB, leads to increased expression of TNF, adhesion molecules, and chemokines (100). Infusion of antioxidants or glutathione limits injury by reducing oxidative stress and by inducing atrial natriuretic peptide that acts on KC independently of formation of reactive oxygen species (101). In this context, NO derived from KCs or other sources may be protective (102), leading to little formation of peroxynitrite. Since KCs function as antigen-presenting cells and express MHC class II antigens (103), KCs may then have a role in the pathogenesis of biliary damage after transplantation (104). In addition, KC-derived ROS and cytokines contribute to damage during ischemia–reperfusion (105), and interference with KC activation leads to modulation of injury. Interestingly, KC may also be implicated in the generation of immune tolerance, contributing to apoptosis of immunoreactive T cells (106). 14 Marra and Parola

Liver regeneration. The capacity of the liver to regenerate is critical after hepatic surgery or in split-liver hepatic transplantation. Activation of KCs is necessary for optimal liver regeneration through release of TNF and IL-6, which activate mitogenic pathways, such as NF-κBand STAT-3, in regenerating hepatocytes (107). Interaction with leucocytes and complement activation are believed to take part in KC activation in this context. Portal hypertension. Thromboxane A2 is a vasoconstrictor metabolite of arachidonic acid that is produced by KCs in response to proinﬂammatory stimuli or endothelin-1 infusion, and aggravates portal hypertension (108). Liver cancer. KC depletion before infusion of tumor cells results in increased tumor development (109). KCs have been shown to phagocy- tose tumor cells (110) and may inﬂuence the growth of cancer inducing NK cell-mediated cytotoxicity via production of IL-12 (109). In addition, nitric oxide production by KCs may lead to cytotoxicity toward tumor cells, and KC cytotoxicity against metastatic cells is enhanced by stimulation with interferon-γ (111). In contrast, once metastases have developed, secretion of proteases and angiogenic factors by KCs may actually result in tumor progression.

4. HEPATIC STELLATE CELLS The first mention of hepatic stellate cells (HSCs) is by von Kupffer, who observed perisinusoidal star-shaped cells that were believed to belong to the perivascular nervous network. In the early 1970s, Kenjiro Wake concluded that the star-shaped phagocytes described by Kupffer were identical to the fat-storing cells described by Ito and that the lipid droplets were largely composed of retinoid esters. The real physiological and pathophysiological relevance of HSCs has emerged only in the past 20 years, with the recognition of the role of HSC in hepatic fibrosis and, more generally, in hepatic repair. HSCs have been referred to as lipocytes, fat-storing cells, or perisinusoidal stellate cells, until an international group of investigators, in 1996, made the recommendation to refer to this cell type as “hepatic stellate cell” (112). Hepatic stellate cells are located in the space of Disse in close contact with hepatocytes and sinusoidal endothelial cells. Although the total number of HSCs constitutes a small percentage of the total number of liver cells (approximately 5–8%), their disposition is sufficient to cover the entire hepatic sinusoidal microcirculatory network. HSCs possess subendothelial processes that ensure a strong connection with the sinusoidal endothelium and ultrastructure and vitamin A content vary according to their location within the liver lobule (113). Vitamin Cells in the Liver—Functions in Health and Disease 15

A-containing cytoplasmic lipid droplets represent the most relevant ultrastructural feature of HSCs in adult normal liver, a feature related to the main known physiological function of HSCs, the hepatic storage of retinyl esters. HSCs are equipped with abundant microtubules and microfilaments, possibly functioning as the cytoskeleton of dendritic processes and playing a role in lipid synthesis and/or transport. The subendothelial processes possess actin-like filaments, suggesting that they may contribute to reinforce the endothelial lining and/or enhance the efficiency of contraction of sinusoidal capillaries (49). A tremendous advancement in this field has been represented by the development of techniques for isolation and culture of HSCs from human or rodent livers, which are an excellent model for investigating their biological characteristics (114). Isolation procedures include a first step aimed at obtaining a suspension of nonparenchymal liver cells, followed by further purification based on the low buoyant density of these cells due to their large fat content (115). However, the presence of lipid droplets is a key feature of only a portion of the total HSC population present in the liver lobule. Therefore, effort has been directed to the identification of cytoskeletal or surface markers able to differentiate HSCs from other nonparenchymal liver cells. In this connection, smooth muscle isoform of α-actin (α-SMA) represents a reliable marker for the identification of “activated” HSCs (114), as it is not expressed in HSCs early after isolation but only after some time in culture or during fibrogenesis in vivo (Fig. 4). Consequently, α-SMA is commonly employed as a marker of HSC activation. The embryonic origin of HSCs is still a matter of debate although they have been originally considered as mesenchymal cells depending on their morphology and positivity for desmin, vimentin, and α-SMA. However, when HSCs were found to contain neural markers, it was speculated that HSCs could be of neuroectodermal origin (116). Other studies have suggested that hepatocytes and HSCs may derive from a common endodermal precursor (117), or even, as shown recently by studies in humans and animal models, from bone marrow precursors (118).

4.1. HSC in Hepatic Physiology and Pathophysiology Retinoid storage and metabolism. In mammals, about 50–80% of total retinol is under normal circumstances stored in the liver, and HSCs play a key role in the metabolism and storage of retinoids (119). Chylomicron-remnant retinyl esters are taken up by hepatocytes and, after binding with speciﬁc retinoid-binding proteins, these compounds are transferred to neighboring HSCs (119), where uptake, storage, 16 Marra and Parola

a b

c d

Fig. 4. Immunohistochemistry for α-SMA identifying hepatic myofibroblasts, likely derived from activation of hepatic stellate cells, in a biopsy of a cirrhotic human patient (chronic HCV infection). In all panels, myofibroblasts are identified by the brown staining in fibrogenic septae. and mobilization of retinoids are regulated by intracellular retinoid- binding proteins. Retinyl esters are a major component of the total lipid mass present in the lipid droplets of HSCs, together with triacylglycerol. Physiological conditions that require an increased utilization of retinoids at the periphery will result in the mobilization of these compounds from HSCs, which also express nuclear retinoid receptors, including retinoic acid receptors (RARs) α, β,andγ, and retinoid X receptors (RXRs) α and β, but not γ (120). HSC and normal extracellular matrix homeostasis. In normal liver, the ECM constitutes about 0.5% of liver wet weight and is constituted by minor amounts of several noncollagenous components, such as fibronectin, laminin, nidogen (entactin), tenascin, undulin, proteoglycans, and hyaluronic acid. The space of Disse, where HSCs are located, is a virtual space constituted by an ECM network composed of type IV collagen, associated with noncollagenous components, allowing an optimal diffusion between the hepatocytes and the bloodstream. During active liver fibrogenesis, HSCs become the major ECM-producing cell type, with a predominant production of collagen I (121). Normal ECM turnover implies that the synthesis of new individual components is Cells in the Liver—Functions in Health and Disease 17 associated with their continuous slow degradation. In normal liver, HSCs and possibly other sinusoidal cells may contribute to the continuous remodeling of the ECM of the space of Disse by producing matrix metalloproteinase-2 (gelatinase or type IV collagenase) (122). HSC as liver-specific pericytes. The possible role of HSCs as liver- specific pericytes is suggested by their anatomical location, ultrastructural features, and the close relationship with the autonomous nervous system. Interestingly, cultured HSCs contract in response to several vasoconstricting stimuli (123), although their activated phenotype may resemble transitional or myofibroblast-like cells rather than quiescent HSCs, suggesting that contractility may be more likely a feature of HSCs in fibrotic liver. Whether HSCs contract in normal liver tissue is still debated, with some observations arguing against the role of HSCs in the regulation of sinusoidal blood flow (124) and other studies that evaluated hepatic microcirculation by intravital microscopy techniques suggesting that HSCs may contribute to sinusoidal tone regulation (125). HSC and fibrogenesis. Liver fibrosis is a multicellular, integrated process that requires a close cross-talk between hepatocytes, cholangiocytes, and nonparenchymal cells, including infiltrating inflammatory cells, Kupffer cells, HSCs, and sinusoidal endothelial cells (126). All forms of fibrogenesis arise in the context of tissue damage, where hepatocytes and nonparenchymal cells provide signals that target HSCs and other fibrogenic myofibroblasts (MFs), leading to ECM accumulation. Activation of HSCs is considered a major source of MF during liver damage (126). Besides HSCs, other ECM-producing cells have been recently identified, including fibroblasts and myofibroblasts of the portal tract, smooth muscle cells localized in vessel walls, and MF localized around the centrilobular vein. It is also increasingly evident that the relative participation of these different cell types is dependent on the development of distinct patterns of fibrosis (127). Finally, several lines of evidence indicate that cells recruited from the bone marrow, such as mesenchymal stem cells and CD45-expressing fibrocytes, contribute to the fibrogenic process. The relative participation of these cell types is dependent on the etiology and the development of distinct patterns of fibrosis (127). An important concept in the pathophysiology of fibrosis is the process of “activation,” whereby HSCs acquire an MF-like phenotype and become fibrogenic (128)(Fig.5). HSC activation requires changes in gene transcription that are triggered by oxidative stress-related products and soluble mediators released by neighboring cells. Once HSCs and/or other cells have acquired an MF-like phenotype, the fibrogenic process is maintained and amplified through a number of biological actions that 18 Marra and Parola

Increased Deposition of fibrillar proliferation/survival Quiescent HSC matrix Activation

Inhibition of matrix Phagocytosis of degradation apoptotic bodies

Secretion of pro Cell migration inflammatory factors

Myofibroblastic HSC

Contraction Angiogenesis

Fig. 5. Biological actions associated with the activation process of hepatic stellate cells. are directed to the efficient execution of the wound healing response (see the Chapter 5). This is achieved via expression and secretion of collagen types I and III, together with secretion of other matrix components such as fibronectin and proteoglycans. TGF-β1 represents the key cytokine in stimulating this process, via activation of intracellular molecules of the Smad family (129). Activated HSCs also show increased proliferation, survival, and migration in response to several factors, of which the most potent is platelet-derived growth factor (PDGF) (123). Directional HSC migration is also regulated by proteins of the chemokine family (130). The process of cell migration is accompanied by profound modifications in the actin cytoskeleton, particularly evident when cells are exposed to PDGF. Matrix degradation. Accumulation of ECM depends not only on increased deposition, but also on decreased removal, based on the action of matrix metalloproteinase (MMPs). Activated HSCs highly express tissue inhibitors of metalloprotease-1 (131) and metalloprotease-2 (131), which bind and inactivate MMPs, thus resulting in a net profibrogenic effect (132). In addition, TIMP-1 has been shown to be a survival factor for stellate cells, providing an additional profibrogenic Cells in the Liver—Functions in Health and Disease 19 action in the context of a fibrotic liver. For these reasons, inhibition of TIMP’s action may be viewed as a promising antifibrotic target. Similarly, HSCs express several components of the plasmin system, which is also involved in the regulation of matrix degradation (133). HSC contraction. Contraction, induced by vasoactive agents such as endothelin-1, angiotensin II, or thrombin (123), has a great relevance in the pathogenesis of portal hypertension, contributing to the “reversible” component of the increase in intrahepatic flow (134). It is important to note that the process of cell contraction may be counterregulated by nitric oxide or carbon monoxide, which induces relaxation of HSC contraction and reduces portal pressure (135). Modulation of inflammation. Activated HSCs secrete cytokines and chemokines that amplify the inflammatory process and lead to recruitment of inflammatory cells (136). The chemokines monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-8 are among the principal chemoattractants expressed by these cells. Activation of the master inflammatory transcription factor, NF-κB, is the main molecular mechanism underlying upregulation of chemokine expression in response to proinflammatory cytokines including IL-1 or TNF. Angiogenesis. Fibrosis is tightly associated with angiogenesis, the formation of new blood vessels from preexisting ones. Angiogenesis has been shown to occur in experimental and human forms of chronic liver injury (137), and activated HSCs have been found to contribute to the angiogenic process through secretion of factors such as vascular endothelial growth factor or angiopoietin-1. These cytokines are expressed in response to hypoxia as well as to factors such as PDGF or leptin (138). HSC and the stem cell niche. A recently identified function of HSCs is related to the maintenance of the stem cell niche, where bipotent hepatic progenitor cells that may acquire characteristics of hepatocytes or cholangiocytes are located. In conditions of liver damage, HSC activation may contribute to the disruption of the HSC niche, leading to altered maturation and differentiation of stem cells (139).

5. SINUSOIDAL ENDOTHELIAL CELLS Liver sinusoidal endothelial cells (LSECs) are highly specialized and very thin cells that line hepatic sinusoids and separate the sinusoidal blood, derived primarily from portal vein, from parenchymal liver cells. LSECs have been recognized as a distinct cell type just in 1972 by Eddie Wisse opening the way to studies that delineated the dynamic and multiple role of these cells with time. In this section, a number of crucial 20 Marra and Parola aspects of LSECs will be recalled which have relevant physiological and pathophysiological implications (140).

5.1. Fenestration, Ultrafiltration, and the Scavenger Role of LSECs LSEC fenestrations, which represent about 5–10% of the surface of these cells, are pores of approximately 50–150 nm of diameter that are typically grouped in clusters formed by several fenestrations (i.e., the liver sieve plates) and lack either a basal lamina or a diaphragm. Fenestrations are heterogenous with slightly larger fenestrations found in the periportal sinusoids and greater porosity in pericentral sinusoids (141–143) and allow plasma and a wide range of related substrates (mainly plasma proteins like albumin and small lipoproteins or chylomicrons remnants) to pass into the space of Disse, with only cells and larger lipoproteins, mainly larger chylomicrons (CLMs), being retained in the sinusoids by the size of fenestrations (1, 144). Of relevance, vascular endothelial growth factor (VEGF) released by hepatocytes and HSCs is the most relevant stimulus regulating LSEC fenestrations as well as endocytic function (1, 145). Indeed, LSECs act as a very efficient scavenger endothelium that uses clathrin-mediated endocytosis to clear endogenous as well as exogenous molecules from sinusoids (145, 146), including viral particles. The list of waste macromolecules removed by LSECs from systemic circulation includes: (a) mannose- or scavenger receptor mediated endocytosis (147, 148)of macromolecules from connective tissue turnover, including collagen α-chain, procollagen C- or N-terminal peptides (such as PICP, PINP, and PIIINP), hyaluronic acid, and chondroitin sulfate; (b) scavenger receptor mediated endocytosis of oxidized as well as acety- lated low-density lipoproteins (LDLs) and advanced glycation end products (AGEs); (c) Fc-γ receptor type IIb2 mediated removal of immune complexes and microbial CpG motifs ((144, 145) and references therein). Any significant change in the structure of sinusoidal endothelium and injury of LSECs will then significantly affect bidirectional transfer of substrates between hepatocytes and sinusoidal blood as well as endocytic function of LSECs. In particular, injured sinusoids and then LSECs may lose porosity and scavenger function by a process commonly defined as capillarization (Fig. 6). Injury to LSECs, even leading to their detachment, may occur in conditions of ischemia–reperfusion injury, early sinusoidal obstruction syndrome, or early acetaminophen toxicity (reviewed in (149)). Moreover, the sinusoids may be obstructed by fibrosis, particularly Cells in the Liver—Functions in Health and Disease 21

Chronic damage Capillarization (loss of fenestrations and of Normal liver scavenger functions) Presence of Barrier to oxygen fenestrations causing hepatocyte Endocytosis hypoxia Scavenging of Sinusoidal Expression of macromolecules, endothelial cells polypeptide factors oxidized LDL, immune involved in liver wound complexes healing Synthesis of nitric oxide Expression of Participation in proinflammatory immune response cytokines and adhesion molecules for leukocytes Reduced synthesis of nitric oxide Neoangiogenesis

Fig. 6. Summary of the characteristics of sinusoidal endothelial cells in the normal and injured liver. the pattern of perisinusoidal ﬁbrosis found in alcoholic and nonalcoholic steatohepatitis (ASH and NASH). Interestingly, in most of these microvascular injuries, the changes to the sinusoids are the primary event that may lead to hepatocyte hypoxia and, with time, liver dysfunction and disruption of portal circulation.

5.2. LSECs as a Source of Biologically Active Mediators In the last 15 years, it has become increasingly obvious that LSECs, particularly when either activated or damaged under conditions of acute and chronic liver injury, may actively contribute to the overall pathophysiological scenario by synthesizing and releasing growth factors, cytokines, chemokines, and other mediators. Once again the list of biological mediators released by LSECs is impressive (145, 150, 151) and includes (a) polypeptide cytokines deeply involved in wound healing and ﬁbrogen- esis like platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β1 (TGF-β1), basic ﬁbroblast growth factor (bFGF), and insulin-like growth factor type 1 (IGF-1), all potentially able to modulate the response of HSCs; LSECs can also release, after liver injury, hepatocyte growth factor (HGF) (152) as well express receptors for several of the mentioned polypeptides, including PDGF-Rβ, EGF-R, and c-met (153); 22 Marra and Parola

(b) proinﬂammatory cytokines and related mediators, including mainly interleukin-1 (IL-1); (c) vasoactive peptides and mediators that are likely to play a relevant role particularly under condition of chronic liver injury, including nitric oxide (NO), endothelins, prostanoids, and prostaglandins; (d) although to a lesser extent than hepatocytes and other nonparenchymal cells, LSECs have been described to contribute to generate reactive oxygen species.

5.3. LSECs and Oxygen Tension (Ischemia–Reperfusion Injury, Angiogenesis) Tissue hypoxia is very common in several pathological conditions affecting liver parenchyma, and indeed LSECs represent the primary target of ischemia–reperfusion injury following liver preservation during OLT. Exposure to hypoxic conditions or to conditions that lead to hypoxia (e.g., high blood alcohol levels) can result in gene reprogramming through the action of hypoxia-inducible factors (HIFs) although the normal oxygen tension in hepatic sinusoids (approximately 5%) is considerably lower to the atmospheric one (145). LSECs have an obvious major role in pathological angiogenesis, and indeed hypoxic hepatocytes and HSCs secrete growth factors and chemokines (137, 154, 155), mainly VEGF-A, that stimulate the endothelial cells to break out of their stable position in the sinusoids to jointly coordi- nate sprouting, branching, and new lumenized network formation—an hypoxia-stimulated process designed to restore normal blood and oxygen supply in which endothelial cell–cell communication, as for tip and stalk EC, via the Notch pathway plays a major role (156). Evidence for high number of endothelial cells in portal tracts as well as the characteristic presence of new microvascular structures has been described in all experimental and clinical conditions of CLDs and indeed neoangiogenesis has a major impact on vascular changes in the progression toward cirrhosis irrespective of etiology (137, 154, 155).

5.4. Interactions of LSECs with Leucocytes and Cancer Cells Interactions between leucocytes or cancer cells and LSECs are known to be involved in the pathogenesis of liver injury (152). In these cell-to-cell interactions, a wide spectrum of adhesion molecules play a key role, and their expression is mainly regulated by inﬂam- matory cytokines such as interleukin-1, tumor necrosis factor-α,and interferon-γ (3, 150). LSECs in normal liver express intercellular adhesion molecule-1, intercellular adhesion molecule-2, leucocyte function-associated antigen-3, very late antigen-5, and CD44. In patients with acute or chronic liver disease, intercellular adhesion Cells in the Liver—Functions in Health and Disease 23 molecule-1 and vascular cell adhesion molecule-1 expression are markedly enhanced in the inﬂamed liver tissue and selectins are not present in normal conditions, but are induced after lipopolysaccharide administration ((3) and references therein). Where metastasis of colorectal tumors is concerned, it has been proposed that single tumor cells get stuck when entering the liver sinusoids because of their size exceeding the diameter of a sinusoid; after plugging, their adhesion molecules might react with the surface molecules of the LSECs, enabling them to extravasate and enter the liver parenchyma as a crucial step in early stages of hepatic metastasis ((156) and references therein).

5.5. The Putative Role of LSECs in Immune Response LSECs can promote active antigen uptake through the expression of Fc-γ receptor and pattern recognition receptors (i.e., mannose and scavenger receptors) and then should be considered as cells taking part in the innate immune response. It has been reported that LSECs may even promote antigen presentation by presenting MHC class I and II antigens and costimulatory molecules such as CD40, CD80, andCD86(157), although this issue is still debated. Indeed, differently from other more conventional antigen-presenting cells, LSECs failed to induce differentiation of naive CD4+ T cells toward a Th1 phenotype, a feature that is associated with the production of negative immunomodulatory cytokines in LSECs—primed T cells upon restimulation have been suggested to signiﬁcantly contribute to the unique hepatic immune tolerance (55).

6. CONCLUSIONS The liver has a complex structure, and all the different cell types present in the tissue provide a contribution in physiological conditions, and especially in condition of disease. A better understanding of the function of each cell type and of the cross talk among them, especially in conditions of disease, is opening new perspectives for the diagnosis and treatment of liver diseases.

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Alexander Sendensky and Jean-François Dufour

CONTENTS BILE ACIDS AND BILE THE LIVER AS A FACTORY THE LIVER AS A DETOXIFIER THE LIVER AS A FILTER REFERENCES

Key Words: Bile acids: synthesis and metabolism, Bile acids: BESP, ASBT, Bile acids: signaling FXR, TGR5, Metabolism: Protein, Albumin, aminotransferase, Metabolism: Autophagy, neoglucogenesis, glycogen, Metabolism: Lipid metabolism / Metabolism regulation, Metabolism: SREBP-1, CPT-1, PPAR-g, LXR, mTOR, AMPK, nuclear receptors (f1), Metabolism: Iron, hepcidin, Copper, ATP7A, Detoxification: phases, cytochromes, Detoxification: MRP2 / Bilirubin detoxification, Detoxification: Alcohol / Ammonium, Detoxification: Glutamate / Urea/Ornithin-cycle, Liver immunology: filter function (f2), Liver immunology: cells and functions, Liver immunology: immunotolerance

1. BILE ACIDS AND BILE In the terminal ileum, bile acids present in the lumen are recuperated and returned to the liver where they are taken up into hepatocytes and excreted into the bile again. This enterohepatic circulation retains over 95% of the bile acids. Each day, only 400–500 mg of bile acids are produced, balancing the small physiologic fecal loss (excretion into urine is normally negligible). In 24 h, approximately 12–25 g of bile acids are secreted into the intestine, turning the whole pool over up to 10 times a

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_2, C Springer Science+Business Media, LLC 2011

33 34 Sendensky and Dufour day. Cholesterol is the starting molecule in the synthesis of bile acids. Conversion of cholesterol into bile acids occurs via two pathways: the classical (or neutral) pathway and the alternative (or acidic) pathway. The classical pathway contributes 75% of the bile acid pool. Reactions leading to primary bile acids, cholic acid and chenodeoxycholic acid, include initiation (hydroxylation in position 7), modification of the sterol ring, oxidation, shortening of the side chain, and conjugation with glycine or taurine. Once secreted into the intestinal lumen, the anaerobic flora metabolizes the primary bile acids into secondary bile acids. The major reaction is 7α-dehydroxylation to give deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid. Secondary bile acids are reabsorbed by the enterohepatic circulation and reconjugated within the hepatocytes before they are secreted into the bile system. Once transported back to the liver, secondary bile acids can be further processed to form tertiary bile acids such as sul- folithocholic acid and ursodeoxycholic acid, which normally contribute marginally to the bile acid pool. Bile acids are derived from cholesterol and their excretion facilitates biliary cholesterol excretion, influencing cholesterol homeostasis. Resins binding bile acids in the intestinal lumen increase their fecal output, stimulate synthesis of bile acids, and, indirectly, act as hypocholesterolemic agents. In contrast, cholestatic liver diseases are characterized by hypercholesterolemia. Conjugated bile acids have powerful detergent-like properties that are important in stabilizing the physical state of bile and in promoting fat digestion and absorption. Bile acids support digestion of nutritional components by formation of micelles and activation/stabilization of enzymes such as pancreatic lipase, phospholipase A, and Pancreatic cholesterol esterase. Micelle formation relies on the amphiphilic nature of bile acids, which are hydrophile on one end while lipophile on the other. This mechanism allows biliary excretion of lipophilic compounds such as cholesterol. To prevent cell damage by formation of micelles while transporting bile acids inside the cell, bile acids bind to specific intracellular transport proteins. Physiologically 600-ml bile is produced daily. It consists of 400-ml canalicular bile formed in the bile canaliculi between hepatocytes and 200-ml ductular bile collected in the bile ducts lined up by cholangiocytes. Hepatocytes and cholangiocytes are polarized cells with basolateral sides and an apical side. Several ATP-dependent pumps are embedded into the canalicular membrane of the hepatocytes at their apical side. These pumps accumulate bile acids, phospholipids, and organic anions in the canalicular bile. Bile salt export pump (BSEP) is one of them, permitting the excretion of conjugated bile acids against a concentration gradient (1). Intestinal recycling of bile acids occurs via Liver Physiology 35 aNa+-dependent carrier (apical sodium bile acid transporter (ASBT)) located on the apical side of enterocytes in the terminal ileum as well as on the apical side of hepatocytes and cholangiocytes. Organic solute and steroid transporters (Ostα,Ostβ) have been shown to be essential transporters on the basolateral side of enterocytes and cholangiocytes. These bile acids are taken up back into the hepatocytes by another Na+-dependent transporter, Na+-Taurocholate cotransporting polypeptide (NTCP). This system avoids precious cholesterol metabolites to be lost with feces and also permits a cross talk between the intestine and the liver. Bile acids are now recognized to be important signaling molecules linking feeding to metabolism regulation (2). Their increased intestinal presence postprandially informs adjacent transmitters and metabolic pathways of the availability of nutrients. Bile acids bind and activate a specific G-protein-coupled receptor, TGR5 (also called GPBAR1, membrane bile-acid receptor or BG37) as well as an intracellular receptor, FXR (farnesoid X receptor). FXR belongs to the group of nuclear hormone receptors and functions as a transcription factor. FXR affects not only bile acid metabolism, but also cholesterol metabolism, triglyceride metabolism, and glucose metabolism. In liver, kidney, and intestinal tissues, FXR hinders accumulation of bile acids and thereby prevents toxic damage. In the liver, FXR intensifies bile acid conjugation which consecutively increases bile flow by enhanced excretion of bile acids from hepatocytes into bile canaliculi. In the intestine, FXR activation leads to increased expression of the ileal bile acid binding proteins (I-BABP, FABP6), of the basolateral bile acid transporters and of the secreted growth factor, fibroblast growth factor 19 (FGF19). Bile acids influence energy homeostasis via the TGR5 pathway. Furthermore, after cellular uptake bile acids exert direct signaling functions in cholangiocytes and hepatocytes via calcium, PKC, MEK, ERK, and PI3K pathways, altering gene expression, cell proliferation, apoptosis, and secretion.

2. THE LIVER AS A FACTORY Protein metabolism. In contrast to muscle cells, which synthesize protein for their own use, hepatocytes synthesize proteins of importance altruistically for the whole organism. The majority of the circulating proteins are synthesized by hepatocytes. These proteins comprise cargo proteins (e.g., albumin, transferrin, ceruloplasmin, haptoglobin, lipoproteins), immune-related proteins (proteins of the complement system, acute-phase proteins), and coagulation factors. 36 Sendensky and Dufour

C-reactive protein is an acute-phase protein, whose hepatocellular production is massively stimulated by cytokines such as IL-6 and IL-1. Albumin is the most abundant plasma protein maintaining intravascular oncotic pressure; its determination reflects the synthesis capacity of the liver over the past few weeks since its half-life is 21 days. To assess the hepatocellular synthesis capacity for a shorter time (hours), the determination of the coagulation factors is appropriate. Aminotransferases transfer an amino group from a donor molecule to a recipient molecule. Aspartate aminotransferase facilitates the conversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate, and vice versa, whereas alanine aminotransferase facilitates the conversion of alanine and α-ketoglutarate to pyruvate and glutamate, and vice versa. AST can be cytosolic and mitochondrial, whereas ALT is strictly cytosolic. These enzymes are intensively expressed in cells involved in physiologic protein metabolism, particularly hepatocytes and muscle cells. Elevated serum aminotransferase levels are nonspecific markers for hepatocellular damage. Proteins are degraded by two major pathways: the autophagic– lysosomal pathway and the ubiquitin–proteasome-related pathway. Autophagy engulfs part of the cytoplasm in vacuoles whose content is digested by lysosomal enzymes after fusion with lysosomes. In the ubiquitin–proteasome pathway, proteins are tagged for degradation by enzymatic linkage with ubiquitin residues. Carbohydrate metabolism. To maintain blood glucose levels within physiologic range, the liver functions as recipient, store, donator, and creator. Up to 90% of the intestinally absorbed glucose is taken up by the liver. Glucose passes membranes via glucose transporters (GLUT family of transporters; GLUT-2, 9, and 10 are expressed in the liver). Once in the cytoplasm, glucose is phosphorylated by hexokinase or glucokinase to access cellular metabolism. Glucokinase is expressed only in the liver and phosphorylates only glucose. Glucokinase activity is particularly important postprandially since its velocity is maximal at much higher concentrations of glucose than hexokinase. Glucose- 6-phosphate is sequentially transformed into glucose-1-phosphate by phosphoglucomutase and into uridine-diphosphate-glucose by glucose- 1-phosphouridyltransferase to be finally stored as glycogen. The arborescent structure of glycogen with a central anchor protein termed glycogenin links up to 50,000 molecules of glucose while keeping them easily accessible for reintegration into metabolism. Glucose-6- phosphate is not solely the initial compound for glycolysis; it can also enter the pentose phosphate pathways via glucose-6-phosphate dehydrogenase to produce NADPH and precursors for nucleotides. Other carbohydrates like fructose and galactose are enzymatically transformed to join the glycolysis pathway. Liver Physiology 37

When glucose blood levels drop, glucagon and adrenaline stimulate via cAMP a protein phosphorylase reverting glycogen to glucose- 1-phosphate (α-glycanphosphorylase) and to glucose-6-phosphate (phosphoglucomutase). G-6-P is converted to glucose by glucose-6- phosphatase. Once glycogen storage has been emptied, glucose needs to be synthesized from other sources. Two third of the glucose derived from neoglucogenesis is synthesized from lactate, which results from anaerobic metabolism and can be supplied to the liver by the muscles. Glucose can also be produced from amino acids, mostly alanine, and from glycerine which is a degradation product of triglycerides. Gluconeogenesis is triggered by hormonal signals. Glucagon increases gluconeogenesis in the short term, while glucosteroids enhance gluconeogenesis in the long term. Insulin inhibits gluconeogenesis. A hallmark of hepatic insulin resistance is the failure of insulin to inhibit hepatic glucose output. Lipid metabolism. Within each liver lobule, there is zonation of the metabolic functions. The periportal zone is where oxidative energy metabolism, amino acid catabolism, cholesterol metabolism, and fatty acid β-oxidation take place, whereas the perivenous zone is where de novo lipid synthesis, ketogenesis and xenobiotic metabolism occur. Liposynthesis occurs by esterification of free fatty acids via acetyl-CoA and glycerol and is driven by glycerophosphate acyltransferase (GPAT), which is activated by nutritional status and insulin and inhibited by glucagon. De novo lipogenesis of free fatty acids from acetyl-CoA is regulated by insulin via activation of sterol regulatory element- binding protein-1c (SREBP-1c), which controls the transcription of lipogenic enzymes such as fatty acid synthase. Insulin stimulates the conversion of carboxyl-CoA to malonyl-CoA, a key regulator for the distribution of free fatty acids toward esterification or oxidation. Low levels of malonyl-CoA direct free fatty acids to the mitochondriae and β-oxidation via carnitine palmitoyltransferase-1 (CPT-1), an outer mitochondrial membrane enzyme. High levels of malonyl-CoA inhibit CPT-1, thus enhancing esterification of free fatty acid into triglycerides. Fatty acids can also be oxidized in peroxisomes (β-oxidation) and microsomes (ω-oxidation). Triglycerides stimulate apolipoprotein B (Apo-B) synthesis and are secreted as VLDL-Apo-B. Insulin inhibits Apo-B synthesis and impairs secretion of triglycerides as VLDL. The regulators. AMP-dependent protein kinase (AMPK) and mammalian target of rapamycin (mTOR) adapt hepatocellular metabolism to energy status. Activated AMPK switches energy-consuming anabolic lipogenic pathways to ATP-producing catabolic pathways (3). Multiple cues activate AMPK; hypoxia, ATP depletion, starv- ing, chronic alcohol consumption, oxidative stress, adiponectin, leptin, and drugs such as metformin or thiazolinediones. AMPK 38 Sendensky and Dufour

Carbohydrates Peptide translation Ribosome biogenesis

Apoptosis GLUT-4 mTOR Translocation to membrane Proteins Transcription regulation

CREB eEF-2-Kinase Blocking peptide elongation G-6-P-ase expression AMPK PEPCK expression

HMGR (Rate-limiting) cholesterol synthesis SREBP GPAT activity

Lipids

Fig. 1. AMPK influences several main metabolic processes as central turntable. mTOR: mammalian target of rapamycin, a protein complex regulating cell cycle and growth. GLUT-4: insulin-dependent transmembrane glucose transporter protein. CREB: c-AMP response element binding protein, a nuclear transcription regulator. PEPCK: phosphoenolpyruvat carboxykinase, speed- limiting gluconeogenesis enzyme, catalyzing metabolism from oxalacetat via guanosintriphosphat to phosphoenolpyruvat. SREBP: sterol regulatory element binding protein, a transcription factor regulating cholesterol metabolism via activation of gene translation. GPAT: glycerol-3-phosphate acyltransferase, the initial step enzyme in glycerolipid synthesis. HMGR: HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis via the mevalonate pathway. controls acetyl-CoA-carboxylase 1 reducing lipogenesis, acetyl-CoA- carboxylase 2 increasing fat oxidation, HMG-CoA-reductase lowering cholesterol synthesis, or mTOR lowering protein synthesis (Fig. 1). Peroxisome proliferator-activated receptors (PPARs) are transcription factors essential for the regulation of cell differentiation and metabolism (4). PPARs sense lipid signals and are to be considered “lipostats”: endogenous fatty acids activate PPAR-α, while leukotrienes and prostaglandins activate PPAR-γ. They are also the targets of several metabolic drugs. Fibrates activate PPAR-α and glitazones activate PPAR-γ.PPAR-α stimulates hepatocellular fatty acid uptake and catabolism. PPAR-γ is highly expressed in adipose tissue, where it regulates adipogenesis and adipose tissue integrity. PPAR-γ is usually Liver Physiology 39 poorly expressed in the liver, but its levels increase significantly during lipid accumulation in both hepatocytes and stellate cells. Activation of hepatic PPAR-γ decreases steatosis and reduces profibrogenic processes. LXR is a nuclear receptor whose ligands are oxysterols. LXR is involved in the regulation of cholesterol, bile acid, and triglyceride metabolism as well as in inflammatory response and energy balance. LXR stimulates cholesterol synthesis and biliary secretion. LXR activates SREBP-1c inducing lipogenesis. LXR promotes glucose utilization by inhibiting expression of glucose-6-phosphatase and induction of glucokinase expression. Iron. The liver regulates iron homeostasis and is the main body store for iron. Iron is taken up by enterocytes in a highly regulated manner, since it is not excreted and loss of iron is not controlled. Intestinal iron absorption is regulated by hepcidine, which is mainly produced by hepatocytes and to a lesser amount by adipocytes and macrophages. Hepcidine concentrations increase under inflammatory conditions or iron overload and decrease in case of anemia or hypoxic conditions (5). Expression of hepcidine is activated by bone mor- phogenic protein, which is controlled by hemojuvelin (HJV), HFE, and transferrin receptor 2 (Tfr-2) proteins. Hepcidine inhibits the expression of the ferroportin transporter, a membrane transporter protein releasing iron from the enterocyte. Once released from the enterocyte, iron binds to transferrin, the main iron transport protein of the body. Iron uptake into the hepatocytes is mediated by transferrin receptor 1 (Tfr-1). Tfr-1 is upregulated by hypoxia-inducible factor, IL-2, mitogens, growth factors, or other cytokines. Proliferating cells, in need of iron for growth, express more Tfr-1. HFE, the defective protein in hereditary hemochromatosis, competes with transferrin for binding to Tfr-1. Transferrin is also endocytosed via Trf-2, but with an affinity 25–30 times lower; Trf-2 seems to act as a transferrin saturation sensor. Copper. Copper is essential for life as it plays a key role as a cofactor for various enzymes. As copper is cytotoxic, it is accompanied by specific protector proteins, which carry and transfer copper to its intracellular destination. At the level of the plasma membrane, copper-transporting ATPases (Cu-ATPases) with two isoforms (ATP7A and ATP7B) play a central role in copper homeostasis by supporting transmembranous copper exchange. ATP7A is responsible for copper transport across the basolateral membrane of enterocytes into the circulation. ATP7B expressed in hepatocytes is responsible for copper excretion into bile. ATP7B deficiency leads to Wilson’s disease with intracellular copper accumulation (5). 40 Sendensky and Dufour

3. THE LIVER AS A DETOXIFIER The liver is the central organ for detoxification of exo- and endogenous substances. While water-soluble substances can be excreted by the kidneys, lipophilic substances have to be transformed in the hepatocytes before excretion. Biotransformations within the liver include not only detoxification, but also activation of certain compounds (e.g., prodrugs). Detoxification processing can be divided into three phases. In a first phase, lipophilic substances are conjugated with an additional reactive group enhancing the polarity of the molecule. These groups most often consist of either –NH2, –COOH, –OH, or –SH groups. Conjugation is achieved by oxidation/hydroxylation, reduction, or hydrolysis, depending on the group to be added. Clinical importance of these processes has been shown best for the microsomal mixed-functional monooxy- genases, which contain the cytochromes P450. Cytochromes P450 consist of several dozens of enzymes—among others those metabolizing drugs such as the CYP3A4, which influences pharmacokinetics and interactions of many drugs. The large number of cytochrome isoenzymes explains the stunning diversity in individual drug metabolization. Phase I reaction may be sufficient to render substances hydrophilic and enhance kidney excretion. The second phase conjugates phase I products with other liver- derived substances such as glucuronic acid, amino acids, activated sulfuric acid or mercapturic acid. The newly generated conjugate provides an increased hydrophilicity due to its most often acid characteristics and therefore can be excreted more easily by the kidneys or into the intestinal lumen by bile excretion. The third phase consists of transmembrane transporters. Noxious compounds conjugated with charged moieties such as glucuronide, glutathione, and sulfate are subsequently pumped into bile across the canalicular membrane by different ATP-binding cassette (ABC) transporters. These involve ABCC2 (MRP2), which largely transports organic anions; ABCG2 (breast cancer-related protein (BCRP)), which transports many charged and uncharged compounds; and ABCB1 (MDR1 P-glycoprotein), which mainly transports uncharged or cationic amphiphilic compounds. Conjugated compounds can also be transported back into the blood by pumps such as ABCC3, ABCC4, and ABCC5, resulting in urinary excretion after filtration or active excretion in the kidney.

3.1. Specific Detoxification Pathways Bilirubin. Bilirubin concentration in the serum consists of a balance of pigment production and elimination. An end product of heme and Liver Physiology 41 hemoproteins, most bilirubin reaches the bloodstream from the spleen, entering the liver via the portal vein. Hepatocyte uptake happens Na+ independent, by organic anion transporter proteins (OATPs) in a glutathione countertransport manner at the sinusoidal surface of the hepatocyte. Intracellular bilirubin is linked to ligandin and Z-protein, specific cytosolic proteins, thus preventing intracellular toxicity. Glucuronidation for excretion takes place in the smooth endoplasmic reticulum by the rate-limiting enzyme uridine diphosphoglucuronate- glucuronosyl transferase (UDP-GT), resulting in hydrophilic bilirubin glucuronide. Excretion into the bile is ATP-dependent as transmembrane efflux is provided by conjugated export pump MRP2 (see above). Small amounts of bilirubin are secreted to the plasma via MRP3. Within the intestinal tract, bile-derived bilirubin is metabolized by gut bacteria via β-glucuronidase for oxidation to stercobilin, which is excreted within feces or in small amounts by the kidneys after reuptake by small intestinal endothelium and further metabolization to urobilirubin (6). Alcohol. The mainstay of alcohol degradation consists of the alcohol dehydrogenase enzyme, though hepatocytes own a microsomal oxidative system located within the ER and catalase within the peroxisomes. The presence of different isoenzymes of ADH explains the individu- ally different capability to cope with ingested alcohol, furthermore, as ADH activity is maximally saturated from 0.3 to 0.5‰ and cannot be upregulated or induced by chronic exposition. ADH metabolizes alcohol to aldehyde acetate, which is highly toxic and has to be further degraded within the microsomes by aldehyde dehydrogenase to acetate acid. Acetate acid is then integrated as acetyl-CoA into the citric acid cycle as well as into the lipid acid cycle and the cholesterin synthesis. ADH is a zinc-depending enzyme, a feature relevant in chronic alcohol abuse, as chronic alcohol consumption most often leads to zinc deficiency. The degradation of alcohol is highly oxygen-dependent and may consume up to 90% of the whole hepatocellular oxygen uptake, meanwhile inhibiting or affecting other oxygen-dependent processes. In chronic alcohol consumption, alcohol specific ADH cannot be induced, whereas the microsomal oxidative system in the ER consisting of cytochrome P450 isoenzymes, primarily unspecific for alcohol, can be upregulated and therefore becomes more and more important as consumption of higher amounts endures. Alcohol induces CYP2E1 subtype, which releases reactive oxygen species and contributes to oxidative stress. Finally, alcohol can also be degraded by catalase, a peroxisomal enzyme degrading H2O2 into water and O2 and reducing alcohol to acetaldehyde only if higher concentrations occur (>1‰) (7). 42 Sendensky and Dufour

+ Ammonium. Ammonium (NH4 ) derives mainly from the colonic bacterial flora by degradation of proteins and urea. The liver produces and metabolizes ammonium within the urea/ornithine cycle. Urinary ammonium excretion amounts to approximately 20–40 mmol/l urine. Ammonium detoxification in the liver is dependent on two systems: the urea/ornithine cycle, which is the mainstay of ammonium detoxification, and the glutamate cycle, which is not liver-specific. In the urea/ornithine cycle, which is liver-specific, ammonium and bicarbonate are conjugated into the mitochondria by carbamylphosphate synthetase to form carbamylphosphate. Carbamylphosphate is transformed to citrulline via the ornithine carbamylphosphate transferase. Citrulline is further metabolized within the cytoplasm via arginine for urea production providing ornithine as a spin-off. The glutamate cycle conjugates ammonium with α-ketoglutaric acid to produce glutamine, which represents the nontoxic transport form of ammonium. The urea/ornithine cycle depends on high ammonium concentrations and is therefore located in the periportal area and detoxifies the bulk of the portal venous ammonium load. It is vulnerable to exogenous/intestinal toxic substances. The glutamine synthesis is located perivenously and due to its high affinity is less dependent on ammonium concentrations. Importantly, the urea/ornithine cycle and the glutamate cycle are linked to the plasma bicarbonate level as bicarbonate acts as substrate for urea production and glutamine synthesis is dependent on plasma pH levels. Hepatic urea synthesis is a major pathway for the removal of metabolically generated bicarbonate (8).

4. THE LIVER AS A FILTER The liver is receiving two third of its blood supply from the intestine. This blood full of nutrients contains many antigens, which are filtered through the hepatic sinusoids by cells of the innate immunity system. The innate immunity system is the first line of defense against pathogens recognizing them via pattern recognition receptors such as the toll-like receptors. The liver is enriched with cells of the innate immune system including Kupffer cells (KCs), dendritic cells (DCs), and natural killer (NK) cells (9). Lipopolysaccharides (LPS), which derive from the cell wall of gram-negative bacteria, are present in concentrations up to 1 ng/ml in the portal blood, whereas LPSs are not detectable in the peripheral blood because they have been cleared Liver Physiology 43 in the liver. Liver sinusoidal endothelial cells (LSECs), KCs, and DCs function as antigen-presenting cells (APCs). The KCs are mobile macrophages which position themselves within the sinusoids to contact circulating lymphocytes and engage antigens. KCs are activated by various bacterial antigen stimuli such as LPS and bacterial super- antigens. Once activated, KCs produce cytokines (IL-6, TNF, IL-12, and IL-18), influencing the function of other cell types present in their vicinity (hepatocytes, LSECs, and NKs). IL-1β, IL-6, TNF-α, and leukotrienes recruit neutrophils. Neutrophils phagocyte bacterial antigens presented by APCs and secrete cytokines to stimulate other innate immune cells and promote attraction and activation of CD4+ and CD8+ cells. Neutrophil recruitment can significantly contribute to liver injury (10). LSECs express mannose and scavenger receptors and antigen- uptake molecules. LSECs also support immune pathways by expressing costimulatory CD 40, CD 80, and CD86, similar to mature DCs. Receptor-mediated uptake of antigens and MHC class II expression is downregulated by TNF-α and IL-10, while activation of the mannose receptor (e.g., by bacterial walls) induces expression of IL-12, IL-1β, IL-6, und TNF-α. LSECs are affected by aging, leading to age- related pseudocapillarization of the sinusoids which is characterized by the loss of fenestration and deposition of collagen in the space of Dissé. NK and NKT cells, which are identified by expression of CD56, have the ability to quickly produce high amounts of cytokines. Their strategic localization in the sinusoids enables NK and NKT cells together with KCs and LSECs to provide an effective first-line innate immune defense against invading pathogens, toxins, food antigens, and circulating tumor cells (11). The liver is exposed to millions of antigens and exobiotics. If every contact would stimulate the immune system, the liver would be in a permanent state of inflammation. Therefore, one of the important functions of the hepatic immune system is the promotion of active tolerance. KCs are crucial for the development of hepatic antigen tolerance. Depletion of KCs impairs antigen tolerance leading to upregulation of T cells (12). Transformation of CD4 T cells to different T-helper (Th) cells or regulatory T (Treg) cells expressing different chemokines (Th1: IFN-γ, Th2: IL-4, IL-10, Th17: IL-17) plays a key role in liver immunotolerance. Short-term inhibition of T-cell stimulation by CTLA-4 and long-term inhibition by PD-1 are nonredundant mechanisms of enduring hepatic immunotolerance (13) (Fig. 2). 44 Sendensky and Dufour

+ + CD4 CD8 CD4+

CD8+

Immune filter/ tolerance CD4+ CD8+

CD4+CD25+Foxp3+

CD4+

KC DC CD4+

PD-L1 HSC CTLA-4 INF-β LSEC IL-10 Clonal deletion of CD8+/CD4+ by FaSL, TRAIL via KC/DC CD8+ Deviation of TH1/Th17 to Th2/Th0 + Hepatocyte CD8 TGF-β-dependent selection of CD4+Treg

Fig. 2. Immunofunctions of the liver. Mechanical filter function for portal venous inflow due to diameter of the sinusoids. Mechanisms of tolerance: CD4+CD25+Foxp3+ Treg cells (graveyard/killing field theory) regulate CD8+ and CD4+ numbers by cell contact, as do Th1 and Th3 cells. Hepatic immune deviation via LSEC “veto” and DC, suppressing IFN-γ-producing Th1-CD4 cells while engaging IL-4 and IL-10 producing Th2-CD4 cells. Furthermore, LSECs, KCs, hepatocytes, and stellate cells produce PD-L1 and CTLA-4, thus suppressing CD4- and CD8-cell function up to induction of apoptosis.

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1. Dawson PA, Lan T, Rao A. Bile acid transporters. J Lipid Res 2009;50:2340–57. 2. Thomas C, Pellicciari R, Pruzanski M, Auwerx J, Schoonjans K. Targeting bile- acid signalling for metabolic diseases. Nat Rev Drug Discov 2008;7:678–93. Liver Physiology 45

3. Viollet B, Foretz M, Guigas B, Horman S, Dentin R, Bertrand L, Hue L, et al. Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders. J Physiol 2006;574:41–53. 4. Genolet R, Michalik L, Wahli W. PPARs. In: Dufour J-F, Clavien PA, eds. Signaling Pathways in Liver Dieases, Springer Publishing, Heidelberg (Germany). 2005. 5. Lalioti V, Muruais G, Tsuchiya Y, Pulido D, Sandoval IV. Molecular mechanisms of copper homeostasis. Front Biosci 2009;14:4878–903. 6. Kamisako T, Kobayashi Y, Takeuchi K, Ishihara T, Higuchi K, Tanaka Y, Gabazza EC, et al. Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance. J Gastroenterol 2000;35:659–64. 7. Lieber CS. Metabolism of alcohol. Clin Liver Dis 2005;9:1–35. 8. Haussinger D. Liver regulation of acid-base balance. Miner Electrolyte Metab 1997;23:249–52. 9. Bhogal RH, Afford SC. Immune cell communication and signaling systems in liver diseases In: Dufour J-F, Clavien PA, eds. Signaling in Liver Diseases. 2nd ed. New York: Springer, 2009. 10. Ramaiah SK, Jaeschke H. Role of neutrophils in the pathogenesis of acute inﬂammatory liver injury. Toxicol Pathol 2007;35:757–66. 11. Notas G, Kisseleva T, Brenner D. NK and NKT cells in liver injury and ﬁbrosis. Clin Immunol 2009;130:16–26. 12. Racanelli V, Rehermann B. The liver as an immunological organ. Hepatology 2006;43:S54–62. 13. Fife BT, Bluestone JA. Control of peripheral T-cell tolerance and autoimmunity via the CTLA-4 and PD-1 pathways. Immunol Rev 2008;224:166–82.

Assessment of Liver Function in Clinical Practice

Hamed Khalili, MD, Barham Abu Dayyeh, MD, and Lawrence S. Friedman, MD

CONTENTS COMMONLY USED LIVER BIOCHEMICAL TESTS PATTERNS OF ABNORMAL LIVER BIOCHEMICAL TESTS EVA L UAT I O N F O R DRUG-INDUCED LIVER INJURY TESTS OF HEPATIC SYNTHETIC FUNCTION LIVER BIOPSY AND NONINVASIVE MARKERS OF LIVER FIBROSIS QUANTITATIVE LIVER BIOCHEMICAL TESTS CHILD–TURCOTTE–PUGH SCORE AND MODEL FOR END-STAGE LIVER DISEASE SCORE REFERENCES

Key Words: Alanine aminotransferase, Albumin, Alkaline phosphatase, Blood group, Isoenzyme, Pregnancy, Zinc deﬁciency, Aminotransferase, Normal values, AST:ALT ratio, ALT–LDH ratio, Antimitochondrial antibodies, AST-to-platelet ratio index, Benign recurrent, Intrahepatic cholestasis, Bilirubin, Biosynthesis, Delta bilirubin, Child–Turcotte–Pugh score, Cholestasis, Drug-induced liver injury, FibroTest (FibroSure), Fibroscan, Gamma glutamyl transpeptidase, Granulomatous liver disease, HALT-C model,

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_3, C Springer Science+Business Media, LLC 2011

47 48 Khalili et al.

Hyperbilirubinemia, Hy’s rule, Liver biochemical tests, Liver biopsy, indications, limitations, Liver function tests, Model for end-stage liver disease, Naranjo Adverse Drug Reactions Probability Scale, 5 Nucleotidase, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progressive familial intrahepatic cholestasis, Prothrombin time, Quantitative liver biochemical tests, Roussel Uclaf Causality Assessment Method, Van den Bergh method, vanishing bile duct syndrome, Wilson’s disease

A broad array of biochemical tests are used to assess the various functions of the liver and evaluate patients with suspected or established liver disease. These tests are collectively referred to as “liver function tests,” a term that is often criticized because the most commonly used tests—the aminotransferases and alkaline phosphatase—are not true measures of liver synthetic, excretory, or metabolic function (1). Rather, these values normally indicate hepatocyte damage or liver inflammation or infiltration. Although the term “liver function tests” is widely used in the medical literature, an alternative, perhaps preferable, term is “liver biochemical tests.” In clinical practice, liver biochemical tests are used to screen for the presence of liver disease as well as to determine etiology, severity, prognosis, and response to therapy; the pattern of abnormalities of liver biochemical tests may be characteristic of a specific or nonspecific disease. Abnormal liver biochemical test results may be the first indication of subclinical liver disease, and interpretation of them should be done in the context of an accurately obtained history and carefully performed physical examination. Pattern recognition and characteristic fluctuations may facilitate a pragmatic clinical evaluation that includes specific disease markers, imaging studies, and liver biopsy. In interpreting abnormalities in liver biochemical test results, one needs to understand their limitations. No liver biochemical test can evaluate the complete functional capacity of the liver. In addition, the limited sensitivities and specificities of individual tests make it important to use a battery of tests to evaluate a patient for liver disease.

1. COMMONLY USED LIVER BIOCHEMICAL TESTS 1.1. Aminotransferases Serum aminotransferases are considered the most sensitive markers of acute liver injury. These tests have been available since the 1950s (2). Alanine aminotransferase (ALT, formerly serum glutamic pyruvic transaminase or SGPT) and aspartate aminotransferase (AST, formerly serum glutamic oxaloacetic transaminase or SGOT) catalyze the transfer of the α-amino groups of alanine and L-aspartic acid, respectively, to the α-keto group of ketoglutaric acid. AST is predominantly a Assessment of Liver Function in Clinical Practice 49 mitochondrial enzyme and is also found in skeletal and cardiac muscles, brain, kidney, erythrocytes, and lung (3). ALT is a cytosolic enzyme that is more specific to the liver but found in other organs as well. Aminotransferases have no known function in serum, with relatively short half-lives in the order of days. Their elevation signifies active injury to the liver or other organs containing them; the level of elevation does not correlate with severity but may have diagnostic value (3, 4). Normal values vary among different laboratories, but values that have gained general acceptance are ≤30 U/L for men and ≤19 U/L for women. There is no standardized reference range for the upper limits of normal, and the variation of the upper limits of normal among laboratories is related to technical issues, with each laboratory responsible for identifying a normal range that is based on either the local population or the original standard range published in the 1950s. The normal values of aminotranferases may need to be adjusted based on sex and body mass index, but the significant of these changes remains unknown (5–8). Excessive fast-food intake can raise serum ALT levels within weeks (9), and initiation of acetaminophen use in a dose of 4 g daily often causes a rise in serum ALT levels acutely, with a subsequent return to normal despite continued use of acetaminophen (10). Coffee consumption (especially with caffeine) reduces serum ALT and AST activity (11). There is controversy as to whether serum ALT levels correlate with mortality and risk of coronary artery disease (12). Values below the normal range seem to have no important clinical consequences.

1.2. Alkaline Phosphatase Alkaline phosphatase is a group of isoenzymes that hydrolyze a number of phosphate esters and thereby generate inorganic phosphate at an alkaline pH for uptake by tissues. These enzymes are dependent on zinc for activity. They are distributed widely throughout the body, with the most clinically important isoenzymes found in the liver and bone. Other isoenzymes are found in the intestine, first-trimester placenta, and kidneys (13–15). The exact function of alkaline phosphatase is unknown but has been speculated to relate to detoxification of lipopolysaccharide (16). The enzyme is found predominantly on the canalicular membrane of hepatocytes. Elevation of the serum alkaline phosphatase level in liver disease seems to result from induction of synthesis of the enzyme by bile acids (17). The metabolism of alkaline phosphatase is likely not mediated by the liver or biliary system. A number of physiologic variables influence the normal range of alkaline phosphatase levels. People with blood groups O and B tend 50 Khalili et al. to have higher levels of circulating intestinal alkaline phosphatase, an effect that is more pronounced after consumption of a fatty meal (18). Values of alkaline phosphatase in serum can increase to as much as two times the upper limits of normal during the third trimester of pregnancy because of placental production of the enzyme (19, 20). Serum levels of alkaline phosphatase also correlate with bone growth, and adolescents can have alkaline phosphatase levels twice as high as those in adults (21). After age 60, levels of alkaline phosphatase in serum rise again, more so in women than in men. A number of diseases are associated with a low level of alkaline phosphatase, including Wilson’s disease (in which copper displaces zinc as a cofactor), perni- cous anemia, congenital hypophosphatemia, hypothyroidism, and zinc deficiency (22).

1.3. Gamma Glutamyl Transpeptidase Gamma glutamyl transpeptidase (GGTP) catalyzes the transfer of gamma glutamyl from peptides (such as glutathione) to other amino acids. GGTP is found in the cell membranes of a wide distribution of tissues, including liver (both hepatocytes and cholangiocytes), kidney, pancreas, spleen, heart, brain, and seminal vesicles. In the liver, GGTP is found in hepatocytes and epithelial cells of bile ducts. The primary use of serum GGTP measurements is to determine the source of an isolated elevation of the serum alkaline phosphatase level, because GGTP is not found in bone (23). GGTP is considered the most sensitive marker of biliary tract disease. Its ubiquitous nature (except in bone), together with induction of the enzyme by a number of drugs, most notably phenytoin and alcohol (24), make GGTP relatively nonspeciﬁc in clinical practice.

1.4. 5 Nucleotidase 5 Nucleotidase (5 NT) catalyzes the hydrolysis of nucleotides by removing a phosphate group from the 5 position of the pentose ring. 5 NT is associated with the canalicular and sinusoidal plasma membranes; its exact function is undeﬁned. 5 NT is also found in intestine, brain, heart, blood vessels, and endocrine pancreas (25). Despite wide distribution of the enzyme, elevation of the serum level of 5 NT seems to be fairly speciﬁc for a hepatobiliary source, because only liver tissue can secrete the enzyme into serum. Its primary use is to distinguish liver from bone as the source of an alkaline phosphatase elevation. Assessment of Liver Function in Clinical Practice 51

1.5. Bilirubin Bilirubin is primarily derived from the breakdown of heme. The initial steps of bilirubin metabolism occur in reticuloendothelial cells, predominantly in the spleen. Heme is converted to biliverdin by the microsomal enzyme heme oxygenase. Biliverdin is then converted to bilirubin by the cytosolic enzyme biliverdin reductase (26). In order for water-insoluble bilirubin to be transported in blood, it needs to be solubilized by binding to albumin; the bilirubin–albumin complex passes readily through the fenestrations in the endothelium lining the hepatic sinusoids into the space of Disse, where the bilirubin dissociates from albumin and is taken up by hepatocytes. The enzyme uridine-5-diphosphate (UDP)-glucuronyl transferase, found in the endoplasmic reticulum, conjugates bilirubin to glucuronic acid to produce bilirubin monoglucuronide and diglucuronide to make bilirubin water soluble (27). Conjugated billirubin is excreted into the bile canaliculi. Serum levels of bilirubin are measured routinely using the van den Bergh method, which permits photometric detection of the azo deriva- tives of bilirubin obtained by a reaction with the diazonium ion of sulfanilic acid (28). Conjugated bilirubin reacts directly with the diazo reagent to allow rapid measurement within 30–60 s. Total bilirubin is measured after addition of an accelerator, such as caffeine or methanol. The indirect bilirubin level is calculated as the difference between the total and direct bilirubin levels. Measurement of bilirubin levels by high-performance liquid chromatography suggests that the van den Bergh method overestimates conjugated bilirubin levels, which constitute only 4–5% of total bilirubin in normal serum. In cases of prolonged or severe elevation of the serum bilirubin level, delta bilirubin is formed by covalent bonding of conjugated bilirubin to albumin (29). This reaction signiﬁcantly increases the half-life of bilirubin from 4 h to 14–21 days (equal to the half-life of albumin).

2. PATTERNS OF ABNORMAL LIVER BIOCHEMICAL TESTS The liver is the largest discrete organ in the body, with a complex structure and function that is central to multiple physiologic processes—including blood filtration and storage; metabolism; formation of bile; synthesis of albumin, coagulation factors, and other proteins; and storage of iron and vitamins (see Chapter “Physiology of Liver Function”). Because of the complexity of the liver, no single laboratory or imaging test is sufficient to assess the total functional capacity 52 Khalili et al. of the liver. When used appropriately, however, liver biochemical testing can identify liver injury, suggest an underlying etiology, estimate severity, and monitor response to therapy. Given the large number of current (and novel) liver biochemical tests, it is often useful to organize them into the following categories: (a) tests that indicate direct hepatocellular damage, reflected by a predominant elevation of the serum aminotransferase (AST and ALT) levels; (b) tests that reflect cholestasis, with a predominant elevation of the serum alkaline phosphatase level; (c) tests that measure the capacity of the liver to transport organic anions and clear endogenous or exogenous substances from the circulation, such as the serum bilirubin level; (d) tests that estimate the synthetic function of the liver, such as the serum albumin level and prothrombin time; and (e) emerging tests that evaluate the severity and reversibility of liver injury, directly or indirectly. Additional biochemical and serologic blood tests are performed to determine the cause of abnormal liver biochemical test results.

2.1. Hepatocellular Pattern Serum aminotransferase elevations suggest hepatocellular injury but are nonspecific and are seen in many forms of liver injury. The magnitude and specific pattern of aminotransferase elevations in the clinical context can suggest a particular diagnose and direct further testing. Marked elevations of serum aminotransferase levels (>1,000 U/L) are often seen in acute viral hepatitis (A–E and herpes simplex virus), toxin- or drug-induced liver injury, ischemic hepatitis, autoimmune hepatitis, acute Wilson’s disease, acute Budd–Chiari syndrome, and acute obstruction of the biliary tract and after hepatic artery ligation (Table 1). More commonly, aminotransferase elevations are mild (less than fivefold the upper limit of normal) and occur in asymptomatic persons, who are discovered to have elevations on routine screening blood tests (Table 2). An evaluation of such mild elevations should be initiated if the elevations persist on repeat testing (in one third of cases the elevations may resolve) and should be interpreted in the context of the patient’s history and specific clinical presentation. Disorders that fall into this category include nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease, drug- and toxin-induced liver injury, chronic hepatitis B (occasionally with D) and C, acute Epstein–Barr virus (EBV) and cytomegalovirus (CMV) infections, hemochromatosis, alpha-1 antitrypsin deficiency, Wilson’s disease, autoimmune hepatitis, and celiac disease. The ratio of the AST level to ALT level in serum is often helpful diagnostically. Typically, most causes of mildly elevated aminotransferase Assessment of Liver Function in Clinical Practice 53

Table 1 Causes of marked aminotransferase elevations (>1000 U/L) and commonly used diagnostic tests

Cause Tests

Acute viral hepatitis IgM anti-HAV HBsAg, IgM anti-HBc Anti-HCV, HCV RNA Anti-HDV (if HBsAg positive) Consider anti-HEV (e.g., in travelers to endemic area) Consider PCR for HSV in plasma or skin lesions Toxins and drugs Acetaminophen level; other drug levels if indicated Ischemic hepatitis LDH Tests of renal function Autoimmune hepatitis ANA SMA Anti-SLA Anti-LKM SPEP Simplified diagnostic criteriaa Liver biopsy Fulminant Wilson’s disease Alkaline phosphatase/bilirubin ratio <4 AST:ALT ratio >2.2 Slit-lamp exam for Kayser–Fleischer rings Serum copper level Tests for hemolysis Tests for renal tubular dysfunction (glycosuria, hypophosphatemia, low serum uric acid) 24-h urinary copper Acute Budd–Chiari syndrome Abdominal ultrasonography with Doppler analysis of hepatic veinsb Biliary obstruction and hepatic Abdominal ultrasonography with Doppler artery ligation analysis ALT, alanine aminotransferase; ANA, antinuclear antibodies; anti-HAV, antibody to hepatitis A virus; anti-HBc, antibody to hepatitis B core antigen; anti-HDV, antibody to hepatitis D virus; anti-HEV, antibody to hepatitis E virus; anti-LKM, liver–kidney microsomal type 1 antibody; anti-SLA, antibody to soluble liver antigen; AST, aspartate aminotransferase; HBsAg, hepatitis B surface antigen; HSV, herpes simplex virus; LDH, lactate dehydrogenase; PCR, polymerase chain reaction; RNA, ribonucleic acid; SMA, smooth muscle antibodies, SPEP, serum protein electrophoresis aHennes EM, Zeniya M, Czaja AJ, et al. Simplified criteria for the diagnosis of autoimmune hepatitis. Hepatology 2008;48:169–76 bPlessier A, Valla DC. Budd-Chiari syndrome. Semin Liver Dis 2008;28:259–69 54 Khalili et al. a alcoholics) hepatic Wilson’s disease) Brain MRI or CT Consider genetic testing Liver biopsy with quantitative iron level AST:ALT ratio (usually >2 in alcoholic liver disease) Liver biopsy with hepatic copperSerum measurement copper Kayser–Fleischer rings (present in only 50% of patients with Table 2 Causes of mild chronic aminotransferase elevations (less than fivefold the upper limit of normal) and commonly used diagnostic tests Alcohol and drugs NA GGTP, carbohydrate-deficient transferrin (often elevated in CauseHepatitis CHepatitis BHemochromatosis Fe, Initial TIBC, tests Anti-HCV ferritin HBsAg, anti-HBc, anti-HBs HBeAg, anti-HBe, HBV DNA HFE gene mutations (C282Y and H63D) Subsequent tests HCV RNA and genotype NAFLDAutoimmune hepatitisAlpha-1 antitrypsinWilson’s disease ANA, SMA, and SPEP Alpha-1 antitrypsin level NA Ceruloplasmin Anti-SLA, anti-LKM, liver biopsy Protease inhibitor phenotyping 24-h urinary copper Liver imaging and liver biopsy Assessment of Liver Function in Clinical Practice 55 y b uncertain; their absence excludes the diagnosis of celiac disease Table 2 Small intestinal biopsy Consider testing for HLA-DQ2 or HLA-DQ8 when diagnosis is (Continued) TTG antibodies IgA level Kaukinen K, Partanen J, Maki M, et al. HLA-DQ typing in the diagnosis of celiac disease. Am J Gastroenterol 2002;97:695–9 Gow PJ, Smallwood RA, Angus PW, et al. Diagnosis of Wilson’s disease: an experience over three decades. Gut 2000;46:415–19. ALT, alanine aminotransferase; ANA, antinuclear antibodies; anti-HBe, hepatitis B e antibody; anti-HCV, antibody to hepatitis C virus; anti-LKM, a b liver–kidney microsomal antibody; anti-SLA, antibody to soluble liver antigen; anti-HBc, antibody to hepatitis B core antigen; anti-HBs, antibod to hepatitis B surfaceantigen; HBsAg, antigen; hepatitis AST, B aspartate surfaceimmunoglobulin aminotransferase; A; antigen; MRI, HCV, ATP, hepatitis magnetic adenosine C resonance triphosphate;serum virus; imaging; protein CT, NA, GGTP, electrophoresis; gamma not computed SMA, glutamyl applicable; tomography; smooth NAFLD, transpeptidase; muscle nonalcoholic HBeAg, HLA, antibodies; fatty human hepatitis TIBC, liver leucocyte total disease; B antigen; iron-binding RNA, e ribonucleic IgA, capacity; acid; TTG, SPEP, tissue transglutaminase CauseCeliac disease IgA anti-endomysial and Initial tests Subsequent tests 56 Khalili et al. levels, with the exception of alcoholic liver disease and muscle disorders, result in an AST:ALT ratio of less than 1. As chronic liver disease progresses to cirrhosis, however, this ratio becomes greater than 1 as a result of impaired functional hepatic blood flow and decreased hepatic sinusoidal uptake of AST (30). This evolution, as well as the thrombocytopenia often seen in patients with advanced liver fibrosis or cirrhosis, was the basis for the development of simple noninvasive markers of liver fibrosis, including the AST-to-platelet ratio index (APRI) and the Hepatitis C Antiviral Long-term Treatment against Hepatitis C (HALT-C) model, which utilizes the platelet count, AST:ALT ratio, and international normalized ratio (INR) (see 4.1 Prothrombin Time, later) (31, 32). Both models were derived and validated in a cohort of patients chronically infected with hepatitis C virus (HCV); however, their utility can also extend to NAFLD (33). Chronic alcohol use results in a deficiency of pyridoxal-5-phosphate required for ALT synthesis. Therefore, the AST:ALT ratio is often greater than 2 in patients with alcoholic liver disease (34). Striking elevations of serum levels of lactate dehydrogenase (LDH) are observed in ischemic hepatitis caused by shock or heart failure. The elevations can be explained by two mechanisms: leakage of the enzyme from damaged hepatocytes and transformation of pyruvate to lactate under anaerobic conditions, which is characteristic of conditions that cause ischemic hepatitis (35). Because of the short half-life of LDH in serum, LDH rises to high levels in serum shortly after an ischemic insult to the liver, with a return to normal more quickly than that of other liver biochemical test levels, such as ALT, after resolution of the insult (36). The ALT–LDH ratio is typically less than 1.5 (37), and the ALT–LDH index (serum ALT/(serum LDH – median of normal LDH) has been shown to have some value in predicting the early prognosis of acute liver injury; an ALT–LDH index value of less than 3 at 3 days predicts a favorable outcome (38). If an evaluation fails to identify the etiology of a patient’s aminotransferase elevations and extrahepatic causes, such as muscle disease, thyroid disorders, and adrenal insufficiency, are excluded, a liver biopsy for histologic evaluation is usually recommended (see Liver Biopsy and Noninvasive Markers of Liver Fibrosis, later). The most likely etiologies identified by biopsy are steatosis and steatohepatitis, but in about 10% of cases, an unexpected diagnosis is identified (39).

2.2. Cholestatic Pattern Cholestasis refers to acute or chronic impairment in bile ﬂow. Cholestasis may be caused by obstruction to bile ﬂow within the liver (intrahepatic) (Table 3) or outside the liver (extrahepatic) (Table 4). In Assessment of Liver Function in Clinical Practice 57

Table 3 Causes of intrahepatic cholestasis

Drugsa Bland cholestasis Anabolic steroids Estrogens Cholestatic hepatitis Angiotensin-converting enzyme inhibitors (captopril, enalapril) Antibiotics (amoxicillin–clavulanic acid, ketoconazole) Azathioprine Chlorpromazine Nonsteroidal anti-inﬂammatory drugs (sulindac, piroxicam) Vanishing bile duct syndrome Amoxicillin–clavulanic acid Chlorpromazine Dicloxacillin Erythromycin Flucloxacillin Granulomatous hepatitis Allopurinol Antibiotics (sulfonamides) Antiepileptics (carbamazepine, phenytoin) Cardiovascular agents (hydralazine, procainamide, quinidine) Phenylbutazone Primary biliary cirrhosis Primary sclerosing cholangitis Granulomatous liver disease Brucellosis Crohn’s disease Fungal infections (histoplasmosis, coccidiodomycosis) Heavy metal exposure (beryllium, copper) Hodgkin’s disease Idiopathic granulomatous hepatitis Leprosy Qfever Sarcoidosis Schistosomiasis Tuberculosis, Mycobacterium avium complex, Bacillus Calmette–Guerin 58 Khalili et al.

Table 3 (Continued)

Viral hepatitis Cytomegalovirus infection Epstein–Barr virus infection Hepatitis A Hepatitis B (and D) Hepatitis C Idiopathic adult ductopenia Genetic conditions Progressive familial intrahepatic cholestasis Type 1 (Byler’s disease) Type 2 Type 3 Benign recurrent intrahepatic cholestasis Type 1 Type 2 Cystic ﬁbrosis Malignancy Hepatocellular carcinoma Metastatic disease Non-Hodgkin’s lymphoma Paraneoplastic syndrome Prostate cancer Renal cancer Inﬁltrative liver disease Amyloidosis Lymphoma Intrahepatic cholestasis of pregnancy Total parenteral nutrition Graft-versus-host disease Sepsis

aCategorized by histologic pattern Source: Adapted from Pratt DS. Liver chemisty and function tests. In: Feldman, Friedman LS, Brandt LJ, eds. Sleisenger and Fordtran’s Gastrointestinal and Liver Disease: Pathophysiology/Diagnosis/Management, 9th ed., 2010, p. 1233. general, the liver biochemical test level pattern consistent with cholestasis is a serum alkaline phosphatase elevation out of proportion to the level of aminotransferase elevations. The serum bilirubin level may be elevated in addition to the alkaline phosphatase level. Assessment of Liver Function in Clinical Practice 59

Table 4 Extrahepatic causes of cholestatic liver enzyme elevations in adults

Intrinsic Autoimmune pancreatitis Cholangiocarcinoma Choledocholithiasis Immune-mediated duct injury Infections AIDS cholangiopathy Cytomegalovirus Cryptosporidium Microsporidia Ascaris lumbricoides Ampullary cancer Primary sclerosing cholangitis Extrinsic Gallbladder cancer Metastases, including portal adenopathy from metastases Mirizzi’s syndromea Pancreatic cancer Pancreatic pseudocyst Pancreatitis

AIDS, acquired immunodeﬁciency syndrome. aCompression of common hepatic duct by a stone in the neck of the gallbladder. Source: Adapted from Pratt DS. Liver chemisty and function tests. In: Feldman, Friedman LS, Brandt LJ, eds. Sleisenger and Fordtran’s Gastrointestinal and Liver Disease: Pathophysiology/Diagnosis/Management, 9th ed., 2010, p. 1233.

The first step in the evaluation of a patient with an isolated alkaline phosphatase elevation is to determine the tissue source, which can be accomplished most accurately by fractionating alkaline phosphatase using electrophoresis; each isoenzyme of alkaline phosphatase has a different mobility. Alternatively, a serum GGTP or 5 NT level can be measured; elevation of either suggests a hepatobiliary source of alkaline phosphatase, as occurs in up to two thirds of cases. The level of increase in the serum alkaline phosphatase may help determine the chronicity of disease. In more than 75% of cases, a greater than fourfold increase in the level of alkaline phosphatase is consistent with chronic cholestasis, whether intra- or extrahepatic. Imaging of the biliary tree, often initially by ultrasound, helps distinguish intrahepatic from extrahepatic 60 Khalili et al. cholestasis; absence of biliary ductal dilatation suggests an intrahepatic cause of cholestasis. In evaluating patients with intrahepatic cholestasis, particular attention needs to be focused on careful history taking, because medications are the most common causes of intrahepatic cholestasis. Information about use of over-the-counter medications, herbal preparations, pre- scription drugs, or illicit drugs, as well as the temporal relation of use with the elevated liver biochemical test levels, should be obtained (see Evaluation for Drug-Induced Liver Injury, later). Table 3 lists the medications most commonly associated with intrahepatic cholestasis. Diagnosis of medication-induced intrahepatic cholestasis can generally be made without the need for a liver biopsy. Liver biochemical tests should normalize on withdrawal of the offending agent, although the rate of normalization may be slow. A number of autoimmune diseases may be associated with intrahepatic cholestasis (see Table 3). Primary biliary cirrhosis (PBC) is an autoimmune disorder that predominantly affects middle-aged women and is characterized by T-cell-mediated destruction of intrahepatic ducts. In up to 95% of cases, antimitochondrial antibodies (AMAs) are found, and detection of AMAs in a patient with cholestasis is diagnostic of PBC (40). Primary sclerosing cholangitis (PSC) is a disease characterized by inflammation and fibrosis of intra- or extraheptic bile ducts (or both). PSC is associated with inflammatory bowel disease (usually ulcerative colitis) in 70% of cases and is found most commonly in men aged 20–50 years. The diagnosis of PSC is suggested by multiple intra- or extrahepatic biliary strictures on magnetic resonance cholangiopancreatography (MRCP) or endoscopic retrograde cholangiopancreatography (ERCP). PSC is a risk factor for cholangiocarcinoma. Intrahepatic cholestasis can also result from hepatic metastases and other infiltrative diseases of the liver, presumably because of localized biliary obstruction and damage. A cholestatic pattern can occur as part of a paraneoplastic syndrome in the absence of obvious evidence of hepatic metastasis in patients with renal carcinoma (Stauffer syndrome) and Hodgkin’s lymphoma. Granulomatous liver disease commonly presents with cholestasis. Hepatic granulomas are seen in 2.5–10% of liver biopsy specimens (41). Common causes of hepatic granulomas in the United States are sarcoidosis and tuberculosis; less common causes include syphilis and fungal infections (specifically histoplasmosis), in addition to PBC. Hepatic sarcoidosis is diagnosed on the basis of extrahepatic manifestations and the presence of granulomas on a liver biopsy Assessment of Liver Function in Clinical Practice 61 specimen. Hepatic sarcoidosis tends to be a benign disease, and its occurrence is infrequently an indication for treatment. Tuberculous hepatitis is the most common infectious cause of hepatic granulomas and is an indication for treatment. In hospitalized patients, high serum alkaline phosphastase levels (>1,000 U/L) may be caused by malignant intrahepatic biliary obtruc- tion, sepsis, ischemic cholangiopathy, and the acquired immunodeficiency syndrome with active infection (42). Infiltrative disorders like amyloidosis also cause such striking elevations. Among infectious causes of hepatitis, a number of bacterial and viral infections, including leptospirosis, EBV infection, and CMV infection, may present with a predominantly cholestatic (or mixed hepatocellular–cholestatic) pattern of liver biochemical test levels. Mild elevations of the alkaline phosphatase level in hospitalized patients with cirrhosis or heart failure are nonspecific and tend to be transient. Intrahepatic cholestasis can be familial with presentations ranging from benign disease that presents in adulthood to a progressive disorder that presents early in childhood. Progressive familial intrahepatic cholestasis (PFIC) refers to a heterogeneous group of autosomal recessive disorders of childhood that are caused by mutations in genes that encode hepatocellular transport proteins (43). In the presence of dilated intra- or extrahepatic biliary ducts on initial imaging, various causes of cholestasis should be considered (see Table 4). Evaluation of extrahepatic causes of cholestasis normally includes ERCP to look for stones, strictures, or a tumor, obtain tissue samples and cytology specimens, and relieve obstruction by therapeutic maneuvers. Endoscopic ultrasonography is also a useful diagnostic test in cases of biliary obstruction.

2.3. Hyperbilirubinemia The serum bilirubin level may be elevated in both hepatocellular and cholestatic disorders and therefore is not necessarily helpful in differentiating between the two. Dark-colored urine, however, indicates the presence of bilirubinuria and thus conjugated hyperbilirubinemia. Hyperbilirubinemia may be the result of excessive breakdown of hemoglobin, as occurs with hemolysis, impaired hepatocellular uptake of bilirubin as a result of reduced UDP-glucuronyl transferase activity, defective conjugation of bilirubin, reduced excretion of bilirubin into the bile canaliculi, as in disorders that affect multidrug resistance- associated protein (MRP2) activity, and hepatocyte or bile duct injury, as described earlier. 62 Khalili et al.

Disorders that cause hyperbilirubinemia can be divided into those associated with predominantly unconjugated hyperbilirubinemia (in which less than 15% of the total bilirubin is conjugated) and those associated with both unconjugated and conjugated hyperbilirubinemia (in which greater than 15% of total bilirubin is conjugated). These disorders are listed in Table 5.

Table 5 Causes of hyperbilirubinemia

Unconjugated hyperbilirubinemia Mixed hyperbilirubinemia (less than 15% conjugated) (more than 15% conjugated)

Hemolysis Intrahepatic cholestasis Glucose-6-phosphate Cirrhosis dehydrogenase deficiency Hepatitis Hypersplenism Medications and toxins Immune-mediated hemolysis Primary biliary cirrhosis Paroxysmal nocturnal Extrahepatic biliary hemoglobinuria obstruction Sickle cell anemia Choledocholithiasis Spherocytosis and elliptocytosis Neoplasm Toxins Primary sclerosing Ineffective erythropoesis cholangitis Cobalamin deficiency Stricture Folate deficiency Other causes Profound iron deficiency Dubin–Johnson or Rotor’s Thalassemia syndrome Resorption of large hematoma Sepsis Impaired hepatocellular uptake Total parenteral nutrition Crigler–Najjar syndrome Type I: absence of UDP-glucuronyl transferase activity Type II: <10% UDP-glucuronyl transferase activity Gilbert’s syndrome (polymorphism in the TATA box of the gene encoding UDP-glucuronyl transferase) Drugs: probenecid, ribavirin, rifampin Shunt hyperbilirubinemia UDP, uridine-5 -diphosphate Assessment of Liver Function in Clinical Practice 63

Clinically, apparent jaundice is seen when the serum bilirubin level exceeds 3 mg/dL. In patients with hemolysis, the serum total bilirubin level does not usually exceed 6 mg/dL. High bilirubin levels (>30 mg/dL) can be seen in patients with hemolysis and concomitant renal failure or biliary obstruction and in patients with hemoglobinopathies such as sickle cell anemia in whom obstructive jaundice or acute hepatitis develops.

3. EVALUATION FOR DRUG-INDUCED LIVER INJURY Drug-induced liver injury (DILI) is a common cause of elevated liver biochemical test levels. The spectrum of liver injury and clinical presentations of patients with DILI is variable and includes acute hepatitis, fatty liver, cholestasis, granulomas, tumors, vascular lesions, fibrosis and cirrhosis, and acute liver failure (44). According to the Councils for International Organizations of Medical Sciences (CIOMS), the pattern of the biochemical abnormalities observed with DILI can be classified as hepatocellular, cholestatic, or mixed (45). In clinical practice, the diagnosis of DILI is often challenging, and the course in an individual patient is variable and depends on the patient’s age, ethnicity, genetic polymorphisms in drug-metabolizing enzymes, concomitant liver disease, nutritional status, and alcohol intake. Common culprits include acetaminophen, anti-inflammatory drugs, antiretroviral drugs, antibiotics, lipid-lowering agents, antitu- berculosis agents, anticonvulsants, cancer medications, and herbal supplements. Multiple scales have been developed to aid in the diagnosis of DILI. These include the Naranjo Adverse Drug Reactions Probability Scale (NADRPS) and the Roussel Uclaf Causality Assessment Method (RUCAM) (46, 47). In clinical practice, however, these scales are not widely used because of their lack of sensitivity and their complexity, as well as the requirement for rechallenge with the offending agent as a diagnostic criterion, which is often not feasible. Therefore, most clinicians still use their clinical judgment to diagnose DILI. A high index of suspicion for the diagnosis of DILI should be maintained in any patient with unexplained liver biochemical test abnormalities, especially when the clinician observes a temporal relation between the patient’s exposure to the drug and the liver biochemical test abnormalities, the presence of rash or eosinophilia, or a mixed hepatocellular and cholestatic pattern of abnormalities and when other causes of the liver biochemical test abnormalities are excluded. In Japan, the peripheral blood of patients with suspected DILI has been tested for leucocyte migration or lymphocyte stimulation using 64 Khalili et al. the leucocyte migration test (LMT) or drug lymphocyte stimulation test (DLST), respectively, after challenge with a solution of the suspected drug. The variable sensitivities and specificities of the tests have discouraged their widespread use in clinical practice (48). Liver biopsy is often performed in suspected cases of DILI. In the majority of cases, the liver biopsy specimen is not diagnostic for DILI but excludes other forms of liver disease, such as autoimmune hepatitis. Histopathologic features observed in patients with DILI include demarcated perivenular necrosis, minimal hepatitis with canalicular cholestasis, a poorly developed portal inflammatory reaction, abundant polymorphonuclear neutrophils, abundant eosinophils, and epithelioid cell granulomas (49). The prognosis of patients with DILI and the natural history of the liver injury after the cessation or continuation of a suspected offending are not well established. Hy’s rule [or Hy’s law] (after the late Hyman Zimmerman, MD) states that elevation of liver enzymes (an AST or ALT level of more than 3 times the upper limit of normal or an alkaline phosphatase level of more than 1.5 times the upper limit of normal) in combination with an elevated bilirubin level (more than three times the upper limit of normal) at any time after a new drug is started implies serious liver injury with a mortality rate of at least 10% (50). Several reports in the literature have confirmed high mortality rates associated with DILI and jaundice, as predicted by Hy’s rule (51, 52).

4. TESTS OF HEPATIC SYNTHETIC FUNCTION 4.1. Prothrombin Time The liver synthesizes all the coagulation factors except factor VIII, which is produced by endothelial cells. Parameters used to quantify blood coagulation include the prothombin time, partial thromboplastin time (PTT), and INR (see Chapter “Haemostasis Abnormalities in Chronic Liver Failure”). The prothrombin time is measured as the rate at which prothrombin is converted to thrombin. This reaction is dependent on coagulation factors I, II, V, VII, and X; therefore, deficiency in any one of these factors leads to a prolonged prothrombin time. The INR standardizes the prothrombin time measurements according to the thromboplastin reagent used in each laboratory. The INR is commonly reported with the prothrombin time. The INR was designed to standardize prothrombin time measurements in patients taking warfarin. The prothrombin time measurement varies depending on the type of analytical system used. This variation is exclusively the result of differences among manufacturer’s batches of tissue factor used Assessment of Liver Function in Clinical Practice 65 to perform the test. In order to normalize these measurements, each manufacturer assigns an international sensitivity index (ISI) value to the tissue factor used in the test. The ISI value indicates how a particular batch of tissue factor compares with an internationally standardized sample. The ISI is usually between 1.0 and 2.0. INR is subsequently calculated as the ratio of a patient’s prothrombin time to a normal (control) sample, adjusted for the ISI value for the analytical system used. The validity of using the ISI in measuring the INR in patients with chronic liver disease has come under scrutiny, however, because ISI values have only been validated in patients on chronic anticoagulation. Two studies have demonstrated that the standard method to calculate INR in patients with chronic liver disease is not accurate, with the recommendation that specific ISI and INR determinations using control patients with liver disease be used to eliminate interlaboratory variability in calculating the INR in patients with cirrhosis (53, 54). A prolonged prothrombin time is not specific for liver disease and can be seen with vitamin K deficiency, sepsis (disseminated intravascular coagulation), and congenital clotting deficiencies. Because factors II, VII, IX, and X are dependent on vitamin K for their function, measurement of vitamin K levels can help distinguish vitamin K deficiency from liver disease. In clinical practice, however, partial correction of a prolonged prothrombin time (specifically, a 30% improvement in the prothrombin time) following intravenous administration of vitamin K confirms vitamin K deficiency (55). Oral vitamin K is not absorbed by the small intestine in patients with obstructive jaundice and therefore should be avoided in patients with jaundice. Considering the short half-life of the coagulation factors, specifically factor VII, in serum, the prothrombin time is a good measure of acute liver injury. The prothrombin time is used to predict the outcome of patients with acute alcoholic hepatitis and acute liver failure. A prolonged prothrombin time is associated with a poor long-term outcome in patients with chronic liver disease and an increase in perioperative mortality in patients with liver disease who undergo surgery (see Child–Turcotte–Pugh Score and Model for End-Stage Liver Disease Score, later). On the other hand, prolongation of the prothrombin time in patients with liver disease does not correlate with the risk of bleeding, because of counterbalancing alterations in levels of factors that contribute to fibrinolysis (56). The PTT is used to assess the intrinsic coagulation pathway. In clinical practice, the PTT is used to measure the degree of anticoagulation on heparin. Although the PTT can be elevated in patients with advanced liver disease, it is not considered an accurate measure of coagulopathy. 66 Khalili et al.

4.2. Albumin Albumin accounts for 75% of plasma oncotic pressure and quantitatively is the most important plasma protein. It is produced exclusively by hepatocytes. The average adult produces 12–15 g of albumin daily, an amount that accounts for less than 5% of the total albumin pool of the body (57). The long half-life (∼20 days) of albumin, together with the large storage pool in the body, makes albumin an unreliable indicator of hepatic synthetic function in patients with acute liver injury. On the other hand, in patients with chronic liver disease, the serum albumin level is a good reﬂection of hepatic synthetic function. The presence of ascites in patients with cirrhosis can decrease the albumin concentration in serum substantially secondary to an increase in the volume of distribution. Nonhepatic causes of hypoalbuminemia include nephrotic syndrome, protein-losing enteropathy, and disorders associated with a chronic systemic inﬂammatory response. Globulins form the other main component of plasma proteins. Globulin levels are elevated in patients with chronic liver disease probably as a result of the inability of reticuloendothelial cells of the hepatic sinusoids to clear intestinal antigens from the portal circulation. Autoimmune hepatitis is characterized by marked elevations in serum globulin levels, usually immunoglobulin (Ig) M and IgG. The globulin levels tend to normalize with therapy. In alcoholic cirrhosis, elevations in serum IgA levels are observed (58), in contrast to cryptogenic cirrhosis in which IgG levels are predominantly elevated. In patients with PBC, levels of IgM are elevated. Although hyperglobulinemia is seen commonly in patients with chronic liver disease, it seems to play no role in the evaluation of etiology or severity.

5. LIVER BIOPSY AND NONINVASIVE MARKERS OF LIVER FIBROSIS Liver biopsy plays an important role in the evaluation of patients with liver disease, despite some limitations and its invasive nature. Indications for and contraindications to liver biopsy are listed in Table 6. Liver biopsy is generally considered the gold standard for the assessment of hepatic ﬁbrosis and cirrhosis. In patients with chronic hepatitis C, the stage of liver ﬁbrosis provides important prognostic information and contributes information relevant to the selection of patients for antiviral treatment with peginterferon and ribavirin (59, 60). Furthermore, liver biopsy remains critical in the categorization of NAFLD into steatosis, with a generally benign natural history course, Assessment of Liver Function in Clinical Practice 67

Table 6 Liver biopsy: indications and contraindications

Indications Contraindications

Evaluation of abnormal liver Absolute biochemical test levels Coagulopathy Evaluation and staging of chronic Prothrombin time >3–4 s over hepatitis control 3 Identification and staging of Platelets <60,000/mm alcoholic liver disease Prolonged bleeding time Evaluation of fever of unknown Unavailability of blood transfusion origin support Evaluation of the type and extent of Presumed hemangioma drug-induced liver injury Identification and determination of Relative Ascites nature of intrahepatic masses Infections in right pleural cavity Diagnosis of multisystem infiltrative Infection below right diaphragm disorders Suspected echinococcal disease Evaluation and staging of cholestatic liver disease Recognition of systemic inflammatory or granulomatous disorders Screening of relatives of patients with familial diseases Acquisition of tissue to culture infectious agents Evaluation of effectiveness of therapies for liver diseases Evaluation of status of the liver graft following transplantation

and steatohepatitis, which is often progressive and can lead to cirrhosis (61). Finally, liver biopsy is crucial for the determination of the nature of liver injury after liver transplantation and the diagnosis of graft rejection. Limitations of liver biopsy include complications resulting from its invasive nature, sampling error, and interobserver variability in interpreting the results. Complications of liver biopsy include pain, which is reported in up to 30% of patients; bleeding; bile peritonitis; puncture of a lung, the colon, or a kidney; formation of an intrahepatic arteriovenous ﬁstula; infection; and seeding of the biopsy tract with tumor. 68 Khalili et al.

Serious complications requiring hospital admission occur in 1–5% of patients, with an associated mortality rate of 0.1–0.01% (62). Cirrhosis may be missed, when present, in 10–30% of cases and is found in one lobe of the liver, but not the other, in 14.5% of cases (63, 64). Although most studies have shown excellent interobserver reproducibility for the staging of fibrosis, the assessment of hepatic inflammatory activity is more variable (65). Because of the limitations of liver biopsy, newer noninvasive tests and prediction models for liver fibrosis based on serum biochemical markers have been developed, with variable sensitivities and specificities (Table 7)(32, 66–84). These tests may be direct and indirect

Table 7 Noninvasive tests for liver ﬁbrosis

Area under the receiver Reference operating curve (predictive accuracy)a

Indirect tests NAFLD Fibrosis 0.88 66 Score Forns indexb 0.86 67 FIB-4c 0.85 68 FibroTest 0.84 69 (FibroSure)d FibroIndexe 0.83 70 APRI 0.80 71 Model 3 (HALT-C 0.78 32 Trial)f Platelets count 0.71 72 AST:ALT ratio 0.57 72

Direct tests Hyaluronic acid 0.86 73 YKL-40 0.81 74 Matrix 0.71 75 metalloproteinases and inhibitors (TIMP-1 and MMP-2) PIIINP 0.69 73 Type I and Type IV NA 76, 77 collagens Assessment of Liver Function in Clinical Practice 69

Table 7 (Continued)

Area under the receiver Reference operating curve (predictive accuracy)a

Laminin NA 78 TGF-β NA 79 Combined tests Fibrometerg 0.88 80 Hepascoreh 0.85 81 Fibrospect IIi 0.83 82 Enhanced liver 0.78 83 ﬁbrosis (ELF)j SHASTA index NA 84

aRange, 0–1 bPlatelets, gamma glutamyl transpeptidase, age, cholesterol cAge, AST, ALT, platelet count d α2-Macroglobulin, haptoglobulin, apoliprotein A1, bilirubin, gamma glutamyl transpeptidase eAST, platelet count, gamma globulins fHyaluronic acid, TIMP-1, platelet count g Hyaluronic acid, AST, platelet count, prothrombin time, α2-macroglobulin, urea, age h Age, gender, hyaluronic acid, α2-macroglobulin, bilirubin i Hyaluronic acid, TIMP-1, α2-macroglobulin jHyaluronic acid, PIIINP, TIMP-1 ALT, alanine aminotransferase; APRI, aspartate aminotransferase-to-platelet ratio index; AST, aspartate aminotransferase; HALT-C, Long-term Treatment against Hepatitis C; NAFLD, nonalcoholic fatty liver disease; MMP-2, matrix metalloproteinase 2; NA, not available; PIIINP, procollagen III amino terminal peptide; SHASTA, serum levels of hyaluronic acid, albumin, and AST; TGF, transforming growth factor; TIMP-1, tissue inhibitor of matrix metalloproteinases measures of hepatic fibrosis. Direct tests measure extracellular liver matrix proteins, and their concentrations correlate with the stage of liver fibrosis. Indirect tests utilize routine laboratory parameters as surrogate markers of liver fibrosis. Progressive accumulation of collagen in the liver parenchyma causes the stiffness of the liver to increase in chronic liver disease. Transient elastography (Fibroscan) utilizes vibration waves applied through a transcutaneous probe to the right thoracic wall at the level of the right liver lobe to estimate the stiffness of the liver parenchyma by measuring the velocity of the propagation of vibration waves. The measurements are expressed in kilopascals, with a range from 2.5 to 74 kPa (85). The 70 Khalili et al. test has a high negative predictive value for excluding cirrhosis, but its accuracy for diagnosing significant fibrosis is affected by the patient’s body mass index and waist circumference and by operator experience (86).

6. QUANTITATIVE LIVER BIOCHEMICAL TESTS Hepatic metabolic function may be quantified with the use of various agents that are excreted or detoxified by the liver and whose clearance can be measured (Table 8)(87). These tests have gained acceptance in Europe but are not used in clinical practice in the United States, in part because the precise roles and significance of the tests remain unclear. In addition, the tests are generally expensive and labor intensive. It has been suggested that quantitative liver biochemical tests may be useful surrogate markers for endpoints in controlled clinical trials.

Table 8 Quantitative liver biochemical tests

Test Method Analysis Interactions Safety

Indocyanine Liver plasma ﬂow Simple Few Uncertain green measurement; serum clearance collected at 3.6 and 49 min Aminopyrine Breath test repeated Complex Many Uncertain breath test three times; 10-min intervals Galactose IV administration of Simple None Safe elimination galactose; serum capacity collected at 5, 25, and 45 min Caffeine breath Oral caffeine; breath Simple Many Safe test sample collected before and after 60 min Lidocaine IV lidocaine; seruma Simple Many Uncertain metabolite collected at 15 and test 30 min

aFor monoethylglycinexylidide IV, intravenous Assessment of Liver Function in Clinical Practice 71

7. CHILD–TURCOTTE–PUGH SCORE AND MODEL FOR END-STAGE LIVER DISEASE SCORE The Child (or Child–Turcotte) score was initially developed by Child and Turcotte in 1964 to risk-stratify patients undergoing portacaval shunt surgery. The initial scoring system was criticized because of ambiguity of some of the parameters (e.g., degree of encephalopathy or malnutrition). In 1972, Pugh and colleagues modified the Child score to its current form, now termed the Child–Turcotte–Pugh (CTP) score. The CTP scoring system is based on five parameters: serum albumin, serum bilirubin, prothrombin time, ascites, and encephalopathy; the score is calculated by adding the points assigned to each of these parameters. On the basis of the CTP score, patients are categorized into one of three Child (or Child–Pugh) classes: A, B, or C (Table 9). The CTP score has traditionally been used by clinicians to predict mortality in patients with cirrhosis. In addition, the CTP score has been validated as a predictor of perioperative mortality in patients with cirrhosis undergoing nonshunt surgery, with perioperative mortality rates for patients with Child’s class A, B, and C cirrhosis estimated to be 10, 30–31%, and 76–82%, respectively (88, 89), although lower rates have been reported in a 2010 study (90). The CTP scoring system has a number of limitations, including the subjectivity of some of the parameters, limited discriminating capacity among patients with chronic liver disease, and use of variables such as albumin and prothrombin time with significant interlaboratory variation.

Table 9 Child–Turcotte–Pugh classiﬁcation

Score 12 3 Bilirubin (mg/dL) <2.0 2.0–3.0 >3.0 Albumin (g/dL) >3.5 2.8–3.5 <2.8 Ascites None Easily controlled Poorly controlled Encephalopathy None Mild Moderate/severe Prothrombin time 1–3 4–6 >6 (seconds prolonged) Total score Child–Pugh class 5–6 A 7–9 B 10–15 C 72 Khalili et al.

The Model for End-stage Liver Disease (MELD) score was initially developed to predict the outcome of patients with cirrhosis undergoing transjugular portosystemic shunt (TIPS) placement (91). The model has since been validated to assess the survival of patients with advanced liver disease. The MELD score is comprised of three variables: INR, serum creatinine, and total bilirubin. A mathematical model is used to calculate the MELD score, with a working score range of 6–40. Besides its use in assessing the prognosis of patients with advanced liver disease, the MELD score has been shown to correlate well with mortality in cirrhotic patients undergoing surgery (90). The United Network of Organ Sharing uses the MELD score for prioritizing the allocation of donor organs for liver transplantation.

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55. Lord JW, Andrus W. Differentiation of intrahepatic and extrahepatic jaundice. Response of the plasma prothrombin to intramuscular injemction of menadione (2-methyl-1, 4-naphthoquinone) as a diagnostic aid. Arch Intern Med 1941; 68:199. 56. Tripodi A, Caldwell SH, Hoffman M, et al. Review article: the prothrombin time test as a measure of bleeding risk and prognosis in liver disease. Aliment Pharmacol Ther 2007;26:141–8. 57. Quinlan GJ, Martin GS, Evans TW. Albumin: biochemical properties and therapeutic potential. Hepatology 2005;41:1211–19. 58. Martin DM, Vroon DH, Nasrallah SM. Value of serum immunoglobulins in the diagnosis of liver disease. Liver 1984;4:214–18. 59. Yano M, Kumada H, Kage M, et al. The long-term pathological evolution of chronic hepatitis C. Hepatology 1996;23:1334–40. 60. Strader DB, Wright T, Thomas DL, et al. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004;39:1147–71. 61. Vuppalanchi R, Chalasani N. Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis: selected practical issues in their evaluation and management. Hepatology 2009;49:306–17. 62. Thampanitchawong P, Piratvisuth T. Liver biopsy: complications and risk factors. World J Gastroenterol 1999;5:301–4. 63. Regev A, Berho M, Jeffers LJ, et al. Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterol 2002;97:2614–18. 64. Maharaj B, Maharaj RJ, Leary WP, et al. Sampling variability and its influence on the diagnostic yield of percutaneous needle biopsy of the liver. Lancet 1986;1: 523–5. 65. Goldin RD, Goldin JG, Burt AD, et al. Intra-observer and inter-observer variation in the histopathological assessment of chronic viral hepatitis. J Hepatol 1996;25: 649–54. 66. Angulo P, Hui JM, Marchesini G, et al. The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD. Hepatology 2007;45:846–54. 67. Forns X, Ampurdanès S, Llovet JM, et al. Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model. Hepatology 2002;36(4 Pt 1):986–92. 68. Vallet-Pichard A, Mallet V, Nalpas B, et al. FIB-4: an inexpensive and accurate marker of fibrosis in HCV infection. Comparison with liver biopsy and fibrotest. Hepatology 2007;46:32–6. 69. Halfon P, Bourliere M, Deydier R, et al. Independent prospective multicenter validation of biochemical markers (fibrotest-actitest) for the prediction of liver fibrosis and activity in patients with chronic hepatitis C: the fibropaca study. Am J Gastroenterol 2006;101:547–55. 70. Koda M, Matunaga Y, Kawakami M, et al. FibroIndex, a practical index for predicting significant fibrosis in patients with chronic hepatitis C. Hepatology 2007;45:297–306. 71. Wai CT, Greenson JK, Fontana RJ, et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology 2003;38:518–26. 72. Lackner C, Struber G, Liegl B, et al. Comparison and validation of simple noninvasive tests for prediction of fibrosis in chronic hepatitis C. Hepatology 2005;41:1376–82. 76 Khalili et al.

73. Guéchot J, Laudat A, Loria A, et al. Diagnostic accuracy of hyaluronan and type III procollagen amino-terminal peptide serum assays as markers of liver fibrosis in chronic viral hepatitis C evaluated by ROC curve analysis. Clin Chem 1996;42: 558–63. 74. Saitou Y, Shiraki K, Yamanaka Y, et al. Noninvasive estimation of liver fibrosis and response to interferon therapy by a serum fibrogenesis marker, YKL-40, in patients with HCV-associated liver disease. World J Gastroenterol 2005;11: 476–81. 75. Boeker KH, Haberkorn CI, Michels D, et al. Diagnostic potential of circulating TIMP-1 and MMP-2 as markers of liver fibrosis in patients with chronic hepatitis C. Clin Chim Acta 2002;316:71–81. 76. Hahn E, Wick G, Pencev D, et al. Distribution of basement membrane proteins in normal and fibrotic human liver: collagen type IV, laminin, and fibronectin. Gut 1980;21:63–71. 77. Trinchet JC, Hartmann DJ, Pateron D, et al. Serum type I collagen and N-terminal peptide of type III procollagen in chronic hepatitis. Relationship to liver histology and conventional liver tests. J Hepatol 1991;12:139–44. 78. Misaki M, Shima T, Yano Y, et al. Basement membrane-related and type III procollagen-related antigens in serum of patients with chronic viral liver disease. Clin Chem 1990;36:522–4. 79. Nelson DR, Gonzalez-Peralta RP, Qian K, et al. Transforming growth factor-beta 1 in chronic hepatitis C. J Viral Hepat 1997;4:29–35. 80. Calès P, Oberti F, Michalak S, et al. A novel panel of blood markers to assess the degree of liver fibrosis. Hepatology 2005;42:1373–81. 81. Adams LA, Bulsara M, Rossi E, et al. Hepascore: an accurate validated predictor of liver fibrosis in chronic hepatitis C infection. Clin Chem 2005;51:1867–73. 82. Patel K, Gordon SC, Jacobson I, et al. Evaluation of a panel of non-invasive serum markers to differentiate mild from moderate-to-advanced liver fibrosis in chronic hepatitis C patients. J Hepatol 2004; 41:935–42. 83. Rosenberg WM, Voelker M, Thiel R, et al. European Liver Fibrosis Group. Serum markers detect the presence of liver fibrosis: a cohort study. Gastroenterology 2004;127:1704–13. 84. Kelleher TB, Mehta SH, Bhaskar R, et al. Prediction of hepatic fibrosis in HIV/HCV co-infected patients using serum fibrosis markers: the SHASTA index. J Hepatol 2005;43:78–84. 85. Del Poggio P, Colombo S. Is transient elastography a useful tool for screening liver disease? World J Gastroenterol 2009 Mar 28;15:1409–14. 86. Castéra L, Foucher J, Bernard PH, et al. Pitfalls of liver stiffness measurement: a 5-year prospective study of 13,369 examinations. Hepatology 2009 [Epub ahead of print]. 87. Morris-Stiff G, Gomez D, Prasad R. Quantitative assessment of hepatic function and its relevance to the liver surgeon. J Gastrointest Surg 2009;13:374–85. 88. Mansour A, Watson W, Shayani V, et al. Abdominal operations in patients with cirrhosis: still a major surgical challenge. Surgery 1997;122:730–5; discussion 735–6. 89. Garrison RN, Cryer HM, Howard DA, et al. Clarification of risk factors for abdominal operations in patients with hepatic cirrhosis. Ann Surg 1984;199:648–55. 90. Telem DA, Schiano T, Goldstone R, et al. Factors that predict outcome of abdominal operations in patients with advanced cirrhosis. Clin Gastroenterol Hepatol 2010;8:451–7. 91. Kamath PS, Kim WR. The model for end-stage liver disease (MELD). Advanced Liver Disease Study Group. Hepatology 2007;45:797–805. Physiology of the Splanchnic and Hepatic Circulations

Gautam Mehta, Juan-Carlos García-Pagán, and Jaime Bosch

CONTENTS INTRODUCTION ANATOMY OF THE SPLANCHNIC CIRCULATION REGULATION OF SPLANCHNIC BLOOD FLOW POSTPRANDIAL HYPEREMIA LOCAL REGULATION OF SPLANCHNIC BLOOD FLOW AUTOREGULATION OF SPLANCHNIC BLOOD FLOW EXTRINSIC REGULATION OF SPLANCHNIC BLOOD FLOW THE HEPATIC ARTERIAL BUFFER SYSTEM REGULATION OF SINUSOIDAL BLOOD FLOW SUMMARY REFERENCES

Key Words: Celiac trunk, Superior mesenteric artery, Inferior mesenteric artery, Splanchnic blood ﬂow, Indocyanine green, Postprandial hyperemia, Myogenic control, Autoregulation, Hepatic arterial buffer, Portal vein, Liver blood ﬂow, Adenosine, Space of Mall, Hepatic stellate cell, Nitric oxide

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_4, C Springer Science+Business Media, LLC 2011

77 78 Mehta et al.

1. INTRODUCTION The splanchnic and hepatic circulations account for over a quarter of cardiac output and are highly specialized to meet the demands of these complex and complementary organs. The hepatic circulation is fundamental to liver function, and the splanchnic circulation participates in the control of systemic hemodynamics as well as perfusion of the intestine. While blood ﬂow to these organs is anatomically and physiologically related, the regulatory mechanisms of these circulatory systems are uniquely adapted to preserve organ function. This chapter reviews the anatomy and physiology of the splanchnic and hepatic circulations, with reference to physiological studies in animals and humans. The effects of liver failure on these circulatory systems are covered in later chapters of this book.

2. ANATOMY OF THE SPLANCHNIC CIRCULATION The arterial blood supply to the splanchnic organs originates from the celiac trunk and the superior and inferior mesenteric arteries. The celiac trunk supplies the stomach, liver, and spleen, while the superior mesenteric artery supplies the entire small intestine, proximal colon, and pancreas. The inferior mesenteric artery supplies the remainder of the colon and rectum except the distal rectum, which is supplied by the internal iliac arteries (Fig. 1). Along the mesenteric border of the intestine, the arterial and venous branches anastomose to form multiple arcades, eventually giving rise to vasa recta which branch to encircle the intestine and ultimately enter the circular muscle forming a plexus in the submucosa. The branching pattern of the intestinal microvasculature was described by Bohlen and Gore (1) and is tailored to the absorptive

Fig. 1. Anatomy of the portal venous (panel A) and splanchnic (panel B) circulations. Physiology of the Splanchnic and Hepatic Circulations 79 and secretory functions of the organ. The vasculature can accommodate variable rates of flow, has a large surface area for absorption, and has greatest blood flow in the areas of functional importance, such as the submucosa, mucosa, and small intestinal villi. Increased splanchnic blood flow is accommodated by recruitment of vessels, diverting blood to the tips of intestinal villi (2, 3). The artery and vein run in parallel along the villus, leading to a countercurrent mechanism of oxygen exchange. However, this contributes to a descending gradient of PO2 from the base of the villus to the tip, which may render the mucosa susceptible to ischemia in the context of decreased splanchnic flow. Splanchnic hypoperfusion has been suggested to be a trigger for gut ischemia–reperfusion injury, with a resultant loss of gut barrier function with bacterial endotoxemia and systemic inflammation (see Chapter “SIRS, bacterial infections, and alterations of the immune system”) (4).

Fig. 2. Indocyanine green (ICG) method of measuring hepatosplanchnic blood ﬂow. ICG is a nontoxic dye, which is exclusively metabolized by the liver in humans. Following an intravenous infusion at a rate (R) below the metabolic capacity of the liver, a steady state is reached within 1 h. ICG levels are then measured in the hepatic vein (HV) and peripheral blood. Since ICG is not metabolized elsewhere, the ICG concentration in peripheral blood is equivalent to that in the hepatic artery (HA) or the portal vein (PV). Therefore, hepatic blood ﬂow can be calculated by the Fick principle: Flow = R/(ICGPeripheral – ICGHV). 80 Mehta et al.

Studies of pressure and flow in the splanchnic circulation in humans are technically limited, due to the diversity of vessels and mixing of the arterial and portal venous circulations within the liver. However, since hepatic venous outflow from the liver represents the sum of hepatic arterial inflow and portal venous inflow from the splanchnic circulation, total hepatosplanchnic blood flow can be estimated by sampling from the hepatic vein. Applying the Fick principle to substances that are exclusively metabolized by the liver and are distributed in plasma (e.g., indocyanine green), hepatosplanchnic flow can be calculated from concentrations of the marker substance in peripheral and hepatic venous blood (Fig. 2)(5). By contrast, elegant animal experiments have explored the regulation of the splanchnic and hepatic circulations by measuring pressure and flow in isolated areas of the vasculature.

3. REGULATION OF SPLANCHNIC BLOOD FLOW The metabolic demands of the GIT are variable according to feeding habit, and the regulation of splanchnic blood flow is adapted to meet these varied demands. Blood flow to the intestines increases in proportion to regional oxygen consumption (6), and this increase in splanchnic perfusion is obtained through either increased cardiac output, or a redistribution of cardiac output, or a combination of both mechanisms. The blood flow to the GIT is regulated by intrinsic and extrinsic mechanisms. The intrinsic factors are local metabolic control and myogenic control, and locally produced vasoactive substances. The extrinsic factors include neural innervation, circulating vasoactive substances, and systemic hemodynamics.

4. POSTPRANDIAL HYPEREMIA A marked increase in splanchnic blood flow is seen following a meal (6). Increased sympathetic outflow mediates an initial increase in cardiac output during the anticipatory phase of digestion, prior to the ingestion of food, along with increased splanchnic and renal vascular resistance (7, 8). These hemodynamic variables return to baseline within 5–30 min after eating, followed by an increase in gastrointestinal blood flow during the absorption phase of digestion, 30–90 min postprandially. Blood flow increases sequentially to the stomach and proximal small intestine, between 30 and 90 min after eating, and to the ileum after 45–120 min. Colonic blood flow decreases Physiology of the Splanchnic and Hepatic Circulations 81 transiently 30 min after eating, due to contractions elicited by the gas- trocolic reflex. Intestinal blood flow increases by up to 200%, remains elevated for 2–3 h, and is accommodated by recruitment of mucosal and submucosal vessels (9, 10). The mechanism of this functional adaptation to splanchnic blood flow remains unclear, but has been suggested to relate to luminal stimuli activating neural, humoral, and paracrine systems. Chou and Coatney performed a series of seminal experiments in the 1970s and 1980s, using radiolabeled microspheres in canine intestine, to explore the role of intestinal luminal contents in vascular regulation (11). They demonstrated that the presence of undigested food within the lumen does not elicit a hyperemia, whereas digested food significantly increases blood flow, suggesting that hydrolytic products of digestion initiate the hyperemia (12). Subsequent studies showed that the most potent mediators of intestinal hyperemia are glucose and long-chain fatty acids (13). Bile has an effect by rendering glucose and long-chain fatty acids vasoactive, and also through a direct vasodilator effect of luminal bile acids, which is blocked by the bile acid sequestrant colestyramine (14, 15).

5. LOCAL REGULATION OF SPLANCHNIC BLOOD FLOW Chou and Coatney also proposed several other possible mechanisms of regulation of intestinal blood ﬂow, including the vasoactive effects of gastrointestinal hormones and peptides, the presence of local non- metabolic vasoactive mediators and of metabolic vasoactive mediators (11). A number of gastrointestinal hormones have vasoactive properties, including gastrin, VIP, CCK, secretin, and glucagons. Although these hormones do not appear to have a vasoactive effect at physiological circulating doses, it is possible that they act in a paracrine manner such that the effects are related to tissue levels rather than circulating levels (16, 17). The small intestine is also capable of producing serotonin, histamine, bradykinin, and prostaglandins in response to a wide range of stimuli (18–20). Modulation of these mediators has been shown to affect postprandial hyperemia, but similarly their physiological role remains to be established.

6. AUTOREGULATION OF SPLANCHNIC BLOOD FLOW By contrast, the role of products of oxidative metabolism has long been recognized in the local control of blood flow in the cardiac and skeletal muscle vascular beds (21). Adenosine is a ubiquitous product 82 Mehta et al. of oxidative cellular metabolism that also acts as a potent local vasodilator. The intestinal circulation is weakly autoregulated compared with the cerebral, coronary, and renal circulations, although the accumulation of adenosine is one likely mechanism for this pressure–flow autoregulation (see section 8. The Hepatic Arterial Buffer System) (22). Therefore, decreased intestinal perfusion will cause a local accumulation of adenosine, leading to local vasodilatation and increased flow. However, splanchnic vessels also demonstrate a degree of myogenic control, which plays a role in pressure–flow autoregulation (23). In the context of elevated venous pressure, the rise in intravascular pressure at the arteriolar level leads to vasoconstriction and decreased flow. The metabolic mechanism is directed toward maintaining constant oxygen delivery, whereas the myogenic mechanism is directed towards maintaining constant intravascular pressure. It is likely that both these mechanisms play a role in maintaining constant blood flow under different circumstances—for example, the myogenic mechanism ensures that intestinal ischemia does not occur in the context of acute increases in portal pressure.

7. EXTRINSIC REGULATION OF SPLANCHNIC BLOOD FLOW Splanchnic blood vessels are richly innervated by sympathetic nerves from the prevertebral sympathetic ganglia, and the enteric nervous system which innervates the gastrointestinal mucosa (24). The sympathetic nerves are the predominant neural supply to splanchnic arterioles, forming a meshwork of perivascular nerves. No parasympathetic vasodilator innervation to the arterioles of the small intestine has been demonstrated, although the colonic circulation receives parasympathetic supply from the vagal and pelvic nerves. Therefore, the extrinsic neural control of intestinal blood flow is predominantly through sympathetic vasoconstriction, mediated by α-adrenoceptors. Sympathetic activity reduces intestinal blood flow by increasing the vascular resistance of arterioles and veins. This plays a role in the regulation of systemic hemodynamics during exercise or shock, by shifting blood from the splanchnic circulation to the systemic circulation. Similarly, circulating catecholamines, through α-adrenoceptor stimulation, will decrease splanchnic blood flow in the context of altered systemic hemodynamics. Circulating vasopressin and angiotensin II are also potent vasoconstrictors that reduce blood flow and increase vascular resistance in all gastrointestinal organs. However, the role of these regulatory systems under other physiological conditions remains unclear—indeed, Chou Physiology of the Splanchnic and Hepatic Circulations 83 and his colleague have demonstrated that the enteric nervous system plays no role in the postprandial hyperemic response, which is more likely regulated by endocrine and paracrine stimuli (25).

8. THE HEPATIC ARTERIAL BUFFER SYSTEM The blood supply of the liver is unique in terms of its dual supply from the hepatic artery and the portal vein, and the nature of the sinusoidal microcirculation. Unlike intestinal blood flow, which is highly variable depending on the metabolic demands of the organ, the hepatic circulation is adapted to maintain constant blood flow. This is essential to maintain hepatic function, particularly regulatory roles such as the metabolism of hormones and drugs, which are dependent on constant hepatocyte blood flow. Although the liver receives over a quarter of cardiac output, the majority of this is from portal drainage of the splanchnic circulation and is therefore highly variable depending on intestinal blood flow and metabolism. While the liver is not capable of directly influencing portal blood flow, several mechanisms exist to regulate overall liver blood flow in the face of variable portal flow. An effect of reduced portal flow on hepatic arterial flow was first described in 1911 by Burton-Opitz (26), although the existence of a hepatic arterial buffer response (HABR), independent of metabolic autoregulation, was not proposed until 1981 by Lautt (27). A series of elegant feline experiments demonstrated that hepatic arterial blood flow varies inversely with variations in portal flow, and that this response is independent of hepatic metabolic demands (28). In an anesthetized cat model, hepatic oxygen supply was reduced by the isovolemic hemodilution of extracorporeal blood with Ringer’s solution and dextran, to reduce hematocrit and decrease oxygen content to two-thirds of control values. The intestine responded as expected, showing vasodilatation, probably as a result of local accumulation of metabolites. However, the hepatic artery did not vasodilate, but constricted to maintain overall liver blood flow, suggesting that the buffer response was dependent on portal flow rather than hepatic metabolites. The same model was used to alter hepatic oxygen demand, by using dinitrophenol to stimulate oxygen use or SKF 525A (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride) to inhibit hepatocyte metabolism. Again, the intestinal circulation showed metabolism-related changes in flow, but hepatic arterial flow showed no tendency to change in correlation with alterations in hepatic oxygen demand. 84 Mehta et al.

Subsequent work by Lautt and colleagues established the role of adenosine in the HABR. Adenosine was shown to be a potent dila- tor of the hepatic artery and when given into the portal circulation caused dose-dependent arteriolar vasodilatation, demonstrating that portal flow has access to the arteriolar circulation (29). Moreover, potentiation of the HABR was seen when intravascular levels of adenosine were increased with dipyridamole (29), and selective inhibition of the HABR was seen the adenosine antagonist 8-phenyltheophylline (30). Therefore, a model was proposed whereby the concentration of adenosine surrounding the terminal branches of the hepatic arterioles and portal venules controls hepatic arterial blood flow. A decrease in portal flow results in reduced washout of adenosine leading to a compensatory vasodilatation of the hepatic artery and increase in liver blood flow. This mechanism initially appears inconsistent with the statement that the HABR is independent of metabolic demand or oxygen supply to the liver, since adenosine is a product of oxidative metabolism. However, adenosine appears to be secreted at a constant rate in the space of Mall—a fluid-filled space within the portal triad surrounding the hepatic arterioles and the portal venules (Fig. 3)(31). Adenosine

Fig. 3. Anatomy of hepatic and sinusoidal circulations. Physiology of the Splanchnic and Hepatic Circulations 85 production in this space is thought to be through oxygen-independent demethylation of S-adenosyl-homocysteine, which is the mechanism of basal adenosine production in the heart. Although the exact site of interaction of the hepatic artery and the portal vein has not been identified, it occurs at a localized level within the hepatic vasculature, proximal to the sinusoidal circulation. Richter et al. conducted rodent experiments of liver blood flow and in vivo intravital microscopy of the sinusoidal microcirculation (32). The site of resistance to hepatic arteriolar inflow was localized by monitoring the changes in diameter of the terminal hepatic arterioles, terminal hepatic venules and sinusoidal capillaries in response to the HABR. These vessels did not show any change in diameter, suggesting that the site of action of the HABR is proximal to the sinusoidal bed. Moreover, in this study, flow to the terminal portal venules was buffered despite selective ligation of portal venous inflow, suggesting the presence of shunts between hepatic arterioles and portal venules. These shunts may augment the function of the HABR in preserving constant sinusoidal blood flow despite variations in portal or hepatic arterial perfusion. Thus, the hepatic microcirculation, while itself tightly regulated, can be considered independent to the control of overall liver blood flow. A recent body of work has demonstrated the importance of endogenous vasoactive agents; vasodilator gases such as nitric oxide (NO) (33), carbon monoxide (CO), and hydrogen sulfide (H2S) (34, 35); and activated hepatic stellate cells (HSCs) (36) as regulators of intrahepatic vascular tone. These mediators are key regulators of sinusoidal blood flow and are relevant to the pathogenesis of endothelial dysfunction and portal hypertension in cirrhosis (see below). However, in the physiological state, resistance in the sinusoidal circulation is very low; therefore, the arteriolar resistance vessels are the primary site of regulation of sinusoidal flow. Additionally, the anatomical arrangement of these inlet vessels precludes the diffusion of products from sinusoidal endothelium to the presinusoidal resistance vessels.

9. REGULATION OF SINUSOIDAL BLOOD FLOW Sinusoidal endothelial cells (SECs) have distinct characteristics to other vascular endothelial cells, due to their unique anatomical and functional adaptations (37). SECs do not possess a basement membrane and are also fenestrated on their surface, allowing selective access of macromolecules and immune cells to the space of Disse and to hepatocytes, facilitating metabolic and immune functions. Furthermore, the sinusoidal bed is characterized by the presence of HSCs in the space of Disse, which possess long cytoplasmic processes embracing 86 Mehta et al.

Hepatic arterial flow regulated by: Portal venous flow (HABR) Sinusoidal flow regulated by: Hepatic stellate cell contractility Vasodilators: Nitric oxide Carbon monoxide Hydrogen sulfide Prostaglandins Vasoconstrictors: Endothelins Angiotensin Splanchnic flow regulated by: Catecholamines Luminal stimuli (bile, fatty acids, glucose) Thromboxane Paracrine mediators Leukotrienes Autoregulation Extrinsic neural regulation

Fig. 4. Factors regulating hepatic arterial, splanchnic, and sinusoidal blood flow. the sinusoidal endothelium. In normal liver, HSCs serve a variety of functions including the storage and release of vitamin A, the regulation of extracellular matrix turnover in the space of Disse, and the secretion of several growth factors including hepatocyte growth factor, vascular endothelial growth factor, endothelin-1, and transforming growth factor-beta (38). In the context of cirrhosis, intrahepatic vascular tone is increased, in association with sinusoidal endothelial dysfunction and activation of quiescent HSCs (39). Sinusoidal endothelial dysfunction is characterized by decreased bioavailability of endothelial vasodilators such as NO (33) and increased production of endothelial vasoconstrictors such as endothelin-1 and thromboxane A2 (40, 41). The vasoactive agents mediate their effect on intrahepatic vascular tone through the activity of activated HSCs. When activated, HSCs undergo a process of transdifferentiation to a myofibroblastic phenotype with contractile properties. Therefore, these activated HSCs can mediate vasoconstriction in sinusoidal capillaries and postcapillary venules with no smooth muscle layer. In response to liver injury, the activated HSCs modulate intrahepatic resistance by active contraction in response to the imbalance of endothelial vasoconstrictors and vasodilators (42). However, there is less evidence that these pathways play a role in the physiological regulation of sinusoidal blood flow. For example, although some studies have demonstrated that modulation of vasoactive agents such as endothelin and CO causes changes in sinusoidal diameter at locations of HSCs (34, 43), other studies have suggested that these changes actually occur outside the hepatic sinusoid (44, 45). Physiology of the Splanchnic and Hepatic Circulations 87

By contrast, there is good evidence that NO plays a role in the regulation of vascular tone in the normal liver. NO is a gaseous molecule generated from the amino acid L-arginine by the NO synthase (NOS) enzymes. The endothelial NOS (eNOS) isoform is the form predominantly expressed by the endothelial cells of the arteriolar resistance vessels and by SECs. A series of experiments performed in isolated, perfused rodent livers demonstrated that NO maintains basal intrahepatic tone within the isolated liver, and that NO from SECs is responsible for resistance in the sinusoidal circulation. Mittal et al. found that a specific NOS inhibitor significantly increased portal pressure in normal, isolated rat liver (46). Similarly, the effect of norepinephrine on portal pressure in a concentration–effect curve was enhanced by the addition of a NOS inhibitor. Studies by Shah and colleagues demonstrated the presence of eNOS in isolated SECs and showed that exposure of SECs to flow increased NO release (47). Subsequent work has confirmed that NO regulates several important vascular functions such as remodeling, angiogenesis as well as vascular tone. Indeed, a decrease in the bioavailability of intrahepatic NO is a well-established feature of liver injury and portal hypertension (39).

10. SUMMARY The splanchnic and hepatic circulation are highly specialized vascular beds, tailored to the functions of the intestines and the liver. It is clear that the splanchnic circulation is adapted to accommodate large variations in blood flow according to digestion or systemic hemodynamics, whereas the hepatic circulation is tightly regulated to maintain constant sinusoidal flow. Hepatic blood flow has different levels of control—from regional control of hepatic arterial perfusion by the HABR to arteriolar resistance controlling blood flow to the sinusoids. The sinusoidal circulation is normally low resistance under physiological conditions, although this may also become a site of resistance due to HSC activation or endothelial dysfunction in liver injury. More recently, Lautt has suggested a further level of control through systemic blood volume. In a similar fashion to the role of adenosine in the space of Mall leading to the HABR, Lautt proposes that adenosine in the space of Mall may activate sensory nerves activating a “hepatorenal reflex” leading to sodium and water retention (31). This response would serve a physiological purpose to increase blood volume in the case of decreased portal flow; however, in cirrhosis this may lead to salt and water retention due to intrahepatic shunting activating the hepatorenal reflex. This hypothesis deserves further attention. 88 Mehta et al.

Our knowledge of the regulation of splanchnic and hepatic blood flow has increased dramatically over recent decades, although further work is needed to elucidate the complex molecular and signalling mechanisms behind these regulatory pathways. While animal studies have been the foundation of progress in this area, the investigator must remain aware of differences between species. Developments in MRI technology may facilitate studies addressing splanchnic and hepatic blood flow in humans, thus avoiding the need for invasive flow measurements. Future work must be directed towards translating these findings to molecular mechanisms at the bench and ultimately to novel therapeutic targets in human.

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Lars P. Bechmann and Scott L. Friedman

CONTENTS INTRODUCTION BIOCHEMICAL AND STRUCTURAL FEATURES OF HEPATIC FIBROGENESIS BIOLOGY OF FIBROGENIC CELLS IN LIVER RESOLUTION OF FIBROSIS DISEASE-SPECIFIC PATTERNS OF FIBROSIS CLINICAL ASPECTS OF FIBROSIS—STAGING AND QUANTIFICATION TREATMENT OF FIBROSIS REFERENCES

Key Words: Fibrogenesis, Hepatic stellate cells, Cirrhosis

1. INTRODUCTION Hepatic fibrosis following either acute or chronic liver injury represents the accumulation of interstitial or scar-like extracellular matrix (ECM). As fibrosis progresses, distortion of hepatic architecture and formation of septae, or broad bands of scar, begin to encircle nodules of hepatocytes. The late stage of fibrosis, or “cirrhosis,” is associated with alterations in microvascular structure, impaired liver function and portal hypertension, and its complications. These include ascites, encephalopathy, synthetic dysfunction, and impaired metabolic

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_5, C Springer Science+Business Media, LLC 2011

91 92 Bechmann and Friedman capacity, as well as an increased risk for the development of hepatocellular carcinoma (HCC). The composition of ECM in the fibrotic liver is similar to that of other fibrosing parenchyma, including lung and kidney, and is also similar among different etiologies of liver disease. Typically, fibrosis requires years or decades to become clinically apparent, but notable exceptions in which cirrhosis develops over months include pediatric liver diseases (biliary atresia), some forms of drug-induced liver disease, and viral hepatitis associated with immunosuppression.

2. BIOCHEMICAL AND STRUCTURAL FEATURES OF HEPATIC FIBROGENESIS During the course of fibrogenesis, the quality, quantity, and localization of ECM components in the liver undergo dramatic changes in which interstitial collagens (types I and III) replace the normal low-density type IV collagen-containing matrix in the subendothelial space of Disse (1). These interstitial collagens distribute primarily in the connective tissue septae surrounding regenerative hepatic nodules. Cirrhotic livers may contain up to six times more collagen and proteo- glycan than healthy organs. Additionally, laminin, collagen type IV, and other nonfibril-forming collagens are increased, as well as matrix glyco- conjugates, including proteoglycans, fibronectin, and hyaluronic acid. The fibrous septae become progressively resistant to proteolysis by matrix metalloproteinases (MMPs) because of increased cross-linking of collagen fibrils. These changes lead to impaired porosity of the endothelial barrier, in which the fenestrae of normal sinusoidal endothelial cells are decreased in number and size, and in which a discontinuous basement membrane on the basal side of sinusoidal endothelial cells is replaced by a continuous basement membrane. Reduced endothelial porosity and intrahepatic shunts impair the free exchange of metabolites between the interlobular hepatocytes and the perfused plasma in the liver. These changes in the microarchitecture of the liver contribute directly to increased portal venous resistance and consecutively increased portal pressure.

3. BIOLOGY OF FIBROGENIC CELLS IN LIVER The hepatic stellate cell (HSC) is a resident perisinusoidal cell that can undergo activation into a proliferative, contractile, migratory, fibrogenic, and inflammatory phenotype upon liver injury. As the main Fibrosis as a Major Mechanism of Chronic Liver Disease 93 storage site of vitamin A (retinoids), HSCs lose their vitamin A storing capacity upon activation and become a major source of ECM in the injured liver. Recent studies have underscored the heterogeneity of ECM-producing cells (see below). However, the key issue is not necessarily where fibrogenic cells arise from, but whether they express ECM molecules, as well as production of inflammatory and fibrogenic mediators. HSC activation can be divided into two phases: initiation, with early changes in gene expression and phenotype, resulting from paracrine stimulation, primarily due to changes in surrounding ECM, as well as exposure to lipid peroxides and products of damaged hepatocytes, and perpetuation, which results from the effects of these stimuli on maintaining the activated phenotype and generating fibrosis (see Fig. 1). Liver cell (i.e., hepatocyte) injury leads to extracellular release of free radicals, intracellular constituents, and/or cytokines and signaling molecules (2). Oxidant stress-mediated necrosis and HSC activation may underlie many liver diseases. Although necrosis is considered a classical inflammatory and fibrogenic stimulus, recent findings also implicate apoptosis in the fibrogenic response (3). HSC engulfment of apoptotic bodies, released from hepatocytes, is fibrogenic in culture, and Fas-mediated hepatocyte apoptosis is also fibrogenic in experimental animals (4). Because liver injury is typically associated with the infiltration of inflammatory cells, the resident macrophages (Kupffer cells) and natural killer cells may initiate local inflammation before the arrival of extrahepatic or circulating inflammatory cells. In alcoholic liver disease, a high-fat diet can multiply the effects of reactive oxygen species on Kupffer cell-mediated HSC activation via TNF-α induction and reduced glutathione (5). Activation of Kupffer cells also mediates leptin-induced fibrosis in NAFLD (6). Other lymphocyte populations (NK, iNK-T cells) have a more complex response in liver injury, in which some subsets are antifibrogenic, while others are profibrogenic, depending on the stage of and nature of hepatic damage (7, 8). The contributions of the immune system to liver fibrosis are gaining increased attention as a result of the observation that patients coinfected with hepatitis C virus (HCV) and HIV, as well as those who are immunosuppressed after liver transplantation, have accelerated fibrosis (9). The increased rate of fibrosis in patients coinfected with HCV and HIV might be partially explained by a reduced CD4/CD8 ratio, because CD8+ cells may be relatively fibrogenic compared with CD4+ cells. In addition, increased microbial translocation from the gut to the liver may also increase the likelihood of progression to cirrhosis (10, 11). Recent data additionally suggest that HSCs may be infected and activated directly by HIV (12). Moreover, key cytokines participating in 94 Bechmann and Friedman

Initiation Perpetuation

Injury Proliferation Contractility Oxidative stress PDGF Apoptotic bodies ET-1 VEGF NO LPS FGF Paracrine stimuli Fibrogenesis

MMP-2&9; MT-1-MMP Altered matrix TIMP-1,2 degradation Reversion PDGF Chemokines Resolution Chemokines Adenosine TLR ligands HSC TIMP-1,2 chemotaxis TRAIL Fas T cells B cells NK cells NK-T cells Apoptosis Inflammatory signaling

Fig. 1. Pathways of hepatic stellate cell activation. Features of stellate cell activation can be distinguished between those that stimulate initiation and those that contribute to perpetuation. Initiation is provoked by soluble stimuli that include oxidant stress signals (reactive oxygen intermediates), apoptotic bodies, lipopolysaccharide (LPS), and paracrine stimuli from neighboring cell types including hepatic macrophages (Kupffer cells), sinusoidal endothelium, and hepatocytes. Perpetuation follows, characterized by a number of specific phenotypic changes including proliferation, contractility, fibrogenesis, altered matrix degradation, chemotaxis, and inflammatory signaling. FGF, fibroblast growth factor; ET-1, endothelin-1; NK, natural killer; NO, nitric oxide; MT, membrane type. Modified with permission from Friedman (91). lymphocyte–HSC interactions may be dysregulated, including CXCR3, CXCR4, IL-10, and FasL (13, 14). Finally, ingestion of lymphocytes by HSCs in hepatic fibrosis may also contribute to the course of hepatic fibrosis (15). Key soluble stimuli regulating HSC activation have been well established, including transforming growth factor-beta (TGF-β), platelet- derived growth factor (PDGF), and endothelin-1 (ET-1). Recently, CCN2 (previously known as CTGF), a downstream target of TGFβ1, Fibrosis as a Major Mechanism of Chronic Liver Disease 95 has also been recognized as a fibrogenic signal (16). Additional TGF-β signaling molecules, including the receptor ALK4/5/7, broaden the number of potential regulators (17). Within the nucleus, a growing number of transcription factors regulate HSC behavior, including peroxisomal proliferator-activated receptors (PPARs), retinoid receptors, NF-κB, Jun D, Krüppel-like factor 6, and Foxf1 (18). A range of general and cell-type-specific membrane receptors and signaling pathways control HSC biology, including receptor tyrosine kinases, chemokine receptors, and integrins (19). As noted above, fibrogenic cells derive not only from resident stellate cells, but also from portal fibroblasts, circulating fibrocytes, bone marrow, and epithelial–mesenchymal cell transition (EMT) (20–22). In cholestatic liver diseases and ischemia, portal fibroblasts may be especially important (23). However, the quantitative contribution to fibrogenesis of non-stellate cell-derived fibroblasts remains unclear. In chronic liver injury, progressive recruitment of bone marrow-derived cells may occur over time. The role of bone marrow-derived cells in fibrogenesis is not entirely clarified; however, as on one hand these cell populations may be fibrogenic, yet bone marrow-derived endothelial progenitor cells can also be antifibrotic (24). Another emerging source of fibrogenic cells is EMT in which adult hepatocytes or biliary epithelium transdifferentiates into fibrogenic cells. This phenomenon has been extensively characterized in kidney and lung fibrosis, and then subsequently in animal models and human samples of liver fibrosis, as well as in the context of carcinogenesis. Interestingly, key cytokines regulating EMT also drive activation of HSCs, including TGF-β, Ras, Smad-7, and Shh (25–27).

4. RESOLUTION OF FIBROSIS The observations that hepatic fibrosis and even cirrhosis may regress have provoked new strategies for developing antifibrotic therapies based on efforts to mimic natural pathways of fibrosis resolution (28). Enzymes controlling matrix degradation comprise a family of MMPs, which specifically degrade collagens and noncollagenous ECM substrates. HSCs are a key source of MMP-2, MMP-9, and stromelysin (MMP-3). In early liver injury, MMP-2 degrades the low-density basement membrane present in the subendothelial space (29). Its replacement with fibril-forming matrix impairs hepatocyte differentiation and function. A major determinant of progressive fibrosis is failure to degrade the fibril-forming or interstitial scar matrix. Inactivation of proteases by binding to tissue inhibitors of metalloproteinases (TIMPs) is an important locus of control, because sustained production of these 96 Bechmann and Friedman proteins during liver injury could inhibit the activity of interstitial col- lagenases, leading to reduced degradation of the accumulating matrix. MMP-1 is the main protease that can degrade type I collagen, the principal collagen in fibrotic liver (30). Pathways of MMP-1 regulation are multiple and include chemokine receptor 2 (CCR2) among many others (31). In addition to its role in inhibiting proteases, TIMP-1 is also antiapoptotic towards HSCs, and thus its expression preserves the population of activated HSC by preventing their clearance (32). During fibrosis resolution, clearance of activated HSCs by apoptosis results from downregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) (33). In support of this conclusion, administration of TIMP-neutralizing antibodies delays regression of liver fibrosis in experimental animals (34). Additionally, senescence of activated HSCs may contribute to fibrosis resolution (35); however, it is uncertain whether senescence is truly distinct from apoptosis (36). As noted above, reversibility also depends upon the relative solubil- ity of collagen, which in turn reflects the cross-linking of collagen and the maturation of hepatic scar through the action of tissue transglutaminase and specific metalloproteinases (ADAMTS2). The long-standing clinical dogma that the slower the pace of injury, the less reversible the scar, is borne out by animal studies in which even advanced fibrosis of short duration is reversible and is primarily limited by the extent of collagen cross-linking (37). Clinically, increased septal thickness and smaller nodule size, both of which reflect more advanced stages of fibrosis, are significant predictors of worse clinical outcomes in patients with advanced liver disease (38). Hepatic macrophages, in addition to HSCs, are important regulators of matrix remodeling. In mouse models, macrophages augment fibrogenesis during progression of liver fibrosis, whereas during resolution they hasten matrix degradation, in part through increased production of MMP-13 (39).

5. DISEASE-SPECIFIC PATTERNS OF FIBROSIS The accelerating prevalence of childhood and adult obesity in the United States and Western Europe is associated with an alarming increase in NAFLD (nonalcoholic fatty liver disease), and progression to NASH (nonalcoholic steatohepatitis), with consequent ﬁbrosis and cirrhosis (40). Free fatty acids, which accumulate in NAFLD, are indirect activators of HSCs in vitro (41). Adipokines can also mediate hepatic manifestations of obesity and ﬁbrogenesis. For example, leptin promotes HSC activation and enhances TIMP-1 expression (42). Fibrosis as a Major Mechanism of Chronic Liver Disease 97

Concurrently, downregulation of adiponectin in obesity can amplify the fibrogenic activity of leptin (43). In support of this conclusion, mice lacking adiponectin have enhanced fibrosis after toxic liver injury (44). Cannabinoid receptors also mediate steatosis and, in ethanol-fed mice, activated HSCs generate endogenous cannabinoids that provoke fatty liver (45). In HCV infection, HSCs may be a direct viral target, since activated HSCs express putative HCV receptors (e.g., CD180), and transgenic expression of HCV proteins in culture induces HSC proliferation with the release of both CTGF and MMP-2 (46, 47). Furthermore, hepatocytes harboring replicating HCV in culture produce fibrogenic stimuli toward HSCs (48). Only one study has examined HBV-specific pathways of fibrogenesis, which suggests that the HBV X protein may activate HSCs, and HDV superinfection further accelerates fibrogenesis (49).

6. CLINICAL ASPECTS OF FIBROSIS—STAGING AND QUANTIFICATION Liver biopsy using connective tissue stains remains the gold standard for assessing the extent and pattern of fibrosis, although the procedure is associated with potential clinical complications and is prone to sampling error and interobserver variability (see Table 1)(50). Complementing liver histology, the quantification of key fibrogenic genes by real-time quantitative PCR in liver tissue might reveal early evidence of regression even before the matrix content has changed (51). This approach would still require a tissue specimen, however, and needs further validation. There has been significant progress in the development of noninvasive tests of fibrosis, which will be essential as early biomarkers of efficacy in antifibrotic clinical trials and to guide clinical usage (52). A number of serum assays (i.e., FibroSpectTM, FiboMaxTM, FibroTestTM) have good correlation with histological diagnosis in several chronic liver disease entities (53, 54). These assays typically include combined measurement of several circulating matrix proteins and/or serum biochemistries, which generate a calculated algorithm that is correlated with fibrosis stage. More recently, a technique for gly- comic analysis (i.e., quantification of specific glycoproteins) has been developed, but wider validation is necessary (55). Imaging methods (CT, MRI, PET, radionuclide receptor scanning) can assess intrahepatic blood flow patterns, organ texture, or possibly the mass of activated HSCs (56, 57). Another evolving approach is the 98 Bechmann and Friedman ). 90 fibrosis fibrosis Perisinusoidal or Peri portal (a) mild, zone 3 (b) moderate, zone 3 (c) portal/periportal Periportal and perisinusoidal Bridging fibrosis expansion: mild fibrosis moderate fibrosis septae: severe fibrosis Fibrous portal Few bridges or septae: Numerous bridges or Cirrhosis Cirrhosis Table 1 o / Common histological staging systems for fibrosis fibrous septae portal fields; w/or w portal fields; w/or w/o fibrous septae portal fields; w/occasional portal–portal bridging portal fields; w/marked bridging w/occasional nodules Fibrous expansion of some expansion w/: with; w/o: without. The Histology Activity Score, reported by Knodell, includes only three stages, while the ISHAK score differentiates six stages. The METAVIR Stage Knodell01 No fibrosis Fibrous portal ISHAK No fibrosis METAVIR (F0-4) No fibrosis NASH clinical research group No fibrosis 23 Bridging fibrosis Fibrous expansion of most Fibrous expansion of most 45 Cirrhosis6 Fibrous expansion of most Marked bridging Cirrhosis score is a simple, widely applied four-stage scoring system. Kleiner et al. quantified fibrosis in NAFLD with a seven-stage system ( Fibrosis as a Major Mechanism of Chronic Liver Disease 99 quantification of liver stiffness, using transient elastography (58). This method has been widely studied and is a useful noninvasive tool that is highly sensitive for detecting cirrhosis. Collectively, all the noninvasive approaches can accurately distinguish between patients with little or no fibrosis and those with advanced disease. However, they are less reliable at discriminating intermediate stages of fibrosis and especially cirrhosis, and their value in individual patient management over time needs to be established (see Fig. 2).

METAVIR F4 F1-F3

HVPG >5 ≥10 ≥12 ≥20

Development Worse Varices Clinical None None of ascites VH, prognosis formation HE in VH Stage Compensated Compensated Compensated Decompensated (stage 1) (stage 2) (stages 3/4)

Insoluble Fibrogenesis Scar Acellular scar Biology scar and small and Neovasc. X-linking Nodule size nodules

Fig. 2 Cirrhosis is a series of progressive stages, not a single stage. Within the spectrum of cirrhosis, the disease is characterized by progressive increases in hepatic venous pressure gradient (HVPG), decompensation, and matrix cross-linking, associated with shrinking nodule size, thickened septae, and enhanced risk of decompensation. For each 1-mm increase in HVPG, the risk of decompensation increases by 11%. Concepts presented here are not rigor- ously supported by primary data for all features, but rather are intended to convey the progressive changes that underlie deterioration in patients with chronic hepatic injury and ﬁbrosis. Stages are based on data from D’Amico et al. (92). HE, hepatic encephalopathy; VH, variceal hemorrhage. Modiﬁed with permission from Friedman (91).

7. TREATMENT OF FIBROSIS Current and evolving therapeutic approaches are built primarily on the pathways of fibrosis and its resolution. They include cure of the primary disease, directly decreasing HSC activation; neutralization of proliferative, fibrogenic, contractile, and/or proinflammatory responses; or induction of apoptosis of HSC. The most effective way to eliminate hepatic fibrosis is to clear the primary cause of liver disease. Among others, this includes abstinence 100 Bechmann and Friedman in alcoholic liver disease, clearance or suppression of HBV or HCV in chronic viral hepatitis, as well as weight loss or bariatric surgery in patients with NASH. Moreover, obesity associated with hepatic steatosis accelerates fibrosis in viral liver diseases, and thus weight loss may slow progression in these patients as well (59). Importantly, clearance of HCV with pegylated interferon and ribavirin can lead to significant improvement in fibrosis and even cirrhosis (60). In contrast, maintenance interferon monotherapy in the absence of antiviral clearance had no impact on fibrosis in the recent large US HALT-C trial, although serum fibrotic parameters improved under therapy in a subanalysis from this study (61, 62). A number of agents have anti-inflammatory activity; for example, corticosteroids have been used for decades to treat autoimmune hepatitis. Pentoxyphylline may exert its antifibrotic activity by downregulating TNF-α signaling (63). The renin–angiotensin system may also amplify inflammation, and angiotensin receptor antagonists have reproducible antifibrogenic activity in several animal studies, with human trials underway (64, 65). Ursodeoxycholic acid (UDCA) has a beneficial effect on fibrosis in primary biliary cirrhosis. Similarly, a nitric oxide-releasing derivative of UDCA reduces inflammation, fibrosis, and portal pressure in an animal model (66). Interestingly, UDCA also activates the pregnane X receptor (PXR), which has antifibrotic properties (67). More recently, ligands of the farnesoid X receptor (FXR) have been developed, which are also antifibrotic in animal models (68). Several other new classes of drugs are in preclinical studies or clinical trials. These include hepatocyte growth factor (HGF) deletion variants and mimetics, as well as insulin-like growth factor, and a small-molecule caspase inhibitor that improves AST levels in patients with chronic HCV (69, 70). Reducing the activation of quiescent HSCs is an attractive target, given its central role in the fibrotic response. The most practical approach is to reduce oxidant stress. Antioxidants suppress fibrogenesis in some, but not all studies of experimental fibrogenesis (71, 72). The variability of effect could reflect the variable potency or stability of antioxidants, since this approach has a strong rationale. Silymarin, a natural flavonoid, has sparked interest as a potential antifibrotic therapy and improved survival advantage in alcoholic cirrhotics (73, 74). PPARγ nuclear receptors are expressed in HSCs, and synthetic PPARγ ligands downregulate HSC activation (75). Given their widespread use in diabetes, clinical trials of PPARγ agonists are now being tested in clinical trials in both NASH and HCV (76). Leptin is produced by activated HSCs, and animals deficient in leptin have reduced hepatic injury Fibrosis as a Major Mechanism of Chronic Liver Disease 101 and fibrosis (77). Adiponectin, a natural counterregulator to leptin, may become a useful antifibrotic agent, particularly in NASH (78). Antagonists to cytokines and their receptors, in particular PDGF, FGF, and TGF-β, are promising targets and are undergoing clinical trials in other tissues (33). The success in developing Imatinib (GlivecR ), a small-molecule tyrosine kinase antagonist in leukemia, might as well be suitable for treating liver fibrosis as it is antifibrotic in experimental liver fibrosis (79). Similarly, the efficacy of the small-molecule VEGF receptor antagonist sunitinib in HCC has been complemented by evidence of antifibrotic activity in an animal model (80). Additional small molecules have been evaluated, including selective inhibitor of Rho- mediated focal adhesions, (81) and antisense cDNA to PDGF B chain (82), among many others. Inhibition of matrix production has been the primary target of most antifibrotic therapies to date. Inhibitors of collagen synthesis such as HOE 077, which blocks the enzyme prolyl hydroxylase, were among the first antifibrotic compounds tested in liver, but success with this agent has been modest (83). Whereas colchicine showed no benefit in alcoholic cirrhosis, TGF-β antagonists are now being extensively tested in a range of diseases, as they have the dual effect of inhibiting matrix production and accelerating its degradation (84). Rapamycin, an immunosuppressive drug used following liver transplantation, has the added benefit of inhibiting HSC proliferation; however, an increased risk in hepatic artery thrombosis has been reported. Other antifibrotic compounds tested include relaxin and bosentan (85, 86). Pathways of fibrosis regression are increasingly clear (see above) and have led to novel approaches to clear activated stellate cells in liver injury and fibrosis. In animal studies, gliotoxin provokes selective apoptosis of stellate cells in culture and in vivo, leading to reduced fibrosis (87). Inhibition of Iκκβ (I kappa kappa beta), whose net effect is to increase NF-κB (NF-kappaB) signaling in stellate cells, may accelerate apoptosis (88). Apoptosis can also be provoked by disruption of integrin-mediated adhesion or through use of TRAIL ligands (89). HSCs contain several families of apoptotic mediators, including Fas/FasL, TNF receptors, nerve growth factor receptors, and Bcl/Bax, so that additional targets to promote apoptosis will likely be exploited in the future. In summary, while there are not yet any approved antifibrotic therapies for clinical use, the rapid progress in uncovering pathways of fibrosis in liver injury has led to dozens of new approaches that are likely to prove beneficial. Combined with improved methods of noninvasive fibrosis detection, success in treating hepatic fibrosis is anticipated. 102 Bechmann and Friedman

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Stem Cells and Chronic Liver Failure: Potential New Therapeutics

Aiwu Ruth He, Arun Thenappan, Feras J. Abdul Khalek, and Lopa Mishra

CONTENTS INTRODUCTION STEM CELL POPULATIONS FOR CELL-BASED THERAPY HEPATIC STEM CELLS IN ADULT LIVER HEPATIC STEM CELL ACTIVATION IDENTIFICATION AND ISOLATION OF HEPATIC STEM CELLS HEPATIC STEM CELLS AND CANCER CELL-BASED THERAPY FOR CHRONIC LIVER DISEASE CONCLUSION REFERENCES

Key Words: Hepatic progenitor/stem cells, Liver failure, Cell-based therapy

1. INTRODUCTION Fulminant hepatic failure is a disease with high mortality. Prior to orthotopic liver transplantation (OLT), the mortality rate was greater than 80%. With improved intensive care, however, several series report

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_6, C Springer Science+Business Media, LLC 2011

109 110 He et al. a survival rate of 60% (1). At present, standard therapy is liver transplantation and approximately 6% of OLTs performed in the United States are for fulminant hepatic failure. The increasing shortage of donor organs, however, has contributed to the development of several cell-based therapies for end-stage liver disease. Currently, several distinct cell populations have been suggested as potential sources for liver regeneration. The first are in vivo mature hepatocytes. Hepatocytes are the primary driver of liver regeneration following acute injury and are capable of restoring a liver that has lost as much as 75% of its tissue mass. Cryopreserved hepatocytes for transplantation are now readily available, but a sufficient number (approximately 10–15% of liver mass) is necessary to not only regenerate, but also sustain metabolic function (2). Moreover, hepatocyte engraftment following transplantation remains a significant challenge to its further development and adoption. Similarly, much work has now focused on the generation of hepatocytes from embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, or bone-marrow-derived hematopoietic stem cells (HSCs). In vitro studies have demonstrated the ability to differentiate ES or iPS cells into endoderm, hepatic endoderm, hepatoblasts, and then mature hepatocytes under special culture conditions (3). It has yet to be demonstrated, however, whether in vitro differentiated hepatocytes are capable of maintaining sufficient metabolic function and possess the same regenerative capacity in vivo as native in vivo hepatocytes. In addition, like mature hepatocytes, engraftment remains a significant challenge. Another candidate for cell-based therapy is adult hepatic progenitor/stem cells, which are referred to as facultative or reserve stem cells. Several studies have described the activation of the progenitor cell compartment in adult human liver, especially in the setting of chronic liver failure. The major goals in evaluating potential hepatic progenitor/stem cell populations are effectively isolating and purifying these cells from primary tissue and then demonstrating engraftment and adequate metabolic and regenerative function in vivo. Currently, research has focused on the identification of potential hepatic progenitor/stem cell populations, including evaluation of potential biomarkers and analysis of the signaling pathways that activate stem cell proliferation. In this chapter, we briefly describe the stem cell populations with potential for cell-based therapy. We then discuss current research on the identification, activation, and isolation of adult hepatic progenitor/stem cells, especially in the setting of chronic liver disease. We then discuss the potential and challenges facing cell-based therapy for liver failure. Stem Cells and Chronic Liver Failure 111

2. STEM CELL POPULATIONS FOR CELL-BASED THERAPY Stem cells are characterized by the properties of self-renewal, pluripotency, and longevity, and are classified as embryonic or adult (4). Adult stem cells are tissue specific, while embryonic stem cells (ESCs) are pluripotent cells that originate from the blastocyst inner cell mass. ESCs give rise to somatic stem cells that further differentiate into multipotent tissue-specific stem cells. Adult stem cells have a limited proliferative capacity and give rise to cell types within a particular cell lineage, while ESCs can proliferate indefinitely and retain their potential to form all the tissues of a developing organism. In fact, pluripotency is the guiding principle of ESC biology and contributes to the ultimate goal of using them in the clinic as stem cell therapies (5). Currently, ESC therapy has been shown to reverse signs of paralysis, improve diabetes, and significantly repair infarcted heart muscle in experimental animal models (6–8). Several studies have also described the differentiation of human ESCs into cells that display hepatocyte-like characteristics (9–11). Bioethical concerns, however, remain a challenge and have hindered ESC research. Recently, research has significantly focused on inducing somatic cells to become pluripotent. Induced pluripotent stem (iPS) cells were generated by introducing four factors—Oct3/4, Sox2, c-Myc, and Klf4 or Oct3/4, Sox2, Nanog, and Lin28—into somatic cells under ESC culture conditions (12–14). Subsequent studies have demonstrated the ability to remove the inducing genes (15), the necessity of introducing only one or two factors in certain cell types (16, 17), and the generation of iPS cells by chemical induction (18, 19). iPS cells are nearly identical to ESCs in pluripotency and differentiation capability and have already been shown in vitro to differentiate into mesodermal-derived cardiomyocytes and ectodermal-derived neuronal lineages. Recently, studies have also demonstrated complete differentiation of iPS cells into endodermal-derived hepatocytes (20, 21). These cells exhibit hepatic morphology, express hepatocyte markers, secrete plasma proteins, and support CYP1A2 and CYP3A4 activity, necessary for drug and toxin metabolism. Moreover, iPS cells derived from mice demonstrated retention of the full potential for fetal liver development, with generation of all hepatic cell types in iPS-derived embryos. These “proof-of-concept” studies lay the groundwork for future studies and demonstrate the enormous potential for iPS cells in cell-based therapy for liver failure. Adult stem cells are another potential source for cell-based therapy. Significant challenges remain, however, in their definitive identification and isolation, especially in the liver. Recent studies describe an 112 He et al. array of potential hepatic stem cell markers, demonstrate key signaling pathways involved in hepatic stem cell activation and proliferation, and describe several potential protocols for hepatic stem cell isolation. We discuss the current research related to hepatic stem cells.

3. HEPATIC STEM CELLS IN ADULT LIVER The liver bud develops on embryonic day (ED) 8.5, as tissue-specific foregut endodermal stem cells proliferate and differentiate under the influence of signals from the septum transversum mesenchyme (22, 23). Coordinated signaling of fibroblast growth factors (FGFs) from cardiac mesoderm (FGF 1, 2, and 8) and bone morphogenetic proteins (BMPs) from the septum transversum mesenchyme (BMP 2, 4, 5, and 7) cooperatively induce liver bud development from endoderm via mitogen-activated protein kinase (MAPK) (ERK 1 and 2) (24–26). The liver bud then gives rise to cells destined to become bipotential liver stem cells and express α-fetoprotein (AFP), albumin (Alb), and later cytokeratins (CKs) 7 and 19 (22, 27). Prior to ED16, bipotential liver stem cells differentiate along the hepatocytic (AFP+/Alb+)and cholangiocytic (CK19+) cell lineages (24). Following liver development, hepatic stem cells are scarce in adult human liver. Immature epithelial cells, however, have been described in the smallest terminal branches of the biliary tree known as the canals of Hering (28). Referred to as “oval cells” in rodents, they were first observed by Farber in 1956 in the livers of rats exposed to chemical carcinogens and have subsequently been described in rats with chronic ethanol or iron-induced liver injury and following partial hepatectomy in retrorsine-injured livers (29–32). Subsequent analysis of rodent models identified oval cells as possessing markers of adult hepatocytes (albumin), bile duct cells (cytokeratins 7 and 19, OV-6) (33), fetal hepatoblasts (AFP) (34), and hematopoietic stem cells (Thy-1, Sca-1, c-kit) (35, 36). Activated oval cells express transcription factors (hepatic nuclear factors and CCAAT/enhancer-binding proteins) that mimic the expression pattern during embryonic liver development (37)and transplantation of oval cells in FAH Aeexon5 mice resulted in liver repopulation with an efficiency equivalent to mature hepatocytes, suggesting that oval cells are a candidate source for hepatic stem cells (38). Activation of oval cells, referred to as a “ductular reaction,” has been subsequently demonstrated in the livers of humans with chronic liver injury in the setting of prolonged necrosis, cirrhosis, and inflammatory disease (39, 40). The ductular reaction involves proliferation of bipotential oval cells that are then capable of differentiating into mature hepatocytes and biliary cells. Intermediate hepatocytes, with Stem Cells and Chronic Liver Failure 113 a phenotype between oval cells and mature hepatocytes, have similarly been described in patients with severe inflammatory hepatitis (41, 42), and the degree of oval cell and intermediate hepatocyte proliferation appears to correlate with the degree of inflammation and fibrosis observed in diseases such as chronic hepatitis, hemochromatosis, and nonalcoholic steatohepatitis (33, 43). In addition to oval cells, several other candidate sources of hepatic stem cells have been described. Small hepatocytes, less than half the size of mature hepatocytes, isolated from rats demonstrate a high proliferative capacity and in vitro, express major bile salt and organic anion transporters in a sequence that mirrors developing rat liver, suggesting that they may represent a type of intermediate hepatocyte or are derived from embryonic hepatoblasts (44–47). Other studies have also suggested hepatic stellate cells. Stellate cells express epithelial cell markers E-cadherin and cytokeratins as well as the stem cell marker CD133. CD133-positive stellate cells have subsequently been shown to differentiate into endothelial cells and hepatocytes, and stellate cells contribute to parenchymal and nonparenchymal cell reconstitution following liver injury (48–55). Similarly, mesenchymal stem cells (MSCs) from the bone marrow have been shown to migrate to injured liver and differentiate into stellate cells and myofibroblasts in the context of liver fibrosis (56). Following transplantation in rodent models, mesenchymal stem cells differentiated into hepatocyte-like cells and a recent study of male to female bone marrow transplant patients found that 70% of fibrogenic hepatic myofibroblasts were derived from transplanted marrow cells (57). A population of hepatic stem cells expressing markers of mesenchymal stem cells, AFP, albumin, CK 8, and CK 18, but not markers for hematopoietic stem cells have also been isolated from normal adult liver and shown to differentiate into mature hepatocyte-like cells in vitro (58). In fact, a recent study using a label retention assay in acetaminophen-treated mice identified four potential hepatic stem cell niches: the canals of Hering, intralobular bile ducts, periductal “null” mononuclear cells, and peribiliary hepatocytes. These results suggest that the liver has a flexible system of regeneration, rather than a single stem cell and location (35, 59). Significant questions remain, however, concerning the key signaling pathways responsible for hepatic stem cell activation and the proper identification and isolation of hepatic stem cell populations.

4. HEPATIC STEM CELL ACTIVATION Overwhelming liver injury, chronic liver injury, or large-scale hepatocyte senescence results in activation of hepatic stem cells. Activation appears to be multifactorial and likely involves the inflammatory 114 He et al. response and several key signaling pathways. Several soluble factors, secreted during the inflammatory response, including IFN-γ, IFN-α, TNF-α, lymphotoxin-β, IL-15, TWEAK, and TGF-β, have been shown to regulate oval cell proliferation (60–64). Studies of CCl4-damaged livers with activated CXCR4-expressing oval cells demonstrate significant upregulation of the chemokine SDF-1 by hepatocytes in response to blocked DNA synthesis (65). SDF-1 subsequently attracts CXCR4+ T cells that express TWEAK. TWEAK interaction with its receptor Fn14 on oval cells then stimulates oval cell proliferation (61). Similarly, oval cell proliferation in wild-type mice is characterized by recruitment of Kupffer, NK, NKT, and T cells with production of proinflammatory cytokines IFN-γ and TNF-α that are mitogenic for oval cells. These results demonstrate significant cross talk between the immune system and the regenerating liver. Similarly, Wnt/β-catenin signaling plays a key role in hepatic biology, including liver development, growth, regeneration, and hepatocellular cancer (HCC) pathogenesis. In the absence of Wnt, cytosolic β-catenin, which is normally bound to membranous E-cadherin, interacts with a complex consisting of the tumor suppressor protein APC (adenomatous polyposis coli), glycogen synthase kinase 3β (gsk3β), and axin. β-Catenin is consequently serine phosphorylated, recognized by an E3 ubiquitin ligase and then degraded (66). In the presence of Wnt binding to its receptor Frizzled (FZ), however, the kinase activity of the APC/gsk3β/axin complex is blocked and β-catenin remains unphosphorylated and accumulates in the nucleus. β-Catenin then binds to the transcription factor TCF4 and can activate downstream target genes such as epidermal growth factor receptor, cyclin D1, c-Myc, glutamine synthetase, and cytochrome P450 2E (67). Studies of oval cell proliferation in mice administered a DDC diet or 2-AAF, followed by partial hepatectomy, demonstrated immunoflourescent colocalization of several Wnt ligands and activated nuclear β-catenin in proliferating oval cells. Perioval hepatocytes demonstrate increased Wnt-1 expression, which may act in a paracrine fashion on the FZ-2 receptor of oval cells, and purified Wnt3a in oval cells stimulates cell cycle entry and β-catenin activation. Subsequent analysis of β-catenin null mice fed a DDC diet demonstrated diminished A6-positive oval cell proliferation, suggesting that Wnt/β-catenin plays a key role in the activation of hepatic stem cells (68, 69). Hedgehog (Hh) signaling through its receptor patched (PTC) also activates transcription of genes regulating the fate of various progenitor cells and is necessary for endodermal commitment and hepatic development. Mature liver epithelial cells lack Hh signaling. Recent studies using a PTC-LacZ mouse, however, have demonstrated the Stem Cells and Chronic Liver Failure 115 presence of Hh-positive and Hh-responsive cells in the portal tracts of adult mice. Subsequent analysis of a murine hepatic progenitor cell line (OV) demonstrated a greater than 35000-fold increase in PTC expression as compared to a well-differentiated hepatic cell line. Isolation of EpCAM-positive hepatic stem cells from human livers also demonstrated enriched Indian Hedgehog (IHh) and PTC transcripts compared to the EpCAM-negative fraction. Subsequent treatment with an Hh-specific blocker resulted in significant necrosis and apoptosis, suggesting that Hh activity influences hepatic stem cell survival, and Hh signaling is preserved in hepatic stem cells from fetal development through adulthood (70). Recent evidence also suggests that disruption of TGF-β signaling results in activation of hepatic stem cells during liver regeneration. Analysis of liver biopsy specimens from recipients of living donor grafts less than 6 weeks posttransplant demonstrated a streaming pattern of hepatocytes expressing progenitor cell markers Oct3/4 and AFP with expression of TGF-β signaling components—the type II receptor (TBRII) and the Smad3/4-adaptor protein β-2 spectrin (β2SP) primarily localized to the portal tract. Examination of specimens from 3 to 4 months posttransplant, when regeneration is nearly complete, however, demonstrated a spatial expansion of TBRII and β2SP from the periportal to the pericentral zone with an expanded remaining pool of Oct3/4-positive cells localized to the portal tract. Further analysis of liver regeneration in β2SP knockout mice (β2SP+–) demonstrated a striking fourfold expanded population of Oct3/4/AFP/CK-19-positive cells with activated Wnt signaling (71). Disrupted TGF-β signaling has also been described in stem cell-like cells in HCC. Exami Disrupted TGF-β signaling has also been shown that in stem cell-like cells in hepatocellular cancer (HCC). Examination of human HCC tumor specimens demonstrated a small strongly positive cluster of cells expressing the Oct3/4 but was negative for TBRII and β2SP in 90% of samples. Cells with this phenotype have not been observed in surveys of normal or regenerating liver, suggesting that Oct3/4-positive human HCC cells which have lost TGF-β signaling proteins may represent a population of cancer stem cells that can give rise to HCC (72). Resistance to the growth inhibitory effects of TGF-β has also been shown to allow oval cells to proliferate under conditions inhibitory to hepatocytes. Quiescent liver demonstrates modest TGF-β levels, but liver injury stimulates a significant increase in TGF-β production by nonparenchymal cells and TGF-β is a potent profibrogenic cytokine leading to hepatic fibrosis. Examination of oval cells in the livers of rats fed a CDE diet demonstrated substantially reduced phospho- Smad2 expression and increased proliferation. In vitro experiments 116 He et al. with primary hepatocytes, AML12 hepatocyte cell line, and two oval cell lines LE/2 and LE/6 subsequently demonstrated greater sensitivity of hepatocytes to TGF-β growth inhibition, suggesting cell-specific responsive to TGF-β and that oval cells proliferate in an environment inhibitory to hepatocyte proliferation (64).

5. IDENTIFICATION AND ISOLATION OF HEPATIC STEM CELLS Isolation of hepatic stem cells has relied on identification of cell- specific markers and a range of markers have been proposed. Several markers identifying oval cells by immunohistochemistry have been described in rodent models. Oval cells share molecular markers with adult hepatocytes (albumin, cytokeratins 8 and 18), bile duct cells (cytokeratins 7 and 19, OV-6, A6) (33), fetal hepatoblasts (AFP) (34), and hematopoietic stem cells (Thy-1, Sca-1, c-kit) (35, 36). Several studies have also suggested that hepatic stem cells express a “side population” (SP) phenotype similar to multipotent hematopoietic stem cells with expression of proteins such as ABCG2, an ATP-binding cassette transporter (73). A detailed list of hepatic stem cell markers described in the literature is presented in Table 1. The extensiveness and variety of markers listed, however, demonstrate the significant challenge of isolating these cells and subsequently confirming their nature as true stem cells with the ability to regenerate hepatic cell types. Definitive experiments involving serial transplantation of marker-positive cells isolated from primary liver tissue with reconstitution of liver cells have been described in cells isolated from HCC specimens. Isolation and serial transplantation of hepatic stem cells from chronic liver failure have yet to be definitively demonstrated.

6. HEPATIC STEM CELLS AND CANCER Recent work also demonstrates that hepatic stem cells most likely can give rise to HCC as well as cholangiocarcinomas (39, 74). Several studies have shown a stem cell phenotype in a substantial number of HCC tumor specimens. Detailed immunophenotyping of HCCs revealed that 28–50% of HCCs express markers of progenitor cells such as CK 7 and CK 19. These tumors also consist of cells that have an intermediate phenotype between stem cells and mature hepatocytes. In fact, HCCs that express hepatocyte and biliary cell markers such as albumin, CK 7, and CK 19 carry a significantly poorer prognosis and higher recurrence after surgical resection and liver transplantation Stem Cells and Chronic Liver Failure 117 ) 36 ) ), ( ) ) 58 124 121 128 ), ( ) ) +) ), ( ), ( ) ), ( 127 121 121 58 117 ) 123 119 123 ) ) ), ( ), ( ) ), ( ), ( ), ( 58 ) ) ), ( ) ), ( 72 ) ), ( 121 126 119 58 72 72 128 119 121 119 122 122 33 58 ,+), ( ) ) ), ( ), ( +), ( ), ( ) ), ( ), ( ), ( ∗ ), ( ), ( ), ( ), ( ), ( ), ( ), ( ), ( 122 72 120 125 121 120 120 72 119 119 120 119 120 129 58 120 77 117 118 Table 1 Phenotypic expression of human hepatic stem cells ++ ++/– ( ND – ( +NDND( N+( +ND+ ++ +++ ( + ( +NDND( ++ + ( ++ + ( +NDND( ++ + ( +/– ND ND ( ++ + ( ++ + ( diff. + – ( +– +++ ( + ( ++ + ( +/– ND ND ( -Catenin (CTNNB1) CD133 (PROM1) CD90 (THY1) SMAD4 TGFBR2 ELF (SPTBN1) Wnt β STAT3 NOTCH1 ABCG2 KIT Oct3/Oct4 (POU5F1) NANOG CK 7 (KRT7) CK 19 (KRT19) CK 18 (KRT18) CK 8 (KRT8) BMI1 ProteinEpCAM (TACSTD1) Hepatic stem cell Cholangiocytic lineage Hepatocytic lineage References 118 He et al. ), low ) ses of 121 ), 137 ), ) ), ( ) ) 77 ), ( 118 58 131 117 ), ( 128 36 ) ) ) ) ), ( )( ), ( ), ( ) ), ( ), ( 121 130 129 58 132 135 58 119 119 121 128 ), ( 136 . Studies demonstrating ), ( ), ( ), ( ), ( ) ) ) ), ( ) ), ( ), ( ), ( ), ( ), ( ∗ ), ( 117 58 58 ( ( ( 120 121 133 134 58 124 125 132 121 120 120 120 125 ), which are provided in parentheses. http://www.genenames.org Table 1 (continued) –NDND( lw ( ND+low– +/––+ – ND ( – ND – ( ( +NDND( ND+– ++NDND( ND ( ND ( +/– – + ( +– + ( +NDND( ++/–+/–( Cholangiocytic and hepatocytic lineages were defined based on morphology and/or expression of CK 19 (cholangiocytic marker) or albumin and -fetoprotein (hepatocytic marker). ND, not determined/reported; +, positive expression; –, negative expression; ++, high/over expression; low, CD45 SMO GCTM-5 MET NCAM1 CK14 (KRT14) Ki-67 (MKI67) HepPar1 CD34 ProteinAFP Hepatic stem cell Cholangiocytic lineage Hepatocytic lineage References ALB CD44 OV6 All sources are listedstudies from denoted by most #. recent Studies to demonstrating serial past transplantability for of reference. cells indicating Strict potential immunohistochemical “stemness” and are denoted confocal by labeling was adhered to in the following in vivo expression of markers are denoted by +. Studies in which “stemness” was studied by mechanistic genetic studies are denoted by %. α expression; diff., positive differential (lowthe or official high) human expression; gene +/–, symbols contradictory from supporting the evidence. HUGO All Gene proteins Nomenclature are Committee listed ( as common alia Stem Cells and Chronic Liver Failure 119

(75). Fifty-five percent of small dysplastic foci (less than 1 mm in size), which represent the earliest premalignant lesions, are comprised of stem cells and intermediate hepatocytes (76), and a side population of cells, with characteristics of both hepatocytic and cholangiocytic lineages, in the human HCC cell lines huh7 and PLC/PRF/5 cells was found to give rise to persistently aggressive tumors upon serial transplantation in immunodeficient NOD/SCID mice (77). These studies suggest that transformation of hepatic stem cells, perhaps in the context of chronic liver inflammation, may contribute to tumorigenesis and the resistance of many tumors to standard chemotherapy.

7. CELL-BASED THERAPY FOR CHRONIC LIVER DISEASE Progress in understanding the mechanisms of liver injury and regeneration and in vitro generation of hepatocytes from stem cells has provided a new therapeutic opportunity for patients with liver failure. Early study of hepatocyte transplant carried out in the 1970s by infus- ing hepatocytes into the spleen demonstrated the capacity for cell-based liver regeneration (78). Further preclinical investigation of hepatocyte transplantation was carried out in animal models of inborn errors of metabolism including the Gunn rat for Crigler–Najjar syndrome type 1 (79), Spf–ash mice for ornithine transcarbamylase deficiency (80), Nagase analbuminemic rats for hypoalbuminemia (81), mice with histidinemia (82), the long-Evan’s rats for Wilson’s disease (83), fumarylacetoacetate hydrolase knockout mice for tyrosinemia type 1 (84), Mdr2 knockouts for PFIC (84, 85), Watanabe rabbits with hypercholesterolemia (86), and dogs with hyperuricosemia (87). Hepatocyte transplantation in these models appeared to achieve medium- to long- term improvement in biochemical abnormalities. Furthermore, hepatocyte transplantation in rodent models in acute liver failure induced by D-galactosamine (88), dimethylnitrosamine (89), 90% hepatectomy (90), or ischemic injury (91) demonstrated marked improvement in long-term survival. These studies demonstrated “proof of principle” and suggested that hepatocyte transplantation may cure or alleviate congenital metabolic diseases of the liver. The clinical hepatocyte transplantation experience, however, has been more limited and confined to several small series of patients. For example, it has been reported that a 10-year-old girl with severe unconjugated hyperbilirubinemia at birth received an infusion of isolated hepatocytes through the portal vein equivalent to 5% of the parenchymal mass, and this led to a 60% reduction in bilirubin level 120 He et al. and a reduced need for phototherapy (92). Similarly, hepatocyte transplantation has been described in the treatment of human glycogen storage disease type 1a (93) and the urea cycle disorder arginosucci- nate lysate deficiency, in which a course of 11 hepatocyte transplants in a 3-year-old patient achieved a peak of 19% donor hepatocytes with sustained engraftment and tissue enzyme activities (94). Infusion of isolated human adult hepatocytes into two children with inherited severe factor VII deficiency improved the coagulation defect and markedly decreased the requirement for exogenous recombinant factor VII (rFVIIa) to approximately 20% of that before cell transplantation (95). Isolated hepatocyte transplantation in a male infant with severe ornithine transcarbamylase (OTC) deficiency also resulted in temporary relief of hyperammonemia and protein intolerance, although this metabolic stability was lost after 11 days likely secondary to rejection of the transplanted cells (96). Moreover, there are close to 40 reported cases of hepatocyte transplantations in patients with acute failure caused by medication overdose, polysubstance abuse, or poison or viral infusion (2, 97–102). Patients generally received infusions of 2.8×107 to 3.9×1010 numbers of hepatocytes. About 18% of patients subsequently recovered without need for OLT, while 21% of patients ended up receiving OLT between day 1 and day 35 following hepatocyte transplant, and 61% of patients died. A few case series of hepatocyte transplantation in patients with chronic liver disease have also been reported, but with generally poor results that are attributed to disruption of liver architecture seen in chronic liver disease leading to difficulties in hepatocyte engraftment (98, 99). These small studies, however, demonstrate the challenges with hepatocyte transplantation and are difficult to interpret secondary to the heterogeneity of the disorders treated, the limited number of patients per series, and the lack of true control groups. In the absence of any randomized controlled trials, it is difficult to comment on the true efficacy of hepatocyte transplantation at this time. The use of hepatocytes derived from stem cells, however, may theoretically possess several advantages over transplantation of mature hepatocytes. First, the use of stem/progenitor cells, which can be expanded in vivo, can come from extrahepatic sources, can generate hepatocyte-like cells, and would circumvent the shortage of mature hepatocytes. Stem cells may be derived from four potential sources: ES cells, iPS cells, hematopoeitic stem cells, or adult hepatic stem cells. Second, stem cells may yield better long-term hepatic repopulation due to their ability to generate newly differentiated hepatocytes. ES cells can be generated from the inner cell mass of human embryos and are totipotent and easily expandable. ES cells, however, Stem Cells and Chronic Liver Failure 121 are allogeneic, and transplantation of hepatocyte-like cells differentiated from ES cells continue to require immunosuppression. Moreover, ethical concerns over the source of ES cells may limit their widespread use. Several studies have utilized mouse-derived ES cells (103, 104), but the percentage of engrafted donor cells was small (92)andthe totipotent potential of ES cells resulted in teratoma formation in some animal models (105). This has led to increased preference for iPS cells. Unlike ES cells, iPS cells provide a resource for cell-based therapy without human leucocyte antigen (HLA) mismatching. Recently, there is also considerable excitement on the potential therapeutic utility of bone marrow in cell-based therapy. Several studies have demonstrated engraftment and clonal expansion of bone marrow- derived cells within the liver (106–110). Others have also reported fusion between donor and recipient cells, including human cord blood with sublethally irradiated mouse hepatocytes (111) and mouse bone marrow with mouse hepatocytes after CCl4 injury (112). Several studies have also utilized bone marrow-derived mesenchymal stem cells (MSCs) for liver disease, although this remains controversial, since rather than differentiating into hepatocytes these cells facilitate repair after their infusion with a subpopulation of hepatocytes (111). A recent series reports three patients subjected to intraportal administration of autologous CD133(+) bone marrow-derived MSCs subsequent to portal venous embolization of the right liver segments had a mean 2.5-fold increase in the left lateral hepatic segments compared with a group of three consecutive patients without application of MSCs (113). Several challenges to bone marrow-derived cell therapy for chronic liver disease, however, still exist including the fact that hepatocyte replacement levels after bone marrow transplantation are generally low (less than 0.01%) (114–116) and hepatic differentiation from bone marrow-derived cells has not been analyzed at the clonal level. To date, it appears that extrahepatic cells are not directly involved in liver regeneration but may be a suitable tool to correct a metabolic defect by fusion-mediated additive gene transfer. Adult hepatic stem cells as a source for cell-based therapy in chronic liver disease also face significant challenges. The primary is difficulty in their precise identification and isolation. Recently, studies isolating EpCAM-positive cells in 0.5–2.5% of liver parenchyma demonstrate expansion with greater than 150 population doublings in a serum-free, defined medium with maintenance of phenotypic stability (117). Following transfer to STO feeders (mouse fibroblast-derived feeder cells), EpCAM-positive cells then gave rise to hepatoblasts, and transplantation of freshly isolated EpCAM-positive cells or cells expanded in culture into NOD/SCID mice resulted in mature liver 122 He et al. tissue expressing human-specific proteins. However, whether the hepatocyte differentiated from the EpCAM-positive cells can carry out the metabolic function as endogenous mature hepatocytes are still to be determined.

8. CONCLUSION Cell-based therapy possesses great therapeutic potential for liver failure. Further development in primary hepatocyte engraftment, in the differentiation of hepatocytes from iPS cells, and the identiﬁca- tion, isolation, and differentiation of adult hepatic stem cells to mature hepatocytes suggests that clinically feasible methods to induce liver repopulation and growth could be developed.

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The Role of Inﬂammatory Mediators in Liver Failure

Joan Clària, Marta López-Parra, Esther Titos, and Ana González-Périz

CONTENTS INTRODUCTION CYTOKINES REACTIVE OXYGEN SPECIES ARACHIDONIC ACID-DERIVED LIPID MEDIATORS SUMMARY REFERENCES

Key Words: Cytokines, TNF-α, Reactive oxygen species, Lipid mediators, Cyclooxygenase, 5-Lipoxygenase

1. INTRODUCTION In response to tissue trauma, viral invasion, or an insult of any etiology, the liver develops a localized inflammatory response, which serves to destroy, dilute, or wall off the injurious agent and the injured tissue. Under some circumstances, however, an excessive inflammatory response causes extensive liver damage and triggers acute liver failure. In other circumstances, the insult persists and inflammation is not properly resolved, becoming chronic and ultimately leading to the formation of tissue scar, fibrosis, and cirrhosis. Although the temporal pattern and cellular events that orchestrate the inflammatory response differ between acute and chronic liver injury, both cases share

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_7, C Springer Science+Business Media, LLC 2011

131 132 Joan Clària et al. common inflammatory pathways and mediators. From a pathophysiological and pharmacological point of view, perhaps the most relevant inflammatory mediators are cytokines such as tumor necrosis factor (TNF)-α and interleukin-6 (IL-6), reactive oxygen species including superoxide anion (O2–) and peroxynitrite (ONOO–), and small bioactive lipid mediators derived from arachidonic acid (i.e., eicosanoids). In this chapter, we review the most recent advances in the knowledge of these inflammatory mediators with especial emphasis on their pharmacological modulation.

2. CYTOKINES Cytokines are low-molecular-weight proteins involved in cellular communication synthesized and secreted by almost every cell in our organism, including most liver cell types (1). Once released, cytokines interact with specific receptors in their target cells where they induce multiple responses in both an autocrine and paracrine fashion (i.e., interacting with the same cell or with the neighboring cells). Many cytokines act synergistically either by binding to the same cell-surface receptor or by exerting multiple overlapping effects (1). Moreover, cytokines tend to have pleiotropic functions that may alter different cell functions such as proliferation, migration, adhesion, and apoptosis, although they are best known by their immunomodulating actions. Cytokines are classified according to their specific activities in different subgroups, which include TNF-α, interleukins (ILs) (currently 18 different interleukins), growth factors (i.e., transforming growth factor (TGF) family), interferons, and chemokines. The production of cytokines by cells of the inflammatory and immune systems is triggered during activation of the inflammatory cascade. According to their role in inflammation, cytokines can be broadly divided into those with primarily proinflammatory actions such as TNF-α, IL-1β, and IL-6, which promote the further liberation of cytokines and cause the classical signs of inflammation, and those with anti-inflammatory actions such as IL-4, IL-10, and IL-13 (2). In this chapter, we will focus on TNF-α and IL-6 because they represent the two most extensively studied cytokines in the context of inflammatory liver injury.

2.1. TNF-α TNF-α is a multifunctional cytokine that can regulate many cellular and biological processes such as immune function, cell differentiation, proliferation, apoptosis, and energy metabolism, although this cytokine is better known by its prominent role in the regulation of inflammatory The Role of Inflammatory Mediators in Liver Failure 133 pathways (3). TNF-α secretion is induced by conserved structural elements common to microbial pathogens such as lipopolysaccharide (LPS), which is recognized by Toll-like receptors present on the surface of immune cells (3). Once secreted, TNF-α exerts its biological effects through binding to structurally related receptor proteins known as the TNF receptor superfamily that embraces at least 12 different receptors. In the liver scenario, the effects of TNF-α appear to be mainly mediated by interaction with two membrane receptors: TNF-R1 and TNF-R2 (4). The biological activities of TNF-α also appear to be mediated through the convergence of NF-κB and NF-AT activating pathways (3). In most circumstances, TNF-α is able to activate other cytokine networks including the release of IL-1 and IL-6, thereby amplifying inflammatory response and tissue injury (4). Studies in patients and animal models have strongly implicated TNF-α in the induction of liver injury by triggering destruction of the liver parenchyma. In this regard, serum levels of TNF-α,TNF-R1, and TNF-R2 are markedly increased in patients with fulminant hepatic failure, and these serum levels directly correlate with disease severity (5, 6). TNF-α has also been implicated in the pathogenesis of liver allograft rejection (7), chronic hepatitis B virus infection (8), and more especially in alcoholic hepatitis (9). In addition, TNF-α together with interferon (IFN)-γ play a major role in the pathogenesis of autoimmune liver disease and cholestasis (10, 11). Genetic association studies have firmly established a direct link between TNF- α promoter polymorphisms and the risk of liver allograft rejection (12), advanced alcoholic liver disease (13), and fulminant hepatitis (14). It has also been demonstrated that TNF-α is a key mediator of liver injury in many experimental models of liver disease. For example, TNF-α appears to be involved in carbon tetrachloride (CCl4)- induced liver damage and TNF-R1/TNF-R2-deficient mice are resistant to the development of histological fibrosis after 8 weeks of CCl4 treatment (15). In the D-galactosamine model, TNF-α induces activation of caspases and produces subsequent hepatocyte apoptosis, infiltration of leucocytes and macrophages, finally leading to death (16). TNF- R1 plays an essential role in this experimental model, since mice lacking this TNF-α receptor are resistant to D-galactosamine-induced damage (17). Given its role in liver injury, there is a rationale for the potential use of anti-TNF-α therapies in liver diseases. At present, there are three different agents approved by international regulatory authorities that either bind directly to TNF-α or block TNF-α signaling: infliximab, adalimumab, and etanercept. Infliximab, a chimeric monoclonal antibody cA2 against TNF-α, is currently used in the treatment of severe active 134 Joan Clària et al.

Crohn’s disease and rheumatoid arthritis and is a representative member of this class of drugs (18, 19). Although single dose of infliximab is associated with significant improvements in parameters of severity and survival in patients with severe alcoholic hepatitis (20, 21), adverse effects including increased incidence of severe infections remain a serious concern (22, 23). On the other hand, adalimumab and etanercept are TNF-α receptor antagonists of therapeutic use in rheumatoid arthritis, Crohn’s disease, psoriasis, and ankylosing spondylitis, although their efficacy in liver diseases has not yet been demonstrated.

2.2. IL-6 Cytokine Family In addition to IL-6, the IL-6 cytokine family comprises other cytokines such as IL-11, oncostatin M, ciliary neurotropic factor, and cardiotrophin-1 (24). The presence of increased serum and intrahepatic IL-6 levels has been reported in patients with acute and chronic liver diseases (25). Although IL-6 is a proinﬂammatory cytokine, its role in liver disease is still intriguing because it appears to have essential functions in protecting this organ during acute or chronic injury. Indeed, IL-6 is one of the most important mediators of the hepatic acute-phase response and potently increases the synthesis of positive acute-phase proteins such as C-reactive protein, α2-macroglobulins, and serum amyloid in this organ after acute physiological stress (26, 27). Similar to IL-6, cardiotrophin-1, another member of the interleukin-6 family, has been shown to be an essential endogenous defense of the liver against injury (28).

3. REACTIVE OXYGEN SPECIES Most biological processes such as energy generation by mitochondria and detoxification reactions inevitably generate free radicals. Free radicals are unstable molecules with an unpaired electron that readily react with organic substrates such as lipids, proteins, and DNA, damaging our cells and tissues (29). The most common free radicals are products of oxygen metabolism known as reactive oxygen species (ROS), whose most representative members are O2– and ONOO–. ROS actively participate in the progression of liver inflammation and injury, and an overproduction of free radicals and an increase in hepatic lipoperox- idation have been reported in alcoholic liver disease, liver cirrhosis, and steatohepatitis (30, 31). Consistent with this, the administration of antioxidants (i.e., vitamin E or S-adenosyl-methionine (SAME)) as drug coadjuvants efficiently ameliorates oxidative stress and liver injury in experimental models of liver disease (32, 33). The Role of Inflammatory Mediators in Liver Failure 135

4. ARACHIDONIC ACID-DERIVED LIPID MEDIATORS Small bioactive lipid mediators originating from the cleavage of structural lipid components of cellular membranes constitute one of the most well-established classes of endogenous regulators of inflammation. A paradigmatic example of this class of inflammatory lipid mediators is the large family of small lipid mediators, collectively known as eicosanoids, which are generated from the essential omega-6 polyun- saturated fatty acid arachidonic acid. In general terms, eicosanoid biosynthesis is initiated by the activation of phospholipase A2 and the release of arachidonic acid from membrane phospholipids in response to the interaction of a stimulus with a receptor on the cell surface (34). Free arachidonic acid is then available as a substrate for the intracellular biosynthesis of eicosanoids through two major enzymatic routes, namely, the cyclooxygenase (COX) pathway and the lipoxygenase (LO) pathway. The COX pathway results in the formation of prostaglandins (PGs) and thromboxane (TXA2), which are known for their powerful physiological properties and their critical role in inflammatory response (35, 36)(Fig.1). On the other hand, the LO pathway comprises three major LOs, designated 5-LO, 12-LO, and 15-LO, of which 5-LO converts arachidonic acid into 5(S)-hydroxyeicosatetraenoic acid (5-HETE) and leukotrienes (LTs), a consolidated pharmacological target in inflammation (35, 36)(Fig.1). Alternatively, arachidonic acid can be converted through free radical-catalyzed peroxidation to a unique series of PG-like compounds, without the direct action of COX enzymes (37)(Fig.1). These nonclassical prostanoids are generically known as isoprostanes, of which 8-epi-PGF2α is the most relevant in terms of biological activity and is one of the most accurate markers of oxidative stress (37).

4.1. COX Pathway COX is the key enzyme in the biosynthesis of PGs from arachidonic acid (38). There are two distinct isozymes of COX, designated COX-1 and COX-2. Although the products generated by these two isozymes are the same, COX-1 is a constitutive enzyme expressed in virtually all cells, whereas COX-2 has limited expression in most tissues but is induced by inflammatory mediators (i.e., IL-1, TNF-α, IFN-γ and LPS) and is, thus, responsible for inflammatory response (39, 40). Both COX isozymes sequentially transform arachidonic acid into PGG2 and, subsequently, into PGH2, which is finally converted by specific synthases into PGs of the D2,E2,F2,andI2 series as well as into TXA2 (Fig. 1). The biosynthesis of COX products is cell specific and any given cell 136 Joan Clària et al.

Fig. 1. Biosynthesis of arachidonic acid-derived lipid mediators. Upon activation of phospholipase A2 (PLA2), arachidonic acid (AA) is released from membrane phospholipids and converted into biologically active eicosanoids by the cyclooxygenase (COX) and 5-lipoxygenase (5-LO) pathways. The COX pathway comprises two isoforms (i.e., COX-1 and COX-2) that oxidize AA into prostaglandin (PG) G2, which is further reduced to PGH2, a highly unstable endoperoxide that is rapidly converted by speciﬁc synthases to PGs of the E2,F2, and D2 series and also to PGI2 (prostacyclin) and thromboxane (TX) A2.PGI2,TXA2, and PGD2 are hydrolyzed to 6-keto-PGF1α,TXB2, 12,14 and 15-deoxy- -PGJ2 (15d-PGJ2), respectively. On the other hand, the 5-LO pathway comprises a dioxygenase (5-LO) which is activated upon interaction with the 5-LO-activating protein (FLAP) and catalyzes the oxygenation of the 5-carbon atom of arachidonic acid resulting in the formation of 5- hydroperoxyeicosatetraenoic acid (5-HpETE). 5-HpETE can be converted to 5-HETE or give rise to the unstable allylic epoxide leukotriene (LT) A4, which is either hydrolyzed by LTA4 hydrolase into LTB4 or converted by LTC4 synthase into LTC4/LTD4/LTE4. type tends to specialize in the formation of one of these eicosanoids as its major product. For example, endothelial cells mainly produce PGI2 (prostacyclin) from PGH2 by means of PGI synthase, and platelets release TXA2 from PGH2 through the action of TX synthase. Both The Role of Inﬂammatory Mediators in Liver Failure 137

PGI2 and TXA2 have a very short half-life and are rapidly hydrolyzed to the inactive compounds 6-keto-PGF1α and TXB2, respectively (38). PGH2 can be alternatively converted into PGF2α by PGF synthase, which is mainly expressed in the uterus. PGH2 is also converted into PGD2 by the action of PGD synthase, of which two distinct types have been identified: lipocalin-type PGD synthase and hematopoietic-type PGD synthase (36). PGD2 is readily dehydrated to the cyclopentenone 12,14 PGs of the J2 series (PGJ2 and 15-deoxy- -PGJ2 (15d-PGJ2)) (see below). PGE2 is formed by the enzyme PGE synthase (PGES) present in virtually every cell type. There are three different PGES isoforms (mPGES-1, cPGES-1, and mPGES-2), of which mPGES-1 was the first to be identified and characterized (41). Owing to their instability, PGs and TXA2 exert their functions mainly in the proximity of their sites of synthesis. Thus, they typically act as autocrine or paracrine hormones, maintaining homeostasis within their cells of origin or in neighboring cells in the tissue. Ten different types and subtypes of receptors, which belong to the G protein-coupled rhodopsin-type receptor superfamily of seven transmembrane domains, mediate the biological effects of PGs (42). Four of the receptor subtypes bind PGE2 (EP1, EP2, EP3, and EP4), two bind PGD2 (DP1 and DP2), two bind TXA2 (TPα and TPβ), and the rest are single receptors for PGF2α and PGI2 (FP and IP, respectively) (42). The COX pathway as a whole offers an unprecedented number of therapeutic opportunities, especially in the area of inflammation. Both COX isozymes (COX-1 and COX-2) are nonspecifically inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), whereas COX-2 is the target of a series of compounds, generically known as COXIBs that are specifically designed to block inflammation without affecting COX-1-dependent PG biosynthesis (43). The use of COXIBs may be of particular interest for combating inflammation in patients with cirrhosis and ascites, in which renal function is critically dependent on COX-1-derived PGs (44–47). In addition, the pharmaceutical industry has also drawn attention to agonists and antagonists acting on specific prostanoid receptors. This strategy may provide advantages in terms of safety over COX inhibitors, although progress in this field has been scarce, mainly because of the existence of such a large number of prostanoid receptors and their function similarity. Nevertheless, the recent cloning and characterization of specific prostanoid receptors have facilitated the development of synthetic agonists and antagonists for some of these receptors. Most of these compounds have proven to be very useful in the identification of the biological role of a given prostanoid receptor and some have shown therapeutic potential. These R include PGE2 analogs such as misoprostol (Cytotec ), an EP3/EP2 138 Joan Clària et al. agonist used as an adjunct to COX inhibitor therapy to reduce gastric irritation and bleeding, and alprostadil (EdexR ), an EP4/EP2 agonist used for erectile dysfunction. Other compounds include a PGI2 analog iloprost (VentavisR ) used in pulmonary hypertension; travopost (TravatanR ), an FP agonist marketed for the treatment of glaucoma; and AA-2114 (SeratrodastR ) and BAY-U-3405 (RamatrobanR ), which are orally active TX receptor antagonists now available for the treatment of asthma (48). The liver possesses a remarkable ability to produce PGs. The major PGs produced by the liver are in this rank order: PGD2,PGE2,and TXA2 (49). Although all liver cell types are capable of synthesizing these eicosanoids, Kupffer cells, the liver resident macrophages, are quantitatively most important. In the context of liver disease, the definitive role of PGs is still under discussion. On one hand, PGs appear to exert cytoprotective actions in the liver, and infusion of PG analogs such as misoprostol was shown to improve survival in fulminant hepatic failure and subfulminant hepatic failure due to hepatitis B infection or caused by acetaminophen overdosage (50, 51). However, later studies failed to show a beneficial effect of misoprostol in acute liver failure (52, 53). On the other hand, PGs appear to play a pathogenic role in chronic liver injury. Consistent with this, COX-2 expression and PG biosynthesis are markedly upregulated in progressive liver disease in chronic hepatitis C virus infection and in patients with cirrhosis (54–56). Interestingly, the hepatitis C virus has been shown to induce COX-2 expression and PGE2 formation in hepatocytes (57). A pathogenic role for COX-2-derived PGs has also been reported in experimental models including CCl4-induced liver injury, alcoholic hepatitis, and nonalcoholic steatohepatitis (58–60). Working with the CCl4 model, our group has demonstrated that selective COX-2 inhibition is associated with remarkable anti-inflammatory and antifibrogenic effects (58). Similar findings have been reported by an elegant study in mice with transgenic expression of COX-2 in the liver, which exhibit increased hepatic PGE2 levels and more prominent liver tissue damage than control mice after the injection of LPS/D-galactosamine (61). Conclusive evidence on the pathogenic role of COX-2-derived PGs has recently been provided by Paik and coworkers who demonstrated remarkable protective effects of up to three different COX-2 inhibitors in two models of liver injury (i.e., bile duct ligation and thioacetamide injection) (62). The status of the hepatic circulation is an important determinant of hepatocellular viability. An increased production of vasoconstrictors in response to hepatic stress induces microcirculatory disturbance and subsequent hepatocellular damage. Among the various The Role of Inflammatory Mediators in Liver Failure 139 vasoconstrictors, TXA2 has been postulated as one of the most potent and effective vasoconstrictive mediators for portal circulation in the liver in two different experimental models of liver injury: CCl4-induced cirrhosis and bile duct ligation (63, 64). Specifically, cirrhotic rat livers exhibit an increased expression of TX synthase, the terminal enzyme that transforms PGH2 to TXA2, and increased production of TXA2 (63). This enhanced vasoconstrictor prostanoid production in cirrhotic livers has been ascribed to occur in sinusoidal endothelial cells and to be mainly dependent on COX-1 activity (65). Attenuation of intrahepatic resistance and hepatic damage has been observed after COX-1 inhibition or TXA2 receptor blockade but not after TX synthase inhibition (63, 65–67), although these changes have not been later confirmed in bile duct-ligated rats (67).

4.2. Cyclopentenone PGs Cyclopentenone PGs (cyPGs) are products of the nonenzymatic dehydration of PGs. cyPGs are structurally defined by the presence of a highly reactive α,β-unsaturated carbonyl moiety in the cyclopentenone ring (68). From a biological point of view, the most relevant cyPGs are those derived from the dehydration of PGD2, including the PGs of the 12 J2 series: PGJ2, -PGJ2 and 15d-PGJ2. Unlike other PGs, to date, no specific surface receptor has been identified for cyPGs. Instead, 15d- PGJ2 appears to exert its effects through binding and activation of the nuclear receptor superfamily of ligand-activated transcription factors. Indeed, 15d-PGJ2 is a natural ligand of PPARγ and a negative regulator of NF-κB transcriptional activity (69, 70). The biological effects of cyPGs in vivo remain controversial. On one hand, 15d-PGJ2 has been shown to display immunomodulatory, anti-inflammatory, antiproliferative and antiapoptotic properties (68, 71); but on the other hand, 15d-PGJ2 has been shown the ability to promote apoptosis in endothelial cells, myofibroblasts, and synoviocytes (58, 72, 73). Although the definitive role of cyPGs is still under discussion, our laboratory has demonstrated the presence of increased levels of 15d-PGJ2 in livers from rats with CCl4-induced liver injury (58), whereas a significant induction of cellular damage in hepatocytes exposed to this cyPG has been reported elsewhere (74).

4.3. Isoprostanes The lipid components of cellular membranes are exposed to the damaging actions of free radicals and lipid peroxidative agents. Some years ago, the group of Morrow and Roberts were the first to demonstrate 140 Joan Clària et al. the formation of a series of PG-like compounds named isoprostanes formed in vivo and in vitro by free radical-catalyzed peroxidation of phospholipid-bound arachidonic acid (75). Formation of isoprostanes involves oxidation of arachidonic acid to form four different PGG2- like bicyclic endoperoxide intermediates which are then reduced by glutathione to four series of isoprostanes collectively known as F2-isoprostanes, because they are isomeric to the COX-derived PGF2α, being 8-epi-PGF2α the most representative isomer (76). The discovery of these PG-like compounds independent of the COX pathway initiated a new epoch on the significance and detection of nonenzymatic lipid peroxidation products (76). Unlike COX-derived PGs, isoprostanes are initially formed in situ in esterified form from membrane phospholipids and are, subsequently, released into the circulation (76). Moreover, isoprostanes are less reactive than other lipid peroxidation products such as lipoperoxides and aldehydes and can be easily detected in plasma and urine (76). Consequently, measurement of isoprostanes is now considered to be the gold standard for the assessment of oxidative stress status in a number of pathologies. Indeed, elevated levels of F2-isoprostanes have been reported in diabetes, chronic obstructive lung disease, allergic asthma, atherosclerosis, ischemia–reperfusion injury, and liver diseases (76, 77). Among the latter, F2-isoprostanes have been confirmed to be reliable markers of tissue injury in alcoholic liver disease, hepatorenal syndrome, hepatitis C, biliary cirrhosis, and liver transplantation (76, 78). Interestingly, alcohol ingestion dose dependently increases urinary excretion of F2- isoprostanes in healthy volunteers (79). Several studies have also revealed that both plasma and urinary levels of F2-isoprostanes increase significantly in well-established models of liver injury. In this regard, the first observation that lipid peroxidation generated F2-isoprostanes wasmadeusingtheCCl4 model of liver injury (80). Measuring F2-isoprostanes by using this model and also after administration of antioxidants (i.e., vitamin E) has been revealed as a unique tool in the establishment of F2-isoprostanes as biomarkers of oxidative stress and tissue injury (76). In addition to being established markers of oxidative stress injury, F2- isoprostanes possess potent biological effects and, thus, may serve as pathologic mediators through their vasoconstrictive and inflammatory properties. Indeed, F2-isoprostanes and 8-epi-PGF2α, in particular, have well-known vasoconstrictive actions in a variety of organs and tissues including liver, lung, and kidney, mediated by putative binding to the vascular TXA2 receptors (76). On the other hand, although the The Role of Inflammatory Mediators in Liver Failure 141 inflammatory properties of F2-isoprostanes are limited, the addition of nanomolar concentrations of 8-epi-PGF2α has been shown to induce a marked increase in DNA and collagen synthesis in activated hepatic stellate cells (77).

4.4. 5-LO Pathway 5-LO is the key enzyme in the biosynthesis of LTs. Upon cellular activation, 5-LO translocates to the nuclear envelope where it interacts with the 5-LO-activating protein (FLAP), a transfer protein that facilitates the conversion of free arachidonic acid into 5-HpETE (35, 81), which is subsequently reduced either to 5-HETE or to the highly unstable allylic epoxide LTA4. Once formed, LTA4 is rapidly transformed either to LTB4 via stereoselective hydration by LTA4 hydrolase or to LTC4 through glutathione conjugation catalyzed by LTC4 synthase (35, 81). Sequential metabolic reactions catalyzed by γ-glutamyl transferase and a specific membrane-bound dipeptidase convert LTC4 into LTD4 and LTE4, respectively. Together LTC4,D4,andE4 are termed cysteinyl- leukotrienes (cys-LTs) which, in the past, were referred to as the slow-reacting substances of anaphylaxis. Once formed, 5-LO-derived products exert their biological effects via activation of G-protein- coupled receptors. To date, two LTB4 and two cys-LT receptors have been cloned (82). The B-LT1 receptor and the recently characterized B-LT2 receptors bind LTB4 with high and low affinities, respectively. The B-LT1 receptor is mainly located on leucocytes and its activation elicits a remarkable chemotactic response, whereas the B-LT2 receptor displays a widespread tissue distribution pattern and its function is currently unknown (82). The two types of cys-LT receptors, cys-LT1 and cys-LT2, bind LTC4 and LTD4. Cys-LT1 is found in airway smooth muscle cells and vascular endothelial cells and its activation promotes vasoconstriction and cell adherence (82). Cys-LT2 is distributed within pulmonary veins, the spleen, Purkinje fibers of the heart, and the adrenal gland, and its function remains unknown (82). The 5-LO pathway leading to LT formation is a major proinflammatory pathway. 5-LO is basically expressed in inflammatory cells and their products are involved in the pathogenesis of many inflammatory disorders (35, 81). LTB4, for example, has remarkable chemotactic activity on neutrophils and eosinophils and promotes neutrophil chemotaxis and adhesion to vascular endothelium through specific integrins. In addition, cys-LTs are eosinophil chemoattractants, cause plasma leakage from postcapillary venules, and induce synthesis and release 142 Joan Clària et al. of proinflammatory mediators including IL-8 and platelet-activating factor (35, 81). A number of pharmacological agents targeting the 5-LO pathway are currently available to treat inflammatory conditions such as asthma, ulcerative colitis, arthritis, and psoriasis. These agents are generically known as LT-modifying drugs and include 5-LO inhibitors, FLAP inhibitors, and cys-LT receptor antagonists. Direct 5-LO inhibitors such as zileuton (ZyfloR ) have shown limited applicability and are only marketed for the prevention and treatment of chronic asthma in adults and children 12 years of age or older (83). FLAP inhibitors have recently attracted much attention because linkage analysis has demonstrated that the gene encoding for FLAP confers a higher risk of myocardial infarction and stroke (84). A very potent and selective FLAP inhibitor, DG-031 (VeliflaponR ), has shown efficacy in preventing heart attacks or strokes in patients with a history of unstable angina or myocardial infarction (85). A similar compound, DG-051, which targets LTA4 hydrolase, is currently being tested in these patients. Finally, orally active receptor antagonists directed against the cys- LT1 receptor including montelukast (SingulairR ), pranlukast (UltairR ), and zafirlukast (AccolateR ) have been marketed in the past few years (83, 86). These agents have demonstrated significant beneficial actions in exercise-induced asthma, allergic rhinitis, and cardiocerebrovascu- lar disease (83, 86). On the other hand, LTB4 receptor antagonists have shown to be efficacious in preclinical models of arthritis and atherosclerosis (87, 88). In the liver, Kupffer cells are the predominant source of 5-LO products (89). Indeed, among the different cell types within the liver, Kupffer cells are the only cell type endowed with the complete enzymatic machinery (i.e., 5-LO, FLAP, LTA4 hydrolase, and LTC4 synthase) necessary for the biosynthesis of LTs (Fig. 2a) (90). Nevertheless, transcellular biosynthesis of cys-LT can be produced through cooperation between Kupffer cells and hepatocytes, since the latter are rich in LTC4 synthase and can transform the intermediate epoxide LTA4 released by Kupffer cells into LTC4/D4/E4 (90). In the context of liver diseases, there are compelling evidence of the participation of 5-LO products in the pathogenesis of acute and chronic liver injury. In this regard, increased urinary excretion of LTs, the levels of which correlate with the severity of liver disease, has been demonstrated in patients with acute alcohol intoxication, in cirrhotic patients with and without ascites, and in patients with intrahepatic cholestasis and obstructive jaundice (91–94). There are also evidence supporting that 5-LO products are specific mediators of inflammation and cell damage in response to endotoxin/D-galactosamine challenge (95). In another model of hepatoxicity in which rats were treated The Role of Inflammatory Mediators in Liver Failure 143

Fig. 2. Role of lipid mediators derived from the 5-lipoxygenase (5-LO) pathway in liver injury. a Expression of enzymes of the 5-LO pathway (i.e. 5-LO, FLAP and LTC4 synthase) in liver cells. KC, Kupffer cells; HSC, hepatic stellate cells; H, hepatocytes; m: size marker; c+: positive control (RNA from macrophages). b Expression of 5-LO and formation of LTC4/D4/E4 in samples of hepatic tissue from control (CT) and CCl4-induced cirrhotic (CH) rats. c The FLAP inhibitor Bay-X-1005 reduces hepatocellular necrosis and hepatic LTC4/D4/E4 levels in CCl4-treated rats. d The FLAP inhibitor Bay-X-1005 reduces liver ﬁbrosis, assessed by analysis of tissue sections stained with H&E (X25) and Masson’s trichrome (X125) and hepatic hydroxyproline content by RP-HPLC in CCl4-treated rats. Reprinted from Titos et al. (90, 103, 104) with permission from Elsevier, The Journal of the Federation of American Societies for Experimental Biology and The Society for Leukocyte Biology, respectively.

with LPS and Propionibacterium acnes, inhibition of LT synthesis reduced massive hepatocyte necrosis (96), while in the hepatotoxin α-naphthylisothiocyanate model, 5-LO inhibition with zileuton did not attenuate liver damage (97). In addition, AA-861, a potent and selective 5-LO inhibitor, was effective in reducing chronic liver injury in mice and intrahepatic vascular resistance in rats (98, 99), while the cys-LT1 receptor antagonist montelukast exhibited remarkable protection against liver failure (100, 101). More conclusive results have been reported by Titos et al. in the CCl4 model of liver injury, in which 5-LO mRNA expression and hepatic levels of cys-LTs were found to be markedly increased (Fig. 2b) (90). Similar ﬁndings were reported in rats with thioacetamide-induced ﬁbrosis (102). In the 144 Joan Clària et al.

CCl4 model, inhibition of the 5-LO pathway with either a nonredox type 5-LO inhibitor or a potent FLAP inhibitor significantly reduced hepatic necroinflammatory damage and fibrosis (Fig. 2c, d) (103–105). Amelioration of necroinflammatory liver injury and fibrogenesis has also been observed in cholestatic rats treated with the cys-LT1 receptor antagonist montelukast (106). Moreover, mice bearing a targeted deletion of the 5-LO gene are protected against CCl4-induced necroinflammation (105). Interestingly, dual inhibition of the 5-LO and COX-2 pathways appears to provide a higher profile of protection against CCl4- induced necroinflammation in mice (105). Since the 5-LO pathway is essential for cell survival and its expression in the liver is basically restricted to Kupffer cells (90), the observed protection against necroinflammatory liver injury exerted by 5-LO inhibition could be partially mediated by depletion of hepatic macrophages through induction of apoptosis (105). Finally, 5-LO inhibitors as well as FLAP inhibitors have shown potent protective effects in hepatic ischemia/reperfusion injury (107, 108).

5. SUMMARY In summary, in the past few years, several lines of evidence have firmly established that a number of inflammatory mediators including cytokines (i.e., TNF-α), reactive oxygen species, and specially small inflammatory lipid mediators derived from arachidonic acid through the activity of the COX and 5-LO pathways are pathogenic factors involved in liver injury. A list of these inflammatory mediators together with other mediators not discussed in this chapter such as platelet- activating factor (PAF), plasminogen activator-1 (PAI-1), fibronectin, or complement factor C3/C5a is summarized in Table 1. In addition, the pharmacological manipulation of the biological effects of TNF-α as well as the modulation of the eicosanoid cascade represent potential targets for the design and discovery of new therapeutic molecules in inflammatory liver injury. Balancing oxidative stress status and intervention of the formation and actions of the nonenzymatic lipid peroxidation products derived from arachidonic acid (i.e., F2-isoprostanes) should also be contemplated. A list of the investigational and marketed drugs targeting these inflammatory pathways and their effects on liver injury is summarized in Table 2. The Role of Inflammatory Mediators in Liver Failure 145

Table 1 List of the most relevant inﬂammatory mediators implicated in liver injury

Inﬂammatory Main source within Reported actions mediators the liver

Cytokines TNF-α Kupffer cells Inﬂammation and tissue injury IL-1 Kupffer cells, Inﬂammation HSCs, SECs IL-6 Kupffer cells, HSCs Hepatic acute-phase response Cardiotrophin-1 Unknown Cytoprotection

Reactive oxygen species (ROS) O2– Kupffer cells Inﬂammation and tissue injury ONOO– Unknown Inﬂammation and tissue injury

Arachidonic acid-derived lipid mediators PGD2 Kupffer cells Unknown 15d-PGJ2 Kupffer cells Hepatocyte apoptosis, HSC apoptosis PGF2α Kupffer cells Vasoconstriction PGE2 Kupffer cells, Controversial: inflammation or hepatocytes, cytoprotection HSCs TXA2 SECs Vasoconstriction, hepatocellular damage LTB4 Kupffer cells Inflammatory cell recruitment, inflammation LTC4/LTD4/LTE4 Kupffer cells Vasoconstriction, inflammation F2-Isoprostanes Hepatocytes, Oxidative stress markers, (8-epi-PGF2α) Kupffer cells, vasoconstriction HSCs, SECs Others PAF Kupffer cells, Inflammation, tissue injury hepatocytes PAI-1 Unknown Tissue injury Fibronectin HSCs Controversial protective actions C3/C5a Kupffer cells, Inflammation, tissue repair hepatocytes

C3/C5a, complement factors; HSCs, hepatic stellate cells; IL, interleukin; LT, – – leukotriene; O2 , superoxide anion; ONOO , peroxinitrite anion; PAI-1, plasminogen activator-1; PAF, platelet-activating factor; PG, prostaglandin; SEC, sinusoidal endothelial cells; TNF, tumor necrosis factor; TX, thromboxane 146 Joan Clària et al. ) 53 , 52 ) ). No effects ( 62 51 , ) , ) 58 23 50 , – 66 , 47 20 – 64 44 infections ( Increased survival but increased incidence of severe Table 2 Yes No No No Yes No effects ( Yes No No ) No Yes fibrosis. Decreased incidence of renal damaging ) Yes Yes Cytoprotection ( ) Yes Yes Prevention of necroinflammatory liver injury and α )NoYes )YesYes Cytotec Flosulide Edex Celebrex (Furegrelate) List of investigational and marketed drugs targeting inflammatory mediators and pathways in liver failure Ozagrel Analogs synthase inhibitors 2 2 Misoprostol ( Celecoxib ( CGP-28238 ( NS-389 No Yes effects ( Infliximab Adalimumab Etanercept DFU No Yes U-63557A OKY-046 ( Alprostadil ( TargetDrugs Targeting TNF- Drugs Targeting the COX pathway COX-2 inhibitors Tested patients Animal models Reported effects (References) PGE TXA The Role of Inflammatory Mediators in Liver Failure 147 ). No ) 66 , 107 , 64 , 105 ) 63 , 99 108 , , 98 101 , 100 ) 108 ) , 67 104 , 103 effects ( Table 2 (continued) No Yes Protection liver failure ( ) No Yes Reduction of necroinflammation, fibrosis, I/R injury )NoYes Veliflapon ) )NoYes Ramatroban Singulair List of investigational and marketed drugs targeting inflammatory mediators and pathways in liver failure Zyflo receptor antagonists 2 SQ-29548 No Yes Reduction portal perfusion pressure ( BAY-X-1005 (RG-031) ( MK-886 No Yes ( BAY-U-3405 ( DG-051 No No Montelukast ( AA-861CJ-13,610 No No Yes Yes Reduction intrahepatic resistance, I/R injury, necroinflammation, fibrosis ( Zilueton ( TargetTXA Tested patients Animal models Reported effects (References) FLAP inhibitors LTA4 hydrolase inhibitors Cys-LT1 receptor antagonists Drugs targeting the 5-LO pathway 5-LO inhibitors 148 Joan Clària et al.

ACKNOWLEDGEMENTS Our research is supported by grants from the Ministerio de Ciencia e Innovación (SAF 09/08767). CIBERehd is funded by the Instituto de Salud Carlos III.

REFERENCES

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Genomics of the Liver in Health and Disease

Konstantinos N. Lazaridis, MD

CONTENTS INTRODUCTION SINGLE-GENE VS.COMPLEX DISEASES STRUCTURE AND VARIATION OF THE HUMAN GENOME RELATION OF GENETIC VARIATION TO DISEASE PHENOTYPES STUDY DESIGNS TO DISSECT DISEASE-CAUSING GENETIC VARIANTS THE HUMAN HAPLOTYPE MAP ETHICAL,LEGAL, AND SOCIAL IMPLICATIONS OF HUMAN GENOMICS SUMMARY GLOSSARY REFERENCES

Key Words: Bioethics, Candidate genes, Complex diseases, Familial aggregation, Genetic diversity, Genetic information, Genome-wide Association Studies, Genomics, Haplotype, Haplotype block, Human haplotype map, Single nucleotide polymorphisms, Single-gene diseases, Susceptibility alleles, Tag SNPs

Abbreviations HGP human genome project SNP single nucleotide polymorphism

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_8, C Springer Science+Business Media, LLC 2011

155 156 Lazaridis

1. INTRODUCTION In 2003, the complete sequence of the human genome became a reality (1, 2). This was the result of an international effort known as the Human Genome Project (HGP). The knowledge gained by completion of the HGP coupled with the emerge of the discipline of genomics have already fueled basic and translational studies to better understand the pathogenesis of liver disease as well as to improve their prognosis and therapies. Hepatologists are expected to directly or indirectly inﬂu- ence the course and application of genomics in liver disease. This is because hepatologists are experts in assessing and classifying pertinent phenotypes and traits related to liver. In the next sections, we discuss (i) the differences between single- gene and multifactorial diseases; (ii) the structure and variation of the human genome; and (iii) the effect of genetic variation to disease phenotypes. A basic glossary of genomics terms is included at the end of the chapter.

2. SINGLE-GENE VS. COMPLEX DISEASES In general terms, there are three categories of genetic disorders: chromosomal, single-gene (i.e., Mendelian), and complex (i.e., multifactorial). Chromosomal diseases are the outcome of deletion or addition of intact chromosomes or segments. Many chromosomal disorders lead to spontaneous abortions or miscarriages because lack of or gain of aberrant segment(s) or entire chromosomes, are usually non- ﬁtting with life. In clinical practice, the majority of hepatologists do not encounter patients with chromosomal disease. Single-gene diseases (i.e., Mendelian) exhibit familial patterns of inheritance (i.e., autosomal recessive, autosomal dominant, or X-linked), and phenotypic expression of the disease is caused by a few rare mutations of a single gene. Single-gene diseases are not frequent in the population, the most frequent being hereditary hemochromatosis, which affects one of every 300 individuals. The genetic basis of Mendelian diseases is simple because of the direct correspondence of a speciﬁc genotype to a phenotype (Fig. 1)(3). Complex diseases, such as alcoholic liver disease, nonalcoholic steatohepatitis, and primary biliary cirrhosis, to mention a few, are considered multifactorial in etiology. These diseases are caused by interaction of several genes or genetic variants with environment factors and might elucidate a modest genetic effect on disease phenotype (4)(Fig.2). As a result, the direct correspondence of a genotype to a phenotype that describes a Mendelian disease does not exist in complex diseases. This notion may also explain the Genomics of the Liver in Health and Disease 157

Mendelian diseases

Mutation Genotype

Gene

Dominant, recessive, or X-linked inheritance Phenotype

Fig. 1. In Mendelian diseases, a single gene is responsible for a disorder, and the disease phenotype follows a predicted inheritance pattern (i.e, autosomal dominant, autosomal recessive, or X-linked). In a family, all affected members carry exactly the same mutation. Mendelian diseases are characterized by a close correspondence of a genotype to a phenotype.

Complex diseases Genetic variant Genetic variant Genetic variant

Gene B

...Gene X Gene A

Environment

Phenotype

Fig. 2. In complex diseases, multiple genetic variants interact with each other along with the environment to cause the disease phenotype. Each genetic variant and the environment have a small effect on the phenotype. Because of the contribution of several genetic variants and environmental factors, complex diseases are heterogeneous in their pathogenesis, progression, and response to treatment. 158 Lazaridis heterogeneity of complex disease etiology and variation of phenotypes (i.e., disease manifestation(s), progression and response to treatment). Although complex diseases demonstrate familial aggregation (i.e., the risk of disease among relatives of the proband is greater than the estimated risk in the general population), this inheritance is not predictable as is in Mendelian disease (5). Complex diseases are the majority of illnesses that we, as hepatologists, evaluate daily in clinical practice and this is the type of disorders in which genomic discoveries will have the highest impact (5).

3. STRUCTURE AND VARIATION OF THE HUMAN GENOME Based on the HGP, we now know that (i) the number of genes in the human genome is about 30,000; (ii) genes are unevenly spaced across the 23 chromosomes; (iii) less than 2% of genomic DNA encodes for proteins; (iv) more than 50% of the genomic DNA consists of repetitive sequences of yet unknown function; (v) the majority of human genes undergo alternative splicing—a molecular mechanism to generate isoforms of proteins with different functional capacities (see glossary); and (vi) genomic regions that code for proteins account for less than 50% of DNA that has been conserved over 70 million years since the divergence of human and mouse (1, 2). This implies that noncoding regions of our genome have been subjected to evolutionary selection. Human displays relatively limited genetic diversity (i.e., variation or polymorphism) because of its relatively young age (∼100,000–125,000 years) and thus, the genetic material was transmitted through a small number (∼5,000) of generations from our ancestral origins (3). Genetic variation contributes in both health and disease, and a better understanding of genetic polymorphisms and biological function of gene(s) will provide us with unparalleled insights into human biology (4). With the exception of monozygotic (i.e., identical) twins, any two human beings share 99.9% of their genomic sequence (1, 2). However, this difference of 0.1% that translates to approximately three million genetic variants coupled with an individual’s environmental exposures (i.e., household, lifestyles, habits, etc.) will determine the variation we observe in health (e.g., body weight) or disease (e.g., alcoholic liver disease, gallstone disease, primary sclerosing cholangitis). One of the aims of HGP was to develop a comprehensive catalog of the millions of existing human genetic variants. The most common of these variants are single nucleotide polymorphisms (SNPs) (1, 2), which are currently available in public databases (http://www.ncbi.nlm.nih.gov/projects/SNP). In fact, in every 500–1,000 base pairs of human genome sequence, there Genomics of the Liver in Health and Disease 159 is an SNP where alternates of nucleotides can exist. For example, an SNP such as a C/T (i.e., abbreviation for Cytosine or Thymine) is a nucleotide location that can harbor one of two alleles (C or T). The more frequent allele of an SNP in a population is called the major allele, rendering the other one the minor allele (6). The location of each SNP within the genome may determine its functional signiﬁcance. SNPs located within or in proximity to a gene are more likely to have an impact on gene function, particularly if they introduce a stop codon or change an amino acid moiety of a protein. SNPs populating intergenic regions are thought to have nonfunctional consequences on gene(s), but they can serve as useful genetic markers in disease-mapping studies and population genetics (6). Other less common variations of the genome include microsatellites and insertions/deletions (7). SNPs are likely the most important genetic variants in the human genome because of their high frequency. They are also easily assayed using automated, high-throughput approaches to score them. SNPs mainly represent genetic markers of biologic diversity. In some cases, they can be the cause of a healthy or disease trait. As inherited markers

Linkage disequilibrium around an ancestral SNP

Ancestral chromosome

Contemporary chromosomes

Fig. 3. The position of a genetic variant (i.e., SNP) is shown with an arrow on an ancestral chromosome. Because of meiotic recombination that occurs over thousands of generations, contemporary chromosomes have variable length segments of the common ancestral chromosome (regions shown in white) that flank the original SNP (i.e., arrow), while new chromosomal sections introduced by recombination are depicted in gray. Thus, genetic markers (i.e., SNPs) that are in physical proximity will remain associated with the original SNP, even as recombination restricts the extent of the association region over time. 160 Lazaridis of variation, SNPs may be in close proximity to a genetic factor that causes a disease. When recombination on a chromosome between an SNP and a disease-causing allele has taken place, such an SNP and the actual genetic factor are said to be in linkage disequilibrium and form a haplotype (Fig. 3)(8). An international effort has defined SNP-based haplotypes (i.e., combinations of SNP alleles found at neighboring loci on the same chromosomal segment, which tend to be transmitted together from generation to generation). These chromosomal regions (termed haplotype blocks) represent stretches of 25,000–35,000 base pairs in length across human genome (9). More importantly, although many SNPs could be present in a haplotype block, only a few (termed tag SNPs) are important to define each block and its haplotypes. SNPs and SNP-based haplotype methods are powerful approaches to identify the genetic basis for complex disease including liver disorders.

4. RELATION OF GENETIC VARIATION TO DISEASE PHENOTYPES 4.1. A. The Common Disease–Common Variant Hypothesis The common disease–common variant hypothesis is based on the fact that the present human population of six billion people represents a global expansion that occurred ∼100,000 years ago from a single sub- Saharan African founding population of relatively small size (∼10,000 people). Thus, the current human population shares a number of alleles from this small group of founders. The hypothesis proposes that alleles present before the global expansion and divergence of humans contribute significantly to predisposition (i.e., susceptibility alleles) of common complex disease. Such alleles may bestow moderate risk to common disease and should occur at relatively high frequencies (i.e., higher than 1%) in the present human population (10). This high frequency of alleles implies that association studies (see below) in large population cohorts will lead to identifying the susceptibility alleles of common complex diseases. The presence of haplotype blocks in the human genome and the fact that a limited number of common haplotypes account for the majority of current haplotypes (9) suggests that association studies with representative SNPs (i.e., tag SNPs) (see below) will identify common haplotypes associated with predisposition to common complex disease. This hypothesis is the scientific basis for developing a genome-wide human haplotype map that describes all major haplotypes and the specific SNPs (tag SNPs) that define them (9). Genomics of the Liver in Health and Disease 161

4.2. B. The Common Disease–Rare Allele Hypothesis An opposing view proposes that most complex diseases are caused by rather rare than frequent alleles (11, 12). The hypothesis predicts extensive allelic and locus heterogeneity at complex disease loci (i.e., different alleles at the same locus and alleles at numerous different loci independently cause the same disease phenotype). Furthermore, more than 99% of the variants predisposing to complex diseases arose following the global expansion and divergence of the human population (11). If this hypothesis is true, genome-wide association studies in a heterogeneous population that search for susceptibility alleles of common complex disease will be fruitless. Similarly, the current construction of a haplotype map based on common alleles (i.e., Human Haplotype Map) (see below) would be inadequate to deﬁne the variants of common complex diseases.

5. STUDY DESIGNS TO DISSECT DISEASE-CAUSING GENETIC VARIANTS 5.1. Candidate–Gene Approaches Given the challenges of linkage strategies to identify the causal genes for complex diseases, alternative approaches, such as association analyses, have been employed. Association analysis is based on a case–control design that searches for a statistical correlation between particular genetic variant(s) and a disease or disease trait (13). Large association studies possess greater statistical power than linkage methods to detect genes that have a small effect on the disease phenotype (14). The genetic variants (i.e., SNPs) may be located on genes (i.e., candidate genes) or distributed throughout the genome. One association study design that evaluates genetic variant(s) of biologically plausible candidate genes would follow this general procedure (15, 16): (i) hypothesized genes that may be involved in the pathogenesis of a disease of interest are suggested; (ii) functional genetic variants with or in close proximity to coding regions, 5 and 3 untranslated regions, and intron/exon boundaries of the candidate genes are identified; (iii) subjects are ascertained including careful definition of the disease phenotype in cases and well-matched, unrelated, unaffected individuals; (iv) cases and controls are genotyped; and (v) statistical analysis is performed to determine whether there is an association between the examined variants and the phenotype of disease. Candidate-gene approaches can be limited by population 162 Lazaridis stratification biases and reproducibility (16). Absence of reproducibility is because the initial reports may be based on small sample size (i.e., less than 200 patients), differences in study design, or heterogeneity of disease locus (i.e., affected individuals possess causal variants at different loci).

5.2. Genome-Wide Association Studies Another approach based on the association strategy is called genome- wide association. In these studies, hundreds of thousands of specific SNPs that encompass the entire genome are analyzed in patients (i.e., cases) and unrelated normal individuals (i.e., controls). Linkage disequilibrium analysis is then used to map the genomic region identifying susceptibility genes or variants. This method is unbiased with respect to specific genes or regions of the genome; however, it may be biased due to population stratification. Since 2005, successful application of genome-wide associations studies (GWAS) resulted in demonstrating strong evidence of association between liver disease (e.g., fatty liver, primary biliary cirrhosis) and traits (e.g., response to therapy for chronic hepatitis C) with potentially causal SNPs. A catalog of published GWAS is provided at the National Human Genome Research Institute web site (http://www.genome.gov/26525384). These discoveries offer promise to dissect the pathogenesis of, predict individual susceptibility to, and apply personalized therapy to disease. Nevertheless, several issues make the application of these genomics discoveries inadequate in clinical practice. For example, contribution of each associated genetic locus to disease is small for the individual carrying the risk allele (i.e., odds ratio of 1.5 or less) (17). Also, strong association does not necessarily guarantee perfect differentiation between affected and healthy individuals. Moreover, the reported associations of GWAS with disease may not be the causal one. Perhaps the main contribution of GWAS is to identify regions of the genome and/or pathways that contribute to human disease. GWAS have no power to discover rare variants of the genome that also affect illness. To discover these uncommon polymorphisms, resequence of human genomes has to take place. Finally, healthcare providers have limited knowledge to interpret these tests for the benefit and education of their patients.

6.THEHUMANHAPLOTYPEMAP The HGP elucidated the complete sequence of the human genome. Despite this milestone, the greatest challenges remain ahead. The translation of this sequence data collection into discoveries that allow the identification of genes and genetic variants in health and disease Genomics of the Liver in Health and Disease 163 will be an enormous task for scientists, physicians, and other health professionals for years to come. Genetic variants such as SNPs can affect an individual’s overall phenotype as well as predispose this person to disease. A group of SNPs located on the same chromosome can be inherited together as a block (4). About 200,000–400,000 blocks may exist in the human genome (9). Although each block contains several thousand SNPs, a small number of tag SNPs will be adequate to identify most blocks in the genome as well as discriminate among the majority of haplotypes that exist in a block. This data is now included in the HapMap (4). The HapMap is a navigator of haplotype blocks, along with the specific SNPs that will define the haplotypes present in each block. This approach limits the number of SNPs needed to pursue a whole-genome association study. Because haplotypes differ among populations of different origins, the HapMap data has focused on common SNPs and haplotypes in four large (i.e., 200–400 individuals), geographically distinct ethnic groups, namely, Japanese, Han Chinese, Yoruba of Nigeria, and US residents with ancestry derived from northern and western Europeans.

7. ETHICAL, LEGAL, AND SOCIAL IMPLICATIONS OF HUMAN GENOMICS Physicians are responsible to protect the confidentiality of their patients’ medical record and to practice medicine in a safe manner: pri- mum non nocere. However, genetic information is discrete from other types of data gathering (i.e., demographic, social, medical) because it has implications for future risk of disease in an individual and likely his/her relatives. The relevant values of bioethics, including the principles of beneficence, respect for autonomy, privacy, confidentiality, and equity, all apply to genetic testing. These principles have implications for determining how clinicians should approach and manage the genetic information with patients and their relatives. The legal and social ramifications of genetic predisposition testing are multiple and interrelated. Genetic information should be considered confidential. Genetic results should be released only to patients, and health professionals should exercise all precautions to prevent unauthorized disclosure to third parties. Physicians are often asked to disclose benefits/risks of genetic testing, to maintain confidentiality of genetic information, and to warn of inherited genetic risk to patients and their family members. To address the ethical, legal, and social implications of knowing our genetic predisposition is a multifaceted task. Physicians and health care providers have to be educated on how to interpret and commu- nicate genetic information to patients and relatives, to help them to 164 Lazaridis make informed decisions regarding their health. Public health agencies should focus on determining when genetic data and tests are trustworthy for routine clinical use. Society has to create laws and monitor their implementation to prevent the inappropriate use of genetic information.

8. SUMMARY Genomics will likely inﬂuence the practice of medicine for years to come. The overall aim is to be able to better predict the risk of an individual to develop complex disease, so that preventive interventions can be applied and, if needed, treatment can be optimized (Fig. 4). To achieve this goal, three steps appear to be essential. We have to better understand the structure (i.e., variation) and function of the human genome. Also, genetic epidemiology studies are needed to dissect the inherited susceptibility variants and environmental factors accounting for contribution to disease. Finally, functional assessment of the associated variants is needed for veriﬁcation and translation of these discoveries into clinical tests and development of innovative pharmacological targets to effectively treat disease.

The impact of genomics in medicine

Disease with genetic component

Identify casual gene(s)/genetic variant

Diagnostic Understand basic tests biological defect

Gene Prevention Drug therapy therapy

Fig. 4. Genomics will likely lead to better understanding the genetic variation as a cause of disease. This knowledge will improve our diagnosis, treatment, and hopefully prevention of human illnesses.

GLOSSARY Allele an alternative form of DNA sequence. Autosomes all human chromosomes but the sex chromosomes (i.e., X and Y) and mitochondrial DNA. Genomics of the Liver in Health and Disease 165

Codon a three-base nucleotide sequence that specifies an amino acid. Epigenetic an expression describing nonmutational phenomena, such as methylation and histone modification that alter gene expression. Euchromatin the gene-rich regions of the genome. Exon a transcribed region of a gene that codes for a protein. Genotype a person’s genetic structure, as reflected by his/her DNA sequence. Haplotype the combination of alleles found at adjacent loci on the same chromosomal segment. Heterochromatin the gene-poor regions of the genome composed of repetitive DNA sequences. Heterozygous having two different alleles at a specific autosomal gene locus. Homozygous having two identical alleles at a specific autosomal gene locus. Intron a nontranscribed region of a gene that does not code for a protein. Linkage the tendency of DNA sequences at specific loci to be inherited together as a consequence of their physical proximity on a single chromosome. Linkage analysis a method to trace and measure the cosegre- gation of a disease in a family with marker loci. Loci plural of locus—the physical location of a gene. Linkage disequilibrium particular alleles at two or more neighboring loci show allelic association if they occur together with frequencies significantly different from those predicted from the individual allele frequencies. Microsatellites small run (usually less than 0.1 kb in length) of tandem repeats with a very simple DNA sequence, commonly 1–4 base pairs. 166 Lazaridis

Mutation an inherited modification in the sequence of genomic DNA. Mutation, frame-shift mutation caused by deletion or insertion of nucleotides (i.e., DNA bases) resulting in an altered open-reading frame of a gene and usually to a truncated protein. Mutation, missense a single nucleotide (i.e., DNA base) substitution leading to a codon that defines an alternative amino acid of a protein. Mutation, nonsense a single nucleotide (i.e., DNA base) substitution resulting in a stop codon that causes truncation of a protein. Mutation, silence a single nucleotide (i.e., DNA base) substitution that causes no change in the amino acid of a protein. Penetrance the likelihood that a person carrying a particular mutant gene will have an altered phenotype. Phenotype the observable features or expressions of a specific gene(s), environmental factors, or both. Polymorphism any variation of two more alleles (i.e., variant). Recombination a natural phenomenon during which regions between pairs of equivalent chromosome, and thus, DNA are exchanged. Relative risk ratio the risk of a sibling to develop a disease if of a sibling (λs) his/her biological brother or sister is already affected. The λs is calculated by dividing the prevalence of a disease among siblings from the prevalence of the disease in the general population. SNPs single nucleotide polymorphisms—any polymorphism (i.e., variation) due to the difference at a single nucleotide between two or more genomes. SNPs are the most common variation in the genome sequence; the human genome contains approximately ten million SNPs. Although less informative than microsatellites, SNPs are more amenable to large-scale automated scoring. Genomics of the Liver in Health and Disease 167

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Part II Effects of Liver Failure on Organ Systems

Hepatic Encephalopathy and Alterations of Cerebral Function

Juan Córdoba and Rita García-Martinez

CONTENTS INTRODUCTION EFFECTS OF LIVER FAILURE ON BRAIN FUNCTION MECHANISMS BY WHICH LIVER FAILURE INDUCES HE PRINCIPLES OF TREATMENT REFERENCES

Key Words: Hepatic encephalopathy, Ammonia, Energy impairment, Brain edema, Hyponatremia, Acute-on-chronic liver failure

1. INTRODUCTION Liver failure is characterized by the induction of a series of abnormalities of brain function which are included under the term hepatic encephalopathy (HE). The neurological manifestations are very variable and can be acute, chronic, or subclinical (minimal HE) (1). In addition, HE can be associated with cirrhosis, fulminant hepatitis, or portosystemic bypass without intrinsic liver disease. In patients with cirrhosis, the most common underlying disease, HE, may be precipitated by an extrahepatic factor (gastrointestinal bleeding, infection, TIPS, disturbances of electrolytes, constipation) or be secondary to an acute exacerbation of a liver disease (acute-on-chronic). Distinction

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_9, C Springer Science+Business Media, LLC 2011

171 172 Córdoba and Martinez between both situations is clinically and pathophysiologically relevant. Precipitating factors appear to have a major role in increasing the exposure of the brain to toxins, and HE resolves usually rapidly with the correction of the precipitating factor (2). In patients with acute-on-chronic liver failure, there are additional aspects of major pathophysiological importance: systemic inﬂammatory response, circulatory dysfunction, and failure of other organs that can cause directly disturbances of brain function (3).

2. EFFECTS OF LIVER FAILURE ON BRAIN FUNCTION 2.1. Disturbances of Neurotransmission HE, as other forms of metabolic encephalopathy, results in abnormalities in neurotransmission (4). This hypothesis is supported by its potential reversibility and by the lack of neuronal damage. Multiple abnormalities of neurotransmitter systems have been described in animal models of HE, including disturbances in the excitatory glutamatergic (5) and inhibitory GABAergic (6) neurotransmitter systems. Some supportive data are provided by studies on autopsied material (7) and by neuroimaging techniques. However, it is very difﬁcult to relate the complexity of the disturbances of neurotransmission to the neurological manifestations. Several therapeutic attempts have been conducted to restore disturbances in neurotransmission with speciﬁc drugs, but the results have not been remarkable (8).

2.2. Injury to Astrocytes Astrocytes are the cells of the central nervous system that are affected in HE (9). The distinctive neuropathological alteration is the Alzheimer type II astrocytic change, probably a chronically degenerated astrocyte secondary to cellular swelling. Glutamine is generated in the astrocytes during the detoxification of ammonia through the amidation of glutamate. The accumulation of glutamine may cause an increase in intracellular osmolality (10) or induce mitochondrial injury by the activation of the mitochondrial permeability transition (11). Factors that precipitate HE, such as inflammation, hyponatremia, and benzo- diacepines, can exacerbate swelling (12). The change in the state of cellular hydration causes impairment of several metabolic pathways and has been proposed to be responsible for brain edema and for the neurological manifestations of HE (13). Mechanisms by which abnormal glial cells can influence neuronal function include interaction with glutamate reuptake (14) and activation of peripheral-type benzo- diacepine receptors, causing increased synthesis of neurosteroids that Hepatic Encephalopathy and Alterations of Cerebral Function 173 are powerful ligands of the neuronal GABAA receptor (an inhibitory neurotransmitter) (15).

2.3. Energy Impairment The brain is the tissue with the highest energy requirements of the body and depends entirely on the process of glycolysis and respiration within its own cells to synthesize its energy demands. In HE in humans, a decrease in consumption of oxygen and glucose is accompanied by a parallel decrease in cerebral blood ﬂow (16). It is not possible to separate whether the decrease in oxygen consumption is the cause or the consequence of encephalopathy. The current interpretation is that, as in other metabolic encephalopathies, energy impairment is secondary to the decrease in neuronal function. However, a direct effect of ammonia on energy metabolism causing neuronal disturbances is also possible (17). In fulminant hepatic failure, and possibly in acute-on-chronic liver failure, disturbances in energy metabolism may have an important participation in the clinical picture. In patients with acute liver failure increases in brain lactate, identiﬁed by brain microdialysis, are followed by surges of high intracranial pressure (18). Furthermore, an increase in plasma lactate is a well-recognized prognostic factor in fulminant and in acute-on-chronic liver failure (19). Experimental models have shown that the increase in brain lactate is of newly synthesized origin and par- allels the increase of brain water in the intracellular compartment (20). Ammonia may impair glycolysis, because it inhibits α-ketoglutarate dehydrogenase, the rate-limiting enzyme of the tricarboxylic acid cycle (21), and may have a direct toxic effect on the mitochondria (11). An alternative explanation is that lactate is generated aerobically by excessive glutamatergic activation (22), which may be induced by excessive amidation of glutamate, secondarily to an increase in brain ammonia (Fig. 1). Irrespective of the mechanism being involved, a drop of brain pH can cause injury at multiple levels, including astrocyte swelling (23).

2.4. Brain Edema Brain edema is now recognized as an element that is present in acute and in chronic liver failure and can be identiﬁed by indirect (24)and direct techniques of magnetic resonance (25). The main factor involved in the generation of brain swelling is the increase in plasma ammonia (26). Other factors, such as hyponatremia, may enhance the effects of ammonia on brain swelling (27). An intriguing ﬁnding is the different distribution of water in the brain in acute and in chronic liver 174 Córdoba and Martinez

glc NH3

a pyr 3 NADH NAD+ lac ATP TCA ATP ATP gln GS 2 1 ATP b K+

4 Na+

glu

Fig. 1. Interaction between ammonia, the glutamate–glutamine cycle, and energy metabolism in astrocytes and neurons. Glutamate (Glu, a neurotransmitter) is reuptaken from the synaptic cleft by the astrocyte (1) in cotransport with Na+ (2). The excess Na+ is interchanged with K+ through a Na/K pump (3). Ammonium (NH3) enters the astrocyte from blood through passive diffusion and combines to glutamate to synthesize glutamine (gln), a reaction (4) that is catalyzed by glutamine synthetase (GS). Glutamine is transported to the neuron where it is transformed again to glutamate, a major neurotransmitter, and closes the glutamine–glutamate cycle. Glucose (glc) enters the astrocyte from blood (a) and is transformed into pyruvate (pyr) to produce ATP in mitochondria. A significant amount of pyruvate is transformed to lactate (lac) that is shuttled to the neuron (b). Lactate reaches the neuron where it is transformed into pyruvate to provide ATP through the tricarboxyilic acid cycle (TCA). failure, suggesting different pathogenetic mechanisms. In acute liver failure, brain water is mostly located in the intracellular space (28), while in chronic liver failure is mostly extracellular (29). This difference suggests a role for increased blood–brain barrier permeability in the chronic situation, which may be mediated through inflammatory mediators. Brain edema has been proposed to have major consequences on neuronal function (13), but good evidences are lacking. The increase in the volume of the brain inside a rigid skull can cause intracranial hypertension, which is responsible for a significant number of deaths in fulminant hepatic failure. Compensatory mechanisms that Hepatic Encephalopathy and Alterations of Cerebral Function 175 require chronic induction, a lower rise in plasma ammonia, and a smaller brain volume explain why intracranial hypertension is sel- dom seen in cirrhosis (30). In fulminant hepatic failure, and possibly in acute-on-chronic liver failure, cerebral vasodilatation and loss of autoregulation may worsen brain swelling (31); measures that inhibit cerebral vasodilatation decrease intracranial pressure (32).

2.5. Brain Atrophy Different neuroimaging techniques have shown brain atrophy in more than half of the patients with cirrhosis and chronic HE (33). The prevalence is higher among alcoholic patients, because alcohol causes a dose-related decrease in brain size that is aggravated by a poor nutritional status but is partially reversible with abstinence (34). Similarly, the chronic exposure to neurotoxins involved in the pathogenesis of HE could lead to loss of brain tissue that can explain the persistence of neuropsychological deﬁcits after liver transplant (35).

3. MECHANISMS BY WHICH LIVER FAILURE INDUCES HE Liver failure causes an increase in the exposure of the brain to several substances that under normal circumstances are efficiently metabolized by the liver; those substances that have a high “first-pass” metabolism are the most important, as shown by the major role of portosystemic shunting in the development of HE. In addition, other factors that are commonly present in patients with liver failure and may worsen neurological function are inflammation, circulatory derangements, nutritional deficits, comorbidities, and failure of other organs.

3.1. Ammonia Toxicity Ammonia has been historically viewed as the most important factor in the genesis of HE (Fig. 2). In normal conditions, ammonia is produced by the gut and an important amount is of bacterial origin (36). The concentration of ammonia in portal blood is high, and a high degree of extraction occurs in the liver (37). Ammonia levels are high in patients with HE (38), specially among those with large portosystemic shunts. Similarly, effects on blood ammonia and brain metabolites are seen in shunts secondary to portal vein thrombosis (39) or of congenital origin. In addition to the intestine and the liver, kidney and muscle contribute to regulate the arterial ammonia level (40). In muscle, ammonia is transformed into glutamine through the action of glutamine synthetase. 176 Córdoba and Martinez

Brain BBB Neuron Astrocyte Gln Gln Mitochondria Muscle

Glu NH3 Glu Synapse Shunts

Liver NH3 Gln Urea Kidney Colonic Diet flora Gnase Intestine

Urea

Fig. 2. Interorgan ammonia trafficking and metabolism. Ammonia is generated in the intestines from nitrogenous compounds from the diet, deamination of glutamine by glutaminase, and metabolism of nitrogenous substances by colonic flora. In normal circumstances, most ammonia is metabolized to urea in the liver. Portal-systemic shunts and liver failure cause a rise in blood ammonia that may affect brain function by inducing several disturbances in astrocytes that may impair mitochondria and the glutamate–glutamine trafficking between neurons and astrocytes. Skeletal muscle is capable to decrease blood ammonia by metabolizing ammonia to glutamine. Kidney has also an important role in determining blood ammonia by excreting urea in the urine and generating ammonia. NH3, ammonia; Glu, glutamate; Gln, glutamine; GNASE, glutaminase; BBB, blood–brain barrier.

The ability of the muscle to “ﬁx” appreciable amounts of blood-borne ammonia becomes important to regulate arterial ammonia in case of liver failure and highlights the importance of maintaining an adequate muscle mass. Patients with HE have an increased diffusion of ammonia into the brain in relation to an increase in arterial ammonia (41). In the brain, ammonia is metabolized to glutamine in astrocytes, where ammonia or glutamine exerts their toxic effects. Recent data provide more information on the mechanisms by which ammonia causes neuronal disturbances, but a complete explanation is still lacking (42). Signs of oxidative stress, such as protein tyrosin nitration, have been found in several experimental preparations. In addition to injury to proteins, ammonia induces RNA oxidation, which may have multiple Hepatic Encephalopathy and Alterations of Cerebral Function 177 consequences in neurotransmission and postsynaptic protein synthesis. Such changes may also underlie the pathologically altered oscillatory networks in the brain of HE patients in vivo, as detected by magnetoencephalography (43).

3.2. Inflammation Patients with acute and acute-on-chronic liver failure develop frequently a marked activation of inflammatory mediators (44). The presence of a systemic inflammatory response syndrome has been linked to the development of HE in fulminant hepatic failure (45) and in cirrhosis (46). The activation of inflammatory mediators, such as cytokines, may modulate the effect of neurotoxins on the brain. The accompany- ing impairment in renal function can increase circulatory urea levels, with subsequent colonic generation of ammonia via urease-containing bacteria. Peripheral inflammation may signal the brain through the activation of vagal afferents (47). Other mechanisms of transduction of signals into brain are binding of cytokines to receptors in cerebral endothelial cells or direct access of cytokines into brain tissue at sites lacking blood–brain barrier (such as the circumventricular organs) (48). Inflammation may also be directly induced in the brain. Microglial activation and induction of synthesis of proinflammatory cytokines have been shown in experimental models (49). Neuroinflammation has an important role in many neurological diseases. In HE, activation of inflammation in brain tissue may increase blood–brain barrier permeability, result in the generation of intracerebral mediators (such as nitric oxide and prostanoids), and cause astrocytic swelling (13, 50).

3.3. Circulatory Dysfunction Patients with liver failure, especially those with acute and acute- on-chronic liver failure, exhibit commonly circulatory disturbances characterized by low arterial pressure, low peripheral vascular resistance, and high cardiac index (51). It has been hypothesized that this circulatory dysfunction participates in the pathogenesis of neuronal disturbances (52). There is a close parallelism between renal and cerebral circulation in liver failure. In advanced liver failure, both territories lose the property of vascular autoregulation (53). In patients with cirrhosis and ascites, there is renal and cerebral vasoconstriction, which are probably related to arterial hypotension and to the overactivity of vasoconstrictor systems (54). The clinical experience also links renal failure to HE. In patients with advanced cirrhosis, an increase in serum creatinine and a decrease in serum sodium are the two most important factors involved in the recurrence of HE (55). The experience with patients 178 Córdoba and Martinez with cirrhosis and organic nephropathies suggest that the mechanisms involved in recurrence of HE do not simply correspond to a decrease in the excretion of urea in urine. The increase in creatinine in advanced cirrhosis identiﬁes the presence of circulatory dysfunction (56). It is probably that circulatory dysfunction is a key mechanism in precipitating HE in patients with advanced liver failure, especially among those with acute-on-chronic liver failure.

4. PRINCIPLES OF TREATMENT HE is a manifestation of severe liver failure; its treatment cannot be separated from the treatment of liver failure, which requires a series of supportive measures, including the general management of a patient with change in mental status. Several measures speciﬁcally designed to treat HE appear to be beneﬁcial (57), although many of them have not undergone proper assessment in good clinical trials and have been criticized (58). Treatment of precipitating factors (Table 1)isamain- stay of management, which requires their active search and continuous monitoring.

4.1. Nutritional Measures Intake of large amounts of proteins should be avoided because they can precipitate HE. However, the classically recommendation of restricting dietary protein intake is no longer valid (59). In patients with cirrhosis, a low-protein diet does not improve the outcome of acute HE (60) and does not reduce its recurrence (61). Protracted nitrogen restriction may be harmful, as witnessed in patients with acute alcoholic hepatitis (62). The current recommendation is to give a diet that contains a normal amount of proteins (0.8–1.2 g/kg/d). Severe malnutrition, which is common among patients with cirrhosis, is associated with a poor short-term prognosis. A positive nitrogenous balance may improve encephalopathy by promoting hepatic regeneration and increasing the capacity of muscle to detoxify ammonia (39). However, improvement in nutritional status in patients with cirrhosis is difﬁcult. A high-protein intake (>1.2 g/kg/d) may be necessary to maintain nitrogen balance, but can increase blood ammonia and may precipitate HE (63). Modifying the composition of the diet and increasing its calorie/nitrogen ratio may improve tolerance to protein. At isonitrogenous levels, vegetable and dairy products cause less encephalopathy than does meat (64). A high calorie-to-nitrogen ratio, which is characteristic of casein-based and vegetable diets, reduces gluconeogenesis and has anabolic effects on the utilization of dietary Hepatic Encephalopathy and Alterations of Cerebral Function 179

Table 1 Precipitating factors for HE

Precipitating Possible effects Mechanism of Associated factor action coprecipitant

Sepsis Increase in blood Protein catabolism Azotemia ammonia Activation of Arterial Enhancement of the cytokines hypotension effects of putative toxins on the CNS Gastrointestinal Impairment in liver Hepatic Infection bleeding function hypoperfusion Anemia Increase in blood Nitrogen load Arterial ammonia Disturbances of hypotension plasma amino acids Hypokalemia Increase in blood Ammonia ammonia generation Azotemia Increase in blood Ammonia ammonia generation Dehydration Increase in blood Hepatic Hypokalemia ammonia hypoperfusion Azotemia Diuretics Increase in blood Hypokalemia ammonia Azotemia Dehydration Acute hepatitis Impairment in liver Liver injury function Activation of Enhancement of effects cytokines on the CNS Surgery Impairment in liver Hepatic Anesthetics function hypoperfusion Constipation Increase in blood Ammonia ammonia generation by enteric ﬂora Large protein Increase in blood Nitrogen load intake ammonia Psychoactive Enhancement of effects Activation of drugs on the CNS inhibitory neurotransmission

CNS, Central nervous system 180 Córdoba and Martinez proteins. The beneﬁts of vegetable-based diets have also been related to the presence of nonabsorbable ﬁber that is metabolized by colonic bacteria. Branched-chain amino acids show anticatabolic effects in patients with chronic liver diseases, probably due to their ability to serve as an energy substitute for muscle, and because of their actions on muscle protein synthesis and degradation (65).

4.2. Decreasing the Production of Toxins: Prebiotics, Probiotics, and Antibiotics The observation of a relationship between portosystemic shunting, constipation, and HE leads to the concept that the intestinal flora is an important source of toxins. This was followed by the introduction of intestinal cleansing, prebiotics, probiotics, and antibiotics to treat HE. The goals of these treatments are to increase fecal nitrogen excretion, reduce the generation of ammonia by fecal flora, and decrease the amount of ammonia that reaches portal blood (66). This can be achieved by promoting the growth of saccharolytic flora with little urease activity and reducing the bulk of proteolytic flora. Another possible beneficial effect of modifying the enteric flora is reducing translocation of intestinal bacteria; a decrease in plasma endotoxin has been shown with prebiotics and probiotics (67). Translocation of bacterial products may activate inflammatory mediators (68), worsen hemodynamic parameters, and favor the development of HE. Lactulose (a nonabsorbable disaccharide) is a prebiotic that was first introduced with the idea of increasing the amount of Lactobacillus bifidus in the enteric flora. The mechanism of action is more complex. Administered orally, lactulose and lactitol (a similar nonabsorbable dis- sacharide) are not broken down by intestinal disaccharidases and reach the cecum, where they are metabolized by enteric bacteria to lactate and acetate (69). These metabolites cause a drop in cecal pH, which is critical for the drugs to be effective, and is associated with cathar- sis. A similar effect can be obtained by giving different combinations of probiotics that are enriched in lactobacillus or in other “healthy” species (70). The efficacy of prebiotics and probiotics in decreasing blood ammonia and improving minimal HE is similar at short term (71). However, probiotics lack the cathartic effect of nonabsorbable disaccharides and fiber, which make them better tolerable, but could also limit their efficacy. Another alternative that has been proposed is administration of acarbose, a drug that inhibits glucose absorption and modifies the enteric flora. In one study, treatment with acarbose decreased ammonia and improved HE (72). Hepatic Encephalopathy and Alterations of Cerebral Function 181

Several antibiotics that reduce the gram-negative bacilli population have been introduced in the treatment of HE. Neomycin and rifaxim, two antibiotics that are poorly absorbed and decrease blood ammonia, are commonly prescribed as alternatives to nonabsorbable disaccharides (73). In addition to decreasing enteric flora, neomycin causes a reduction in mucosal glutaminase activity and thereby decreases the ability of the mucosa to consume glutamine and produce ammonia (74). There are concerns that long-term therapy with neomycin could result in intestinal malabsorption and renal or auditory toxicity, because it is an aminoglycoside. Rifaximin appears safer for prolonged therapy (75). Therapies aimed at reducing the production of toxins by the intestinal flora are by far the most commonly used and better studied. Unfortunately, few placebo controlled trials have been conducted. The best results have been observed for treatment of minimal HE (58)and for prevention of recurrence of HE (76). Combination of different therapies may exert some synergism, but the available data are scarce (77). In patients with cirrhosis and an acute episode of HE, the major aims of therapy are controlling the precipitating factor and improving liver function. In this circumstance, many patients receive broad-spectrum antibiotics. The administration of drugs that decrease the intestinal production of toxins may have only a marginal benefit (78).

4.3. New Therapies The current burden of illness and hospitalization for HE is very high (79), indicating that there is a need for better therapies. New goals of therapy are achieved with drugs that reduce blood ammonia without interfering with enteric flora, new measures for precipitating factors, and liver-support devices. The generation of ammonia in the small intestine may be reduced by inhibiting glutaminase (80). However, since glutamine is a major energetic substrate of the intestine, this may result in serious adverse effects (81). An alternative mechanism to decrease ammonia is to increase the disposal by stimulating the synthesis of nontoxic nitrogenous compounds. Muscle may become an important organ to enhance ammonia detoxification by conversion to glutamine. L-ornithine– L-aspartate, which has undergone clinical evaluation (82) and is available in several countries, and L-ornithine–L-phenylacetate(83), now under clinical investigation, provide intermediates for glutamine synthesis and decrease plasma ammonia. Exacerbation of circulatory dysfunction (identified by an increase in creatinine) and hyponatremia are the two most important risk factors for 182 Córdoba and Martinez the development of HE (55). Aquaretic drugs increase plasma ammonia and have shown some promise in improving minimal HE (84). Their putative mechanism of action is through diminishing astrocyte swelling. It is possible that patients with ascites and hyponatremia treated with aquaretics could experience fewer episodes of HE. The administration of albumin alone or combined with vasoconstrictors have shown to be beneficial in preventing circulatory dysfunction in patients with cirrhosis (56) and may secondarily reduce the incidence of HE. In patients with diuretic-induced HE, patients treated with albumin showed a better outcome than those treated with another volume expander (85). The physicochemical characteristics of albumin and the observation of improvement in parameters of oxidative stress suggest that treatment with albumin may decrease the effects of toxins on circulatory, renal, and neurological function (86). Liver support devices, such as the Molecular Adsorbents Recirculating System (MARS), might also play a role. MARS improves the grade of HE (87) independently of changes in ammonia and cytokines, suggesting that other toxins, such as oxygen-based free radicals, might be important.

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Bacterial Translocation and Alterations of the Digestive System

Reiner Wiest

CONTENTS PATHOLOGICAL BACTERIAL TRANSLOCATION IN CHRONIC LIVER FAILURE:DEFINITION AND DIAGNOSIS OF BACTERIAL TRANSLOCATION PATHOPHYSIOLOGY OF PATHOLOGICAL BACTERIAL TRANSLOCATION PATHOLOGICAL BT AND ITS POTENTIAL CONSEQUENCES REFERENCES

Key Words: Bacterial translocation, Caspase-activating and recruitment domain 15, Clostridium difﬁcile-associated disease, Colony-forming units, Dendritic cells, Gut-associated lymphoid tissue, Gastrointestinal tract, Hepatopulmonary syndrome, Intestinal bacterial overgrowth, Lipopolysaccharide, Mesenteric lymph nodes, Nitric oxide, Polymerase chain reaction, Portal hypertensive gastropathy, Portal hypertensive duodenopathy, Reticuloendothelial system, Spontaneous bacterial peritonitis, Tight junctions, Tumor necrosis factor

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_10, C Springer Science+Business Media, LLC 2011

189 190 Wiest

1. PATHOLOGICAL BACTERIAL TRANSLOCATION IN CHRONIC LIVER FAILURE: DEFINITION AND DIAGNOSIS OF BACTERIAL TRANSLOCATION Bacterial translocation (BT) has initially been defined as the migration of viable microorganisms from the intestinal lumen to mesenteric lymph nodes (MLNs) and other extraintestinal organs and sites (1). This definition has later been refined by including all microbial translocation, defined as the passage of both viable and nonviable microbes and microbial products such as endotoxin across an even anatomically intact intestinal barrier. Limited BT to MLN is a physiological phenomenon that has been proposed to be essential for development and maintenance of tolerance against the intestinal flora (2). However, any increase in rate and severity of BT may be deleterious for the patient and thus should be termed “pathological BT.” In experimental cirrhosis and portal hypertension, BT is usually assessed as viable bacteria being culturable in mesenteric lymph nodes (3–6). In these studies the whole chain of MLN is dissected, homog- enized, and cultured. Moreover, utilizing green-fluorescent-protein- marked Escherichia coli and using intravital microscopy translocation of E. coli across the intestinal mucosa could be visualized after its administration into the lumen of a surgically created small bowel pouch in cirrhotic rats (7). By this approach, a vastly shorter transit time in cirrhotic rats as compared to control rats could be demonstrated. Studies of BT in humans are limited because of the need for surgery and the removal of usually only one MLN in conditions that may alter the results (e.g., perioperative antibiotics). Although the rate of positive MLN cultures appears to be higher in Child C cirrhotic patients, BT has not been predictive of the development of postoperative infections (8). Alternative approaches to diagnosing BT in humans have thus been postulated. BT can be considered the primary event in the genesis of endotoxemia in cirrhosis. While endotoxemia is present in cirrhotic rats with BT, only low and in most cases negligible concentrations of endotoxin are detectable in cirrhotic rats without BT (9). The higher endotoxin levels in splanchnic blood of cirrhotic animals and the strong correlation between MLN and blood endotoxin support a gut origin of endotoxin. This is further supported by the finding in patients subjected to shunt surgery of higher portal than peripheral venous endotoxin levels (10). Besides endotoxin, TNF has been proposed as indirect surrogate marker of pathological BT. TNF levels in MLN seem to have a better correlation with Child status and with the development of bacterial infections (11); however, this method still requires surgery. In BT and Alterations of the Digestive System 191 contrast, the plasma levels of lipopolysaccharide (LPS)-binding protein (LBP), produced by the liver in response to endotoxin (gram-negative infections), remain increased for up to 72 h after transient bacteremia or endotoxinemia and thus reflect a long-term memory of gram-negative bacterial translocation (12). LBP levels were found to be increased in a subset of patients, with advanced cirrhosis being predictive for the development of severe bacterial infections (13). Over the last couple of years, PCR-based detection of bacterial DNA (bactDNA) has been proposed as a surrogate marker for BT, since it has been detected in blood and ascites of about a third of patients with cirrhosis and culture-negative ascites (14, 15). Lending validity to this test, sequential testing shows that bactDNA from subsequent samples was identical to the one detected in a first sample (16). However, methodological concerns remain, particularly since a variety of bacterial species have been reported to be detectable even in healthy volunteers (17, 18), inherent to a technique that amplifies eubacterial PCR bactDNA contamination in the pre-PCR workup and/or at the time of DNA extraction (19). Furthermore, at best multicentric studies are needed to validate this and other molecular modalities.

2. PATHOPHYSIOLOGY OF PATHOLOGICAL BACTERIAL TRANSLOCATION Three factors have been implicated in the development of BT (20), all of which have been found to be present in cirrhosis (Fig. 1): intestinal bacterial overgrowth (IBO), increased intestinal permeability, and impaired immunity. Intestinal bacterial overgrowth (IBO). The upper gastrointestinal (GI) tract in healthy conditions is sparsely populated with bacteria; this may well be the reason for the known lower resistance to bacterial translocation in the upper GI tract, since this compartment is not made to host vast amounts of bacteria. In contrast, from the ileum on there is a sharp increase in microbial density, from 105 colony-forming units (CFU)/ml in the jejunum to 108 in distal ileum and cecum, up to 1012 in the colon (21). Therefore, IBO is deﬁned as more than 105 CFU/ml in the duodenum. Intestinal anaerobic bacteria normally outnumber aerobic bacteria by 100:1 to 1000:1, despite which anaerobes very rarely translocate (22). In contrast, aerobic gram-negative bacteria translocate easily and even across a histologically intact intestinal epithelium (23–26). Moreover, anaerobic bacteria limit the colonization and overgrowth of other potentially invasive microbes, thereby 192 Wiest

Luminal factors Bacterial overgrowth Colonization factors Virulence Zonula occludens Bacteria (tight junctions) Attachment Aerobic Zonula adherens IgA Anaerobic Bile Mucins Mucus Chloride Desmosome

Penetration Gap junction Oxidative stress Mucosal acidosis Enterocytes ATP depletion

Immune response Lamina propria Lymphocytes Local GALT T-cell activation Macrophages Cellular recuritment of macrophages/neutrophils Blood vessel Hematogenous/portal Chemokine/cytokine release Systemic Lymphatic vessel Lymphogenous/MLN Reticuloendothelial system Liver, spleen, lung

Fig. 1. Pathophysiology of bacterial translocation. Modified after Wiest and Garcia-Tsao (205). GALT, Gut-associated lymphoid tissue; MLN, mesenteric lymph node; ATP, adenosine triphosphate. confining potentially pathogenic bacteria. In fact, selective elimination of anaerobic bacteria facilitates intestinal bacterial overgrowth and translocation of facultative bacteria (26). Bacteria that translocate most readily are facultative intracellular pathogens (e.g., Salmonella, Listeria) that are known to resist phagocytic killing to various degrees. In contrast, normal enteric species (commensals) are easily killed after phagocytosis, surviving only when host defenses are impaired. Gram-negative bacteria (specifically E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and other Enterobacteriaceae), enterococci, and other streptococci have been found to be the most adept at translo- cating to MLN (22). Interestingly, these species are those that most frequently cause infection in cirrhotic patients (27). Special strains of E. coli have been shown to translocate more efficiently, probably as a result of a greater ability to adhere to the intestinal mucosa (28). Moreover, differences in virulence among strains may lead to greater resistance against host defense mechanisms allowing for a more efficient survival and dissemination (29, 30). Bacterial overgrowth is one of the main factors that promote BT. A direct relationship between numbers of a specific bacterial strain populating a segment of the intestine and numbers of viable bacteria of this strain present in MLN has been demonstrated in mice (31), particularly when adherent bacteria are involved (32). BT and Alterations of the Digestive System 193

Experimental studies demonstrate that rats with cirrhosis, ascites, and BT have significantly higher rates of IBO compared to animals without BT (33, 34). Importantly, in the absence of IBO (i.e., with bacterial counts within two standard deviations from the mean bacterial count of normal rats), BT occurs rarely (0–11%) and at rates comparable to normal rats. However, since BT does not occur in up to half the animals with IBO, it appears that IBO is necessary but not sufficient for BT to occur and that other factors play the most important role. In humans, IBO has been shown to be more prevalent in cirrhotic patients than in healthy controls, particularly in those with more severe liver disease (35) and in those with a prior history of spontaneous bacterial peritonitis (SBP) (36). These studies have raised concerns regarding the use of breath tests in the diagnosis of IBO, but studies in which IBO is assessed by quantitative culture of jejunal aspirates have also shown high IBO rates of 43% (33) and 61% (37) in cirrhotic patients. Interestingly, in one of these studies (37), development of SBP did not correlate with IBO but did correlate with ascitic fluid protein (marker of decreased local immunity) and with serum bilirubin (marker of poor liver function), underscoring the importance of a decreased immune status as the main factor in the pathogenesis of BT in cirrhosis. IBO in cirrhosis has been related to a delayed intestinal transit time that has been shown to occur in cirrhotic rats, particularly in those with BT (34), and in patients with cirrhosis (38), particularly in those with more severe liver disease (39). In fact, cisapride has been shown to reduce BT rates being associated with lower jejunal, but not cecal, bacterial counts (40) pointing toward the upper GI tract as main site of pathological BT to occur. Another factor contributing to the development of IBO is hypo- and achlorhdria which have been observed in cirrhotics, resulting in higher pH in the small intestine and thus lowering bacterial defense and enabling bacterial overgrowth (41). In the same context, the use of proton pump inhibitors (PPIs) has been found to be associated with IBO (42) and retrospective case–control studies revealed its association with the development of SBP (43). PPIs also increase the risk of enteric infections per se including Clostridium difficile-associated disease (CDAD) (44) for which hospitalized cirrhotic patients are particularly predisposed (45). The development of CDAD in cirrhotics predicts a poor prognosis, since mortality in patients with cirrhosis and CDAD is higher than that in patients with either disease alone (even after controlling for age, comorbid conditions, and other complications of cirrhosis) (45). Of note, more than 60% of cases on PPI had no good indication for PPI therapy (46) and therefore should be used very careful in high-risk patients such as cirrhotic patients, avoiding its use except for indications with proven benefit. In addition, antibiotic use 194 Wiest

(either outpatient, e.g., as prophylaxis for SBP or in hospital) was identified as risk factor for CDAD in hospitalized cirrhotic patients (46). Thus, any hospitalized cirrhotic patient requiring antibiotics should be monitored for the development of CDAD. Increased Intestinal permeability. The intestinal mucosal barrier includes both secretory and physical preventive measures against the penetration of microbes. Secretory components include mucins, chloride, antimicrobial peptides, etc. whose role in promoting BT has not been addressed adequately in cirrhosis. However, fecal excretion of secretory IgA has been observed to be decreased in some patients with cirrhosis (47). Moreover, bile protects against BT by inhibiting IBO, having trophic effects on the intestinal mucosa (48), decreasing epithelial internalization of enteric bacteria (49), exerting detergent actions with antiadherence effects, and binding and neutralizing endotoxins (50, 51). Therefore, the absence of bile in the intestine has been shown to facilitate BT (52–55) and to enhance endotoxin-induced BT (56). In cirrhosis, marked decreases in intestinal intraluminal concentrations of bile acids have been ascribed to decreased secretion and increased deconjugation by enteric bacteria. Conjugated bile acids activate farnesoid X receptor (FXR), a nuclear transcription factor, in the distal small intestine inducing the expression of gene products that promote antimicrobial defense (inhibiting bacterial overgrowth) and epithelial integrity (preventing mucosal injury) (57). Indeed, intestinal mucosal FXR overexpression prevents bacterial translocation in bile duct-ligated animals revealing a central role of FXR in protecting the distal small intestine from bacterial invasion. The physical part of the intestinal barrier is the mucosal monolayer of epithelium itself. Specialized cell–cell junctional complexes allow for selective paracellular permeability (tight junctions, TJs), maintain intercellular adhesion (intermediated junctions and desmosomes), and permit intercellular communication (gap junctions). At the apicolateral epithelial surface, TJs maintain a permeability seal restricting paracellular movement of even very small (2 kDa) molecules, thereby preventing the transepithelial movement of not only bacteria, but also macromolecules such as lipopolysaccharide (LPS). However, BT of living bacteria does not occur via the paracellular but is mediated via transcytosis (58). In cirrhosis, data on TJ proteins and function as well as transcytosis are lacking. Ultrastructurally, dilated extracellular spaces between neighboring enterocytes and a reduced number of microvilli have been noted, although appearance of TJs in distal duodenum is unaltered in patients with advanced cirrhosis (59). Most interestingly, distension of intercellular space has been found to correlate best with serum aldosterone, indicating a role for plasma volume BT and Alterations of the Digestive System 195 in this scenario (60). In fact, acute plasma volume expansion results in marked widening of the intercellular space (61). Experimental cirrhosis results in oxidative stress of the mucosa of the small intestine as determined by increased xanthine oxidase activity and altered antioxidant status, increased lipid peroxidation of the brush border membranes, and abnormal intestinal transport (62). These are changes similar to those described in endotoxin-induced damage, in which BT is also increased (63). The association of cirrhosis with oxidative damage of the intestinal mucosa is further supported in another study in which malonaldehyde, a marker of lipid peroxidation, was found to be significantly higher in ileal and cecal (but not in jejunal) mucosa of cirrhotic rats, mainly in those with ascites and BT (64). Other factors that may contribute to this impairment in barrier function at the epithelial level are increased levels of nitric oxide, tumor necrosis factor and interferon- γ, all known to be present in increased levels in advanced cirrhosis (65–67). Each of these mediators has been shown to directly impair epithelial barrier function (68, 69). However, even in the presence of mucosal injury, the strain of bacteria (pathogenic vs. nonpathogenic) appears to be a more important determinant of BT (28). Regarding functional changes, intestinal permeability has been shown to be increased in cirrhotic rats with ascites, particularly in those with BT (34, 70). However, while BT occurred in 13/15 (87%) cirrhotic rats with both IBO and increased intestinal permeability, it did not occur in any of six animals with increased intestinal permeability alone (34). Furthermore, elimination of IBO alone, without changes in intestinal permeability, led to a decrease in BT. This further suggests that IBO is more important than increased gut permeability in promoting BT. Studies of intestinal permeability in patients with cirrhosis using differential sugar absorption are controversial. While some show no differences in permeability between cirrhotic patients and controls (71), others observe increased permeability in advanced cirrhosis, mainly in those with sepsis or spontaneous bacteremia/SBP within 10 days of the test. As sepsis itself can induce intestinal permeability changes, these results are difficult to interpret. Impaired immunity. For translocation to become clinically significant, namely e.g. to lead to SBP or bacteremia, a failure of local and systemic immune defenses should also be present. That is, in a healthy, nonimmunocompromised host, translocated bacteria may reach MLN or portal blood but they will usually be phagocytosed and killed prior to multiplication and seeding of systemic blood and other sites. As for the local defense at the level of the gut, the intestinal tract contains essentially every type of leucocyte involved in immune response. The “gut” mucosal immune system consists of the gut-associated lymphoid tissue 196 Wiest

(GALT), the largest immunological organ of the body, which comprises four lymphoid compartments: Peyer’s patches, lamina propria lymphocytes (including dendritic cells, DCs), intraepithelial lymphocytes, and MLN. Microbial colonization of the gastrointestinal tract affects GALT composition and function (72). Intestinal commensals interact with the gut epithelium and trigger both innate and adaptive immune responses. Data on the GALT in cirrhosis are sparse and just starting to evolve. In experimental cirrhosis, expansion and activation of Th1 cells and monocytes in MLN has been noted (73). Moreover, BT is associated with mononuclear cell infiltrate in the lamina propria as well as severe submucosal and mesenteric inflammation, particularly at the cecal level (4, 74, 75). Although it is uncertain whether inflammation is the cause or the result of BT, in animal models of severe intestinal inflammation viable bacteria appear in the portal circulation even before they appear in efferent intestinal lymph (76), suggesting that inflammation may influence the route of translocation by damaging intercellular associations and allowing access to submucosal capillaries. In this context, genetic polymorphisms related to nucleotide-binding site and leucine- rich repeat (NBS–LRR) proteins, involved in intracellular recognition of microbes and their products, namely, the caspase-activating and recruitment domain-15/nucleotide oligomerization domain 2 (CARD 15/ NOD2) gene, have been implicated in the pathogenesis of mucosal inflammation in Crohn’s disease (77, 78) and in gastrointestinal graft- versus-host disease (79), conditions associated with increased BT. In fact, the occurrence of SBP was likewise observed to be significantly increased in cirrhotic patients carrying NOD2 risk alleles (80). Moreover, detection of any NOD2 risk allele was found to represent the strongest independent predictor of death in these patients. Besides such polymorphisms, deficiencies in host defense beyond the intestine are well accepted in cirrhosis. Importantly, cirrhosis is accompanied by an impaired reticuloendothelial system (RES) activity. The RES is the main defensive system against bacteremia and other infections acquired through a hematogenous route. Most of the RES activity is located in the liver where Kupffer cells (tissue macrophages) are the major component. In cirrhosis, RES activity is impaired because of portosystemic shunting that bypasses the liver (thereby escaping the action of the RES) and because of an impaired phagocytic activity of Kupffer cells. It has been shown that cirrhotic patients with a decreased RES activity develop spontaneous bacteremia and SBP at a higher rate than patients with normal RES activity (81). Bypassing the RES through portosystemic shunting is an important mechanism that explains not only the failure to clear portal or systemic bacteria in cirrhosis, but also the failure to clear other bacterial products such as endotoxins and cytokines. BT and Alterations of the Digestive System 197

The even higher risk of infection in cirrhotic patients with GI hemorrhage is thought to be secondary to multiple factors, among them a further decrease in RES activity (82, 83) and a higher BT rate (84). Moreover, beyond gut and liver, cirrhosis is accompanied by impairment of systemic host defense mechanisms such as a decrease in chemotaxis, phagocytosis, and bactericidal activity of peripheral blood mononuclear cells (PMNs) (85–88). Decreased receptor-independent (intrinsic) phagocytosis, particularly for E. coli, and decreased opsonic activity have been postulated as mediators of this cellular dysfunction. Impaired tuftsin activity, known to modulate biological activities of phagocytic cells, is reduced in cirrhotic patients and is associated with a higher incidence of bacterial infections (89). Reduced complement levels as well as defects in Fcy-receptors on mononuclear cells may contribute to decreased opsonic activity (90–92). In cirrhotic patients, low ascites complement levels correlate with decreased opsonic activity, decreased bactericidal activity, and increased risk for SBP (93, 94). Also, low serum C3 levels independently predict infection and correlate with a poor liver synthetic function (95). Interestingly, peripheral blood mononuclear cells but not resident spleen mononuclear cells exhibit a normal Fcy-receptor function and expression in vitro (96), emphasiz- ing the importance of distinguishing between systemic/circulating and local/resident cellular mechanisms of innate immunity.

3. PATHOLOGICAL BT AND ITS POTENTIAL CONSEQUENCES Pathological BT in advanced liver cirrhosis with impaired host defense represents a disruption of the normal host/ﬂora equilibrium that may lead to a self-perpetuating vicious circle affecting many organ systems (Fig. 2). BT has been postulated as the main mechanism in the pathogenesis of spontaneous infections in cirrhosis as well as the hyperdynamic circulatory state, a key factor in the growth of varices and in the development of ascites. In addition, even independent of the development of any overt bacterial infection, bacterial products as well as the BT-associated proinﬂammatory cytokine response may have severe clinical consequences in advanced liver cirrhosis triggering and/ or exacerbating hepatic dysfunction, hepatic encephalopathy, hepatorenal syndrome as well as hepatopulmonary syndrome. Finally, translocation of bacterial DNA associates with poor survival (97) which may well be due to an increased risk of acute-on-chronic liver failure pointing toward a major role of bacterial products in modulating intrahepatic signaling pathways. 198 Wiest

Hepatic Clearence encephalopathy capacity SBP Hepatorenal Inflamm- syndrome BT ation Variceal bleeding? LPS, TNF, IL-6, IL-1, CpGs, Cachexia? LTA, NOx, etc. etc. Hepato Potentiationi pulmonary Risik — chance for development Risik — chance for Splanachnic vasodilatation development Risik — chance for syndrome?

Degree of hyperdynamic circulatory syndorme

Fig. 2. Pathological bacterial translocation as motor of a potential vicious circle in chronic liver failure. Modiﬁed after Wiest and Schölmerich (206). BT, Bacterial translocation; SBP, spontaneous bacterial peritonitis; LPS, lipopolysaccharide; IL, interleukin; TNA, tumor necrosis factor; LTA.

BT and spontaneous infections. Spontaneous bacterial infections in chronic liver disease are the result of failure of the gut to contain bacteria and failure of the immune system to kill the bacteria once they have escaped the gut. Pathological BT has a potent impact on the natural course of the disease and is widely accepted to be the most important event in the development of bacterial infections in patients with advanced cirrhosis and ascites. Older studies assessing the etiology and types of bacterial infections in cirrhotic patients showed that the most common infections were community acquired, mainly urinary tract infections, SBP and pneumonia, 70–80% of which were caused by gram-negative bacilli, mainly E. coli, suggesting that the gut was the main source of bacteria. The spectrum of bacteria causing infection in cirrhosis in more recent series shows a significantly higher rate of gram- positive cocci infections, probably due to an increase in the number of therapeutic invasive procedures (98) and to the use of chronic antibiotic prophylaxis (99, 100). However, the most common infections, SBP and urinary tract infection, are still mostly caused by gram-negative bacteria (101) and thus are thought to be due to BT. In fact, molecular evidence clearly demonstrates the presence of identical strains of bacteria in stool, MLN, and ascites in the majority of cases of SBP in experimental BT and Alterations of the Digestive System 199 cirrhosis (6). Also in patients with cirrhosis and culture-negative, nonneutrocytic ascites bacterial DNA was detected simultaneously in blood and ascitic fluid, and DNA sequencing revealed the similarity of bacterial strain in both locations, indicating that the bacterial DNA present in ascites and blood originated from the same single clone (102). However, presence of bacterial DNA was not found to increase the risk of endogenous spontaneous infections including SBP (97). This underscores that translocation of bacterial DNA has to be separated from pathological translocation of viable culturable bacteria not only in its pathogenesis but also in its clinical impact. BT and cytokine storm. The well-known priming of mononuclear cells to produce excessive amounts of proinflammatory cytokines in conjunction with a marked decrease in clearance capacity in advanced cirrhosis can result in a “cytokine storm” induced by pathological BT. In fact, activation of peripheral mononuclear cells with a marked increase in LPS-induced TNF expression has been reported in cirrhotic patients with ascites (103–105). Moreover, increased TNF production by MLN has also been reported in end-stage cirrhotic patients and has been associated with an increased incidence of bacterial infections after liver transplantation (11). Moreover, the presence of bactDNA in decompensated cirrhosis was found to induce the complement system activation (106) and correlate with a higher synthesis of NO by peritoneal macrophages/monocytes and higher cytokine production (107, 108), leading to enhanced levels of ascitic and serum TNF (109). Important to emphasize is the fact that bactDNA can be derived from gram-negative as well as gram-positive bacteria. With respect to the degree and type of inflammatory response, the presence of bactDNA from gram-positive bacteria is associated with a similar increase in ascitic and serum levels of TNF, IL-6, and NOx as bactDNA from gram- negative bacteria being not related to LPS (110, 111). In fact, overall the degree of soluble immune response induced by bacterial DNA in cirrhotic patients with noninfected ascites has been suggested to be comparable to patients with SBP (112). BT and hemodynamic disturbances. The level of proinflammatory cytokines is higher in ascitic fluid as compared to serum or other compartments in case of SBP (113), indicating that the splanchnic circulation faces the highest levels of those cytokines also known to trigger vasodilation. The enhanced susceptibility of the splanchnic circulation for any vasodilative stimulus in the decompensated stage of disease points toward the potent clinical impact of any “pathological BT.” In experimental cirrhosis, the presence of BT has been shown to result in further significant impairment of mesenteric vascular contractility and hyporesponsiveness to endogenous vasoconstrictors (9, 67). Moreover, 200 Wiest increased serum levels of LBP have been reported to identify a subpopulation of patients with enhanced levels of proinflammatory cytokines being associated with a more severe hyperdynamic circulatory syndrome (114). Indeed, selective gut decontamination has been shown to ameliorate the hyperdynamic circulation in experimental as well as human cirrhosis (67, 114–116). Considering the “forward” component of portal hypertension due to increased portal venous inflow associated with the hyperdynamic splanchnic circulation, BT could also aggravate portal hypertension. Although this has not been evidenced so far in human cirrhosis, preliminary data indicate that BT worsens intrahepatic endothelial dysfunction since postprandial increase in portal pressure was found to be significantly increased in bactDNA-positive patients (117). BT and distant organ failure. The potential detrimental intrahepatic effects of gut-derived products include acceleration of fibrogenesis (118), modulation of liver injury, and hepatic dysfunction (119, 120). These effects are not restricted to endotoxin and products of gram- negative bacteria but also to gram-positive bacteria and bacterial DNA (121, 122). In fact, the presence of bactDNA (simultaneous in blood and ascites) in a cirrhotic patient with ascites associates with an increased 30-day mortality which appears to be mainly due to the development of acute-on-chronic liver failure (97). In addition, the gut and its flora has long been known to reflect one of the main sources of ammonia leading to and aggravating hepatic encephalopathy. Moreover, other gut-derived toxins implicated in HE are γ-aminobutyric acid (GABA) and benzodiazepine (BZD)-like substances (123), both of which may also be produced by specific colonic bacteria (124, 125). The importance of the intestinal flora and most likely pathological BT in the pathogenesis of HE is supported by studies showing that the total colectomy leads to a significant decrease in baseline and protein-induced ammonia production (126) and reverses cases of medically intractable HE (127). However, HE recurrence occurs in this setting probably as a result of colonization of small bowel (128). In addition, primary prophylaxis of SBP utilizing norfloxacin in high-risk cirrhotic patients prevents also the development of hepatorenal syndrome most likely via inhibition of pathological BT (129). Finally, experimental data shed light on the gut–lung axis. Prevention of gram-negative bacterial translocation has been shown to ameliorate the severity of hepatopulmonary syndrome in experimental cirrhosis (130). Experimental shock-induced lung injury has been shown to be prevented by prior surgical mesenteric lymphatic division, indicating that gut-derived mediators carried in the mesenteric lymph are involved in pulmonary injury (131). However, these interesting findings have not been verified so far in cirrhotic patients. BT and Alterations of the Digestive System 201

3.1. Macro- and Microscopic Changes in the Digestive System in Chronic Liver Failure Portal hypertension is associated with a variety of mucosal and vascular alterations seen at endoscopy and being subject to multiple histomorphological and pathophysiological evaluations. In general, depending on the cohort studied, these findings appear to be more prevalent in advanced stages of disease and increasing severity of portal hypertension. However, no investigation so far did assess the whole gastrointestinal tract concomitantly at once as for macroscopic changes and thus stomach, duodenum/small intestinum, and colon are discussed seperately. Most changes described are vascular phenomena including capillary congestion with dilated mucosal vessels, ectasia, increased number of small vessels, and arterialization of venules with erythema, red macula and telangiectasia, and/or angiogenesis, but signs of inflammation have also been described in cirrhosis (75, 132, 133). Most interestingly, with respect to the potential role of pathological BT for these vascular changes, experimental evidence links bacterial DNA with angiogenesis (134). CpG motifs known to be present in the genome of many bacteria and viruses but not in mammalian DNA trigger the formation of new blood vessels (134). However, the potential impact of pathological BT in patients with advanced liver cirrhosis for the well-accepted increase in splanchnic angiogenesis is not known so far. The term “congestive gastropathy” was first used by McCormack et al. who reported mucosal and submucosal vascular dilatation without signs of significant inflammatory changes in patients with portal hypertension (135). This was intended to differentiate this entity from the gastritis seen in absence of liver disease in which chronic inflammatory cells predominate. Congestive gastropathy is now named portal hypertensive gastropathy (PHG) and is characterized by endoscopic features including mosaic pattern and a proximal distribution. Moreover, mild PHG reflects a mosaic pattern without redness, whereas severe PHG is represented by red signs superimposed on the mosaic pattern. Morphological studies have demonstrated that the histological lesions characteristic of this condition consist of enlarged mucosal and submucosal vessels with little or no inflammatory infiltrate or epithelial erosion. PHG has been realized as potential source of gastrointestinal bleeding. Although incidence rates of 1–14% are low, bleeding may be severe and fatal (136). The prevalence of PHG has been reported to be variable in cirrhosis ranging between 11 and 93% (137–140). A recent study investigating 222 cirrhotic patients with mild portal hypertension and no history of gastrointestinal bleeding reported a prevalence 202 Wiest of 22% of PHG at baseline increasing up to 50% at 3 years of follow- up (136). Although portal hypertension is believed to be essential for its development, as its severity is correlated with portal hypertension (136, 141), the pathogenesis is not yet completely understood. Factors including splanchnic hyperemia with local disturbances in the regulation of vascular tone, humoral factors, and congestion induced by blockade of gastric blood drainage have been suggested as contributing factors. Moreover, gastric specimen from cirrhotic patients with PHG revealed impaired energy metabolism and reduced intracellular mucin content in comparison with noncirrhotic controls (142), potentially limiting gastric mucosal defense mechanisms. In addition to PHG, increased susceptibility of the gastric mucosa to injury by noxious factors has been reported in portal hypertension. In fact, the prevalence of peptic ulcer in cirrhotic patients, reported from endoscopic screening studies, is approximately 5–20% as compared to only 2–4% of the general population (143). In addition, not only increased frequency but also enhanced bleeding complications associated with mucosal injury do occur during portal hypertension. This is reflected in the observed increment in gastric bleeding rate per area of injury induced by oral NSAIDs in portal hypertension, indicating that the lesions appearing bleed more than those observed in normal mucosa (144). Although Helicobacter pylori is also a risk factor in cirrhotic patients, its incidence does not seem to be increased in cirrhosis (145). However, particularly advanced cirrhosis and liver insufficiency predispose to peptic lesions, since Child classes B and C are independent predictors for the development of peptic ulcers in multivariate logistic regression analysis (145). Structural and physiological changes mediating mucosal damage and impairing mucosal healing response to injury in advanced cirrhosis may include reduction of potential difference in gastric mucosa (146), impairment of bicarbonate secretion (147, 148), impairment of gastric oxygenation (149), suppression of endogenous prostaglandin production, and excessive NO production (150–152)as well as increased oxidative stress due to reduced levels of glutathione peroxidase, superoxide dismutase, and catalase (153). Endoscopic features of portal hypertensive duodenopathy (PHD) are found in 8–50% of cirrhotic patients with portal hypertension, but histopathological changes are seen in much more cases reaching 85% of cirrhotic patients assessed (154, 155). In addition to vascular changes stated above, nonvascular changes such as increased apoptosis, fibromuscular proliferation, increase in intraepithelial lymphocytes, and shortened and atrophic villi with decreased villous/crypt ratio have also been reported (155, 156). Interestingly, some of these changes did correlate closely with changes in brush border enzymes as well as cell BT and Alterations of the Digestive System 203 and membrane enzymes (157). Moreover, due to the introduction of capsule endoscopy data on mucosal alterations in the whole small intestine reflecting portal hypertensive enteropathy in cirrhotic patients are accumulating. These changes include also inflammatory-like abnormalities (edema, erythema, granularity, and friability) as well as vascular lesions (158). In fact, portal hypertensive enteropathy has been reported to be detected in up to 63% of capsule endoscopies being performed in cirrhotic patients with chronic anemia and history of variceal bleed (159). Macroscopic impression of edema is mediated most likely by the increase in interstitial hydration due to marked increases in intestinal capillary filtration caused by portal hypertension. In fact, it has been proposed that in case of chronic severe portal hypertension, intestinal interstitial fluid content may be increased up to 40% (160). Mucosal changes in the colon in portal hypertension have been shown to be similar to that seen in the upper GI tract (156, 161, 162). As for the presence of inflammatory changes represented by a more or less focal increase in neutrophils in the lamina propria, however, prevalence ranges from 10% to more than 50%(163) and thus appears to be higher than in the upper GI tract. As indicators for the presence of intestinal inflammation, fecal calprotectin or PMN-elastase has been used and in fact and is found to be increased in cirrhotic patients (47, 164). In contrast to portal hypertensive colopathy which is rarely a cause of significant bleeding, the most common sites of lower gastrointestinal bleeding are hemorrhoids and rectal varices, which have reported prevalence of 28–63% and 0–44%, respectively (165, 166). The degree of portal hypertension and/or disease severity has been reported to associate with hemorrhoids but not with rectal varices (167).

3.2. Functional Changes in the Digestive System and Nutrition in Chronic Liver Failure Other than macroscopic changes, advanced chronic liver disease can affect almost any function of the digestive tract. This includes alterations in gastric emptying and acidity, prolonged intestinal transit, increased small intestinal water secretion, enhanced lymph flow, malabsorption (168–170), intestinal protein loss (171, 172), and alterations in release of gut-derived hormones. As for the latter, please see Chapter “Genomics of the Liver in Health and Disease” on metabolomics in chronic liver failure. The exact relation of such dysfunction with pathological BT, however, is not known. Delayed gastric emptying with enhanced gastric accomodation and prolonged small intestinal transit time has been frequently reported and 204 Wiest appears to correlate with gastrointestinal symptoms (early satiety, postprandial fullness, nausea, etc.) as well as postprandial hyperglycemia, hyperinsulinemia, and hypoghrelinemia (173, 174). These alterations may be mediated at least in part by autonomic dysfunction (175). As for the small intestine, multiple aspects of nutrient absorption are dysfunctional in advanced cirrhosis. Fecal concentrations of albumin, transferrin, and α1-antitrypsin have been proposed as marker for intestinal protein loss and are found to be increased in cirrhotic patients (47). Thus, TIPS insertion has been shown to markedly ameliorate fecal excretion of albumin, IgG, and α1-antitrypsin in cirrhotic patients with protein-losing enteropathy (176). Intestinal transport of sugars and amino acids is disturbed in experimental cirrhosis (177–179) and inhibition of the activity of the membrane enzymes alkaline phosphatase and aminopeptidase, as well as the activity of succinic dehydrogenase and reduced galactose transport (177), has been reported in experimental cirrhosis (180). In contrast, an enhanced intestinal glutaminase activity is present in liver cirrhosis and may contribute essentially to the increase in ammonia following an oral glutamine challenge (181). Glutaminase is the main glutamine-catabolizing enzyme in the small intestine and glutamine is the main respiratory fuel of intestinal cells (182). The mechanism by which glutaminase activity can be increased in cirrhosis remains to be delineated but may be due to enhanced glutamine load associated with splanchnic hyperemia or could be activated by glucagon and/or angiotensin II (183). Multiple evidence for fat malabsorption in chronic liver failure exists and steatorrhea may be present—if investigated thoroughly—in up to 50% of patients (168). Explanations proposed for this malabsorption include (a) reduced pool size of bile acids resulting in the inability to form micelles, (b) bacterial deconjugation of bile salts in the small intestine due to IBO, and (c) portal hypertension-associated edema and intestinal malfunction. In addition, triglyceride levels in the small intestine of experimental as well as human cirrhosis are significantly decreased, probably because of low intestinal apolipoprotein A-IV (184). Also, fatty acid transport is altered and in contrast to healthy conditions in cirrhosis portal absorption of long-chain fatty acids is observed and increases inflow of fat into the liver in advanced diseases (185). Chronic portal hypertension by increasing intestinal capillary pressure enhances capillary filtration coefficient and thus lymph flow (capillary filtration rate) up to three to four times as compared to healthy conditions (160, 170). This increment in intestinal lymph flow is known to increase with rising venous pressure, and its protein content lowers with increasing hydrostatic pressures (170). Moreover, the BT and Alterations of the Digestive System 205 number of lymphatic vessels in the mesentery in portal hypertension is vastly increased and may represent a specific adaptation to long-standing edemagenic stress (186, 187). Consecutively, increased interstitial fluid pressure opposes the increase in intestinal capillary pressure, and transcapillary oncotic pressure gradient remains stable. In fact, this and the compensatory increase in lymph flow may explain why diarrhea is not a prominent feature of cirrhosis despite mucosal edema. Not only the intestine is affected by liver cirrhosis, but also the pancreas and biliary system are known to be affected. Cirrhosis is associated with a high prevalence of gall stones, particularly in advanced decompensated stages resulting from hypersplenism, altered hepatic metabolism as well as reduced gall bladder contractility and its lack of coordination with gastric kinetics (188, 189). Moreover, a relationship with intestinal barrier dysfunction for formation of pigment gallstones has been proposed (190, 191). Finally, abnormalities of histology of the pancreas in patients with chronic liver disease have been reported, but the incidence is disputed, as is the relationship to the etiology of disease, since it is most frequently observed in alcoholic conditions. As for pancreatic function, increased volume of pancreatic secretion often with diminished bicarbonate concentration (168) and impaired exocrine pancreatic function has been reported in patients with chronic liver disease, particularly of alcoholic etiopathogenesis (192, 193). Cachexia is a prominent symptom in liver cirrhosis (194) with deleterious consequences for morbidity and mortality, as the degree of malnutrition has been shown to correlate inversely with survival and to compromise liver transplantation results (195, 196). Pathogenesis of cachexia in advanced cirrhosis is multifactorial and may include complex metabolic disorders, catabolism, malassimilation, and malnutrition. Moreover, most cachectic conditions are associated with underlying inflammatory processes mediated at least in part by increased levels of proinflammatory cytokines (194, 197). These cytokines are associated with anorexia and depression and play a role in hypermetabolism, protein catabolism, and insulin resistance. The cytokine receptors sTNF-RI, sTNF-RII, and sCD14 have been shown to be higher in patients with cachectic liver cirrhosis and to be related with the resting energy expenditure corrected by body cell mass. In this context, pathological BT may play as well a perpetuating role. In animal models, starvation and malnutrition per se promote bacterial overgrowth, diminish intestinal mucin production, decrease global gut IgA levels, cause mucosal atrophy increasing intestinal permeability, attenuate the T and B lymphocyte cell number and function in Peyer’s patches and lamina propria, and accelerate oxidative stress (198–200). Therefore, 206 Wiest malnutrition by itself has been shown to aggravate BT (201) and could fuel the proinflammatory process further aggravating the cachectic process. In addition, decreased food intake is frequently observed in advanced cirrhosis and contributes to the negative energy balance in liver cirrhosis (202). TIPS insertion has clearly been shown to increase body cell mass, evidencing an improvement in nutritional status after portal decompression (203). This points toward a key role of portal hypertension and associated changes in intestinal nutrient absorption as well as improved food intake due to relief from abdominal symptoms and protein anabolism. In patients with liver cirrhosis, the severity of gastrointestinal symptoms is related to both recent weight loss and severity of disease and thus, not surprisingly, is associated with health- related quality of life (204). An adequate daily energy and protein supply should be ensured in patients with liver cirrhosis, which is higher than in the normal population because of hypermetabolism and higher amino acid turnover.

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J. Macnaughtan, V. Stadlbauer, R.P. Mookerjee, and R. Jalan

CONTENTS INTRODUCTION ROLE OF SIRS IN CHRONIC LIVER DISEASE BACTERIAL INFECTIONS IN LIVER CIRRHOSIS PATHOPHYSIOLOGY OF IMMUNE DYSFUNCTION POTENTIAL THERAPEUTIC STRATEGIES SUMMARY REFERENCES

Key Words: Cirrhosis, Liver failure, Sepsis, Systemic inﬂammatory response, Acute-on-chronic liver failure

1. INTRODUCTION The World Health Organization estimates that liver cirrhosis will be the ninth most common cause of death in the Western world by 2015 (1). In a signiﬁcant proportion of these patients, death is related to multiple organ failure. The term acute on chronic liver failure (ACLF) refers to the development of acute hepatic dysfunction in previously well- compensated cirrhotic patients following a precipitating event such as

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_11, C Springer Science+Business Media, LLC 2011

219 220 Macnaughtan et al. sepsis, variceal hemorrhage, or alcoholic hepatitis (2). This commonly manifests as jaundice, hepatic encephalopathy, and/or the hepatorenal syndrome. It is well known that patients with liver cirrhosis showing signs of decompensation (e.g., refractory ascites, hepatic encephalopathy, coagulation disorders) have a higher mortality. In a large cohort study, 6-year mortality in decompensated cirrhotics was 79% (3). A more recent study confirmed these results, showing a 3-month mortality of 13% with a good predictive utility of Child–Pugh and MELD score (4). Admission to ICU is associated with a short-term mortality of between 46 and 89%. (5–20). Current data demonstrates that the occurrence of single organ failure in patients with a defined severity of liver disease indicates a poor prognosis. It is the degree of end-organ failure rather than the nature of the precipitating event that is the chief determinant of outcome (21, 22). Infection is however an important precipitant of the systemic inflammatory response syndrome, a central feature of the pathogenesis of ACLF.

2. ROLE OF SIRS IN CHRONIC LIVER DISEASE The systemic inflammatory response syndrome (SIRS) is a generalized inflammatory process, frequently observed in the presence of infection, but also complicates noninfectious insults, such as trauma, shock, ischemic organ damage, or immune-mediated injury. SIRS is defined as the presence of two or more of the following: temperature >38◦Cor<36◦C, heart rate >90 beats/min, respiratory rate >20 per min or PaCO2 <32 mmHg; and white blood cells >12,000 cells/mm3 or <4000 cells/mm3. SIRS is a well-recognized feature of acute liver failure in which its presence is associated with a worse prognosis. SIRS is not exclusively precipitated by infection in acute liver failure, but septic patients exhibit a more pronounced SIRS response with more rapid progression of encephalopathy, reducing the chances of transplantation and conferring a poorer prognosis (23, 24). Identification of the SIRS syndrome in chronic liver disease might be complicated by one or more of the following factors: leucopenia secondary to hypersplenism, baseline tachycardia due to a hyperdynamic circulation, hyperventilation consequent on hepatic encephalopathy, or blunted elevation of body temperature (25). The presence of SIRS exacerbates the effect of ammonia in cirrhosis (26) and is associated with leucocyte activation (27). The development of SIRS in patients with cirrhosis and variceal bleeding (28, 29) or the development of acute SIRS, Bacterial Infections, and Alterations of the Immune System 221 functional renal impairment (30) has been recognized as a negative predictor for outcome. The occurrence of SIRS in the context of ACLF is associated with a higher mortality (31). The presence of SIRS, whether associated with infection or not, is an important outcome predictor and can be used to identify patients at high risk of organ failure. Studies are required to determine the effect of established treatments of common primary insults on the SIRS process.

3. BACTERIAL INFECTIONS IN LIVER CIRRHOSIS Infection is a common feature of ACLF, complicates the natural history, and is associated with signiﬁcant morbidity and mortality (12). About 30–50% of hospital admissions of cirrhotic patients are indicated for sepsis and a further 15–35% will develop nosocomial infections as compared to 5–7% in the general hospital population (25, 32, 33). Patients with alcoholic cirrhosis show a 6.3-fold increased mortality risk from an episode of bacteremia (34). Mortality is associated with the severity of disease, lack of resolution of infection, and associated complications of cirrhosis, in particular variceal hemorrhage, rather than with underlying etiology (35, 36, 37). Bacterial infections in liver cirrhosis are associated with an increased mortality and commonly precipitate organ failure, accounting for 30–50% of deaths in this patient group (38). Overall in-hospital mortality rates of cirrhotic patients with infection are approximately 15%, more than double those without infection (25). The presence of septic shock on presentation is a signiﬁcant determinant of outcome. The mortality rate of cirrhotic patients with severe bacterial infection in the absence of septic shock is approximately 30% but rises to 60–100% in the context of hemodynamic compromise (39). Mackle et al. found sepsis to be a feature of 39% of ICU admissions with alcoholic cirrhosis (40). ICU and in-hospital mortality rates were 78 and 88%, respectively. Infection is an important independent risk factor for early rebleeding following variceal hemorrhage. Once both episodes have been overcome, no associated increased risk with infection has been found (35, 41, 42). Sepsis is also a risk factor for the development of type I hepatorenal syndrome which is associated with a poor prognosis and occurs in 18% of cirrhotic patients with ascites (43). Bacterial infections are also a well recognized precipitant of hepatic encephalopathy. Screening and treatment of infection is one of the main principles of therapy (42). While in principle cirrhosis can predispose to sepsis from a broad range of pathogens, distinctive patterns of bacteremias are commonly observed. Often bacteremias will be monomicrobial, frequently due 222 Macnaughtan et al. to gram-negative organisms. This is thought to be due to bacterial translocation from the gastrointestinal tract frequently manifesting as spontaneous bacterial peritonitis or bacteremia of obscure origin (44). Gram-positive bacteremias are increasing in prevalence due to increased antibiotic use and more invasive procedures. Cirrhosis is a risk factor for the development of severe pneumococcal sepsis in which MELD score is a good predictor of mortality (45). Pneumococcal bacteremia in cirrhotics is more commonly associated with spontaneous bacterial peritonitis than pneumonia.

4. PATHOPHYSIOLOGY OF IMMUNE DYSFUNCTION 4.1. Innate Immunity For nearly 30 years attempts have been made to identify immune dysfunction in cirrhosis with most studies focusing on innate immunity. Neutrophils are an essential component of the innate immune response and central to the pathogenesis of alcoholic hepatitis (46). Data on neutrophil function in alcoholic hepatitis or cirrhosis is paradoxical with some studies suggesting neutrophil priming, indicating a readiness to respond to bacterial challenge (47), as measured by hydrogen peroxide overproduction, decreased L-selectin expression (48), and high levels of neutrophil elastase (49). Other studies showed decreased neutrophil phagocytic capacity (50) correlating with disease severity (51–54), whereas others found increased phagocytosis in rats with portal hypertension and bacterial translocation (55). This paradox (increased priming but also decreased phagocytic capacity) has been partly resolved by the observation that neutrophils in cirrhosis and alcoholic hepatitis are fully activated in the peripheral blood, where they normally should be in a resting state and have markedly reduce phagocytic capacity in the same patient. This has two important consequences. First, the production of free oxygen radicals in the peripheral blood can result in cellular injury. Second neutrophils become energy depleted due to a high oxidative burst, rendering them unable to respond to further bacterial stimuli due to impaired phagocytosis. The clinical importance of these findings is demonstrated by the observation that an elevated resting oxidative burst and decreased phagocytic capacity correlated with increased infection rate and mortality (56). It was further shown in this study that this immune defect is caused by a serum factor and not by a primary cellular defect. Since endotoxin is known to be elevated in patients with alcoholic hepatitis (56) and has the capability SIRS, Bacterial Infections, and Alterations of the Immune System 223 to both prime (47, 58) and activate neutrophils (59, 60), it is likely that endotoxin causes the neutrophil dysfunction observed. This hypothesis is further supported by the observation that ex vivo removal of endotoxin restores neutrophil function (56). Other aspects of the innate immune response are also defective in advanced liver disease. Several studies also show “immune paralysis” in monocytes, which is similar to the immune defect observed in sepsis (61). Expression of surface markers, such as HLA-DR on monocytes changes during the course of disease in ACLF and has been described as a prognostic marker (62, 63). Endotoxin again plays an important role in this process (64). Macrophage opsonophagocytic dysfunction has been shown to occur in chronic liver disease (65) with decreased macrophage Fc gamma receptor expression observed in patients with alcoholic cirrhosis. This was found to correlate with severe bacterial infection (66). NK cells are found in abundance in the liver and play a key role in viral clearance and tumor suppression via cytotoxic effector mechanisms. They are implicated in autoimmune liver disease, viral hepatitis, and hepatocellular carcinoma. They also contribute to the innate immune response to bacteria, and bacterial products are known to be NK cell ligands. NK cells have however been implicated in hepatocellular injury in response to bacterial infection. This is thought to be mediated by TLR2 (67, 68). It is generally accepted that intrinsic cellular defects have a minor contribution to the immunopathology of ACLF (69). Alterations in humoral factors in addition to endotoxin important for innate immune system function have been reported. These include defects in opsoniza- tion (65) which have been attributed to a decrease in protein synthesis due to the liver insufficiency compounded by malnutrition. In cirrhosis malnutrition correlates with a decrease in interleukin-1 production, suggesting an important role of nutrition in cell-mediated immune response (70). The impact of nutrition on immune function is well described in several diseases, and the therapeutic use of functional food is subject of several studies (71). Multiple cytokine expression abnormalities have been described in chronic liver disease and remain the subject of much ongoing investigation. IL-6 and TNF-α are important cytokines implicated in the immunopathology of cirrhosis. Levels of both are significantly elevated in cirrhotic patients and are associated with an increased risk of sepsis, in particular TNF-α (72). Complement deficiencies involving both the classical and alternative pathways are also observed in cirrhosis (73). C3 deficiency is a significant predictor of mortality (74). 224 Macnaughtan et al.

4.2. Gut–Liver Axis As described above, endotoxemia plays a key role in precipitating immunopathology in ACLF. Endotoxemia in patients with cirrhosis occurs due to bacterial translocation of organisms derived from the enteric flora (44). Bacterial translocation rates in cirrhosis are determined by bacterial overgrowth and increased gut permeability, in addition to the dysfunctional immune responses previously described. The liver is the major site for endotoxin clearance in the body. In health, endotoxin is present in the portal circulation but is normally cleared by Kupffer cells preventing systemic endotoxemia (75). In liver failure, however, there is diminished reticuloendothelial activity with reduced elimination of endotoxin in the context of hepatocyte dysfunction (76). This effect is further compounded by reduced immune surveillance by the Kupffer cells as a consequence of portosystemic shunting away from the liver (77). Studies have shown that not only endotoxin, but also other bacterial products, such as bacterial DNA, which have been found in plasma and ascitic fluid of cirrhotic patients (78). Bacterial DNA is not only a marker for bacterial translocation (79) but also a prognostic indicator (80). Bacterial DNA induces a proinflammatory state and mimics the immune response seen in patients with spontaneous bacterial peritonitis (81, 82), but interestingly, this seems to be unrelated to endotoxin (83).

4.3. Adaptive Immune Response Abnormalities of the adaptive immune response in cirrhosis have been less well studied. Absolute numbers of naïve and memory T lymphocytes are known to be reduced with a higher proportion of memory T cells expressing apoptotic markers. The percentage of activated T lymphocytes is however increased with mesenteric Th1 polarization observed in animal models of cirrhosis (84). This occurs in concert with an increase in monocytes within mesenteric lymph nodes and blood with characteristic production of IFN and TNF-α. Interestingly, this effect is attenuated by intestinal decontamination with antibiotics, highlighting the influence of the gut microflora on the immune profile. Recent data suggests that chronic antigenic stimulation may result in modulation of the adaptive immune response to induce a state of “permissive antigenemia.” Marquez et al. observed an increase in “exhausted” cytotoxic T cell populations (CD8+CD45RO+CD57+) with an increase in expression of apoptotic markers (CD95) (85). These immunosenescent cells are known to be unable to proliferate in response to a new antigenic load. In addition, expanded populations of regulatory T-cell populations were observed within the cirrhotic groups SIRS, Bacterial Infections, and Alterations of the Immune System 225 contributing to the downregulation of the immune response. Together, this data suggests an exhausted adaptive immune response in cirrhosis resulting in tolerance of endotoxemia.

4.4. Toll-like Receptors Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated, molecular patterns and promote the activation of leucocytes. Neutrophils express all known TLRs except TLR3. The most widely studied TLRs in liver cirrhosis are TLR2, -4, and -9, because they respond to bacterial products which are thought to be important in the pathogenesis of immune dysfunction. TLR4 is the designated endotoxin receptor, as well as TRL2, which also senses pep- tidoglycans (products of gram-positive bacteria). TLR9 can be found intracellularly and is activated by bacterial DNA (86). Activated TLRs enable neutrophil recruitment and lead to the generation of reactive oxygen species; regulation of phagocytosis; and secretion of chemokines, cytokines, and antimicrobial products. In alcoholic hepatitis, neutrophil dysfunction is associated with endotoxemia and increased incidence of infection (87). In the same patient group, an overexpression of TRL2, -4, and -9, most likely due to the presence of endotoxin and other bacterial products, has been observed (88). Constant stimulation of peripheral blood mononuclear cells (PBMCs) with endotoxin leads to the development of endotoxin tolerance, which is characterized by reduced capacity to produce proinflammatory cytokines (89). In contrast to the findings on neutrophils alone, investigations of TLR2 and -4 in PBMCs showed that TLR2 is significantly upregulated in patients with high endotoxin levels in contrast to TLR4, which is downregulated. (90). This indicates that TLRs are important players in orchestrating an immune response and may react differently on different cell types. In a human study, overexpression of TLR2 but not TLR4 was found on PBMCs of cirrhotic patients, with TLR2 expression correlating with circulating levels of the proinflammatory cytokines TNF-α and sTNFR (91). TLR9 has been implicated in mediating the hypergammaglobulinemia observed in alcoholic cirrhosis (92). This lends more weight to the hypothesis that not only endotoxin but also different bacterial products may play a role in immune dysfunction in cirrhosis. From recent studies in patients with cirrhosis and noninfected ascites, it is known that 34% have detectable levels of bacterial DNA, with a close correlation between the level of bacterial DNA and the inflammatory response, also supporting the concept that the 226 Macnaughtan et al. presence of bacterial products (endotoxin, bacterial DNA, and possibly other substances) is related to the inflammatory response seen in these patients (82).

4.5. Role of Albumin Albumin, previously considered to function mainly as a plasma expander in liver disease, has recently been discovered to have effects over and above volume expansion, such as reduction in severity of hepatic encephalopathy and improved survival of cirrhotic patients with spontaneous bacterial peritonitis and hepatorenal syndrome treated with albumin (93–95). Albumin is known to be involved in fatty acid transport, metal chelation, drug binding, and antioxidant activity (96), and therefore might act as a potent detoxiﬁcation agent, especially in the context of liver disease, where protein-bound, potentially toxic substances are increased and albumin levels are decreased. Profound alterations in the oxidative state (97) and the function of albumin with respect to fatty acid and free metal-binding capacity have been found in patients with cirrhosis and ACLF (98). The interaction of endotoxin and albumin is thought to be of importance in the development of immune dysfunction in liver disease. Fukui and colleagues showed that plasma possesses endotoxin-inactivating action and that this action is most probably carried out by the endotoxin binding and inactivating action of albumin. The endotoxin-inactivating rate of a patient’s plasma correlates with the endotoxin-binding capacity of albumin. Albumin also inhibits endotoxin function in the LAL assay and endotoxin-induced IL-1 secretion of macrophages (99–101). Despite these observations, it has been shown that the interaction of endotoxin and albumin is important for delivery of endotoxin to cellular acceptors, implying that albumin is an essential component for the formation of biologically active endotoxin species. These active endotoxin–albumin complexes are then recognized by cell surface receptors (102). So far studies trying to resolve this paradox are lacking.

4.6. Models of Immunopathology in ACLF The immunological response in sepsis is multimodal, characterized by an initial proinflammatory process (SIRS) followed by a mixed anti-inflammatory response syndrome and then by a compensatory anti-inflammatory response syndrome (CARS) (103). Early mortality has been attributed to cytokine storm-mediated events. Experimental strategies targeting this hyperinflammatory phase have however been associated with variable outcomes and mortality largely occurring in SIRS, Bacterial Infections, and Alterations of the Immune System 227

SIRS De g ree of imm u ne a

cti Recovery va tion

CARS

Death

Fig. 1. Hypothetical model of immune dysfunction in acute-on-chronic liver failure (ACLF). The red line depicts the proinflammatory SIRS and anti- inflammatory CARS phases of the physiological immune response. These phases are exaggerated in ACLF survivors (green line) but restoration of baseline immune function is thought to be achieved. Mortality from ACLF (blue line) appears to be particularly associated with a more pronounced anti-inflammatory CARS response. septic patients who are immune suppressed (103). Hotchkiss et al. found that most deaths secondary to sepsis occurred in the prolonged state of immune suppression (104). It may be that a similar picture underlies the immunopathology of ACLF (Fig. 1). As with sepsis, a state of immunological dissonance is observed with an increase in both pro- and anti-inflammatory cytokines. TNF-α and IL-6 are both known to be raised in cirrhosis, yet levels of IL-10, an anti-inflammatory cytokine, have also been found to be elevated and correlated with endotoxemia. As with sepsis, it is in the state of immune paralysis that many of these patients develop organ dysfunction and die. It may be that future therapeutic strategies should be directed toward this phase of the immune response.

5. POTENTIAL THERAPEUTIC STRATEGIES Immune failure, leading to bacterial infections in cirrhosis, is multifactorial and might not be reversed by isolated interventions. Disease severity is so far the only common denominator for the risk of infection (105). Today the main hypothesis for immune dysfunction in 228 Macnaughtan et al.

Hepatic dysfunction Liver Transplantation + Portal hypertension

Albumin exchange Antibiotics Albumin column Probiotics

↓Albumin ↓Protein ↓Endotoxemia Chronic ↓Immune function synthesis antigenemia surveillance

Immune dysfunction/paralysis

Innate immune dysfunction Adaptive immune dysfunction

Neutrophil Monocyte ?NK cell Exhausted ↑Apoptosis ↓Abililty to dysfunction dysfunction dysfunction T cells proliferate

↓Phagocytosis Loss of DR ↑Resting burst ↑TLR2 TLR ↑TLR2, -4, -9 antagonists

Fig. 2. Pathophysiology of immune dysfunction in acute-on-chronic liver failure. The presence of underlying hepatic dysfunction and portal hypertension results in the alteration of multiple factors which affect both the innate and adaptive immune responses. The red text indicates targeted therapeutic interventions. cirrhosis and ACLF is based on the concept of increased bacterial products (e.g., endotoxin, bacterial DNA) due to increased gut permeability and/or changes in gut ﬂora as well as problems in the elimination of the bacterial products by the liver (Fig. 2). Therefore, several attempts have been made to decrease gut permeability and/or alter gut ﬂora. Selective intestinal decontamination with antibiotics has an important role in therapy. Liver cirrhosis is one of the very few clinical conditions requiring antibiotic prophylaxis, in the context of either variceal hemorrhage or spontaneous bacterial peritonitis (106). Antibiotic therapy also ameliorates the cellular changes on monocytes found in patients with evidence of elevated circulating bacterial products (107). Antibiotic use is however increasingly associated with resistance and therefore other strategies need to be considered. SIRS, Bacterial Infections, and Alterations of the Immune System 229

Probiotics have been demonstrated to modulate gut flora (44, 108) and have shown positive effects on liver injury in experimental models of liver disease, depending on the bacterial species used (109). In patients with liver cirrhosis, probiotics have been shown to decrease hepatic encephalopathy (110, 111), improve liver biochemistry (112), and decrease the rate of infections after liver transplantation (113). Several meta-analyses have already supported the benefit of probiotics in preventing infections in the general hospital population, (114–116); however, the exact mechanism is still largely speculative. It has been suggested that a healthy flora promotes the integrity of the gut defense barrier by normalizing intestinal permeability and thereby controls intestinal inflammatory responses by modulating the release of cytokines (117). In a small clinical study, it has been shown that food supplementation with Lactobacillus casei Shirota improves neutrophil phagocytic capacity and modulates cytokine production and TLR expression (118). Another interesting aspect is the fact that malnutrition leads to acquired immune deficiency (119). Oral supplementation of branched- chain amino acids restores neutrophil phagocytosis in decompensated cirrhotic patients (120). High circulating levels of pro- and anti-inflammatory cytokines are involved in the pathogenesis of ACLF, possibly contributing to immune paralysis (110). Strategies to attempt the removal of systemic cytokines have been studied. Artificial liver support systems (albumin dialysis, Molecular Adsorbent Recirculating System (MARS), fractionated plasma separation, Prometheus) are designed to remove protein-bound substances from the blood of patients with ACLF. Although these systems are able to remove cytokines from the serum, the actual serum levels remain unaffected, likely due to a higher rate of production than can be removed by the current devices (121–123). Not much is known about the influence of artificial liver support on immune function. Preliminary data suggests that MARS could potentially influence the inflammatory response of ACLF patients by decreasing neutrophil activation and thereby restoring neutrophil function, possibly through endotoxin removal (124). If patients with ACLF have uncontrolled infection, artificial liver support seems not to confer significant advantage. Bacteremia was found to be a strong negative prognostic factor for outcome in patients with ACLF treated with MARS (100% mortality); therefore, it has been recommended that culture-positive patients should not be treated with MARS (125). Prometheus has been shown to cause a reversible drop in mean arterial pressure in patients with signs of systemic inflammation (123). 230 Macnaughtan et al.

Recently, modulation of TLR function has gained considerable interest. Several small-molecule inhibitors of TLR4 have been discovered and are undergoing human testing (126), but to date to the best of our knowledge no studies in liver cirrhosis are underway. But when considering the fact that TLR overexpression seems to be the consequence and not the cause of immune dysfunction, it is questionable if TLR4 inhibition would really improve immune function in the setting of cirrhosis (87). However, addition of human albumin, which is known to scavenge endotoxin, in pathophysiologically relevant concentrations, prevented an increase in resting burst, normalized chemokine receptor expression, and phagocytic dysfunction. This was associated with prevention of increases in TLR2, -4, and -9 expressions, providing support for the concept of using albumin and/or other endotoxin removal strategies to prevent neutrophil dysfunction in severe alcoholic hepatitis. The capacity of albumin to bind endotoxin could also be an explanation for the effect seen by MARS therapy (124). To date it remains unclear if it will ever be possible to use this interesting strategy in patients, since preclinical data is not convincing yet (127). A further, rather experimental, approach is the use of granulocyte colony-stimulating factor, which has been shown to improve neutrophil transmigration in patients with advanced cirrhosis (128). It is also likely that the use of granulocyte colony-stimulating factor would worsen the situation in liver cirrhosis, since a higher number of activated neutrophils would cause even more harm to different organs by the increase in free oxygen radicals. Finally, since the only curative therapy in end-stage liver disease is liver transplantation, innate immune dysfunction should improve thereafter. Although immunosuppression is necessary after transplantation, there is evidence that liver transplantation improves neutrophil function (129).

6. SUMMARY Liver cirrhosis and ACLF are characterized by a dysfunctional immune response. This frequently manifests as a systemic inflammatory immune response syndrome with paradoxical functional immunoparesis. This process appears to be driven by an increase in circulating bacterial products, most commonly endotoxemia as a consequence of bacterial translocation. Increased susceptibility to infection leads to severe complications frequently resulting in multiple organ failure and death in a large proportion of patients. Potential therapeutic strategies involve the use of antibiotics or probiotics to modulate gut flora, SIRS, Bacterial Infections, and Alterations of the Immune System 231 albumin or albumin dialysis to remove toxins from the bloodstream, TLR modulation, or the use of granulocyte colony-stimulating factor. Early antibiotic use in SBP and variceal hemorrhage has been clinically validated and early data suggests benefit from probiotic therapy. Further clinical studies are however required to further describe the immunopathology of ACLF and mechanisms of bacterial translocation to develop interventional strategies to impact on the natural history of a condition with a currently unacceptably high mortality.

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Jens H. Henriksen, MD

CONTENTS INTRODUCTION EXTRACELLULAR FLUID VOLUME REGULATION DISTRIBUTION AND REGULATION OF PLASMA VOLUME NEUROHUMORAL REGULATION HEPATIC NEPHROPATHY AND THE HEPATORENAL SYNDROMES CONCLUSION REFERENCES

Key Words: Aqaporin water channels, Argenin vasopressin, Blood volume, Central and arterial blood volume, Cardiac output, Glomerular ﬁltration rate, Hepatorenal syndrome, Hepatic venous pressure gradient, Ascites, Plasma renin activity, Renal blood ﬂow, Sympathetic nervous system, Sodium retention, Systemic vascular resistance, Vasodilatation, Vasopressin-2 receptor

Abbreviations

ACE Angiotensin converting enzyme AQP2 Aquaporine-2 water channels AVP Arginine vasopressin BV Blood volume cAMP Cyclic adenosine monophosphate

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_12, C Springer Science+Business Media, LLC 2011

239 240 Henriksen

CBV Central and arterial blood volume CCl4 Carbon tetrachloride CGRP Calcitonin gene-related peptide CO Cardiac output ET Endothelin-1 HR Heart rate GFR Glomerular filtration rate HRS-1 Hepatorenal syndrome type-1 HRS-2 Hepatorenal syndrome type-2 HVPG Hepatic venous pressure gradient MAP Mean arterial pressure PA Ascitic fluid hydrostatic pressure Pc Capillary hydrostatic pressure πA Ascitic fluid oncotic (colloid osmotic) pressure πg Oncotic pressure in endothelial gap πp Plasma oncotic pressure PRA Plasma renin activity PV Plasma volume RAAS Renin angiotensin aldosterone system RBF Renal blood flow SBP Spontaneous bacterial peritonitis SNS Sympathetic nervous system SVR Systemic vascular resistance V2 Vasopressin-2 receptors

1. INTRODUCTION Normal fluid homoeostasis includes dynamic shifts in water, crystalloids, and proteins between the various compartments of the body (1–3). The fluid dynamics are controlled by refined mechanisms that include water and solute intake, renal handling, haemodynamic/oncotic forces, and neurohumoral regulation (4, 5). Fluid retention is a characteristic feature in the progressive state of cirrhosis, where the accumulation of fluid in the peritoneal cavity occurs in a substantial number of patients (6–8). Over the last decade, much effort has been devoted to the role of vasodilation, abnormal blood volume distribution, hyperdynamic cardiovascular decompensation, and kidney dysfunction in the formation of hepatic ascites (9–15). A considerable advance in the understanding of the microvascular dynamics and pathophysiology of the extravascular volume regulation has been achieved by studies with indicators of different molecular size (16–19). Advances in the insight of the dynamics of lymphatics and the peritoneal space Extracellular Fluid and Renal Function 241 have in part been attained by studies on patients undergoing peritoneal dialysis (20, 21). Renal dysfunction in patients with advanced cirrhosis has been intensively studied over several decades (22–27). The present chapter will deal with the regulation of the extracellular fluid volume, kidney function, and aspects of the pathophysiology of the hepatorenal syndromes. Some cornerstones and recent advances in the understanding of the normal state, especially with respect to the regulation of transvascular exchange at the local level, are also considered.

2. EXTRACELLULAR FLUID VOLUME REGULATION 2.1. Plasma and Interstitial Space Dynamics The balance between intravascular and extravascular fluid is governed by the Starling forces, i.e. microvascular permeability/hydraulic conductivity, capillary and interstitial fluid hydrostatic pressure, plasma and interstitial fluid oncotic pressure, and lymphatic drainage (28–31). The normal hepatosplanchnic vascular bed is a low-pressure system with a small pressure gradient of 2–5 mmHg across the sinusoids of the liver and another small up-stream in pressure from the portal vein to the intestinal capillaries (32). Surplus capillary filtrate from the liver and gastrointestinal tract drains through hepatic and intestinal lymphatics into the thoracic duct. The peritoneal space is mainly drained through lymphatics on the abdominal site of the diaphragm into the right lymphatic duct. From a structural point of view the normal blood- lymph-barrier consists of the capillary endothelial cells, a basement membrane, and a connective tissue space, draining into the terminal lymphatics. The microcirculation in the normal liver has discontinuous capillaries (sinusoids) with large fenestra without a basement membrane (33, 34). The exchange of material is diffusive and filtra- tive/convective (35, 36). Selectivity in the capillaries is only present for very large proteins (17, 33). In the normal liver there is a close agreement between measurements obtained by morphometric and physiological methods (33, 37). Owing to the large size of the fenestra in the liver sinusoids and the bulk carriage of fluid with a high protein content, there is no significant oncotic pressure gradient across the sinusoids or the liver blood-lymph-barrier (33). The microcirculation in the gastrointestinal tract has fenestrated capillaries with a basement membrane that is relatively impermeable to macromolecules, but very permeable to water and smaller molecules. Intestinal capillaries show permselectivity with characteristic sieving (30, 38). The intestinal lymph-blood protein ratio is normally relatively 242 Henriksen low in accordance with the relatively tight capillary membrane here (39). The blood capillaries in the peritoneal membrane are of the continuous type with a basement membrane like those of skeletal muscles, subcutaneous, and cutaneous tissue. Several studies on transperitoneal dynamics have been carried out during the last decade, especially in uraemic patients undergoing peritoneal dialyses (21, 40, 41). The peritoneal clearance of small substances like glucose and creatinine, is in the order of 5–15 ml/min (20, 21, 40, 42). Water and low molecular solutes have a major direct transperitoneal transport, whereas proteins and other high molecular substances are transported back into the blood stream, via the subdiaphragmatic lymphatics (36, 43–45). In chronic liver disease, an increase in hepatic vascular resistance, mainly located postsinusoidally, and an increase in mesenteric inflow of blood are important in the dysregulation of the extracellular fluid volume (46). Hydrostatic pressure in the sinusoids is increased and almost identical with portal pressure (47). This favours an overall outward movement of fluid, see Figure 1. In addition, the sinusoidal lining is tightened, the so-called capillarisation, with appearance of a basement membrane and collagen deposits in the perisinusoidal space (33, 37, 48). Investigations on protein kinetics (10) and electron-microscopic studies indicate a decrease in the number, rather than the size, of the sinusoidal fenestra (37, 49). This implies that the sinusoidal permselectivity remains small in cirrhosis, and consequently the effective oncotic pressure gradient across the sinusoids remains low (17, 33). However, the tightening of the sinusoidal wall decreases the transvascular permeability and hydraulic conductivity, a morphological change that represents some counterweight against the increased sinusoidal pressure with respect to transvascular fluid and solute transport. Kinetic studies have shown substantially elevated blood to lymph transport of water, solutes, and protein (16, 49), and direct measurements on lymph vessels have accordingly shown an enlarged thoracic duct with substantially increased lymph flow in cirrhosis (39, 50). When the transsinusoidal filtration exceeds the transport capacity of the lymph vessels, surplus fluid will pass into the peritoneal space. Even in this case, however, the peritoneal transport rate is relatively small (less than 5 per cent of the lymph flow) compared with that entering directly into the hepatic lymphatics and thoracic duct (16, 36). Most of the intraperitoneal protein in the ascitic fluid comes from the liver via transsinusoidal filtration (16, 17). The presence of small and large proteins in proportion to their concentration in plasma is in keeping with bulk carriage into the peritoneal space (17, 33). Low molecular solutes and water are transported directly across the gastrointestinal peritoneal lining dominated by a protein-poor fluid that will equilibrate with Extracellular Fluid and Renal Function 243

Fig. 1. Illustration of hepatic and gastrointestinal circulation in relation to transperitoneal transport and lymphatic reabsorption in patients with cirrhosis and ascites. Arterial supply red, venous return blue, lymphatics clear. Transport rates are: a bulk transport from hepatic sinusoids to peritoneal space, b lymphatic drainage of peritoneal space through right lymphatic duct, c thoracic duct drainage of hepatic and gastrointestinal lymph, and d direct transperitoneal transport of water and low molecular substances. the protein-rich fluid, originating from the transsinusoidal filtration, according to the Starling forces (51). Kinetics of the transperitoneal transport are illustrated in Figure 2. The porosity of the capillary membrane in the peritoneal lining and subperitoneal tissue is currently under debate (21, 41). Two-pore and three-pore models are suggested and transvascular transport of water through fenestra and transport proteins (aquaporins) is currently being considered (21, 52). Also the heterogeneity of the interstitial space close to the capillary is important for the transport and size of the oncotic pressure gradient across the blood interstitial barrier (41, 53, 54). These aspects are present and future topics of research, see Figure 3. The presence of protein in the peritoneal space is an important requirement for peritoneal fluid sequestration. Introduction of water 244 Henriksen

Fig. 2. Transport from the blood stream into the peritoneal space in cirrhosis. I. Low molecular substances (like glucose, amino acids, vitamins, etc.): disappearance from plasma (P) and appearance in the ascitic fluid (A) after intravenous tracer injection. The slope of the ascitic curve relative to the plasma concentration indicates the netto transport rate (5–15 ml/min). It is seen that several hours pass before the concentration in ascitic fluid equals that of plasma. II. Transport of high molecular substance (albumin) from plasma (P) into the ascitic fluid (A) after intravenous tracer injection. As with the low molecular substances, the slope of the ascitic curve relative to the plasma concentration indicates the transport rate (0.3 ml/min). Note that the overall transport rate of low molecular substances is orders of magnitudes higher than the protein transport. This holds true for the transport rate from the peritoneal space back into the blood stream as well. These aspects are important to paracentesis, albumin substitution, diuretic treatment and antibiotic therapy.

into the serous cavities is followed by a complete and fast net reabsorption, as noticed already by Starling more than 100 years ago (28). Absorption of isotonic protein-free fluid is also relatively fast, owing to the effect of plasma oncotic pressure (55, 56). By contrast, protein- rich fluid is absorbed very slowly (16, 36). When protein is introduced into the peritoneal space, as in cirrhosis, fluid and high-molecular solute movements take place in different areas as already described, and ascitic fluid is generated with a relative protein composition similar to that of plasma (17, 57), but the protein concentration in ascitic fluid is much lower than that in plasma, because of the hydrostatic/oncotic equilibration of an almost protein-free fluid over the large peritoneal surface area of the gastrointestinal tract (5, 58, 59). The low concentration of ascitic fluid protein will, in part, counteract the elevated portal/intestinal capillary pressure (the so-called protein wash down) (60). Thus, the effective ascitic oncotic pressure gradient may be looked upon as a mirror image of the increased portal pressure (5), see Figure 4. Extracellular Fluid and Renal Function 245

Fig. 3. Transvascular transport in different capillaries. Schematic illustration of transport from plasma into ascitic fluid versus transport from plasma into the interstitial space. The ascitic fluid is mixed with almost uniform protein concentration (which can be directly determined after diagnostic paracentesis) giving ideal conditions for the Starling forces to operate. Pc hydrostatic pressure in capillary, PA hydrostatic pressure in ascitic fluid, πp oncotic pressure in plasma, πA oncotic pressure in ascitic fluid. With respect to the interstitial space it has recently been shown that there may be restrictions to proteins related to the transvascular transport and interstitial “mixing” which may give a much higher oncotic pressure in the interstitial fluid directly related to the endothelial gap. Thus πg may be considerable higher than πI whereas the hydrostatic pressure Pg is close to PI. This means that the resorptive effect of the plasma oncotic pressure (πp) is reduced, and may explain why steady state reabsorption in the venous end of the capillary may be much reduced especially with low πp. Weather this phenomenon is present for peritoneal capillaries is yet unknown, but the high reabsorption rate during regression of ascitic fluid may indicate that it is of minor importance.

It is important to note ascitic fluid with low opsonity and low protein, present in patients with large volume ascites and highly elevated portal pressure, because of increased risk of spontaneous bacterial peritonitis (SBP) (61–63). Transport rates into the peritoneal space and back into the blood stream are not only of academic interest, but are also important for therapy (16) (Figure 1 and 2). Thus, for reasons mentioned above, mobilisation of fluid by diuretic treatment should be adjusted to the rate of inflow into the peritoneal cavity (55, 56, 58). The astonishing amelioration of ascites after surgical implantation of a peritoneal venous shunt that works as an artificial mega-lymphatic 246 Henriksen

Fig. 4. Transperitoneal Starling forces in cirrhosis. The effective oncotic pressure (ascitic fluid (πA) minus plasma (πp) oncotic pressure) can be considered as a “mirror image” of the portal pressure, as indicated by the hepatic venous pressure gradient (HVPG). This means that protein “wash down” takes place in the ascitic fluid, and that the Starling forces are almost in complete operation with a small overweight of filtration, when HVPG is below approximately 20 mmHg. When HVPG is above 20 mmHg the protein in the ascitic fluid is almost completely “washed down”, and filtration prevails. Each horizontal line represents a single patient with two measurements made; continuous line: HVPG and discontinuous line (πA-πp). illustrates the importance of effective drainage from the peritoneal cavity (64). Another important way of removing ascitic fluid and peritoneal protein is by paracentesis (65). Several controlled clinical trials have proved the efficacy of this treatment (65–70). Owing to the size and balance of transport dynamics, paracentesis, and especially repeated paracentesis, should never be performed without giving the patient a plasma expander, as this procedure will otherwise lead to a further reduction in the effective circulating albumin mass and further effective arterial hypovolaemia (65, 67–71). The microvascular fluid exchange may be influenced by several modulators, such as recruitment of capillaries, altered balance between precapillary and postcapillary vascular tone, changed microvascular permeability, and altered receptor status (72, 73). The sinusoids of the liver probably do not exhibit recruitment like the capillaries in the intestine, peritoneal membrane, and skeletal muscle. Vasodilatation in the splanchnic capillaries will favour increased permeability and elevated hydrostatic capillary pressure here (9, 46). The sympathetic Extracellular Fluid and Renal Function 247 and parasympathetic nervous system may modulate the vascular tone in the splanchnic area and in the liver (74, 75). This holds true in the normal state and in patients with cirrhosis. There are indications that increased sympathetic nervous activity may raise portal venous pressure and alpha- and beta-adrenergic blockers may often reduce portal and sinusoidal pressure (76–78). In this way a change in autonomic tone may contribute to a change in transsinusoidal and transcapillary intestinal filtration. Contractile elements in the lining of the sinusoidal fenestra have been described in animal experiments, and in vitro growth of sinusoidal endothelial cells has shown a modulating effect of different vasoactive substances (79, 80). Consequently, neurohumoral modulators participate in the dysregulation of transsinusoidal fluid exchange.

3. DISTRIBUTION AND REGULATION OF PLASMA VOLUME 3.1. Splanchnic and Peripheral Vasodilatation Splanchnic vasodilatation in cirrhosis is well documented and generates increased transvascular filtration, owing to higher intracap- illary hydrostatic pressure and, to some extent, also increased capillary permeability, because of the larger microvascular surface area (9, 18, 19, 46, 57, 81). In contrast to the splanchnic circulation, the peripheral circulation may be variably vasodilated and vasoconstricted in patients with cirrhosis (82–87). The renal circulation is typically vasoconstricted, owing to a highly elevated sympathetic nervous tone and elevated angiotensin II and endothelin 1 (74, 88–93). In addition, intrarenal vasodilators may be disturbed (94). In skeletal muscle, subcutaneous and cutaneous tissue there are indications of different vascular tone with areas with microvascular dilation and constriction (15). The role of this heterogeneous pattern in the overall control of extravascular fluid volume is not clear at present, but elevated venous pressure and reduced plasma oncotic pressure are most likely important in the declive oedema in patients with cirrhosis (58, 59, 95). The transition of fluid from plasma into the interstitial spaces depends highly on the overall fluid status and the distribution of the volume within the circulation medium (2, 8, 29, 96, 97). Albumin and other plasma proteins leave the intravascular space via a rather slow transcapillary movement (30, 35, 65, 98). This means that oncotic material, primarily introduced into the vascular space, is transferred slowly into the interstitial space with the end result that a little more albumin is 248 Henriksen located in the interstitial space than in the vascular volume in the normal condition (31). In patients with cirrhosis this distribution is reverse, owing to interstitial space protein wash-down caused by the increased transvascular and lymphatic fluid exchange (16). The volume distribution is not even within the vascular space as arteries and capillaries contain much less of the circulating medium than do veins and the splanchnic system (31, 99). In patients with cirrhosis, especially at the more advanced stages, there is an increase in total vascular compliance, and the arterial compliance is also increased in these patients (12, 14, 83, 100, 101). This means that a volume load has a relatively small effect on the intravascular pressure, and that the distribution of the added volume may be different in the various vascular beds (97). Animal studies of experimental cirrhosis and portal hypertension, as well as investigations in patients with cirrhosis, have revealed that the blood volume in the splanchnic organs is substantially increased (23, 99, 102). The overall blood volume is increased, but the central blood volume is decreased or normal in cirrhosis (103–105). As shown by a dynamic indicator dilution technique, the central and arterial blood volume (that is the volume in the heart cavities, lungs, and central arterial tree) is decreased in patients with cirrhosis, especially in those with advanced disease (97, 103). In agreement with the directly measured, reduced effective arterial blood volume, highly elevated homoeostatic markers, such as plasma renin activity (PRA), circulating catecholamines, aldosterone, and vasopressin, indicate that patients with advanced cirrhosis have functional hypovolaemia (3, 75, 82, 90, 106, 107). As plasma atrial natriuretic peptide may be normal or increased (76, 108), the overall picture points towards a situation with a reduced effective arterial blood volume and varying degrees of reduced central blood volume, depending on the absence or presence of cirrhotic cardiomyopathy (109–111). The cause of the reduced effective arterial blood volume is largely the splanchnic vasodilation with reduced overall systemic vascular resistance and increased vascular compliance (3, 82, 112, 113). These findings are explained by the arterial vasodilation theory, which implies a reduced effective arterial blood volume and increased non-central blood volume consequent on vasodilatation (7, 82, 91, 113). Intimately related with the vasodilatation, which occurs before sodium retention and plasma volume expansion (91), is the increased cardiac output and low arterial blood pressure (15, 23, 100, 114). The cardiac output is increased, but not enough to maintain a normally high arterial blood pressure (97, 115). The mechanisms counteracting the effective arterial hypovolaemia include activation of all available vasopressor systems, see Figure 5. Extracellular Fluid and Renal Function 249

Fig. 5. Relationship between portal hypertension, splanchnic vasodilatation, abnormal blood volume distribution, activation of vasoconstrictor systems, renal vasoconstriction and renal sodium-water retention in cirrhosis. HVPG: hepatic venous pressure gradient, PSS: portosystemic shunting, MAP: mean arterial pressure, HR: heart rate, CO: cardiac output, RBF: renal blood ﬂow, GFR: glomerular ﬁltration rate, SVR: systemic vascular resistance, VDS: vasodilatator sensitivity, VCS: vasoconstrictor sensitivity, CBV: central and arterial blood volume, COMP: vascular compliance, PV: plasma volume, BV: blood volume, RAAS: renin, angiotensin, aldosterone system, SNS: sympathetic nervous system, AVP: Arginin vasopressin, ET: endothelin-1.

3.2. Dynamic Coupling Between the Heart and Central Arterial Tree Several recent studies in patients with heart failure and arterioscle- rotic heart disease have indicated that the coupling between the left ventricle and the central arterial tree (ascendant aorta and aortic arch) is very important for the load of the left ventricle and thereby the cardiac performance, fluid dynamics, and neurohumeral regulation (116, 117). In patients with cirrhosis, especially in those with advanced disease, the arterial afterload is reduced. This is brought about by an overall reduction in the systemic vascular resistance. However, recent years have also shown that the arterial compliance is substantially increased (12, 14, 100). This is caused by both structural and functional alterations of the arteries (118) and also by the low or low normal arterial blood pressure in these patients (115). Arterial pulse wave velocity is reduced 250 Henriksen and the back reflections of the arterial pulse are delayed (100). This provides protection against strain to the left ventricle, and recent investigations of the arterial pulse in cirrhosis by fast Fourier analysis have confirmed this (119). Bringing the arterial blood pressure back to normal level with a vasoconstrictor like terlipressin increases the afterload and the pulse velocity (120, 121). This can unmask a latent left ventricular failure, and recent studies of left ventricular performance, wall motion, and wall thickness have indicated that normal arterial blood pressure and normal arterial compliance (i.e. normal afterload) unmask a latent left ventricular dysfunction (122, 123). The presence of latent and manifest cardiac dysfunction and failure will aggravate the effective central arterial underfilling, with further activation of neurohumeral counterregulatory mechanisms, and aggravate renal dysfunction with avid sodium water retention and upcome of the hepatorenal syndrome at the end stage (111, 124–128).

4. NEUROHUMORAL REGULATION Sinusoidal and portal hypertension in cirrhosis is related to a defective nitric oxide production (9, 46, 81). However, endogenous vasoconstrictors like endothelin 1, angiotensin II, and catecholamines may also increase the sinusoidal vascular resistance in addition to structural changes (85, 92, 129, 130). However, the coupling between portal- sinusoidal hypertension and the genesis and perpetuation of systemic and splanchnic vasodilatation is not completely understood (9, 85). It may be caused either by direct neurohumoral signals from the liver (131) or by an overproduction of circulating vasodilators induced by shear stress. Several findings suggest that the splanchnic vasodilatation precedes renal sodium and water retention (9, 112, 113). In experimental and clinical portal hypertension, splanchnic vasodilatation leads to reduced systemic vascular resistance, decreased effective arterial blood volume, and a reduction in arterial blood pressure (97, 103, 100). This brings about activation of potent systemic vasoconstrictor systems (15), and the haemodynamic consequences include a hyperdynamic systemic circulation (14, 97, 132). Recently, it became clear that in advanced cirrhosis further underfilling of the arterial circulation is also secondary to a reduction of the increased cardiac output as described in patients with renal failure and spontaneous bacterial peritonitis (125–127, 133, 134). The highly activated sympathetic nervous system, RAAS, and increased circulating vasopressin contribute to sodium-water retention, extracellular fluid volume dysregulation, and formation of oedema and ascites. However, these systems are not only highly activated, they are Extracellular Fluid and Renal Function 251 also dysfunctional (108, 135, 136). Thus, a down-regulation of beta- adrenergic receptors has been described (123). Parasympathetic and sympathetic dysfunction is present at several levels: Central nervous system, peripheral nerves, pre-synaptic, synaptic, and post-synaptic (137, 138). In early cirrhosis the RAAS is normal or slightly activated (90), but in some patients it may be suppressed, suggesting a primary sodium-water retention (23). One should remember that normal plasma renin activity in a patient with expanded plasma volume indicates RAAS overactivity (11, 15, 82), see Table 1. In advanced cirrhosis, high activation of the RAAS is the typical finding (90). Reduced sensitivity to aldosterone in the renal tubules has been described (139, 140), and smooth muscle cells from patients with cirrhosis and animals with experimental cirrhosis have a reduced sensitivity to noradrenaline, angiotensin II and vasopressin (85). A characteristic feature of human and experimental cirrhosis is lack of mineralocorticoid escape (23, 141). Nitric oxide is an important vasodilator in the systemic vasodilatation (130, 142), with an up-regulation of the nitric oxide synthesis (9, 46, 85, 130). Calcitonin gene-related peptide (CGRP) and adrenomedullin are potent neuropeptide vasodilators that have been found to be increased, especially in advanced cirrhosis with ascites and the hepatorenal syndrome (143–145). Systemic vasodilatation has also been related to

Table 1 Evidence of reduced central and effective arterial ﬁlling in patients with cirrhosis

Cirrhosis Controls

Central and arterial blood 0.25±0.04∗ (n=60) 0.34±0.10 (n=22) volume relative to blood volume (fraction, CBV/BV) Central and arterial blood 11.8±2.9∗ (n=60) 19.4±4.4 (n=22) volume relative to cardiac output (sec, CBV/CO) Plasma volume relative to 3.41∗ (n=89) 7.6 (n=32) plasma renin activity −15 2 2 (10 ml /pg, PV/PRA) ∗ Mean ± SD. Cirrhosis vs controls: p <0.001 In most clinical cases an increased plasma renin activity (PRA) will be taken as evidence of reduced arterial filling in patients with cirrhosis CBV central and arterial blood volume, BV blood volume, PV plasma volume, CO cardiac output Data from References Henriksen et al. (12), Moller et al. (105, 132) 252 Henriksen resistance to vasopressors. An impaired response to vasoconstrictors is related to changes in receptor affinity, downregulation of receptors and post-receptor defects that may be related to increased expression of nitric oxide (46, 85). The pathophysiology and implications of arterial vasodilatation are complex. Definite experimental and clinical evidence shows that it precedes the activation of counterregulatory neurohumoral activation and the renal sodium and water retention in cirrhosis. Therefore it plays a primary and major role in both local control of extracellular fluid volume dysregulation and overall sodium water dynamics in cirrhosis.

4.1. Kidney Function Characteristic changes in renal function follow hepatic insufficiency, especially in patients with cirrhosis. Renal dysfunction with relation to extracellular volume regulation is considered in the following. Renal sodium and water retention in advanced cirrhosis is the most avid seen in human pathophysiology (91). A twenty-four hour sodium excretion of only one mmol or even less is not exceptional. However, it must be realised that all parts of the nephron exhibit altered physiology in cirrhosis, and it is in the main a functional type without characteristic structural changes (127, 146). The characteristic renal dysfunction here is a reduced renal blood flow (RBF), reduced glomerular filtration rate (GFR), increased proximal tubular sodium reabsorption, increased distal tubular sodium reabsorption, and reduced urinary water excretion, owing to enhanced distal tubular and collecting duct reabsorption of water (97, 147, 148). It should be noted that owing to altered metabolism, body composition and dietary sodium intake, and excess alcoholic intake, some adaptive changes in renal function may also take place in patients with cirrhosis (22). By substituting carbohydrates and lipids by isocaloric ethanol, rats show a characteristic reduction in GFR with a reduced area of the glomerular membrane (149). Conversely, increased protein intake will increase GFR, and reduced protein intake will reduce GFR (22). In the ageing person, GFR decreases concordantly with the reduction in skeletal muscle mass leaving the serum creatinine concentration unchanged. A long term reduction in the sodium intake may also reduce RBF and GFR. Thus, in the cirrhotic patient with a high ethanol intake, low intake of protein and sodium, reduced skeletal muscle mass, and intake of diuretics, reduced kidney function would be expected for purely adaptive reasons (6, 24). However, the changes in cirrhosis are far more pronounced and constitute a terminal stage in advanced disease Extracellular Fluid and Renal Function 253 with functional renal failure, either progressing quickly as in the hepatorenal syndrome type 1 (HRS-1), or more slowly in the hepatorenal syndrome type 2 (HRS-2) (7, 24, 26, 125).

4.2. Renal Blood Flow (RBF)

In certain types of experimental cirrhosis (CCl4 in rats, dimethylnitrosamine in dogs), the RBF may be unchanged or even increased (150, 151). Increased RBF and hyperfiltration have also been described in some patients with early pre-ascitic cirrhosis (86, 147). However, at present there is no general agreement as to the fraction of cirrhotic patients with this phenomenon (10, 51, 90, 152). A characteristic change in moderate and advanced cirrhosis is a reduction in RBF (148). This is brought about by constriction in both the afferent and the efferent arteriole (146). In early cirrhosis the efferent arteriole may be more constricted than the afferent, thus reducing perfusion more than filtration (increased filtration fraction). Enhanced sympathetic nervous activity, elicited by effective arterial hypovolaemia, reduces the RBF, owing to alpha-adrenergic stimulation (76, 135). Similarly, an inverse relation between renal venous noradrenaline and RBF and between the central blood volume and renal venous noradrenaline has been described (146). The beta-adrenoceptors are also stimulated, which give rise to increased renin production with elevated angiotensin II, which also reduces the renal perfusion. Circulating endothelin 1, a very powerful peptide vasoconstrictor of renal and coronary arteries, may be increased and contribute to the vasoconstriction seen in renal dysfunction and the hepatorenal syndrome (92). Thus, Moore and co-workers reported a higher concentration of endothelin 1 in the renal vein compared to the artery in patients with the hepatorenal syndrome (93). Moreover, endogenous renal vasodilators like the prostaglandins are reduced in patients with cirrhosis (94). In addition, it has recently been shown that calcium/polyvalent cation receptors may be down-regulated in the smooth muscle of renal vessels, with a potentially increased renal vasoconstriction as the outcome (153–156). Another mechanism of reduced RBF is the lowering of the arterial blood pressure and the reduced effective renal perfusion pressure, owing to the increased renal venous pressure in some cirrhotic patients, because of the presence of ascites and elevated pressure in the inferior vena cava (95, 157–159). The effective renal perfusion pressure may thus be reduced from 85 mmHg in the normal condition to 50 mmHg or even less in advanced cirrhosis. 254 Henriksen

The normal response to activation of renal sympathetic nerves is increased renin secretion, increased proximal tubular reabsorption of sodium, and with intensified sympathetic activity the RBF and GFR will decrease (91). Renal hypoperfusion is, at least initially, a physiological response to changes in the systemic circulation with effective arterial hypovolaemia (159). The increased activity of plasma renin and plasma noradrenaline concentrations correlates inversely with the reduction in the RBF and GFR (76). As angiotensin II chiefly acts on the efferent arteriole, ACE inhibition may induce a significant reduction in the GFR and filtration fraction and further reduction in sodium excretion, even in the absence of any change in arterial blood pressure or RBF (159).

4.3. Glomerular Filtration Rate (GFR) GFR is governed by a balance in the constriction of the afferent and efferent arterioles of the nephron. Thus, if the afferent arteriole is constricted, the GFR may be relatively more affected than RBF. When a simultaneously high tone is present in the efferent arteriole, the filtration pressure will be maintained (160). In such case, the filtration fraction will be high, as found in early ascitic patients and patients without the hepatorenal syndrome (146, 167). Severely decompensated patients and patients with the hepatorenal syndrome have a very high sympathetic nervous activity. These patients are characterised by a very low RBF, especially in the cortical area, substantially decreased GFR, and low filtration fraction, indicating preferential constriction of the afferent arterioles (91, 148). In advanced decompensated cirrhosis, the kidneys appear to function like bilateral Goldblatt kidneys (158). Keeping up the arterial blood pressure results, as judged from earlier experiments with ornipressin (148) or more recent experiments with terlipressin (107, 121, 162), in a substantial increase in GRF and reduction in serum creatinine.

4.4. Proximal Tubules In the normal condition, about 80% of filtered sodium is reabsorbed in the proximal tubules (163–165), and the preurine is isotonic when it leaves the proximal tubules (166). In cirrhosis, the proximal tubular reabsorption fraction (i.e. sodium reabsorption as a fraction of the filtered sodium load) is normal or increased (167), which indicates that the distal sodium delivery is reduced (107, 147, 168, 169). The increase in proximal sodium reabsorption is in part brought about by enhanced alpha-adrenergic receptor activity (135). Various animal models of cirrhosis (CCl4, bile duct ligation in rats) may give somewhat different results (150, 168). Particularly in experimental CCl4 cirrhosis Extracellular Fluid and Renal Function 255 in rats, hyperfiltration has been reported with high sodium reabsorption in proximal and distal tubules (170). However, in advanced human cirrhosis with diuretic-resistant or diuretic-intractable ascites, failure of natriuresis is often bound to enhanced proximal sodium reabsorption, which may sometimes be overcome by the supra-addictive effect of a thiazide diuretic (146, 159).

4.5. Thick Ascending Limb of Henle’s Loop and Distal Tubules Increased distal sodium reabsorption has been described in patients with cirrhosis and animals with experimental cirrhosis (91, 171). This has been attributed to the effect of increased circulating aldosterone (3, 147), increased sensitivity of the distal tubules to aldosterone (140, 172), down-regulation of the enzyme 11-beta-hydroxy-steroid dehydrogenase with inappropriate activation of tubular sodium reabsorption by endogenous glucocorticoids (173), or down-regulation of renal calcium/polyvalent cation sensing receptors with up-regulation of tubular sodium retaining channels (153, 156). Most likely a combination of different mechanisms exists, especially in advanced cirrhosis (174).

4.6. Collecting Ducts Stimulation of vasopressin-2 receptors (V2 receptors) activates adenylate cyclase (3, 175). Increased intracellular cyclic adenosine monophosphate (cAMP) increases the water transport in the collecting ducts due to an increased number of aquaporine 2 water channels (AQP2) in the apical cellular membrane (152). The short- term up-regulation involves increased intracellular recycles of AQP2 molecules to the apical membrane. Increased intracellular cAMP stimulates also genomic transcription in the long-term up-regulation of AQP2. Thus, by stimulation of V2 receptors, endogenous arginine- vasopressin (AVP) increases the number of AQP2 and thereby enhances collecting duct reabsorption of water with reduced free water clearance as the outcome (176, 177). In patients with cirrhosis, there is a non-osmotic stimulation of neu- ropituitary release of AVP with increased stimulation of collecting duct V2 receptors and enhanced transport of water from the urine back to the plasma, reducing the free water clearance, even in the presence of hyponatraemia (3, 175, 178). Although most of the APQ2 is recycled and endocytosed into ductal epithelial vesicles, a small part spills over into the urine, where the content of APQ2 may reﬂect the stimulation of V2 receptors and thereby the increased action of AVP on the collecting ducts (175, 179). 256 Henriksen

5. HEPATIC NEPHROPATHY AND THE HEPATORENAL SYNDROMES In the early stage of cirrhosis, impairment of renal function is evidenced by reduced renal sodium excretion after acute administration of a sodium chloride load or change in body position (90). A later reduction in the RBF and GFR with increased renal sodium and water reabsorption in parallel with the reduction in liver function takes place (146, 148). In addition these patients have reduced free water clearance and may develop reduced serum sodium or a full-blown dilutional hyponatraemia with a serum sodium concentration below 125 mmol/l (27, 107, 178, 180). In the end stage, progressive reduction in the RBF and GFR leads to the development of the hepatorenal syndrome (HRS). Two types of HRS have been defined, depending on how fast the renal failure progresses (24, 26, 125). The effective arterial hypovolaemia and the massive activation of vasoconstrictor systems are central to the pathogenesis of HSR (125, 146, 161, 181). A prerequisite for the development of HRS is advanced liver disease with severely disturbed liver function and sinusoidal-portal hypertension. Normalisation of renal function and regulation of extracellular fluid volume after orthotopic liver transplantation may indicate that the liver is directly involved in the disturbance of renal function. The existence of a hepatorenal reflex has been described in animal studies, but has been debated for years in man (135, 182). The presence of a hepatorenal reflex in man is supported by observations of reduced RBF following a rise in portal pressure and an increase in the renal release of endothelin 1 (93, 183). In healthy subjects, the renal autoregulation maintains normal renal perfusion in spite of alterations in arterial blood pressure, provided it is above approximately 70 mmHg, see Figure 6. Below this threshold RBF is directly related to the renal perfusion pressure, which equals the arterial mean pressure as long as the renal venous pressure is low (184). In animals with increased sympathetic nervous activity or infusion of sympathomimetic agents, the renal autoregulation curve shifts towards the right side (184). It is well known that enhanced renal sympathetic nervous activity in man will also shift the renal autoregulation curve to the right (135). In patients with cirrhosis, there are indications that the relation between RBF and renal perfusion pressure shifts to the right with a lower RBF, even in the case of normal renal perfusion pressure (185, 186), see Figure 6. This means that even with a low normal or normal arterial blood pressure, patients with cirrhosis may be very sensitive to a further reduction in the mean arterial blood pressure and thereby renal perfusion pressure. This is especially true in the presence of ascites, where renal venous pressure is elevated (95), further reducing the effective renal perfusion pressure. In the presence of Extracellular Fluid and Renal Function 257

Fig. 6. I. Autoregulation of renal perfusion in dogs. Y-axis: renal blood flow (RBF), X-axis renal perfusion pressure (= mean arterial blood pressure minus renal venous pressure). Control, animals after infusion of phenoxybenzamine (alpha-adrenergic blocker) and methoxamine (alpha-adrenergic stimulator), and animals after methoxamine alone. Adopted from Langaard et al. (184). After alpha-adrenergic stimulation RBF decreases and the renal autoregulation curve is shifted to the right. II. Renal blood flow (RBF) in relation to mean arterial blood pressure (MAP) in control subjects (normal subjects and patients without cirrhosis) and patients with cirrhosis. It is seen that the autoregulation curve in patients with cirrhosis is shifted to the right and that the level of RBF is lower in patients with cirrhosis compared to the controls like in the experimental situation with alpha-adrenergic stimulation. Adopted from Henriksen et al. (185, 186). cirrhotic cardiomyopathy, with diastolic and systolic dysfunction, and spontaneous bacterial peritonitis (both of which are frequently associated with HRS) the low arterial blood pressure may further decrease the renal perfusion (111, 125, 126). In patients with advanced liver disease the arterial blood pressure and effective renal perfusion pressure may be so low that elevation of the arterial blood pressure with vasopressin (terlipressin) or another vasoconstrictor may increase the RBF and thereby improve renal function, simply by moving up the first part of the renal autoregulation curve (51, 107, 158, 159, 162, 187). Prevention of effective arterial hypovolaemia, arterial hypotension, and cardiac dysfunction are therefore important targets for therapy and prevention of the HRS. The GFR is reduced below 30 ml/min in patients with the HRS. The amount of sodium reaching the distal nephron is reduced and may explain why diuretics such as furosemide and spironolactone may be of limited value in these patients (159, 188). The low free water clearance consequent on massive V2 receptor stimulation leads to dilutional 258 Henriksen hyponatraemia (25, 111). The use of V2 receptor antagonists and opioid antagonists are important issues for future potential therapy of dilutional hyponatraemia (27, 125).

6. CONCLUSION Dysregulation of the extracellular fluid volume in cirrhosis is important for organ failure, fluid homoeostasis, and kidney dysfunction. It results from combined humoral, nervous, and haemodynamic changes. The renal and cardiovascular complications in cirrhosis are part of a multiorgan syndrome that affects survival. Caution should be exercised with respect to complications like bleeding and infections and to such stressful procedures as intensive diuretic treatment, large volume paracentesis without volume expansion, and surgery. Liver transplantation has been shown to reverse fluid retention and renal and cardiovascular dysfunction in patients with cirrhosis. Further research should be directed towards a better understanding of the complex mechanisms underlying the regulatory, renal, and haemodynamic dysfunctions in liver disease, as this may be the key to treatment.

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The Heart in Chronic Liver Failure

Hongqun Liu, Soon Woo Nam, and Samuel S. Lee

CONTENTS CLINICAL FEATURES CONSEQUENCES OF CIRRHOTIC CARDIOMYOPATHY (TABLE 1) POSSIBLE PATHOGENIC MECHANISMS TREATMENT REFERENCES

Key Words: Cirrhotic cardiomyopathy, Portal hypertension, Cardiovascular, Heart failure, Cardiac, Systolic, Diastolic, QT interval, Cirrhosis, Chronic liver failure, Bile duct ligation

Cirrhosis and chronic liver failure are associated with several cardiovascular abnormalities which include hyperdynamic circulation, characterized as increased cardiac output and decreased peripheral vascular resistance and arterial pressure. Despite the baseline increase in cardiac output, cardiac function is abnormal in several respects such as attenuated systolic and diastolic contractile responses to pharmacological or physiological stress, electrophysiological repolarization changes including prolonged QT interval, and enlargement or hypertrophy of cardiac chambers. This constellation of cardiac abnormalities is termed cirrhotic cardiomyopathy. It has been suggested that cirrhotic cardiomyopathy has a role in the pathogenesis of cardiac dysfunction and even overt heart failure after transjugular intrahepatic portosystemic

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_13, C Springer Science+Business Media, LLC 2011

269 270 Liu et al. shunt placement, major surgery, and liver transplantation. Cardiac dysfunction contributes to morbidity and mortality after liver transplantation, even in many patients who have no prior history of cardiac disease. Depressed cardiac contractility contributes to the pathogenesis of hepatorenal syndrome, especially in patients with spontaneous bacterial peritonitis. Cirrhotic cardiomyopathy is now well recognized as a clinically relevant entity.

1. CLINICAL FEATURES 1.1. Blunted Contractile Response to Stimuli Raised cardiac output is a characteristic of cirrhotic cardiomyopathy and is due to increases in both heart rate and ventricular stroke volume. This increased cardiac output may lead to the mistaken presumption that cardiac function is intact in cirrhotic patients. Rather, cardiac response to various stressful conditions can be abnormal. Mikulic et al. (1) showed a decreased inotropic effect of the β-adrenergic agonist dobutamine in patients with alcoholic cirrhosis. Ramond et al. (2) demonstrated decreased chronotropic responses after isoproterenol administration in cirrhotic patients. Cirrhotic rats also showed lower maximal heart rate responses and had significantly reduced myocardial β1 receptor density (3). Therefore, downregulation of myocardial β-adrenergic receptor density can result in blunted chronotropic response to β-adrenergic stimulation. Patients with cirrhosis are known to have decreased exercise tolerance, and this may be due to a blunted systolic responsiveness. An attenuated ability to increase cardiac contractility with exercise was demonstrated initially by Kelbaek et al. (4) and later confirmed by numerous studies (5, 6). An inappropriately low cardiac output in response to tachycardiac stress is a relatively unreliable index of systolic dysfunction, as cardiac output is affected by many extracardiac factors including the loading conditions of the heart, the degree of peripheral vasodilation or constriction, and the neurohumoral regulatory systems such as sympathetic tone. However, animal studies using isolated perfused cardiomyocytes from cirrhotic Rats induced by bile duct ligation (BDL) show clear systolic dysfunction with significantly slower rates of contraction, as measured by the peak shortening velocity or time to achieve half-maximal contraction (7)(Fig.1). Diastolic relaxation is also impaired in cirrhotic hearts when the passive elastic properties of the myocardium are decreased secondary to factors such as changes in the extracellular collagen and subsequent stiffening of the left ventricle (8). A significantly reduced E The Heart in Chronic Liver Failure 271

# 10 0.5 ** # 8 0.4

0.3 6 * m/s) μ ( 4 0.2 Time to peak 50% (s)

Maximum systolic velocity Maximum 2 0.1

0 0.0 Sham Sham + PDTCBDL BDL + PDTC BDL + Bay 11-7082 Sham Sham + PDTCBDL BDL + PDTC BDL + Bay 11-7082

Fig. 1. Systolic contractility in isolated cardiomyocytes. Effect of NF-κB inhibitors. Isoproterenol-stimulated systolic velocity in isolated cardiomyocytes. The left panel shows maximal systolic contraction velocity. The right panel shows time to attain half-maximal contraction. Both measures were signiﬁcantly slower in cirrhotic rats compared with the shams-control group (∗P<0.05, ∗∗P<0.01). Treatment of the cirrhotic group with PDTC or Bay 11-7082 signiﬁcantly reversed the reduced systolic contractility in BDL rats (# P<0.05 compared with BDL group) (n=6 in each group). BDL, bile duct ligated; PDTC, pyrrolidine dithiocarbamate (reproduced from Liu and Lee (7)).

(the velocity of blood flow from the atrium to the ventricle during early diastole)/A (the velocity of blood flow during late diastole) ratio on echocardiography is also observed in cirrhotic patients. Finucci et al. (9) demonstrated higher late diastolic flow velocities and resultant decreased E/A ratios in cirrhotic patients compared with control subjects. 1.2. Electrophysiological Abnormalities Three electrophysiological abnormalities have been observed in cirrhotic hearts (5, 6): QT interval prolongation, chronotropic incompetence, and electromechanical dyssynchrony. QT prolongation is a result of abnormal myocardial repolarization and may be associated with a higher risk of torsade de pointes (10) ventricular tachycardia, at least theoretically. However, whether there is truly an increased risk of torsade de pointes in patients with cirrhosis remains unknown. In a large population of unselected patients with cirrhosis, Bernardi et al. (11) showed that the corrected QT interval (QTc) was prolonged. Among the cirrhotic patients, 46.8% had prolonged QTc, compared to only 5.4% in controls. QTc length was not influenced by the etiology of cirrhosis but correlated with Child–Pugh score, liver function tests such as prothrombin activity and serum bilirubin, and plasma norepinephrine levels. Multivariate analysis showed that only Child–Pugh score and plasma norepinephrine levels were independently correlated with QTc duration. Patients with QTc prolongation had a significantly lower survival rate than those with normal QTc. Similar findings were 272 Liu et al. reported by Mohamed et al. (12). In that study, during the follow-up period, QTc interval further lengthened in those patients whose liver function worsened. Liver transplantation reversed the prolonged QT interval (12). Chronotropic incompetence is the inability of the heart to meet various physiological and pharmacological stimuli with appropriate tachycardiac responses. This has been demonstrated in many studies. For example, Kelbaek et al. (4) reported this phenomenon in patients with alcoholic cirrhosis. Grose et al. (13) also showed exercise-induced chronotropic incompetence in patients with alcoholic and non-alcoholic cirrhosis. Electromechanical dyssynchrony refers to a disruption of the normally very tightly regulated time interval between electrocardiographic systole (onset of the Q wave) and mechanical systole (opening of the aortic and pulmonic valves). Henriksen et al. (14) described this curi- ous phenomenon in patients with cirrhosis, showing that the difference in time between both events was significantly greater in those with a prolonged QTc interval. The clinical significance of this phenomenon remains unclear at present.

1.3. Abnormal Structure and Histology There is no consistent pattern of cardiac chamber and wall dimen- sions in patients with cirrhosis. Perhaps the only point of agreement is that the changes, if any, are essentially on the left side of the heart. In the absence of another condition such as portopulmonary hypertension, the right ventricle and atrium are normal in size and thickness. Some degree of left atrial enlargement has been reported by most but not all studies (5, 6, 15) and has generally been ascribed to the increased total blood volume or some other factor associated with hyperdynamic circulation. Whether left ventricular hypertrophy exists is more controversial. An example of a positive study is that of Wong et al., who demonstrated by echocardiography that left atrial size, interventricular septal thickness, and left ventricular relative wall thickness are significantly increased in cirrhotic patients compared with a control group (16). However, other studies have found no LV hypertrophy. The heterogeneity of patients with cirrhosis and chronic liver failure may explain part of the incon- sistency. In particular, alcohol may play a large complicating role here, not only because alcoholic cardiotoxicity is a well-known phenomenon, but also because arterial hypertension (and thus LV hypertrophy) is precipitated or aggravated by chronic alcoholism. A few animal studies tend to support the pro-hypertrophy camp. Caramelo et al. (17) revealed that the heart weight was significantly The Heart in Chronic Liver Failure 273 higher in CCl4-induced cirrhotic rats. Left ventricular hypertrophy and normal right ventricle were reported in bile duct ligated (BDL)-cirrhotic rats (18). The histomorphological alterations of the heart in cirrhosis are mild and non-specific. Abnormal cardiac histology was first reported more than five decades ago and subsequently confirmed in several studies (19–21). The histological changes include cardiomyocyte edema, fibrosis, exudation, nuclear vacuolation, and unusual pigmentation. Virtually all these autopsy studies are many decades old, at a time when alcohol was thought to be essentially the only cause of cirrhosis. Thus it remains unclear which changes might be due to cirrhosis per se and which are due to the known toxic effects of chronic alcoholism on the heart.

2. CONSEQUENCES OF CIRRHOTIC CARDIOMYOPATHY (TABLE 1) 2.1. Abnormal Renal Function Peripheral vasodilatation resulting in decreased effective circulating volume is thought to be the major pathogenic mechanism responsible for renal sodium and water retention in cirrhosis (22, 23). Despite increased total blood and plasma volumes, the decreased effective volume leads to the kidney sensing a hypovolemic state and thus con- serving salt and water to maintain volume homeostasis. For many years, the primary, if not the only, focus of study has been the dilated peripheral vasculature. However, the pump at the center of the circulation had been ignored until very recently. In order for the “effective circulating volume” to be decreased, a relative or absolute degree of pump insufficiency must also be playing a role. Firm evidence for or against a role of cirrhotic cardiomyopathy in the direct pathogenesis of ascites formation remains lacking. The complexity of cardiovascular regulation and its dysfunction in cirrhosis continue to hinder the testing of this hypothesis. Specifically, patients and animal models with cirrhosis always show peripheral vasodilatation, so separating out the effects of cardiodepression vs. vasodilatation in the genesis of renal sodium/water retention is problematic. The extreme end of the spectrum of renal sodium/water retention is hepatorenal syndrome (HRS) in which the kidney functionally shuts down, with oliguria and elaboration of a virtually sodium-free urine. Several lines of evidence from patient studies indirectly but strongly suggest that cardiac dysfunction plays a major contributory or permissive role in the genesis of HRS. A seminal study in this regard was done by Ruiz-del-Arbol et al. (24). In 23 patients recovering from 274 Liu et al. ) ) ) 30 30 27 ) 29 ( Cazzaniga et al. Rabie et al. ( Krag et al. ( Rabie et al. ( not A >1 / E A / E <1 had slow ascites A / response to TIPS-augmented preload affects or predicts survival 28 post-TIPS available E the lowest CO had the highest mortality at all time points examined clearance compared to the group with Suggests that diastolic Large two-center study; day Of 101 patients, the 41 with Of 24 patients, the group with Table 1 <1 at day 28 post-TIPS <1 at baseline predicted <1 at baseline associated A A A only predictor of 1-year survival 1-year survival in 101 patients with slow/poor diuresis post-TIPS at 1, 3, and 12post-TIPS months / / / Evidence of effectE Comments Reference E E Low CO predicted mortality Clinical consequences of cirrhotic cardiomyopathy TIPS insertion TIPS insertion TIPS insertion TIPS insertion consequence Clinical Mortality after Mortality after Poor diuresis after Mortality after dysfunction output or systolic dysfunction problem Cardiac Diastolic Low cardiac The Heart in Chronic Liver Failure 275 ) ) ) ) 24 26 33 35 ) 35 et al. ( et al. ( et al. ( Donovan et al. ( ( Ruiz-del-Arbol Ruiz-del-Arbol Sampathkumar Many studies Donovan et al. HRS; this group had lower CO which declined further after infection resolution also more pronounced in the HRS group be higher than the 1–6% reported in these two studies no large prospective study to date may be subclinical Of 23 patients, 8 developed Peripheral vasodilatation was True prevalence of LVF may Probably subclinical but Clinical significance unclear; , ratio of early- to late-diastolic filling wave velocity. CO, cardiac output. A / E ) and 6% Table 1 33 (Continued) ) patients 35 resolution associated with development of HRS-1 on to develop HRS during follow-up had lower CO 1% (7/754) ( (4/71) ( post-transplant with advanced cirrhosis edema noted in 56% of patients in the first postoperative month Low CO after infection Evidence of effect Comments Reference Of 66 patients, 27 who went Reversible LVF occurred in Found in 30–60% of patients Radiographic pulmonary aggravate HRS post-SBP hepatorenal syndrome post-liver transplantation? “torsade de pointes” arrhythmia? post-liver transplantation? consequence Precipitate or Clinical Precipitate Poor outcomes Unclear; risk of Poor outcomes Abbreviations: TIPS, transjugular intrahepatic portosystemic shunt. ventricular failure prolongation ventricular failure problem Cardiac Overt left QTc Mild left SBP, spontaneous bacterial peritonitis. HRS, hepatorenal syndrome. LVF, left ventricular failure. QTc, corrected QT interval 276 Liu et al. an episode of spontaneous bacterial peritonitis (SBP), 8 went on to develop HRS. The HRS group differed from the non-HRS group in having a lower baseline cardiac output that declined further after infection resolution. Arterial pressure also dropped in the HRS group, allowing the speculation (25) that the inadequate ventricular contractile response led to decreased renal perfusion. Another longitudinal study from the same group, following a cohort of 66 patients with end-stage liver failure, found that the 27 who developed HRS during the follow-up period differed from those who did not, in having a lower cardiac output and higher portal pressures and evidence of more marked peripheral vasodilatation (26). A recent study by Krag et al. (27) shows similar findings in 24 patients with cirrhosis and ascites. Those with the lowest cardiac output (<1.5 L/min/m2) also had the lowest renal blood flow and glomerular filtration rate and, even more importantly, showed the poorest survival at 3, 9, and 12 months of follow-up. The low cardiac output group also had a significantly higher chance of developing type 1 HRS. On multivariate analysis, low cardiac output was the only independent factor that could predict survival. Even the MELD score failed to predict survival.

2.2. Responses and Survival After TIPS Insertion Insertion of a transjugular intrahepatic portosystemic stent-shunt (TIPS) carries significant hemodynamic consequences, diverting a large volume of portal blood to the systemic circulation and thus dramatically increasing the cardiac preload. However, the decompression of the portal hypertension is beneficial to patients with either resistant or recurrent variceal bleeding or refractory/resistant ascites; thus TIPS continues to be a popular therapy. Recent evidence offers the unexpected suggestion that in addition to its beneficial therapeutic effects, TIPS can be viewed as a type of diagnostic cardiac “stress test.” To elaborate, a subset of patients can be identified by the cardiac diastolic response to the TIPS: those in whom the increased preload embarrasses the left ventricle that cannot easily handle the extra diastolic filling volume (28). This manifests as diastolic dysfunction which can be measured by a variety of methods, the simplest being the E/A ratio. Cazzaniga et al. in an important study found that the blunted ventricular diastolic response to TIPS insertion, measured at 4 weeks after the procedure and manifesting as an E/A <1, was the only independent predictor of mortality at 1 year (29). In this respect, even the MELD and Child–Pugh scores, as well as other parameters of liver functional reserve, failed to predict mortality. A dual-center study also reported that in 101 TIPS patients, a baseline E/A <1 predicted both survival at 1 year and a slow or poor The Heart in Chronic Liver Failure 277 diuretic response to the TIPS (30). In this study, the E/A data after TIPS insertion were not available. However, the demonstration that diastolic dysfunction is associated with a poor diuretic response to TIPS and the aforementioned study by Krag et al. (27) showing poor renal function in those with the lowest cardiac output together offer the first indirect evidence that pump failure may contribute to ascites formation in advanced cirrhosis. The HRS and TIPS studies detailed above strongly suggest that cirrhotic cardiomyopathy by attenuating the contractile response to the cardiac challenges of bacterial infection and TIPS-increased preload, respectively, plays a key pathogenic role in these two situations.

2.3. Liver Transplantation Liver transplantation is considered to be the “acme” of surgery – it is the longest-duration and most complex of all the routine procedures done on the human body. Thus it also poses an enormous cardiovascular stress during the perioperative and early postoperative periods. The cardiac complications during this period have been reviewed in detail (31, 32) and will only be briefly summarized here. A potential clinical consequence of cirrhotic cardiomyopathy was first noticed by anesthetists and intensivists responsible for the perioperative care of transplant recipients. Sampathkumar et al. reported that about 1% (7/754) of transplant recipients had an unexplained reversible dilated left ventricular failure in the postoperative period (33). Although they did not invoke cirrhotic cardiomyopathy as a likely cause, in retro- spect, we believe that these patients indeed were manifesting evidence of this syndrome under the stress of transplantation. Indeed, if one looks carefully, evidence of cardiac dysfunction can be found in the immediate postoperative period (34), as well as during the first postoperative month. In that time interval, Donovan et al. documented the presence of radiographic pulmonary edema, which was usually subclinical in up to 56% of transplant recipients (35). Prospective studies using sophisti- cated methods such as cardiac MRI will likely be needed to determine the exact prevalence of postoperative mild or severe heart failure in transplant recipients. Methods of predicting which patients are at risk of developing postoperative cardiac complications are also currently imperfect (36) and will require careful prospective studies. The “good news” about cirrhotic cardiomyopathy and liver transplantation is that most if not all the cardiac abnormalities probably regress after successful transplantation. Torregrosa et al. reported that several indices of cardiac function regressed or normalized within 6–12 months after liver transplantation (37). 278 Liu et al.

3. POSSIBLE PATHOGENIC MECHANISMS Over the past two decades, a primary focus of our laboratory has been the elucidation of pathogenic cellular and molecular mechanisms underlying cirrhotic cardiomyopathy, mainly using the BDL rat model of biliary cirrhosis. These studies have been previously reviewed (5, 6, 38); herein we brieﬂy summarize the major positive ﬁndings. Although an initial glance may suggest that there are abnormalities of many different regulatory systems working in isolation, we believe that most of these regulatory systems described below are interrelated and the complex interconnections between them continue to be studied in our laboratory and elsewhere.

3.1. β-Adrenergic Receptor Function The primary positive stimulus for cardiomyocyte contractility is the β-adrenergic receptor system (Fig. 2). In our experimental rat cirrhosis models, several abnormalities in the β-adrenergic signaling pathway have been identiﬁed. First, the ventricular β-adrenergic receptor density is decreased in experimental animals (3). Sarcolemmal plasma membrane Gs protein expression is reduced (39). Membrane-bound adenylate cyclase activity is attenuated with resultant decreased cAMP generation (39). Finally, membrane PKA activity is downregulated (unpublished data). All these factors negatively impact cardiomyocyte contractility.

3.2. Membrane Physicochemical Changes Fluorescent lipid probes inserted in a membrane can be used to estimate the ability of lipid and protein moieties in the membrane to move in various directions. This ability to move is called membrane fluidity (40). A normal biophysical membrane environment is essential for many receptors, including the β-adrenoceptor, to function properly (41). We found that membrane fluidity is decreased in cardiomyocytes from BDL rats, associated with an increase in the membrane cholesterol content and cholesterol/phospholipid ratio. Changes in cholesterol content can affect myocardial contractility, excitability, and conduc- tion properties. The activities of cardiac sarcolemmal enzymes such as Na+-K+-ATPase, Mg2+-ATPase, and Ca2+ pump ATPase as well as Ca2+-dependent K+ channels and Na+-Ca2+ exchanger can be altered by changes in the membrane cholesterol content (42). Restoration of normal values of membrane fluidity in vitro in BDL cardiomyocytes also restored adenylate cyclase activity (43). This suggests that at least part The Heart in Chronic Liver Failure 279

Fig. 2. β-Adrenergic-stimulated myocyte contraction. Cardiomyocyte contractility is mainly regulated by β-adrenergic stimulation. The binding of either adrenaline or noradrenaline to the β-adrenergic receptor leads to the interaction of the receptor with a binding protein known as the Gs or the G-stimulatory protein. This, in turn, leads to the activation of another membrane-bound protein, adenylate cyclase (AC). The net result is the production of cyclic AMP (cAMP) from adenosine triphosphate (ATP). cAMP then stimulates protein kinase A (PKA) which phosphorylates the L-type calcium channel and increases the probability and duration of the open state of the channels and also phosphorylates the cardiac ryanodine receptor (RyR, SR, Sarcoplasmic reticulum) thereby increasing calcium release from RyR. This promotes the influx of calcium into the cytosol of the cardiomyocyte, causing myofibrillar actin–myosin cross-linking, which results in cellular contraction. of β-adrenergic receptor dysfunction is due to the abnormal membrane lipid milieu around the receptor, i.e., decreased fluidity.

3.3. Cellular Calcium Kinetics Cardiac dysfunction can be due to alterations in Ca2+ handling in the myocyte. These alterations usually lead to depressed cardiac Ca2+ transients and thus decreased contractility. There are two sources of calcium which determine cardiomyocyte contractility: extracellular and intracellular (RyR). Extracellular calcium enters the cell through membrane calcium channels, mainly voltage-dependent calcium channels (VDCC). The main component of VDCC is the L-type Ca2+ channels, also called dihydropyridine receptors (DHPRs). We showed that L-type calcium channel currents (ICa, L) in BDL myocytes are sig- niﬁcantly less than that in controls (44). These electrophysiological 280 Liu et al. data conﬁrm that the membrane L-type calcium channel function is attenuated. Furthermore, L-type calcium channel protein expression is quantitatively decreased in BDL cardiomyocytes compared with controls (44). As for intracellular calcium kinetics, the sarcoplasmic reticulum releases calcium when stimulated by ryanodine or caffeine; thus this has been termed the ryanodine-release receptor (RyR). However, RyR binding experiments showed no difference between BDL and sham controls (44), nor was there any difference in mRNA transcription and protein expression of RyR and sarcoplasmic reticulum Ca2+-ATPase (SERCA2). These results thus indicate that the abnormality of calcium delivery is in the cardiomyocyte plasma membrane, whereas the intracellular calcium system appears intact.

3.4. Nitric Oxide The evanescent gas nitric oxide (NO) plays important roles in normal physiology and also mediates pathophysiological changes in many different cells and tissues. It transduces its signal primarily by stimulating membrane-bound guanylate cyclase to produce its second messenger cGMP. NO contributes to physiological regulation of normal myocardial function, mainly by counteracting β-adrenergic stimulation (45). cGMP is a negative inotrope, decreasing calcium transients in cardiac myocytes by several different mechanisms. It is generally accepted that NO is overproduced in cirrhotic patients and animal models (46–49). Increased NO may stem from augmented activity of one or more of the three NO synthase isoforms: the endothelial constitutive NOS (eNOS) upregulated due to hyperdynamic circulation with resultant increased shear stress; the inducible NOS isoform (iNOS) stimulated by the increased levels of cytokines such as interleukins and TNF-α; and the constitutive neuronal NOS (nNOS), which is becoming increasingly recognized as a signiﬁcant pathogenic mediator in conditions such as hyperdynamic circulation (50) and mesenteric venous abnormalities in cirrhosis (51). The role of NO in the cirrhotic heart has been reviewed (52). In BDL- cirrhotic rats, Van Obbergh et al. reported that inhibition of NOS with NG-monomethyl-L–arginine signiﬁcantly increases ventricular systolic pressure and the peak rate of rise of left ventricular pressure in isolated cirrhotic hearts but not controls (53). We showed an increased iNOS mRNA transcription and protein expression in the hearts of BDL- cirrhotic rats, without any change in eNOS mRNA or protein (54). Immunohistochemistry localized the iNOS protein mainly to cardiomyocytes. NO generated by an exogenous NO donor, S-nitroso-N-acetyl The Heart in Chronic Liver Failure 281 penicillamine, also diminished contractile force in papillary muscles isolated from control rats, whereas the NOS inhibitor, nitro-L-arginine methyl ester (L-NAME), normalized the decreased isolated papillary muscle contractility in cirrhotic rats (54). These results suggest a pathogenic role for NO in the induction of cirrhotic cardiomyopathy, mediated by the iNOS isoform.

3.5. Carbon Monoxide There are two isoforms of heme oxygenase (HO): inducible (HO-1, also known as heat-shock protein-32) and constitutive (HO-2). HO catalyzes the oxidation of heme to biologically active molecules: iron, biliverdin, and the short-lived gas carbon monoxide (CO). CO, like NO, plays a number of physiological roles. Like NO, CO has been shown to increase cGMP levels by activating guanylate cyclase (55, 56). In an experimental canine model of right-sided congestive heart failure, HO-1 gene transcription was increased in the right ventricle, but not in the left (57). We demonstrated that HO-1 mRNA transcription and protein expression and cGMP levels are increased in cirrhotic left ventricles compared with sham controls (58). The HO inhibitor zinc protoporphyrin IX (ZnPP) normalized the increased cGMP level in the cirrhotic rat heart and reversed the decreased maximal contractility in cirrhotic papillary muscles. In contrast, copper protoporphyrin IX (CuPP), an analog of ZnPP without HO inhibiting effects, has no impact on cardiac contractility. These results suggest that the HO–CO–cGMP pathway is involved in the pathogenesis of cirrhotic cardiomyopathy (58). However, because NO is a much more powerful stimulator of guanylate cyclase than CO, the exact physiological and pathophysiological signiﬁcance of CO has been questioned (59). There is also a complex cross talk between the NO and CO systems, and their interrelationship in cirrhotic cardiomyopathy remains unknown.

3.6. Endocannabinoids Endogenous cannabinoids or endocannabinoids are known to have a negative inotropic effect on cardiac contractility in both humans (60) and rats (61). It is thought that bacterial lipopolysaccharides (endotoxin) increase endocannabinoid levels. Endotoxemia is common in advanced cirrhosis due to the leaky gut syndrome; thus it is plausible that the elevated endotoxin levels stimulate the production or decrease the degradation of anandamide and possibly other endocannabinoids or endocannabinoid-like substances. Indeed the plasma level of anandamide is known to be increased in cirrhosis (62). 282 Liu et al.

We recently demonstrated a major role for increased local cardiac production of endocannabinoids in cirrhotic cardiomyopathy (63). This conclusion is based on the restoration of blunted contractile response of isolated left ventricular papillary muscles from BDL-cirrhotic rats after preincubation with a CB1 antagonist, AM251. Additionally, endocannabinoid reuptake blockers (VDM11 and AM404) enhance the relaxant response of cirrhotic papillary muscles to higher frequencies of contraction in an AM251-sensitive fashion, suggesting an increase in the local production of endocannabinoids acting through CB1 receptors. Other in vitro evidence suggests a mainly neuronal source for the local production of endocannabinoids, as these effects were mostly abolished by pretreatment with the neurotoxin tetrodotoxin (63).

3.7. NF-κB and Cytokines Nuclear factor-κB(NF-κB) proteins are a family of transcription factors that regulate cellular responses. NF-κB modulates the synthesis of proinflammatory cytokines such as TNF-α and interleukin 1β. These cytokines are well documented to be cardiac contractile inhibitors. Deleting NF-κB improves cardiac contractility in heart failure (7, 64). Our study showed that in the cirrhotic rat heart, NF-κB and TNF-α were significantly increased and cardiac contractility was significantly attenuated. Pyrrolidine dithiocarbamate and Bay 11-7082, which are NF-κB inhibitors, significantly reduced NF-κB activity, decreased TNF-α concentration, and improved cardiac contractile function (7)(Fig.1). These results suggest that TNF-α-related myocardial dysfunction in cirrhosis is also NF-κB dependent.

3.8. Myoﬁlament Proteins Ultimately contraction is due to actin–myosin cross-bridging. Myosin heavy chains (MHC) are the site of actin and ATP binding and are thus known as the “motor” of cardiac contraction. Expression of α-andβ-MHC, the two functionally distinct cardiac MHC isoforms, is tightly regulated (65, 66). α-MHC contracts and relaxes faster than β-MHC, but the latter uses less ATP to function. Relative expression of these isoforms is changed in cardiac failure (67). For example, in murine heart failure, a shift from normally predominant α-MHC toward β-MHC is observed (30). Recent work demonstrates that α-MHC expression is also downregulated in human heart failure (31–33). Whether the MHC isoform shift is a pathogenic or compensatory response remains debated. Our preliminary data indicate that an α-MHC to β-MHC shift also occurs in the BDL rat ventricle (unpublished observations), suggesting that MHC isoform shifts may play a pathogenic role. The Heart in Chronic Liver Failure 283

4. TREATMENT At present, there is no definitive treatment recommendation for cirrhotic cardiomyopathy. In large part, this is due to lack of formal studies in this area. As the condition is often subclinical, many patients will require no active intervention. However, in some who develop more overt heart failure or who show impaired cardiac response to a major intervention such as TIPS or surgery, certain measures may be tried. Some drugs used for the management of the complications of cirrhosis can be applied to improve cardiac and/or liver functions. Clinical deterioration under conditions that challenge the cardiovascular system might be due to or aggravated by cirrhotic cardiomyopathy, and this should be considered when searching for causes for deterioration in patients with end-stage chronic liver failure. β-Blockers such as nadolol and propranolol are widely used to decrease the risk of variceal bleeding (68, 69). Non-selective β-blockers such as propranolol act by decreasing cardiac output and splanchnic arterial blood flow (70). Henriksen et al. (71) showed that acute administration of propranolol reduces the electrocardiographic QT interval. On the other hand, chronic β-blocker therapy of 1–3 months with nadolol in 30 patients with cirrhosis produced more complex effects. In those with normal QTc interval, it lengthened the interval slightly but significantly, whereas in those with prolonged QTc, it shortened the interval, but only when using the Bazett correction formula (72). We believe that chronic β-blockade probably does indeed shorten QTc in those with a prolonged interval. Whether β-blockade may prove useful in long-term cardioprotection by improving contractility indices, as has been suggested for non-cirrhotic congestive heart failure, remains unclear at present. Diuretics are effective in the management of fluid retention of liver cirrhosis by decreasing reabsorption of sodium and water, decreasing total blood volume, and lowering portal hypertension (73). Diuretics acting by aldosterone blockade also may potentially improve diastolic dysfunction and have some antifibrotic properties; therefore there is some interest in this line of treatment in cirrhotic cardiomyopathy (74). Pozzi et al. reported a 6-month trial of the anti-aldosterone drug potassium canrenoate in patients with end-stage liver failure (74). The treatment showed several favorable trends in cardiac chamber/wall sizes and diastolic function, which did not quite reach statistical significance. The authors suggest that a longer treatment duration is needed to show a definite beneficial effect. This avenue seems promising. Non-pharmacological management measures such as bed rest, improved nutrition, and oxygen supplementation can be applied to patients with cirrhotic cardiomyopathy (5). Again this suggestion is not 284 Liu et al. based on a controlled trial but rather is empirical. As mentioned previously, cardiac function improves after orthotropic liver transplantation (37). Thus transplantation appears to cure not just the liver disease but also the associated cirrhotic cardiomyopathy (75).

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Armando Tripodi

CONTENTS INTRODUCTION PRIMARY HAEMOSTASIS COAGULATION FIBRINOLYSIS CONCLUSIVE REMARKS REFERENCES

Key Words: Thrombin, Thrombomodulin, Protein C, Antithrombin, Factor II, Factor VIII, Platelets, Thrombocytopenia, Hemorrhage, Thrombosis, Fibrinolysis, Primary haemostasis, Coagulation, Thrombin generation, Prothrombin time

1. INTRODUCTION Chronic liver disease is characterized by a complex hemostatic defect which affects primary haemostasis (platelets–vessel wall interaction), coagulation (fibrinogen-to-fibrin conversion), and fibrinolysis (clot dis- solution). Because of this, chronic liver disease has become over the years the epitome of acquired hemorrhagic disease, and the concept of the causal relationship of abnormal haemostasis tests and bleeding has been widely accepted as shown by the common practice of screening patients with haemostasis tests and treating those with abnormal values in order to correct the identified abnormalities prior to liver biopsy or other potentially hemorrhagic procedures. However, evidence from the

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_14, C Springer Science+Business Media, LLC 2011

289 290 Tripodi literature and clinical practice shows that this might not hold true. The aim of this chapter is to focus on the main aspects of primary haemostasis, coagulation, and ﬁbrinolysis that may be used as arguments to dispute this paradigm.

2. PRIMARY HAEMOSTASIS Primary haemostasis defines the interaction of platelets and vessel wall at the site of vascular injury. This interaction is mediated by the adhesive multimeric plasma protein von Willebrand factor (VWF) that binds specific receptors constitutively expressed on the platelet membrane on one side and collagen exposed by the injured endothelium on the other side (Fig. 1a). The adhesion is followed by a change in platelet shape and secretion of substances such as adenosine diphosphate (ADP) and thromboxane A2 (TXA2), which are able to recruit other platelets and trigger fibrinogen- or VWF-mediated platelet aggregation (Fig. 1b). Activated platelets are also able to expose on their surfaces negatively charged phospholipids (phosphatidylserine) that function as receptors for plasma coagulation factors, thus facilitating

Fig. 1. Platelet adhesion at the site of injury and aggregation with one another (a). Platelet plug consolidation (b) and platelets expressing procoagulant activity on their surface with subsequent thrombin generation (c). Procoagulant factors are represented by roman numbers. P-serine, phosphatidylserine. Haemostasis Abnormalities in Chronic Liver Failure 291 the activation of coagulation and thrombin formation (Fig. 1c). In addition to converting fibrinogen to fibrin, thrombin also acts as a potent agonist for platelet recruitment at the site of injury. Impairment in any of the above mechanisms may lead to defective primary haemostasis and possible bleeding risk. Chronic liver disease has been shown over the years to be associated with variable thrombocytopenia and/or thrombocytopathy. The former is due to increased destruction of platelets or increased splenic and hepatic sequestration (1) and the latter is due to defective TXA2 synthesis, storage pool deficiency, or defects of platelet glycoprotein Ib or other factors (2–11). The bleeding time (BT), which has long been considered as the global test for primary haemostasis, is prolonged in up to 40% of patients with cirrhosis (12). However, the practical relevance of the BT prolongation in patients with cirrhosis is not clearly defined. Boberg et al. showed that the bleeding time prolongation (>12 min) was associated with a fivefold decrease of hemoglobin after liver biopsy (13), but the extent of clinical bleeding was not investigated. Subsequent studies showed that desmopressin, a synthetic analogue of the antidiuretic hormone l-arginine vasopressin able to shorten the BT in patients with VWF disease (14), when given intravenously or subcutaneously was also able to shorten the prolonged BT in patients with cirrhosis (15–17), but again the clinical relevance of this finding was not established. On the other hand, studies with clinical end points showed that desmopressin infusion in patients with cirrhosis was unable to control bleeding. One of these studies showed that the addition of desmopressin to the standard treatment (i.e., terlipressin) in patients with cirrhosis did not affect the extent of bleeding from esophageal varices (18). Another study showed that the treatment with desmopressin was unable to control bleeding and transfusion requirement in patients with cirrhosis undergoing hepatectomy (19). The above information may be taken as evidence that the defect of primary haemostasis as investigated by the BT is not relevant from the clinical standpoint. In a recent study of platelet adhesion in cirrhosis assessed under flow conditions, Lisman et al. found that highly elevated levels of VWF, which are typically found in patients with cirrhosis, contribute to the induction of primary haemostasis (20). Accordingly, increased VWF may compensate for the defect of platelet numbers and functions in these patients (20). One may, therefore, conclude that although defects of primary haemostasis in cirrhotics may have a causative role in the occurrence of bleeding, the BT once regarded as the test of choice to investigate primary haemostasis is probably not predictive of bleeding and should be abandoned. The logical conclusion is that if one wishes to rely on laboratory testing for primary haemostasis to predict bleeding in chronic liver disease, laboratory methods should go 292 Tripodi beyond the BT. Results from the literature showed that other surrogate tests for the BT in this setting such as the platelet function assay (PFA- 100) are also of little clinical value. Perhaps, new methods carried out under flow conditions (20) would be more appropriate, but they require extensive investigation.

3. COAGULATION 3.1. Pathophysiology of Coagulation Coagulation is defined as the chain of reactions that, through the activation of plasmatic coagulation factors, leads to thrombin generation (Fig. 2a). Thrombin in turn converts fibrinogen into fibrin, which is eventually stabilized by activated factor XIII. This is a highly integrated cellular/humoral process initiated when plasma activated factor VII (FVIIa) comes into contact with its specific cell receptor tissue factor exposed on cellular surfaces at sites of vascular injury. The complex FVIIa–tissue factor activates factor X, which in turn catalyzes the factor Va-mediated generation of thrombin from factor II (prothrombin). The complex FVIIa–tissue factor is also able to activate factor IX, which interacts with activated factor VIII on activated platelets. This complex generates additional amounts of activated factor X, thus amplifying the process of thrombin generation. In normal conditions the activity of the procoagulant factors as well as thrombin generation is tightly controlled by the anticoagulant system, which includes antithrombin, protein C/S, and the tissue factor pathway inhibitor (TFPI) (Fig. 2b). The balance

Fig. 2. Schematic representation of coagulation with thrombin generation (a) and inhibition (b). Procoagulant factors are represented by roman numbers. TF, tissue factor; AT, antithrombin; PC, protein C; TM, thrombomodulin; APC-PS, activated protein C–protein S; TFPI, tissue factor pathway inhibitor. Haemostasis Abnormalities in Chronic Liver Failure 293 between the pro- and anticoagulant factors prevents unwanted thrombin generation and ﬁbrin deposition, whereas its disruption results in hypo- or hypercoagulability.

3.2. Hypocoagulability As mentioned above chronic liver disease is characterized by relatively low levels of circulating pro- and anticoagulant factors. This is due to the poor liver synthetic capacity and also due to the defective postribosomal carboxylation of coagulation factors, mediated by vitamin K, which makes mature coagulation factors able to sustain normal coagulation. Notable exceptions to this rule are factor VIII and VWF which are both elevated in patients with chronic liver disease. The reasons for these elevations are poorly understood. VWF levels are high in acute liver disease (21) presumably because of endothelial cell dysfunction that mobilizes VWF from these cells where it is stored. In patients with cirrhosis VWF is even higher (22)andevi- dence has been provided that this is due to an increased liver synthetic capacity (23). Conversely, factor VIII is apparently increased as a consequence of decreased clearance from the circulation (24). Although the mechanisms are not precisely known, it has been postulated that the reduced clearance may be due to a combination of effects brought about by VWF and by the low-density lipoprotein receptor-related protein (LRP). VWF functions in vivo as the carrier protein for circulating factor VIII which is, therefore, protected against the proteolytic inactivation mediated by plasma proteases and premature clearance (24). Thus, increased levels of VWF, typically found in patients with cirrhosis, explain the relatively elevated levels of circulating factor VIII (24). LRP, on the other hand, is a multiligand receptor that plays a role in the cellular uptake and degradation of plasma factor VIII (24). LRP is highly expressed in normal liver, but much less in cirrhosis (24). This might be responsible for the reduced clearance of factor VIII from the circulation, thus explaining the elevated levels typically found in this condition. Whatever the reasons, the high levels of VWF and factor VIII might play a crucial role in primary haemostasis (see above) and in the balance of coagulation (see below) in patients with chronic liver disease. As mentioned, in patients with chronic liver disease, in addition to the procoagulant factors the anticoagulant factors are also decreased to some extent. Notably, protein C and antithrombin which are the two most important naturally occurring anticoagulants are reduced progressively from patients classified as Child class A to Child class C, reaching in the latter class values that are even lower 294 Tripodi than those found in patients with congenital deficiencies of the two proteins (25). Historically, the complex coagulation defect observed in patients with chronic liver disease has been investigated by means of the conventional global coagulation tests prothrombin time (PT) and activated partial thromboplastin time (APTT). Arbitrary cutoff values (especially for the PT) have been incorporated into clinical guidelines meant to guide prophylactic replacement of coagulation factors (either with fresh frozen plasma or with concentrates) in patients prior to liver biopsy or other potentially hemorrhagic procedures (26). However, it is well known from the literature (27) and clinical practice that the PT [and its derivative the international normalized ratio (INR)] is not predictive of bleeding in patients with cirrhosis who are about to undergo liver biopsy and other potentially hemorrhagic procedures or those who are at risk of or are actively bleeding from the gastrointestinal tract. This apparent paradox may be due to the fact that the PT, because of its design, might not be suited to truly represent the balance of coagulation as it occurs in vivo, especially in patients with chronic liver disease, a condition characterized by a partial deficiency of the procoagulant factors, but also by a concomitant deficiency of the naturally occurring anticoagulants (28). The concomitant decrease of both coagulation drivers might result in a restored balance. According to this concept the PT test and its congeners would be responsive to the procoagulant factors, but much less to the concomitant decrease of the naturally occurring anticoagulants, especially protein C (28). Plasma protein C downregulates thrombin generation by inhibiting the activated forms of factor VIII and V (29). However, in order to express its full anticoagulant activity, protein C needs to be activated by thrombin in cooperation with thrombomodulin (29). Thrombomodulin is a transmembrane receptor located on endothelial cells (29). It is of interest to note that plasmas and reagents used to perform the PT test do not contain sufficient amounts of thrombomodulin and cannot be truly representative of the balance of the pro- vs. the anticoagulants operating in vivo. As a matter of fact PT and APTT are useful laboratory tools to investigate congenital coagulopathies due to deficiencies of procoagulant factors (hemophilia and allied disorders), conditions where the anticoagulant factors are normal, but much less to investigate acquired coagulopathies, conditions where both pro- and anticoagulant factors are reduced (28). This hypothesis was tested by means of newly developed thrombin generation tests performed under in vitro conditions mimicking more closely than the PT what occurs in vivo. These tests are based on the activation of plasma coagulation by small amounts Haemostasis Abnormalities in Chronic Liver Failure 295 of tissue factor and negatively charged phospholipids as exogenous triggers (30, 31). Thrombin generation (that is dependent on the procoagulant drivers) and decay (that is dependent on the anticoagulant drivers) are eventually recorded as a curve (thrombin generated vs. time) and the area under the curve, which is also called endogenous thrombin potential (ETP), is taken as an index of the total amount of thrombin that can be generated under the specified experimental conditions depending on the balance between the pro- and anticoagulant drivers operating in plasma (30, 31). The application of the above technique showed that patients with compensated cirrhosis generate less thrombin than healthy subjects when the test was run in the absence of thrombomodulin (32). This is not unexpected as it reflects the partial deficiency of procoagulant factors that is not counterbalanced by the concomitant deficiency of the anticoagulants, especially protein C that is not fully activated in the absence of thrombomodulin. As a matter of fact, patients with cirrhosis generated as much thrombin as healthy subjects when the test was run in the presence of thrombomodulin. This has been taken as an indication that the balance of coagulation in cirrhosis is restored by the concomitant deficiency of pro- and anticoagulant factors (32). Thrombin generation in the above study was measured in platelet-free plasma. Under these assay conditions, thrombin is generated solely as a function of pro- and anticoagulant factors and does not account for platelets which are known to contribute significantly through their procoagulant activity mediated by the exposure of negatively charged phospholipids on their membranes (33). Another study investigated such an effect by measuring thrombin generation under the same assay conditions, but in platelet-rich plasma (34). In these experiments exogenous phospholipids were omitted from the assay and platelet numbers from patients and controls were adjusted by dilutions of the autologous platelet- free plasma into autologous platelet-rich plasma to a standard count of 100 × 109/L or to the original platelet count as found in whole blood for each patient and control (34). When thrombin generation was measured in the absence of thrombomodulin in plasma with platelet numbers adjusted to a standard count, patients generated significantly less thrombin than control subjects, but (as for platelet-free plasma) when the test was performed in the presence of thrombomodulin the differences were abrogated (34). On the contrary, when the test was performed in plasma with platelet numbers adjusted to the original whole blood count, thrombin generation was significantly lower in patients than in controls regardless of the addition of thrombomodulin (34). Interestingly, patients with the lowest platelet count generated the least amounts of thrombin (34). These experiments confirm the role played by platelets 296 Tripodi in thrombin generation and indicate that platelets from patients with cirrhosis are qualitatively suitable to support normal thrombin generation provided that they are in sufficient numbers. Analyses of the correlation between thrombin generation and platelet numbers allowed estimation of the minimum number of platelets needed to secure near-normal thrombin generation and this corresponded to 56 × 109/L (34). This number is close to the threshold value used in common practice for liver biopsy or intracranial pressure monitor placement (35). The above findings may have important practical implications. First, they provide evidence to question the usefulness of conventional coagulation tests such as the PT as tools to assess the hemorrhagic risk in this patient population. Whether thrombin generation tests or other global tests such as thromboelastography (36) are suited to this task should be investigated in appropriate clinical studies. Second, they provide support to the recent findings that the use of procoagulant agents such as the activated recombinant factor VII is not effective in controlling bleeding in patients with active variceal bleeding (37, 38)or in patients undergoing hepatectomy (39, 40) in spite of the fact that the drug is able to shorten the abnormal prolonged PT in the vast majority of treated patients (37). Perhaps, transfusion of platelets or treatment with the agonist of the thrombopoietin receptor (1) would be more effective by providing a suitable phospholipid surface to complement the normal thrombin generation provided by plasma, thus securing normal coagulation even in those patients with severe thrombocytopenia (41).

3.3. Hypercoagulability The restored balance of coagulation afforded by the concomitant reduction of the pro- and anticoagulant factors may explain why patients with chronic liver disease are not protected from thrombotic events which have been observed in these patients as shown by a retrospective study that looked at the clinical records of patients admitted to a large tertiary-care teaching hospital over an 8-year period (42) and recently confirmed by a larger nationwide population-based case–control study (43). The first study was aimed at determining the incidence and predictors of venous thromboembolism (VTE) including deep vein thrombosis and pulmonary embolism in hospitalized patients with cirrhosis. Approximately 0.5% of all the patients with chronic liver disease admitted to the hospital had VTE; the PT-INR and the platelet counts did not predict thrombosis, whereas low levels of albumin did (42). The second study determined the incident cases of VTE Haemostasis Abnormalities in Chronic Liver Failure 297 from 1980 to 2005 using population-based clinical records from the Danish National Registry of Patients and from the Civil Registration System. From the data of 99,444 patients with VTE and 496,872 population controls included in the study, it was established that patients with liver disease had an increased relative risk of VTE ranging from 1.74 (95% CI 1.54–1.95) for cirrhosis to 1.87 (95% CI 1.73–2.03) for noncirrhotic liver disease and the risks were higher for deep vein thrombosis than for pulmonary embolism. In the analysis restricted to the patients and controls with unprovoked VTE, the risk was even higher [2.06 (95% CI 1.79–2.38) for cirrhosis and 2.10 (95% CI 1.91– 2.31) for noncirrhotic liver disease] (43). From the above observations it is clear that patients with liver disease have a substantial increased risk of VTE despite the prolongation of conventional coagulation tests. This paradox may be explained if one considers that patients with chronic liver disease generate normal or even higher than normal thrombin (32, 34) and may occasionally be exposed to acquired or genetic risk factors leading to thrombosis. Portal vein thrombosis is in fact not an infrequent event (44, 45), especially in cirrhotics who are candidates for liver transplantation (46) or who are carriers of prothrombotic gain-offunction mutations such as factor V Leiden mutation or the prothrombin G20210A mutation (47). Therefore, the concept of considering the coagulopathy solely on the basis of the prolonged traditional coagulation tests does not hold true and should be reconsidered. A recent study investigated whether plasma from cirrhotic patients has an imbalance of pro- vs. anticoagulation factors that could be identified by laboratory testing (25). The study was based on the investigation of plasma from 134 cirrhotic patients and 131 healthy subjects as controls for the levels of the single pro- and anticoagulant factors and for thrombin generation in the presence or absence of thrombomodulin (25). Thrombin generation was assessed as endogenous thrombin potential (ETP) and the ratio of values measured with:without thrombomodulin was calculated thereafter. This ratio can be taken as an index of hypercoagulability (the higher the ratio the greater the hypercoagulability) and is in fact increased in plasma from patients with clinical conditions known to be at increased risk of VTE such as those who are carriers of congenital defects of the protein C anticoagulant pathway (i.e., factor V Leiden or deficiencies of protein C/S). The median ratio of thrombin generation (with:without thrombomodulin) was higher in patients (0.80, range: 0.51–1.06) than controls (0.66, range: 0.17–0.95), indicating that cirrhotic patients are resistant to the action of thrombomodulin (25). This resistance resulted in greater hypercoagulability of plasma 298 Tripodi from patients of Child class C than of class A or B. The hypercoagulability of plasma from patients of Child class C (0.86, range: 0.70–1.06) was slightly greater than that observed under the same experimental conditions in patients with congenital protein C deficiency (0.76, range: 0.60–0.93) (25). Interestingly, the levels of factor VIII, which is one of the most potent procoagulant drivers involved in the amplification of thrombin generation, increased progressively with Child–Pugh score (from Child class A to C) (25). Conversely, the levels of protein C, which is one of the most potent naturally occurring anticoagulant drivers involved in quenching thrombin generation, showed the opposite trend (25). The above observations are consistent with the hypothesis that the hypercoagulability of plasma from patients with cirrhosis appears to result from the increased levels of factor VIII combined with the concomitant decreased levels of protein C, which are typical features of patients with cirrhosis. These findings might explain the risk of venous thromboembolism in patients with chronic liver disease and suggest that treating patients with cirrhosis to prevent recurrence of venous thromboembolism with heparin or oral anticoagulants (44), once regarded as a strong contraindications in this category of patients, is plausible.

4. FIBRINOLYSIS Fibrinolysis is a tightly integrated system operating whenever fibrin deposition occurs. It includes the proenzyme plasminogen that can be converted into the active enzyme plasmin, which in turn degrades fibrin into degradation products (Fig. 3). The plasminogen to plasmin conversion is triggered by activators such as the tissue plasminogen activator (tPA) and the urokinase plasminogen activator (uPA), and counteracted by antiactivators such as the specific inhibitors of tPA (mainly PAI-1) and plasmin inhibitors (Fig. 3). Recently, a new inhibitor, called thrombin-activatable fibrinolysis inhibitor (TAFI), has been described. It is a procarboxypeptidase synthesized by the liver, which is activated by thrombin or plasmin and mediates the removal of lysine residues from fibrin, thus preventing the binding and activation of plasminogen which do occur on its surface. The correct balance of fibrinolysis is crucial to prevent unwanted plasmin generation and the perturbation of this balance may result in hyper- or hypofibrinolysis. Although the occurrence of hyperfibrinolysis in patients with cirrhosis has been advocated, its role in the bleeding events observed in these patients is still debated (35). The reasons for this uncertainty, as for primary haemostasis and coagulation, probably rest on the lack of appropriate laboratory tests for its evaluation. Most observations are Haemostasis Abnormalities in Chronic Liver Failure 299

Fibrinolysis Histidine-rich-glycoprotein

Plasminogen

Instrinsic activation (FXlla, Kal, etc.) TAFI Tissue plasminogen activator Tissue plasminogen activator inhibitor Urokinase Prourokinase

Plasmin Plasmin inhibitor

Fibrin Fibrin degradation products

Fig. 3. Schematic representation of fibrinolysis. Solid and broken arrows represent activators and inhibitors, respectively. TAFI, thrombin-activatable fibrinolysis inhibitor. based on the measurement of the individual components rather than on the evaluation of the overall fibrinolysis activity which takes into account the balance of pro- and antifibrinolytic factors. As a matter of fact, cirrhosis has been associated with reduced levels of plasminogen, plasmin inhibitor, factor XIII, or TAFI, but also with increased levels of tPA or its inhibitor PAI-1 (48–53). Special attention has been paid to TAFI (53), arguing that decreased levels of this inhibitor might explain the condition of hyperfibrinolysis associated with cirrhosis. Recently, Lisman et al. (54) evaluated this hypothesis by measuring the individual components of fibrinolysis as well as by employing a global test to assess the overall plasmatic fibrinolytic capacity. They concluded that the TAFI deficiency observed in patients with cirrhosis does not trans- late into an increased plasma fibrinolysis as shown by the results of the global test, suggesting (as for coagulation) that the balance of fibrinolysis is restored by the concomitant reduction of pro- and antifibrinolytic factors (50). Opposite conclusions were reported by Colucci et al. (55). This controversy has not yet been resolved and might be explained by the different global fibrinolysis assays employed in the two studies, thus leaving room for further investigation in order to clarify the role of fibrinolysis in this clinical condition. However, the measurements of the individual components of the fibrinolytic pathway are unlikely to help 300 Tripodi and probably lead to an overestimation of the role played by hyperfibrinolysis in chronic liver disease. Future efforts should be aimed at designing global tests, which should then be evaluated in clinical trials to assess the extent of fibrinolysis derangement and its role in the bleeding risk of patients with chronic liver disease.

5. CONCLUSIVE REMARKS In summary, the aforementioned observations support the following considerations. First, the abnormality of haemostasis in compensated chronic liver disease is more a myth than a reality because the balance of coagulation is somewhat restored by the concomitant reduction of pro- and anticoagulants that are typical features of this clinical condition. The concept that patients with chronic liver disease behave as normal subjects with respect to the coagulation balance is also supported by the evidence that they are at increased risk of thrombosis particularly at mesenteric and portal veins. Second, the PT test, which has been used for a long time as an index of bleeding in chronic liver disease, should be replaced by more suitable tests representing the balance of coagulation as it occurs in vivo. Until then, the risk of bleeding in patients with chronic liver disease should be based on clinical rather than on laboratory criteria. Third, the long held belief of correcting the abnormal PT test prior to liver biopsy should be reconsidered. Recently, the position paper of the American Association for the Study of Liver Diseases (AASLD) (41) while recommends platelet transfusion to correct severe thrombocytopenia, also warns on the indiscriminate use of the prophylaxis or rescue therapy based on plasma, ﬁbrinolysis inhibitors, or recombinant factors. However, the risk of bleeding in patients with chronic liver disease should not be underscored, especially in decompensated patients. When this happens culprit(s) other than abnormalities of haemostasis should probably be looked for. It should be realized that although restored, the balance of coagulation or ﬁbrinolysis in chronic liver disease is not as stable as in healthy subjects and may, therefore, be easily perturbed, especially if there are other underlying pathological conditions. In addition to severe thrombocytopenia, other potential candidates that may perturb the balance and trigger bleeding are the hemodynamic alterations subsequent to portal hypertension, endothelial dysfunction, recurrent bacterial infections leading to disseminated intravascular coagulation and development of endogenous heparinoids (56), or renal failure. Perhaps, interventions aimed at correcting these abnormalities might be more effective to stop bleeding than correcting the hemostatic derangement. Haemostasis Abnormalities in Chronic Liver Failure 301

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The Systemic and Splanchnic Circulations

Yasuko Iwakiri, Ph.D.

CONTENTS INTRODUCTION CONCLUSION REFERENCES

Key Words: Portal hypertension, Arterial vasodilatation

1. INTRODUCTION Splanchnic and systemic hemodynamic abnormalities are common in patients with portal hypertension with liver cirrhosis (1–4). Portal hypertension is characterized by an increase in portal venous pressure due to an increase in intrahepatic resistance. Upon development of portal hypertension, portosystemic collaterals, diverting portal blood to the systemic circulation, are formed. Furthermore, portal hypertension leads to the development of the hyperdynamic systemic circulatory syndrome characterized by reduced arterial pressure, decreased peripheral vascular resistance, and increased cardiac output. It is known that the splanchnic arterial vasodilatation (i.e., vasodilatation in mesenteric arteries) is a prerequisite for the development of this syndrome (5, 6). Locally produced vasodilator molecules mediate these processes. However, in recent years, studies using experimental animals have strongly suggested the importance of the intestinal microcirculation as a “sensing organ” of small changes in portal pressure and initial site of

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_15, C Springer Science+Business Media, LLC 2011

305 306 Iwakiri

Chronic hepatitis Liver cirrhosis Alcohol abuse Other etiologies Worsen Portal hypertension Portal pressure

Intestinal microcirculation Splanchnic/systemic circulation Vasodilator molecules

Arterial vasodilatation

Hyperdynamic circulatory syndrome 1. Mean arterial pressure 2. Systemic vascular resistance 3. Cardiac output 4. Splanchnic blood flow

Complications Gastroesophageal varices Ascites

Fig. 1. Overview of the development of the hyperdynamic circulation in liver cirrhosis.

vasodilatation (7). Such initial vasodilatation may contribute to exacerbation of portal hypertension in the very early stages of liver cirrhosis. Figure 1 summarizes hemodynamic events in portal hypertension. This chapter will briefly cover these findings. In the 1980s, discovery of nitric oxide (NO) as a novel vasodilator molecule (8, 9) significantly advanced our understanding of the mechanisms of arterial vasodilatation in the splanchnic and systemic circulations (1). NO produced by endothelial cells is a major contributor to arterial vasodilatation in both the splanchnic and systemic circulations (10). In recent years, other vasodilator molecules, as well as factors involved in the induction of vasodilator molecules, have been identified. Interestingly, a reduced response to vasoconstrictive molecules has also been shown to contribute to arterial vasodilatation in the splanchnic and systemic circulations in portal hypertension (11). The goal of this chapter is to give an overview of the etiology and processes involved in the development of arterial vasodilatation in the intestinal, splanchnic, and systemic circulations in liver cirrhosis with portal hypertension. Thus this chapter will discuss (1) vasodilatation in the intestinal, splanchnic, and systemic circulations, and (2) molecular mechanisms of vasodilatation. The Systemic and Splanchnic Circulations 307

1.1. Vasodilatation in the Intestinal, Splanchnic, and Systemic Circulations 1.1.1. INTESTINAL AND SPLANCHNIC CIRCULATIONS Splanchnic arterial vasodilatation is an essential process for the development of the hyperdynamic systemic circulatory syndrome (5, 6). However, recent discoveries have revealed that production of vasodilator molecules occurs within the intestinal microcirculation even earlier than in the splanchnic arterial circulation (7). This was demonstrated by using a surgical model of portal hypertensive rats. The surgical technique used to generate this model is called partial portal vein ligation (PVL) (1). It is accomplished by inserting different size needles into the lumen of the portal vein (released after the ligation is completed), creating luminal openings of various diameters, thereby mimicking different stages of portal hypertension. This method allows us to study the effects of differences in portal pressure on local vasculatures as well as on hemodynamics, without influencing liver function (1). When mild portal hypertension (in which portal pressure is too low to induce splanchnic arterial vasodilatation) was generated, there was a significant increase in the production of intestinal vascular endothelial growth factor (VEGF) with a subsequent increase in expression of endothelial NO synthase (eNOS), in the intestinal microcirculation (7)(Fig.2). VEGF is one of the most potent angiogenic factors (12). It is able to increase eNOS expression (13) and activation via phosphorylation at Ser1177 (human) (14–17). This model of mild portal hypertension is most likely representative of the portal pressure changes observed in early-stage cirrhosis, during which portal hypertension generally progresses slowly. This finding suggests that small increases in portal pressure, most likely characteristic of early-stage cirrhosis, may first be sensed by the intestinal microcirculation (Fig. 3). Thus, the ability of the intestinal microcirculation to sense small changes in portal pressure provides a physical stimulus that upregulates VEGF expression and, consequently, eNOS expression. Furthermore, increased intestinal VEGF also enhances angiogenesis, leading to increased portal blood inflow and a rise in portal pressure, worsening portal hypertension (Fig. 2). Upon reaching a certain level, portal pressure induces vasodilatation in the splanchnic circulation, possibly by increased shear stress due to an increase in blood flow. A study using rats with severe portal hypertension induced by PVL supports these mechanisms, since rats with severe portal hypertension showed eNOS upregulation in both the splanchnic arterial circulation (18) and intestinal microcirculation (19). Studies have demonstrated that VEGF receptor 2 (VEGFR2) blockers such as Sunitinib (20) and Sorafenib (21) ameliorate portal 308 Iwakiri

Fig. 2. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) as potential therapeutic targets to ameliorate portal hypertension. VEGFR2 is a cell surface tyrosine kinase receptor. Sunitinib (SU11248) is an oral, small molecule, multitargeting receptor tyrosine kinase inhibitor. It inhibits several important receptors, including VEGFR2, platelet-derived growth factor receptors, and KIT (also called CD117 or c-kit receptor). Sorafenib is an inhibitor of tyrosine protein kinases and is known to inhibit downstream signaling pathways of receptors, including VEGFR2 and PEDF. In an animal model of cirrhosis and portal hypertension, both Sunitinib and Sorafenib have been demonstrated to ameliorate collateral formation (i.e., angiogenesis) and portal hypertension (20, 21). eNOS, endothelial nitric oxide synthase; NO, nitric oxide.

Fig. 3. Portal pressure is sensed at different vascular locations depending on the severity of portal hypertension. Local NO production is affected by severity of portal hypertension. The intestinal microcirculation may be the most sensitive to changes in portal pressure. hypertension, suggesting that VEGFR2 could be a therapeutic target for portal hypertension in cirrhosis.

1.1.2. THE SYSTEMIC CIRCULATION The systemic circulatory abnormalities seem to occur secondarily to changes seen in regional vascular beds. It is well known that any changes in peripheral vascular resistance are rapidly compensated for The Systemic and Splanchnic Circulations 309 by changes in cardiac output (22). In portal hypertension the progressive reduction in peripheral resistance is slower, and compensatory mechanisms, such as sodium and water retention causing expansion of the plasma volume, play a fundamental role in perpetuating and aggravating the hyperdynamic syndrome (1, 4). As discussed previously, initial vasodilatation occurs in the intestinal microcirculation, followed by the splanchnic arterial circulation and ﬁnally in the systemic arterial circulation (7)(Fig.3). Although vasodilatation is fundamental to the development of the hyperdynamic circulatory syndrome, and systemic arterial vasodilatation may help sustain arterial hypotension, it is known that expansion of the plasma volume and portosystemic shunting are also essential factors in the development of this syndrome (23, 24).

1.2. Molecular Mechanisms of Vasodilatation Figure 4 summarizes vasodilator molecules and factors that are known to be involved in arterial vasodilatation in the splanchnic and systemic circulations in portal hypertension (10). Endothelial cells are the source of those vasodilator molecules, which generally diffuse into underlying smooth muscle cells and cause relaxation. Among those vasodilator molecules, NO is a primary contributor to the excessive vasodilatation observed in the splanchnic and systemic arterial circulations in portal hypertension (1, 2, 10, 25).

1.2.1. ROLE OF NITRIC OXIDE (NO) Nitric oxide, an endothelial-derived relaxing factor, is a key player in the arterial vasodilatation of the splanchnic and systemic circulations which leads to the hyperdynamic circulatory syndrome in portal hypertension (25–29). NO causes vasodilatation by stimulating soluble guanylyl cyclase (sGC) to generate cyclic guanosine monophosphate (cGMP) in vascular smooth muscle cells (8). NO synthases (NOSs): NO is synthesized by a family of three NOSs which are constitutively expressed isoforms: endothelial NOS (eNOS) (9), neuronal NOS (nNOS) (30, 31), and inducible NOS (iNOS) (32). Among these isoforms, eNOS is the major enzymatic source of vascular NO overproduction in the splanchnic arterial circulation (25, 33, 34). Recent evidence has suggested that nNOS, found in neuronal and vascular smooth muscle cells, is also upregulated in the mesenteric artery (35, 36) as well as in the aorta (37), and plays a role in the development/maintenance of the hyperdynamic splanchnic circulation in experimental cirrhosis (37). iNOS is synthesized de novo in a variety of cell types, including macrophages and vascular smooth muscle cells, 310 Iwakiri

Shear Stress Adrenomedullin Agonists Endothelial cells VEGF TNFα

Caveolin-1 IP3 eNOS GTP- Cys cyclohydrolase I NO

PO4 Akt 2+ Ca CaM BH4 eNOS HO-1 COX CSE EDHF AA Hsp90 Ethanolamine

Anandamide NO CO PGI2 H2S

KCa CB1R sGC AC Hyperpolarization

Smooth cGMP cAMP muscle cells

Vasodilatation

Fig. 4. Molecules and factors that are involved in arterial vasodilatation in splanchnic and systemic circulations. Growth factors and cytokines, such as adrenomedullin, vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha (TNFα), or physical stimuli, such as shear stress, stimulate Akt (also known as protein kinase B) which directly phosphorylates and activates endothelial nitric oxide (NO) synthase (eNOS). eNOS is calcium (Ca2+)/calmodulin (CaM)-dependent and requires cofactors such as tetrahydrobiopterin (BH4) for its activity. Heat shock protein 90 (Hsp90), a chaperone abundantly expressed in many cell types, is one of the positive regulators of eNOS. Recently, it was reported that the formation of nitrosothiol on a specific cysteine (Cys) residue by NO, a process known as S-nitrosylation, negatively regulates eNOS activity (44, 101). Anandamide (arachidonyl ethanolamide) is an endogenous lipid ligand, derived from arachidonic acid (AA) (57). Similar to NO, carbon monoxide (CO), synthesized by heme oxygenase-1 (HO-1), causes vasodilatation by activating soluble guanylate cyclase (sGC) to generate cyclic guanosine monophosphate (cGMP) in vascular smooth muscle cells. Prostacyclin (PGI2) is synthesized by cyclooxygenase (COX) and elicits smooth muscle relaxation by stimulating adenylate cyclase (AC) and generation of cyclic adenosine monophosphate (cAMP) (1). Hydrogen sulfide (H2S), synthesized by cystathionine-γ-lyase (CSE), opens KATP channels, leading to vasodilatation (83, 86, 87). IP3: Inositol trisphosphate; EDHF: Endothelium-derived hyperpolarizing factor; CB1R: Cannabinoid 1 receptor. only after induction by endotoxin lipopolysaccharides and inflammatory cytokines (38). Interestingly, despite the presence of endotoxemia in cirrhosis, iNOS is not detected in the splanchnic arterial vasculature in either portal vein-ligated or CCl4 cirrhotic rats (26, 27, 39, 40). The Systemic and Splanchnic Circulations 311

However, increased iNOS expression was observed in aorta isolated from biliary cirrhotic rats (41, 42). eNOS regulation: eNOS is regulated by complex protein–protein interactions, cofactors, and posttranslational modifications, such as phosphorylation (43), S-nitrosylation (44, 45), and acetylation (46). While regulation of eNOS by S-nitrosylation and acetylation has been demonstrated recently, it is not known whether these modifications of eNOS play a role in the hyperactive eNOS production observed in arterial splanchnic and systemic circulations in portal hypertension. Cofactors: eNOS is Ca2+/calmodulin (CaM)-dependent and requires cofactors such as tetrahydrobiopterin (BH4) for its activity (43). In cirrhotic rats, an increase in circulating endotoxin activates GTP- cyclohydrolase I, which increases BH4 production in mesenteric arteries. This increase in BH4 is associated with enhanced eNOS activity and eNOS-derived NO overproduction in the mesenteric arterial beds (47). Protein–protein interactions: eNOS is regulated by complex protein– protein interactions and posttranslational modifications (43). Among positive regulator proteins, Hsp90 (48) and the serine/threonine kinase Akt/PKB (protein kinase B) contribute to the activation of eNOS in the splanchnic arterial circulation in portal hypertensive rats (18). Akt/PKB directly phosphorylates eNOS at Ser1177 (human) or Ser1179 (bovine) and enhances its ability to generate NO (14–17). Various forms of stimuli, such as VEGF, inflammatory cytokines, and mechanical forces through shear stress, stimulate the production of NO by this mechanism (phosphorylation) (14–16) in portal hypertension (18, 33, 41). In the intestinal microcirculation, VEGF upregulates eNOS protein expression (7), leading to the development of the hyperdynamic circulatory syndrome (19).

1.2.2. ROLE OF CARBON MONOXIDE (CO) Similar to NO, CO is an endogenously produced gas molecule that activates sGC, resulting in increased production of cGMP (49), thereby regulating vascular tone in a manner similar to NO (49, 50). CO is produced from the breakdown of heme to biliverdin via the activity of the enzyme heme oxygenase (HO). Two isoforms of HO have been iden- tiﬁed: HO-1 and HO-2. HO-1 is inducible by multiple agents, while HO-2 is constitutively expressed (51). A progressively increased expression of HO-1 was found in aorta and mesenteric arteries of biliary cirrhotic rats, while no change was observed in HO-2 expression. Acute intraperitoneal injection of zinc protoporphyrin (ZnPP), a selective inhibitor of HO activity, to biliary cirrhotic rats (at a dose that normalizes aortic HO activity) ameliorates 312 Iwakiri the hyperdynamic circulatory syndrome. These results suggest a role for CO in arterial vasodilatation in portal hypertension (52).

1.2.3. ROLE OF PROSTACYCLIN (PGI2) PGI2 is synthesized by cyclooxygenase (COX) and released from endothelial cells. This molecule elicits smooth muscle relaxation by stimulating adenylyl cyclase (AC), which then leads to the production of cyclic adenosine monophosphate (cAMP) and subsequent vasodilatation (53). An increase in circulating PGI2 has been observed in cirrhotic patients (54) and portal hypertensive rabbits (55). Based on the above observations, a contributory role for PGI2 in the hyperdynamic circulatory syndrome has been suggested (54–56).

1.2.4. ROLE OF ENDOCANNABINOIDS Endogenous cannabinoids (or endocannabinoids) is a collective term used to describe a novel class of endogenous lipid ligands, including anandamide (arachidonyl ethanolamide) (57, 58). Endocannabinoids, through their binding to the CB1 receptor, cause hypotension. It has been shown that anandamide is increased in cirrhotic monocytes and that overactivation of CB1 receptors within the mesenteric vasculature may well contribute to the development of splanchnic vasodilatation and portal hypertension (59). The blockade of CB1 receptors by the antagonist SR141716A not only increases mean arterial pressure (59, 60) and peripheral resistance (60), but also reduces mesenteric blood ﬂow in rats with CCl4-induced cirrhosis (59). Under the inﬂuence of the same inhibitor, biliary cirrhotic rats show increased vascular tone in the mesenteric arteries (61). CB1 receptors are present in vascular endothelial cells, and activation of endothelial CB1 receptors leads to increased production of NO which causes subsequent arterial hypotension (59, 62, 63). On the other hand, a study by Ros et al. (60)showed that anandamide-induced hypotension is NO-independent. Therefore, it is currently still unclear whether the vasodilatation mediated by endocannabinoids is NO-dependent.

1.2.5. OTHER IMPORTANT VASODILATOR MOLECULES Endothelium-derived hyperpolarizing factor (EDHF): The molecular identity of EDHF is still under debate. The major molecules being considered to explain EDHF-mediated vasodilatations are (1) arachidonic acid metabolites (64–67), (2) the monovalent cation K+ (68), (3)gap junctions (69–71), and (4) hydrogen peroxide (72–76). EDHF seems to be more prominent in smaller arteries and arterioles than in larger arteries. This observation has been made in a number of vascular beds, The Systemic and Splanchnic Circulations 313 including those of the mesentery, cerebrum, ear, and stomach (77–81). It has been shown that EDHF is present in mesenteric arteries isolated from biliary cirrhotic rats (82). Hydrogen sulﬁde (H2S): A growing body of evidence suggests that H2S is a potent endogenous vasodilator in the aorta (83, 84) and mesenteric arteries (85). H2S is synthesized endogenously from L-cysteine – mainly by the activity of two enzymes, cystathionine-γ-lyase and cystathionine-β-synthase (83, 86). An intravenous bolus injection of H2S was shown to transiently decrease the blood pressure of rats by 12–20 mmHg. It is known that H2S-mediated vasodilatation occurs through the opening of KATP channels. Interestingly, unlike NO or CO, H2S relaxes vascular tissues independently of the activation of the cGMP pathway (87). The role of H2S in the hyperdynamic syndrome of cirrhosis has not been studied.

1.2.6. FACTORS THAT INDUCE AND SUSTAIN VASODILATATION Vascular endothelial growth factor (VEGF): VEGF is one of the most potent inducers of angiogenesis (12). It regulates vasomotor tone via eNOS induction. Upon binding to its receptor on endothelial cells, VEGF induces signaling cascades that activate Akt/protein kinase B followed by subsequent activation of eNOS through phosphorylation at Ser1177 (human). As previously mentioned, VEGFR2 has been effectively targeted to reduce portosystemic collateral formation and decrease portal pressure in an experimental model of portal hypertension (19). Tumor necrosis factor alpha (TNFα): TNFα, produced by mononuclear cells upon activation by bacterial endotoxins, is found in increased levels in portal hypertension (88, 89) and is a well-known mediator of NO release (90). Antagonism of TNFα using an anti-TNFα neutralizing antibody, or inhibition of TNFα synthesis by thalidomide, reduces the severity of the hyperdynamic circulation in portal hypertensive rats (89, 91). It has been suggested that TNFα stimulates the gene expression and activity of the key enzyme for the regulation of BH4 biosynthesis, GTP-cyclohydrolase I, in endothelial cells (92, 93); this enhanced BH4 production then directly increases eNOS-derived NO bioavailability (92, 94). On the other hand, it has recently been shown that in biliary cirrhotic rats, TNFα, through the activation of iNOS in lung and aorta, plays a role in the development of the hyperdynamic circulatory and hepatopulmonary syndromes (95). Adrenomedullin: Adrenomedullin is a potent vasodilatory peptide composed of 52 amino acids in human and 50 amino acid residues in rat. In cirrhotic liver patients, there is an increase in 314 Iwakiri circulating adrenomedullin levels (96, 97) associated with increased plasma nitrite (a stable NO metabolite) and plasma volume expansion. These increased adrenomedullin levels have been found to inversely correlate with peripheral resistance (97). The administration of anti- adrenomedullin antibodies prevents the occurrence of the hyperdynamic response in early sepsis (98) and ameliorates a reduced contractile response to phenylephrine in aorta isolated from cirrhotic rats (99). Adrenomedullin phosphorylates and activates Akt, and increases cGMP production in rat aorta, an indicator of NO production. It has been suggested that adrenomedullin-mediated vasorelaxation occurs through the production of NO (100). Decreased response to vasoconstrictor molecules: The balance between vasodilatation and vasoconstriction is in favor of vasodilatation in arteries of the splanchnic and systemic circulations in portal hypertension. In isolated superior mesenteric arterial beds and in aorta from portal hypertensive rats, a decreased response to vasoconstrictors has been recognized (18, 33). Thus, it is thought that this reduced contractile response may also contribute to the persistent vasodilatation observed in the splanchnic and systemic arterial circulations in portal hypertension. This decrease in contractile response seems to be attributable to impaired signaling pathways for vasoconstriction in smooth muscle cells (11). It remains unknown how this phenotypic change occurs in the smooth muscle cells of arteries in the splanchnic and systemic circulations in portal hypertension.

2. CONCLUSION In the last 50 years signiﬁcant advances have been made in our knowledge concerning mechanisms of arterial vasodilatation in splanchnic and systemic circulations in portal hypertension. It has been concluded that the observed arterial vasodilatation underlying the hyperdynamic circulatory syndrome of the splanchnic and systemic circulations is due to “hyperactive endothelial cells,” characterized by excess NO production. This is totally contrary to the so-called “endothelial dysfunction” observed in many other diseases. In this regard, portal hypertension could be considered as a unique type of vascular abnormality. Our basic scientiﬁc and clinical knowledge in the area of mechanisms of arterial vasodilatation in liver disease and their effect on hemodynamic circulatory systems will increase as the foundation presented within this chapter is built upon and expanded. Hopefully, such studies will lead to a fruitful understanding of general vascular biological principles, as well as to knowledge of therapeutic value. The Systemic and Splanchnic Circulations 315

ACKNOWLEDGMENT My apologies to colleagues whose references were omitted for the sake of brevity or whose contributions were not cited in this chapter. Dr. Iwakiri is supported by NIH/NIDDK K01 award (DK067933), NIH/NIDDK R01 award (DK082600), and Clinical Translational Scholar Award from Yale Center for Clinical Investigation (UL1RR024139).

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Hepatic Microcirculation

Chittaranjan Routray and Vijay Shah

CONTENTS INTRODUCTION PHYSIOLOGY OF REGULATION OF HEPATIC MICROCIRCULATION PATHOPHYSIOLOGY OF HEPATIC MICROCIRCULATION EFFECT OF TREATMENT ON HEPATIC PERFUSION IN SEPSIS AND SHOCK CONCLUSION REFERENCES

Key Words: Endotoxins, Sepsis, Sinusoidal endothelium, Nitric oxide, Endothelin-1, Tumor necrosis factor-α, Toll-like receptor, Transforming growth factor-β, Hepatic stellate cells, Portal resistance Drug Names: Bosentan, Dobutamine, Dopamine, N-acetylcysteine

1. INTRODUCTION Hepatic circulation is considered to be a low-resistance system, which accommodates about 30% of the total cardiac output. Like pulmonary circulation, mammalian liver also gets dual blood supply, from both portal vein and hepatic artery. About 80% of the blood supply to the liver is poorly oxygenated venous blood from portal vein and the rest is hepatic arterial blood (1). Blood ﬂow to the liver, as in other organs, is maintained as well as regulated per demand due to a well-balanced interplay of neuroendocrine and paracrine pathways.

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_16, C Springer Science+Business Media, LLC 2011

323 324 Routray and Shah

This adaptive regulatory mechanism may fail to maintain adequate perfusion to tissues which vary depending upon the systemic exposure to different situations such as stress or disruptive processes (i.e., circulatory insufficiency (e.g., MI), sepsis or endotoxemia, trauma and extensive burn). Therefore, this chapter is intended to describe the different pathophysiological mechanisms of altered hepatic microcirculation which occurs in cirrhosis, ischemia, and sepsis, beginning with a brief introduction about the regulation of microcirculatory flow in the liver. This chapter will also emphasize the effects of sepsis on microcirculation along with a brief summary of the biochemical behavior of some important molecules, endothelin-1, nitric oxide, carbon monoxide, and thromboxane, which play an important role in the regulation of hepatic microcirculation. As we know, the gastrointestinal tract is the most important source of toxins and metabolic wastes. For example, endotoxins and ammonia produced by the luminal bacteria play a major role in the development of septicemia and multiorgan failure. Thus, interestingly, the gastrointestinal tract acts as a primary barrier against luminal toxins and microorganisms. The gut and hepatic macrophages act as the first barrier against the spread of the translocated bacteria and their toxic products to the systemic circulation (2). The initiation of an event of deranged homeostasis, where bacteria and endotoxins leak into the systemic circulation, is believed to be due to insufficient splanchnic blood flow in a condition of increased metabolic demand (3). In clinical practice, microcirculation is of particular importance especially while taking care of critically ill patients, where monitoring the vitals of microcirculation can help identify patients at risk of further organ damage, so that a timely intervention can be made. Thus, in recent days, microcirculation has been given much attention in critical care medicine. This chapter will cover the following to have a better understanding of the conditions that affect microcirculation. (1) Cellular anatomy in brief (2) Regulation of hepatic microcirculation (3) Microcirculation in (a) cirrhosis, (b) ischemia, and (c) sepsis (4) Different treatment modalities toward sepsis-induced liver failure

2. PHYSIOLOGY OF REGULATION OF HEPATIC MICROCIRCULATION Hepatic microvascular subunit (HMS) is considered to be a conical microvascular subunit of classic lobule containing a group of sinusoids, Hepatic Microcirculation 325 and the concept of such a functional and structural unit is recently being supported by scanning electron microscopy and in vivo microscopic studies. Liver cells, which are the active participants of hepatic microcirculation, are the endothelial cells present in the sinusoids, portal venules, hepatic arterioles, hepatic venules, and contractile cells, also known as the hepatic stellate cells (HSCs). There are specific vascular mediators like nitric oxide (NO), endothelin (ET), and carbon monoxide (CO), which regulate the function of hepatic circulation. Several noxious stimuli (e.g., alcohol, endotoxins, carbon tetrachloride) can impair the hepatic vascular function in the absence of any structural changes, and they do so by the production of cytokines from Kupffer cells up to a certain extent. However, the vascular endothelium plays a dynamic role in the regulation of hepatic blood flow, which differs phenotypi- cally compared to the blood flow regulation in other regional vascular beds. Sinusoids are the principal vessels, which are not only involved in the transvascular exchange of nutrients and particulate matters or toxins between sinusoidal blood and cells of hepatic parenchyma, but are also the principal site for regulation of blood flow through the microcirculation in both normal and injured liver. The fenestrated sinusoidal endothelium, which is the invagination of endothelial plasma membrane across the endothelium, regulates the sinusoidal blood flow by the Ca2+-actinomycin-mediated contraction and dilation of the fenestrae. Immunoelectron microscopy and biochemical studies have also shown that caveolin-1 (a major structural protein of caveolae) and nitric oxide synthase (eNOS) coexist in the plasma membrane of sinusoidal endothelial cells and play a role in the locoregional regulation of sinusoidal blood flow in a NO-dependent manner (4, 5). HSCs and Kupffer cells also play an important role in regulating the diameter of sinusoids by regulating the velocity of blood flow in these specialized vessels. The vasoactive molecules of NO, CO, and ET regulate hepatic microvascular resistance. Although endothelial cells can produce these vasoactive substances, the absence of traditional underlying smooth muscle puts into question the precise mechanism of sinusoidal tone modulation. In such a case, HSCs become an interesting target for researchers as they may function as sinusoidal effector cells which may play a role in maintaining the vascular tone and pressure in hepatic microcirculation. Although the factors involved in autoregulation of sinusoidal blood flow and their relation to hepatic structure and function are not completely understood, in recent days, much has been revealed with the help of electron microscopy and in vivo microscopic studies. 326 Routray and Shah

3. PATHOPHYSIOLOGY OF HEPATIC MICROCIRCULATION Although we have come a long way since human blood circulation was established by William Harvey and Malpighi, it has always been a challenging task to study the microcirculation in most of the organs and also in the liver (6). Disturbance in microcirculation is responsible for hepatic failure in conditions such as alcoholic liver disease, fatty infiltration, cirrhosis, and sepsis. Primarily, however, hepatocellular dysfunction occurs due to ischemia and sepsis. Even though endotoxemia can produce multiple organ failure, it is often said that the cause of organ failure is not known, although multiple organ failure is the primary cause of death in patients with sepsis. Sepsis can cause alteration in hepatic microcirculation and thus produce an alteration in liver function. The role of hepatic blood flow in the development of sepsis-induced hepatic failure still remains a controversial topic. Many studies have concluded that the blood flow to liver declines in sepsis and thus parenchymal ischemia plays a role in hepatic dysfunction. At the same time, other experimental studies have indicated that hepatic blood flow increases in sepsis patients, suggesting the involvement of a more complex process in the pathophysiology of hepatic dysfunction. A study by Dahn et al. predicted that hepatic dysfunction is more likely to be due to hypoxic and ischemic events in patients with sepsis rather than due to increase in blood flow. However, this study had a drawback as it could not prove the role of any single factor in regulating the parenchymal oxygen tension (7). In recent days, many experimental studies have presented very challenging facts, trying to explain the mechanism responsible for producing microvascular alteration and liver failure. Some of these include (1) architectural modification in fibrosis/cirrhosis, (2) ischemia or perfusion deficit due to systemic hypotension and a reduced mean arterial pressure (MAP) caused by endotoxemia as well as impaired microperfusion due to decreased flow, (3) activation of clotting cascade and coagulation abnormality such as disseminated intravascular coagulation and thrombosis in sepsis, (4) impaired microperfusion due to tissue edema by increased capillary permeability, (5) mitochondrial dysfunction which might be due to NO overproduction, (6) induction of cytotoxic enzymes, free radicals, and apoptosis, (7) opening of arteriovenous shunts, and (8) increased blood viscosity and decreased red blood cell deformability (Fig. 1)(8). Hepatic Microcirculation 327

• Capillarization of sinusoids Cirrhosis • Portosystemic shunts • Regenerative nodules

• Endothelial and Kupffer cell activation • Upregulation of iNOS • Increased microvascular permeability and tissue edema Disturbed Sepsis • Activation of coagulation cascade • Decreased RBC deformability microcirculation • Openeing of AV shunts and redistribution and of blood flow • Vasoconstriction and hepatic failure Obstruction to blood flow Ischemia • Vasodilation and hypotension • Hypoxia and hepatic dysfunction

• Fatty infiltration and mechanical Steatosis obstruction

Fig. 1. Disturbed hepatic microcirculation and hepatic failure due to cirrhosis, ischemia, sepsis, and steatosis and their involved mechanisms. Hepatic microcirculation is affected by cirrhosis, sepsis, and steatotic liver diseases. These affect the microcirculation either by mechanical effects or by modulating tissue permeability, iNOS regulation, AV shunting, RBC rheology, and coagulation pathway. iNOS, inducible nitric oxide synthase. AV, arterio–venous; RBC, red blood cells.

3.1. Microcirculation in Cirrhosis Cirrhosis is a condition with increased intrahepatic vascular resistance and is an important component of portal hypertension. It is a chronic process which leads to continual parenchymal destruction, reparative hyperplasia of hepatocytes, and deposition of collagen within the space of Disse leading to overgrowth of fibrous tissue. The most important microcirculatory change in cirrhosis is deposition of collagen I in the space of Disse with the appearance of basement membrane beneath the sinusoidal endothelial cells and the formation of portohep- atic shunts. There is perisinusoidal deposition of glycosaminoglycans, type I and type IV collagen, and laminin along with a decrease in the number and diameter of the fenestrae of sinusoidal endothelium, which slows the blood flow through microcirculation. There is capillarization of sinusoids restricting the diffusion of albumin and other protein-bound substrates from the plasma to space of Disse, impairing exchange between sinusoidal blood and hepatocytes and thereby contributing to liver failure (9, 10). Although the exact cause of hepatic 328 Routray and Shah failure in cirrhosis is unclear, infection is the most common condition associated with the acute deterioration of liver function in preexisting chronic liver diseases. A diseased liver is more prone to sepsis and therefore more likely to fail, often called acute on chronic hepatic failure, in other words, an acute deterioration of liver function in a preexisting chronic liver disease. In a clinical setting, gram-negative bacterial infection accounts for most of the acute decompensation of hepatic function in cirrhotic patients. Studies have pointed to microcirculation as the principal contributing element because impaired hepatic microcirculation can result in impaired nutrient and oxygen exchange irrespective of the metabolic load or metabolic capacity of the hepatocytes (“intact cell hypothesis”) (11). Therefore, the overall effect is slowing of blood flow leading to prolonged contact between the blood and tissue, which permits additional extraction of oxygen. Eventually this elevation in arteriovenous oxygen difference in the absence of an increased consumption leads to hypoxic damage. In such a scenario, a bacterial leak from the intestine may provoke the recruitment of inflammatory cells, flooding the microenvironment with fibrogenic cytokines. In an attempt to repair the damage, HSCs generate scar tissue which can interfere with the blood flow within the hepatic microcirculatory unit leading to further liver injury. Therefore, an increase in the recruitment of HSCs and an increased production of constrictors like ET and reduced synthesis of dilators such as NO by endothelial cells in total conspire to increase the hepatoportal vascular resistance significantly. 3.2. Pathophysiology of Hepatic Microcirculation Due to Ischemia Ischemia of the liver can occur in several clinically significant conditions such as hemorrhage, sepsis, and iatrogenic causes (i.e., resection of a large hepatic tumor or transplant surgery). Ischemia creates a state of hyperdynamic circulation due to hypoxia resulting in cellular injury and, when followed by restoration of circulation, it is subjected to even more obvious damage than that induced by ischemia (12). Both ischemia and reperfusion lead to induction of a significant inflammatory process in the liver which causes Kupffer cell-mediated activation of endothelial cells and neutrophils followed by generation of cytokines (TNF-α) and a reactive oxygen species, resulting in further interruption of blood flow and tissue injury. TNF-α activates mitogen-activated protein kinase (MAPK) cascade which is known to be involved in the pathogenesis of ischemia–reperfusion injury (13). MAPK, after being activated, further stimulates the production of TNF-α leading to a progressive hepatic dysfunction. Pentoxifylline has been shown to increase the sinusoidal blood flow by inhibiting TNF-α (14). Hepatic Microcirculation 329

Another molecule of importance which contributes significantly to ischemic liver injury is CO. CO activates guanylate cyclase and produces dilation of sinusoids by relaxation of HSCs. CO is produced in the liver by heme oxygenase (HO). HO-1 is mainly produced by Kupffer cells, and HO-2 is expressed in hepatocytes. Due to the anatomical orientation, CO produced by the HO-2 can directly modulate the contractility of stellate cells. CO produced by HO-1 maintains the hepatic microvascular tone in a relaxed state; on the other hand, blockade of CO production results in an increase in portal resistance and reduction in sinusoidal blood flow (15). HO-generated CO has a protective role in hepatic microcirculation from conditions of stress because HO inhibitors have been found to produce hepatoportal vascular derangement and hepatocellular reductive stress. Experimental studies have shown HO-1 induction in hepatocytes, Kupffer cells, and intestines of portal hypertensive rats undergoing partial portal vein ligation suggesting a possible role of CO in the regulation of hepatoportal hemodynamics. Another experimental study in a murine model has shown that exogenous exposure to CO during early hours of ischemia or systemic inflammation protected hepatic microcirculation and improved impaired hepatocellular integrity as well as its redox state (16).

3.3. Microcirculatory Failure in Hepatic Steatosis Steatotic liver diseases are associated with abnormal accumulation of triglycerides in hepatocytes. They have been classiﬁed histologically into microvesicular and macrovesicular steatosis. Triacylglycerol accumulates as an intracytoplasmic fat droplet in the hepatocytes leading to an increase in hepatocellular volume, which may induce distortion and narrowing of liver sinusoids consequently altering hepatic blood ﬂow and microcirculation. It has also been suggested that a balance between ET and NO is very crucial in maintaining sinusoidal perfusion in steatotic liver diseases (17, 18).

3.4. Pathophysiology of Hepatic Microcirculation in Sepsis The condition of endotoxemia is associated with increased delivery of endotoxin to the portal circulation, which in turn stimulates the Kupffer cells and sinusoidal endothelial cells to produce proinﬂamma- tory cytokines along with secretion of other vasoactive molecules (19). Vasoactive molecules such as NO (a vasodilator), endothelin-1 (ET-1, a potent vasoconstrictor), and CO (a vasodilator) are known to regulate the vascular tone and are found to be highly expressed during sepsis (Fig. 2)(20). In the early hours of sepsis, these active molecules can produce hepatosplanchnic hypoperfusion due to vasoconstriction, 330 Routray and Shah

Fig. 2. Vasoactive molecules actively involved in the maintenance of hepatic microvascular tone. Vasoactive substances involved in the regulation of hepatic microcirculation are nitric oxide (NO) – vasoconstrictor, carbon monoxide (CO) – vasodilator, endothelin-1 (ET-1) – vasoconstrictor, and tumor necrosis factor (TNF-α) – vasoconstrictor. MAP, mean arterial pressure; KC, Kupffer cell; IL-1, interleukin-1; RBC, red blood cell; ROS, reactive oxygen species. producing a rise in the biological markers of liver damage (SGOT, SGPT, LDH, and bilirubin) (21). Microcirculatory dysfunction and the mitochondrial redox state have been restored in Kupffer cell-depleted animal models, which explains the potential initiative role of Kupffer cells in sepsis-induced hepatic injury. This also opens up an avenue for extensive research into the beneficial role of Kupffer cell modulation in hepatoportal hemodynamics (22). A high circulating level of endotoxin activates the sinusoidal endothelial cells and Kupffer cells leading to an increased interaction between neutrophils, platelets, and endothelium, causing impaired microperfusion and hepatocellular damage. In the mean process, the sinusoidal endothelial cells undergo structural modification which causes leakage of plasma, albumin, and neutrophils into the interstitium; this leads to tissue edema and further interruption of microcirculatory flow. The endothelial cells also lose their normal anticoagulant property and express cell adhesion molecules which, along with platelets and white blood cells, produce plugging of the sinusoids with fibrin clots. This reduces the sinusoidal perfusion area and blood flow, culminating in microperfusion failure and hepatocyte damage (23). Experimental study models have been designed in rodents and canines, where hepatic perfusion is studied by continuous endotoxin Hepatic Microcirculation 331 challenge by intravenous infusion. However, few studies have examined hepatic perfusion in a human sepsis model. The isolated perfused liver is increasingly being used to study the effects of endotoxin on liver microcirculation because it is a good model with no interference of cardiovascular or endocrine system (24). All these models consistently suggest that hepatosplanchnic circulation is highly increased in volunteers injected with endotoxin (25). A similar effect was observed in canine liver models, irrespective of whether endotoxin was injected through portal vein or hepatic artery (26). Sepsis produces a hyperdynamic circulatory state in the liver which is again due to the endotoxin-induced overproduction of inflammatory cytokines. Endotoxin activates several signaling pathways which collectively influence microcirculation.

3.5. Role of Toll-Like Receptor-4 (TLR4) Signaling In vivo experiments have explained the role of Toll-like receptors by lipopolysaccharide (LPS) challenge to animal models. LPS challenge activates Toll-like receptor-4 (TLR4) (member of a family of receptors that recognize pathogen-associated molecular pattern) which ultimately increases the expression of the transcription factor NF-κB, a key molecule that regulates the synthesis of proinflammatory cytokines like TNF-α.TNF-α acts as the central mediator of endotoxin-induced hepatic dysfunction by promoting adhesion of leukocytes and platelets to hepatic microvessels and impairing sinusoidal perfusion (27). TNF- α also upregulates Fas ligand-mediated apoptotic pathway causing endothelial cell and hepatocyte dysfunction, the key components of hepatic microcirculatory unit (Fig. 3). However, this pathway is deficient in normal hepatocytes where NF-κB essentially acts as a member of antiapoptotic pathway. Recent studies have reported the role of TLR- 4 in fibrosis associated angiogenesis in liver, suggesting that TLR-4 could regulate neo-angiogenesis and thus could contribute to vascular remodelling in the liver (28). Following liver injury, the endotoxin level increases in portal circulation due to the breach in the protective barrier against bacterial translocation and changes in intestinal permeability, which in turn stimulate the Kupffer cells leading to the activation of HSCs (the main precursors of pericytes in the liver). 3.6. Role of TGF-β Signaling Activated HSCs express TLR4 and thus are highly responsive to low concentrations of LPS. Activation of TLR4 in quiescent HSCs (concurrent with the upregulation of chemokine secretion) induces chemotaxis of Kupffer cells and downregulates the TGF-β pseudore- ceptor (BMP) and the activin membrane-bound inhibitor (Bambi) augmenting collagen production and thus fibrogenesis. Therefore, 332 Routray and Shah

LPS

MD2 TLR4 CD14

MyD88 TRIF

Caspase-8 IRAK-4 IKK

Apoptosis

P38/MAPK NF-κB

Cytokines TNF-α (vasoconstriction, leukocyte adhesion, platelet aggregation, apoptosis, hepatocyte dysfunction)

Fig. 3. Role of TLR signaling pathway in hepatic microcirculation. TLR4 receptors, after exposure to LPS, get activated and form a membrane-bound complex (TLR4–MD2–CD14), starting a cascade of events leading to the generation of transcription factor NF-κB which regulates the biosynthesis of TNF-α, the key cytokine in hepatic microcirculation. LPS, lipopoly saccha- ride; TLR4, toll-like receptor-4; CD-14, cluster of differentiation-14; MYD88, myeloid differentiation-88; TRIF, TIR-domain-containing adaptor-inducing interferon-β;NF-κβ, nuclear factor kappa-beta; P38/MAPK, mitrogen associated protein kinase. MD2, Lymphocyte antigen 96; CD 14, Cluster of differentiation 14; IKK, Inhibitor of NF-κB Kinase; IRAK, Interleukin-1 receptor associated kinase. Bambi is downregulated by LPS and its sensitization to TGF-β is mediated by MyD88–NF-κB-dependent pathway. Thus, TLR4-mediated downregulation of Bambi is essential to allow Kupffer cells to fully activate HSCs in a TGF-β-dependent manner thereby facilitating hepatic ﬁbrosis and affecting microcirculation in liver (29).

3.7. Role of MAPK Signaling Pathway MAPK is known to play a potential role in regulating the inﬂammatory process. It has been shown that phospho-P38 MAPK regulates the Hepatic Microcirculation 333 hepatocellular expression of TNF-α in septic mice. In LPS-induced endotoxemia, MAPK regulates the adhesion and rolling of leukocytes in hepatic microvascular endothelium and activates the cascade of events to generate TNF-α. Inhibition of P38 MAPK with SB 239063 has been shown to protect against LPS-induced leukocyte–endothelium interaction and signiﬁcantly reduce LPS-induced microvascular reaction and hepatocellular damage (30).

3.8. Role of Cyclooxygenase Pathway (TXA2) The microcirculatory dysfunction by TNF-α is elicited by an arachidonic acid metabolite thromboxane (TXA2) and is facilitated by thromboxane-prostanoid (TP) receptor signaling. In turn, TXA2 has been shown to enhance liver injury by modulating TNF-α production (31). TXA2, which exerts a vasoconstrictive effect by acting on HSC, has also been shown to increase portal pressure in endotoxic and cholestatic liver (31–34). Increased production of TXA2 in the liver in response to endotoxins is mainly from Kupffer cells, although endothelial cells have also been shown to release TXA2 in response to LPS administration (Fig. 4).

Endotoxemia Cirrhosis LPS

Kupffer cells

TXA2

Platelet Leukocyte Vasoconstriction TNF-α production aggregaation adhesion

Microcirculatory failure, Hepatic dysfuction

Fig. 4. Role of thromboxane A2 (TXA2). Endotoxin activates the Kupffer cells stimulating the generation of TXA2, which causes vasoconstriction, platelet aggregation, and adherence of leukocytes to the vessel wall, slowing the microcirculatory ﬂow. TXA2 also stimulate the production of tumor necrosis factor-α (TNF-α), thus potentiating its action. LPS, lipopolysaccharide. 334 Routray and Shah

3.9. Role of Endothelin-1 and Nitric Oxide Hepatic hemodynamic abnormality is also linked to the endotoxin- induced upregulation of ET-1 and NO synthase in sinusoidal endothelial cells. It has been found that endothelin stimulates synthesis of NO in endothelial cells. At a lower concentration, it produces relaxation of isolated blood vessels. In contrast, NO inhibits the synthesis of ET-1 in endothelium. This explains how the balance between ET and NO is maintained and why an endotoxin-challenged alteration in their synthesis can be critical for development of a diseased process (Fig. 5). ET-1 has been found to reduce the sinusoidal diameter and decrease sinusoidal blood ﬂow along with increasing the total portal resistance in normal rat liver upon exogenous administration (35, 36). It is also been found to be elevated in the portal and systemic circulation in LPS- induced endotoxemia. Such potentiation in action and enhancement in response of ET-1 toward LPS, which occurs primarily at the sinusoidal and presinusoidal levels, can contribute to endotoxin-induced

Endotoxin Endotoxin

Hepatocyte dysfunction

Endothelin-1 NOS CO

TXA2 TNF-α

HSC

Vasoconstriction Vasoconstriction Vasodilation

RBC and platelet from deposition aggregation Mitochondrial dysfunction and enzyme inhibition Microcirulatory failure Protects

Fig. 5. Regulation of hepatic microcirculation. Endotoxin activates the sinusoidal endothelial cells and the Kupffer cells present along the endothelial lining to start a cascade of events eventually leading to the generation of NO, ET-1, TNF-α, and TXA2.TNF-α and TXA2 act in a coordinated fashion to produce vasoconstriction, platelet aggregation, and an increase in the microvascular resistance. NO produces alteration in microcirculation by vasodilation and it can cause hepatic injury by mitochondrial dysfunction. ET-1, secreted by endothelial cells after exposure to endotoxin, is a potent vasoconstrictor and increases portal resistance. TXA2, Thromboxane A2;TNF-α, Tumor necrosis factor – alpha; NOS, Nitric Oxide Synthase; CO, Carbon Monoxide; HSC, Hepatic stellate cell. Hepatic Microcirculation 335 microcirculatory failure because endothelin receptor antagonists like bosentan and tezosentan have been found to limit liver injury in endotoxin-challenged cirrhotic rats (37). In a study using isolated perfused rabbit liver, Wang et al. have shown that ET-1-induced hepatic vasoconstriction occurs almost exclusively in the portal vein (38). Although there is no clear mechanism for such selective action of ET-1 on presinusoidal vessels or portal vein, it may be ascribed to the contractility of HSCs. Zhang et al. using a high-power intravital microscopy and portal pressure measurement techniques have clearly shown that the increase in portal venous pressure and the decrease in sinusoidal diameter are due to contractility of HSCs, which are widely present throughout sinusoids (39). The study by Pannen et al. showed that portal venous contractile response to ET-1 was significantly enhanced in the rat liver following pretreatment with endotoxin. The sinusoids constricted with a greater magnitude in response to ET-1, resulting in a significant decrease in the sinusoidal flow in LPS- stimulated animals compared with control animals (40). Although the exact mechanism of the hyperresponsiveness of the portal circulation to ET-1 is not clear, a number of studies explained the possible role of ET- 1-induced increased release of other vasoconstrictors like thromboxane A2, which are secreted by the hepatic Kupffer cells in response to LPS (40, 41). TXA2 was found to be significantly higher in mice with bile duct ligation compared to normal (sham) mice, potentiating the action of ET-1. Several studies have also suggested that sepsis-induced organ failure may be due to a higher concentration of NO in septic patients causing mitochondrial dysfunction, as cellular use of oxygen is dependent on mitochondrial phosphorylation reaction (42). The microorganism- associated molecular pattern (MMAP)-induced inflammatory process upregulates the expression of inducible nitric oxide synthase (iNOS), producing vasodilation mediated by cGMP and reducing global hepatic perfusion which results in tissue hypoxia (43, 44). Although NO is generated by both eNOS and iNOS in liver, NO derived from eNOS helps ensure the hepatic microvascular blood flow by vasodilation, platelet antiaggregating effects, and neutralization of superoxide anion (45, 46). The role of NO in hepatic microcirculation during endotoxic shock has always been a controversial issue. The NO production from macrophages, Kupffer cells, and endothelial cells increases during sepsis. Some experimental studies have shown that inhibition of NO in septic animals caused a deterioration of liver function, suggesting a protective role of NO toward hepatic microcirculation (47), whereas some have also reported that NO plays a role in causing liver injury after ischemia and reperfusion and plays a toxic role in endotoxic shock (48). 336 Routray and Shah

The toxic effects of NO are due to inhibition of NADH: ubiquinone oxidoreductase, cis-aconitase, glyceraldehyde-3-phosphate dehydrogenase, and ribonucleotide reductase (48). Furthermore, overproduction of NO is also reported to cause hypotensive tissue injury (49). Similarly, when endotoxin-treated rabbits were treated with NO inhibitors, a detrimental effect on hepatic circulation was noted which resulted in an increase in mortality most likely due to hepatic hypoperfusion whereas the NO donors L-arginine and linsidomine improved hepatic blood ﬂow (50).

3.10. Role of Rheological Behavior of Red Blood Cells Increased red blood cell (RBC) aggregation and reduced deformability of the spherical shape may contribute to impaired cellular oxygen supply, which may be due to upregulation of adhesion molecules and reduced content of sialic acid (SA) found on RBCs in septic patients. There are few studies explaining the cause of reduced SA in RBCs; reduced SA content in RBCs is most likely due to the increased secretion of sialidase enzyme by leukocytes in septic patients. A better understanding of the rheological behavior of RBCs in septic patients might help to reduce the microvascular collapse and organ failure in ICU patients (51).

3.11. Role of Normal Coagulation Cascade In a setting of sepsis, activated sinusoidal endothelial cells lose their normal ability to maintain an anticoagulant state and invite massive amounts of platelets and white blood cells to adhere to them, resulting in plugging of the sinusoid with ﬁbrin clots and red blood cells. Eventually, this results in a fall in the sinusoidal ﬂow velocity further reducing the perfusion. In the presence of a massive endotoxin load, the coagulopathy continues culminating in microvascular collapse and hepatocellular failure.

3.12. Role of Arteriovenous Shunts in Sepsis Another possible mechanism which may play a role in tissue perfusion defect is the arteriovenous shunting of blood in sepsis. The hepatic microvascular unit may shut down in response to the endotoxic load, promoting the shunting of oxygen transport from arterial to venous compartment. It has been found that intrahepatic anastomoses shunt blood away from the sinusoids in both cirrhotic patients and animal models with or without sepsis, resulting in tissue dysoxia. In cirrhotic patients with portal hypertension, shunts are usually found Hepatic Microcirculation 337 between portal vein and hepatic veins. AV shunting plays an important role in sepsis-induced multiorgan failure through the proposed mechanisms: (a) shunting through the anatomical anastomosis, where blood flows from arterioles to venules through shunts thereby bypassing the microcirculation and leaving the microvascular bed hypoxic, and (b) there is also a redistribution of intrahepatic circulation by channeling of blood away from contracted to dilated vessels, creating a net decrease in sinusoidal perfusion area (52). Sepsis-induced alteration in the microcirculatory architecture, formation of microemboli, and tissue edema make it difficult for the blood flow to reach the microcirculatory units. This results in functional shunting of flow toward healthy microcirculatory units from those that are compromised by endotoxemia (“vascular steal phenomenon”) and eventually produces irreversible loss of function (53).

4. EFFECT OF TREATMENT ON HEPATIC PERFUSION IN SEPSIS AND SHOCK The primary focus of treatment toward organ failure in severe sepsis and septic shock is to control infection and to maintain adequate oxygenation as well as tissue perfusion by using vasopressors and transfusion (21). Although the idea of using a vasopressor is to maintain adequate blood pressure, it can also maintain an adequate cardiac output especially while using inotropes like dobutamine. Use of vasopressors has been shown to produce less organ failure (54); however, its impact on hepatic failure has not been evaluated. In a study by Guerin et al., it was shown that although dopamine increases mesenteric blood flow, it also causes negative hepatic energy balance at high doses (55). Since excessive NO is found to be involved in sepsis-induced hepatic failure, inhibition of NOS becomes an interesting target for treatment options available to prevent or restore liver function in sepsis. However, nonselective NOS inhibition was found to have detrimental effects on hepatic perfusion and oxygen transport due to the fact that NO, in addition to being a vasodilator, also has antiplatelet properties. Thus, blockade of NOS resulted in vessel occlusion and a more severe reduction in sinusoidal blood flow (45, 56). The selective iNOS inhibition was found to show equivocal results. However, NO donor studies suggested that low-to-moderate levels of NO in experimental sepsis models may protect sinusoidal endothelial cells and increase the overall hepatic perfusion (57). Similarly ET-1 is also considered a good therapeutic target. However, a study in a porcine septic shock model showed 338 Routray and Shah that the endothelin receptor antagonist bosentan increased microcirculatory blood flow in all organs except liver, suggesting that blockade of ET-1 receptor could not dilate hepatic microcirculation effectively (37). Since reactive oxygen species are considered to cause hepatic dysfunction, N-acetylcysteine (NAC), an antioxidant, has also been studied in this regard. In early clinical sepsis, high doses of NAC were found to increase hepatic blood flow, decrease lactate levels, and improve liver function (58–60). Another study showed that pre- and posttreatment with antioxidants in sepsis models protected against liver damage and improved sinusoidal perfusion (61). Although selective iNOS inhibition and antioxidants are seen to improve sinusoidal perfusion and protect against liver damage, none was reproducible in clinical trials.

5. CONCLUSION Prevention of sepsis-induced multiorgan failure is crucial because current attempts to restore sepsis-induced microcirculation function are often inadequate. It demands more research, not only to understand the pathophysiological and biochemical factors involved in regulating the hepatic microcirculation, but also to identify those speciﬁc targets which are actually responsible for microcirculatory failure in septic patients, which can be intervened in a timely fashion to reduce mortality and morbidity.

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Angiogenesis and Vascular Growth in Liver Diseases

Manuel Morales-Ruiz, Sònia Tugues, and Wladimiro Jiménez

CONTENTS INTRODUCTION LONG-TERM STRUCTURAL CHANGES OF THE HEPATIC ANGIOARCHITECTURE IN CHRONIC LIVER DISEASES LONG-TERM STRUCTURAL CHANGES OF SPLANCHNIC AND SYSTEMIC ANGIOARCHITECTURE IN CHRONIC LIVER DISEASES CONCLUSIONS REFERENCES

Key Words: Angiogenesis, Fibrosis, Chronic liver disease, Endothelial cells, Vascular remodeling, Portal hypertension

1. INTRODUCTION Chronic liver disease (CLD) can be deﬁned as a complex pathophysiological process of the liver that involves a progressive destruction and regeneration of the liver parenchyma leading to ﬁbro- sis, cirrhosis, and an increasing risk of hepatocellular carcinoma (HCC). Viral hepatitis and alcohol are considered the two most important etiologies for CLD, although nonalcoholic fatty liver disease (NAFLD) is also increasingly being recognized as a common cause. A common

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_17, C Springer Science+Business Media, LLC 2011

343 344 Morales-Ruiz et al. complication of these patients is the presence of a hyperdynamic circulatory syndrome characterized by high cardiac output and low mean arterial pressure, all occurring in the setting of reduced systemic vascular resistance (1). Underlying these hemodynamic abnormalities there is a profound alteration in the hepatic parenchyma due to the formation of scarring areas that hinder the sinusoidal intrahepatic circulation. This phenomenon is responsible for a decreased hepatic perfusion and a rise in the intrahepatic resistance to portal blood flow. In this pathophysiological context, the hepatic and splanchnic vasculature reorganize their architecture to supply an adequate blood flow to tissues by two different mechanisms. The first mechanism involves modulation of vascular tone by the action of vasoactive substances and the second mechanism contemplates the induction of long-term structural changes (i.e., angiogenesis, development of portosystemic shunts, vascular remodeling). Although all these vascular modifications represent a rescue system against the increased intrahepatic resistance, the inability of this adaptative process to efficiently couple these hemodynamic abnormalities results in major complications, such as ascites and variceal bleeding (2). The process through which this pathology induces long-term vascular changes is still not completely understood; however, such information would be of potential benefit for CLD patients. Therefore, the aim of this chapter is to provide the general reader with an update of recent investigations in this pathophysiological aspect of CLD. A close association between HCC and vascular growth has also been reported to play a role in tumor cell proliferation. As a result, antiangiogenic treatment has emerged as a new approach to treat advanced HCC (3). Nonetheless, this has previously been reviewed (4) and is therefore beyond the scope of this chapter.

2. LONG-TERM STRUCTURAL CHANGES OF THE HEPATIC ANGIOARCHITECTURE IN CHRONIC LIVER DISEASES The liver receives about 15–25% of the cardiac output via two sources of blood supply, the hepatic artery that delivers arterial blood and provides 25–30% of the hepatic blood volume, and the portal vein that carries the circulation from the stomach and abdominal organs and represents two-thirds of the liver blood supply. This portal blood carries not only nutrients but also various contaminants (drugs, toxins, bacteria, by-products of blood cell recycling), which have been absorbed through the intestinal mucosa or produced in the spleen and Angiogenesis and Vascular Growth in Liver Diseases 345 must be efficiently metabolized by hepatocytes. Blood from both the portal vein and the hepatic artery is drained into the hepatic sinusoids and then passes out of the liver through the hepatic vein (5, 6). Thanks to the pioneering work done by Wisse et al. on the ultrastructure of liver sinusoidal endothelial cells (LSECs) (7, 8), we now know that the liver sinusoid is a specific capillary network system which differs from other capillaries in the body because of its discontinu- ity, the absence of basal membrane, and the presence of fenestration in the endothelial cell surface. The fenestrae are arranged in structures that are called sieve plates, which are approximately 0.1 μmin diameter and comprise 20–50 aggregated pores. These differential characteristics make LSECs a highly differentiated endothelial cell type that allows an efficient exchange of metabolites between blood and hepatocytes. In physiological conditions, hepatic resistance remains within normal values by the action of vasoactive molecules and through the morphological components characteristic of the LSECs, as mentioned above (9). However, in chronic liver diseases, several studies have described that hepatic vascular compliance decreases. In this context, LSECs seem to contribute to this phenomenon by undergoing molecular and structural changes such as an impaired endothelial nitric oxide (eNOS) activation (10–12) and a progressive loss of fenestrae, concomitant to the formation of a basal lamina recovering the sinusoidal endothelium (13, 14). These structural changes are called sinusoidal capillarization. Additionally, in an elegant study it has recently been shown that platelet-derived growth factor (PDGF) signaling through ephrin- 2 stimulates hepatic stellate cell (HSC) coverage of sinusoids in vivo (15). All these changes compromise the exchange of metabolites and oxygen with the parenchyma. Apart from these in-depth changes occurring in the sinusoidal angioarchitecture, it is currently well accepted that CLD is also associated with liver angiogenesis. For instance, liver neovascularization significantly increases during the progression of several chronic liver diseases such as hepatitis C, biliary cirrhosis, autoimmune hepatitis, or alcoholic cirrhosis. This neovasculature, mostly sprouting of arterial branches, is mainly located within the fibrotic areas and allows arterioportal and portosystemic anastomoses (16–22). In this context, one major question arises that merits consideration for designing future therapeutic strategies: What underlying cellular and molecular mechanisms drive these changes in the liver angioarchitecture? To answer this question we need to understand the tight link between inflammation and angiogenesis. Angiogenesis is of major importance under the circumstances of adult tissue repair and it is also a 346 Morales-Ruiz et al. hallmark of inflammatory processes where both phenomena are closely integrated (23). The link between inflammation and angiogenesis can be understood considering that many inflammatory mediators, which in chronic liver diseases are released by leukocytes and damaged hepatocytes (i.e., tumor necrosis factor-α, interleukin-1 (IL-1), IL-6, IL-8, prostaglandins), have direct angiogenic activities, and they may also indirectly stimulate other cells to produce potent angiogenic factors (24–27). Angiogenesis, in turn, contributes to the perpetuation and the amplification of the inflammatory state due to the expression of adhesion molecules and cytokines such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and IL-6 in the neovasculature which promote the recruitment of inflammatory cells (28, 29). The inflammatory response can also be accentuated by new vessels that constitute an entrance pathway for the continuous recruitment of inflammatory cells, as well as oxygen and nutrients, to hypoxic areas that have been developed in this chronic inflammatory context. Accordingly, several evidences have demonstrated that neovascularization is essential for the persistence of different pathological situations associated with inflammation such as psoriasis, rheumatoid arthritis, diabetes, Crohn’s disease, and cancer among others (30). One major molecular link between chronic inflammation and angiogenesis is hypoxia. In addition to a wide range of growth factors and cytokines, hypoxia is one of the major stimulators of vascular endothelial growth factor-A (VEGF-A) production through both gene transcription and mRNA stabilization, thereby providing compensatory mechanisms by which tissues can bypass an inadequate O2 supply (31). However, the activation of hypoxia-inducible factor-1 (HIF-1) is associated not only with the activation of angiogenic factors but also with the upregulation of proinflammatory genes such as cyclooxygenase-2 (COX2), NF-κB, and interferon regulatory factor-1 (IRF-1) (32, 33), further emphasiz- ing the close association between inflammation and angiogenesis. Local hypoxia is a well-known event in cirrhotic livers as is the close colocalization between hypoxia and the expression of angiogenic factors such as VEGF-A, placental growth factor (PlGF), and angiopoietin-2 (21, 22). These observations suggest that, proinflammatory cytokines may initially contribute to the modest and local upregulation of angiogenic factors in the liver but after extensive fibrosis, and as a result of hypoxic conditions, there is a significant overexpression of angiogenic factors in the whole cirrhotic organ that maintain chronic inflammation and the fibrogenic process. Leptin, which is also a factor regulated by hypoxia, is another common stimulus for both inflammation and angiogenesis that stimulates the increased production of monocyte chemotactic protein-1 (MCP-1), VEGF-A, and angiopoietin-1 and, additionally, Angiogenesis and Vascular Growth in Liver Diseases 347 activates NF-κb and HIF-1α in human HSCs (34). Thus, the exploration of these molecular targets is without any doubt a promising strategy to design new therapeutic approaches for chronic liver diseases. Some preclinical investigations have suggested that angiogenesis inhibitors (Table 1) may interfere with the progression of fibrosis. In fact, studies in experimental models of cirrhosis have shown that angiogenic inhibitors such as TNP-470, neutralizing monoclonal antibody anti-VEGF receptors (VEGFRs), and adenovirus expressing the extracellular domain of Tie2 effectively decrease liver fibrosis (35–37). Another interesting therapeutic candidate as an inhibitor of angiogenesis in cirrhosis is Sunitinib. This indolinone molecule was designed to have a broad selectivity for the split kinase family of receptor tyrosine kinases (RTKs), including KIT and FLT3 (38), and it has demonstrated efficacy as a potent antitumor and antiangiogenic agent in clinical trials. Its antiangiogenic efficacy is based on the fact that Sunitinib is also an effective inhibitor of VEGFRs and PDGF receptors (PDGFRs), both of which are essential for angiogenesis development (39). Apart from angiogenesis, the inhibition of PDGFR has an additional therapeutic interest considering that PDGF is one of the most potent mitogenic, profibrogenic, and chemotactic agents for HSCs (40). These cells over- express the receptor β subunit (PDGFR-β) when they are activated (41) and play a key role in the development and progression of hepatic fibrosis. In this context, cirrhotic rats treated with Sunitinib showed a significant decrease in hepatic vascular density, inflammatory infiltrate, and fibrosis (22). This beneficial effect of Sunitinib is likely due to the decrease in the number of hepatic vessels expressing VCAM-1 and ICAM-1. The importance of these findings lies in the fact that the nature of the inflammatory response is regulated by the induction of adhesion molecules in endothelial cells, which is necessary for the recruitment of inflammatory cells to sites of inflammation. Among these adhesion molecules, VCAM-1 and ICAM-1 appear to play a major role in the adhesion and transmigration of macrophages and lymphocytes across endothelial cells in a variety of acute and chronic inflammatory diseases (28, 29). Therefore, this study demonstrated that there is a significant synergistic effect between inflammation and angiogenesis in cirrhotic livers. In addition, Sunitinib treatment was also able to decrease portal pressure in cirrhotic rats (Fig. 1). Several properties of Sunitinib must be considered to explain this beneficial effect on portal hypertension. For instance, considerable evidence has demonstrated that HSCs are the primary source of extracellular matrix accumulation in livers, and PDGFR signaling is known to have a major role in this process. Therefore, it is likely that Sunitinib decreases α-smooth muscle 348 Morales-Ruiz et al. angiogenic factors activity angiogenic factors angiogenic factors proliferation migration cell growth cell growth angiogenic factors Interference with some Inhibition of cellular Inhibition of endothelial , and PlGF Interference with some 121 ,VEGF 165 165 cells cells Not yet knownTropomyosin in endothelial VEGF Inhibition of endothelial Table 1 Antiangiogenic drugs and their mechanism of action fragment of plasminogen of collagen XVIII Ig/2IgVEGFR1/3IgVEGFR2) Tyrosine kinase inhibitor PDGFR, c-kit, and abl1/2 Inhibition of kinase Monoclonal antibodyOligonucleotide VEGF VEGF Interference with some Fumagillin analogue Not known (cyclins A and D1) Inhibition of cellular Monoclonal antibodyFusion protein (Fc VEGF Interference with some (Regeneron (Novartis) (Genentech Inc.) (Eyetech Pharmaceuticals) Pharmaceuticals) (Genentech Inc.) Inc.) STI157/Imatinib Ranibizumab/Lucentis Pegaptanib/Macugen InhibitorTNP-470 (TAP Type Target Mechanism of action Thrombospondin-1Angiostatin Endogenous inhibitorEndostatinBevacizumab/Avastin CD36-expressing Endogenous endothelial inhibitor/internal VEGF Trap Endogenous inhibitor/fragment Angiogenesis and Vascular Growth in Liver Diseases 349 activity and inhibition of cell growth Inhibition of kinase activity Inhibition of kinase activity Inhibition of kinase activity FLT3 PDGFR c-kit Table 1 (Continued) Tyrosine kinase inhibitor VEGFR, PDGFR, KIT, and Tyrosine kinase inhibitorTyrosine kinase Raf, inhibitor VEGFR, and PDGFR VEGFR Inhibition of kinase activity Tyrosine kinase inhibitorTyrosine kinase VEGFR2 inhibitor VEGFR and mTOR Inhibition of kinase activity Inhibition of kinase Inhibition of kinase activity Tyrosine kinase inhibitor VEGFR1–3, PDGFR, and Tyrosine kinase inhibitorTyrosine kinase VEGFR2 inhibitor and EGFRTyrosine kinase EGFR inhibitor Inhibition EGFR of kinase activity Inhibition of kinase activity Inhibition of kinase activity (Pfizer Inc.) Tyrosine kinase inhibitor VEGFR2, FGFR, and (Pfizer Inc.) (Bayer Inc.) (AstraZeneca Inc.) (Pfizer Inf.) (Novartis) (Novartis) (AstraZeneca Inc.) (Genentech Inc.) (AstraZeneca Inc.) InhibitorSU11248/Sunitinib Type Target Mechanism of action BAY43-9006/Sorafenib AZD2171/Cediranib SU5416/Semaxinib SU6668 Everolimus/Afinitor PTK787/Vatalanib ZD6474/Vandetanib Erlotinib/Tarceva ZD1839/Gefitinib 350 Morales-Ruiz et al. functions integrin-mediated cell functions degradation degradation degradation degradation Inhibition of ECM 3 Inhibition of integrin β v v Inhibition of α α MMP14 Table 1 (Continued) Hydroxamic acid MMP2 and MMP9 Inhibition of ECM Hydroxamic acidDerived from hydroxamic acid MMP2, MMP16, MMP3, MMP8, MMP9, and and MMP12 Inhibition of ECM (Bayer Inc.) Butanoic acid analogue MMP2 and MMP9 Inhibition of ECM (British Biotech Inc.) Biotech Inc.) (Pfizer Inc.) Cilengitide (Merck) Cyclic RGD peptide Integrin InhibitorMarimastat (British Type Target Mechanism of action Batimastat BAY12-9566 AG3340/Prinomastat Vitaxin (MedImmune) Monoclonal antibody Integrin Angiogenesis and Vascular Growth in Liver Diseases 351

B C D 10 20 15 8 15 10 6 10 4 5 PP (mmHg) -SMA positive area -SMA positive 5

α 2

0 00 % of fibrotic positive area % of fibrotic positive % of

Vehicle Vehicle Vehicle SU11248 SU11248 SU11248

Fig. 1. Cirrhotic rats treated with Sunitinib showed a significant decrease in HSC activation, extracellular matrix accumulation, and portal pressure. In (A), liver sections from cirrhotic rats were immunostained for α-SMA. Abundant α-SMA staining (brown) indicates the presence of activated HSCs in cirrhosis (a). In Sunitinib-treated cirrhotic rats, the number of α-SMA positive cells was significantly reduced (b). Abundant collagen deposition (blue) indicates the presence of liver fibrosis (c). In Sunitinib-treated cirrhotic rats, collagen deposition was significantly reduced (d). In (B) and (C), the α-SMA and fibrotic positive areas were quantitatively measured. In (D), Sunitinib significantly decreased portal pressure (PP) in cirrhotic rats. In (B), (C), and (D), ∗ denotes P<0.01 compared with cirrhotic rats not treated with Sunitinib. Reprinted from Hepatology 2007;46:1919–26 with permission. actin (α-SMA) and extracellular matrix accumulation in cirrhotic livers through inhibition of the PDGF signaling pathway in HSCs. The increase in portal blood flow is another important component contributing to portal hypertension. Significant evidence has suggested that the increase in portal blood flow is not only due to splanchnic vasodilation but also due to an enlargement of the splanchnic vascular tree caused by angiogenesis (11, 42, 43). Therefore, it may be anticipated that the significant inhibition of angiogenesis promoted by Sunitinib 352 Morales-Ruiz et al. in this anatomical region would be translated into a decrease in portal blood flow in cirrhotic rats. This study demonstrated that multitarget-based therapies against angiogenesis, inflammation, and fibrosis might be beneficial in the treatment of cirrhosis. The validity of this approach has recently been confirmed by a similar study performed in cirrhotic rats in which the authors showed that Sorafenib, another multitargeted inhibitor of RTKs that targets Raf, PDGF, VEGF, and c-kit signaling pathways, produced a remarkable reduction in liver angiogenesis, hepatic fibrosis, and inflammatory infiltrate as well as a significant decrease in portal pressure (44).

3. LONG-TERM STRUCTURAL CHANGES OF SPLANCHNIC AND SYSTEMIC ANGIOARCHITECTURE IN CHRONIC LIVER DISEASES 3.1. Long-Term Structural Changes of Splanchnic Angioarchitecture in Chronic Liver Diseases Important angiogenic processes clearly take place in cirrhotic livers with or without superimposed hypervascular tumor such as hepatocellular carcinoma. However, the questions as to whether angiogenesis also occurs in the splanchnic area and what its contribution to the development of collateral circulation may be have been the subject of discussion during the last decades. The confirmation that in the condition of portal hypertension there is a much more dynamic rearrangement of the angioarchitecture than previously thought is supported by several observations showing that portal hypertensive rats do experience angiogenic processes. For instance, prominent persistent abnormalities in the microangioarchitecture of gastric mucosa of portal vein ligated rats have been described, which could explain the hypertrophic gastropathy observed in cirrhotic patients (45). Moreover, in vivo mesenteric angiogenesis assays have shown that portal hypertension is accompanied by a significant increase in NO-dependent angiogenesis (46, 47), in agreement with NO being recognized as an important in vivo and in vitro angiogenic molecule. Intravital microscopy studies have also revealed that an extensive neovascularization occurs in the rat hepatic arterial system following partial portal vein ligation (48). Furthermore, increased angiogenesis and permeability have recently been reported in peritoneal circulation of rats with portal hypertension and cirrhosis (49). Other indirect evidence supporting the hypothesis that angiogenesis may also occur in the splanchnic area of cirrhotic patients is based on the observation that ascites of cirrhotic patients behaves as a powerful Angiogenesis and Vascular Growth in Liver Diseases 353 inducer of angiogenesis. The activation of the PI3K/Akt signaling pathway by fibronectin seems to be a major contributor to this phenomenon (50). The development of portosystemic collateral vessels between the portal vasculature and the systemic venous system is one of the major causes of complications of portal hypertension. The origin of this collateral venous system has been a matter of discussion as has whether collateral formation is exclusively dependent on an increase in portal venous inflow. In this regard, early work by Halvorsen and Myking (51) proposed that collateral vessels arise from the passive dilation of preexisting venous channels in portal vein ligated rats. However, this conclusion has recently been balanced by studies demonstrating a more dynamic control of collateral growth by VEGF and PDGF. In this regard, the treatment of portal hypertensive rats with a neutralizing antibody to VEGF receptor 2, SU5416, and rapamycin (all blockers of the VEGF signaling pathway) as well as imatinib (which targets ABL, c-kit, and PDGF receptors) has proven to efficiently decrease the formation of portosystemic collateral vessels and attenuate the hyperdynamic splanchnic circulation (42, 52, 53). In concordance with these studies, another member of the VEGF family, the placental growth factor (PlGF), has recently been shown to be upregulated in the splanchnic microvasculature of portal hypertensive mice. Furthermore, PlGF deficiency in portal hypertensive mice has been associated with a significant decrease in splanchnic angiogenesis and with a reduction in portosystemic shunting as well as mesenteric artery flow (54). Therefore, the list of active molecular modulators of collateral vessels is actively growing and altogether sustains the concept of the existence of a dynamic collateral growth in the context of portal hypertension.

3.2. Vascular Remodeling in Conductance Vessels Reduced arterial pressure, high cardiac output, low peripheral resistance, endothelial dysfunction, and altered vascular reactivity are the characteristic features of advanced liver disease. In most cases this marked cardiovascular dysfunction develops over a long period of time, thus making vascular remodeling processes extremely likely in patients with decompensated cirrhosis. As endothelium-derived NO plays a central role in regulating the structure of the vessel wall (55), it may be hypothesized that this vasoactive molecule also contributes to vascular remodeling in CLD. This contention is further supported by numerous investigations showing increased NO-dependent vasorelaxation (56), higher production of endothelium-derived NO (57), and increased 354 Morales-Ruiz et al. vascular expression of eNOS mRNA and protein (58) in humans and rats with cirrhosis. Studies performed in rats with CCl4-induced cirrhosis and ascites have demonstrated that these animals undergo an intense process of vascular remodeling (59). The most remarkable features of this phenomenon are a decrease in the thickness and the total area of the vascular wall. In addition, NOS inhibition in cirrhotic rats resulted in a signiﬁcant improvement in the architectural distortions of arterial vessels, higher arterial pressure and peripheral resistance, and lower arterial compliance compared to cirrhotic rats receiving vehicle (Fig. 2).

Fig. 2. Vascular remodeling in cirrhotic rats. (a) Photomicrographs of representative cross sections of aorta from a control and a cirrhotic rat with ascites. Note the marked reduction in wall thickness and the diminution in the number of nuclei in the cirrhotic vessel (original magniﬁcation: ×200). (b)Wall thickness and total wall area in control rats and in cirrhotic rats with ascites. THORAC, thoracic aorta; ABDOM, abdominal aorta; MES, mesenteric artery; REN, renal artery. Bars represent mean±SE. Reprinted from Am J Pathol 2003;162:1985–93 with permission. Angiogenesis and Vascular Growth in Liver Diseases 355

These results raise the hypothesis that hyperdynamic circulation in cirrhosis causes a chronic increase in endothelium-derived NO that promotes important long-term architectural changes in blood vessels and that may further contribute to the already described unresponsiveness of endogenous vasoconstrictors in CLD patients.

4. CONCLUSIONS Despite all the relevant preclinical knowledge regarding the angiogenic pathways activated in chronic liver diseases, only clinical trials targeting angiogenesis in HCC have been made. Excluding HCC, the spectrum of angiogenic factors that should be inhibited to produce a therapeutic impact on liver diseases is currently under study. Since numerous evidences suggest that inﬂammation is linked with pathological angiogenesis in several diseases, a broad spectrum of multitargeted antiangiogenic drugs may have therapeutic potential in chronic liver diseases. Nevertheless, according to the literature this therapeutic strategy may be associated with adverse events (60). Therefore, the question is restricted not only to the selection of angiogenic inhibitors to be used in the context of portal hypertension or liver dysfunction but also to the selection of antiangiogenic compounds with reduced side effects. Recent preclinical studies have suggested that targeting PlGF activity may present such a proﬁle. However, further clinical studies are needed to validate the therapeutic potential and safety of these strategies. Regarding the importance of angiogenesis in many pathophysiological processes, interest in the use of angiogenesis inhibitors in liver disease remains high. Therefore the challenge is there and we look forward to scrutinizing the results obtained with these new therapeutic strategies.

ACKNOWLEDGEMENTS This work was supported by grants from Ministerio de Ciencia e Innovación (SAF 2003-02597, SAF 2006-07053, SAF 2007-63069, and SAF 2009-08839), Programa Ramón y Cajal, Fondo de Investigación Sanitaria (01/1514 and 04/1198), Fundació la Marató de TV3 (000610), and AGAUR (2009 SGR 1496). CIBERehd is funded by Instituto de Salud Carlos III del Ministerio de Ciencia e Innovación. REFERENCES

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Pulmonary Alterations in Chronic Liver Failure

Michael J. Krowka, MD and Aynur Okcay, MD

CONTENTS INTRODUCTION EFFECTS OF ASCITES,ENCEPHALOPATHY, AND SLEEP-DISORDERED BREATHING ARTERIAL OXYGENATION AND PULMONARY FUNCTION TESTING HEPATIC HYDROTHORAX PULMONARY VASCULOPATHIES – THE DILEMMA REFERENCES

Key Words: Sleep-disordered breathing, Hypocapnia, Atelectasis, Hyper- ventilation, Hypoxemia, Hepatopulmonary syndrome, Portopulmonary hypertension, Hepatic hydrothorax, Pulmonary function, Dyspnea, Oximetry, DLCO, Vasculopathy, Echocardiography, Arterial blood gas, Liver transplantation, Prostacyclin, Endothelin, Right heart catheterization

1. INTRODUCTION Chronic liver failure can have profound effects on the lungs. Hepatic dysfunction affects the process of breathing (when awake or during sleep), impairs lung mechanics, and may cause dramatic changes in pulmonary circulation. These pulmonary alterations commonly result in symptoms of fatigue (possibly related to sleep-related breathing

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_18, C Springer Science+Business Media, LLC 2011

361 362 Krowka and Okcay abnormalities) and dyspnea which can signiﬁcantly affect the quality of life in addition to direct consequences of chronic liver failure. Effects on the pulmonary circulation signiﬁcantly worsen survival and independently alter liver transplant consideration and outcomes. The pathophysiology of chronic liver failure is characterized by combinations of arterial hypoxemia (rest, exercise, and sleep), abnormal pulmonary function testing, and unique pulmonary vasculopathies, the last including the hepatopulmonary syndrome (vascular dilatation resulting in hypoxemia) and portopulmonary hypertension (vascular obstruction leading to right heart failure) (1). The purpose of this chapter is to concisely review the mechanism/diagnosis/management of the more common pulmonary alterations and syndromes experienced in caring for patients with chronic liver failure (Table 1).

Table 1 Common pulmonary alterations in chronic liver failure

Abnormal arterial blood gases Arterial hypoxemia Chronic respiratory alkalosis Abnormal pulmonary function testing Restrictive physiology (decreased lung volumes) Reduced DLCO Sleep-disordered breathing Nocturnal hypoxemia Obstructive sleep apnea Central sleep apnea Hepatic hydrothorax Pulmonary vasculopathies Hepatopulmonary syndrome (HPS) Portopulmonary hypertension (POPH) DLCO=single breath diffusing capacity for carbon monoxide – a measure of efﬁciency of gas exchange within the lung.

2. EFFECTS OF ASCITES, ENCEPHALOPATHY, AND SLEEP-DISORDERED BREATHING The pulmonary effects of massive ascites and varying degrees of encephalopathy seen in chronic liver disease on respiratory function are not trivial. Reductions (up to 20% from baseline) in total lung capacity (TLC) and forced vital capacity (FVC) have been documented Pulmonary Alterations in Chronic Liver Failure 363 consequences of massive or tense ascites as the hemidiaphragms are pushed caudally, contribute to basilar lung atelectasis, and have limited excursion with inspiration (2). Dyspnea results from increased rigidity of the diaphragms and thoracic and abdominal walls. These effects may be more pronounced when the patient assumes the supine position. Large-volume (2–8 l) or total paracentesis can immediately (within 2 h) improve such ventilatory function parameters and in some cases improve arterial oxygenation (by reducing the degree of lung atelectasis). Ascites along with massive hepatomegaly can have a dramatic effect on diaphragm position and function. The combination of massive ascites and obstructive sleep apnea is of clinical importance since treatment of the former may dramatically improve the latter (3). Encephalopathy, as a metabolic consequence of hepatic dysfunction, not only predisposes to pulmonary aspiration and pneumonitis especially in the elderly (age >65 years), but may also be associated with abnormal breathing and sleep patterns in all patients (4, 5). Hyperventilation characterized by hypocapnia (↓PaCO2) with respiratory alkalosis (↑pH) is a common arterial blood gas finding noted in liver disease, especially in cirrhotic patients with advanced hepatic encephalopathy. It has been suggested that elevated serum levels of ammonia, progesterone, and estradiol stimulate ventilation, a process facilitated by the altered blood–brain barrier present in cirrhotic patients. Moreover, progesterone receptors in the central nervous system may be increased by excessive estradiol (6). Abnormal circadian rhythms due to encephalopathy are well documented and such sleep alteration may contribute to fatigue. Sleep-disordered breathing (SDB; shorter total sleep time, long induction period, frequent nocturnal waking, and daytime sleepiness) in liver disease is historically well documented as an early sign of hepatic encephalopathy (5). The prevalence, severity, and clinical implications of associated nocturnal hypoxemia with sleep-related disordered breathing have gained interest in recent years. A mechanism of significant interest involving numerous vascular beds is that of endothelial dysfunction that can occur in association with SDB (7). The finding of frequent, abnormal overnight oximetry patterns in 52% of patients being considered for liver transplantation has been reported in a prospective Mayo Clinic study of 152 consecutive LT candidates (Fig. 1). Oxygen saturation drops greater than 4% were documented in 25% of patients using a standardized index that suggested significant sleep-disordered breathing patterns (8). A significant relationship with massive ascites or encephalopathy was noted. Additionally, there 364 Krowka and Okcay

1a 1c

100 175 100 175

90 150 90 150 88 88

80 125 80 125

70 100 70 100 Saturation (%) Saturation (%) Saturation 2 60 75 2 O O 60 75 Heart (beats/min) rate Heart (beats/min) rate

50 50 50 50 Heart Rate Heart Rate 40 25 20:00 21:00 22:00 23:00 0:00 1:00 2:00 3:00 4:00 5:00 40 25 21:00 22:00 23:00 0:00 1:00 2:00 3:00 4:00 5:00 Time (h:min) Time (h:min) 1b 1d 100 175 100 175

90 150 88 90 150 88

80 125 80 125

70 100 70 100 Saturation (%) Saturation (%) Saturation 2 60 2 60 75

O 75 O Heart (beats/min) rate Heart (beats/min) rate

50 50 50 50 Heart Rate Heart Rate 40 25 40 25 19:00 20:00 21:00 22:00 23:00 0:00 1:00 2:00 3:00 4:00 5:00 22:00 23:00 0:00 1:00 2:00 3:00 4:00 5:00 Time (h:min) Time (h:min)

Fig. 1. Overnight oximetry (OvOx) patterns measuring oxygen saturations and pulse rate as documented in chronic liver failure. Normal body mass index (BMI) <25. (a) Normal OvOx from a 27-year-old female, BMI=39.6, liver transplant candidate due to glycogen storage disease (Von Gierke’s disease). (b) Fifty-year-old male, BMI=28.8, liver transplant candidate due to autoimmune hepatitis. OvOx showed numerous desaturations reaching as low as 74% in spite of using continuous positive assisted pressure (CPAP) nasal mask. (c) Fifty-year-old male, BMI=27.8, liver transplant candidate due to hepatitis B. OvOx was consistent with sleep-related disordered breathing despite normal ESS=3 (Epworth Sleepiness Scale). (d) Forty-ﬁve-year-old female, BMI=25.3, liver transplant candidate due to alcoholic cirrhosis, complicated by moderate portopulmonary hypertension. Abnormal OvOx suggesting a need for nocturnal oxygen and further testing for sleep-related disordered breathing.

appeared to be no relationship with increased body mass index (BMI) or the Epworth Sleepiness Scale, suggesting the need to explore alternative hypotheses to explain this hepatic dysfunction–sleep relationship. Disease-speciﬁc sleep abnormalities and severe fatigue have been documented in primary biliary cirrhosis (9) and nonalcoholic fatty liver disease (10–13). The importance of sleep-related issues in such patients, especially in those who have nocturnal hypoxemia (with or without obstructive sleep apnea), relates to the potential reversibility of fatigue, worsening hepatic function (14), exacerbation of liver–lung vascular Pulmonary Alterations in Chronic Liver Failure 365 syndrome (see in later discussion), and prevention of cardiovascular events such as atrial ﬁbrillation and sudden death.

3. ARTERIAL OXYGENATION AND PULMONARY FUNCTION TESTING Hypoxemia is common in chronic liver failure, not only due to syndromes mentioned in this chapter, but also caused by the effects of active or previous smoking, chronic obstructive pulmonary diseases (such as alpha-1 antitrypsin-related emphysema), and pulmonary fibrosis that frequently occurs in such patients (1). Often unappreci- ated, hypoxemia often worsens with activity and sleep. Quantifying the degree of and identifying major reasons for reduced arterial oxygenation (hypoxemia) is important in terms of prognosis (in the case of hepatopulmonary syndrome – see below) and minimizing morbidity. Noninvasively, finger pulse oximetry is an excellent screening method to detect hypoxemia. Hemoglobin saturations less than 92% in the sitting position at rest should trigger further study (arterial blood gas, exercise saturations, and overnight oximetry). An arterial blood gas (ABG) obtained by radial artery puncture is the gold standard to characterize hypoxemia by measuring the partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2) (the latter accounting for the rate of breathing). Breathing room air, PaO2 less than 80 mmHg, or a calculated alveolar–arterial oxygen gradient of >20 mmHg (see Table 2)atany age should be considered abnormal in the setting of chronic liver failure. For the majority of clinical purposes, the measured PaO2 is sufficient to characterize hypoxemia. The most common pulmonary function test (PFT) abnormality in chronic liver failure is a reduced DLCO (single breath diffusing capacity for carbon monoxide) (15). DLCO measures the efficiency of oxygen–carbon dioxide gas exchange within the lungs. Decreased DLCO is a nonspecific finding, very common (∼50%) in liver transplant candidates, but also seen independently in pulmonary vascular disorders, interstitial lung disease, and chronic obstructive lung disease that may complicate liver disorders. A current explanation for decreased DLCO in liver disease relates to the reduced transit time of the pulmonary capillary blood volume due to the high cardiac output state that follows splanchnic vasodilatation (hence carbon monoxide diffusion into the capillary bed is impaired) (15). PFTs measuring lung volumes, expiratory airflows, and DLCO are advised in chronic liver failure patients who smoke, those with alpha-1 antitrypsin deficiency, and dyspneic patients with abnormal chest radiographs. 366 Krowka and Okcay

Table 2 Hepatopulmonary syndrome: diagnostic and prognostic criteria

Diagnostic 1. Portal hypertension (clinical diagnosis) 2. Hypoxemia via arterial blood gasa a. PaO2 <80 mmHg; or b. A-a oxygen gradient >20 mmHg 3. Pulmonary vascular dilatation - Positive, delayed contrast echocardiographyb - Brain uptake >6% after 99mTc MAA lung scanning Prognostic (worse survival) -PaO2 <60 mmHg - Brain uptake >20% - Association with Child C aArterial blood gas obtained in the sitting position, breathing room air. bMicrobubbles appear in the left atrium >3–4 cardiac cycles following right heart opaciﬁcation after peripheral vein injection of agitated saline. PaO2 = partial pressure of oxygen in arterial blood (measured);PAO2 = partial pressure of oxygen in the alveoli (calculated); PaCO2 = partial pressure of carbon dioxide in arterial blood (measured); A-a oxygen gradient = PAO2−PaO2,where PAO2 = 150−(PaCO2/0.8).

4. HEPATIC HYDROTHORAX Pleural effusion that develops from any pathophysiology causing ascites is referred to as hepatic hydrothorax (HH). Pleural effusion occurs in 5–10% of those with chronic liver failure, and the fluid is usually unilateral (right hemithorax 85%; left hemithorax 13%), transudative (total protein <2.9 mg/dl, serum to pleural fluid albumin gradient >1.1 g/dl, cholesterol <45 mg/dl), sometimes chylous (triglyceride >110 g/dl), and uncommonly a manifestation of metastatic hepatocellular carcinoma (positive cytology) (16). The pleural fluid accumulates due to the passage of ascitic fluid from the peritoneum through small (<1 cm) diaphragmatic fenestrations. Negative inspiratory pleural pressure facilitates the peritoneal–pleural fluid flow even in the setting of clinically absent abdominal ascites. Injection of 99mTc sulfur colloid into the peritoneal cavity with uptake in the pleural space within 4 h confirms the origin of the pleural fluid in uncertain cases. Diagnostic thoracentesis should be performed when suspected HH is associated with fever or pleuritic pain, or in the setting of malignancy. Therapeutic thoracentesis is usually beneficial for symptomatic HH that occupies more than 50% of the hemithorax; no more than 2 l should be removed at any one time due to the risk of painful mediastinal shifts Pulmonary Alterations in Chronic Liver Failure 367 or re-expansion pulmonary edema. Repeat thoracenteses may be necessary for symptomatic relief, but chest tube insertion is not advised. Such intervention is frequently associated with complications (renal dysfunction, electrolyte imbalances, and infection) and in the largest single institution study to date, failure to remove a chest tube increased mortality in patients with increasingly severe liver disease. Thoracoscopic pleurodesis (using abrasion or talc slurry) or repair of diaphragmatic fenestrations can be successful in controlling the accumulation of HH in highly selected patients. Transjugular intrahepatic portosystemic shunts (TIPS) have demonstrated effective temporary relief of HH (unresponsive to standard management of ascites – salt restriction and diuretics) in up to 80% of patients (Fig. 2a, b). Mortality within the first 2 months of TIPS has occurred in up to 25% of patients. Hepatic encephalopathy, shunt occlusion, and acute increases in right ventricular preload may be significant limitations in selected patients. As a temporary intervention for refractory HH, TIPS should be considered a bridge to the definitive treatment – liver transplantation. Transplant outcomes for refractory HH are reportedly no different from non-HH outcome (17). A significant complication of HH is that of pleural fluid infection referred to as spontaneous bacterial empyema (SBE). SBE is quite

Fig. 2. Moderate right-sided hepatic hydrothorax. (a) Pretransjugular intrahepatic portosystemic shunt (TIPS) in a patient with refractory abdominal ascites and transudative pleural effusion. (b) Two weeks post-TIPS (white arrow) demonstrating near-total resolution of the pleural effusion. 368 Krowka and Okcay uncommon, and can occur without either clinical ascites (∼25%) or spontaneous bacterial peritonitis (∼ 43%) (16). It has been successfully managed effectively without the use of chest tubes since most of the fluid is free-flowing in 24 SBE episodes (16 patients) reported from Spain. Not an empyema in the true sense of the word (no pus and pH is usually above 7.2), bacteria have been cultured from the pleural fluid, such as Enterobacter, Escherichia coli, Streptococcus, Klebsiella, and Pseudomonas. Mortality associated with SBE treatment was reported to be 20% (18).

5. PULMONARY VASCULOPATHIES – THE DILEMMA In genetically susceptible individuals, it is hypothesized that portal hypertension creates a circulatory and metabolic environment (dysfunctional hepatic clearance causing excess of potential vasoactive factors or the absence of stabilizing hepatic factors) that adversely affects the pulmonary arterial/capillary bed (1). Increased levels of local or circulating mediators such as nitric oxide, vascular endothelial growth factor (VEGF), and endothelin-1 have been considered as key factors in altering the pulmonary vascular bed in patients with any cause of portal hypertension. The current clinical dilemma from a pathophysiological perspective is why some individuals with portal hypertension go on to develop hepatopulmonary syndrome, yet others develop portopulmonary hypertension – two clinically and pathologically distinct pulmonary vasculopathies requiring different treatment approaches and having dissimilar prognoses (19).

5.1. Hepatopulmonary Syndrome (HPS) The triad of portal hypertension, pulmonary vascular dilatation, and arterial hypoxemia characterizes HPS (1, 20). HPS affects both children and adults, and approximately 10–20% of all liver transplant candidates fulfill generally accepted diagnostic criteria. Dyspnea, finger clubbing, and spider angiomas of the skin should alert the clinician to HPS, but these findings are not universal. The key pathophysiological concept is that arterial hypoxemia develops due to the altered ventilation–perfusion relationship at the pulmonary alveolar-capillary level. It is hypothesized that three vascular processes occur: true dilatation of existing capillaries (an upregulation of endothelin B vasodilating receptors and increased nitric oxide-mediated vasodilatation), proliferation of dilated “new” capillaries (angiogenesis), and the creation or opening of dormant arterial–venous pathways that bypasss the alveolar gas exchange unit (20–22). The result is that during normal ventilation, Pulmonary Alterations in Chronic Liver Failure 369 oxygen molecules do not fully reach the deoxygenated venous blood that traverses the pulmonary vascular bed. A rapid transit time due to the hyperdynamic circulatory state caused by chronic liver failure exacerbates this abnormal diffusion of oxygen into the blood. The degree of the arterial hypoxemia breathing room air may be severe (PaO2 <50 mmHg) and worsen in the upright position (orthodeoxia), with exercise and during sleep (20, 23). Response to supplemental oxygen (nasal cannula) is usually favorable and may improve the symptoms of dyspnea; however, some individuals may have a very limited response to 100% inspired O2. Such individuals have either discrete anatomic arteriovenous communications or severe diffuse dilatation of the pulmonary vascular bed, or may have concomitant pulmonary nonvascular processes (pulmonary fibrosis, sarcoidosis, chronic obstructive pulmonary disorders) that occur in up to 30% of HPS patients (20). The existence of pulmonary vascular dilatations is best detected noninvasively by saline, contrast-enhanced echocardiography, 99mtechnitium macroaggregated lung perfusion scanning with brain imaging, or pulmonary angiography. Echocardiography and lung scanning are used with the same diagnostic principle: following peripheral vein administration, there is the passage of either agitated saline-induced microbubbles (>15 μm in diameter) or radiolabeled albumin particles (>20 μm in diameter) through abnormally dilated capillaries (normal size 8 μm diameter) or arteriovenous communications greater than 3 mm in size. Following such abnormal passage, transthoracic or transesophageal echocardiography can image microbubble opacification in the left heart or brain uptake of 99mTc can be detected (see Fig. 3). Pulmonary angiography is usually conducted only if hypoxemia is severe (PaO2 <50 mmHg) and poorly responsive to supplemental oxygen (PaO2 <300 breathing 100% inspired oxygen), to look for discrete arteriovenous communications that might be amenable to coil embolotherapy. The pulmonary hemodynamics in HPS reflects the hyperdynamic circulatory state of chronic liver failure with high cardiac output and normal or reduced vascular resistance to pulmonary arterial flow (20). Screening for HPS should be done in all liver transplant candidates and can be accomplished by finger pulse oximetry (<92% considered abnormal) or arterial blood gases; the latter remains the gold standard since pulse oximetry saturations can vary considerably with breathing and pulse patterns. Patients with PaO2 <50 mmHg should be considered to have HPS until proven otherwise. Those with hypoxemia should then undergo either contrast echocardiography or lung perfusion scanning to determine the existence of pulmonary vascular dilatation. Diagnostic criteria of HPS are shown in Table 2. 370 Krowka and Okcay

Fig. 3. Pulmonary vascular dilatation demonstrated with (a) transthoracic contrast-enhanced echocardiography using agitated saline showing delayed (after the appearance of 3–4 cardiac cycles in the right heart) microbubble opaciﬁcation in the left heart and (b) 99mTc macroaggregated albumin lung– brain scanning showing abnormal uptake over the brain (>6%), quantiﬁed by using geometric means of right and left side brain images and estimating a 13% cardiac output to the brain.

An array of medical treatments to improve arterial oxygenation (aside from supplemental oxygen) has failed to result in consistent or long-term improvement of hypoxemia. Although increased exhaled nitric oxide has been demonstrated in HPS patients, the use of L-NAME (nitro-L-arginine methyl ester) to block the effect of NO has not been shown to improve the hypoxemia of HPS (24). Rarely, HPS may spontaneously resolve in patients with alcoholic cirrhosis with complete alcohol abstinence. The use of TIPS has been controversial and is not recommended as treatment for HPS. The prognosis of HPS is poor, especially in Child C severity of liver disease (25, 26). Cadaveric or living donor liver transplantation remains the treatment of choice for HPS. HPS can totally resolve with successful LT and no intraoperative deaths due to severe hypoxemia have been reported to date (19, 20). Prognosis is shown in Fig. 4a. Higher priority for LT has been granted in the United States when PaO2 <60 mmHg due to HPS as a means to prevent post-LT mortality, since the severity of pre-LT hypoxemia (PaO2 <50 mmHg) has been related to transplant hospitalization mortality (27). The post-LT survival is signiﬁcantly better compared to those who are not transplanted. The time to resolution of HPS after LT is related to the severity of hypoxemia prior to LT and is measured in months (28). HPS patients are at higher risk for LT, Pulmonary Alterations in Chronic Liver Failure 371

4b n = 75 POPH MPAP >25 mm Hg (49; 27–86) –5 1.0 PVR >240 dynes.s.cm (521; 241–1285) TPG >12 mm Hg (37; 14–77)

0.8

LT + PAH Rx (n = 9)

0.6 5-year survival 0.4 (P<0.02) Surviving 45% vs 14%

0.2 LT alone (n = 4) No treatment PAH Rx alone (n = 43) (n = 19) 0.0 050100150200

Survival from Dx (mo)

Fig. 4. Kaplan–Meier survival curves from the Mayo Clinic Pulmonary Vascular Complications of Liver Disease database. (a) Hepatopulmonary syndrome patients (n = 61) stratiﬁed by initial PaO2 and transplant outcomes. Overall 5-year survival with LT was 70% and that without LT 20%. (b) Portopulmonary hypertension patients (n = 75) stratiﬁed by treatment. Five-year survival was 45% with medical treatment alone (continuous 24/7 prostacyclin infusion) versus 14% without treatment. LT, liver transplantation; MPAP, mean pulmonary artery pressure; PVR, pulmonary vascular resistance; TPG, transpulmonary pressure gradient; PAHRx, pulmonary arterial hypertension treatment. 372 Krowka and Okcay

99m especially if PaO2 <50 mmHg or Tc MAA scans demonstrate >20% uptake over the brain (20). It is not uncommon for hypoxemia to worsen in the immediately post-LT (24–72 h) due to volume shifts and atelectasis. Paradoxically, the temporary use of inhaled nitric oxide to improve ventilation–perfusion matching has been beneﬁcial in such instances to improve hypoxemia (20).

5.2. Portopulmonary Hypertension (POPH) In contrast to HPS, a syndrome characterized by degrees of arterial hypoxemia, POPH is a hemodynamic problem with minimal oxygenation abnormality that causes varying degrees of pulmonary artery hypertension and subsequent right heart failure if not treated. Early manifestations are nonspecific including exertional dyspnea, but as the pulmonary hypertension worsens, syncope and chest pain may occur. POPH is uncommon, occurring in 3.5–8.5% of all liver transplant candidates with greater frequency in females and those with autoimmune-associated liver disease (29, 30).Thereappearstobeno relationship with the severity of portal hypertension, Child–Turcotte– Pugh, or the model for end-stage liver disease scores, but genetic risk factors may exist (31,32). The diagnosis must be established by right heart catheterization, since other causes of pulmonary hypertension may exist that are related to excess volume and simply the high flow state of chronic liver failure. Screening chronic liver failure patients for any cause of pulmonary hypertension is best accomplished by transthoracic echocardiography looking specifically at right ventricle systolic pressure estimates and the size/function of the right ventricle. However, even in the setting of an abnormal screening echocardiogram, approximately 35% of patients will not have true POPH, but rather have high flow or increased left atrial volumes (excess volume, diastolic or systolic heart failure) that are not necessarily associated with increased pulmonary vascular resistance to arterial flow (33). Diagnostic criteria of POPH are given in Table 3. The pathophysiology of POPH is related to the lack of prostacyclin synthase in the pulmonary endothelial cells, increased circulating endothelin-1, and the possible role of concomitant sleep-disordered breathing (29). Endothelial dysfunction, smooth muscle proliferation, accumulation of platelet aggregates within pulmonary capillaries, in situ thrombosis, and vasoconstriction result in increased resistance to pulmonary arterial flow. These changes occur in the setting of the high-flow circulatory state, so the finding of increased cardiac output is distinctive in the earlier stages. As the vasculopathy progresses, the Pulmonary Alterations in Chronic Liver Failure 373

Table 3 Portopulmonary hypertension: diagnostic and prognostic criteria

Diagnostic 1.Portal hypertension (clinical diagnosis) 2.Via right heart catheterization a.MPAP >25 mmHg b.PVR >240dyn s /cm5 or >3 Wood units c.TPG >12 mmHg Prognostic (worse survival) - Cardiac output <4 l/min - MPAP >35 mmHg (worse transplant outcome)

PVR (dyn s/cm5) = (MPAP−PAOP) × 80 CO TPG (mmHg) = (MPAP–PAOP) where MPAP = mean pulmonary artery pressure; PVR = pulmonary vascular resistance; TPG = transpulmonary pressure gradient; CO= cardiac output; PAOP = pulmonary artery occlusion pressure (same as pulmonary capillary wedge pressure). For PVR in Wood units, do not multiply the numerator by 80. resistance to flow increases, mean pulmonary artery pressures increase up to a point when the right ventricle begins to fail, and cardiac output begins to decline. It is of particular importance to recognize that a markedly increased MPAP and PVR with a “normal” cardiac output may portend a poor prognosis, especially if the right ventricle is dilated or enlarged by echocardiography (29). The prognosis of POPH without any pulmonary vasomodulating therapy is dismal with a 14% versus 45% 5-year survival for those treated; about half of the deaths are directly related to right heart failure (34). Patients with lower cardiac outputs appear to have poorer prognosis (31). POPH is considered within the group 1 classification of pulmonary hypertension, an important observation since such patients are appropriate candidates for a variety of pulmonary vasomodulating therapies that fall within three broad classes: prostacyclin replacement, endothelin receptor antagonist, and phosphodiesterase inhibition (35). In addition to improving pulmonary hemodynamics, the use of such medications may facilitate liver transplantation in selected patients who otherwise might be denied because to the severity of their pulmonary hypertension (36–42). Encouraging experience (decreased MPAP and reduced PVR) with the selective endothelin receptor antagonist ambisentan awaits further confirmation with an upcoming, multicenter, placebo-controlled trial in POPH (43). All of the experiences regarding pulmonary vasomodulating medications to 374 Krowka and Okcay facilitate LT have been small series or case reports – no controlled trials have been attempted to date. Unlike HPS with complete resolution of the syndrome following successful LT, the outcome of POPH post-LT is less optimistic and problematic. Without pre-LT pulmonary vasomodulating therapy, transplant hospitalization mortality was 35% in 36 patients reported from a multicenter LT database (19)(Fig.4b). Despite aggressive medical treatments with the newer agents, progression of pulmonary hypertension post-LT has been reported. Yet, more than 50% of POPH patients were unable to completely eliminate pulmonary vasomodulating drugs after LT, therefore realizing complete resolution of the syndrome. Although specific pre-LT prognostic factors have not been clearly identified in retrospective studies, MPAP >35 mmHg and lower cardiac outputs have been related to mortality (19, 29). MPAP >50 mmHg remains an absolute contraindication to LT at many centers. If significant pulmonary hemodynamic improvement with vasomodulating therapy can be accomplished, higher priority for LT has been espoused in an attempt to definitively treat portal hypertension and prevent further evolution of POPH (44). In highly selected patients unresponsive to POPH medical treatments, combined liver–lung transplantation has been accomplished (45).

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10. Newton JL, Gibson J, Tomlinnson M, et al. Fatigue in primary biliary cirrhosis is associated with excessive daytime somnolence. Hepatology 2006; 44: 91–8. 11. Singh H, Pollock R, Uhanova J, Kryger M, Hawkins K, Minuk GY. Symptoms of obstructive sleep apnea in patients with nonalcoholic fatty liver disease. Dig Dis Sci 2005; 50: 2338–43. 12. Newton JL, Jones DE, Henderson E, Kane L, Wilton K, Burt AD, et al. Fatigue in non-alcoholic fatty liver disease (NAFLD) is signiﬁcant and associates with inactivity and excessive daytime sleepiness but not with liver disease severity or insulin resistance. Gut 2008; 57: 807–13. 13. Kallwitz ER, Herdegen J, Madura J, et al. Liver enzymes and histology in obese patients with obstructive sleep apnea. J Clin Gastroenterology 2007; 41: 918–21. 14. Tanne F, Gagnadoux F, Chazouilleres O, et al. Chronic liver injury during obstructive sleep apnea. Hepatology 2005; 41: 1290–5. 15. Degano B, Mittaine M, Guenard H, et al. Nitric oxide and carbon monoxide lung transfer in patients with advanced liver cirrhosis. J Appl Phys 2009; 107: 139–43. 16. Kinasewitz GT, Kaddissi JI. Hepatic hydrothorax. Curr Opin Pulm Med 2003; 9: 261–5. 17. Xiol X, Tremosa G, Castellote J, et al. Liver transplantation in patients with hepatic hydrothorax. Transpl Int 2005; 18: 672–5. 18. Liu LU, Haddadin HA, Bodian CA, et al. Outcome analysis of cirrhotic patients undergoing chest tube placement. Chest 2004; 126: 142–8. 19. Krowka MJ, Mandell MS, Ramsay MAE, et al. Hepatopulmonary syndrome and portopulmonary hypertension: a report of the multicenter liver transplant database. Liver Transpl 2004; 10: 174–82. 20. Rodriguez-Roisin R, Krowka MJ. Hepatopulmonary syndrome: a liver-induced lung vascular disorder. N Engl J Med 2008; 358: 2378–87. 21. Zhang J, Luo B, Tang L, et al. Pulmonary angiogenesis in a rat model of hepatopulmonary syndrome. Gastroenterology 2009; 136: 1070–80. 22. Luo B, Lui L, Tang L, et al. Increase pulmonary vascular endothelin B receptor expression and responsiveness to endothelin-1 in cirrhotic and portal hypertensive rats: a potential mechanism for experimental hepatopulmonary syndrome. J Hepatol 2003; 38: 556–63. 23. Palma DT, Philips GM, Arguedas MR, et al. Oxygen desaturation during sleep in hepatopulmonary syndrome. Hepatology 2008; 47: 1257–63. 24. Gomez FP, Barbera JA, Roca J, et al. Effects of nebulized N(G)arginine methyl ester in patients with hepatopulmonary syndrome. Hepatology 2006; 43: 1084–91. 25. Schenk P, Schoniger-Hekele M, Fuhrman V, et al. Prognostic signiﬁcance of the hepatopulmonary syndrome in patients with cirrhosis. Gastroenterology 2003; 125: 1042–52. 26. Swanson KL, Wiesner RH, Krowka MJ. Natural history of hepatopulmonary syndrome: impact of liver transplantation. Hepatology 2005; 1122–9. 27. Fallon MB, Mulligan DC, Gish RG, Krowka MJ. MELD exception for hepatopulmonary syndrome. Liver Transpl 2006; 12 (Suppl): S105–7. 28. Taille C, Cadranel J, Bellocq A, et al. Liver transplantation for hepatopulmonary syndrome: a ten-year experience in Paris, France. Transplantation 2003; 75: 1482–9. 29. Porres-Aguilar M, Zuckerman MJ, Figuerosa-Casas JB, Krowka MJ. Portopulmonary hypertension: state of the art. Ann Hepatol 2008; 7: 321–30. 30. Le Pavec J, Souza R, Herve P, et al. Portopulmonary hypertension: survival and prognostic factors. Am J Respir Crit Care Med 2008; 178; 637–43. 376 Krowka and Okcay

31. Kawut SM, Krowka MJ, Trotter F, et al. Clinical risk factors for portopulmonary hypertension. Hepatology 2008; 48: 196–203. 32. Roberts KE, Fallon MB, Krowka MJ, et al. Genetic risk factors for portopulmonary hypertension in advanced liver disease. Am J Respir Crit Care Med 2009; 179: 835–42. 33. Krowka MJ, Swanson KL, Frantz RP, et al. Portopulmonary hypertension: results from a 10-year screening algorithm. Hepatology 2006; 44: 1502–10. 34. Swanson KL, Krowka MJ, Wiesner RH, et al. Survival in portopulmonary hypertension: Mayo Clinic experience categorized by treatment groups. Am J Transpl 2008; 8: 2445–53. 35. Simonneau G, Robbins IM, Beghetti M, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Card 2009; 54 (Suppl): S44–55. 36. Hoeper MM, Seyfarth HJ, Hoeffken G, et al. Experience with inhaled iloprost and bosentan in portopulmonary hypertension. Eur Respir J 2007; 30: 1096–102. 37. Sussman N, Kaza V, Barshes N, et al. Successful liver transplantation following medical management of portopulmonary hypertension: a single center series. Am J Transpl 2006; 6: 2177–82. 38. Fix OK, Bass NM, DeMarco T, Merriman RB. Long-term follow-up of portopulmonary hypertension: effect of treatment with epoprostenol. Liver Transpl 2007; 13: 875–85. 39. Ashfaq M, Chinnakoatla S, Rogers L, et al. The treatment impact of portopulmonary hypertension on survival after liver transplantation. Am J Transpl 2007; 7: 1258–64. 40. Hemmes AR, Robbins IM. Sildenafil monotherapy in portopulmonary hypertension can facilitate liver transplantation. Liver Transpl 2009; 15: 15–9. 41. Gough MS, White RJ. Sildenafil is associated with improved hemodynamics in liver transplantation candidates with pulmonary hypertension. Liver Transpl 2009; 15: 30–8. 42. Sakai T, Planinsic RM, Matier M, et al. Initial experience using continuous intravenous treprostinil to manage pulmonary artery hypertension in end-stage liver disease. Transpl Int 2009; 22: 554–61. 43. Cartin-Ceba R, Wiesner RH, Krowka MJ. Safety and efficacy of ambrisentan for the therapy of portopulmonary hypertension. Hepatology 2009; 50 (Suppl): A95 (abstract). 44. Krowka MJ, Fallon MB, Gish Mulligan, at al. MELD exception for portopulmonary hypertension. Liver Transpl 2006; 12 (Suppl): S114–6. 45. Grannas G, Neipp M, Hoeper M, et al. Indications for and outcomes after combined lung and liver transplantation: a single-center experience on 13 consecutive cases. Transplantation 2008; 85: 524–31. Adrenal Function in Chronic Liver Failure

Javier Fernández and Juan Acevedo

CONTENTS HYPOTHALAMIC-PITUITARY-ADRENAL AXIS IN CRITICAL ILLNESS EFFECTS OF CORTISOL DURING CRITICAL ILLNESS ACHALLENGING AND CONTROVERSIAL ISSUE:CLINICAL DIAGNOSIS OF RELATIVE ADRENAL INSUFFICIENCY ADRENAL INSUFFICIENCY IN CRITICAL ILLNESS IN THE GENERAL POPULATION ADRENAL INSUFFICIENCY IN SEVERE SEPSIS OR SEPTIC SHOCK IN CIRRHOSIS ADRENAL FUNCTION IN OTHER POPULATIONS OF PATIENTS WITH CIRRHOSIS POSSIBLE MECHANISMS OF RELATIVE ADRENAL INSUFFICIENCY IN CIRRHOSIS OTHER UNSOLVED QUESTIONS REFERENCES

Key Words: Relative adrenal insufﬁciency, RAI, Adrenal function, Adrenal dysfunction, Hypothalamic-pituitary-adrenal axis, Critical illness, Corticotropin test, Cortisol, Hydrocortisone, Cholesterol, Septic shock, Shock reversal, Survival

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_19, C Springer Science+Business Media, LLC 2011

377 378 Fernández and Acevedo

Adequate adrenal function is fundamental to survive critical illness. Cortisol is vital in the host adaptation to stress. It is essential to maintain the normal vascular tone, endothelial integrity, vascular permeability, and the distribution of total body water within the vascular compartment (1–5). Consequently, the failure of an appropriate adrenal response in the setting of critical illness, an abnormality known as relative or functional adrenal insufﬁciency (RAI), may have important clinical consequences. Such patients still secrete cortisol and corticotropin in the early phases of critical illness, but less than expected during acute stress. Several studies performed in the general population showed that RAI contributes to vascular hyporesponsiveness in septic shock and increases mortality (6–14). The effect of steroid supplementation on the clinical outcome of these patients is controversial. Initial papers suggested that this therapy improved shock reversal and hospital mortality. New data, however, raise doubts about the usefulness of this treatment. Data on the clinical relevance of adrenal dysfunction in critically ill cirrhotic patients are scarce but suggest that RAI is very frequent in patients with septic shock and is associated with disease severity and poor prognosis. The impact of this entity on other clinical decompensations of cirrhosis is currently unknown. This chapter will focus on adrenal dysfunction in critically ill cirrhotic patients.

1. HYPOTHALAMIC-PITUITARY-ADRENAL AXIS IN CRITICAL ILLNESS In the early phase of critical illness most patients with severe sepsis, trauma, major surgery, or burns display high plasma cortisol levels, reﬂecting the host adaptation of the hypothalamic-pituitary-adrenal axis to stress (see Fig. 1)(1–5, 8). The activation of the system is initiated by the action of cytokines and other factors on the hypotha- lamus promoting the release of corticotropin-releasing hormone (CRH) and vasopressin (ADH) (1, 2). These hormones stimulate corticotropin secretion (ACTH) by the pituitary gland, which in turn increases cortisol secretion by the adrenal glands (see Fig. 2). In addition, levels of cortisol-binding protein decrease rapidly and substantially (1, 2, 8), in part as a result of elastase-induced cleavage, resulting in proportionally much higher increase in the circulating plasma levels of free cortisol, which is the active component of the system (15). The half-life of cortisol in the blood is also increased during stress owing to a decreased rate of hepatic extraction and renal enzymatic inactivation of cortisol to cortisone by 11β-hydroxysteroid dehydrogenase (1). Moreover, during acute illness the negative feedback of cortisol on the release of Adrenal Function in Chronic Liver Failure 379

50 Severe sepsis g/dL)

μ 40 (n = 15)

30 Controls (n = 15)

Serum cortisol ( 10

0 0 12345678Days

Fig. 1. Serum cortisol levels during acute illness. Patients with severe sepsis and normal adrenal function presented signiﬁcantly higher serum cortisol concentrations than normal subjects at admission and for more than 1 week.

Stress

Cytokines, bacterial products CRH + ADH +

ACTH

Cytokines Cortisol

Immunomodulatory and Metabolic Cardiovascular anti-inflamatory effects effects system

Fig. 2. Hypothalamic-pituitary-adrenal axis in critical illness. CRH, corticotropin-releasing hormone; ADH, antidiuretic hormone or vasopressin; ACTH, corticotropin.

CRH and ACTH is depressed (1, 3, 8, 16), and this is important to keep a sustained activation of the hypothalamic-pituitary-adrenal axis. Finally, there is an increase in the number and sensitivity of glucocorticoid receptors (1, 2). Therefore, during critical illness an integrated 380 Fernández and Acevedo multilevel response exists to optimize the effect of cortisol in the peripheral tissues. Cytokines (e.g., tumor necrosis factor, interleukins, and macrophage migration inhibitory factor) and bacterial products can modulate the response of the hypothalamic-pituitary-adrenal axis at each level (1) (see Fig. 2). The acute phase of critical illness typically lasts for a few hours or days. In the prolonged phase of the disease (weeks to months), there is a dissociation between high plasma cortisol levels and low ACTH levels, suggesting non-ACTH-mediated mechanism for regulation of the adrenal cortex (17). Cytokines and other circulating factors may suppress ACTH synthesis and secretion. Cortisol levels slowly decrease and reach normal levels only in the recovery phase of the disease. Cortisol-binding protein levels recover in the chronic phase of illness. Metabolically, there is a suppression or delay of the anabolic processes, resulting in typical features of prolonged critical care such as cachexia, including breakdown of muscle tissue, loss of lean body mass, polyneuropathy, generalized tissue wasting, and dystrophy (17). Theoretically, the persistent elevation of cortisol levels is beneﬁcial in prolonged critical illness but may also increase the susceptibility of these patients to infectious complications. Alternatively, the risk for the development of RAI may increase in the chronic phase of the disease and may predispose to an adverse outcome.

2. EFFECTS OF CORTISOL DURING CRITICAL ILLNESS Cortisol is essential for the adaptation and maintenance of stress homeostasis during critical illness. It is a pluripotent hormone that acts on all tissues to regulate numerous aspects of metabolism, growth, and physiological functioning, being in this way essential for survival in critical illness (see Fig. 3)(1, 17). Cortisol is a powerful immunosuppressive hormone and modulates the inﬂammatory response and cytokine production, thus protecting the organism against excessive inﬂammation (8, 18–20). By this mechanism cortisol inhibits the production of nitric oxide and other mediators in septic shock (20). A direct inhibitory effect of cortisol on the inducible form of nitric oxide synthase has also been demonstrated (21). Not surprisingly, cortisol is an important factor in the maintenance of vascular tone and permeability in severe sepsis. Moreover, cortisol increases the vascular and cardiac response to the renin–angiotensin and the sympathetic nervous systems (13–20), the two most important homeostatic mechanisms in the maintenance of arterial pressure during severe sepsis. Moreover, metabolically, hypercortisolism induces glycogen, fat, and protein catabolism and delays anabolic pathways so that energy becomes available to vital organs (1). Adrenal Function in Chronic Liver Failure 381

Cortisol Immune and inflammatory Metabolic reaction effects Potent immunosuppressive -Catabolism of glycogen, and anti-inflammatory hormone: fat and proteins cytokines, nitric -Delays anabolic pathways oxide...

Cardiovascular system -Retention of intravascular fluid -Increase in the cardiac and vascular response to catecholamins and angiotensin

Fig. 3. Biological effects of cortisol. This pluripotent hormone is essential to survive critical illness.

3. A CHALLENGING AND CONTROVERSIAL ISSUE: CLINICAL DIAGNOSIS OF RELATIVE ADRENAL INSUFFICIENCY As stated before, RAI is defined as the failure of an appropriate adrenal response in the setting of critical illness. This state of functional adrenal insufficiency is characterized by an inadequate production of cortisol, although high in terms of absolute value, with respect to the peripheral demands (1, 6–14, 17). The importance of recognizing inadequate adrenal function may be critical since there is evidence that RAI is associated with a poor outcome in critically ill patients. However, the diagnosis of RAI is not possible on clinical grounds (1, 2, 17)and nowadays relies on the determination of total cortisol levels. The short corticotropin stimulation test is the dynamic probe most commonly used to evaluate adrenal function in critical illness (1, 17). It consists of the measurement of total plasma cortisol levels prior and 60 min after the intravenous administration of 250 μg of synthetic adrenocorticotropic hormone. Although there is no consensus on the diagnostic criteria of this entity, a reduction in the response to the corticotropin test, namely an absolute increment of the serum cortisol level lower than 9 μg/dL, is considered the main diagnostic criterion of RAI in patients with baseline cortisol concentration below 35 μg/dL (2). RAI is also diagnosed when baseline cortisol concentrations are low. Two different cutoff values have been used in the literature, 9 and 15 μg/dL (2, 22). Finally, some authors also consider a cortisol level after corticotropin 382 Fernández and Acevedo administration (peak cortisol) lower than 18 μg/dL as diagnostic of adrenal insufficiency (22). Several guidelines and reviews published until 2008 recommended the assessment of adrenal function in patients with septic shock (corticotropin test) in order to identify those patients with RAI who should receive hydrocortisone supplementation. However, the evaluation of adrenal function in septic shock using this dynamic test is nowadays discouraged based on the results of the CORTICUS study. In this recent randomized controlled trial comparing hydrocortisone therapy vs. placebo in patients with septic shock, no significant differences in survival at 28 days were observed either in nonresponders to corticotropin (39% in the hydrocortisone group vs. 36% in the placebo group) or in responders (29% in both groups) (23). As a consequence, current guidelines do not recommend the use of corticotropin test to guide steroid therapy in critically ill patients with septic shock (24, 25).

4. ADRENAL INSUFFICIENCY IN CRITICAL ILLNESS IN THE GENERAL POPULATION During the past few years several studies have shown that septic shock is frequently associated with RAI in the general population (6–13). It has also been reported that patients with septic shock and RAI show resistance to vasoconstrictor drugs (19), a higher incidence of refractory shock (8), and very high hospital mortality rate (6, 10, 12, 13). These data were the basis for the use of steroids at high doses during short periods of time in patients with septic shock. However, this treatment was abandoned because it was associated with a further increase in mortality, mainly related to the development of secondary infections (26). The immunosuppressive effects of steroids administered at supraphysiological doses accounted for this increase in mortality. In contrast, several trials (7, 27, 28) and two recent meta- analyses (26, 29) have shown that the administration of low doses of hydrocortisone (stress doses) during several days improves shock reversal and survival in these patients. A recent RCT confirmed that steroid administration accelerates the reversal of septic shock but failed to show a survival benefit associated with this treatment. A higher incidence of secondary infections in the hydrocortisone group, including new episodes of septic shock, explains the lack of positive effects of steroid therapy on survival (23). It should be noted, however, that although this trial was the largest in the field of corticosteroid therapy for sepsis, it was insufficiently powered to draw firm conclusions on the effect of hydrocortisone on survival of patients with septic shock. In this regard, Adrenal Function in Chronic Liver Failure 383 the same authors of the paper suggest that hydrocortisone therapy has a role in the management of patients with septic shock who are treated early after the onset of this complication and who remain hypotensive despite the administration of high doses of vasopressors (23–25).

5. ADRENAL INSUFFICIENCY IN SEVERE SEPSIS OR SEPTIC SHOCK IN CIRRHOSIS Several studies have assessed adrenal function in patients with cirrhosis and severe sepsis or septic shock and suggest that RAI is a frequent event in critically ill cirrhotic patients (51–77%; Table 1) (30–35). Tsai et al. evaluated 101 cirrhotic patients with severe sepsis or septic shock requiring intensive monitoring or treatment (50 and 51 patients, respectively) (30). According to the criteria used in the study (baseline cortisol less than 15 μg/dL or cortisol response less than 9 μg/dL in patients with baseline cortisol concentration below 35 μg/dL), adrenal insufﬁciency was diagnosed in 51% of the patients and was associated with hemodynamic instability, disease severity, and renal failure. Eighty percent of the patients with septic shock and 19% of those without vasopressor support had adrenal insufﬁciency (p<0.001). RAI was also more frequent in patients with advanced liver failure (62% in patients with a Child–Pugh score ≥11 points compared to

Table 1 Incidence of relative adrenal insufﬁciency in critically ill patients with chronic liver failure

Number of Critical illness Incidence patients

Harry et al. (31) 20 Acute or chronic liver 69% disease and septic shock Marik et al. (32) 146 Cirrhosis 66% Tsai et al. (30) 101 Cirrhosis and severe 51% sepsis or septic shock Fernandez et al. (33) 25 Cirrhosis and septic 68% shock Thierry et al. (34) 14 Cirrhosis and septic 77% shock Cheyron et al. (35) 50 Cirrhosis 62% 384 Fernández and Acevedo

32% in those with a score less than 11 points, p<0.001) or renal failure (69% vs. 28%, p<0.001). Moreover, cortisol response was inversely correlated with various scoring systems that evaluated illness severity: the sequential organ failure assessment (SOFA), the Acute Physiology, Age, Chronic Health Evaluation III (APACHE III), and the organ system failure (OSF). Finally, and more importantly, intensive care unit (71% vs. 26%, p<0.001) and hospital mortality rates (81% vs. 37%, p<0.001) were significantly higher in patients who had RAI. In this series of patients, cortisol response to corticotropin administration and APACHE III score were identified as independent predictive factors of hospital mortality. The authors of this study concluded that RAI may be of clinical relevance in critically ill patients with cirrhosis and suggested that the risk and benefits of glucocorticoid treatment in these patients should be prospectively evaluated. Three cohort studies have evaluated the effects of stress doses of hydrocortisone on patients with severe liver failure. The first work consisted of a retrospective comparative study in 40 patients with hypotensive acute or chronic liver failure requiring vasopressor support (31). Twenty patients were treated with a continuous infusion of 300 mg/day of hydrocortisone for 4–5 days. Twenty patients who did not receive hydrocortisone and who were admitted during the same period and matched for the proportion of acute and chronic liver failure, age, sex, and severity of illness served as controls. Adrenal function was assessed in patients receiving hydrocortisone. Baseline cortisol and the response to corticotropin were abnormal in nearly 30 and 70% of patients, respectively. Hydrocortisone reduced vasopressor requirements but was associated with a significant increase in the incidence of infections by resistant bacteria. No difference in hospital survival was observed. The second investigation, a more recent but also a retrospective study, evaluated adrenal function in 146 critically ill patients with chronic liver failure (32). In hemodynamically instable patients with RAI, steroid administration was associated with a more rapid reversion of septic shock. Moreover, mortality rate was lower in those patients with RAI who were treated with steroids (26% vs. 46%, p=0.002). Only one study has assessed the effect of supplemental treatment with low doses of hydrocortisone in a homogeneous population of cirrhotic patients with septic shock and RAI (33). The study prospectively evaluated the effects of steroids on shock resolution and hospital survival in a series of 25 consecutive patients (prospective series). Adrenal function was evaluated by the short corticotropin test within the first 24 h of admission. Patients with RAI received stress doses of intravenous hydrocortisone, 50 mg every 6 h. Treatment was gradually discontinued (reduction of 50 mg per day) when patients did not require Adrenal Function in Chronic Liver Failure 385 vasopressor support to maintain arterial pressure. Data were compared to those obtained from a retrospective series of 50 consecutive cirrhotic patients with septic shock admitted to the same intensive care unit in whom adrenal function was not investigated and who did not receive treatment with steroids. The first relevant finding of this study is the confirmation that RAI is a frequent event in cirrhotic patients with septic shock. The incidence of RAI in the prospective series was 68% (see Table 1). This figure is among the highest previously reported in the literature for septic shock and it is slightly lower than that recently reported for cirrhosis (11–18, 30) . Our data also confirmed that in the setting of septic shock RAI is very difficult to diagnose based on clinical data. Only the presence of advanced liver failure (incidence of adrenal insufficiency of 76% in Child C patients vs. 25% in Child B patients, p=0.08) and a lower heart rate of the patients at admission into the intensive care unit (90±25 vs. 113±24 beats/min, p=0.09) were indicators of adrenal dysfunction in this series of patients. The most important finding of the study was that treatment of septic shock in cirrhosis with low dose of hydrocortisone was associated with a marked increase in the rate of shock reversal and hospital survival. Resolution of septic shock (96% vs. 56%, p=0.001), intensive care unit survival (68% vs. 38%, p=0.03), and hospital survival (64% vs. 32%, p=0.003) were significantly higher in the prospective series (see Figs. 4 and 5). The results of the study also indicate that steroid

ICU admission 140 Hydrocortisone 120

100

80 MAP (mmHg) 60 Heart rate 40 (b.p.m) Noradrenaline 20 (μg/kg/h)

0 0 8 16 24 32 40 48 hours

Fig. 4. Effects of hydrocortisone administration on mean arterial pressure (MAP), heart rate, and vasopressor requirements in a cirrhotic patient with septic shock and relative adrenal insufﬁciency. 386 Fernández and Acevedo

1,0

0,8

0,6 Group 1 (n = 25)

0,4 p = 0.003

0,2 Group 2 (n = 50) Probability of hospital survival Probability

0,0 0 10 20 30 40 50 60 Days

Fig. 5. Probability of hospital survival of two different series of cirrhotic patients with septic shock. In the prospective series (continuous line) adrenal function was assessed and treated. In the retrospective series (broken line) adrenal function was not investigated and no patient received treatment with steroids.

supplementation could contribute to change the natural course of septic shock in these patients. Whereas refractory shock caused the majority of deaths in the retrospective series (20 out of 34 patients), in the prospective series it was acute on chronic liver failure (7 out of 9 patients). Infection by resistant bacteria was not a major problem in our patients receiving hydrocortisone. However, two patients died as a consequence of severe fungal infections in the lungs. Treatment with hydrocortisone, therefore, is not free of complication in cirrhosis and should be used when indicated. In our opinion, all cirrhotic patients with septic shock and RAI should be treated with hydrocortisone. In patients with advanced liver disease (Child–Pugh grade C), with an incidence of adrenal insufﬁciency extremely high, treatment should be initiated immediately after diagnostic testing and could be stopped if results do not indicate the presence of RAI. In patients with relatively preserved hepatic function (Child–Pugh grade A or B), in whom the incidence of RAI is low, treatment should be started after diagnosis. Finally, recent data indicate that the hyperreninemic hypoaldosteronism syndrome is also frequent in critically ill patients with cirrhosis (52%) (35). This abnormality, which reﬂects a transient dysfunction in the adrenal synthesis of mineralocorticoids, seems to be associated Adrenal Function in Chronic Liver Failure 387 with disease severity and poor prognosis in this population of cirrhotic patients.

6. ADRENAL FUNCTION IN OTHER POPULATIONS OF PATIENTS WITH CIRRHOSIS The clinical impact of RAI on other decompensations of cirrhosis is poorly known. Recent data derived from a prospective study performed in our Liver Unit indicate that RAI (delta cortisol <9 μg/dL) is prevalent in decompensated cirrhosis. The prevalence of RAI was 27% in the whole series of noncritically ill cirrhotic patients, 21% in SBP, 24% in non-SBP infections, 46% in patients with sterile ascites, 31% in hepatic encephalopathy, 26% in gastrointestinal bleeding, 29% in hepatorenal syndrome, and 22% in a group of ambulatory patients. No signiﬁcant differences in mortality were observed between patients with and without RAI. The activation of hypothalamic-pituitary-adrenal axis (high free cortisol index at admission) was an independent predictor of poor short-term survival (data submitted to the EASL Meeting, Vienna, 2010).

7. POSSIBLE MECHANISMS OF RELATIVE ADRENAL INSUFFICIENCY IN CIRRHOSIS The mechanisms of RAI in septic shock are not well established either in the general population or in cirrhosis. It may be related to a reduction in adrenal blood ﬂow. On the other hand, very high levels of inﬂammatory cytokines directly inhibit adrenal cortisol synthesis (see Fig. 2). Finally, preexisting conditions of the hypothalamic-pituitary- adrenal axis could be also important (2, 8). In patients with decompensated cirrhosis and ascites, the cytokine response to endotoxin is very much increased (36), and the blood perfusion to extrasplanchnic organs is generally reduced (37). Furthermore, as indicated before, cirrhotic patients may present RAI prior to the infection (30). Finally, we have to consider that cholesterol is the main precursor for adrenal synthesis of steroids. About 80% of circulating cortisol is derived from plasma cholesterol, the remaining being synthesized in situ from acetate and other precursors (see Fig. 6)(32). Experimental studies suggest that high-density lipoprotein (HDL) is the main lipoprotein source for steroid synthesis in the adrenal gland. It is well known that patients with advanced cirrhosis may present a marked decrease in the total cholesterol and in HDL levels. This factor could contribute to the development 388 Fernández and Acevedo

Mineralocorticoid Glucocorticoid Androgen pathway pathway pathway

Cholesterol

ACTH α Pregnenolone 17 -hydroxypregnenolone Dehydroepiandrosterone

Progesterone 17α-hydroxyprogesterone Androstenedione

11-deoxycorticosterone 11-deoxycortisol Testosterone

Corticosterone Cortisol Estrogen

Angiotensin II 18-hydroxycorticosterone

Aldosterone

Fig. 6. Pathways of adrenal steroid biosynthesis in adrenal gland. Cholesterol is the main precursor used by the adrenal cortex in the synthesis of cortisol and aldosterone. ACTH: corticotropin. of RAI (32). Therefore, RAI in cirrhotic patients with severe bacterial infections is probably multifactorial.

8. OTHER UNSOLVED QUESTIONS Several features regarding diagnostic and therapeutic aspects of RAI in critical illness are still unsolved and require further investigations. As stated before, the short corticotropin stimulation test was considered to date the gold standard to assess adrenal function in this setting. However, its usefulness in the management of patients with septic shock is now under discussion. Moreover, there are several methodological problems with the test. First, important interindividual differences have been reported, which complicate the differentiation between normal and abnormal adrenal response, particularly in the course of an acute illness (1, 17). Second, the short corticotropin stimulation test evaluates total plasma levels of cortisol (serum-free cortisol plus the protein-bound fraction of cortisol). The current consensus is that the free cortisol, rather than the protein-bound cortisol fraction, is responsible for the physiological function of the hormone. Considering that more than 90% of circulating cortisol in human serum is bound to proteins (cortisol-binding protein and albumin) it is reasonable to suggest that alterations in the levels of these two binding proteins Adrenal Function in Chronic Liver Failure 389 could affect measured concentrations of serum total cortisol and, thus, the interpretation of the test (15). Critically ill patients frequently develop multiorgan dysfunction and malnutrition, and consequently the concentrations of cortisol-binding protein and albumin are commonly decreased. Therefore, measuring total serum cortisol levels in these patients could be misleading, resulting in the incorrect conclusion that adrenal function is impaired. Recent studies evaluating free cortisol levels in the general population showed that nearly 40% of critically ill patients with hypoproteinemia had subnormal serum total cortisol levels in spite of normal adrenal function (15). In order to avoid these errors in the evaluation of adrenal function, some groups recommend the use of a calculated correction factor, the free cortisol index, defined as the serum total cortisol concentration divided by the serum cortisol-binding protein concentration, as a surrogate marker that better defines glucocorticoid secretion. Finally, because the 250 μg ACTH stimulation test induces supraphysiological ACTH concentrations, the 1 μgACTH stimulation test has been suggested to be more sensitive to diagnose adrenocortical insufficiency (1). Type, doses, and duration of steroid replacement therapy in patients with RAI are also under discussion. As described previously, low-dose glucocorticoid replacement therapy seems to be associated with beneficial effects in patients with septic shock. However, the administration of hydrocortisone at the so-called “stress or replacement doses” still results in several-fold higher total and free cortisol levels. In the opinion of some groups, a reevaluation of the doses of hydrocortisone is required (1, 17). The duration of therapy is also under debate. Some authors recommend 7 days of therapy (7) while others suggest the maintenance of steroid administration until shock resolution (33). Finally, some authors recommend the administration of not only glucocorticoids (hydrocortisone) but also mineralocorticoids (9α-fludrocortisone) in patients with relative adrenal dysfunction (7).

REFERENCES

1. Schuetz P, Müller B. The hypothalamic-pituitary-adrenal axis in critical illness. In: Acute Endocrinology. Endocrinol Metab Clin North Am 2006;35: 823–38. 2. Cooper MS, Stewart PM. Corticosteroid insufﬁciency in acutely ill patients. N Engl J Med 2003;348:727–34. 3. Widmer IE, Puder JJ, König C, et al. Cortisol response in relation to the severity of stress and illness. J Clin Endocrinol Metab 2005;90:4579–86. 4. Reincke M, Allolio B, Wurth G, et al. The hypothalamic-pituitary-adrenal axis in critical illness: response to dexamethasone and corticotropin-releasing hormone. J Clin Endocrinol Metab 1993;77:151–6. 390 Fernández and Acevedo

5. Jurney TH, Cockrell Jr. JL, Lindberg JS, et al. Spectrum of serum cortisol response to ACTH in ICU patients. Correlation with degree of illness and mortality. Chest 1987;92:292–5. 6. Annane D, Sebille V, Troche G, et al. A 3-level prognostic classification in septic shock based on cortisol levels and cortisol response to corticotropin. JAMA 2000;283:1038–45. 7. Annane D, Sebille V, Charpentier C, et al. Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock. JAMA 2002;288:862–71. 8. Beishuizen A, Thijs LG. Relative adrenal insufficiency in intensive care: an identifiable problem requiring treatment? Best Pract Res Clin Endocrinol Metab 2001;15:513–31. 9. Briegel J, Schelling G, Haller M, et al. A comparison of the adrenocortical response during septic shock and after complete recovery. Intensive Care Med 1996;22:894–9. 10. Rothwell PM, Udwadia ZF, Lawler PG. Cortisol response to corticotropin and survival in septic shock. Lancet 1991;337:582–3. 11. Sibbald WJ, Short A, Cohen MP, et al. Variations in adrenocortical responsiveness during severe bacterial infections. Unrecognized adrenocortical insufficiency in severe bacterial infections. Ann Surg 1977;186:29–33. 12. Soni A, Pepper GM, Wyrwinski PM, et al. Adrenal insufficiency occurring during septic shock: incidence, outcome, and relationship to peripheral cytokine levels. Am J Med 1995;98:266–71. 13. Bollaert PE, Fieux F, Charpentier C, et al. Baseline cortisol levels, cortisol response to corticotropin, and prognosis in late septic shock. Shock 2003;19:13–5. 14. Annane D, Bellissant E, Sebille V, et al. Impaired pressor sensitivity to norepinephrine in septic shock patients with and without impaired adrenal function reserve. Br J Clin Pharmacol 1998;46:591–9. 15. Hamrahian AH, Oseni TS, Arafah BM. Measurements of serum free cortisol in critically ill patients. N Engl J Med 2004;350:1629–38. 16. Perrot D, Bonneton A, Dechaud H, et al. Hypercortisolism in septic shock is not suppressible by dexamethasone infusion. Crit Care Med 1993;21:396–401. 17. Langouche L, Van den Berghe G. The dynamic neuroendocrine response to critical illness. In: Acute Endocrinology. Endocrinol Metab Clin North Am 2006;35:777–91. 18. Oppert M, Schindler R, Husung C, et al. Low-dose hydrocortisone improves shock reversal and reduces cytokine levels in early hyperdynamic septic shock. Crit Care Med 2005;33:2457–64. 19. Annane D, Cavaillon JM. Corticosteroid in sepsis: from bench to bedside? Shock 2003;20:197–207. 20. Keh D, Boehnke T, Weber-Cartens S, et al. Immunologic and hemodynamic effects of low dose hydrocortisone in septic shock: a double-blind, randomized, placebo- controlled, crossover study. Am J Respir Crit Care Med 2003;167:512–20. 21. Radomski MW, Palmer RM, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci U S A 1990;87:10043–7. 22. Grinspoon SK, Biller B. Laboratory assessment of adrenal insufficiency. J Clin Endocrinol Metab 1994;79:923–31. 23. Sprung CL, Annane D, Keh D, et al. for the CORTICUS Study Group. Hydrocortisone therapy for patients with septic shock. N Engl J Med 2008;358:111–24. Adrenal Function in Chronic Liver Failure 391

24. Dellinger RP, Levy MM, Carlet JM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock. Crit Care Med 2008;36:296–327. 25. Marik PE, Pastores SM, Annane D, et al. for the American College of Critical Care Medicine. Recommendations for the diagnosis and management of corticosteroid insufficiency in critically ill adult patients: consensus statements from an international task force by the American College of Critical Care Medicine. Crit Care Med 2008;36:1937–49. 26. Minneci PC, Deans KJ, Banks SM, et al. Meta-analysis: the effects of steroids on survival and shock during sepsis depends on the dose. Ann Intern Med 2004;141:47–56. 27. Briegel J, Forst H, Haller M, et al. Stress doses of hydrocortisone reverse hyperdynamic septic shock: a prospective, randomized, double-blind, single-center study. Crit Care Med 1999;27:723–32. 28. Bollaert PE, Charpentier C, Levy B, et al. Reversal of late septic shock with supraphysiologic doses of hydrocortisone. Crit Care Med 1998;26:645–50. 29. Annane D, Bellissant E, Bollaert PE, et al. Corticosteroids for severe sepsis and septic shock: a systematic review and meta-analysis. Br Med J 2004;329:480–9. 30. Tsai MH, Peng YS, Chen YC, et al. Adrenal insufficiency in patients with cirrhosis, severe sepsis and septic shock. Hepatology 2006;43:673–81. 31. Harry R, Auzinger G, Wendon J. The effects of supraphysiological doses of corticosteroids in hypotensive liver failure. Liver Int 2003;23:71–7. 32. Marik PE, Gayowski T, Starzl TE. The hepatoadrenal syndrome: a common yet unrecognized clinical condition. Crit Care Med 2005;33:1254–9. 33. Fernandez J, Escorsell A, Zabalza M, et al. Adrenal insufficiency in patients with cirrhosis and septic shock: effect of treatment with hydrocortisone on survival. Hepatology 2006;44:1288–95. 34. Thierry S, Giroux Leprieur E, Lecuyer L, et al. Echocardiographic features, mortality and adrenal function in patients with cirrhosis and septic shock. Acta Anaesthesiol Scand 2008;52:45–51. 35. Cheyron D, Bouchet B, Cauquelin B, et al. Hyperreninemic hypoaldosteronism syndrome, plasma concentrations of interleukin-6 and outcome in critically ill patients with liver cirrhosis. Intensive Care Med 2008;34:116–24. 36. Navasa M, Follo A, Filella X, et al. Tumor necrosis factor and interleukin-6 in spontaneous bacterial peritonitis in cirrhosis: relationship with the development of renal impairment and mortality. Hepatology 1998;27:1227–32. 37. Guevara M, Bru C, Gines P, et al. Increased cerebrovascular resistance in cirrhotic patients with ascites. Hepatology 1998;28:39–44.

Part III Management of Chronic Liver Failure

Antibiotic Prophylaxis and Management of Bacterial Infections

Joseph K. Lim, MD, Puneeta Tandon, MD, and Guadalupe Garcia-Tsao, MD

CONTENTS EPIDEMIOLOGY OF BACTERIAL INFECTIONS IN CIRRHOSIS CONSEQUENCES OF BACTERIAL INFECTION SPONTANEOUS BACTERIAL INFECTIONS SBP – TREATMENT SBP PROPHYLAXIS NONSPONTANEOUS BACTERIAL INFECTIONS REFERENCES

Key Words: Spontaneous bacterial peritonitis, Bacterial infection, Cirrhosis

1. EPIDEMIOLOGY OF BACTERIAL INFECTIONS IN CIRRHOSIS Recent prospective studies suggest that approximately one-third of cirrhotic patients who are hospitalized develop a bacterial infection either at the time of admission or during the course of their hospitalization (1, 2). This is signiﬁcantly higher than the 5–7% prevalence of infections in hospitalized noncirrhotic patients and is even

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_20, C Springer Science+Business Media, LLC 2011

395 396 Lim et al. higher (∼45%) among cirrhotic patients admitted for gastrointestinal hemorrhage (3, 4). In fact, the most important predictors of bacterial infection in cirrhotic patients are gastrointestinal bleeding and severity of liver disease (5–7). The most common bacterial infections are spontaneous bacterial peritonitis (approximately 25% of all infections), urinary tract infections (20%), pneumonia (15%), and bacteremia (12%) (4). In studies performed almost a decade ago in consecutive hospitalized patients, gram-negative bacteria (GNB) and gram-positive bacteria (GPB) were equally prevalent. However, GNB such as Escherichia coli predominated as the causative organism for SBP and urinary tract infections, whereas GPB were more commonly responsible for pneumonia and were more likely to be identified in nosocomial infections (1, 8). The spectrum of bacteria causing infections in cirrhosis has changed more recently probably as a result of the widespread use of antibiotics and the development of antibiotic- resistant organisms. Among patients who are not receiving selective intestinal decontamination with antibiotics, E. coli, streptococci (primarily pneumococci), and Klebsiella species are responsible for most episodes of SBP (9, 10). However, in individuals receiving antibiotic prophylaxis with quinolones, the relative proportion of GPB is greater, with infections commonly related to streptococci (10). In addition to shifting the microbial spectrum, the broader use of quinolones for SBP prophylaxis has resulted in an increasing incidence of GNB with multidrug resistance. In a cohort of patients evaluated for liver transplantation during 1991–1995 and 1996–2001, the frequency of multiple-antibiotic-resistant bacterial infection was significantly greater in the latter cohort (38.5% vs. 8.3%) (11). More recent data are available from an unpublished study by Acevedo et al. who evaluated 224 hospitalized patients with 500 bacterial infections (12), a third of which were nosocomial. Multiresistant bacteria were identified in a large percentage of nosocomial infections (28% of SBP, 63% of UTI, and 29% of pneumonia cases). The most common multiresistant bacteria were extended-spectrum β-lactamase-producing enterobacteria followed by Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Enterococcus faecium. Prognosis is greatly impacted by infection with a multidrug-resistant organism with increased rates of septic shock and death (11, 12).

2. CONSEQUENCES OF BACTERIAL INFECTION The body’s systemic inﬂammatory response (hyper- or hypothermia, tachycardia, tachypnea, elevated or decreased white cell count or left shift) to a bacterial infection is termed sepsis. Largely due to impaired immune defenses, individuals with cirrhosis, particularly those with Antibiotic Prophylaxis and Management 397 more advanced dysfunction, are at an increased risk of developing an exaggerated response to infection (13, 14). This response is manifest clinically by hypoperfusion or hypotension that is either responsive (severe sepsis) or refractory (septic shock) to intravascular volume expansion (15, 16). In addition to the development of circulatory dysfunction, multiple end organs can be compromised in the setting of sepsis (17). “Acute-on- chronic liver failure,” or the acute deterioration of chronic liver disease, can be precipitated by sepsis and is associated with increased mortality. Renal dysfunction can develop in a third of patients with SBP or other bacterial infections despite treatment with antibiotic therapy (18–21) and is a robust predictor of in-hospital mortality (22). Coagulopathy and thrombocytopenia are well-known features of hepatic dysfunction. These abnormalities can worsen in the setting of infection, with further impairment of platelet function, increased ﬁbri- nolysis, the production of endogenous heparin-like substances, and the consumption of clotting factors (23, 24). Due to these coagulation abnormalities and also due to the increase in portal pressure that occurs with infection, bacterial infection is a recognized precipitant of variceal hemorrhage (25). Moreover, among individuals with variceal hemorrhage, bacterial infections are associated with failure to achieve initial control of bleeding, and an increased risk for early variceal rebleeding (3, 25–27). Bacterial infections can also impact neurological function and they are a recognized precipitant of “septic encephalopathy” in patients with cirrhosis (28–30). Lastly, bacterial infections account for approximately 30% of the deaths in cirrhosis (4) and they double the in-hospital mortality when compared to noninfected patients with cirrhosis (16).

3. SPONTANEOUS BACTERIAL INFECTIONS Spontaneous bacterial infections including spontaneous bacterial peritonitis (SBP), spontaneous bacterial empyema, and spontaneous bacteremia are considered characteristic of cirrhosis. By definition, these are infections that lack an obvious source of bacteria within peritoneal fluid, pleural fluid, or blood, respectively. Of these, SBP is the most common. Although SBP may present with abdominal pain, fever, gastrointestinal symptoms, or symptoms suggestive of hepatic encephalopathy, up to 50% of hospitalized patients with SBP are asymptomatic. Due to this high rate of asymptomatic disease and the proven inferiority of clinical assessment alone as compared to ascitic fluid analysis (31), ascitic fluid evaluation is the gold standard for the diagnosis of SBP. Prompt diagnostic paracentesis is recommended for 398 Lim et al. all patients with cirrhosis and ascites (1) at admission to the hospital, (2) who develop typical symptoms or signs, or (3) who have worsening renal or liver function (32). Given that bacterial organisms are isolated in only half of ascitic fluid cultures, the diagnosis of SBP is based not on bacteriological cultures, but on ascites polymorphonuclear (neutrophil) cell count. An ascitic fluid polymorphonuclear (PMN) cell count >250/mm3 establishes a diagnosis of SBP, with correction of 1 PMN for every 250 red blood cells (RBCs) in the case of a traumatic or bloody tap (RBC >10,000) (32). Newer, faster methods of establishing a diagnosis of SBP have been developed, including urine reagent strips similar to those used to establish a rapid diagnosis of urinary tract infections and meningitis. These azo dye strips evaluate for leukocyte esterase activity of activated granulocytes within peritoneal fluid. Unfortunately their use cannot be recommended due to an unacceptably high false-negative rate of up to 55% (22, 33). In order to increase the chances of identifying a causative bacterial pathogen, ascitic fluid (10 mL) should be inoculated into blood culture bottles. Blood cultures should be obtained at the time of diagnostic paracentesis as approximately 50% of all cases of SBP are associated with bacteremia (9, 32). In addition, secondary peritonitis should be excluded in individuals with a compatible clinical history, an inadequate response to antibiotic therapy within 48 h (clinical worsening or the absence of at least a 25% decrease in the ascitic PMN count), or evidence of a polymicrobial infection (particularly in the presence of anaerobes and/or fungi). Ascitic/serum fluid analyses may be helpful in distinguishing primary from secondary peritonitis. The presence of two of the following criteria are suggestive: ascitic glucose <50 mg/dL, ascitic protein >10 mg/dL, and an ascitic lactic dehydrogenase greater than serum lactic dehydrogenase (34, 35).

4. SBP – TREATMENT 4.1. Basic Principles The development of SBP represents an important event in the natural history of cirrhosis, and is associated with a 1-year survival of 30–50%, a 2-year survival of 25–30%, and a median survival of 9 months (36). As such, all patients should be considered for liver transplant evaluation following an initial episode of SBP. As renal failure represents the most important predictor of mortality, medications or procedures that may deplete the intravascular volume (e.g., diuretics), worsen vasodilatation (large-volume paracentesis, vasodilators), worsen renal vasoconstriction (nonsteroidal anti-inﬂammatory drugs), or nephrotoxins (e.g., aminoglycosides) should be avoided during SBP. Antibiotic Prophylaxis and Management 399

4.2. Accepted Antibiotic Therapies The choice of an appropriate antibiotic requires consideration of safety and efficacy, the potential for multidrug-resistant organisms, and knowledge regarding local antibiotic susceptibility patterns. Based on randomized controlled trials from the 1990s that demonstrated an 80–94% rate of resolution of SBP with intravenous cefotaxime (19, 37–39), third-generation cephalosporins have been the therapy of choice recommended by consensus (32). Similar rates of resolution have been observed with either cefotaxime 2 g IV every 8 h for 10 days vs. 5 days (93% vs. 91%) (37), or cefotaxime 2 g IV every 12 h vs.2gIVevery6h(39). This has led to the recommended dose of cefotaxime of 2 g IV every 12 h for a minimum of 5 days. The intravenous administration of a combination of amoxicillin–clavulanic acid (1 g/200 mg IV every 8 h and later transitioned to 500 mg/125 mg PO every 8 h once the patient has clinically improved) has also been recommended as an acceptable first-line agent as it has demonstrated similar safety and efficacy to cefotaxime in a series from a decade ago (40). In these studies, the response to cefotaxime and to amoxicillin–clavulanic acid was similar in patients taking chronic quinolone prophylaxis and patients not on prophylaxis (10, 40). Recent studies raise concern that both cephalosporins and amoxicillin–clavulanic acid may be losing their effectiveness as first- line agents, particularly in the subgroup of patients with nosocomial SBP (12, 41). In one of these studies published only in abstract form, the rate of infection resolution with empirical antibiotic therapy (third- generation cephalosporins for SBP) was only 44% in nosocomial SBP (12). In a second series of 236 patients with SBP, nosocomial isolates of gram-negative organisms had a 41% rate of resistance to third- generation cephalosporins (42). In this study, independent predictors of resistance to third-generation cephalosporins were the previous use of these antibiotics, nosocomial acquisition of pathogens, and presentation with acute renal failure. Therefore, patients with nosocomial SBP may benefit from broader empirical antibiotic coverage (carbapenem or glycopeptide antibiotics) with subsequent narrowing of the antibiotic spectrum after obtaining identification and susceptibility profile of the causative pathogen (in culture-positive cases). It is unclear whether these recommendations need to be extended to patients presenting with acute renal failure. Moreover, given the increase in antibiotic- resistant organisms, it is highly recommended to repeat an ascitic fluid PMN count after 48 h of therapy and assess for a 25% reduction as a marker of response to therapy. In patients who have not been on prophylaxis with oral quinolones, if the ascitic fluid PMN count has responded to intravenous antibiotics, they can be transitioned to an oral quinolone such as ciprofloxacin or levofloxacin. This approach has 400 Lim et al. been associated with earlier hospitalization discharge and lower costs (43). However, if the ascitic fluid PMN count has not decreased by at least 25% at 48 h, ascitic fluid cultures should be redrawn, antibiotic coverage reassessed, and further investigation for secondary causes of peritonitis should be initiated.

4.3. Role of Albumin The mechanism behind renal dysfunction in SBP is a worsening of the vasodilatory state of cirrhosis with further decrease in effective arterial blood volume. The primary role of albumin in this setting is to increase effective arterial blood volume, but it may also bind endotoxin and other vasodilating substances, and reduce cytokine (TNF-α)and nitric oxide levels (44, 45). The combination of intravenous albumin and antibiotic therapy reduces the rates of renal dysfunction (10% vs. 33%), concomitantly reducing in-hospital mortality (10% vs. 29%) and 3-month mortality (22% vs. 41%) when compared to antibiotics alone (19). The dose of albumin used in this study, 1.5 g/kg body weight intravenously within 6 h of initial diagnosis followed by 1 g/kg body weight intravenously on day 3, was empirical and should probably be adjusted according to the changes in serum creatinine not to exceed 100 g/day (46). Albumin is costly and limited in supply at many medical centers, and should be targeted for the ∼40% of patients who appear to bene- ﬁt most from albumin, that is, those who at the time of SBP diagnosis already have an altered renal function (BUN >30 mg/dL or creatinine >1.0 mg/dL) or those with a serum bilirubin >4 mg/dL (19). Individuals with “low-risk” SBP (BUN <30 mg/dL, creatinine <1.0 mg/dL, bilirubin <4 mg/dL) have a low risk of acute kidney injury or death and albumin is not required in these patients. Plasma expanders such as hydroxyethyl starch do not have the hemodynamic or neurohumoral beneﬁts observed with albumin and would not appear to be appropriate alternatives to albumin (47).

5. SBP PROPHYLAXIS Prophylaxis for SBP is based on the oral administration of nonabsorbable or poorly absorbed antibiotics for selective intestinal decontamination of gram-negative enteric flora. Long-term administration of oral norfloxacin decreases GNB counts in the fecal flora of cirrhotic patients without affecting GPB or anaerobic bacteria (48). However, long-term use of quinolones may result in the development of resistance to both quinolones and trimethoprim/sulfamethoxazole (1), and Antibiotic Prophylaxis and Management 401 therefore prophylaxis should be restricted to individuals at the highest risk of developing SBP or other gram-negative infections. Risk factors that predict the development of SBP include advanced liver disease, an ascitic fluid protein concentration <1 g/dL (36, 49, 50), and a prior episode of SBP (48). Among patients with low ascites fluid protein, a high serum bilirubin (51, 52) and a low platelet count (52) have been identified. However all these risk factors have to be taken in the context of the setting in which the patient with cirrhosis and ascites is situated. According to Table 1, there are three settings in which the usefulness of antibiotic prophylaxis for SBP should be evaluated.

5.1. Prophylaxis in Hospitalized Patients with Gastrointestinal Hemorrhage Cirrhotic patients who are hospitalized with gastrointestinal hemorrhage bear a higher risk of bacterial infections than hospitalized cirrhotic patients without bleeding (45% vs. 33%). Five randomized trials have demonstrated a clear benefit of short-term antibiotics in the prevention of bacterial infections (including SBP) in patients hospitalized with an acute gastrointestinal hemorrhage (53–57). As shown in Table 1, when looking specifically at SBP (which was not the primary end point of these studies), the in-hospital rate was rather low at 13% (representing about a third of all infections) but was even lower (5%) in patients receiving antibiotic prophylaxis (p=0.004) (3). In a meta- analysis of these studies comparing no antibiotic vs. antibiotic therapy, a significant decrease in bacterial infections (45% vs. 14%) and, importantly, a significant decrease in mortality (24% vs. 15%) were observed (3). Therefore, antibiotic prophylaxis is currently an integral part of therapy for patients with cirrhosis presenting with gastrointestinal hemorrhage and should be instituted from admission (58). Regarding the type of antibiotic and route of administration, two of the randomized trials used orally administered poorly absorbable (norfloxacin) or nonabsorbable antibiotics, whereas three trials used intravenous and/or oral fully absorbable antibiotics (ofloxacin, ciprofloxacin, amoxicillin/clavulanate), with varying course durations of 7–10 or 2–3 days following cessation of bleeding. In the meta- analysis, no difference was observed between oral and intravenous antibiotics. A more recent randomized controlled trial in patients with upper gastrointestinal hemorrhage and “advanced” cirrhosis (as defined by at least two of the following features: ascites, severe malnutrition, encephalopathy, or bilirubin >3 mg/dL) compared intravenous ceftriaxone (1 g/day) to oral norfloxacin (400 mg twice daily). Treatment 402 Lim et al. ) c ) ) 67 – ) b 65 68 ( d Yes No ( Yes (3) Uncertain (48, 60 Data obtained from the group randomized to c 2/35 6% 3/159 2% 9/196 5% 11/80 14% Table 1 30% 13% 13% 35% 12 months 10/33 12 months 21/157 In-hospital 26/198 Meta-analysis of five randomized controlled trials. b ± ascites + liver dysfunction + circulatory dysfunction ascites high bilirubin Low-protein Low-protein Setting Time frame No antibiotic Antibiotic prophylaxis Survival benefit References a 3-month survival only. d Rates of SBP in different settings without and with antibiotic prophylaxis (only randomized controlled trials SBP (3) No prior (1)GIbleed (2) Prior SBP 5–6 months 14/40 Primary end point was prevention of bacterial infections in general (not only SBP). These were observed in 45% of the control group vs. a 14% in the antibiotic prophylaxis group. daily norfloxacin. Antibiotic Prophylaxis and Management 403 with ceftriaxone resulted in a lower rate of bacterial infections (11% vs. 26%), but without differences in the rate of SBP (2% ceftriaxone, 5% norfloxacin) or mortality (59). Notably, only 9% of patients met entry criteria, decreasing the generalizability of the results. Differences between these antibiotics could be ascribed either to the type of antibiotic or to the route of administration. The fact that six of seven GNB isolated in the norfloxacin group were quinolone-resistant suggests that the type of bacteria explained differences between antibiotics. Therefore, intravenous ceftriaxone should be preferred in patients previously on norfloxacin prophylaxis or in hospital settings with a high prevalence of infections due to quinolone-resistant organisms and should also be considered in patients with severe liver disease. Oral norfloxacin/ciprofloxacin or intravenous ciprofloxacin can be considered in other patients, with the oral route recommended in patients who are able to take drugs orally. Antibiotics should be used for 7 days (32), or for a lesser period if bleeding has stopped and the patient is being discharged from the hospital.

5.2. Prophylaxis in Patients with Prior Episodes of SBP Following SBP resolution, its recurrence is common, with a 1-year probability of 70% (48). As shown in Table 1, the rate of SBP is highest in the patient population with previous SBP and is significantly reduced with antibiotic prophylaxis using norfloxacin at a dose of 400 mg orally every day (48, 60). A survival benefit has not been demonstrated because this was not an end point of the landmark study (48). Economic analyses have demonstrated that antibiotic prophylaxis for primary prophylaxis is cost-saving when compared with treatment at the time of diagnosis (61, 62). Based on this evidence, antibiotic prophylaxis should be initiated in all patients following an episode of acute SBP. The preferred antibiotic is norfloxacin at a dose of 400 mg orally every day. This daily regimen is strongly preferred over weekly regimens due to superior efficacy and lower rates of quinolone resistance (60, 63). While norfloxacin is preferred in patients who have recovered from an episode of SBP due to GNB or SBP in which an organism was not identified, its efficacy is uncertain when the index SBP was due to GPB or multidrug-resistant organisms. The choice of antibiotic prophylaxis in a patient developing SBP on a quinolone remains unclear, and may vary based on local microbial resistance patterns. Notably, trimethoprim/sulfamethoxazole is not recommend- able in this setting because patients on norfloxacin prophylaxis also haveahighrate(∼44%) of infections due to TMP/SXM-resistant organisms (1). 404 Lim et al.

5.3. Prophylaxis in Patients Without a Prior Episode of SBP In these patients, low ascites total protein is an important predictor of first SBP. This is attributed to decreased levels of proteins critical for opsonic activity in ascitic fluid (64) and was supported by a prospective study which revealed that patients with low ascitic protein levels (<1 g/dL) developed SBP at a rate of 20% over 1 year, whereas patients with normal ascitic protein levels (>1 g/dL) did not develop SBP over a period of 2 years (50). As shown in Table 1, among patients with a low ascites protein, two groups with a very different risk of developing SBP can be identified. In randomized trials including patients with low ascites protein (65–67), the rate of SBP was rather low at 13% at 1 year and, although significantly lower (2%) with the use of antibiotic prophylaxis, there was no significant effect on mortality (40/157 or 25% in control groups vs. 27/159 or 17% in antibiotic groups, p=0.07). One could argue that the risk of creating antibiotic-resistant organisms outweighs the benefits of prophylaxis in this subgroup of patients. On the other hand, a recent placebo-controlled study that included patients with low (<1.5 g/L) ascites protein who also had advanced liver failure (Child score ≥9 and serum bilirubin ≥3 mg/dL) or circulatory dysfunction (serum creatinine ≥1.2 mg/dL, blood urea nitrogen level ≥25 mg/dL, or serum sodium level ≤130 meq/L) demonstrates a much higher rate of first SBP of 30%, but significantly lower on norfloxacin prophylaxis (6%) (68). Importantly, the study also demonstrated a significant decrease in the 1-year probability of developing hepatorenal syndrome and in 3-month mortality (68). Even though less than half of the patients with low ascites protein met entry criteria and the survival benefit did not extend to 1 year, the risk/benefit ratio would probably favor primary antibiotic prophylaxis in this subgroup of patients. Notably, two recent meta-analyses have evaluated trials on primary prophylaxis of SBP, with somewhat differing results (69, 70). Although both show some survival benefit with antibiotics, the interpretation of the results is limited by the inclusion of patients with differential risk for SBP (Table 1). In this age of individualized patient therapy, rather than grouping disparate groups of patients, the goal is to identify subgroups of patients who require distinct therapies.

6. NONSPONTANEOUS BACTERIAL INFECTIONS As recently reviewed by Christou et al. (71), patients with cirrhosis are at risk for other common and uncommon bacterial infections due to their immunocompromised state. Urinary tract infections and respiratory tract infections are the most prevalent “nonspontaneous” Antibiotic Prophylaxis and Management 405 infections (1). Any nonspontaneous bacterial infection that occurs in a noncirrhotic patient can occur in a patient with cirrhosis. However, detailed data regarding the morbidity, mortality, and treatment recommendations associated with these specific infections in cirrhosis are limited. What is clear is that in most cases, Child–Pugh C patients are at the highest risk for infection (71) and that the presence of a bacterial infection in a patient with cirrhosis increases mortality and length of stay in hospital (72). In the absence of specific data on patients with cirrhosis, treatment recommendations for nonspontaneous infections should follow local infectious disease guidelines. Since the rate of multidrug-resistant organisms in cirrhotic patients with nosocomial bacterial infections is increasing, with a lack of response to empirical therapy in 34% of nosocomial acquired urinary tract infections and 17% of nosocomial acquired pneumonia, more broad-spectrum empirical antibiotic coverage is indicated in these patients (12). Similar to SBP, non-SBP infections are associated with impairment of the effective circulating volume and precipitate renal failure in approximately one-third of cirrhotic patients (20). Although there are no data regarding the use of albumin in these patients, it would be preferable to use albumin in patients who have some degree of kidney dysfunction upon diagnosis of the infection. The specific impact of Clostridium difficile-associated illness (CDAD) has recently been evaluated in a case–control analysis of over 83,000 patients with cirrhosis (73). Patients with cirrhosis and CDAD had a significantly higher mortality rate (13.8% vs. 8.2%) and a longer length of stay than cirrhotic patients without CDAD. On a separate analysis of the center’s local database of 162 cirrhotic patients, inpatient antibiotic use and outpatient proton-pump inhibitor use were noted as independent factors predicting CDAD, stressing the importance of the judicious use of these agents clinical practice (74). Interestingly, unlike in other infections, there was no correlation demonstrated between the risk of CDAD and the degree of liver dysfunction, as measured by the MELD and Child–Pugh scores.

6.1. Summary Bacterial infections are a well-recognized complication of cirrhosis, and represent a major source of morbidity and mortality. This risk of infection is highest among individuals who are hospitalized and in those who present with acute upper gastrointestinal hemorrhage. Prophylaxis in at-risk individuals, early diagnosis in hospitalized patients, and aggressive medical management are needed to optimize patient outcomes. Spontaneous bacterial infections (spontaneous 406 Lim et al. bacterial peritonitis, spontaneous bacterial empyema, spontaneous bacteremia) are the most common infections in cirrhosis and are considered characteristic of the disease. In spontaneous bacterial peritonitis (SBP), the mainstay of treatment is antibiotic therapy in all patients and intravenous albumin in those patients at high risk of developing renal failure. Antibiotic prophylaxis for SBP is indicated in patients admitted with gastrointestinal hemorrhage (short-term), in those who have already had an episode of SBP, and in a select group of patients with low ascites protein. Of the nonspontaneous bacterial infections, urinary and respiratory tract infections are the most common and also account for considerable morbidity and mortality, particularly due to the development of renal insufﬁciency. The microbial spectrum of bacteria causing infections in cirrhosis has changed more recently, probably as a result of the widespread use of antibiotics and the development of antibiotic-resistant organisms. As patients with nosocomial acquisition of infection are at a particularly high risk of infection with multidrug- resistant organisms and of nonresponse to empirical antibiotic therapy, coverage should be broadened in this subgroup of patients.

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67. Terg R, Fassio E, Guevara M et al. Ciprofloxacin in primary prophylaxis of spontaneous bacterial peritonitis: a randomized, placebo-controlled study. J Hepatol 2008;48:774–9. 68. Fernandez J, Navasa M, Planas R et al. Primary prophylaxis of spontaneous bacterial peritonitis delays hepatorenal syndrome and improves survival in cirrhosis. Gastroenterology 2007;133:818–24. 69. Loomba R, Wesley R, Bain A et al. Role of fluoroquinolones in the primary prophylaxis of spontaneous bacterial peritonitis: meta-analysis. Clin Gastroenterol Hepatol 2009;7:487–93. 70. Saab S, Hernandez JC, Chi AC et al. Oral antibiotic prophylaxis reduces spontaneous bacterial peritonitis occurrence and improves short-term survival in cirrhosis: a meta-analysis. Am J Gastroenterol 2009;104:993–1001. 71. Christou L, Pappas G, Falagas ME: Bacterial infection-related morbidity and mortality in cirrhosis. Am J Gastroenterol 2007;102:1510–7. 72. Foreman MG, Mannino DM, Moss M. Cirrhosis as a risk factor for sepsis and death: analysis of the National Hospital Discharge Survey. Chest 2003;124:1016–20. 73. Bajaj JS, Ananthakrishnan AN, Hafeezullah M et al. Clostridium difficile is associated with poor outcomes in patients with cirrhosis: a national and tertiary center perspective. Am J Gastroenterol 2010;105:106–13. 74. Garcia-Tsao G, Surawicz CM. Editorial: Clostridium difficile infection: yet another predictor of poor outcome in cirrhosis. Am J Gastroenterol 2010;105:114–6. Management of Ascites and Hyponatremia

Andrés Cárdenas and Pere Ginès

CONTENTS MANAGEMENT OF ASCITES MANAGEMENT OF HYPONATREMIA REFERENCES

Key Words: Cirrhosis, Ascites, Hyponatremia, Diuretics, Large-volume paracentesis, TIPS, Vaptans

Patients with cirrhosis frequently develop disturbances in body fluid regulation that result in an increase in the volume of extracellular fluid which accumulates as ascites and/or edema (1). A large body of evidence indicates that ascites formation is the consequence of the homeostatic activation of vasoconstrictor and sodium-retaining systems triggered by marked arterial vasodilation that occurs in the splanchnic circulation (Fig. 1) (reviewed in chapter 12)(2). Marked abnormalities in the splanchnic microcirculation due to portal hypertension facilitate the accumulation of the retained fluid in the peritoneal cavity (reviewed in chapter 15). Ascites is frequently associated with abnormalities of renal function such as impaired ability to eliminate solute-free water and vasoconstriction of the renal circulation which

Grant support: Some of the studies reported in this chapter have been performed with the support of grants from the Fondo de Investigación Sanitaria, Ministerio de Ciencia e Innovación: Fondo de Instituto de Salud Carlos III, FIS08; PI080126, EC07/90077

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_21, C Springer Science+Business Media, LLC 2011

411 412 Cárdenas and Ginès

Cirrhosis

TIPS Portal hypertension Vasodilator factors (NO, peptides, endocannabinoids, CO) Splanchnic arterial vasodilation Abnormal distribution of blood volume Reduced effective arterial blood volume

Stimulation of antinatriuretic/vasoconstrictor systems

Diuretics Increased tubular sodium reabsorption Positive sodium balance Increased capillary filtration coefficient Sodium retention

Paracentesis Ascites

Fig. 1. Proposed pathogenesis of ascites formation in cirrhosis according to the arterial vasodilatation hypothesis and available therapeutic interventions (bold). TIPS, transjugular intrahepatic portosystemic shunt; NO, nitric oxide; CO, carbon monoxide. may lead to development of dilutional hyponatremia and hepatorenal syndrome (HRS), respectively (1, 3). The aim of this chapter is to review the management of ascites and hyponatremia in cirrhosis. The management of hepatorenal syndrome is reviewed in chapter 22.

1. MANAGEMENT OF ASCITES 1.1. Pharmacological Therapy The aim of the pharmacological therapy of ascites is to achieve a negative sodium balance so that patients can get rid of the excess of extracellular ﬂuid accumulated. The restriction of sodium intake may help reduce the amount of ascites particularly in patients who have marked sodium retention and require relatively high doses of diuretics. The recommended sodium intake for these patients is 5 g of salt/day (which is equivalent to approximately 90 mmol of sodium). In patients with only mild renal sodium retention, such important sodium restriction is probably not necessary because a negative sodium balance in these patients can be achieved easily with low doses of diuretics.

1.2. Choice of Diuretics and Efficacy of Treatment Diuretics eliminate the excess extracellular fluid presenting as ascites and edema by increasing renal sodium excretion. The diuretics most frequently used in patients with cirrhosis and ascites are aldosterone antagonists, mainly spironolactone and potassium canrenoate, drugs that selectively antagonize the sodium-retaining effects of aldosterone in the Management of Ascites and Hyponatremia 413 renal collecting tubules, and loop diuretics, especially furosemide, that inhibit the Na+–K+–2Cl– cotransporter in the loop of Henle. Diuretic therapy is effective in the elimination of ascites in 80–90% of patients with ascites, a percentage that increases up to 95% if only patients without renal failure are considered (4–9). The remaining patients either do not respond to diuretic therapy or develop diuretic-induced adverse effects that prevent the use of high doses of these drugs. This condition is known as refractory ascites (10). Despite the use of diuretics in clinical practice for more than 30 years, a few randomized trials have been reported comparing the efficacy of different diuretic agents in the treatment of ascites (6, 7). In patients without renal failure, the aldosterone antagonist spironolactone at a dose of 150 mg/day (increased to 300 mg/day if there was no response) was shown to be more effective than the loop diuretic furosemide at a dose of 80 mg/day (increased to 160 mg/day if there was no response) (6). This increased efficacy of aldosterone antagonists over loop diuretics has also been suggested in several other studies (4, 8, 9). Based on these findings, aldosterone antagonists are considered the diuretics of choice in the management of cirrhotic ascites. In clinical practice, aldosterone antagonists are frequently given in combination with loop diuretics. Theoretical advantages of this combination over aldosterone antagonists alone include a greater natriuretic potency, earlier onset of diuresis, and less tendency to induce hyperkalemia. Two different diuretic regimes have been proposed. In the first regime, the dose of aldosterone antagonists is increased progressively (from 50–100 up to 400 mg/day of spironolactone) and loop diuretics (furosemide from 20–40 up to 160 mg/day) are added only if no response is achieved with the highest dose of spironolactone. In the second regime, the two drugs are given in combination right from the beginning of therapy. Studies comparing both strategies have shown discrepant findings, which are likely due to differences in the population of patients studied (11, 12). On the basis of a detailed analysis of these studies, it can be recommended that patients with their first episode of ascites, who usually have mild sodium retention, should be treated initially with low doses of spironolactone (50–100 mg/day) without loop diuretics to prevent an excessive diuresis that may lead to renal failure, dehydration, and/or hypovolemic hyponatremia. Ideal weight loss in these patients is approximately 500 g/day in patients without edema and 1000 g/day in patients with edema. By contrast, in patients who have recurrent ascites (usually with more intense sodium retention than that of patients with a first episode of ascites), the recommended initial diuretic treatment should be based on the combination of spironolactone and furosemide (100 and 20–40 mg/day, respectively) 414 Cárdenas and Ginès at increasingly progressive doses if there is no response. The administration of spironolactone alone in patients with recurrent ascites induces a high frequency of hyperkalemia compared to the combination therapy (12).

1.3. Side Effects of Diuretics Diuretics should be used judiciously in cirrhosis because of the significant risk of complications during therapy. Some important and common complications are related to disturbances in fluid and electrolyte balance, including hyponatremia, dehydration, renal impairment, and potassium abnormalities (mainly hyperkalemia as hypokalemia occurs only if patients are treated with loop diuretics alone). Patients should be followed closely in clinic and lab tests including renal function tests and serum and urine electrolytes need to be performed at regular intervals. This is particularly important at the beginning of treatment and when the doses of diuretics have been increased to allow for an early detection of impairment of renal function and electrolyte disturbances. Diuretics should be stopped or their dose reduced if some of these abnormalities are detected. Moreover, patients should be instructed to monitor their weight and reduce the diuretic dose and seek medical advice if there is an excessive decrease in body weight. An excessive weight loss over several days may be associated with dehydration and renal failure. Other frequent complications of diuretic therapy include hepatic encephalopathy, gynecomastia, and muscle cramps (4, 13). Hepatic encephalopathy is frequently observed, particularly in those patients with advanced liver disease; yet predictive factors of this complication have not been established. Hepatic encephalopathy due to diuretic therapy is frequently associated with the occurrence of renal impairment or hyponatremia; however, in some patients, encephalopathy may develop in the absence of these complications. Whether diuretics are responsible for an episode of hepatic encephalopathy in a particular patient is very difficult to ascertain in daily practice because there are no established criteria to diagnose diuretic-induced hepatic encephalopathy. In the absence of diagnostic criteria, a simple rule that can be used in practice is that if there is a chronological relationship between treatment with diuretics and occurrence of hepatic encephalopathy in at least two distinct episodes, then diuretics should be considered the trigger of encephalopathy and treatment discontinued. Spironolactone-induced gynecomastia and mastodynia are common; however, they are usually moderate and do not require discontinuation of the drug. Alternative treatments to spironolactone in those patients who require discontinuation are amiloride and eplerenone. However, both drugs have less Management of Ascites and Hyponatremia 415 natriuretic potency than that of spironolactone and their use may be associated with recurrence of ascites (7, 14). Finally, muscle cramps of variable intensity, sometimes severe, may also occur as an adverse effect of diuretics. Administration of albumin (25 g/week) was reported to be effective in reducing the frequency and intensity of muscle cramps (15).

1.4. Therapeutic Paracentesis Therapeutic paracentesis has progressively replaced diuretics over the past few years as the initial treatment in patients with large ascites. This change in treatment strategy is based on the results of several randomized controlled trials comparing paracentesis associated with plasma volume expansion versus diuretic therapy (16–19). Because paracentesis does not affect renal sodium retention, patients should be given diuretics after paracentesis to avoid reaccumulation of fluid (20). Two aspects concerning the use of therapeutic paracentesis in patients with cirrhosis and ascites are worth mentioning: (1) the population of patients with cirrhosis in whom therapeutic paracentesis should be used; and (2) the use of plasma expanders to prevent disturbances in circulatory function. All recent guidelines agree that therapeutic paracentesis with the administration of albumin is the first-line treatment for all patients with large ascites (21–25). Results of randomized trials indicate that therapeutic paracentesis is faster and more effective than diuretics and in several trials was associated with a lower incidence of adverse events compared with diuretics (16–19). Therefore, on the basis of the available evidence it seems clear that therapeutic paracentesis should be considered the first-line treatment for all patients with large ascites (Table 1). The removal of large volumes of ascitic fluid is associated with circulatory dysfunction characterized by reduction of effective blood volume (26–28). Several lines of evidence indicate that this circulatory dysfunction and/or the mechanisms activated to maintain circulatory homeostasis have harmful effects in cirrhotic patients. First, circulatory dysfunction is associated with rapid reaccumulation of ascites (28). Second, approximately 20% of these patients develop hepatorenal syndrome and/or hypervolemic hyponatremia (26). Third, portal pressure increases in patients developing circulatory dysfunction after paracentesis, probably owing to an increased intrahepatic resistance due to the action of vasoconstrictor systems on the hepatic vascular bed (27). Finally, the development of circulatory dysfunction is associated with shortened survival (28). A randomized trial showed that albumin is more effective than other plasma expanders (dextran-70, polygeline) for the prevention of circulatory dysfunction (28). When 416 Cárdenas and Ginès

Table 1 Recommendations for the management of patients with cirrhosis and large-volume ascites

1. Therapeutic paracentesis plus intravenous albumin (8 g/l of ascites removed). Patients can be treated as outpatients. Hospitalization is recommended for patients with associated complications (i.e., encephalopathy, bacterial infection, gastrointestinal bleeding). 2. After removal of ascitic fluid, start with moderate sodium restriction (90 mmol/day) and diuretics, either aldosterone antagonists alone (i.e., spironolactone, starting dose 50–100 mg/day) in patients with the first episode of ascites or in combination with loop diuretics (i.e., furosemide, starting dose 20–40 mg/day) in patients with recurrent ascites. If patients were on diuretics before the development of large-volume ascites, check compliance with sodium diet and diuretic therapy. Compliant patients should be given doses of diuretics higher than those given before paracentesis in order to prevent the recurrence of ascites. Noncompliant patients should be instructed to comply with therapy. less than 5 l of ascites is removed, dextran-70, polygeline, or saline show efficacy similar to that of albumin. However, albumin is more effective when more than 5 l of ascites are removed (28, 29). Despite this greater efficacy in preventing circulatory dysfunction, randomized trials have not shown differences in the survival of patients treated with albumin compared with those treated with other plasma expanders, probably owing to the relatively low sample size of the studies.

1.5. Transjugular Intrahepatic Portosystemic Shunt (TIPS) TIPS was introduced in clinical practice in the 1990s for the management of refractory variceal bleeding with the aim of creating a portosystemic shunt without the need of surgery. The procedure consists of the placement of an intrahepatic stent between one hepatic vein and the portal vein using a transjugular approach. It soon became evident that in patients with variceal bleeding and ascites treated with TIPS, there was a beneﬁcial effect on renal function, leading to the reduction or elimination of ascites in most patients. Initial studies show that TIPS is effective in preventing recurrence of ascites in patients with refractory ascites. This effect is due to reduction in the activity of sodium-retaining mechanisms and improvement of renal function, which results in an improved response to diuretics (30– 33). The main drawbacks of TIPS include shunt stenosis or obstruction Management of Ascites and Hyponatremia 417

(up to 75% of patients treated with uncovered stents develop stenosis within 6–12 months leading to reaccumulation of ascites in most cases) and a high rate of encephalopathy due to the shunting of blood from the splanchnic to the systemic circulation (34, 35). With the use of the new covered stents, this high frequency of obstruction of the shunt has decreased markedly (35, 36). Other adverse effects include impairment in liver function, hemolytic anemia, and heart failure (30–35). Five randomized trials comparing TIPS using uncovered stents versus repeated therapeutic paracentesis with intravenous albumin in patients with cirrhosis and refractory ascites have been published (37– 41). Although there are some discrepancies between studies, the results can be summarized as follows: (1) TIPS is more effective than therapeutic paracentesis in the prevention of recurrence of ascites (37–41). Nonetheless, normalization of renal sodium homeostasis is not completely achieved and most patients treated with TIPS still require sodium restriction and diuretics during follow-up. Large and difficult- to-treat leg edema is a problem in some patients (39, 40). (2)TIPS reduces the risk of occurrence of hepatorenal syndrome type 1 (39). (3) TIPS is associated with an increased risk of severe hepatic encephalopathy and does not reduce significantly the risk of other complications of cirrhosis, such as gastrointestinal bleeding or spontaneous bacterial peritonitis (39, 40). (4) There is a high rate of TIPS stenosis or obstruction that requires frequent intervention to maintain shunt patency (37–41). However, because all these studies used uncovered stents, the use of covered stents may help reduce such a high frequency of obstruction (36). (5) TIPS does not improve quality of life compared with repeated therapeutic paracentesis with intravenous albumin (40). (6) The cost of TIPS is higher than that of repeated therapeutic paracentesis and intravenous albumin (39). (7) TIPS does not improve either overall or transplant-free survival compared with repeated therapeutic paracentesis with intravenous albumin. Several meta-analyses of these randomized controlled studies concluded that TIPS is better at controlling ascites but does not improve survival compared with paracentesis (42–45). A recent meta-analysis that reviewed individual patient data of three trials (38, 40, 41) indicates that TIPS significantly improves transplant-free survival of cirrhotic patients with refractory ascites (45). Although meta-analysis using individual data seems a more reliable tool compared with standard meta-analysis, it cannot overcome the problems of individual studies, such as inclusion of patients who did not have refractory ascites in two of the studies (38, 41), use of large-volume paracentesis without albumin in one study (38), and use of TIPS as rescue therapy in a large proportion of patients treated with large-volume paracentesis in another study 418 Cárdenas and Ginès

(41). All recent treatment guidelines indicate that repeated therapeutic paracenteses and intravenous albumin are the ﬁrst-line treatment for patients with refractory ascites (21–25) and that TIPS should be considered only in patients who require very frequent paracenteses or in whom they are not effective, without contraindications to therapy. The most important contraindications to TIPS are Child–Pugh score >11, recurrent hepatic encephalopathy, serum bilirubin >3 mg/dl, large hepatocellular carcinoma, severe cardiopulmonary disease, and advanced age. The recommendations for the treatment of refractory ascites based on these conclusions are summarized in Table 2. Table 2 Recommendations for the management of patients with cirrhosis and refractory ascites

1. Therapeutic paracentesis plus intravenous albumin (8 g/l of ascites removed). Repeat paracentesis during follow-up whenever needed. Patients can be treated as outpatients. 2. Patients should be on sodium restriction (90 mmol/day) and maximum tolerated doses of diuretics (spironolactone up to 400 mg/day and furosemide up to 160 mg/day): check urine sodium under diuretic therapy. If urine sodium is greater than 30 meq/day, diuretic therapy may be maintained because it may help to delay the recurrence of ascites. If urine sodium is lower than 30 meq/day or diuretic treatment induces complications, diuretics should be withdrawn. 3. Consider the use of transjugular intrahepatic portosystemic shunt (TIPS) in patients with preserved liver function and with low acceptance of treatment with paracenteses or very frequent paracenteses or in those in whom paracenteses are not effective.

2. MANAGEMENT OF HYPONATREMIA Hyponatremia is common in advanced cirrhosis and is usually related to impaired solute-free water excretion primarily due to increased circulating antidiuretic hormone (arginine vasopressin (AVP)) concentration, which results in a disproportionate retention of water relative to sodium (Fig. 2)(46). Interest in hyponatremia was fostered by studies in the late 1970s and 1980s which showed that hyponatremia is an important prognostic indicator in patients with cirrhosis (47). Recent studies extended these observations and showed that hyponatremia is also an important marker of prognosis in patients with cirrhosis awaiting liver transplantation and may be associated with an increased morbidity, particularly neurological complications, and reduced survival after transplantation Management of Ascites and Hyponatremia 419

Cirrhosis Portal hypertension Splanchnic arterial vasodilation

Reduced effective arterial blood volume

Activation of sodium-retaining systems Increased vasopressin secretion

V receptor 2 V receptors on kidney collecting duct cells antagonists 2 Restricted Increased solute-free water retention fluid intake

Dilutional hyponatremia

Fig. 2. Proposed pathogenesis of hypervolemic hyponatremia in cirrhosis and available therapeutic interventions.

(48–52). Hyponatremia in cirrhosis is usually defined as a reduction in serum sodium concentration below 130 mmol/l (46). Patients with cirrhosis may develop two types of hyponatremia: hypovolemic and hypervolemic. Hypovolemic hyponatremia occurs when there is contraction of extracelullar fluid volume secondary to marked sodium and fluid loss, with body sodium depletion, and is most commonly secondary to excessive diuretic therapy. Hypervolemic hyponatremia occurs when there is a reduction in serum sodium concentration with expansion of the extracellular fluid and plasma volume, with ascites and edema. The first step in the management of hyponatremia in cirrhosis is to identify whether hyponatremia is hypovolemic or hypervolemic, because the management differs markedly according to the type of hyponatremia. The management of hypovolemic hyponatremia consists of identification and treatment of the cause of sodium loss together with the administration of sodium. Hypovolemic hyponatremia will not be considered further in this chapter. The key to the management of hypervolemic hyponatremia is to increase renal solute-free water excretion with the aim of normalizing the increased total body water, which would result in an improvement of serum sodium concentration (Fig. 2). There are several potential advantages of treating hyponatremia in cirrhosis. First, the reversal of hyponatremia would allow patients to drink fluids normally and avoid fluid restriction. Second, since hyponatremia has been shown to be a predisposing factor to hepatic encephalopathy (53, 54), the improvement of serum sodium concentration may help reduce the risk 420 Cárdenas and Ginès of encephalopathy. Third, in patients awaiting liver transplantation, the normalization of serum sodium concentration before transplantation may be associated with a reduction in the frequency and severity of neurological complications after transplantation (52). The available therapeutic methods for the management of hypervolemic hyponatremia are summarized below.

2.1. Fluid Restriction Fluid restriction has been the standard of care for the management of hypervolemic hyponatremia in cirrhosis for many years, because of the lack of alternative effective therapies. There have been no studies specifically assessing the effectiveness of fluid restriction in hypervolemic hyponatremia in cirrhosis. It is the clinical experience that fluid restriction is helpful in preventing a progressive decrease in serum sodium levels, although it is rarely effective in improving serum sodium concentration. This lack of efficacy is probably due to the fact that in practice total daily fluid intake cannot be restricted to less than 1 l per day, an amount that is generally insufficient to cause a negative fluid balance.

2.2. Sodium Chloride Administration The usefulness of hypertonic sodium chloride administration in patients with hypervolemic hyponatremia has not been investigated in randomized studies and available information is very scant (46). Nevertheless, it is the clinical experience that hypertonic sodium chloride has a partial and usually short-lived effect in improving serum sodium concentration in cirrhosis probably because it has no effect on renal solute-free water excretion. Moreover, it has the disadvantage of increasing ascites and edema due to the severe sodium retention present in these patients.

2.3. Albumin Administration A few short-term studies including a low number of patients suggest that the administration of albumin improves serum sodium concentration in patients with hypervolemic hyponatremia (55, 56). This beneﬁcial effect of albumin is probably related to an improvement in circulatory function with suppression of several sodium- and water- retaining systems, including AVP. Further studies in a larger series of patients and for prolonged periods of time are needed to assess the potential beneﬁts of albumin administration on hyponatremia. Management of Ascites and Hyponatremia 421

2.4. AVP Antagonists – the Vaptans In recent years, the pharmacological approach to treatment of hyponatremia moved a step forward with the discovery of vaptans, drugs that are active orally and cause a selective blockade of the V2 receptors of AVP in the principal cells of the collecting ducts (57) (Fig. 3). In healthy subjects, the administration of vaptans induces a marked and dose-dependent increase in urine volume with low urine osmolality due to a marked increase in solute-free water excretion, but without an increase in urinary sodium excretion. Randomized, double-blind, comparative studies have demonstrated that treatment with vaptans for a short period of time (up to 1 month), including tolvaptan, lixivaptan, and satavaptan, improves serum sodium concentration in patients with cirrhosis and hypervolemic hyponatremia (58–61). A small retrospective study suggests that conivaptan, a vaptan that is not only an antagonist of the V2 receptors but also of the V1 receptors of AVP, is also effective (62). The increase in serum sodium concentration occurs within the ﬁrst few days of treatment and normalization of serum sodium concentration has been observed in 27–54% of patients (46). Moreover, in approximately one-third of additional patients, serum sodium increases more than 5 mmol/l but does not reach normal values. In these studies, no signiﬁcant effects have been

3A 3B Blood Urine Blood Urine

Gαs cAMP Gαs cAMP H2O H2O AVP PKA AQP2 H O PKA AQP2 AQP2 2 H O AVP AVP AQP2 2 H O V Receptor V Receptor 2 2 AVP 2 H2O H O 2 H2O H2O H2O H2O H2O H O H2O 2 H2O H2O H O Basolateral Principal cell of the Apical Basolateral Principal cell of the Apical 2 membrane Collecting tubule membrane membrane Collecting tubule membrane

Fig. 3. Schematic drawing of the principal cell of the collecting duct and the intracellular action of arginine vasopressin (AVP). (a) The hormone binds to the V2 receptor in the basolateral membrane and activates adenylate cyclase with generation of cyclic adenosine monophosphate (cAMP). Protein kinase (PKA) is the target of cAMP, which then phosphorylates aquaporin 2 (AQP2). This water channel is translocated from the cytoplasm to the luminal membrane, increasing water permeability and allowing water to be reabsorbed into the capillaries through the basolateral membrane. (b) V2 receptor antagonists (vaptans) block the action of AVP by blocking the coupling of AVP to the V2 receptor. Consequently there is no movement of AQP2 to the apical membrane, leading to aquaresis. 422 Cárdenas and Ginès observed on renal and circulatory function and on the activity of the renin–angiotensin–aldosterone system. The most frequent side effect reported in studies evaluating the vaptans in patients with hyponatremia is thirst, which is related to the pharmacodynamic actions of these drugs. Potential theoretical concerns of the administration of vaptans in patients with cirrhosis include hypernatremia and dehydration (a rapid increase in serum sodium concentration which could theoretically lead to osmotic demyelination syndrome) and renal failure due to depletion of the intravascular volume. In short-term studies hypernatremia (serum sodium >145 mmol/l) occurred in only 2–4% of patients treated (46). Nevertheless, the frequency of this complication may be higher if patients are treated with high doses of vaptans. An important concern is to avoid a rapid increase in serum sodium concentration which could lead to osmotic demyelination syndrome. An increase of serum sodium concentration of greater than 8 mmol/l per day within the first days of therapy has been reported with low and similar frequency in patients treated with vaptans compared to patients treated with placebo, ranging from 4 to 14% in different studies (58, 59, 61). Moreover, osmotic demyelination syndrome has not been reported thus far in any of the studies. Nevertheless, it is important to emphasize that in all studies patients were treated in the hospital for the first days of therapy, had free access to water, and followed strict investigation protocols with daily measurement of serum sodium during the first days of therapy and temporary interruption of drug administration in patients in whom serum sodium increased more than 8 mmol/l per day. In short-term studies, no significant impairment of renal function was found in vaptan-treated groups compared to placebo. Nonetheless, it should be pointed out that in these studies patients were treated for short periods of time, under strict clinical and analytical surveillance, and with low doses of diuretics. Therefore, it is not known whether the frequency of renal impairment could be higher under different study conditions. Finally, vaptans are metabolized by CYP3A enzymes in the liver; therefore drugs that are strong inhibitors of CYP3A, such as ketoconazole, grapefruit juice, and clarithromycin among others, increase the exposure to vaptans and may be associated with larger increases in serum sodium concentration. By contrast, drugs that are inducers of the CYP3A system, such as rifampin, barbiturates, and phenytoin, may decrease the effectiveness of vaptans. A phase 3 randomized double-blind placebo-controlled study comparing the efficacy of long-term treatment with satavaptan in combination with diuretics aimed at preventing ascites recurrence in patients with cirrhosis treated with LVP and not to improve serum sodium concentration showed an increased frequency of complications and Management of Ascites and Hyponatremia 423 reduced survival in patients receiving the drug compared to those receiving placebo. It is not known if this increased mortality during long-term treatment is a class effect or is exclusively related to satavaptan. Therefore, although vaptans are effective and safe in the short-term management of hyponatremia (up to 1 month), their long-term safety remains to be evaluated in future studies. Among the different vaptans, tolvaptan has recently been approved in the United States for the management of severe (<125 mmol/l) hypervolemic hyponatremia and in Europe for the management of SIADH. Conivaptan is also approved in the United States for the short-term (5 days) intravenous treatment of hypervolemic hyponatremia. Treatment with tolvaptan is started with 15 mg/day and titrated progressively to 30 and 60 mg/day, if needed, according to the desired changes in serum sodium concentration. In randomized studies, a slightly increased frequency of gastrointestinal bleeding was reported in patients with cirrhosis and hyponatremia receiving tolvaptan compared

Table 3 Recommendations for the management of hypervolemic hyponatremia in cirrhosis

1. Fluid restriction to 1000–1500 ml/day. 2. If fluid restriction is not effective, tolvaptan is the drug of choice, as there are very limited data on the efficacy of conivaptan. 3. Tolvaptan treatment should be started in the hospital at a starting dose of 15 mg/day. This dose should be given for the first few days and then the dose should be titrated (to 30 and 60 mg/day) to achieve a slow increase in serum sodium concentration. Serum sodium concentration should be monitored closely particularly during the first days of treatment and whenever the dose of the drug is increased. 4. Rapid increases in serum sodium concentration (of greater than 8 mmol/day) should be avoided to prevent the potential occurrence of osmotic demyelination syndrome. 5. Neither fluid restriction nor administration of saline should be used in combination with vaptans to avoid a too rapid increase in serum sodium concentration. 6. Patients may be discharged after serum sodium levels are stable and no further increase in the dose of the drug is required. 7. Treatment with drugs that are either potent inhibitors or inducers of the CYP3A should be avoided. 8. The duration of treatment with tolvaptan is not known. Safety has been established only for short-term treatment (1 month). 424 Cárdenas and Ginès to that in patients treated with placebo. This would require evaluation in future studies. No prospective evaluation on the efficacy and safety of conivaptan has been performed in patients with cirrhosis and hyponatremia. On the basis of available evidence, the recommendations for the management of hypervolemic hyponatremia in cirrhosis are summarized in Table 3.

REFERENCES

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Management of Renal Failure

Vicente Arroyo, MD and Mónica Guevara, MD

CONTENTS INTRODUCTION DIAGNOSIS OF RENAL FAILURE AND HRS IN CIRRHOSIS PATHOGENESIS OF HRS IN CIRRHOSIS TREATMENTS OF TYPE 1 HRS TREATMENTS FOR TYPE 2 HRS REFERENCES

Key Words: Cirrhosis, Ascites, Renal failure, Hepatorenal syndrome, Albumin, Terlipressin

1. INTRODUCTION Renal failure is a common complication of cirrhosis with ascites. The most frequent type of renal failure in cirrhosis is that induced by diuretics. Ascites reabsorption is a rate-limited phenomenon and if diuretics increase urine volume at a rate greater than the passage of ascites from the peritoneal cavity to the systemic circulation, a contraction of the effective plasma volume occurs leading to renal hypoperfusion and reduction in GFR (1). Diuretic-induced renal failure

Supported in part by grants from Fondo de Investigación Sanitaria FIS070443 and 080108. Centro de investigaciones en red de enfermedades hepaticas y digestivas (CIBEREHD) is supported by the Instituto de Salud Carlos III.

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_22, C Springer Science+Business Media, LLC 2011

429 430 Arroyo and Guevara is rapidly reversible after discontinuation of therapy and usually does not progress to more severe types of renal failure, such as hepatorenal syndrome (HRS) or acute tubular necrosis (ATN). Acute bacterial infection is also frequently associated with renal failure (2, 3), and usually reverses after infection resolves. However, renal failure associated with infections may progress to HRS. Hemorrhagic shock due to variceal bleeding or sepsis can produce ATN. Patients with decompensated cirrhosis are also predisposed to develop drug-induced acute renal failure (nonsteroidal anti-inflammatory drugs and nephrotoxic antibiotics). Glomerulonephritis due to IgA deposits in alcoholic cirrhosis or associated with hepatitis B and hepatitis C viral infection is relatively common in patients with compensated and decompensated cirrhosis. HRS is, however, the most characteristic type of renal failure in cirrhosis. The annual incidence of HRS in patients with cirrhosis and ascites has been estimated as 8%. It is characterized by intense renal vasoconstriction, which leads to very low renal perfusion and glomerular filtration rate (GFR). Renal histology shows no lesions sufficient to explain the impairment in renal function. The current chapter offers a review of the pathogenesis, clinical aspects, prevention, and treatment of HRS. The other types of renal failure are only marginally considered. The reader should consult other reviews published recently (4–10) as well as the reports of two consensus conferences on HRS organized by the International Ascites Club in Chicago and San Francisco (11, 12).

2. DIAGNOSIS OF RENAL FAILURE AND HRS IN CIRRHOSIS The first step in the diagnosis of HRS is the demonstration of a reduced GFR, and this is not easy in advanced cirrhosis. The muscle mass, and therefore, the release of creatinine, is reduced in these patients who may present with a normal serum creatinine concentration in the setting of a very low GFR. Therefore, a false-negative diagnosis of renal failure is relatively common (13–15). There is consensus to establish the diagnosis of HRS when serum creatinine has risen above 1.5 mg/dl (11). The second step is the differentiation of HRS from other types of renal failure. For many years, this was based on traditional parameters used to differentiate functional renal failure from acute tubular necrosis (low urine volume and urine sodium concentration and high urine-to-plasma osmolality ratio). However, acute tubular necrosis in patients with cirrhosis and ascites usually occurs with oliguria, low urine sodium concentration, and urine osmolality greater than Management of Renal Failure 431 plasma osmolality (16). On the contrary, relatively high urinary sodium concentration has been reported in patients with HRS (17). Because of the lack of specific tests, diagnosis of HRS is based on the exclusion of other disorders that can cause renal failure in cirrhosis (Table 1)(11). Acute renal failure of prerenal origin due to renal (diuretics) or extrarenal fluid losses should be investigated. If renal failure is secondary to volume depletion, renal function improves rapidly after volume expansion, whereas no improvement occurs in HRS. Even if there is no history of fluid losses, renal function in patients with renal failure should be assessed after diuretic withdrawal and volume replacement to rule out any subtle reduction in plasma volume as the cause of renal failure. The diagnostic criteria of HRS proposed by the International Ascites Club in 2005 consider that volume replacement should be performed with intravenous albumin rather than with saline (11, 12). This proposal is based on a randomized study showing that albumin is more effective as a plasma expander than a saline solution of hydroxyethyl starch in patients with SBP (18). The presence of shock before the onset of renal failure points toward a diagnosis of acute tubular necrosis. Cirrhotic patients with infections may develop transient renal failure, which resolves after resolution of the infection (2, 3). Therefore, HRS in cirrhotic patients with bacterial infections should be diagnosed in patients without septic shock and only if renal failure does not improve following antibiotic administration. Complete resolution of the infection is not currently required for the diagnosis of HRS because it may delay the initiation of treatment. Cirrhotic patients are predisposed to develop renal failure in the setting of treatments with aminoglycosides (19), nonsteroidal anti-inflammatory drugs (20), and

Table 1 Diagnostic criteria of hepatorenal syndrome

1. Cirrhosis with ascites 2. Serum creatinine >133 μmol/l (1.5 mg/dl) 3. No improvement of serum creatinine (decreased to level of 133 μmol/l or less) after at least 2 days with diuretic withdrawal and volume expansion with albumin. The recommended dose of albumin is 1 g/kg of body weight per day up to a maximum of 100 g/day 4. Absence of shock 5. No current or recent treatment with nephrotoxic drugs 6. Absence of parenchymal kidney disease indicated by proteinuria >500 mg/day, microhematuria (>50 red blood cells per high-power field), and/or abnormal renal ultrasonography 432 Arroyo and Guevara vasodilators (renin–angiotensin system inhibitors, prazosin, nitrates) (21). Therefore, treatment with these drugs in the days preceding the diagnosis of renal failure should be ruled out. Finally, renal failure due to glomerulonephritis is associated with proteinuria, hematuria or both, or abnormal renal ultrasonography (small irregular kidneys with abnormal echostructure). There are two types of HRS (12). Type 1 HRS is characterized by a severe and rapidly progressive renal failure, which has been defined as doubling of serum creatinine reaching a level greater than 2.5 mg/dl in less than 2 weeks. Although type 1 HRS may arise spontaneously, it frequently occurs in close relationship with a precipitating factor, such as severe bacterial infection, gastrointestinal hemorrhage, major surgical procedure, or acute hepatitis superimposed on cirrhosis. The association of HRS, spontaneous bacterial peritonitis (SBP), and other bacterial infections has been carefully investigated (2, 3, 22). Type 1 HRS develops in approximately 25% of patients with SBP despite a rapid resolution of the infection. Patients with severe circulatory dysfunction prior to infection or intense inflammatory response (high concentration of polymorphonuclear leukocytes in ascitic fluid and high cytokine levels in plasma and ascitic fluid) are prone to develop type 1 HRS after the infection. Besides renal failure, patients with type 1 HRS associated with SBP show signs and symptoms of rapid and severe deterioration of liver function (jaundice, coagulopathy, and hepatic encephalopathy) and circulatory function (arterial hypotension and very high plasma levels of renin and norepinephrine). It is interesting to note that at variance with SBP, sepsis related with other types of infection in patients with cirrhosis is rarely associated with type 1 HRS. In one study, sepsis unrelated to SBP induced type 1 HRS only in the setting of lack of response to antibiotics (23). In most patients with sepsis unrelated to SBP responding to antibiotics, renal impairment, which was also a frequent event, was reversible. In a second study (24), the prevalence of HRS was 30% in patients with SBP, 19% in patients with severe acute urinary tract infection, and only 4% in patients with sepsis of other origin. Interestingly enough, as in SBP, some patients with severe urinary tract infection developed type 1 HRS despite the resolution of the infection. The mechanism for the higher frequency of HRS in SBP as compared with other bacterial infections is unknown. Without treatment, type 1 HRS is the complication of cirrhosis with the poorest prognosis with a median survival time after the onset of renal failure of only 2 weeks (Fig. 1)(25, 26). Type 2 HRS is characterized by a moderate and slowly progressive renal failure (serum creatinine lower than 2.5 mg/dl) (11, 26). Patients with type 2 HRS show signs of liver failure and arterial hypotension, Management of Renal Failure 433

1,0 Median survival time Type 1 15 days 0,8 Type 2 150 days

0,6

0,4 Type 2 Probability

P < 0,0001 0,2 Type 1

0,0 100 200 300 400 500 600 days

Fig. 1. Survival of patients with cirrhosis after the diagnosis of type 1 or type 2HRS. but to a lesser degree than patients with type 1 HRS. The dominant clinical feature is severe ascites with poor or no response to diuretics (a condition known as refractory ascites). Patients with type 2 HRS are predisposed to develop type 1 HRS following infections or other precipitating events (2, 3, 22). Median survival of patients with type 2 HRS (6 months) is worse than that of patients with nonazotemic cirrhosis with ascites (11, 26, 27)(Fig.1).

3. PATHOGENESIS OF HRS IN CIRRHOSIS The development of portal hypertension in cirrhosis is associated with arterial vasodilation in the splanchnic circulation due to increased local release or production of local vasodilators including nitric oxide, carbon monoxide, and endogenous cannabinoids (28–31). According to the “peripheral arterial vasodilation hypothesis,” HRS would be the extreme expression of this splanchnic arterial vasodilation, which would increase steadily with the progression of the disease (29–32).At the initial phases of cirrhosis, the decrease in systemic vascular resistance is compensated by the development of a hyperdynamic circulation (increased heart rate and cardiac output). However, as the disease progresses and arterial vasodilation increases, the hyperdynamic circulation is insufﬁcient to correct the effective arterial hipovolemia (Fig. 2). Arterial hypotension develops, leading to activation of high-pressure baroreceptors, reﬂex stimulation of the renin–angiotensin and sympathetic nervous systems, increase in arterial pressure to normal or near-normal levels, sodium and water retention, and ascites formation. The stimulation of antidiuretic hormone occurs later during the course of the disease. Patients then develop water retention and dilutional 434 Arroyo and Guevara

Fig. 2. Peripheral arterial vasodilation hypothesis and renal dysfunction in cirrhosis. At the initial phases, when cirrhosis is compensated, the increase in splanchnic arterial vasodilation is compensated by an increase in cardiac output (hyperdynamic circulation). The effective arterial blood volume and the activity of renin–angiotensin system, sympathetic nervous system, and plasma ADH are normal despite a reduction in systemic vascular resistance. With the progression of liver disease, splanchnic arterial vasodilation increases but not the cardiac output. An effective arterial hypovolemia therefore develops leading to activation of the renin–angiotensin and sympathetic nervous systems and ADH. Systemic vascular resistance does not decrease due to vasoconstriction in extrasplanchnic organs. Type 2 HRS could be the extreme expression of renal vasoconstriction. hyponatremia. At this stage of the disease, the renin–angiotensin and sympathetic nervous systems are markedly stimulated and arterial pressure is critically dependent on the vascular effect of the sympathetic nervous activity, angiotensin-II, and antidiuretic hormone. Since the splanchnic circulation is resistant to the effect of angiotensin-II, noradrenaline, and vasopressin due to the local release of vasodilators (33, 34), the maintenance of arterial pressure is related to vasoconstriction in extrasplanchnic vascular territories such as the kidneys, muscle and skin, and brain (35–37). HRS develops at the latest phase of the disease when there is extreme deterioration in effective arterial blood Management of Renal Failure 435 volume and intense stimulation of the renin–angiotensin system, sympathetic nervous system, and antidiuretic hormone, leading to marked renal vasoconstriction and decrease in GFR (32). HRS has traditionally been assumed to develop in the setting of a progression of the hyperdynamic circulation. Despite that, in a few studies assessing cardiovascular function in patients with HRS or refractory ascites, cardiac output was found to be significantly reduced compared to patients without HRS (38, 39). In some cases cardiac output was even lower than in normal subjects. Two recent studies by Ruiz-del-Arbol et al. confirmed this finding (40, 41). In the first study (40), systemic and hepatic hemodynamics and the endogenous vasoactive systems were measured in 23 cirrhotic patients with SBP at the diagnosis of infection and following SBP resolution. Eight patients developed type 1 HRS. The remaining 15 patients did not develop renal failure. Development of type 1 HRS was associated with a significant decrease in mean arterial pressure and a marked stimulation of the renin–angiotensin and sympathetic nervous systems, indicating a severe impairment in effective arterial blood volume. Peripheral vascular resistance did not change despite the intense stimulation of these endogenous vasoconstrictor systems, which is consistent with a progression of the arterial vasodilation already present in these patients. The most important result of the study, however, was the observation of a marked decrease in cardiac output in all cases. These changes were not observed in patients not developing renal failure. Impairment in systemic hemodynamics and type 1 HRS associated with SBP was, therefore, clearly related with the simultaneous occurrence of a decrease in cardiac output and an accentuation of the arterial vasodilation. In this study, patients who developed type 1 HRS showed significantly higher values of cytokines, plasma renin activity, and sympathetic nervous activity and lower cardiac output and glomerular filtration rate at infection diagnosis than patients not developing renal failure. These data confirm previous studies showing that in patients with SBP the severity of the inflammatory response and the degree of impairment of systemic hemodynamics and renal function prior to the infection are important predictors of type 1 HRS (22). The second study (41) consisted of a longitudinal investigation of 66 nonazotemic cirrhotic patients with ascites. Forty percent of patients developed HRS (type 1 or type 2). These patients were studied at inclusion and following the development of HRS. In the initial study, those patients who went on to develop HRS had significantly lower mean arterial pressure and cardiac output, and significantly higher plasma renin activity and norepinephrine concentration compared with those who did not develop HRS. Moreover, those who developed HRS had a further decrease in arterial pressure and cardiac output and increase in renin 436 Arroyo and Guevara and norepinephrine without changes in peripheral vascular resistance. These findings further support that circulatory dysfunction and HRS in cirrhosis are due to both an increase in arterial vasodilation and a decrease in cardiac function (Fig. 3). The results of these studies have recently been confirmed by Krag et al., who showed that development of renal failure and poor outcome in patients with cirrhosis and ascites are related to cardiac system dysfunction leading to reduced cardiac output (42). The mechanism of renal vasoconstriction that causes HRS is complex. Since renal perfusion in decompensated cirrhosis correlates inversely with the activity of the renin–angiotensin and sympathetic nervous systems (43, 44), HRS is thought to be related to the extreme stimulation of these systems. The urinary excretion of prostaglandin E2, 6-keto prostaglandin F1α (a prostacyclin metabolite), and kallikrein is decreased in patients with HRS, which is compatible with a reduced renal production of these vasodilatory substances (45, 46). Renal failure

Fig. 3. Peripheral vasodilation hypothesis (upper graph) and modified peripheral vasodilation hypothesis (lower graph). According to the latter hypothesis, impairment in arterial blood volume in cirrhosis could be the consequence of a progression of splanchnic arterial vasodilation and a decrease in cardiac output. RAAS: renin angiotensin aldosterone system; SNS: sympathetic nervos system; ADH: antidiuretic hormone; HRS: Hepatorenal Syndrome. Management of Renal Failure 437 in HRS could, therefore, be the consequence of an imbalance between the activity of the systemic vasoconstrictor systems and the renal production of vasodilators. Additionally, once renal vasoconstriction develops, it could be amplified by the stimulation of other intrarenal vasoactive systems. For example, renal ischemia increases the generation of angiotensin-II by the juxtaglomerular apparatus, the intrarenal production of adenosine, which, in addition to being a renal vasoconstrictor, potentiates the vascular effect of angiotensin-II, and the synthesis of endothelin (47). Other intrarenal vasoconstrictors that have been implicated in HRS are leukotrienes and F2-isoprostanes (48). Renal vasoconstriction in HRS is, therefore, related to the simultaneous effect of numerous vasoactive mechanisms on the intrarenal circulation. Brachial and femoral blood flows are markedly reduced in patients with HRS, indicating a vasoconstriction in the cutaneous and muscular arterial vascular beds (36). The resistive index in the mean cerebral artery is also increased in these patients, indicating cerebral vasoconstriction (49)(Fig.4). The degree of vasoconstriction in these vascular territories in decompensated cirrhosis (patients with ascites with and without HRS) correlates directly with the degree of renal vasoconstriction and with the plasma levels of renin. Impairment in circulatory function in cirrhosis is therefore associated with generalized nonsplanchnic arterial vasoconstriction (Fig. 5). Patients with type 2 HRS and refractory ascites frequently present with muscle cramps. Although the pathogenesis of this abnormality is unknown, muscle cramps disappear or improve following plasma volume expansion with albumin (50), suggesting that they could be related with this reduction of muscular blood flow. Hepatic encephalopathy is common in patients with HRS. There are many possible mechanisms of this complication, including the precipitating event of HRS, which can also cause hepatic encephalopathy, and the deterioration of hepatic function seen in these patients. Cerebral vasoconstriction, however, could be an additional factor (51). Angiotensin-II, noradrenaline, and vasopressin have powerful effects on the intrahepatic circulation. They produce arterial vasoconstriction and increase the intrahepatic resistance to portal venous flow at different levels (small portal venules, sinusoids, and small hepatic venules). In patients with cirrhosis, these effects are increased due to a reduced intrahepatic synthesis of nitric oxide (52, 53). It is, therefore, not sur- prising that the stimulation of the endogenous vasoactive systems in HRS could be associated with an aggravation of portal hypertension and a marked reduction in hepatic blood flow. This has recently been shown by Ruiz-del-Arbol et al. (41). They studied hepatic hemodynamics in a large series of nonazotemic cirrhotics with tense ascites when 438 Arroyo and Guevara

Fig. 4. (Upper graph) Resistive index in the middle cerebral artery in patients with compensated cirrhosis, patients with ascites, and healthy subjects. (Lower graph) Relationship between the renal resistive index and the resistive index in the middle cerebral artery in cirrhotic patients. they had normal serum creatinine concentration and after a follow-up of several months when patients had developed type 1 or type 2 HRS. The hepatic venous pressure gradient (HVPG) was significantly higher in the follow-up study than in the baseline study in patients developing type 1 HRS. Type 1 HRS was also associated with a dramatic reduction in hepatic blood flow. In patients developing type 2 HRS, significant differences were observed only in the hepatic blood flow. In a second investigation from the same group, hepatic hemodynamics was assessed in patients with SBP at infection diagnosis and following infection resolution (40). There was only a week interval between both studies. HVPG increased markedly in patients who developed type 1 HRS but not in patients who did not develop renal failure. Changes in intrahepatic hemodynamics in the two studies correlated significantly with the Management of Renal Failure 439

Fig. 5. HRS as a part of a multiorgan failure. HRS: hepatorenal syndrome; AII: Angiotensin II; NE: norepinephrine; ADH: antidiuretic hormone. increase in plasma renin activity, suggesting that circulatory dysfunction associated with hepatorenal syndrome and the secondary activation of the endogenous vasoconstrictor systems adversely influences intrahepatic hemodynamics. Acute deterioration of hepatic function is a common event in patients with type 1 HRS. Variceal bleeding is also frequent in patients with severe bacterial infections and HRS. The intense reduction in hepatic blood flow and the increase in portal pressure associated with type 1 HRS could play a role in the development of these complications. Arterial vasodilation is followed by an appropriate response of the vasoactive neurohormonal systems in patients with type 2 HRS. There is a marked increase in the plasma levels of renin and norepinephrine and vasoconstriction in the extrasplanchnic organs that maintains arterial pressure (36, 49). However, the cardiac response is clearly abnormal in these patients. Cardiac output, which should increase in response to the fall in peripheral vascular resistance, decreases. Moreover, despite the intense activation of the sympathetic nervous activity, no change in heart rate is observed (41). These data indicate a clear impairment in cardiac inotropic and chronotropic functions. In patients with type 1 HRS the deterioration of cardiac function is even much more evident. Type 1 HRS occurs in the setting of a severe decrease in cardiac output, which may reach values below normal. The heart rate remains unchanged despite a dramatic activation of the renin–angiotensin and sympathetic nervous systems (41). The pathogenesis of this impaired 440 Arroyo and Guevara cardiac response to arterial vasodilation in HRS is unknown. A specific cardiomyopathy characterized by attenuated systolic and diastolic responses to stress stimuli, electrophysiological repolarization changes, and enlargement and hypertrophy of cardiac chambers is common in patients with advanced cirrhosis (54). This cirrhotic cardiomyopathy has been suggested to play a role in the pathogenesis of heart failure seen after the insertion of a transjugular intrahepatic portosystemic stent-shunt (TIPS) (55, 56), major surgery, or liver transplantation (57, 58)andinHRS(40, 41). Other features, however, suggest that the impairment in the cardiac inotropic function in HRS is not organic but mainly functional in nature and related to a decrease in venous return. First, the reduced cardiac output in patients with HRS occurs in the setting of a decrease in cardiopulmonary pressures, which is compatible with a fall in cardiac preload (41). Second, circulatory dysfunction in HRS can be reversed by the intravenous administration of albumin associated with vasoconstrictors, or after the insertion of a TIPS (4, 59, 60). Both treatments increase venous return and cardiac output. Finally, expansion of plasma volume with albumin is highly effective in the prevention of type 1 HRS in patients with SBP (61). The impairment of cardiac chronotropic function has been attributed to downregulation of β-adrenergic receptors secondary to the chronic stimulation of the sympathetic nervous system. Two recent studies indicate that relative adrenal insufficiency is frequent in patients with HRS associated with sepsis. In the first study (62), adrenal insufficiency was detected in 80% of patients with HRS but only in 34% with serum creatinine below 1.5 mg/dl. Other features associated with adrenal insufficiency were severe liver failure, arterial hypotension and vasopressor dependency, and hospital mortality. Since normal adrenal function is essential for an adequate response of the arterial circulation to endogenous vasoconstrictors, adrenal insufficiency could be an important contributory mechanism of circulatory dysfunction associated with HRS in patients with severe bacterial infections. In the second study (63), data were presented showing that treatment with hydrocortisone in cirrhotic patients with severe sepsis and adrenal insufficiency was associated with rapid improvement in systemic hemodynamics, reduction in vasoconstrictor requirements, and high hospital survival. The mechanism of adrenal dysfunction in cirrhosis with severe sepsis has not been explored. Since adrenal dysfunction is particularly prevalent in patients with HRS, a reduction in adrenal blood flow secondary to regional vasoconstriction is a possible mechanism. Cytokines directly inhibit adrenal cortisol synthesis. The inflammatory reaction associated with bacterial infections is, therefore, another potential mechanism. Management of Renal Failure 441

4. TREATMENTS OF TYPE 1 HRS 4.1. Liver Transplantation Liver transplantation is the treatment of choice of any patient with advanced cirrhosis, including those with type 1 and type 2 HRS (64–67). Immediately after transplantation a further impairment in GFR may be observed and many patients require hemodialysis (35% of patients with HRS as compared with 5% of patients without HRS) (65). Because cyclosporine or tacrolimus may contribute to this impairment in renal function, it has been suggested to delay the administration of these drugs until a recovery of renal function is noted, usually 48–72 h after transplantation. After this initial impairment in renal function, GFR starts to improve and reaches an average of 30–40 ml/min by 1–2 months postoperatively. This moderate renal failure persists during follow-up, is more marked than that observed in transplantation patients without HRS, and is probably due to a greater nephrotoxicity of cyclosporine or tacrolimus in patients with renal impairment prior to transplantation. The hemodynamic and neurohormonal abnormalities associated with HRS disappear within the ﬁrst month after the operation and the patients regain a normal ability to excrete sodium and free water. Patients with HRS who undergo transplantation have more complications, spend more days in the intensive care unit, and have a higher in-hospital mortality rate than transplantation patients without HRS. The long-term survival of patients with HRS who undergo liver transplantation is however good, with a 3-year probability of survival of 60%. This survival rate is only slightly reduced compared with that of transplantation in patients without HRS (which ranges between 70 and 80%) (67, 68). The main problem of liver transplantation in type 1 HRS is its applicability. Due to their extremely short survival, most patients die before transplantation. The introduction of the MELD score for listing has partially solved the problem since patients with HRS are generally allocated a higher priority on the waiting list. Treatment of HRS with vasoconstrictors and albumin (see below) increases survival in a signiﬁcant proportion of patients and, therefore, the number of patients reaching transplantation before death, decreases early morbidity and mortality after transplantation, and prolongs the long-term survival (69).

4.2. Vasoconstrictors and Albumin The administration of vasoconstrictor agents (intravenous terlipressin, or noradrenaline, or oral midodrine) and intravenous albumin infusion is an effective therapy of type 1 HRS. Numerous studies have 442 Arroyo and Guevara shown reversal of HRS (decrease of serum creatinine below 1.5 mg/dl) in 40–60% of patients (70–86). Type 1 HRS may recur after discontinuation of the treatment in a minority of patients but they may respond again after retreatment. These features contrast sharply with studies in patients with type 1 HRS treated with plasma volume expansion alone or associated with vasodilators (dopamine) or octreotide or with peritoneovenous shunting. Reversal of HRS was observed in fewer than 5% of affected patients in these studies. Survival at 3 months is approximately 30% in patients treated with vasoconstrictors and albumin, and 0% in patients using other treatments. Survival of patients responding to vasoconstrictors and albumin is significantly longer than that of patients not responding to this treatment (83, 86). Terlipressin is the most widely used vasoconstrictor agent in type 1 HRS. It is very effective and is associated with a low incidence of side effects. Noradrenaline has also been shown to be effective and safe and there is a randomized controlled trial in a small number of patients with type 1 and type 2 HRS (mainly type 2) indicating that it is as effective as terlipressin (87). Whereas there is a large experience with terlipressin, noradrenaline has been used in only a few studies. Reversal of type 1 HRS in studies in which terlipressin was given alone (25%) (75, 79) was lower than that in studies in which vasoconstrictors were associated with intravenous albumin, suggesting that the albumin is an important component in the pharmacological treatment of type 1 HRS. The beneficial effect of albumin on circulatory and renal function in patients with type 1 HRS is related not only to the expansion of the plasma volume but also to a direct vasoconstrictor effect on the peripheral arterial circulation (88). Terlipressin dosage should be progressive, starting with 0.5 mg/4 h. If serum creatinine does not decrease by more than 30% in 3 days, the dose should be doubled. The maximal dose of terlipressin has not been defined, although there was consensus that patients not responding to a dose of 2 mg/day will not respond to higher doses. Albumin should be given starting with a priming dose of 1 g/kg body weight followed by 20–40 g/day. It is advisable to monitor central venous pressure. In patients responding to therapy, treatment should be kept until normalization of serum creatinine (<1.5 mg/dl). There are preliminary data suggesting that the efficacy of terlipressin may be higher if given as continuous infusion.

4.3. Transjugular Intrahepatic Portosystemic Shunt (TIPS) Three pilot studies have evaluated TIPS in type 1 HRS (59, 60, 82). In the ﬁrst study (60), 14 patients with type 1 HRS and 17 with refractory ascites (some of them with type 2 HRS) not suitable for liver Management of Renal Failure 443 transplantation were treated. Patients with bilirubin >15 mg/dl, Child– Pugh score >12, or hepatic encephalopathy were excluded. Eleven out of the 31 patients developed de novo hepatic encephalopathy or deterioration of previous hepatic encephalopathy. The 3-, 6-, and 12-month survival rates in patients with type 1 HRS were 64, 50, and 20%, respectively. The second study (59) was performed in seven patients (four alcoholics) with type 1 HRS and a Child–Pugh score <12. A marked decrease in serum creatinine was observed in six patients and reversal of HRS in 4 patients. Five patients developed episodes of hepatic encephalopathy after TIPS but they responded satisfactorily to medical treatment. Five patients were alive after 1 month of TIPS but only two after 3 months. The third study (82) was performed in 14 patients with type 1 HRS treated initially with vasoconstrictors (midodrine and octreotide) plus albumin. Reversal of HRS was observed in 10 patients. TIPS was subsequently inserted in 5 of these 10 patients who had bilirubin <5 mg/dl, INR <2, and Child–Pugh score <12. Normalization of GFR was obtained in all patients and they were alive 6–30 months following TIPS. TIPS, therefore, may be an alternative treatment of type 1 HRS in patients with relatively preserved hepatic function.

4.4. Extracorporeal Albumin Dialysis (MARS) Three pilot studies aimed at assessing MARS in patients with type 1 HRS have been reported (89–91). Since MARS incorporates a standard dialysis machine or a continuous veno-venous hemofiltration monitor, the decrease in serum creatinine observed in most patients could be related to the dialysis process. In fact, a recent study has shown that MARS treatment is not associated with an increase in GFR (92). However, clear beneficial effects on systemic hemodynamics and on hepatic encephalopathy were observed. The survival rate 1 and 3 months after treatment was 41 and 34%, respectively. Two randomized controlled trials in a large series of cirrhotic patients with hepatic encephalopathy or acute-on-chronic liver failure (93), many of them with HRS, have demonstrated a clear beneficial effect of MARS on the rate and time of recovery of encephalopathy. Survival, however, was not affected.

5. TREATMENTS FOR TYPE 2 HRS 5.1. Transjugular Intrahepatic Portosystemic Shunt Five trials comparing TIPS vs. paracentesis in patients with refractory and/or recidivant ascites have so far been published (55, 94–97). Unfortunately, very few of these patients had HRS. Therefore, data 444 Arroyo and Guevara from these trials are not valid for the assessment of TIPS in type 2 HRS. There are only two pilot studies specifically assessing TIPS in type 2HRS(60, 87). In one study (87), a marked reduction of serum creatinine was observed in eight out of nine patients, associated with a significant improvement in the control of ascites. Four of these patients died, two within the first month and two 12 and 14 months after the procedure. The remaining five patients had longer survival. No data were given on the type and rate of complications associated to TIPS. The second study included 14 patients with type 1 HRS and 17 with type 2 HRS treated by TIPS (60). A significant improvement in serum creatinine and creatinine clearance was observed in the whole group of 31 patients as well as an improvement in the control of ascites in 24 cases. Six patients developed TIPS dysfunction and 11 developed hepatic encephalopathy during follow-up. The 1-year probability of survival in the 17 patients with type 2 HRS treated by TIPS was 70%. TIPS is therefore effective in reversing type 2 HRS, although more data on complication rate and survival are needed. The introduction of covered stents should be a stimulus to reevaluate the role of TIPS in the management of refractory ascites and type 2 HRS.

5.2. Vasoconstrictors and Albumin Three pilot studies provided data on the effect of terlipressin plus albumin in patients with type 2 HRS (73, 79, 87). Reversal of HRS was obtained in most cases. In one of these studies (87), the course of renal function after stopping treatment was assessed, and HRS recurred in all cases. There were no data on survival. This high prevalence of HRS recurrence has recently been conﬁrmed in a second study of Alessandria et al. (98). In a randomized comparative study of terlipressin vs. noradrenaline in patients with type 1 and type 2 HRS, HRS recurrence was observed in eight patients, ﬁve with type 2 HRS. It appears that vasoconstrictors in type 2 HRS are not as effective as in type 1 HRS due to the high rate of recurrence.

5.3. Prevention of HRS Three randomized controlled studies have shown that HRS can be prevented in speciﬁc clinical settings. The administration of albumin (1.5 g/kg intravenously at infection diagnosis and 1 g/kg 48 h later) to patients with cirrhosis and SBP markedly reduced the incidence of type 1 HRS (10% vs. 33%), hospital mortality rate (10% vs. 29%), and the 3-month mortality rate (22% vs. 41%) (61). Primary prophylaxis of SBP using long-term oral norﬂoxacin in patients with low-protein ascites (<1.5 mg/dl) and serum bilirubin >4 mg/dl associated with a Management of Renal Failure 445

Child–Pugh score >9, or serum creatinine >1.2 mg/dl was associated to a signiﬁcant decrease in 1-year probability of development of SBP (7% vs. 61%) and type 1 HRS (28% vs. 41%) and a signiﬁcant increase in the 3-month and 1-year probability of survival (94% vs. 62% and 60% vs. 48%, respectively) (99). Finally, the administration of the tumor necrosis factor inhibitor pentoxifylline (400 mg 3 times a day) to patients with severe acute alcoholic hepatitis reduced the occurrence of HRS (8% in the pentoxifylline group vs. 35% in the placebo group) and the hospital mortality (24% vs. 46%, respectively) (100).

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49. Guevara M, Bru C, Gines P, Fernandez-Esparrach G, Sort P, Bataller R, Jimenez W, Arroyo V, Rodes J. Increased cerebrovascular resistance in cirrhotic patients with ascites. Hepatology 1998; 28: 39–44. 50. Angeli P, Albino G, Carraro P, Dalla PM, Merkel C, Caregaro L, De BE, Bortoluzzi A, Plebani M, Gatta A. Cirrhosis and muscle cramps: evidence of a causal relationship. Hepatology 1996; 23: 264–273. 51. Keiding S, Sorensen M, Bender D, Munk OL, Ott P, Vilstrup H. Brain metabolism of 13N-ammonia during acute hepatic encephalopathy in cirrhosis measured by positron emission tomography. Hepatology 2006; 43: 42–50. 52. Wiest R, Groszmann R. Nitric oxide and portal hypertension: its role in the regulation of intrahepatic and splanchnic vascular resistance. Semin Liver Dis 1999; 19: 411–26. 53. Iwakiri Y, Groszmann RJ. The hyperdynamic circulation of chronic liver diseases: from the patient to the molecule. Hepatology 2006; 43: S121–131. 54. Alqahtani SA, Fouad TR, Lee SS. Cirrhotic cardiomyopathy. Semin Liver Dis 2008; 28: 59–69. 55. Gines P, Uriz J, Calahorra B, Garcia-Tsao G, Kamath PS, del Arbol LR, Planas R, Bosch J, Arroyo V, Rodes J. Transjugular intrahepatic portosystemic shunting versus paracentesis plus albumin for refractory ascites in cirrhosis. Gastroenterology 2002; 123: 1839–47. 56. Braverman AC, Steiner MA, Picus D, White H. High-output congestive heart failure following transjugular intrahepatic portal–systemic shunting. Chest 1995; 107: 1467–69. 57. Myers RP, Lee SS. Cirrhotic cardiomyopathy and liver transplantation. Liver Transpl 2000; 6: S44–52. 58. Therapondos G, Stanley AJ, Hayes PC. Systemic, portal and renal effects of terlipressin in patients with cirrhotic ascites: pilot study. J Gastroenterol Hepatol 2004; 19: 73–7. 59. Guevara M, Gines P, Bandi JC, Gilabert R, Sort P, Jimenez W, Garcia-Pagan JC, Bosch J, Arroyo V, Rodes J. Transjugular intrahepatic portosystemic shunt in hepatorenal syndrome: effects on renal function and vasoactive systems. Hepatology 1998; 28: 416–22. 60. Brensing KA, Textor J, Perz J, Schiedermaier P, Raab P, Strunk H, Klehr HU, Kramer HJ, Spengler U, Schild H, Sauerbruch T. Long term outcome after transjugular intrahepatic portosystemic stent-shunt in non-transplant cirrhotics with hepatorenal syndrome: a phase II study. Gut 2000; 47: 288–95. 61. Sort P, Navasa M, Arroyo V, Aldeguer X, Planas R, Ruiz-del-Arbol L, Castells L, Vargas V, Soriano G, Guevara M, Gines P, Rodes J. Effect of intravenous albumin on renal impairment and mortality in patients with cirrhosis and spontaneous bacterial peritonitis. N Engl J Med 1999; 341: 403–409. 62. Tsai MH, Peng YS, Chen YC, Liu NJ, Ho YP, Fang JT, Lien JM, Yang C, Chen PC, Wu CS. Adrenal insufﬁciency in patients with cirrhosis, severe sepsis and septic shock. Hepatology 2006; 43: 673–681. 63. Fernandez J, Escorsell A, Zabalza M, Felipe V, Navasa M, Mas A, Lacy AM, Gines P, Arroyo V. Adrenal insufﬁciency in patients with cirrhosis and septic shock: effect of treatment with hydrocortisone on survival. Hepatology 2006; 44: 1288–1295. 64. Rimola A, Gavaler JS, Schade RR, el-Lankany S, Starzl TE, Van Thiel DH. Effects of renal impairment on liver transplantation. Gastroenterology 1987; 93: 148–156. Management of Renal Failure 449

65. Gonwa TA, Morris CA, Goldstein RM, Husberg BS, Klintmalm GB. Long- term survival and renal function following liver transplantation in patients with and without hepatorenal syndrome – experience in 300 patients. Transplantation 1991; 51: 428–430. 66. Seu P, Wilkinson AH, Shaked A, Busuttil RW. The hepatorenal syndrome in liver transplant recipients. Am Surg 1991; 57: 806–809. 67. Gonwa TA, Klintmalm GB, Levy M, Jennings LS, Goldstein RM, Husberg BS. Impact of pretransplant renal function on survival after liver transplantation. Transplantation 1995; 59: 361–365. 68. Nair S, Verma S, Thuluvath PJ. Pretransplant renal function predicts survival in patients undergoing orthotopic liver transplantation. Hepatology 2002; 35: 1179–1185. 69. Restuccia T, Ortega R, Guevara M, Gines P, Alessandria C, Ozdogan O, Navasa M, Rimola A, Garcia-Valdecasas JC, Arroyo V, Rodes J. Effects of treatment of hepatorenal syndrome before transplantation on posttransplantation outcome. A case–control study. J Hepatol 2004; 40: 140–146. 70. Guevara M, Gines P, Fernandez-Esparrach G, Sort P, Salmeron JM, Jimenez W, Arroyo V, Rodes J. Reversibility of hepatorenal syndrome by prolonged administration of ornipressin and plasma volume expansion. Hepatology 1998; 27: 35–41. 71. Angeli P, Volpin R, Gerunda G, Craighero R, Roner P, Merenda R, Amodio P, Sticca A, Caregaro L, Maffei-Faccioli A, Gatta A. Reversal of type 1 hepatorenal syndrome with the administration of midodrine and octreotide. Hepatology 1999; 29: 1690–1697. 72. Gulberg V, Bilzer M, Gerbes AL. Long-term therapy and retreatment of hepatorenal syndrome type 1 with ornipressin and dopamine. Hepatology 1999; 30: 870–875. 73. Uriz J, Gines P, Cardenas A, Sort P, Jimenez W, Salmeron JM, Bataller R, Mas A, Navasa M, Arroyo V, Rodes J. Terlipressin plus albumin infusion: an effective and safe therapy of hepatorenal syndrome. J Hepatol 2000; 33: 43–48. 74. Mulkay JP, Louis H, Donckier V, Bourgeois N, Adler M, Deviere J, Le Moine O. Long-term terlipressin administration improves renal function in cirrhotic patients with type 1 hepatorenal syndrome: a pilot study. Acta Gastroenterol Belg 2001; 64: 15–19. 75. Colle I, Durand F, Pessione F, Rassiat E, Bernuau J, Barriere E, Lebrec D, Valla DC, Moreau R. Clinical course, predictive factors and prognosis in patients with cirrhosis and type 1 hepatorenal syndrome treated with terlipressin: a retrospective analysis. J Gastroenterol Hepatol 2002; 17: 882–888. 76. Duvoux C, Zanditenas D, Hezode C, Chauvat A, Monin JL, Roudot-Thoraval F, Mallat A, Dhumeaux D. Effects of noradrenalin and albumin in patients with type I hepatorenal syndrome: a pilot study. Hepatology 2002; 36: 374–380. 77. Halimi C, Bonnard P, Bernard B, Mathurin P, Mofredj A, di Martino V, Demontis R, Henry-Biabaud E, Fievet P, Opolon P, Poynard T, Cadranel JF. Effect of terlipressin (Glypressin) on hepatorenal syndrome in cirrhotic patients: results of a multicentre pilot study. Eur J Gastroenterol Hepatol 2002;14: 153–158. 78. Moreau R, Durand F, Poynard T, Duhamel C, Cervoni JP, Ichai P, Abergel A, Halimi C, Pauwels M, Bronowicki JP, Giostra E, Fleurot C, Gurnot D, Nouel O, Renard P, Rivoal M, Blanc P, Coumaros D, Ducloux S, Levy S, Pariente A, Perarnau JM, Roche J, Scribe-Outtas M, Valla D, Bernard B, Samuel D, Butel J, Hadengue A, Platek A, Lebrec D, Cadranel JF. Terlipressin in patients with 450 Arroyo and Guevara

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93. Hassanein TI, Tofteng F, Brown Jr. RS, McGuire B, Lynch P, Mehta R, Larsen FS, Gornbein J, Stange J, Blei AT. Randomized controlled study of extracorporeal albumin dialysis for hepatic encephalopathy in advanced cirrhosis. Hepatology 2007;46: 1853–62. 94. Lebrec D, Giuily N, Hadengue A, Vilgrain V, Moreau R, Poynard T, Gadano A, Lassen C, Benhamou JP, Erlinger S. Transjugular intrahepatic portosystemic shunts: comparison with paracentesis in patients with cirrhosis and refractory ascites: a randomized trial. French Group of Clinicians and a Group of Biologists. J Hepatol 1996; 25: 135–144. 95. Rossle M, Ochs A, Gulberg V, Siegerstetter V, Holl J, Deibert P, Olschewski M, Reiser M, Gerbes AL. A comparison of paracentesis and transjugular intrahepatic portosystemic shunting in patients with ascites. N Engl J Med 2000; 342: 1701–1707. 96. Sanyal AJ, Genning C, Reddy KR, Wong F, Kowdley KV, Benner K, McCashland T. The North American study for the treatment of refractory ascites. Gastroenterology 2003; 124: 634–641. 97. Salerno F, Merli M, Riggio O, Cazzaniga M, Valeriano V, Pozzi M, Nicolini A, Salvatori F. Randomized controlled study of TIPS versus paracentesis plus albumin in cirrhosis with severe ascites. Hepatology 2004; 40: 629–635. 98. Alessandria C, Ottobrelli A, bernardi-Venon W, Todros L, Cerenzia MT, Martini S, Balzola F, Morgando A, Rizzetto M, Marzano A. Noradrenalin vs terlipressin in patients with hepatorenal syndrome: a prospective, randomized, unblinded, pilot study. J Hepatol 2007; 47: 499–505. 99. Fernandez J, Navasa M, Planas R, Montoliu S, Monfort D, Soriano G, Vila C, Pardo A, Quintero E, Vargas V, Such J, Gines P, Arroyo V. Primary prophylaxis of spontaneous bacterial peritonitis delays hepatorenal syndrome and improves survival in cirrhosis. Gastroenterology 2007; 133: 818–824. 100. Akriviadis E, Botla R, Briggs W, Han S, Reynolds T, Shakil O. Pentoxifylline improves short-term survival in severe acute alcoholic hepatitis: a double-blind, placebo-controlled trial. Gastroenterology 2000; 119: 1637–48.

Correction of Abnormalities of Haemostasis in Chronic Liver Disease

Marco Senzolo and Andrew Kenneth Burroughs

CONTENTS INTRODUCTION ASSESSMENT OF THE RISK OF BLEEDING INVASIVE PROCEDURES COAGULATION DURING INFECTION AND SEPSIS THERAPY OF HAEMOSTATIC ABNORMALITIES IN LIVER DISEASE ASSESSMENT OF THE RISK OF THROMBOSIS AND CLINICAL USE OF ANTICOAGULATION REFERENCES

Key Words: Cirrhosis, Coagulation, Portal hypertension, Bleeding, Portal vein thrombosis, Haemostasis, Anticoagulation, Liver transplantation, Fibrinolysis, Liver, Platelets, Infection, Invasive procedures

1. INTRODUCTION The liver plays a key role in blood coagulation being involved in both primary and secondary haemostasis (1, 2). It is the site of synthesis of all coagulation factors and their inhibitors except for von

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_23, C Springer Science+Business Media, LLC 2011

453 454 Senzolo and Burroughs

Willebrand factor (vWf) (3). Liver damage is commonly associated with impairment of coagulation, when liver function reserve is poor. The haemostatic system is in a delicate balance between prothrombotic and antithrombotic processes, aiming both to prevent excessive blood loss from injured vessels and to prevent spontaneous thrombosis. Liver failure is accompanied by multiple changes in the haemostatic system, because of reduced plasma levels of procoagulative and anticoagulative clotting factors synthesized by the intact liver (4). Vitamin K deﬁciency may coexist, so that defective clotting factors due to the lack of carboxylation are produced. Moreover during liver failure, there is a reduced capacity to clear activated haemostatic proteins and protein inhibitor complexes from the circulation. Thus the global effect of liver disease with regard to haemostasis is complex, so that patients with advanced liver disease can experience severe bleeding or even thrombotic complications (Table 1). Finally, when marked portal hypertension develops with collateral circulation and secondary splenomegaly, thrombocytopaenia develops due to splenic sequestration, but thrombocytopaenia may also be due to decreased hepatic thrombopoietin synthesis. There is also impaired platelet function. These haemostatic abnormalities do not always lead to spontaneous bleeding, but the onset of complications of cirrhosis such as variceal bleeding or infection/sepsis may lead to worsening of coagulation status. The presence of a baseline consumptive coagulopathy other than that secondary to sepsis or other predisposing causes is disputed.

Table 1 Clinical haemorrhage in patients with liver cirrhosis

Microvascular manifestations Petechiae Bruising, purpura, bleeding at venepuncture sites or after minor injury (brushing, teeth, shaving) Epistaxis Gingival bleeding DIC vs. pseudo-DIC (see text) Macrovascular manifestations Haemorrage during surgery or invasive procedures (TIPS, ICP monitoring, liver biopsy, paracentesis, thoracentesis) Variceal haemorrhage Portal hypertensive gastropathy Peptic ulcer disease Correction of Abnormalities of Haemostasis in Chronic Liver Disease 455

Usually, correction of abnormal coagulation in liver disease is needed only during bleeding or before invasive procedures. When end-stage liver disease occurs, liver transplantation is the only treatment available, which can restore normal haemostasis, and correct genetic defects, such as haemophilia or factor V Leiden mutation contributing to the abnormal coagulation. During liver transplantation, haemorrhage may occur due to the preexisting hypocoagulable state, the collateral circulation caused by portal hypertension and increased ﬁbrinolysis which occurs during this surgery.

2. ASSESSMENT OF THE RISK OF BLEEDING The role played by coagulation defects in the occurrence of bleeding in cirrhosis is still unclear. This is particularly due to the difficulty (and cost) in measuring static procoagulant and anticoagulant activities, and evaluating the balance between the two. In addition there are very few tests which reflect or predict coagulation in vivo. The prothrombin time and INR are poorly correlated with the risk of bleeding during interventional procedures in patients with liver cirrhosis (5). Recently generation of thrombin has been explored in vitro in patients with liver cirrhosis and was found to be normal or even increased in patients with severe liver dysfunction in parallel with the decreased synthesis of anticoagulant factors. However, the in vitro technique has some drawbacks, the major one being that platelets are substituted by phospholipids (4). Recently, the same authors have shown that when platelets were adjusted to correspond to whole-blood count of patients in vivo, patients with cirrhosis generated significantly less thrombin than controls (6) when platelet count was lower than 80,000/μl. The correlation between thrombin generation and bleeding or thrombotic events remains to be explored. Clinical signs of coagulopathy in liver disease depend on the degree of impairment of the haemostatic system. Commonly, minor signs of bleeding tendency are present, such as bleeding gums and epistaxis, but major bleeding can be encountered. The role of haemostatic abnormalities in the risk of variceal bleeding is not clear. Abnormal laboratory tests of haemostasis which can indicate worse hepatic synthesis are more a surrogate of severe disease rather than a risk factor for haemorrhage per se. PT and INR have been demonstrated to be independently associated with the risk of rebleeding in patients with variceal bleeding (often these patients have worse liver function) (7), but are not good prognostic indexes for the first bleeding episode. Hyperfibrinolysis has been shown to be associated, but as yet not causally related to an increased risk of variceal bleeding, in a cohort of 61 cirrhotics. 456 Senzolo and Burroughs

Higher levels of ﬁbrinogen degradation products were associated with a greater risk of variceal bleeding compared to patients without them (odds ratio=8), but Child–Pugh score and endoscopic characteristics of varices remained the most important prognostic factors (8). Recently the role of infection and endogenous heparin-like substances demonstrated by transient eleastography (TEG) has been evaluated in variceal bleeding. Infection may be a trigger factor for bleeding (9) and both infection and heparin-like substances may be mechanisms responsible for the persistence of bleeding in some (10). TEG, which is a quick and reliable method to assess clot formation and lysis (11), also allows detection of heparin-like substances. Studies from our group have shown worsening coagulation during infection due to low molecular weight heparin-like substances detected by TEG (12).

3. INVASIVE PROCEDURES Historically, PT and platelet count have been used to assess the risk of bleeding prior to invasive procedures. Patients with cirrhosis have increased mortality and morbidity during surgery (13), mainly due to increased bleeding in 60% of cases (14, 15). Early studies linked PT to this risk during surgery (PT prolongation >1.5 and >2.5 s associated with 47 and 87% mortality, respectively) (16). Hence platelet count <50,000/mm3 and PT >3 s have been considered relative contraindications to elective surgery (15) and have also been used as thresholds to require platelets and clotting factors prior to invasive procedures. However, the most commonly observed risk factor for bleeding during surgery is the severity of portal hypertension and the presence of collateral veins, but this has been poorly documented in published studies. Hyperfibrinolysis (17) and clotting activation, due to increased tPA levels, have been described in patients undergoing liver resection (18). However, a study performed in patients undergoing laparoscopic liver biopsy failed to demonstrate any correlation between bleeding seen at the hepatic puncture site and the coagulation tests prior to the procedure, so that the degree of injury may be an important factor in the risk of bleeding in this setting (19). Liver biopsy is widely used diagnostically and to grade the severity of liver disease or fibrosis. Moreover it is an essential tool after liver transplantation to diagnose rejection and other causes of graft dysfunction. Bleeding complications occur in 0.35–0.5%, leading to mortality in 0.1% (20). Direct ultrasound guidance is often used to mark the optimal biopsy site, as there may be a reduction of the risk of complications and it is recommended in some guidelines (21). Despite the evidence Correction of Abnormalities of Haemostasis in Chronic Liver Disease 457 that there were no threshold abnormalities of clotting tests associated with the risk of bleeding during laparoscopic liver biopsy (22), thresholds for INR and platelet count are still used to determine the safety for percutaneous liver biopsy in terms of risk of bleeding (23). An audit from the British Society of Gastroenterology (BSG) performed in 1991 showed a doubling of the risk of bleeding in patients with INR ≥1.5, but that only 7.1% of the bleeding occurred with INR greater than 1.5, and 90% occurred with INR ≤1.3 (24). Therefore, a normal INR does not exclude the risk of bleeding complications after liver biopsy. A cut-off for platelet count is difficult to justify from the literature. Most text- books in the UK and the BSG guidelines require platelet count above 80,000/mm3 (25), whereas a survey from the Mayo Clinic suggested 50,000 mm3 asacut-off(26). Current recommendations state that a percutaneous liver biopsy can be done safely without support with platelet counts above 60,000/mm3 (23). Burroughs et al. advocated evaluating the use of bleeding time to assess the risk of bleeding for percutaneous liver biopsy (27), but this has not been taken up in clinical practice. If clotting parameters are outside stipulated ranges, a transjugular liver biopsy can be performed more safely, without plasma or platelet therapy (28), despite the recommendation for such therapy in some guidelines. A plugged liver biopsy (injection of gel foam while the biopsy gun is being withdrawn) is also said to be safer than standard percutaneous biopsy, but it may cause greater risk of bleeding in hypocoagulable patients (20). During minor procedures such as thoracentesis, paracentesis, lumbar puncture or dental extraction performed in patients with liver disease, there are no firm guidelines as to the values of clotting tests to perform these procedures without use of blood products. The largest review on 608 patients with cirrhosis who underwent paracentesis or thoracentesis with mild coagulation abnormalities reported 0.2% as having excessive bleeding requiring transfusion and a 0.02% mortality. There was no correlation with PT, PTT, platelet count and the risk of this bleeding (29). In another study which evaluated paracentesis in 200 cirrhotics with INR ≥3 and PLT count ≤19,000/mm3, no complications were seen, regardless of baseline INR and platelet count (30). Central venous access can be safely performed in patients with abnormalities of haemostatis without blood product infusion – the internal jugular route should be used (31). Platelet transfusion if platelet count is less than 50,000 is sometimes used although evidence for this is not available. If hyperfibrinolysis is present, its correction reduces the risk of bleeding during central venous access (32). A contraindication to using invasive procedures is clinically evident disseminated intravascular coagulation (DIC) or fibrinolysis (33). At 458 Senzolo and Burroughs present, there are no evidence-based reports to establish “safe” coagulation tests for patients to undergo these procedures. However, it is axiomatic, if not more important, to limit such invasive procedures to circumstances in which the perceived benefit of the procedure is clearly greater than the risk, as this risk of bleeding exists and cannot be accurately estimated. The recent findings that many patients with cirrhosis may not be hypocoagulable may well influence recommendations for use of plasma products and platelets in the future. Prospective studies are urgently needed to review the current guidelines in the light of this new evidence.

4. COAGULATION DURING INFECTION AND SEPSIS The overall cumulative incidence of infection in patients with cirrhosis is estimated to be at least 30% (34), and is possibly associated with increased risk of variceal bleeding (9). Infection is associated with early variceal rebleeding and increased mortality from variceal bleeding (35, 36). Prophylactic antibiotic therapy has led to less early rebleeding and better control of bleeding, in two randomized controlled trials (37, 38). Using TEG, 20 cirrhotic patients who experienced early rebleeding were found to have worsening TEG parameters the day before rebleeding (10). Moreover patients with bacterial infection have worse TEG parameters, which can be corrected in vitro by heparinase I, which cleaves heparin-like substances (12)(Fig.1a and b). The presence of heparin-like substances is associated in some with increased anti-Xa activity (39). Heparin-like substances have been detected hours after variceal bleeding in cirrhotic patients (40). Based on these observations, the hypothesis has been put forward that endotoxins and inflammation due to infection can release heparinoids from the endothelium and mast cells (12). One study, as yet not fully reported, showed increased heparan sulphate concentrations in patients with variceal bleeding compared to patients without (41). Moreover sepsis can cause impairment of platelet function, decreasing platelet number and aggregation, due to increased NO production (42). Cytokines, in particular IL-6 and TNF-α which are released during infection, can trigger DIC with hyperfibrinolysis (43). One study showed a strong association between both fragment F1+2 and D-dimer with endotoxaemia. These markers interestingly returned to normal after antibiotic therapy (44). Another report recently showed decreased platelet count and levels of factor VII, X, V and II in cirrhotic patients with severe sepsis, suggesting consumptive coagulopathy (45), whereas a further study found decreased activity of protein C, which is associated with increased fibrinolysis (46). Correction of Abnormalities of Haemostasis in Chronic Liver Disease 459

Native

10 mm

R K Angle MA G EPL LY30 A CI LY60 min min deg mm d/sc % % mm % 29.6 17.2 13.8 55.8 6.3K 0.0 0.0 54.6 0.6

Fig. 1. Presence of heparin-like effect in a patient with cirrhosis and peritoneal infection. Native-TEG (a) and heparinase I-TEG (b) on samples collected at the onset of spontaneous bacterial peritonitis in a patient with liver cirrhosis. A signiﬁcant heparin-like effect was found; the slow rate of coagulation in (a) compared to (b) is shown by the decrease in slope (lower angle of separation). Treatment of the sample in (b) with heparinases increased the rate of coagulation, thus sampling the presence of heparin-like substances.

Prevention of infection during variceal bleeding is a well-defined treatment which has been proven to reduce rebleeding and mortality (37, 47). The effect may also be associated with preventing a worsening of portal haemodynamics. This has been shown indirectly in a single study by Ruiz-del-Arbol et al. in which hepatic venous pressure gradient (HVPG) was higher following spontaneous bacterial peritonitis in some patients even after resolution of infection (48) and confirmed in another study evaluating the effect of intestinal decontamination. HVPG values decreased significantly after intestinal decontamination 460 Senzolo and Burroughs with rifaximin in patients with alcohol-related decompensated cirrhosis and this might have been achieved through significant reduction of plasma endotoxin levels (49). Derangement of coagulation due to infection per se has been shown using TEG, hours after the bleeding (40) and this could be another reason why antibiotics are associated with rebleeding. Infusion of protamine sulphate has been reported aiming to revert the heparin-like effect seen after reperfusion during liver transplantation in cirrhotic patients (50). However, reduction in bleeding and blood unit requirement was not clearly demonstrated. Use of heparinase I in vivo (Neutralase) has been investigated recently, to treat coagulopathy due to the release of exogenous heparin in patients undergoing coronary artery bypass surgery, but it has been shown to worsen the outcome compared to standard protamine treatment (51). Theoretically, the ability of heparinase I to cleave endogenous heparinoids more specifically than protamine could offer an advantage in patients with demonstrable heparin-like effect due to endogenous glycosaminoglycans which are found in cirrhotics with infection or bleeding, but its clinical use, if any, has to be evaluated.

5. THERAPY OF HAEMOSTATIC ABNORMALITIES IN LIVER DISEASE Usually, haemostatic abnormalities in liver disease lead only to mild clinical consequences. However, in particular clinical settings, life-threatening bleeding can occur such as bleeding from varices or peptic ulcers. Signiﬁcant microvascular haemorrhage is unusual, despite multiple coagulation test abnormalities. Therapy for haemostatic abnormalities of liver disease is needed only during variceal or other bleeding, and before and/or during invasive procedures and surgery.

5.1. Vitamin K Intravenous vitamin K injection of 10 mg daily for 1–2 days can correct vitamin K deﬁciency (25) due to cholestasis or malabsorption. However after this period of treatment no increase of PT/INR occurs in patients with chronic liver disease in whom the impairment of liver function is the main cause of decreased synthesis of vitamin K coagulation factors. Despite this fact, there is, in clinical practice, a widespread use of vitamin K in patients with chronic liver disease; Lucena et al. reported in 2002 that among 568 patients followed up in 25 hospitals in Spain an average of 24% were treated with vitamin K (52). In acute liver Correction of Abnormalities of Haemostasis in Chronic Liver Disease 461 failure, about 25% can have subclinical vitamin K deﬁciency which can be corrected by intravenous injection.

5.2. Fresh Frozen Plasma and Transfusion Requirements Fresh frozen plasma (FFP) contains all the clotting factors and can correct the laboratory ﬁnding of an elevated PT effectively, but this correction depends on the volume of FFP and the baseline abnormality of PT. Whether this correction of the PT results in increased haemostasis has yet to be proven. Use of plasma-based blood products has been a dogma for treatment of the coagulopathy of liver disease. However, randomized clinical trials provide little support for this generalized practice. In addition, the laboratory correction is short term (24–48 h), as it depends on the half-life of the clotting factors (especially factor VII) (53). A commonly used indication for FFP infusion is the presence of persistent bleeding in patients with INR ≥2 or PT prolongation greater than 4 s (54). In surgical or invasive procedures, the threshold for suggested administration is 50% of the normal PT (i.e. INR of 2) and should be the target outcome when using replacement therapy. For neurological procedures such as intracranial pressure monitoring during acute liver failure, a threshold of 80% of normal PT range (i.e. an INR of about 1.2–1.3) is suggested (54). During massive blood transfusion, to avoid dilutional decrease of clotting factors for every 2 units of blood, 1 unit of FFP is typically given (55). However, to increase the activity of clotting factors by 1–2%, a dose of 1 ml FFP/kg of body weight is necessary (56). Moreover, because of the short half-life of FFP, infusions every 6–12 h are needed (57). Therefore a high volume is required and thus adequate replacement is difﬁcult in patients with cirrhosis, in whom the intravascular plasma volume is already expanded and in whom ascites may be present. This is also a problem in patients with ALF, in whom increasing plasma volume can lead to increases in intracerebral pressure. Thus FFP administration has limited clinical utility. During variceal bleeding massive transfusions can theoretically worsen bleeding by increasing portal pressure. The few existing human studies on the effect of crystalloids and saline infusion in cirrhotics show no increase in portal pressure after either is infused, despite an increase in central venous pressure after crystalloid infusion (58) and expansion of interstitial space with saline infusion (59). In one study performed in 12 patients with cirrhosis and portal hypertension the immediate restitution of blood volume previously depleted caused a slight increase in hepatic venous pressure gradient only in 4 patients (60). 462 Senzolo and Burroughs

In rats with severe portal hypertension and haemorrhage there are more extreme increases in portal pressure with greater volume of transfusion. When groups with immediate 0, 50 and 100% replacement of the volume of blood lost were compared, the group which received 100% replacement had a higher rate of rebleeding and poorest survival (61). In a recent randomized controlled trial, 214 patients with cirrhosis and gastrointestinal bleeding were randomized to a restrictive (target Hb 7–8 g/dl) or liberal transfusion strategy (target Hb 9 g/dl). In the subgroup of patients who bled from oesophageal varices, the 6 weeks survival without endoscopic failure was better in the restrictive strategy group (62). Moreover in the liberal strategy group a signiﬁcant increase in HVPG was demonstrated (63). In liver transplantation, use of plasma transfusion leads to higher red cell transfusion requirement suggesting that this effect is also related to increasing portal hypertension (64). In patients with INR >1.5, FFP is typically given at the dose of 12–15 ml/kg before liver biopsy, but this practice is not based on any evidence. A transjugular biopsy would seem to be a better alternative if there are abnormalities over the coagulation limits for a standard biopsy (65).

5.3. Prothrombin Complex Prothrombin complex concentrates (PCC) are a mixture of vitamin K-dependent clotting factors at 20-fold higher concentrations than FFP. PCC has been shown to correct to PT <3 s prolonged above controls in about 50% of patients with cirrhosis and coagulopathy (66, 67) with the ﬁrst preparations that were developed. This correction of PT was seen at a mean dose of 1500 ml but was associated with an increase in thrombin–antithrombin complexes but without the occurrence of thrombotic events (68). In addition, other complications including type 2 heparin-induced thrombocytopaenia and thromboembolic complications have been described. The thrombotic complications can theoretically be more important in patients with cirrhosis in whom a concomitant reduction of anticoagulant factor is present.

5.4. Cryoprecipitate Cryoprecipitate contains factors VIII, fibrinogen, vWf, fibronectin and XIII. Because of the small volume (30–50 ml/U/10 kg) required during infusion (69), it can be useful in liver cirrhosis and ALF, but it lacks some coagulation factors and may worsen the imbalance already present in patients with liver disease. Cryoprecipitate is recommended Correction of Abnormalities of Haemostasis in Chronic Liver Disease 463 in some patients with DIC and severe (i.e. <50 mg/dl) hypofibrinogenemia, but cryoprecipitate infusion does not correct the underlying hyperfibrinolytic defect (70). In a recent randomized trial performed in patients with ALF, 5 units of cryoprecipitate were less effective than 4 units of FFP in improving the prothrombin time, and one patient developed pulmonary oedema in the FFP group (71).

5.5. Recombinant Activated Factor VII Recombinant activated factor VII (rFVIIa) was first developed for the treatment of patients with haemophilia A and B who developed inhibitors. The normal FVII:FVIIa ratio in the circulation is 100:1. Following rFVII administration, the circulating total FVIIa activity is increased approximately by 100-fold such that the FVII:FVIIa ratio is 1:1, increasing thrombin generation (72). Theoretically, rFVIIa could have a promising role in the treatment of coagulation disorders in liver disease (73). Indeed rFVIIa has been shown to normalize INR in healthy volunteers treated with acenocoumarol (74), thus leading to the hypothesis of possible use in patients with acquired deficiency of vitamin K-dependent clotting factors. In a preliminary study, a single dose of recombinant factor VIIa was shown to correct prolonged PT in a dose-dependent manner in patients with cirrhosis with bleeding (75). The half-life of factor VIIa is 2.3 h (76). The median duration of normalization of PT was 2, 6 and 12 h following a single dose of 5, 20 and 80 μg/kg, respectively (75). A randomized study using rFVIIa in 71 patients undergoing laparoscopic liver biopsy found no differences in liver bleeding time. Two complications occurred in the rFVIIa group (one DIC and one PVT) (77). However, limited data are available regarding the use of rFVIIa in surgical procedures. In a multicentre double-blind placebo controlled trial, 204 patients undergoing partial hepatectomy due to neoplasia were randomized to receive preoperative injection of either placebo or rFVIIa (20 or 80 μg/kg), followed by a second dose 5 h after surgery, started if the operation took longer than 6 h. The use of the drug did not result in significant reduction in either the number of patients transfused or the number of blood products transfused (78). These data were confirmed by another RCT in 235 cirrhotics undergoing partial hepatectomy. Serious thromboembolic complications occurred in 3–6% in the rFVIIa group and thus this is a major complication which represents a contraindication to its use (79). In ALF, rFVIIa may be useful to normalize PT in the setting of intracranial pressure monitoring, as only a small volume of infusion 464 Senzolo and Burroughs is required. The King’s College group reported on seven patients who had normalization of PT after treatment with rFVIIa before ICP monitoring (80). During variceal bleeding in a randomized controlled trial, 245 patients were randomized to receive 100 μg/kg of rFVIIa or placebo in addition to standard endoscopic and pharmacological treatment. Only a modest and nonsignificant reduction in the early rebleeding rate was observed in a subgroup of Child B and C patients after rFVIIa infusion, although overall there was no difference in the control of bleeding, transfusion and deaths (81). A further trial based solely on 256 patients with cirrhosis, Child B and C patients and variceal bleeding showed no significant effect on rebleeding and mortality at day 5 (82). Another report described initial haemostasis after infusion of rFVIIa in 10 patients, but 6 experienced early rebleeding and all of them died, illustrating the short interval of action of this drug (83). In a cohort of eight patients with acute variceal bleeding uncontrolled with endoscopic and medical therapy, rFVIIa administration achieved haemostasis in 25% after a single dose (84). The efficacy of factor rFVIIa is limited in patients with low fibrinogen (<100 mg/dl) and concomitant administration of low volumes of plasma is required (77). Safety, especially with concerns of possible prothrombotic effects or triggering of DIC, still has to be assessed in large studies in patients with liver disease (85).

5.6. Platelet Transfusions and Treatment for Thrombocytopaenia Based on the work of Tripodi and colleagues, increasing evidence provides guidance for using platelet transfusion in patients with cirrhosis. Adequate thrombin production seems to be present in cirrhosis when platelet counts are approximately 50,000– 60,000/ml or above, whereas optimal levels are seen with counts higher than 100,000/ml (6). However, clinical translation of these laboratory cut-offs is mandatory as interaction of platelets with clotting pathways in vivo needs to be studied. Platelet transfusion is still used, but current clinical practice lacks any evidence-based support. Rheological studies have indicated that normal platelet ﬂow is adversely affected with haematocrit levels less than 25%, supporting the recommendation to maintain this level in patients who have cirrhosis and bleeding (86). One unit for every 10 kg body weight should is typically administered, and platelet count should be checked 1 h after the infusion (69). However no correlation between amelioration of bleeding time, increase in platelet count and enhanced haemostasis has been shown (23). Correction of Abnormalities of Haemostasis in Chronic Liver Disease 465

Recently, there have been two pharmacological approaches to treating thrombocytopaenia. The first approach involves cytokines and growth factors that enhance bone marrow production and the second involves drugs that activate the TPO receptor and promote megakary- ocyte or PLT production (87). Among cytokines, only recombinant IL-11 has been successfully administered in some patients with solid tumours undergoing chemotherapy. One case study has demonstrated that rIL-11 can correct HCV-associated thrombocytopaenia (88). Eltrombopag is an orally bioavailable low molecular weight nonpep- tide growth factor capable of inducing proliferation of megakaryocytes. Eltrombopag has been evaluated in patients with HCV-induced thrombocytopaenia who were candidates for antiviral treatment in an RCT trial. At week 4, platelet counts were increased to 100,000/mm3 or more in a dose-dependent manner among patients for whom these data were available: in none of the 17 patients receiving placebo, in 9 of 12 (75%) receiving 30 mg of eltrombopag, in 15 of 19 (79%) receiving 50 mg of eltrombopag and in 20 of 21 (95%) receiving 75 mg of eltrombopag (P<0.001). Antiviral therapy could be initiated in 49 patients (in 4 of 18 patients receiving placebo, 10 of 14 receiving 30 mg of eltrombopag, 14 of 19 receiving 50 mg of eltrombopag and 21 of 23 receiving 75 mg of eltrombopag). Twelve weeks of antiviral therapy were completed by 36, 53 and 65% of patients receiving 30, 50 and 75 mg of eltrombopag, respectively, but by only 6% of patients in the placebo group (89). However, some of the patients developed severe thrombocytosis, although no serious adverse events were seen. The clinical scenario could be totally different in patients with cirrhosis rather than chronic hepatitis in which the resetting of coagulation leads to a precarious equilibrium with the possibility of thrombotic complications by use of agents which increase platelet counts. Another RCT evaluating eltrombopag in patients with liver cirrhosis and thrombocytopaenia undergoing minor invasive procedures has recently been stopped because of an increased incidence of thrombotic complications in the treatment arm. Splenectomy is generally contraindicated in patients with liver cirrhosis, because of the high mortality rate and a risk of secondary portal vein thrombosis, leading to increased risk of bleeding from oesophageal-gastric varices and more difficult surgery during subsequent liver transplant (90). Splenic embolization with 30–50% reduction in flow can normalize or significantly improve platelet number in some patients with liver cirrhosis (91) but is not widely recommended. Insertion of transjugular intrahepatic portosystemic shunt (TIPS) has been shown to increase, but not to normalize platelet number (92, 93). 466 Senzolo and Burroughs

5.7. Desmopressin Desmopressin (1-deamino-8-D-arginine vasopressin (DDAVP)), an analogue of the antidiuretic hormone, increases plasma level of factor VIII and vWf, probably by increasing the release from endothelial storage sites (94). It can improve bleeding time, enhancing primary haemostasis at the dose of 0.3 μg/kg in patients with liver failure (95). However a randomized trial associating terlipressin and DDAVP in patients with variceal bleeding demonstrated no difference in the control of bleeding and possibly a worsening of the terlipressin action in the DDVAP group (96). In a recent randomized trial, DDVAP failed to decrease blood loss during hepatic resection, despite an increase in factor VIII and vWf (97).

5.8. Antifibrinolytics Hyperfibrinolysis has been correlated with variceal bleeding, but probably contributes more to the risk of bleeding in specific areas such as oral cavity following dental extraction or during particular procedures such as partial hepatectomy or liver transplantation (98). Aprotinin is a serine protease inhibitor which antagonizes various proteases (99). Although the use of aprotinin in liver transplantation has been extensively studied, only one RCT on the use of this drug in partial hepatectomy has been conducted in 97 patients with cirrhosis evaluating blood loss and transfusion requirements. Intraoperative blood loss and blood transfusion were significantly lower in the treatment group compared to placebo (100). However, these data have never been confirmed in nonrandomized studies. The efficacy of nafamostat mesilate which is another serine protease inhibitor was evaluated in 22 patients with HCC who underwent partial hepatic resection. Despite laboratory evidence of suppression of fibrinolysis, no differences in blood loss were demonstrated (101). Epsilon aminocaproic acid (EACA) interferes with plasminogen binding to fibrin and thus inhibits the conversion of plasminogen to plasmin (102). Similar to EACA, tranexamic acid inhibits fibrinolysis, but it is 6–10 times more potent than EACA (103). The use of tranexamic acid administered intravenously just before surgery and followed by 250 mg every 6 h for 3 days was associated with blood transfusion-free hepatectomy in an RCT studying 214 patients (104). In a recent systematic review with meta-analysis of RCT of 23 studies with a total of 1407 cirrhotic patients undergoing liver transplantation, both aprotinin and TA reduced transfusion requirements compared with controls. No increased risk for hepatic artery thrombosis, venous thromboembolic events or perioperative mortality was observed for either drug (105). Correction of Abnormalities of Haemostasis in Chronic Liver Disease 467

Before dental extraction, administration of 4×100 mg of tranexamic acid orally for a week can be used to reduce bleeding tendency. Tranexamic acid has also been shown in some reports to be useful in reducing blood transfusion requirements in patients with cirrhosis and gastric antral vascular ectasia who did not respond to endoscopic treatment, not portal decompression (106).

5.9. Antithrombin III Infusion ATIII infusion is not routinely recommended, even though some studies show increases in the ﬁbrinogen concentration during DIC (107). In liver transplantation, ATIII administration has not been shown to improve blood loss or decrease mortality (108).

6. ASSESSMENT OF THE RISK OF THROMBOSIS AND CLINICAL USE OF ANTICOAGULATION Thrombotic complications can paradoxically occur in cirrhotic patients even if clinically they are perceived as being at risk solely of bleeding. Despite abnormal coagulation tests, patients with cirrhosis should not be viewed as being “anticoagulated”. Wanless et al. hypothesized that portal vein and hepatic vein thrombosis caused histological disease progression in cirrhotic patients. Hepatic veins and portal vein radicle or larger vessels thromboses were found in at least 70 and 36% respectively of explanted livers and were associated with regions of confluent fibrosis (focal parenchymal extinction) (109). Portal vein thrombosis complicates liver cirrhosis in 0.6–15% of cases, leading to worsening of liver function, and mesenteric infarction if the mesenteric vein is involved (110). In these patients early anticoagulation is indicated as it has been shown to result in recanalization of the splanchnic veins in about 50% of cases, and prevent the extension of the thrombus, without causing bleeding complications (111). If high-risk varices are present, beta-blockers may be used for prophylaxis. In Budd–Chiari syndrome (BCS), even if a prothrombotic cause is not identified, anticoagulation should be started immediately after this diagnosis, because many genetic prothrombic defects remain yet to be discovered and acquired disorders common in BCS may prove elusive to diagnose such as polycythemia vera or paroxysmal nocturnal haemoglobinuria (PNH). Early anticoagulation is associated with improved survival. Anticoagulation therapy should continue even after liver transplantation because of the high rate of recurrence and thrombotic complications which can be as high as 30% after OLT, and also because multiple prothrombotic disorders may exist not limited to protein deficiencies (112). 468 Senzolo and Burroughs

There is an increased risk of deep-vein thrombosis and pulmonary embolism in patients with cirrhosis which has not been appreciated until recently reported in epidemiological studies (113). Patients with cholestatic disease, such as PBC and PSC, often exhibit a procoagulant state demonstrated by TEG, and may be prone to thrombosis, but this is not documented in natural history studies perhaps because as the data are not specifically collected (114). In particular, patients with cirrhosis appear to have a higher incidence of unprovoked DVT and pulmonary embolism (PE) compared with the general population. In retrospective studies, the incidence of DVT/PE ranges from 0.5 to 1.9%, similar to patients without comorbidities, but lower than patients with other chronic diseases (i.e. renal or heart disease). Importantly, standard coagulation laboratory parameters are not associated with a risk of developing DVT/PE. However, in a multivariate analysis, serum albumin level was independently associated with the occurrence of thrombosis, suggesting that the risk of thromboembolism is associated with worse liver dysfunction (115). The increased risk of deep-vein thrombosis and pulmonary embolism in patients with liver cirrhosis was also confirmed in a recent population-based study (116). Current guidelines on antithrombotic prophylaxis do not specifically comment on populations with cirrhosis. Despite a lack of specific guidelines during the last decades, there has been a reduction in thromboembolic complications in patients with cirrhosis in the last decades similar to the trend in other groups of patients, due to the increase use of thromboprophylaxis. However, the lack of specific guidelines for thromboembolic prophylaxis in cirrhosis is due to the perceived increased risk of bleeding complications (117). Cirrhosis is thus considered to be a relative contraindication for thromboprophylaxis in some centres. However, based on the work by Tripodi et al., this is likely to be a misconception. In the largest series reporting on the use of anticoagulation in patients with PVT and liver cirrhosis, Francoz et al. describe 19 patients on the waiting list for liver transplantation who underwent anticoagulation therapy, with recanalization in 42% without additional bleeding complications (111). However, only four patients had advanced liver disease (Child C), and the severity of the portal hypertension was not well described by the authors. On the other hand, Garcia Fuster et al. (118) described the results of anticoagulation for DVT in 17 patients with liver cirrhosis. Eleven of 17 patients were treated with LWMH, and six started with LWMH and switched to acenocoumarol thereafter. There was an 83% incidence of bleeding requiring transfusion, and only three patients were able to complete the treatment at 3 months. The grade and severity of portal hypertension was not reported by the authors. The monitoring of oral Correction of Abnormalities of Haemostasis in Chronic Liver Disease 469 anticoagulation in patients with cirrhosis can also be a problem since INR does not reflect the anticoagulant effect of drug in those patients (119). When 38 patients with cirrhosis and portal vein thrombosis (12 Child C, 20 Child B, 6 Child A) were treated with LMWH, independently of the severity of the impairment of coagulation parameters and tailoring the LMWH dose according to platelet count, there were two bleeding complications: one variceal bleeding from non-high-risk oesophageal varices and one intracranial haemorrhage which was self-limited in a patient older than 60 years with no additional risk factors for bleeding (120). Current guidelines for thromboprophylaxis in patients with important risk factors for bleeding state: “for medical patients with risk factors for venous thromboembolism and for whom there is a contraindication to anticoagulant thromboprophylaxis, we recommend the optimal use of mechanical thromboprophylaxis with graduated compression stockings or intermittent pneumatic compression”. The statement has the maximum grade of evidence and strength of recommendation; therefore it should be considered in patients with cirrhosis and high-risk varices who require DVT prophylaxis until such time that studies are conducted in patients with cirrhosis. One disadvantage of the use of LMWH as DVT prophylaxis in patients with cirrhosis is the unpredictable efficacy. LMWH requires antithrombin to exert its anticoagulant function, and antithrombin levels are frequently decreased in these patients. The use of new classes of anticoagulants, notably the antithrombin-independent direct Xa and thrombin inhibitors, may be of benefit in these patients, but these drugs will also need careful evaluation due to the risk of haemorragic complications in this subset of patients.

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Juan G. Abraldes, Jaime Bosch, and Juan Carlos García-Pagan

CONTENTS INTRODUCTION NATURAL HISTORY OF VARICES IN CIRRHOSIS SCREENING FOR VARICES SELECTION OF PATIENTS FOR PROPHYLAXIS TREATMENTS FOR THE PREVENTION OF FIRST BLEEDING:BETA-ADRENERGIC BLOCKERS VS.ENDOSCOPIC BAND LIGATION PREVENTION OF RECURRENT BLEEDING FROM ESOPHAGEAL VARICES THE ACUTE BLEEDING EPISODE TREATMENT OF ACUTE VARICEAL BLEEDING BLEEDING FROM GASTRIC VARICES BLEEDING FROM PORTAL HYPERTENSIVE GASTROPATHY (PHG) REFERENCES

Key Words: Portal hypertension, Endoscopic band ligation, Sclerotherapy, Drug therapy, TIPS

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_24, C Springer Science+Business Media, LLC 2011

477 478 Abraldes et al.

1. INTRODUCTION Variceal bleeding is one of the most common and severe complications of cirrhosis. Even with the current best medical care, mortality from variceal bleeding is still around 15–20%. Moreover, variceal bleeding often leads to deterioration in liver function, and it is a common trigger for other complications of cirrhosis, such as bacterial infections or hepatorenal syndrome. The treatment of portal hypertension includes the prevention of variceal hemorrhage in patients who have varices but never bleed, the treatment of the acute bleeding episode, and the prevention of rebleeding in patients who have survived a bleeding episode from esophageal or gastric varices. The natural history and prognosis in these scenarios are very different and should be considered when selecting therapies, since the hemostatic or prophylactic efﬁcacy of the available treatments is proportional to their invasiveness and adverse effects.

2. NATURAL HISTORY OF VARICES IN CIRRHOSIS When cirrhosis is diagnosed, varices are present in about 30–40% of compensated patients and in 60% of those who present with ascites (1). In those cirrhotic patients who present without varices, the annual incidence of new varices is about 5–10% (2–6). An HVPG over 10 mmHg (6) is the strongest predictor for the development of varices. This is in keeping with the role of portal pressure as the driving force for the development of collaterals. Once developed, varices increase in size from small to large. The reported rate of progression from small to large varices is highly variable, ranging from 5 to 30% per year (1, 5, 7, 8). The factor most consistently associated with variceal progression is baseline Child– Pugh class or worsening of the class during follow-up (5, 7, 9). Other factors include alcoholic etiology of cirrhosis and the presence of red wale markings (5). It has been shown that when HVPG decreases below 12 mmHg (either “spontaneously” or by drug therapy) esophageal varices decrease in size (10, 11). Once varices have been diagnosed, the overall incidence of variceal bleeding is around 25% at 2 years (12). The most important predictive factors of bleeding are variceal size, severity of liver dysfunction expressed by the Child–Pugh classification, and the presence of red wale marks on the variceal wall (13). These risk indicators have been combined in the NIEC index which allows classification of patients into different groups with a predicted 1-year bleeding risk ranging from 6 to 76% (13). Overall, variceal size remains the most useful predictor The Treatment and Prevention of Variceal Bleeding 479 for variceal bleeding. The risk of bleeding is very low (between 1 and 2%) in patients without varices at the first examination, and increases to about 5% per year in those with small varices and to 15% per year in those with medium or large varices (1).

3. SCREENING FOR VARICES The main aim of screening patients for esophageal varices is to identify those requiring prophylactic treatment. The current consensus is that every cirrhotic patient should be screened endoscopically for varices at the time of diagnosis. In patients without varices on initial endoscopy, a second (follow-up) evaluation should be performed after 2–3 years (14). Since endoscopy is invasive, Doppler-US (15–18)or transient elastography measurements by ﬁbroscan (19–21) have been proposed as possible surrogate markers of the presence of esophageal varices. Unfortunately, none of these noninvasive tests has proved to be accurate enough to safely avoid endoscopy (22, 23).

4. SELECTION OF PATIENTS FOR PROPHYLAXIS 4.1. Patients Without Varices Based on the results in experimental models of portal hypertension (24) in which nonselective beta-adrenergic blockers attenuated the development of portosystemic collaterals, these drugs were proposed for preventing the development of varices in patients with cirrhosis. However, in a multicenter study including a large number of compensated cirrhotics without esophageal varices (6), the rate of development of esophageal varices did not differ between patients treated with the nonselective beta-adrenergic blocker timolol or with placebo. Thus, so far, there is no effective treatment to prevent the development of varices.

4.2. Patients with High-Risk Varices In the past, only patients with medium to large varices were considered to have high-risk varices suitable for prophylactic treatment. However, it is well established that small varices with red signs or varices in Child–Pugh class C patients have a bleeding risk that is similar to that of large varices (13). In addition, it has been suggested that beta-adrenergic blockers may reduce the rate of progression from small to large varices, and decrease the incidence of variceal bleeding in patients with small varices (8). Thus, current guidelines recommend initiating nonselective beta-blockers in patients with moderate to large varices, but also in patients with small varices with red signs or if 480 Abraldes et al. the patient belongs to Child–Pugh class C (14). In patients with small varices, if beta-blockers are not initiated, a follow-up endoscopy should be performed every 1–2 years to check for a possible increase in size. This time must be shortened in case of clinical decompensation. No follow-up endoscopy is needed once the patient is on beta-blockers.

5. TREATMENTS FOR THE PREVENTION OF FIRST BLEEDING: BETA-ADRENERGIC BLOCKERS VS. ENDOSCOPIC BAND LIGATION Nonselective beta-adrenergic blockers (propranolol or nadolol) have been shown to reduce the risk of first variceal bleeding (from 24 to 15% after a median follow-up of 2 years) and mortality (from 27 to 23%) (12). However, approximately 25% of cirrhotic patients with high-risk esophageal varices may have either contraindications or intolerance to beta-adrenergic blockers. Additionally, the degree of protection (about 40% relative risk reduction on an intent-to-treat basis) is not ideal. The addition of isosorbide-5-mononitrate (ISMN) increases the HVPG response to beta-adrenergic blockers (25). However, it is less clear whether this translates into a greater clinical efficacy in primary prophylaxis. An open trial showed a lower rate of first bleeding in patients receiving nadolol + isosorbide mononitrate than in those receiving nadolol alone (26, 27). These beneficial effects on bleeding were not confirmed in a subsequent larger and better designed double- blind, placebo-controlled study (28). Currently, in primary prophylaxis it is not recommended to add isosorbide-5-mononitrate to nonselective beta-blockers. Endoscopic band ligation (EBL) is effective in preventing the first variceal bleeding in patients with medium to large varices (29). There is no agreement on how frequently varices should be ligated, the interval varying from every 1 to every 4 weeks (30, 31). A recent trial evaluated the effectiveness and complications of EBL every 2 weeks vs. every 2 months. This trial included patients with and without previous bleeding, although most patients were treated for primary prophylaxis (32). The 2-month interval scheme obtained a higher total eradication rate and lower recurrence rate. No patient in either group bled. Thus, although admittedly weak, current evidence favors at least monthly intervals. Once the varices are eradicated, follow-up endoscopies should be performed at 1–3 months and every 6 months thereafter, and varices should be re-eradicated upon recurrence. This is in marked contrast with prophylaxis with beta-blockers, in which no follow-up endoscopies are needed. The Treatment and Prevention of Variceal Bleeding 481

So far, 17 trials have compared EBL with beta-blockers for the primary prevention of variceal bleeding (30, 31, 33–45). The meta- analysis of these trials shows an advantage of EBL over beta-adrenergic blockers in terms of prevention of ﬁrst bleeding, without differences in mortality (46, 47)(Fig.1). These results, however, deserve several comments. First, most trials were underpowered or lacked any sample size calculation. Moreover, four trials were prematurely stopped

Fig. 1. Meta-analysis (random effects model) of randomized controlled trials comparing endoscopic band ligation (EBL) with beta-adrenergic blockers in the prevention of ﬁrst variceal bleeding. CI: Conﬁdence Interval; RR: Relative Risk. 482 Abraldes et al.

(30, 31, 39, 45), with three of them due to futility. When restricting the meta-analysis to those trials fully published including more than 100 patients there were no differences between beta-blockers and EBL (48). This illustrates that available evidence to favor EBL over beta-blockers is very weak. Another important issue when comparing two different treatment alternatives is the incidence of adverse events (49). Most side effects related to beta-blockers (hypotension, tiredness, breathlessness, impo- tence, insomnia) are easily managed by adjusting the dose or discontinuing the drug and do not require hospital admission (46). On the contrary, side effects related to EBL (most are bleeding episodes directly related to the procedure) frequently required hospitalization and blood transfusion and even might lead to the death of the patient (31, 45). Another variable to consider is patient’s and physician preferences. A recent study evaluated predicted preferences from patients and physicians with an interactive computer task. Sixty-four percent of the patients preferred EBL, based mainly on the possibility of shortness of breath and hypotension caused by nonselective beta-blockers (50). However, the option of switching from beta-blockers to EBL in case of side effects was not considered in the algorithm. Based on all these issues, the 2005 Baveno consensus conference recommended the use of nonselective beta-blockers as the first- choice treatment to prevent first variceal bleeding, while EBL should be offered to patients with contraindications or intolerance to beta- blockers (14). The guidelines of the AASLD and the ACG are slightly different; although they recommend beta-blockers as the first choice in Child A patients with medium/large varices and no red signs, they consider that both beta-blockers and EBL can be used as the first choice in patients with higher risk varices (51). A recent study has compared EBL against carvedilol (52), a nonselective beta-blocker with an intrinsic alpha-adrenergic blocker effect that produces a greater decrease in portal pressure than propranolol (53, 54), in the primary prophylaxis. In this study, carvedilol significantly reduced the incidence of variceal bleeding as compared to EBL (52). Whether carvedilol should be the preferred choice over propranolol, nadolol, or EBL in primary prophylaxis deserves further evaluation (55). The combination of pharmacology and endoscopic therapy has been investigated with contrasting results. In one study, band ligation plus beta-adrenergic blockers offered no benefit in terms of prevention of first bleeding when compared to band ligation alone (56). In a more recent study, combination therapy significantly reduced the occurrence The Treatment and Prevention of Variceal Bleeding 483 of the first episode of variceal bleeding and improved bleeding-related survival in comparison to EBL alone in a group of cirrhotic patients with high-risk esophageal varices on the waiting list for liver transplantation (57). Probably more studies would be required, although these are unlikely to be performed due to the very large number of patients that would be needed.

6. PREVENTION OF RECURRENT BLEEDING FROM ESOPHAGEAL VARICES Patients surviving a ﬁrst episode of variceal bleeding have a very high risk of rebleeding and death. Median rebleeding incidence within 1–2 years in untreated patients is around 60%, whereas mortality is around 33% (12). Therefore, prevention of recurrent bleeding is mandatory (58, 59). Randomized controlled trials for prevention of rebleeding have shown variceal size, Child–Pugh class, continued alcohol abuse, and hepatocellular carcinoma as risk indicators of rebleeding and death (12). In addition, a spontaneous decrease in portal pressure after alcohol abstinence or the pharmacological reduction of portal pressure by more than 20% of baseline values or to below 12 mmHg is associated with a sustained decrease in the risk of rebleeding (10, 60, 61). Available treatments for preventing variceal rebleeding include pharmacological therapy, endoscopic therapy, TIPS, and surgical shunting.

6.1. Drug Therapy Meta-analyses of the published studies have consistently found a marked beneﬁt of beta-adrenergic blockers, in terms of both preventing recurrent bleeding (from 63% in controls to 42% on beta-adrenergic blockers) and reducing mortality (form 27 to 20%) (12). Only two studies (only one published in abstract form (62)) have evaluated whether the association of isosorbide-5-mononitrate improves the efﬁcacy of nonselective beta-blockers administered alone in the prevention of recurrent bleeding with inconsistent results (62, 63). However, indirect data from studies that have compared the combination of beta-blockers plus isosorbide-5-mononitrate vs. endoscopic band ligation in the prevention of rebleeding strongly suggest that this combination drug therapy is the optimal pharmacological therapy. If it is possible to measure the hemodynamic response to beta-adrenergic blockers and a 20% decrease in HVPG or to ≤12 mmHg is achieved with the administration of only beta-blockers, there is probably no rationale to add isosorbide mononitrate (64). 484 Abraldes et al.

6.2. Endoscopic Therapy Endoscopic injection sclerotherapy of esophageal varices reduces both rebleeding and death, but has been replaced by endoscopic band ligation which is superior to sclerotherapy in preventing rebleeding with fewer side effects (65).

6.3. Drug vs. Endoscopic Therapy The meta-analyses of four available trials have shown a similar efficacy in preventing rebleeding and in survival in patients receiving optimal endoscopic treatment (band ligation) or optimized pharmacological treatment (combination of beta-blockers and isosorbide mononitrate) (66–69). Therefore, either beta-adrenergic blockers ± nitrates or banding ligation can be used as first-line treatments for the prevention of variceal rebleeding. If the patient bleeds while on primary prophylaxis with beta-adrenergic blockers, then the choice should be to add banding ligation, maintaining beta-adrenergic blockers. Two recent trials have shown that the association of beta-adrenergic blockers and endoscopic band ligation fares better than band ligation alone (70, 71). This beneficial effect was not confirmed in a third study (72). In addition, another two trials evaluated whether adding EBL to combined pharmacological therapy with nadolol + isosorbide-5-mononitrae improves the efficacy of nadolol + isosorbide- 5-mononitrate alone. Both studies failed to show a clear-cut benefit from adding EBL to drug therapy (73, 74). Therefore, although a recent meta-analysis suggests that the combination of drug therapy with EBL is probably the best strategy for preventing variceal rebleeding (75) more studies are needed to address this issue.

6.4. TIPS in the Prevention of Rebleeding TIPS has proven better than the combination of isosorbide-5- mononitrate and propranolol (76) and better than endoscopic therapy in the prevention of variceal rebleeding (77), with rebleeding rates of 9–23%. However, the high effectiveness in preventing recurrent bleeding is associated with an increased risk of encephalopathy, without a survival benefit. Because of this, TIPS is recommended as rescue therapy for patients who bleed despite adequate medical and endoscopic treatment. TIPS has been compared with surgical shunts in two randomized controlled trials (8 mm portocaval H-graft shunt in one and distal splenorenal shunt (DSRS) in the other) to prevent variceal rebleeding (78, 79). The first study favored surgical shunts, with a significantly lower rebleeding rate and a lower incidence of the composite end point The Treatment and Prevention of Variceal Bleeding 485 of rebleeding, shunt thrombosis, deaths, and need for transplant but without differences in survival. However, no differences in these parameters were observed in a second and larger trial, although at the expense of a higher reintervention rate in the TIPS group (82%) (79). In this study, TIPS was done using bare stents. However, the obstruction and reintervention rates were markedly decreased with the use of polytetrafluoroethylene (PTFE)-covered stents (80). A recent reanalysis of that trial modeling the rates of dysfunction assuming the use of PTFE- covered stents showed that TIPS is more cost-effective than DSRS (81). According to these data, TIPS using PTFE-covered stents represents the rescue therapy of choice for failures of medical and endoscopic treatment.

7. THE ACUTE BLEEDING EPISODE Ruptured esophageal varices are the cause of greater than 70% of all upper gastrointestinal bleeding episodes in patients with portal hypertension (82). Thus, in any cirrhotic patient with acute upper gastrointestinal bleeding, a variceal origin should be suspected. Diagnosis is established at emergency endoscopy based on observing one of the following: (a) active bleeding from a varix (observation of blood spurting or oozing from the varix); (b) white nipple or clot adherent to a varix; (c) presence of varices without other potential sources of bleeding. Endoscopy should always be performed within 12 h of admission and as early as possible.

8. TREATMENT OF ACUTE VARICEAL BLEEDING Acute variceal bleeding should be managed in an intensive care setting by a team of experienced medical staff, including well-trained nurses, clinical hepatologists, endoscopists, interventional radiologists, and surgeons (83). Lack of these facilities demands immediate referral. Decision making should follow the guidelines set up in a written protocol developed to optimize the resources of each center.

8.1. General Management The general management of the bleeding patient is aimed at correcting hypovolemia (with judicious volume replacement and transfusion) and at preventing complications associated with gastrointestinal bleeding (bacterial infections, hepatic decompensation, renal failure), which 486 Abraldes et al. are independent of the cause of the hemorrhage and demand immediate management. Initial resuscitation should be aimed at restoring an appropriate delivery of oxygen to the tissues (which depends on SaO2, cardiac output, and hemoglobin concentration). Airway should be immediately secured, especially in encephalopathic patients, since the patient is at risk of bronchial aspiration of gastric content and blood. This risk is further exacerbated by endoscopic procedures. Endotracheal intubation is mandatory if there is any concern about the safety of the airway. Blood volume replacement should be initiated as soon as possible with plasma expanders, aiming at maintaining systolic blood pressure around 100 mmHg. Avoiding prolonged hypotension is particularly important to prevent infection and renal failure, which are associated with increased risk of rebleeding and death (84). It has been shown that excessive volume expansion may induce rebound increases in portal pressure and rebleeding (85, 86). Blood transfusion should aim at maintaining the hematocrit at 21–24% (Hb 7–8 g/l) (14), except in patients with rapid ongoing bleeding or with ischemic heart disease. An initial study suggested that the use of recombinant activated factor VII (rVIIa, NovosevenR ), which corrects prothrombin time in cirrhotics (87), could significantly improve the results of conventional therapy in patients with moderate and advanced liver failure (stages B and C of the Child–Pugh classification) without increasing the incidence of adverse events (88). However, this has not been confirmed in a more recent trial specifically designed to treat this high-risk population (89). Therefore, there is no indication for the use of rVIIa in the treatment of acute variceal bleeding. Infection is a strong prognostic indicator in acute variceal bleeding (90). The use of prophylactic antibiotics has been shown to reduce both the risk of rebleeding (91) and mortality (92). Therefore, antibiotics should be given to all patients at admission. Quinolones are frequently used (norfloxacin 400 mg/bid) due to their ease of administration and low cost (93). In high-risk patients (hypovolemic shock, ascites, jaundice, or malnutrition) intravenous ceftriaxone (1 g/day) has recently been shown to be superior to oral norfloxacin (94), mainly due to the prevention of infections by quinolone-resistant bacteria. Variceal bleeding can trigger hepatic encephalopathy. However, there are no data to support the prophylactic use of lactulose or lactitol (14).

8.2. Speciﬁc Therapy for Control of Bleeding Initial therapy for acute variceal bleeding is based on the combination of vasoactive drugs with endoscopic therapy. The Treatment and Prevention of Variceal Bleeding 487

8.3. Pharmacological Therapy Terlipressin is a long-acting triglycyl lysine derivative of vasopressin. Clinical studies have consistently shown less frequent and severe side effects with terlipressin than with vasopressin (even if associated with nitroglycerin). Terlipressin may be initiated as early as variceal bleeding is suspected at a dose of 2 mg/4 h for the first 48 h, and it may be maintained for up to 5 days at a dose of 1 mg/4 h to prevent rebleeding (95). Compared with placebo or nonactive treatment, terlipressin significantly improves the rate of control of bleeding and survival (96). This is the only treatment that has been shown to improve prognosis of variceal bleeding in placebo-controlled RCTs and meta-analysis (12, 96). Terlipressin is as effective as other therapies, including endoscopic injection sclerotherapy (EIS), and is safer than vasopressin + nitroglycerin and injection sclerotherapy (12, 95, 96). The most common side effect of this drug is abdominal pain. Serious side effects such as peripheral or myocardial ischemia occur in fewer than 3% of the patients (95). The overall efficacy of terlipressin in controlling acute variceal bleeding at 48 h is 75–80% across trials (96), and 67% at 5 days (95). Somatostatin is empirically used as an initial bolus of 250 μgfol- lowed by a 250 μg/h infusion that is maintained until the achievement of a 24 h bleed-free period. The bolus injection can be repeated if bleeding is uncontrolled. Therapy should be maintained for up to 5 days to prevent early rebleeding (97). Major side effects with somatostatin are rare. Minor side effects, such as nausea, vomiting, and hyperglycemia, occur in up to 30% of patients (97–99). Results of the studies comparing somatostatin vs. placebo are inconclusive (100). However, the efficacy of somatostatin in acute variceal bleeding is suggested by those studies that compare the drug against other therapies such as terlipressin or endoscopic therapy. Indeed, somatostatin has been shown to be equivalent to terlipressin and to endoscopic therapy in the control of acute variceal bleeding, prevention of early rebleeding and mortality, and reduction of the incidence of adverse events (12). The use of higher doses (500 μg/h) causes a greater fall in HVPG and translates into increased clinical efficacy in the subset of patients with active bleeding at emergency endoscopy (99). Octreotide is a somatostatin analogue with longer half-life. The optimal doses are not well determined. It is usually given as an initial bolus of 50 μg,followedbyaninfusionof25or50μg/h (100). The efficacy of octreotide as a single therapy for variceal bleeding is controversial. However, randomized controlled trials using octreotide followed by sclerotherapy have shown a significant benefit in terms of reducing early rebleeding (101). Mortality, however, was not affected 488 Abraldes et al.

(12, 101). These results suggest that octreotide may improve the results of endoscopic therapy but has uncertain effects if used alone.

8.4. Endoscopic Therapy Both sclerotherapy and band ligation (EBL) have been shown to be effective in the control of acute variceal bleeding. Two randomized trials speciﬁcally compared band ligation and sclerotherapy in acute variceal bleeding (102, 103). In one of them, all patients also received somatostatin (103). In eight additional trials, these two modalities were compared both in acute bleeding and in the prevention of rebleeding. Meta-analysis shows that EBL is better than sclerotherapy in the initial control of bleeding, and is associated with less adverse events and improved survival. Additionally, sclerotherapy, but not EBL, may increase portal pressure (104). Therefore EBL is the endoscopic therapy of choice in acute variceal bleeding, although injection sclerotherapy is acceptable if band ligation is not available or technically difﬁcult. Endoscopic therapy can be performed at the time of diagnostic endoscopy, early after admission, provided that a skilled endoscopist is available. This is important since there has been an increased frequency of aspiration pneumonia since emergency endoscopic therapy has become universal practice.

8.5. Current Recommendations for Initial Treatment The current recommendation is to start vasoactive drug therapy early (ideally during the transfer to hospital or on arrival, even if active bleeding is only suspected) and to perform endoscopic band ligation (or injection sclerotherapy if band ligation is technically difﬁcult) after initial resuscitation (14, 51)(Fig.2). No study has directly compared the available drugs in combination with endoscopic therapy. Therefore, terlipressin, somatostatin, octreotide, and vapreotide are all acceptable options. The optimal duration of drug therapy is not well established. Current recommendations are to maintain the drug for 2–5 days (14).

8.6. Rescue Therapies: Tamponade, Surgery, and TIPS In 10–20% of patients, variceal bleeding is unresponsive to initial endoscopic and/or pharmacological treatment. If bleeding is mild and the patient is stable a second endoscopic therapy might be attempted. If this fails, or bleeding is severe, the patient should be offered a derivative treatment, before his or her clinical status deteriorates further. Balloon tamponade achieves hemostasis in 60–90% of variceal bleedings (105) but should be used only in the case of a massive bleeding, for a short period of time (less than 24 h) as a temporal “bridge” The Treatment and Prevention of Variceal Bleeding 489

Fig. 2. Algorithm of the management of acute bleeding from ruptured esophageal varices. TIPS: Transjugular Intrahepatic Portosystemic Shunt. until definite treatment is instituted. Bleeding recurs after deflation in over half of the cases and severe complications are common. A recent report suggests that the use of esophageal covered stents might achieve hemostasis in most patients with refractory bleeding (106), with the advantage over tamponade of less severe complications despite longer periods of treatment. Adequately designed trials should confirm these findings. Both TIPS and surgical shunts are extremely effective in controlling variceal bleeding (control rate approaches 95%), but due to worsening of liver function and encephalopathy mortality remains high (105, 107). TIPS is the first choice, since most patients requiring rescue treatment have advanced liver disease. However, rarely, if ever, will a patient with a Child–Pugh score over 13 survive TIPS. This clearly indicates that some patients do not benefit from TIPS in this setting, and sometimes it is difficult to make a clinical-based decision. Prognostic scores (108) may provide objective parameters to support decisions of not offering invasive treatments in difficult cases. A recent randomized trial explored whether patients with poor prognostic indicators might benefit from a more aggressive therapeutic approach ab initio. Patients with high risk (with HVPG >20 mmHg) were randomized to receive standard therapy or TIPS. Those who underwent early TIPS had significantly less treatment failure and lower 490 Abraldes et al. mortality than patients undergoing standard therapy (109). A more recent trial (still in abstract form) showed that in patients at high risk of failure (Child B with active bleeding or Child C) the performance of an early TIPS dramatically decreases rebleeding and mortality at 1 year, without an increase in encephalopathy rates (110). If confirmed, these results would change the current approach to the management of acute variceal bleeding, favoring a more aggressive approach in those patients at high risk of treatment failure.

9. BLEEDING FROM GASTRIC VARICES Gastric varices develop in approximately 20% of patients with portal hypertension (111). They are the source of 5–10% of all upper digestive bleeding episodes in patients with cirrhosis. The risk of gastric variceal bleeding is lower than that of esophageal variceal bleeding, but gastric variceal bleeding, in particular that from fundal varices, tends to be more severe, to require more transfusions, and to have a higher mortality rate (111). Fundal varices account for 1–3% of all variceal bleeds. The optimal treatment of gastric fundal varices has not been determined, since there are few trials and most data come from retrospective series. The initial treatment is similar to that of esophageal variceal bleeding, including the administration of a vasoactive drug (terlipressin, somatostatin or a somatostatin analogue). Balloon tamponade, with the Linton-Nachlas tube, has been used with limited success (112, 113), but may serve as a bridge to derivative treatments in patients with massive bleeding. Some endoscopic therapies are promising, but quality information is scarce, and most studies include both fundal varices and gastroesophageal varices. Sclerotherapy, variceal obturation with tissue adhesives (“glue injection”), thrombin, band ligation, and application of large detachable snares have been reported (114). Most uncontrolled series using cyanoacrylate report a high rate of control of bleeding (about 90%) (115). Two recent randomized trials compared EBL with cyanoacrylate injection. In one trial cyanoacrylate injection was more effective and safer than EBL in the control of acute bleeding, and was associated with less rebleeding (116). In the other trial both treatments were equally effective in controlling acute bleeding, but rebleeding was more frequent in the ligation group (117). In another study, cyanoacrylate was better than sclerotherapy both in achieving initial hemostasis and in achieving faster variceal obliteration (118). These trials suggest that cyanoacrylate injection is the most effective endoscopic therapy in the control of acute bleeding from gastric fundal varices. This The Treatment and Prevention of Variceal Bleeding 491 technique, however, needs expertise, and may not be feasible during active bleeding. TIPS is very effective in the treatment of bleeding gastric varices, with more than 90% success rate for initial hemostasis and very low rebleeding rate (119, 120). A recent trial has shown that it is more effective than glue injection in preventing rebleeding (121). Surgery is also effective, but with limited applicability in advanced cirrhosis. Another approach is the retrograde intravascular obliteration of spontaneous splenorenal shunts that are frequently present in patients with large fundal varices (122). To date, however, this treatment has not been studied in a controlled trial. The authors’ recommendation in patients with gastric variceal bleeding is to initiate treatment with a vasoactive drug. If bleeding is not controlled and if an expert endoscopist is available, variceal obturation with glue must be performed. In cases of massive bleeding or after failure of previous therapies, TIPS (or surgical shunt in Child A patients) is mandatory. A second attempt at endoscopic therapy should not be allowed in these patients.

10. BLEEDING FROM PORTAL HYPERTENSIVE GASTROPATHY (PHG) Portal hypertensive gastropathy is a macroscopic finding of a characteristic mosaic-like pattern of the gastric mucosa (“mild” PHG), red-point lesions, cherry red spots, and/or black-brown spots (“severe” PHG) (123). These lesions, however, are not entirely specific – they can occur in the absence of portal hypertension. In PHG, there is marked dilatation of the vasculature of the gastric mucosa and submucosa, together with an increased blood flow and tendency to decreased acid secretion. PHG is unrelated to Helicobacter pylori infection. The overall prevalence of PHG in patients with cirrhosis strongly correlates with the severity of the disease and ranges between 11 and 80% (123). The incidence of acute bleeding is low (less than 3% at 3 years) with a mortality of 12.5%, while the incidence of chronic bleeding is 10–15% at 3 years. In acute bleeding from PHG beta-adrenergic blockers, somatostatin, octreotide, vasopressin, terlipressin, and estrogens have been proposed based on their ability to decrease gastric perfusion in this condition (124–127). However, only one uncontrolled study so far has evaluated one of these drugs (somatostatin) in acute bleeding from PHG (128). Hemostasis was achieved in all patients. Nonselective beta-blockers effectively decrease chronic bleeding from PHG (129). TIPS may be effective in preventing bleeding from PHG. 492 Abraldes et al.

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122. Matsumoto A, Hamamoto N, Nomura T, Hongou Y, Arisaka Y, Morikawa H, et al. Balloon-occluded retrograde transvenous obliteration of high risk gastric fundal varices. Am J Gastroenterol 1999 Mar;94(3):643–9. 123. Primignani M, Carpinelli L, Preatoni P, Battaglia G, Carta A, Prada A, et al. Natural history of portal hypertensive gastropathy in patients with liver cirrhosis. The New Italian Endoscopic Club for the study and treatment of esophageal varices (NIEC). Gastroenterology 2000 Jul;119(1):181–7. 124. Panes J, Bordas JM, Pique JM, Garcia-Pagan JC, Feu F, Teres J, et al. Effects of propranolol on gastric mucosal perfusion in cirrhotic patients with portal hypertensive gastropathy. Hepatology 1993 Feb;17(2):213–8. 125. Panes J, Pique JM, Bordas JM, Llach J, Bosch J, Teres J, et al. Reduction of gastric hyperemia by glypressin and vasopressin administration in cirrhotic patients with portal hypertensive gastropathy. Hepatology 1994 Jan;19(1):55–60. 126. Panes J, Pique JM, Bordas JM, Casadevall M, Teres J, Bosch J, et al. Effect of bolus injection and continuous infusion of somatostatin on gastric perfusion in cirrhotic patients with portal-hypertensive gastropathy. Hepatology 1994 Aug;20(2):336–41. 127. Panes J, Casadevall M, Fernandez M, Pique JM, Bosch J, Casamitjana R, et al. Gastric microcirculatory changes of portal-hypertensive rats can be attenuated by long-term estrogen–progestagen treatment. Hepatology 1994 Nov;20(5): 1261–70. 128. Kouroumalis EA, Koutroubakis IE, Manousos ON. Somatostatin for acute severe bleeding from portal hypertensive gastropathy. Eur J Gastroenterol Hepatol 1998 Jun;10(6):509–12. 129. Perez-Ayuso RM, Pique JM, Bosch J, Panes J, Gonzalez A, Perez R, et al. Propranolol in prevention of recurrent bleeding from severe portal hypertensive gastropathy in cirrhosis. Lancet 1991 Jun 15;337(8755):1431–4. Extracorporeal Artiﬁcial Liver Support Systems

Rafael Bañares and María-Vega Catalina

CONTENTS INTRODUCTION:REASONS FOR ARTIFICIAL LIVER SUPPORT PATHOPHYSIOLOGICAL APPROACH TO ARTIFICIAL LIVER SUPPORT AND TYPES OF LIVER SUPPORT SYSTEMS PATHOPHYSIOLOGICAL EFFECTS OF LIVER SUPPORT SYSTEMS CLINICAL EFFICACY OF LIVER SUPPORT SYSTEMS REFERENCES

Key Words: Liver failure, Artiﬁcial liver support, Albumin dialysis, Hepatorenal syndrome, Hepatic encephalopathy, Circulatory dysfunction

1. INTRODUCTION: REASONS FOR ARTIFICIAL LIVER SUPPORT One of the most appealing therapeutic approaches for treating any organ failure is the possibility to provide a temporary artificial support of its function. In some cases (for example kidney failure) dialysis procedures are widely accepted and used, while in others (for example cardiac failure) artificial devices remain largely investigational. In this context, the concept of artificial liver support (ALS) has gained importance in the past decades. Although the prevention and management

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_25, C Springer Science+Business Media, LLC 2011

501 502 Bañares and Catalina of liver failure has improved in recent years, it is still an important cause of morbidity and mortality in Europe and the Unites States (1). A widely accepted classification of liver failure includes three main categories: acute liver failure (ALF), when the liver structure and function are normal before the onset of the symptoms; acute-on-chronic liver failure (ACLF), when an acute injury affects an already damaged liver; and chronic liver failure (CLF), which represents end-stage failure of the liver. The main difference between the first two forms and chronic liver is the potential for recovery to previous levels of liver function in the former (2). Importantly, manifestations of liver failure involve not only the deterioration of liver function, manifested by coagulopathy and jaundice, but also severe impairment of extrahepatic organs, mainly the brain, kidney, and circulatory system. The therapeutic approach for patients with severe liver failure is aimed at stabilizing the patient until the liver spontaneously recovers or until a liver graft is available, because liver transplantation is the only therapeutic measure with a clearly proven survival benefit. However, due to the shortage of organs, a significant proportion of patients die while waiting for liver transplant. Also, patients with ALF and ACLF often have multiorgan dysfunction that precludes liver transplantation. Finally, some patients with ALF or ACLF may have contraindications to liver transplantation including advanced age, alcohol use, drug abuse, etc. These observations clearly emphasize the need for liver supportive strategies. The primary aim of this approach is to facilitate the complete regeneration of liver mass and function, while maintaining hepatic and extrahepatic function. A secondary aim is to provide a bridge to liver transplantation in case that full regeneration of the liver or recovery of previous function is not possible. In the recent years, great efforts have been made in the development of new ALS systems. The aim of this chapter is to review the role of ALS in patients with chronic liver failure with special emphasis in emerging clinical aspects.

2. PATHOPHYSIOLOGICAL APPROACH TO ARTIFICIAL LIVER SUPPORT AND TYPES OF LIVER SUPPORT SYSTEMS Liver cell damage depends on the type, duration, and severity of the noxious agent, which results in cell death from necrosis, apoptosis, or both (3). These cellular changes induce accumulation of various toxic substances such as ammonia, mediators of oxidative stress, bile acids, Extracorporeal Artiﬁcial Liver Support Systems 503

NO, lactate, products of arachidonic acid metabolism, benzodiazepines, indoles, and mercaptans, resulting in increased susceptibility to infections, circulatory disturbances, and end-organ dysfunction. In addition, due to this “toxic” state, secondary liver damage occurs as a consequence of the vicious circle that results from the release of inflammatory mediators, oxidative stress, and sinusoidal endothelial cell damage. Therefore, there are several theoretical pathophysiological approaches to restore liver function in acute liver failure. The first is based on the potential to increase liver regeneration; several studies (3) have shown that stimulation of liver cell regeneration by growth factors such as HGF, EGF, and TGF-α and other mediators is feasible under experimental conditions. However, the clinical application of these approaches is still limited because of the paucity of knowledge regarding the interaction between parenchymal and nonparenchymal cells. It is also uncertain whether manipulation of single systems can really be translated to clinical outcomes in patients with ALF. Another possibility is to increase the metabolic mass, through the use of auxiliary liver transplantation and hepatocyte transplantation, which serves as a bridge to liver transplantation in patients with ALF or until liver function has been restored (4, 5). However these procedures are difficult to perform, require complex logistics, and remain unproven. The most promising approach to artificial liver support is the use of extracorporeal liver support systems. These devices are divided into biological, nonbiological (also called artificial or cell-free techniques), and bioartificial (hybrid techniques) devices (6). Biological devices are based on the perfusion of blood or plasma through an extracorporeal bioreactor loaded with vital liver cells. The aim of these devices is to support the detoxifying and metabolic function of the liver, thus theoretically replacing all failing liver functions. In contrast, nonbiological devices aim to remove from plasma water-soluble and protein-bound toxins through dialysis-derived techniques. Bioartificial or hybrid devices combine the best of both biological and nonbiological techniques. The pros and cons of biological and nonbiological techniques as well as the main characteristics of these devices (2, 7–9)andthe prospects for next-generation bioartificial devices are summarized in Tables 1, 2, 3,and4. From a clinical point of view, extracorporeal nonbiological devices, especially albumin dialysis, have been more extensively analyzed. There are two main systems, the molecular adsorbent recirculation system (MARS) and the fractionated plasma separation and absorption (Prometheus). In contrast to the single-pass albumin techniques in which the albumin-rich dialysate is discharged 504 Bañares and Catalina

Table 1 Mechanisms of action and summary of clinical outcomes of cell-free liver support systems

Type of device Mode of action Outcomes

Hemodialysis (46) Exchange diffusion Improvement of coma, across a no improvement of semipermeable survival membrane Hemofiltration (47) Continuous convective Limited outcome solute removal across a semipermeable membrane High-volume Exchange of high Improvement of plasmapheresis plasma volumes biochemical (48) parameters and clinical status Hemodiafiltration Convection (large Case reports, (49, 50) molecules) and improvement of diffusion (small biochemical molecules) removal parameters and across a neurological status semipermeable membrane Hemoperfusion Perfusion of Removal of toxins, (51) blood/plasma over improvement of charcoal, synthetic mental status, no neutral resins, or anion survival benefit exchange resins Hemodiabsorption Dialysis against a Improvement of (52) combination of biochemical charcoal and ion parameters and exchanger clinical status, no improvement of survival Molecular Removal of Improvement of adsorbent protein-bound and biochemical recirculating water-soluble parameters and system (MARS) substances across a clinical status, survival (30, 32, 34) specialized membrane benefit in short against randomized trials. albumin-enriched Large trials ongoing dialysate Extracorporeal Artificial Liver Support Systems 505

Table 1 (Continued)

Type of device Mode of action Outcomes

FPSA Hemodiafiltration using Improvement of (Prometheus) albumin dialysate biochemical parameters (11, 14) and clinical status Artificial liver Combination of plasma Improvement of support system exchange, biochemical parameters (ALSS) (53) hemoperfusion, and clinical status hemofiltration, and hemodialysis PF-Liver Dialysis Combines Improvement of (54) hemodiabsorption biochemical parameters with push-pull and clinical status sorbent-based apheresis

FPSA, fractionated plasma separation and adsorption

Table 2 Pros and cons of extracorporeal liver support systems

Biological Systems Non-biological systems

–PROS –PROS • Potential to provide • Relatively easy to use synthetic functions • Ameliorates – CONS Pathophysiological • Cell Source findings of ALF • Need for a critical • Potential benefit on bioactive mass survival • Complex technology • More advanced clinical • Xenotranmission development • Cost – CONS • Lack of synthetic functions • Deficiency for specific removal of some compounds • Cost after passing through the dialyzer, in the MARS system, albumin is regenerated in a separate circuit by using low-flux dialysis and different absorbers to “clean” the albumin dialysate that is then used again 506 Bañares and Catalina 9 10 × ) freshly isolated aggregates ) 7 ( 10 AMC-BAL Porcine, No 7 9 10 × freshly isolated or bilirubin columns HBAL (65) Porcine, Charcoal –Small freshly isolated Albumin dialysis organoids ) and van de Kerkhove et al. ( LSS MELS (63, 64) LSS: porcine 8 MELS: human, MELS: freshly isolated 200–230 g Up to 600 g 10 Radial flow bioreactor (RFB) (62) Porcine, No LSS: no 9 10 × Table 3 isolated column previous to bioreactor TECA_HALSS (61) Porcine, freshly – Aggregates Tissue-like freshly isolated cell entra- pped BLSS (60) 70–120 g 10–20 Phase 1 Phase 1 Phase 1 Phase 1 Phase 1 Phase 1 No Charcoal Porcine, Collagen 9 10 × column previous to bioreactor opreserved attached; irregular aggregates (1) Phase 1 Hepat-assist (58, 59) Porcine, cry- Microcarrier (2) RCT RCT hepatoma aggregates (1) Phase 1 Up to 168 h 6 hNo 12 h Charcoal Up to 5 h Up to 24 h 7–74 h 6 h 4–35 h ELAD (56, 57) (2) RCT Characteristics of clinically applied BAL systems (adapted from Chamuleau et al. ( clinical development time detox devices Cell amount 200–400 g 5–7 Phase of Plasma/blood BloodTreatment Additional Plasma Blood Plasma Plasma Plasma Plasma Plasma Cell type C3A Shape of cells Large Extracorporeal Artificial Liver Support Systems 507 , ) in serum ammonia and bilirubin 7 AMC-BAL ( Marked decrease HBAL (65) LSS MELS (63, 64) in serum ammonia Radial flow bioreactor (RFB) (62) Mild decrease Table 3 (Continued) TECA_HALSS (61) decrease in serum ammonia BLSS (60) 46 7 151214 Mild benefit in post hoc analysis adverse events Hepat-assist (58, 59) (1) 10 Survival No relevant (2) 171 survival adverse events ELAD (56, 57) (1) 11 No effect on No relevant (2) 24 BAL, bioartificial liver; BLSS, bioartificial liver support system; ELAD, extracorporeal liver assist device; HBAL, hybrid bioartificial liver; LSS patients results Number of Clinical liver support system; MELS, modularrandomized extracorporeal liver clinical support; trial RFB, radial flow bioreactor; HALSS, hybrid artificial liver support system; RCT, 508 Bañares and Catalina

Table 4 Prospects for the future bioartiﬁcial liver systems (adapted from Park and Lee (9))

Present bioartiﬁcial Next-generation systems bioartiﬁcial system

Hepatocyte source Pig or hepatoma Human hepatocytes from stem cells or humanized pigs Culture method Simple and conventional Liver-like structures with (bioreactor) culture techniques optimum cell–cell interactions, microarchitectured 3-D coculture Treatment time Less than 24 h Week to months Liver function Several vital functions Entire liver functions including bile excretion Target disease Acute liver failure Chronic as well as acute liver failure Safety Procedures to decrease Minimize the risk of zoonosis

for the detoxiﬁcation process. In the Prometheus system, a special albumin-permeable ﬁlter is used. Thus, albumin and the protein-bound toxins pass through the membrane and are then directly removed by special adsorbers within the secondary circuit. The native albumin is subsequently returned to the patient. The process is completed with hemodialysis to eliminate water-soluble toxins.

3. PATHOPHYSIOLOGICAL EFFECTS OF LIVER SUPPORT SYSTEMS 3.1. Pathophysiological Effects of Albumin Dialysis in ACLF Artificial liver support systems have provided a unique opportunity to evaluate the pathological mechanisms associated with acute decompensation of chronic liver failure. In this context, recent studies have analyzed the effect of the different systems of albumin dialysis in ACLF. All published studies have uniformly shown a significant reduction in serum bilirubin and biliary acids and other protein-bound Extracorporeal Artificial Liver Support Systems 509 substances with both the MARS and Prometheus devices (10–12). Early in vitro and in vivo studies with MARS showed that there was an improvement of the amino acid profile, with relative clearance of the aromatic amino acids and an improved ratio of branched-chain amino acids to aromatic amino acids. In a recent study (13) aimed at evaluating the influence of MARS on amino acid profile, the plasma levels of neuroactive amino acids, methionine, glutamine, glutamate, histidine, and taurine decreased during MARS treatment suggesting a favorable effect on the plasma amino acid profile of patients with HE. MARS did not induce significant removal of physiologically important proteins. However, it is associated to a significant decrease of serum levels of albumin-bound toxins, such as fatty acids, followed by bile acids, tryptophan, and bilirubin. Other studies evaluating the clearance capacity of Prometheus device (11, 14) showed a significant decrease of both protein-bound and water-soluble substances after Prometheus therapy. A direct comparison between the detoxifying capacities of both systems has been reported. Both systems significantly decreased serum levels of protein-bound and water-soluble toxins. However, Prometheus produced higher clearances of all tested substances, especially bilirubin (15). In another recent comparative study, MARS and Prometheus devices removed total bile acids to a similar extent (16). Although the precise pathophysiological basis of acute-on-chronic liver failure is unclear, systemic inflammatory response is considered to be implicated (17). Several studies have analyzed the effect of albumin dialysis in this context. Sen et al. (18) have evaluated the effects of albumin dialysis in patients with acute alcoholic hepatitis in a randomized trial. In this study, albumin dialysis was associated with an improvement of encephalopathy without effects on blood pressure and renal function as compared to standard medical therapy. Importantly, these findings were associated to a significant reduction in plasma concentration of NO, without changes in cytokine profile, plasmatic malondialdehyde levels, or plasma ammonia. A recent study has compared the effects of MARS and Prometheus on cytokine profile in patients with ACLF mainly secondary to acute alcoholic hepatitis (19). In this study, although both MARS and Prometheus showed clearance for several cytokines (IL-6, IL-8, IL-10, TNF-α,andsTNF-αR1) neither MARS nor Prometheus device was able to reduce serum concentrations of any cytokine. However, another study (20) has shown that MARS procedure induced a significant decrease in TNF-α, IL-6, and IL-1β.In addition, survival was better in those patients who had a decrease in 510 Bañares and Catalina cytokine level after MARS therapy. Therefore, the influence of albumin dialysis on proinflammatory cytokines is controversial. Plasma from patients with liver failure may contain toxic molecules that cause hepatocyte apoptosis and worsen liver disease, suggesting that removal of proapoptotic factors may be an appropriate therapeutic strategy (21). Several recent studies have explored the potential influence of MARS in modulating serum levels of these cytokines, especially taking into account that other dialysis modalities have shown a potentially deleterious increase in their concentrations. One study has studied pre- and post-MARS serum concentrations of the proinflammatory cytokine IL-18 and its activator caspase-1 without showing any significant change (22); in addition, MARS did not decrease serum levels of other chemokines also implicated in ACLF such as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-3α (MIP-3α). Several studies have analyzed the effects of albumin dialysis on splanchnic and systemic hemodynamics in patients with ACLF. The majority of these studies have shown an improvement in the hyperdynamic circulation of advanced liver failure, with an increase in arterial pressure and systemic vascular resistance with an attenuation of hyper- activated vasoactive systems (23–25). These beneficial hemodynamic effects are not restricted to systemic circulation but can also be observed in the splanchnic circulation. Two studies (23, 26)haveshownthat albumin dialysis with MARS significantly decreases portal pressure as estimated by a reduction in hepatic venous pressure gradient (HVPG). Interestingly, the effect on portal pressure was independent of extracorporeal circulation as shown by the fact that conventional hemofiltration did not induce any change in portal pressure (26). A comparison between the hemodynamic effects of both albumin dialysis systems has recently been completed (24). Although Prometheus induced a significantly greater decrease of serum bilirubin, only MARS was associated with a significant improvement in mean arterial pressure and, interestingly, with an attenuation of serum markers of circulatory dysfunction (plasma renin activity, aldosterone, norepinephrine, vasopressin, and nitrate/nitrite levels). These findings were confirmed in a recent randomized study (27) showing that MARS but not Prometheus increased arterial pressure in patients with advanced liver failure but without acute decompensation. Finally, in a recent study (28) aimed at analyzing functional properties of albumin in patients with acutely decompensated cirrhosis, MARS was not able to modify the detoxifying capacity of albumin. Comparison between physiological and hemodynamic effects of MARS and Prometheus is given in Table 5. Extracorporeal Artificial Liver Support Systems 511

Table 5 Comparison between pathophysiological effects of MARS and FPSA in acute-on-chronic liver failure (15, 24)

Parameter MARS FPSA

Bilirubin ↓↓↓ Biliary acids ↓↓↓ IL-6 No change No change IL-8 No change No change IL-10 No change No change TNF-α No change No change STNF-αR1 No change No change Heart rate No change ↑ MAP ↑ No change Stroke volume ↑ No change SVR ↑ No change Aldosterone ↓ No change Norepinephrine ↓ No change ADH ↓ No change Nitrites/nitrates ↓ No change

MARS, molecular adsorbent recirculating system; FPSA, fractionated plasma separation and adsorption; IL-6, -8, -10, interleukin-6, -8, -10; TNF-α, tumor necrosis factor-α;STNF-αR1, soluble receptor 1 of tumor necrosis factor; MAP, mean arterial pressure; ADH, antidiuretic hormone; SVR, systemic vascular resistance

4. CLINICAL EFFICACY OF LIVER SUPPORT SYSTEMS 4.1. Nonbiological Artificial Devices in Acute-on-Chronic Liver Failure The initial reports with MARS albumin dialysis were in uncontrolled case series in ACLF with bilirubin greater than 20 mg/dL (10, 29–31) (Table 6). In the largest study, in-hospital survival was high and was associated with improvement in serum bilirubin and in end-organ failure (encephalopathy, renal function, and circulatory disturbances) and with the degree of liver failure estimated by Child–Pugh scores (29). There are only three randomized trials with MARS in patients with ACLF. The first study was aimed at evaluating the effect of albumin dialysis in patients with type I hepatorenal syndrome (32). Thirteen patients were randomized to receive either MARS (n=8) or hemodiafiltration (n=5). Patients allocated to MARS therapy had a 512 Bañares and Catalina vs. 6/12) MARS-treated patients No effect on survival Improvement in Brb level Decrease in HE and NO in MARS-treated patients UNOS 2 b: 10/10 alive Mortality reduction (1/12 Observed mortality 27% 3-month survival 50% decrease in Brb level mortality observation In-hospital mortality UNOS 2 a: 7/16 alive 30-day mortality Death rate 75% vs. 100% 3-month mortality Expected mortality 76% Primary: Sustained Pathophysiological Secondary: In-hospital Table 6 26 13 (8 MARS, 8 24 (12 MARS, 18 = = = = = n n 5 control) n n 12 control) n (9 MARS, 9 control) Clinical studies in acute-on-chronic liver failure ) RCT; multicenter ACLF with severe HE Improvement of HE Improvement of HE in ) RCT; two centers ACLF ) RCT; two centers Type I HRS 35 ) Case series ACLF (Brb >20 mg/dl) 34 32 29 ) Case series ACLF in severe AAH 18 ACLF, acute-on-chronic liver failure; RCT, randomized clinical trial; HRS, hepatorenal syndrome; MARS, molecular adsorbent recirculating Author (ref.)Stange et al. ( Design Study population Aim Main results Mitzner et al. ( Heeman et al. ( Jalan (55) Case seriesHassanein et al. ( ACLF in severe AAH Sen et al. ( system; AAH, acute alcoholic hepatitis; Brb, bilirubin; HE, hepatic encephalopathy; NO, nitric oxide Extracorporeal Artificial Liver Support Systems 513 significant decrease in serum bilirubin and creatinine, as compared with hemodiafiltration patients. In addition 7-day survival was significantly prolonged in the albumin dialysis arm (27.5% vs. 0%). Although the results of the study are promising, the small size and the absence of a standardized medical therapy with vasoconstrictors and plasma expansion preclude a definitive evaluation of the efficacy. In addition, in a recent pilot study (33) performed in patients with HRS unresponsive to vasoconstrictor therapy, MARS was not able to improve glomerular filtration rate, although it was associated with a decrease in plasma concentration of NO and an improvement in serum creatinine. The second study was performed in 24 patients with ACLF who were randomly allocated to receive MARS or standard medical therapy (34). The primary end point was a 3-day stable reduction of serum bilirubin below 15 mg/dL; MARS therapy was significantly associated with not only a decrease in serum bilirubin level but also lower mortality. However, the results of this trial regarding mortality should be interpreted with caution as the trial was not designed to detect differences in mortality and the definition of the inclusion criteria was poor. The third study (35) was aimed at evaluating the effects of albumin dialysis in patients with cirrhosis and advanced encephalopathy; the end point of the study was the improvement of encephalopathy assessed by a decrease of at least two grades in the level of encephalopathy. The results of the study indicate that albumin dialysis significantly improved the probability of recovery of hepatic encephalopathy as compared to standard medical therapy, including conventional hemofiltration (58% vs. 37% at 72 h; p=0.045). Interestingly, the survival of patients who reached this end point in both groups was significantly greater than in patients who did not improve the level of hepatic coma, indicating the strong influence of hepatic encephalopathy on survival. The methodology to assess hepatic encephalopathy and the lack of effect of MARS therapy on survival have been criticized (36); however, it is important to remark that the trial was neither designed nor powered to detect differences in survival. Finally one study has explored cost-effectiveness of MARS treatment (37), mainly in acute alcoholic hepatitis. This cohort study, partially coming from one of the aforementioned randomized trials (34), suggested that although MARS was associated with high initial treatment costs, the significantly better survival seen in this study led to reasonable costs per life year gained. These results should be cautiously analyzed due to the very optimistic estimation of long-term survival. Overall, all these studies suggest a potential role of albumin dialysis in patients with ACLF that could be confirmed in the ongoing European 514 Bañares and Catalina large-scale multicenter trials (RELIEF trial with MARS; HELIOS study with Prometheus). One of the most important issues when analyzing any therapeutic measure is safety of the device. This aspect is even more relevant when one considers the baseline conditions of patients with ACLF (advanced liver failure, poor nutrition, risk of infections, coagulation disturbances, etc.) and the nature of albumin dialysis procedures (need of vascular access, extracorporeal circulation, etc.). Although the majority of published studies have shown a reasonable safety profile, several aspects should be analyzed. Doria et al. (38) described an overall 30% incidence of positive blood cultures in ACLF treated by MARS. Importantly, infection was associated with a worsening of hemodynamic profile, the need of vasoactive support, and death. Gram-negative strains were responsible in most cases (78%) although gram-positive bacteria were also detected. In another study (39), 17 out of 83 patients (20.5%) had infection during MARS therapy. Four (23%) patients developed bacteremia, five (29.4%) respiratory infections, three (17.5%) urinary tract infections, one (5.9%) spontaneous bacterial peritonitis, and four (23%) infection in other locations. Although gram-positive isolates were most frequently found (29.4%), gram-negative (11.8%), anaerobic (5.9%), and fungal isolates (17.4%) were also observed. Only ACLF (OR 3.1 (l–9.9)) and the number of sessions (OR 1.5 (1–2.2)) were independently associated with infection in multivariate logistic regression. Ten out of 17 infected patients (59%) died during the hospital stay, 5 of them due to uncontrolled infection. These results confirm the high incidence of bacterial infection in advanced liver disease (40, 41), and its influence on mortality. Therefore, careful surveillance and early therapy of infections should be accomplished during MARS therapy. Clinically relevant coagulation disturbances may be observed in patients treated by albumin dialysis procedures due to the existence of extracorporeal circuits, the need of anticoagulation in order to avoid clotting of the filters, and the risk of infection. In a recent study, Bachli et al. (42) reported a 10% incidence of clinically relevant episodes of coagulopathy with or without bleeding. In addition, a worsening of coagulation parameters was also reported in nonbleed- ing patients. Only patient age, fibrin D-dimer level, and INR were independently associated with coagulopathy, although the positive predictive value for the presence of coagulopathy of the model was low. The risk of coagulation disturbances is also present during albumin dialysis using the Prometheus device. A recent study (43) has shown that albumin dialysis is associated with significant adsorption of procoagulant and anticoagulant factors, with potentially relevant clinical consequences. Extracorporeal Artificial Liver Support Systems 515

All these data suggest that although MARS therapy seems to be safe in patients with advanced liver failure, the possibility of developing severe complications should be taken into account.

4.2. Difficulties in the Evaluation of Clinical Efficacy of Artificial Liver Support One of the greatest difficulties in the development of artificial liver support is the evaluation of its efficacy. Published studies are usually uncontrolled, include a very small number of patients, or represent the report of single-center experience. In addition, the definition of liver failure is not homogeneous among the different trials and they are not adequately designed to detect differences in clinically relevant variables as survival. Furthermore, the two published meta-analyses do not allow firm conclusions; the results of the first meta-analysis, although they suggest a benefit in survival in patients with ACLF, have been criticized because of heterogeneity due to pooling of patients treated for diverse indications, different primary end points, and different treatment protocols (44). The second meta-analysis (45) includes a very small number of studies with a low rate of events, and the negative conclusion regarding survival rate in MARS-treated patients should also be considered as preliminary. Therefore, the available information does not provide an accurate answer to the possible influence of ALS on clinical outcomes of patients with ALF.

4.3. Requirements of a Future Ideal Liver Support System Although in the past decade important advances in the field of ALS have been reached, it is obvious that actual devices are far from ideal. The ideal ALS system should provide metabolic and synthetic liver function. It should also allow regeneration of the injured liver thus influencing the pathophysiological alterations of liver failure. Importantly, this ideal system should also be able to incorporate detoxifying properties and to provide support to end-organ dysfunction, mainly brain, renal, and circulatory derangements. In the case of bioartificial devices, it is essential to improve new hepatocyte cell lines such that they retain good functional capacity and adequate cell mass. From a logistic point of view, the ideal device should be readily available and relatively cheap and easy to use. Efforts should be made to define precisely the target population including appropriate definitions of the disease to be treated, adequate time frame, schedules, and duration of therapy. On the other hand, efficacy should not be assessed by the potential effects on pathophysiological end points or surrogate markers, 516 Bañares and Catalina but by adequately defined clinically relevant variables, preferably survival, in the context of well-designed randomized trials with correct estimation of sample size.

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Issues in Transplantation of Patients with Chronic Liver Failure

Michael D. Leise, W. Ray Kim, and Patrick S. Kamath

CONTENTS INTRODUCTION LIVER TRANSPLANT REFERRAL AND EVA L UAT I O N ALLOCATION OVERVIEW PREDICTION MODELS THE CRITICALLY ILL LIVER PATIENT REFERENCES

Key Words: Child–Turcotte–Pugh (CTP), Model for end-stage liver disease (MELD), Mayo primary biliary cirrhosis score , MELDNa, Survival beneﬁt model, Acute physiology and chronic health evaluation (APACHE), Sequential organ failure assessment (SOFA), Organ system failure (OSF)

1. INTRODUCTION The field of liver transplantation has advanced dramatically since the first successful liver transplant was performed in 1967 (1). Advances in surgical technique, immunosuppression, and care of the critically ill liver patient have resulted in approximate 1-year survival rates of 85–90% and 5-year survival rates of 75% (2). These astonishing figures have led to a major paradigm shift in the care of chronic liver disease patients with the inadvertent consequence of liver transplant being often

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_26, C Springer Science+Business Media, LLC 2011

521 522 Leise et al. considered the only therapeutic approach even in patients with potentially reversible liver disease. A major challenge has been appropriate patient selection in the face of donor liver shortages. Unfortunately, there are no accepted models to determine which patients benefit from intensive care, or from bioartificial liver support, and in whom most treatment, including liver transplantation, is futile. Thus, the early identification of the patient who will benefit the most from liver transplantation needs further study. This chapter will focus on identifying which patient with cirrhosis in the intensive care unit will be best served by liver transplantation, and the evidence behind this decision making process.

2. LIVER TRANSPLANT REFERRAL AND EVALUATION 2.1. Indications The diseases and conditions for which liver transplantation is indicated are diverse. However, in patients with cirrhosis, liver transplantation is indicated in the presence of decompensation (ascites, variceal bleeding, hepatic insufficiency, jaundice, and hepatocellular carcinoma) because the median survival in this group of patients is about 2 years (3–6). Multiple prognostic tools have been developed to stratify patients based on their risk of death. The Child–Turcotte–Pugh (CTP) may be the most well known of these scoring systems (7, 8). The CTP establishes an overall severity of liver disease score of A, B, or C, in a particular patient based on the subjective findings of encephalopathy and ascites in conjunction with the objective measures of albumin, bilirubin, and prothrombin time. Class C (score >10) carries the worst prognosis with approximately 50% of patients dying within a year. Class B (7–9) and class A (5, 6) have an approximately 80% and 100% chance of living 1 year without transplant, respectively. Validated prognostic models for primary biliary cirrhosis and primary sclerosing cholangitis have also been developed, but are disease-specific models and not generalizable to all patients with cirrhosis (9, 10). They are of limited utility in selecting patients for transplantation. The model for end-stage liver disease (MELD) score has been found to accurately predict short-term wait list survival (11). The MELD score is based on bilirubin, creatinine, and international ratio of prothrombin time and ranges from 6 to 40, correlating to 3-month survival rates of 90 and 7%, respectively. At most institutions, the CTP, MELD, or a variant is used to justify listing a patient for liver transplantation. The risk– benefit ratio of liver transplantation is favorable for the patient once a Issues in Transplantation of Patients with Chronic Liver Failure 523

CTP score of 7 or a MELD score of ≥ 15 is reached (12–14). Most experts would agree that referral for liver transplantation for chronic liver disease should be made when CTP>7 or MELD >10, or with the development of complications including ascites, encephalopathy, variceal hemorrhage, progressive jaundice due to hepatic insufﬁciency, and hepatocellular carcinoma (15).

2.2. Pretransplant Evaluation After establishing the necessity of transplantation based on available prognostic tools, a thorough multidisciplinary pretransplantation evaluation should ensue. While each transplant center has its own protocol, the following medical investigations should take place at a minimum (Table 1): history and physical examination; routine blood work including complete blood count, MELD labs, and serum sodium; laboratory studies directed at evaluating liver disease status and basis for disease; evaluation for coexisting or previous infections including hepatitis B, hepatitis C, CMV, and HIV; cardiopulmonary testing with a dobutamine stress echocardiogram; pulmonary function tests; cross-sectional imaging of the abdomen to determine presurgical anatomy and possibility of hepatocellular carcinoma; age-appropriate cancer screening (15). Paramount to the pretransplantation workup is the psychosocial evaluation to determine the patient’s social support system and ability to comply with medical recommendations, as well as to identify any ongoing issues with substance abuse. In patients who are hospitalized with liver failure, this evaluation needs to be carried out on an expedited basis. In this context, it may not be feasible to carry out extensive cardiopulmonary and psychosocial evaluations.

2.3. Contraindications to Transplantation In some cases, a patient may have an appropriate indication for transplant and have MELD or CTP scores that merit evaluation, but may be too sick to tolerate a liver transplant operation. As the field of liver transplantation evolves, so has the list of contraindications to receiving a transplant. By design, many of the elements of the pretransplant evaluation previously discussed can detect absolute and relative contraindications to this surgery. Before any liver transplant evaluation begins, the age of the patient is the first element to consider. In the past, an age >65 has been used as a general cutoff for transplant eligibility. In Europe, liver transplantation in patients >60 year of age has increased from 10 to 20% over the past 15 years (16). Thus, age above 65 alone should not determine candidacy. In most cases, a patient 524 Leise et al. CMV HIV HBV HCV Infectious b a function tests stress echocardiogram (DSE) testing Pulmonary Dobutamine serologies MELD labs (bilirubin, INR, creatinine) Table 1 Chronic liver disease Essential pretransplant evaluation HCC Sodium Coronary arteriography Abdominal CT Age appropriate CBC Imaging Cancer screening Routine blood work Cardiopulmonary Coronary arteriogram recommended if DSE is positive for ischemia DSE recommended for patients >50 years and for patients with risk factors such as diabetes mellitus, chronic smoking, and family history of CT, computed tomography; HCC, hepatocellulara carcinoma; CBC, complete blood count; BUN,b blood urea nitrogen; Cr, creatinine; HBV, physical exam History and coronary disease hepatitis B virus; HCV, hepatitis C virus; CMV, cytomegalovirus; HIV, human immunodeficiency virus Issues in Transplantation of Patients with Chronic Liver Failure 525

Table 2 Contraindications to liver transplantationa

Absolute Relative

Brain stem herniation Advanced age Severe intracranial hypertension Current smoking (ICH >50 mmHg) Class III obesity Current or recent extrahepatic malignancy HIV Severe cardiopulmonary disease Malnutrition and muscle wasting Uncontrolled infection Hepatic angiosarcoma Active substance abuse Persistent noncompliance

ICH, intracranial hypertension aMany absolute and relative contraindications are transplant center dependent over this age who has relatively few or minor comorbidities can still be considered for liver transplantation. The approach to patients with human immunodeficiency virus, cholangiocarcinoma, and portal vein thrombosis has also changed. HIV was once considered an absolute contraindication due to poor outcomes (17–19). However, in the age of highly active antiretroviral therapy (HAART) specialized centers have now shown that similar outcomes can be achieved in this population as compared to the general population (20–23). Similarly, in carefully selected patients, liver transplantation after neoadjuvant chemoradiation for cholangiocarcinoma, and a pretransplant staging laparotomy, can result in 1- and 5-year survival as high as 92 and 82%, respectively. With increasing experience and skill, transplant surgeons can now undertake portal vein thrombectomy in straightforward cases and extra-anatomic venous grafts, arterialization of the portal vein, or cavo- portal hemitransposition when extensive thrombus burden is present. Obesity has become an increasingly common challenge in the transplant environment. In fact, the majority of patients who received liver transplantation in the United States are overweight (BMI >25). Survival may be decreased in patients with obesity, especially class III obesity and morbidity is increased (24). The American Association for the Study of Liver Diseases has recommended that class III obesity (BMI >40) be considered a contraindication for liver transplantation (15, 25). Many absolute contraindications have not changed. These include severe cardiopulmonary disease, active or recent extrahepatic malignancy, hepatic malignancy with macrovascular or diffuse tumor invasion, untreated sepsis, cerebral herniation, and active alcohol or substance abuse. Denying liver transplantation to those very ill patients 526 Leise et al. is medically and ethically justifiable, but patients and families find these decisions difficult to comprehend.

3. ALLOCATION OVERVIEW Once a patient has been deemed appropriate for liver transplantation, he or she is listed at a particular transplant center. The liver transplantation wait list and current selection process can be best understood by having some working knowledge of the evolution of liver transplantation in the United States. In the early 1980s, liver transplant programs were sparse and donor organs were shared as they became available, with no particular system or structure in place. This changed in 1984 with the advent of the Organ Procurement and Transplantation Network (OPTN) created by the US government through the National Organ Transplant Act (NOTA). The OPTN was to be run by a private, not- for-profit entity, namely the United Network for Organ Sharing (UNOS) (26). Creation of the UNOS-operated OPTN marked the beginnings of a standard process for allocation of organs. The first liver allocation models were based on a patient’s level of care with patients in the intensive care unit (ICU) gaining first consideration, followed by hospitalized patients, and then ambulatory patients (27). Ultimately, the discrimina- tory function of this model was easily overwhelmed and time spent on the waiting list became the most influential factor. Many patients were then listed for liver transplantation well before it was medically necessary, in order to accrue enough wait list time to receive a transplant when it did become necessary. In 1998 the acuity of care approach was abandoned in favor of the Child–Turcotte–Pugh (CTP) scoring system. In this system, the highest priority or status 1 was assigned to patients with fulminant hepatic failure, decompensated Wilson’s disease, primary nonfunction of the liver, or hepatic artery thrombosis diagnosed within 7 days of transplant. Next in line were status 2a (CTP score ≥10, admission to the ICU, and estimated <7 days to survive), status 2b (CTP ≥10 or CTP ≥7 in patients with one or more complications of portal hypertension), and status 3 (CTP ≥7) (12). This system had several flaws in that CTP was never validated to predict mortality over time in liver patients with cirrhosis, it contained subjective elements, was not discriminative, and had only limited categories (CTP score 7–15, or nine categories). Here again, waiting time was a dominant factor, but this was not associated with an increased death rate (28). Despite incorporation of the CTP scoring system, inequities continued to exist prompting the US Health and Human Services Department to issue the Final Rule. This legislation called for elimination of waiting time as an allocation criterion for status 2B and 3 patients, along Issues in Transplantation of Patients with Chronic Liver Failure 527 with the development of an objective scoring system for prioritiza- tion of patients for liver transplantation (29, 30). This legislation was ultimately suspended, but the Institute of Medicine investigated organ allocation practices and endorsed these main concepts. In 2000, a model to predict survival in patients undergoing transjugular intrahepatic portosystemic shunts was developed using data from patients at four US medical centers (31). The original model, known as the Mayo end-stage liver disease model, was based on the etiology of liver disease and three biochemical parameters – bilirubin, creatinine, and international normalized ratio of prothrombin (INR). In subsequent studies, the etiology of liver disease fell out of the model as an important predictive variable (11). The UNOS Liver and Intestinal Transplantation Committee decided to assess this prediction tool as a potential basis for liver allocation. The name was changed to the model for end-stage liver disease for the sake of universal acceptance. Additionally, UNOS altered some of the MELD calculations by fix- ing the lower limit of the three variables at 1 to avoid negative scores, and capping the upper limit of creatinine at 4 mg/dL. On 27 February 2002, the MELD was implemented and replaced status 2A, 2B, and 3. Status 1 categorization remained intact to accommodate the most acute liver failure patients. The Status 1/MELD system is the current liver allocation system in the United States.

4. PREDICTION MODELS The hepatologist and liver surgeon are required by society to be soothsayers, a task which is very difficult. Clinical acumen alone is not sufficiently accurate to predict who will require and benefit from liver transplantation. The search for prognostic variables began in the 1960s and 1970s followed by the advent of mathematical models in the late 1980s. When reviewing the many models covered, it is important to note that their use is generally judged by a measure known as the c-statistic, or concordance statistic. The c-statistic measures the discriminative ability of a prognosis tool, or plainly put, the ability to determine whether patients with a higher score will die before patients with a lower score. The c-statistic value ranges from 0 to 1. A c-statistic >0.7 indicates a useful test, and a value >0.8 is reflective of a very good prognostic tool (32).

4.1. Mayo Primary Biliary Cirrhosis Natural History Model The goal of the Mayo primary biliary cirrhosis (PBC) natural history model, also called the Mayo PBC risk score, was to improve 528 Leise et al. selection for and timing of liver transplantation in PBC patients. The model, derived from a trial (n=312) of D-penicillamine in PBC, identified five variables as clinically and statistically significant predictors of survival including age, bilirubin, albumin, prothrombin time, and edema (9). Most importantly, this model could predict that survival with liver transplantation was significantly better than without transplantation. The Mayo PBC model gained popularity because it did not require liver histology and was multiply validated, as opposed to the Yale and European models (33, 34). A drawback was the perceived difficulty in calculating the risk score. An abbreviated risk score, in a format similar to that of Child–Turcotte–Pugh score, was created and validated to address this problem (http://www.mayoclinic.org/gi-rst/models.html). It was recommended that an abbreviated Mayo PBC score of 6 be used for minimal listing criteria, and a score of 7.8 represented the optimal time for transplantation (35). This model is not currently used in making decisions regarding either timing or candidacy for liver transplantation.

4.2. Child–Turcotte–Pugh Child and Turcotte proposed a scoring system in 1964 that risk stratified patients into low, intermediate, and high risk for mortality following portosystemic shunt surgery for bleeding esophageal varices (7). The initial variables comprising this model were albumin, bilirubin, nutritional status, ascites, and encephalopathy. Pugh applied a modified version of this classification scheme to patients undergoing esophageal transection for bleeding varices, by replacing nutritional status with prothrombin time and assigning a numeric value (1–3) to each variable (Table 3)(8). This scoring system leads to the assignment of each patient to category A, B, or C, corresponding to best, moderate, or worst prognosis, respectively. Despite the lack of statistical validation for either the original or modified scoring systems, the CTP score provides the clinician with an easy to use bedside tool for prognostication. Child class A (5–6 points) is reflective of compensated cirrhosis, or cirrhosis without complications of portal hypertension. Two-year survival without liver transplantation for CTP class A cirrhosis is approximately 85%. Child–Turcotte–Pugh class B (7–9) carries an 57% 2-year survival rate, with CTP class C predicting death in approximately 65% of patients at 2 years. Most experts recommend referral for liver transplantation in patients with a CTP score greater than or equal to 7 points (15).There are multiple flaws in the CTP scoring system. Ascites and encephalopathy are subjective findings and can fluctuate with treatment. It has never been clear as to whether a patient should receive a CTP score when ascites or encephalopathy is at its best or worst. Regarding the laboratory components of the CTP, the laboratory value cut-off Issues in Transplantation of Patients with Chronic Liver Failure 529

Table 3 Child–Turcotte–Pugh score

1 point 2 points 3 points Encephalopathy None Grade 1–2 Grade 3–4 Ascites None Mild/moderate Severe Bilirubin (mg/dL) <2 2–3 >3 Albumin (g/dL) >3.5 2.8–3.5 <2.8 Prothrombin time (s)/INR <4/<1.7 4–6/1.7–2.3 >6/>2.3

INR, international normalized ratio 5–6 points, CTP class A; 7–9 points, CTP class B; 10–15 points, CTP class C points were created arbitrarily. This results in a “ceiling effect” as a bilirubin of 3.5 mg/dL and a bilirubin of 35 mg/dL would merit the same score. A similar but opposite phenomenon can be seen with albumin referred to as the “ﬂoor effect.” Intravenous albumin infusions may also confuse the interpretation of the albumin (36). Another drawback is the heterogeneity within the categories. A patient of CTP class C and score of 10 is not distinguished from a patient with a CTP score of 15. Despite these limitations, the Child–Turcotte–Pugh score generates a c-statistic that measures reasonably well with the MELD score, for a variety of liver disease patients (36).

4.3. Model for End-Stage Liver Disease (MELD) and MELD Variants The MELD score is based on three continuous, objective variables: bilirubin, creatinine, and international normalized ratio of prothrombin time. The mathematical formula is MELD = 9.57 × loge(creatinine) + 3.78 × loge(total bilirubin) + 11.2 × loge(INR) + 6.43 (26). Patients are assigned a score based on these three variables, from 6 to 40, corresponding to a 3-month survival of 90 and 7%, respectively. The MELD score has been validated as an accurate predictor of survival not only in patients with cirrhosis, acute liver failure, and alcoholic hepatitis, but also in patients with a variety of complications of chronic liver disease (11, 13, 31, 37–41). Most studies have demonstrated a c-statistic of ≥0.8 with modest superiority over the CTP score. The MELD score was found to predict 3-month mortality in hospitalized patients (c-statistic 0.87), non-cholestatic ambulatory patients (c-statistic 0.80), PBC patients (c-statistic 0.87), and historical cirrhotic patients (c-statistic 0.78) (11). A MELD calculator for clinicians can be found online at http://www.unos.org/resources/meldpeldcalculator.asp. Many countries now use the MELD score to prioritize organ allocation 530 Leise et al. among patients on the liver transplant waiting list (42–45). The advantages of the MELD are its objective and continuous variables, inclusion of renal function, and solid evidence base. In the United States, annual mortality on the waiting list decreased by 15% after going to a MELD- based allocation system and median time to transplantation decreased from 656 to 300 days (13, 46). The disadvantage of this model is the variability in measurement technique in different laboratories. The INR was introduced to standardize the anticoagulation effect of warfarin (47). The thromboplastin agent used is speciﬁc to anticoagulated patients, and it has been suggested that the thromboplastins should be calibrated to patients with liver disease for use in the MELD. The creatinine can be problematic in a number of ways. There are multiple methods of measuring creatinine which result in great variability and, subsequently, variability in the MELD score (48, 49). The creatinine value can be inaccurate with a bilirubin >25. Many labs use a calorimetric alkaline picric Jaffe method that will overestimate the creatinine due to the darker yellow color of serum seen in jaundiced patients (50). Furthermore, females with liver disease have lower GFR than males for the same creatinine value leading to a possible systematic allocation bias (51, 52). The lack of incorporation of clinical data has also been suggested to be a disadvantage of the MELD. The MELD score underestimates quality of life in patients with end-stage liver disease and intractable hepatic encephalopathy (53). This should be contrasted with the fact that a dramatic decrease in the scores of hepatic encephalopathy at liver transplantation was noted after institution of the MELD score. This was felt to represent a by-product of manipulation of Child–Turcotte–Pugh scores to increase the chances of liver transplantation. That is, patients were represented to have a higher grade of encephalopathy than they actually did, so that they would be assigned a higher CTP score (54). Lastly, the MELD score does not predict post liver transplantation survival. Unfortunately, no currently available prediction model can adequately depict posttransplant survival. This is further discussed in the “Survival Beneﬁt Models” section.

4.3.1. MELDNA Hyponatremia has been linked to the presence of ascites, hepatorenal syndrome, and risk of death from liver disease (4, 55–58). It had been suggested that serum sodium was a signiﬁcant predictor of mortality above and beyond the MELD. Perhaps the most promising of new formulations of the “MELD” is that of the MELDNa. The MELDNa data taken from all adult candidates registered through OPTN in 2005 Issues in Transplantation of Patients with Chronic Liver Failure 531

(n=6769) revealed an interaction between serum sodium, MELD, and 3-month mortality. This interaction conﬁrmed that a decrease in serum sodium was associated with risk of death in patients on the waiting list, after adjustment for MELD. Calibration of the MELDNa score on the candidates on the 2006 OPTN list (n=7171) demonstrated that mortality predicted from MELDNa matched observed mortality more so than MELD alone (c-statistic 0.88). This effect is most notable in patients with moderate to low MELD scores and serum sodium on the lower end of the 125–140 mmol/L range. Using this model for organ allocation would have prevented about 33 deaths per year in the United States (59). The United Kingdom currently uses a prediction model that incorporates bilirubin, creatinine, INR, and serum sodium.

4.3.2. DELTA-MELD Another area of investigation examined whether the change in MELD (delta-MELD) scores over a 30-day period was more predictive of 30-day mortality than MELD alone. The hypothesis was that patients with rapidly increasing MELD scores would be at higher risk of death than their counterparts with a stable score of the same severity. Bambha et al. found that the MELD alone was still the single best predictor of mortality, and that much of the delta-MELD’s predictive ability lies within the MELD (60). Additionally, there are inherent biases in this approach in that the sickest patients will have laboratories checked more often. These values will also be reported more often in order to advance the patients’ priority on the liver transplant waiting list. The delta-MELD model does not allow the provider enough lead time for it to be practical in clinical use. Its ability to predict death within a 4-day period is probably related to the actual event of dying.

4.3.3. D-MELD The D-MELD, devised by Halldorson et al., represents the product of donor age and the calculated preoperative MELD score (61). The purpose of such a model was to identify the optimal donor organ and recipient combinations to maximize posttransplant survival. Multiple variables were used including donor age, donation after cardiac death, split/partial liver grafts, African-American race, donor height, donor cause of death from cerebrovascular accident, cold ischemia time, and donor steatosis. Of these variables, donor age over 40 years, and particularly over 60 years, is the predominant donor risk factor. The product of donor age and MELD yields a score that ranges from 40 to 3400. This score was found to be a stronger predictor of posttransplant survival 532 Leise et al. when compared with pretransplant MELD and donor age alone. The authors recommended a risk cap at a D-MELD of 1600 as this was associated with significantly decreased survival. As an example used in this study, a patient with a MELD score of 25 and donor age of 30 would result in a D-MELD score of 750. Expected 4-year survival with this score is 77%. However, if the same patient (MELD = 25) is offered a 66-year-old donor, the D-MELD is 1650 and the expected 4-year survival decreases to 62%. It is anticipated that this type of model would redistribute donor livers from low and high MELD groups to patients with a moderate MELD score (18–29). Specifically, a high MELD (>29)–high donor age match and a low MELD (<15)–high donor age combination would ideally be avoided due to diminished posttransplant survival. Transplant benefit models represent a different philosophi- cal approach than the current “sickest first,” or justice system. Benefit models will be discussed in the next section.

4.4. Survival Benefit Models Three different approaches to allocation of donor organs in liver transplantation exist – the justice, utility, and benefit models (62–64). The justice or medical urgency system allocates organs on the basis of expected waiting list mortality as calculated by the MELD or other similar scoring systems. Opponents of this system argue that patients with the highest risk of waiting list death may also be at the highest risk of posttransplant mortality, thereby shifting mortality from the pre- to posttransplant side without resulting in fewer deaths. The utility model would allow for liver transplantation in patients who are deemed to have the best expected posttransplant survival. An argu- ment against this model is that patients with best waiting list survival may also have the best posttransplant outcomes. The benefit model attempts to take into account both pre- and posttransplant survival and allocates donor organs to those with the largest difference in pre- and posttransplant survival. With the scarcity of donor organs, the goal of this approach is to ensure the best use of a scarce resource on a societal level. In 2009, Merion et al. proposed a model incorpo- rating recipient and donor variables associated with waiting list and posttransplant survival as modeled by Cox regression (65). Recipient factors included creatinine, albumin, sodium, age, diagnosis, diabetes, dialysis, hospitalization status, previous liver transplant, mechanical support, portal vein thrombosis, previous abdominal surgery, and hepatitis C. Donor factors included age, cause of death, donation after cardiac death, cold ischemia time, and regional versus national organ sharing status. The final model was applied to actual candidates on Issues in Transplantation of Patients with Chronic Liver Failure 533 the waiting list in January 2006 and evaluated by a simulated allocation model. The simulated model revealed that the benefit model resulted in a gain of an additional 2223 life-years (over 5 years) as compared to the MELD model. Challengers of the benefit model have several concerns. Benefit models are derived from exceedingly complex statistics and involve numerous variables resulting in difficulty with practical application by the physician on a day to day basis. In contrast, other models such as the MELD and CTP scores can be generated fairly easily and provide the physician a general sense of prognosis (66). The complex statistics of these models must also be thoroughly explored for precision and accuracy. The MELD score does not require accuracy, only precision so that transplant candidates could be ranked in relation to each other. The benefit model will also require accuracy in the prediction of pre- and posttransplant survival in order to calculate differences between the two. At present, the c-statistic for posttransplant models is in the 60% range, with 50% corresponding with what is expected by chance alone (67). Posttransplant survival cannot be predicted by pretransplant variables. Lastly, a benefit model approach may result in broad changes in transplantation practices for patients with certain types of liver disease. For example, hepatitis C patients may receive liver transplants less frequently as posttransplant survival is diminished in this group (66). The future of prediction models in liver disease lies within two schools of thought. One approach is to further refine existing models while the other approach brings new models to the table like the benefit model. Updated transformations of the MELD coefficients and the addition of the serum sodium variable will be the subject of future investigation (68, 69). Benefit models with their inherent complexity will require multiple iterations and validations, but may result in a superior model if those challenges can be overcome.

5. THE CRITICALLY ILL LIVER PATIENT Patients with compensated cirrhosis eventually decompensate, a condition manifested by progressive jaundice, hepatic encephalopathy, gastroesophageal variceal bleeding, or ascites. When decompensation occurs rapidly, multiorgan failure and death may follow. Thus, it becomes important to understand which patients will recover from their critical illness, who will die without liver transplantation, and in whom further therapies are futile. This determination becomes particularly important due to scarcity of donor organs, the pain and suffering caused by continuation of futile therapy, and the burden of expensive 534 Leise et al. treatments such as terlipressin, transjugular intrahepatic portosystemic shunts, and bioartiﬁcial liver devices.

5.1. Prediction Models in the Critically Ill Cirrhotic Patient General predictive scoring systems as well as liver-specific models have been evaluated for the prediction of mortality in cirrhotic patients in the intensive care unit setting. General predictive models include the acute physiology and chronic health evaluation (APACHE) II and III which evaluate severity of illness, whereas other general models like the sequential organ failure assessment (SOFA) and the organ system failure (OSF) quantify organ dysfunction (70–72). There has been uncertainty in applying these models to the care of individual cirrhotic patients. In six studies which focused on cirrhotic patients, APACHE II score had a c-statistic ranging from 0.66 to 0.83 (73–78). The SOFA model demonstrated a c-statistic range of 0.83–0.94 among cirrhotics in the ICU (74, 76–79). The SOFA score also demonstrated superior calibration as compared to the APACHE II in patients from a single center (76, 79, 80). Despite this evidence, disease severity and organ dysfunction scores are cumbersome and not routinely used in practice. Furthermore, the use of these tools may be optimized if 48 h data are incorporated, further limiting the ease of use (81). Liver-specific models to predict death in the ICU include the Child–Turcotte–Pugh and MELD scores. The c-statistic values for these models were 0.71–0.77 and 0.81 (one study), respectively (74, 76–80). In a single study, the MELD score was found to be equivalent to the SOFA score and superior to the APACHE and CTP scoring systems (77). Based on the available data, the general ICU models are superior to the Child–Turcotte–Pugh score. The MELD score requires further exploration and validation in the ICU population. In the United States, the MELD score is still used as a gauge for transplant in the ICU setting, and reasons for admission to the ICU such as myocardial infarction and uncontrolled sepsis provide important contraindications for transplantation. From a practical standpoint, organ failure is the most straightforward tool in the clinician’s arsenal to predict mortality. In a study out of the Royal Free Hospital by Cholongitas and Burroughs, cirrhotics with Zero, 1, 2, or 3 or more failing organ systems had 4, 45, 65, and 90% mortality, respectively (P<0.001) (77). Renal failure represents the most important organ failure, and heralds a particularly poor outcome. The CTP is the poorest performer out of the aforementioned models as it is the only model which does not include a renal function variable. One may conclude that further treatment of cirrhotic patients in the ICU with Issues in Transplantation of Patients with Chronic Liver Failure 535 multiorgan failure is likely to be futile and the patients may be too sick to benefit from liver transplantation.

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Chronic Liver Disease in the Intensive Care

Andrew Slack and Julia Wendon

CONTENTS INTRODUCTION AIRWAY BREATHING RESPIRATORY MECHANICS,ASCITES AND INTRA-ABDOMINAL PRESSURE HEPATOPULMONARY SYNDROME PORTOPULMONARY HYPERTENSION CARDIOVASCULAR SPECIFIC LIVER-RELATED PATHOLOGIES SUMMARY REFERENCES

Key Words: Chronic liver disease, Hepatorenal syndrome, patopulmonary syndrome, Portopulmonary syndrome, Variceal bleeding, Ascites, Spontaneous bacterial peritonitis

1. INTRODUCTION A number of speciﬁc complications are encountered in patients with CLD. These include variceal bleeding, ascites, spontaneous bacterial peritonitis, hypotension from central volume depletion due to excess diuretic use, hepatic encephalopathy (HE) and hepatorenal syndrome (HRS). All these complications can lead to further deterioration that necessitates admission to a critical care environment and can also be

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9_27, C Springer Science+Business Media, LLC 2011

541 542 Slack and Wendon associated with sepsis and multi-organ dysfunction. Indeed, patients with cirrhosis have impaired immune function and have an increased risk of infectious complications. Alcoholics too seem particularly predisposed to sepsis and its associated complications (1). Consequently, the care of patients with CLD is particularly focused on the prevention of these complications and the early treatment of sepsis in order to primarily reduce mortality, but also to lessen the impact on hospital and intensive care unit (ICU) admission length of stay. The mortality prediction scores of Child–Turcotte–Pugh (CTP), model of end-stage liver disease (MELD) and acute physiology and chronic health evaluation (APACHE) II and III scores continue to be scrutinized and all are associated with problems when applied to critically ill patients with CLD (Tables 1, 2,and3).

Table 1 Child–Turcotte–Pugh scoring system (48, 49)

Measure Units 1 point 2 points 3 points

Bilirubin (total) μmol/l <34 (<2) 34–50 (2–3) >50 (>3) (mg/dl) Serum albumin g/l >35 28–35 <28 INR N/A <1.7 1.71–2.2 >2.2 Ascites N/A None Mild Severe Hepatic N/A None Grade I–II (or Grade III–IV (or encephalopathy suppressed refractory) with medication)

Table 2 Child–Turcotte–Pugh scores and 1- and 2-year survival rates

Points Class 1-year survival 2-year survival

5–7 A 100 85 7–9 B 81 57 10–15 C 45 35 Chronic Liver Disease in the Intensive Care 543

Table 3 Model of end-stage liver disease and UK end-stage liver disease scoring system (50, 51)

MELD score 3-month mortality (%) (3.78[Ln serum bilirubin (mg/dL)] + 11.2[Ln INR] + 9.57[Ln serum creatinine (mg/dL)] + 6.43) UKELD (5 × (1.5 × ln(INR) + 0.3 × ln(Creat) + 0.6 × ln(Br) – 13 × ln(Na) + 70)

>40 100 30–39 83 20–29 76 10–19 27 <10 4

The CTP score performs well in describing the extent of any underlying liver disease, but due to a lack of extra-hepatic factors, fails to maintain the same mortality prediction power in the setting of multiorgan failure. Likewise, the APACHE II score fails to encompass all the variables required to reflect both the degree of multi-organ dysfunction and underlying liver disease. MELD fails to incorporate the extra-hepatic and extra-renal factors that can impact on mortality like acute respiratory failure and sepsis. With these issues in mind, the MBRS mortality prediction score taken on day 1 of ICU admission has been shown to outperform these other mortality prediction scores for cirrhotic patients with acute kidney injury (AKI). The score comprises the following variables, MBRS, mean arterial pressure > 80 mmHg, bilirubin >80 μmol/l, acute respiratory failure and sepsis each assigned 1 point and associated with a progressive and significant elevation in mortality among all patients. The overall mortality for patients with CLD who are critically ill with AKI exceeds 80% (2). Those that do survive an ICU admission, but are not eligible for liver transplantation, have a median survival of only 1 month (3). Consequently, these patients present a significant challenge when critically ill, but with early intervention and a good understanding of the specific pathologies encountered, a small but significant number of patients can survive to reach transplantation. In particular, the implementation of early airway control and effective management of extensive variceal haemorrhage achieving haemostasis quickly can be associated with good outcomes and short ICU stays. 544 Slack and Wendon

2. AIRWAY Patients with CLD sometimes need endotracheal intubation to protect the airway and prevent pulmonary aspiration. HE can be associated with marked depression in the level of consciousness that necessitates intubation. Less severe levels of encephalopathy may also require intubation for airway protection in the context of significant haematemesis from variceal haemorrhage. However, intubation is not without risk in patients with CLD, due to the combination of delayed gastric emptying, a result of large-volume ascites causing raised intra-abdominal pressure, and the loss of protective reflexes associated with advanced encephalopathy. The awareness of these factors and implementation of specific manoeuvres commonly employed during a rapid sequence induction in conjunction with the aspiration of gastric contents through in situ nasogastric tubes and maintenance of 30◦ elevation of the head of the bed can help to reduce the risk of aspiration. However, the perceived benefit of prophylactic intubation has not been consistently demonstrated, especially for those patients with HE grade II or less without haematemesis (4). In the setting of large-volume haematemesis, all patients with CLD should be considered for early intubation to prevent the catastrophic consequences of pulmonary aspiration. Over the last 10 years percutaneous tracheostomy has been performed with increasing frequency and has been demonstrated to reduce ICU length of stay (5). It is currently regarded as a cost-effective, safe alternative to the open surgical technique and carries the advantage of being widely practised as a bedside procedure. Current practice in the United Kingdom suggests that most are performed between 6 and 10 days. However, the potential benefit of early (<5 days) versus late (>10 days) tracheostomy in ICU patients remains unclear and will remain so until the results of the Tracman trial are published. Currently, it is widely accepted that re-intubation rates of 15–20% represent an acceptable balance between premature extubation and prolonged ventilation (6). Most patients ventilated for variceal haemorrhage can be extubated early, but in those with persistent encephalopathy, a tracheostomy is likely to be required. The procedure of percutaneous tracheostomy can be performed safely in patients with CLD, with strict attention to the correction of any coagulopathy, assessment of neck vasculature with ultrasonography and visualized cannulation of the tracheal lumen with bronchoscopic guidance (7). After this procedure the cessation of sedation facilitates the weaning of ventilatory support and the reconditioning of generalized and respiratory muscle strength through physiotherapy leads to rehabilitation. Chronic Liver Disease in the Intensive Care 545

3. BREATHING Patients with CLD are more susceptible to both respiratory tract infections and respiratory failure compared to the general population (8). Some common factors that lead to respiratory failure include effusions, VQ mismatch and impaired respiratory dynamics secondary to elevated intra-abdominal pressure due to large-volume ascites. Chronic obstructive pulmonary disease too is seen with increased frequency and this is postulated to be due to the association of cigarette smoking with alcohol consumption. The prevalence of pulmonary tuberculosis in CLD has been quoted to be at around 6%, but this was from a small study in Argentina (9). Both community and nosocomial acquired infections also appear to be encountered with increased frequency in patients with CLD with pneumonia accounting for 18% of these infections (9). Acute respiratory failure occurs to a greater extent in cirrhotics compared to non-cirrhotic patients with an incidence of 4.7% versus 3% with an associated relative risk of 1.4 (10). This is likely to be attributable to the numerous factors speciﬁcally encountered in CLD that can affect the mechanics of ventilation. Also there are two distinct respiratory pathological conditions encountered in CLD that affect the pulmonary vasculature, and these are termed hepatopulmonary and portopulmonary syndrome.

4. RESPIRATORY MECHANICS, ASCITES AND INTRA-ABDOMINAL PRESSURE The accumulation of ascites is the commonest of three of the main complications of cirrhosis, with the other two being HE and variceal bleeding. Ascites develops in up to 50–60% of patients with previously compensated cirrhosis over a 10-year period. Cirrhotic patients who develop ascites have an 85 and 56% probability of survival at 1 and 5 years, respectively, without liver transplantation. The development of ascites is associated not only with a poor quality of life, but also with sepsis, kidney injury and worsening of long-term outcomes (11). It arises due to increases in portal pressures and the neuro-humoral responses that cause a reduction in the excretion of sodium in the urine. A simple combination of dietary salt restriction and diuretics can be an effective treatment strategy in up to 90% of patients. However, a fifth of these patients will become diuretic resistant or intolerant over time (11). All patients with CLD whilst in intensive care are at risk of worsening effusions and ascites, due to the combination of sodium and volume 546 Slack and Wendon loading, increases in vascular permeability, reductions in oncotic pressure and myocardial dysfunction associated with sepsis and cirrhosis (12). The approach to fluid management for critically ill patients with CLD is a difficult balance between optimizing haemodynamics and its deleterious effects; fluid accumulation in the pleural space, subcutaneous tissues and abdominal cavity has an impact on optimizing ventilation. Large-volume ascites displaces the diaphragm, reducing lung volumes and compliance, leading to the impairment of ventilation and gas exchange. Similar disruptions to lung mechanics are associated with hepatic hydrothorax, which can occur in up to 10% of patients with ascites (12). The presence of large volumes of pleural or ascitic fluid mandates setting the positive end expiratory pressure (PEEP) to a level that will offset the derecruitment process preventing segmen- tal atelectasis and therefore maintain optimal lung compliance. The routine measurement of the intra-abdominal pressure with a bladder catheter manometer in conjunction with both a clinical and radiolog- ical assessment of ascites can assist decisions regarding the therapeutic benefits of small- or large-volume paracentesis. Each can result in an immediate benefit to haemodynamic and ventilatory parameters. The relief of intra-abdominal pressure reduces the displacement of the diaphragm and allows the recruitment of basal lung segments. The resultant increases in lung compliance allow a reduction in ventilator peak plateau pressures leading to a fall in intra-thoracic pressure, which in tandem with the fall in intra-abdominal pressure can improve venous return and cardiac output. It should be mentioned though that the initial improvement in haemodynamics can be short-lived and the phenomenon of paracentesis- induced circulatory dysfunction (PICD) can arise as a result of decompression of the splanchnic circulation. The features of this diagnosis are recognized to occur between 48 h and 6 days following a large-volume paracentesis when the blood volume redistributes to the “unloaded splanchnic circulation” resulting in central hypovolaemia. Subsequently, there is the release of vasoactive mediators, which result in significant increases in renin levels that have also been shown to be predictive of acute deterioration in renal function. This activation of neuro-humoral and sympathetic pathways may not become clinically relevant for many critically ill patients with CLD as a significant number will have established multi-organ failure (10). However, it is an important clinical entity to be mindful of in those patients who initially present with intact kidney function. In such cases, large-volume paracentesis should be supported with infusions of albumin and vasopressor analogues, like terlipressin, to avoid the deleterious effects of PICD (13, 14). Chronic Liver Disease in the Intensive Care 547

A common finding in patients with CLD is protein and calorie malnutrition with significant reductions in muscle mass. The metabolism of patients with CLD closely resembles 2–3 days of starvation in a normal healthy individual with the oxidation of fat, increases in gluconeogenesis associated with reduced glucose utilization and glycogenolysis. Protein malnutrition is a key feature in CLD, which can be exacerbated by the significant protein losses encountered with abdominal paracentesis. Protein-rich diets that endeavour to achieve a positive nitrogen balance to limit protein deficits have been linked to a perceived deleterious effect on HE, but this association has not been demonstrated (15). Overall, patients with CLD can have a moderate to severe degree of malnutrition, which is an independent risk factor not only for critical illness mortality, but also for critical illness polymyoneuropathy (CIPMN). The additional risk factors of sepsis, the systemic inflammatory response syndrome (SIRS) and multi-organ failure account for the high prevalence of CIPMN (16). CIPMN impacts greatly on respiratory weaning and rehabilitation and prolongs ICU lengths of stay.

5. HEPATOPULMONARY SYNDROME Hepatopulmonary syndrome (HPS) is related to pulmonary vasodilatation leading to arterial hypoxaemia. The degree of hypoxaemia correlates directly with outcome and can have important implications regarding the timing and risks of liver transplantation. The diagnosis of HPS requires three components to be detected: portal hypertension, arterial hypoxaemia and pulmonary vasodilatation. Arterial hypoxaemia can be multi-factorial in CLD with coexisting pulmonary disease associated with HPS in up to 30% of cases (17). Qualitative assessment of intra-pulmonary shunting is by contrast echocardiography, but quantitative assessment involves quantiﬁcation of uptake in the brain following peripheral injection of technetium- 99m-radiolabelled macro-aggregated albumin. Normally, there is less than 6% uptake in the brain and greater than this demonstrates a sig- niﬁcant degree of pulmonary capillary dilatation. Transaesophageal echocardiography and pulmonary angiography/CT angiography may rarely be required to rule out pulmonary arteriovenous and intra-cardiac shunts. Pulmonary angiography is recommended only when there is severe hypoxaemia and embolization of large arteriovenous shunts is a therapeutic potential. Survival rates for patients with HPS range from 24 to 87 months. Liver transplantation is the only successful treatment with a 5-year survival rate of 76% (17). 548 Slack and Wendon

6. PORTOPULMONARY HYPERTENSION The association between portal and pulmonary hypertension was ﬁrst described in 1951 by Mantz and Craige and was termed portopulmonary hypertension (PPH) (18). The prevalence of PPH is increased sixfold in patients with cirrhosis compared with the general population (19). Genetic variations in oestrogen signalling and cell growth regulators appear to be associated with an increased risk of PPH (20). Doppler echocardiography has a positive predictive value of 30% for the diagnosis of PPH (19). The prognosis from the time of diagnosis and mild symptoms to death is around 15 months (21). The degree of PPH impacts transplant survival with a high mortality for those with mean pulmonary artery pressures greater than 50 mmHg (22).

7. CARDIOVASCULAR The accurate assessment, continuous monitoring and support of the circulation are pivotal to the care of patients who are critically ill. There are many controversies regarding the different modes of monitoring available and the parameters used to guide therapeutic support of both macro- and microcirculations. The initial resuscitation period, classically defined within 6 h of admission for patients with sepsis, is the only period with validated data to support specific treatment goals associated with improved outcome. The Surviving Sepsis Guidelines embraced these data and although it may seem attractive to extend the use of these guidelines to critically ill patients with CLD important differences must be acknowledged. The circulation is hyperdynamic at baseline, with an increased heart rate and cardiac output (CO) and reduced systemic vascular resistance, reflected by variable degrees of arterial hypotension and reduction in the effective arterial blood volume. Increases in intra-abdominal pressure as a result of ascites can compromise kidney perfusion further, especially when above 25 mmHg signifying intra-abdominal hypertension (IAH). The impact on venous return, pressures in portosystemic shunts and on kidney perfusion exacerbates the stimulation of these neuro-humoral pathways mentioned establishing a vicious cycle. Cirrhotic cardiomyopathy is characterized by an increase in CO, electrophysiological abnormalities and attenuation of systolic and diastolic ventricular function with blunted myocardial responses to beta stimulation, exercise and stressful stimuli, like sepsis (23). A blunted response to beta stimulation serves as a poor prognostic marker (23, 24). Chronic Liver Disease in the Intensive Care 549

Patients with CLD admitted to the ICU should be considered for early invasive cardiac monitoring, especially when unresponsive to initial resuscitative measures. It is also advisable to perform an echocardiogram, if resources allow, to interrogate myocardial function, ventricular filling and to estimate pulmonary pressures. The early implementation of haemodynamic monitoring focuses the initial assessment on determining whether the intravascular compartment is adequately filled and associated with an adequate cardiac output. In addition to those parameters derived from haemodynamic devices there are other important parameters, such as blood lactate, mixed venous and central venous oxygen saturation that are surrogate markers of oxygen delivery and tissue perfusion. Both mixed and central venous oxygen saturation have been used to assess global tissue perfusion with the mixed venous oxygen saturation (Sv02) shown to predict mortality in sepsis. Furthermore, the surrogate marker of Sv02, the central venous oxygen saturation (Scv02), has been demonstrated to be associated with a reduced mortality when above >70% during the initial resuscitation period of sepsis. Scv02 is not a validated measure of global tissue perfusion in patients with CLD, pertaining to the elevated baseline cardiac output and possible tissue dysoxia. There are also many potential confounders associated with this parameter in patients with CLD. In particular, there is an increased prevalence of complex regional circulations, portosystemic and pulmonary arteriovenous shunts and also an increased incidence of a patent foramen ovale; all have the potential to affect the utility of these measures. An elevation in serum lactate is another established independent marker of poor prognosis regardless of the underlying cause. Responses to treatment of the lactate levels appear to be of greater relevance than absolute numbers. The central venous pressure (CVP) too is a poor measure of intravascular filling due to the effect of changes in intra-abdominal and intra-thoracic pressure. Ascites, hydrothorax and variations in lung compliance all affect positive pressure ventilation, which impacts on intra-thoracic and central venous pressure measurements. Thus, CVP is an unreliable measure of central volume and should not be used in isolation to determine intravascular filling status.

8. SPECIFIC LIVER-RELATED PATHOLOGIES 8.1. Acute Kidney Injury (AKI) The assessment of kidney function has for many years relied on the measurement of the concentration of serum creatinine (SCr). Patients with CLD have a significantly lower baseline SCr concentration than 550 Slack and Wendon the general population (35–75 μmol/l) (25). The RIFLE classification for AKI highlights the importance of an individual’s baseline SCr and that small changes are associated with significant increases in hospital mortality (26). Newer classification for AKI has reduced the potential impact variations in baseline SCr can have on the interpretation of acute changes in kidney function. The incidence of AKI in hospitalized patients with CLD is around 20% (27). There are three main causes of AKI in CLD: volume- responsive pre-renal failure, volume-unresponsive pre-renal failure with tubular dysfunction, known as acute tubular necrosis (ATN) and hepatorenal syndrome (HRS) type 1, with prevalence rates of 68, 33 and 25% respectively (28). Of note, these three clinical scenarios should only be considered once acute kidney parenchymal disease, obstructive uropathy and unrecognized chronic renal disease have been excluded. This can be achieved by performing an ultrasound of the kidneys, dipstick urine analysis assessing the presence of haematuria and proteinuria in conjunction with appropriate same day serological testing for antibodies against the glomerular basement membrane and those diagnostic for the small-vessel vasculitides, when other clinical features suggest such diagnoses are possible. Contrast nephropathy is an important cause of kidney injury in patients with decompensated CLD with three times the risk in those with ascites (29). Patients may be given 5 ml/kg of sodium-containing intravenous fluids 1 h prior to contrast administered with some evidence suggesting superior outcomes with isotonic sodium bicarbonate (30). Spontaneous bacterial peritonitis (SBP) affects approximately 20% of patients with decompensated CLD and is a leading precipitant, up to 30% of cases, of HRS/AKI. Hypotension should always prompt the meticulous assessment of gastrointestinal bleeding from variceal haemorrhage, an eminently treatable cause and a detailed search for sepsis with a thorough interrogation of the drug chart to omit medications that will further compromise blood pressure or be potentially nephrotoxic. Established beneficial treatments include fluid resuscitation, vasopressor analogue use, albumin infusions and the omission of nephrotoxic drugs (31, 32).

8.2. Hepatorenal Syndrome HRS occurs in about 4% of patients admitted with decompensated cirrhosis, the cumulative probability being 18% at 1 year, increasing to 39% at 5 years (33). Numerous animal studies provide an increasing amount of evidence that challenges the ischaemia centric concepts of AKI. It seems likely Chronic Liver Disease in the Intensive Care 551 that AKI in sepsis is a distinct entity with evidence confirming a fall in renal vascular resistance, increases in renal blood flow leading to renal venous congestion and microvascular dysfunction within the kidney. Cellular metabolism at the mitochondrial level along with other measures of bioenergetics appears to be well preserved (34). The therapeutic studies on HRS-1 in CLD have concentrated on the use of various vasopressors and albumin. In the United States the splanchnic vasopressors studied have included alpha agonists like midodrine (35), noradrenaline and the somatostatin analogue octreotide and in Europe the focus has been on the V1 vasopressin agonist terlipressin. There has been one prospective, randomized, double-blind, placebo- controlled clinical trial of terlipressin performed in patients with type 1 HRS. Patients were randomized to terlipressin (1 mg every 6 h) or placebo, with albumin in both groups. Terlipressin was superior to placebo with reversal rates for HRS-1 of 34 and 13%, respectively. Transplantation-free survival was similar between study groups, but HRS reversal significantly improved survival at day 180 (13). There may be additional improvement in renal function seen following TIPSS in patients with HRS-1 (36).

8.3. Renal Replacement Therapy The use of renal replacement therapy (RRT) in the ICU continues to be the focus of much debate. The issues range from the mode, timing of initiation, indications for initiation, dose, anticoagulation use and the perception that continuous compared to intermittent regimens improve outcomes. There is little evidence available to clearly delineate any of these issues. Furthermore, the standard ultraﬁltration dose of 35 ml /kg/h is being increasingly questioned; despite this, some important aspects to delivering RRT in the ICU in patients with CLD need to be outlined. RRT often needs to be tailored to address the issues of ﬂuid management and the profound metabolic derangements often encountered. However, RRT should always be implemented with a continued appreciation for the severity of the underlying chronic condition. It is also important to appreciate the effect the mode, dose and inter- ruptions or intermittent regimens of RRT have on drug elimination. In critical illness the marked changes in the pharmacokinetics and pharmacodynamics of drugs require where possible the use of close drug monitoring. In the absence of drug monitoring, antibiotic prescriptions should aim to “overdose” treatments with a low toxicity. Furthermore, any changes in the dose of RRT warrant the adjustment of antibiotic doses to compensate for increases in drug clearance. 552 Slack and Wendon

The anticoagulation of RRT circuits in patients with CLD remains a problem, despite the frequent occurrence of coagulation and platelet abnormalities. Patients with CLD can have a complex array of acquired coagulopathies with defective synthesis of all clotting factors except for factor VIII and von Willebrand factor. Conventional investigations of coagulation have been shown to be poor at reflecting the true coagulation profile encountered in CLD. These patients often have increased amounts of thrombin and various reductions in both procoagulant and anticoagulant factors, like protein C and antithrombin. Thromboelastography (TEG) has been used during liver transplantation to assist decision regarding clotting support and it has been shown that it can also be useful in patients with stable CLD. It is also likely that the TEG is also of use in patients with CLD in the ICU setting to help determine defects in the clotting pathways that result in both procoagulant and anticoagulant states (37). Currently, prostacyclin is frequently used, because its short half-life confers an excellent safety profile in those with thrombocytopaenia and the systemic effects are not realized when delivered directly into the extracorporeal circuit. Citrate anticoagulation is contraindicated in patients with significant hepatic dysfunction, due to problems associated with citrate accumulation, namely acid–base and electrolyte disturbances, due to electrolyte chelation and the accumulation of citrate–calcium complexes. However, there has been a small study that reported minimal side effects when using citrate anticoagulation during slow extended dialysis in 10 sessions for seven patients with advanced CLD and AKI (38). However, citrate anticoagulation demands close monitoring and experience to be utilized safely. It continues to be contraindicated in patients with severe liver dysfunction, but this small study suggests that there may be a role for this form of extracorporeal anticoagulation in the future.

8.4. Variceal Haemorrhage Variceal haemorrhage (VH) is a frequent complication of cirrhosis and is associated with a mortality of 15–20%, which is primarily related to the severity of the underlying liver disease (39). The mortality prediction scores of Child–Turcotte–Pugh (CP) and model of end-stage liver disease (MELD) score have both demonstrated good performance in predicting outcomes for patients with CLD following VH (Tables 1, 2, and 3). There has been a signiﬁcant improvement in the survival of these patients over the past 25 years, which has largely been achieved through better prophylaxis using both medical therapies for portal hypertension and endoscopic banding surveillance strategies. Chronic Liver Disease in the Intensive Care 553

Acute variceal haemorrhage requires the early assessment of portal vein patency with ultrasonography, and a good history to determine the severity of the underlying CLD can help to delineate the therapeutic options available. The initial support should focus on the correction of any coagulopathy, platelet support for thrombocytopaenia, haemodynamic resuscitation and the administration of empirical antibiotic therapies, the last shown to decrease the risk of subsequent bleeding. Several studies have evaluated two key interventions used to achieve haemostasis and prevent re-bleeding, namely, endoscopic variceal ligation (EVL) and portal pressure reduction with vasopressin or somatostatin analogues. Terlipressin appears to perform as well as octreotide in attaining haemostasis for acute variceal bleeding. Terlipressin should normally be continued for 5 days; however, low-dose terlipressin in combination with EVL performs better than terlipressin alone in the prevention of early re-bleeding (40, 41). The early consideration of intubation and airway protection, which has been discussed in detail earlier, can also improve tolerance of the Sengstaken Blakemore tube (SBT). This is an important adjunct to haemostatic therapy and can be used while other definitive interventions are planned. However, a SBT should not be in situ for >12–24 h as pressure necrosis can occur. It is usually unnecessary to inflate the oesophageal balloon if the gastric balloon is well positioned by reducing blood flow to oesophageal varices. Fluid, ideally a mixture of water and contrast agent, should be instilled into the gastric balloon and the position maintained by taping to the face or a helmet to ensure constant and appropriate traction at the gastroesophageal junction. The failure to achieve haemostasis often hinges on the location of the varices and the availability of appropriate endoscopic expertise. Endoscopic sclerotherapy is often required for gastric varices and the availability of such expertise can be variable. This demands that referral to centres where such expertise is available is made early to ensure haemostasis is achieved in shortest time possible. In those patients who have failed endoscopic therapy, discussions regarding the suitability of TIPSS shunting should be undertaken. In the acute setting it is important to ensure that patients who may undergo TIPSS have normal right ventricular function. There is a suggestion that portal pressures greater than 20 mmHg are associated with high risk of re-bleed and increased mortality without a TIPSS shunt. The deaths that occur following VH can be divided into early and late, the distinction between the two made at 6 weeks. Early deaths are related exclusively to the initial bleeding insult and failure to establish haemostasis, with late deaths attributed to the severity of the underlying liver disease. Consequently, the severity of liver disease 554 Slack and Wendon and volume of bleeding broadly determine prognosis. Haemorrhage severity indicators that predict failure to achieve haemostasis include active bleeding at endoscopy, transfusion requirements, haematocrit and blood pressure. Several studies have evaluated the number of death in the early and late groups, which is around 48% compared 52%, respectively (42).

8.5. Hepatic Encephalopathy Hepatic encephalopathy (HE) occurs in up to 30–40% of patients with CLD and in 10–15% undergoing TIPSS. HE is associated with a poor prognosis, and survival rates from the first episode of acute HE are 42 and 23% at 1 and 3 years, respectively (43). The assessment of HE has largely depended on the use of the West Haven criteria. However, the Glasgow coma scale (GCS), commonly used to assess level of consciousness, is another useful tool and measure widely used and understood. It can aid decisions regarding the escalation of care especially when the GCS is less than 8 implying blunting of airway protective reflexes and a need for intubation. The pathogenesis of HE is incompletely understood and the clinical course can be very variable and unpredictable (44). Ammonia plays a central role in the pathogenesis. PET scanning has elegantly confirmed the role of ammonia by demonstrating intense uptake in the brain in those patients with HE (45). Consequently, it is important to understand the inter-organ handling of ammonia in order to appreciate the available therapeutic options. The main site for ammoniagenesis is the gut, where glutamine is absorbed and converted by glutaminase to glutamate and ammonia. The detoxification of ammonia occurs in the liver and skeletal muscle and as liver disease advances there are significant changes to these pathways. With advancing liver disease, functional liver parenchyma volumes reduce and increases in portosystemic shunts result in a reduction in the conversion of ammonia to urea and consequently high levels of ammonia within the portal vein. In normal subjects, 50% of ammonia detoxification is handled by skeletal muscle and as CLD advances the activity of muscle glutamate synthetase increases. However, the correlation between ammonia blood concentration and the clinical manifestations of HE has not been consistently established. This has raised the suspicion that other factors are involved and the close correlation of sepsis with HE has led investigators to explore the role of inflammation. Recent studies have demonstrated significantly elevated levels of inflammatory cytokines in those with minimal HE compared to those without (45–47)(Fig.1). Chronic Liver Disease in the Intensive Care 555

Fig. 1. The relationship between ammonia, inﬂammation, neurotransmitter imbalance and cerebral blood ﬂow on the development and manifestation of HE. BBB, Blood brain barner; TNFα, tumor necrosis factor-α; IL, interleukin; GABA, γ-aminobutinic acid.

The main therapeutic options for hyperammonaemia include the avoidance of constipation with the use of enemas, laxatives and in particular, lactulose, which can all help to reduce the production and absorption of glutamine from the gut. Lactulose, though, can be associated with signiﬁcant gut distension, which can exacerbate any degree of ileus or increase in intra-abdominal pressure. Lactulose enemas often need to be used in patients in the ICU, especially when oral intake is not possible. Hypo-osmolar states like hyponatraemia should also be avoided because of the association with reduced levels of astrocyte myoinosi- tol, an organic osmolyte. Hypokalaemia also causes an increase in the renal production of ammonia and if a metabolic alkalosis is established + + NH3 is liberated from NH4 , a cation which can pass across the blood– brain barrier. Alternative therapies include L-ornithine, L-aspirate and benzodiazepine antagonists, which have all been shown to have some beneﬁt. The small molecular weight, 17 Da, of ammonia makes its clearance with RRT possible. However, only a few studies in children with urea 556 Slack and Wendon cycle disorders have demonstrated the effective clearance of ammonia using RRT. The association of sepsis, HE and HRS should encourage further studies examining the potential role of RRT in the management of hyperammonaemia.

9. SUMMARY In CLD, all organ systems can display a variable degree of dysfunction and this is often on the background of a chronic and profound lack of physiological reserve. Additionally, the immunoparesis of CLD increases the susceptibility for sepsis, which can be a feature of all the four common complications of CLD – HE, HRS, variceal bleeding and ascites. The initial management of critically ill patients with CLD should be focused on the identification and treatment of sepsis; if identification proves elusive then the early use of empirical antibiotic therapy should be implemented. Other precipitants of deterioration necessitate prompt treatment to prevent further deterioration in other vital organ systems. The optimization of the circulation is particularly crucial and can respond to simple manoeuvres, but early invasive haemodynamic monitoring to guide therapy, due to the difficulties of assessing the circulation, is recommended. As patients with CLD have a limited physiological reserve and often a poor prognosis when critically ill, added importance is extended to the early delivery of interventions and potential transfer into the critical care environment. Subsequently, a pragmatic assessment of the rate of response to therapy along with an understanding of the extent of the underlying CLD can all assist in determining likely outcomes and influence decisions regarding the appropriateness of both the continuation and if necessary the escalation of therapy.

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Subject Index

A liver failure, 486 Acetaminophen-mediated injury, 13 prophylactic antibiotics, 486 ACLF, see Acute-on-chronic liver failure quinolones, 486 (ACLF) secured airway, 486 Activated partial thromboplastin time pharmacological therapy (APTT), 294 bolus injection, 487 Acute Budd–Chiari syndrome, 52 efﬁcacy, 487 Acute kidney injury (AKI), 543 placebo-controlled RCTs, 487 Acute liver failure (ALF), 502 placebo/nonactive treatment, 487 acetaminophen overdose, 7 sclerotherapy, 487 Acute obstruction of biliary tract, 52 side effects, 487 Acute-on-chronic liver failure (ACLF), somatostatin analogue, 487 219–220, 502 triglycyl lysine vasopressin Acute physiology and chronic health derivative, 487 evaluation (APACHE), 542 rescue therapies Acute variceal bleeding treatment balloon tamponade, 488 bleeding control therapy encephalopathy mortality rates, 489 vasoactive drugs and endoscopic endoscopic/pharmacological therapy combination, 486 treatment, 488 endoscopic therapy esophageal stents, 489 diagnostic time, 488 hemostasis, 489 meta-analysis, 488 prognostic scores, 489 modalities, 488 therapeutic approach, 489 sclerotherapy and band ligation, 488 TIPS and surgical shunts, 489 initial treatment recommendations Acute Wilson’s disease, 52 optimal duration, 488 Adenosine diphosphate (ADP), 290 vasoactive drug therapy, 488 Adiponectin, 13 management Adrenal function and chronic liver acute variceal bleeding failure, 378 treatment, 486 acute stress, 378 blood transfusion, 486 adrenal steroid biosynthesis pathways, blood volume replacement, 486 388 correcting hypovolemia, 485 challenging and controversial issue endoscopic procedure, 486 adrenal response, 381 infection and failure, 486 cutoff values, 381 initial resuscitation, 486 guidelines and reviews, 382 intravenous ceftriaxone, 486 hydrocortisone therapy, 382 lactulose/lactitol, 486 insufﬁciency, 381

From: Clinical Gastroenterology: Chronic Liver Failure, Edited by: P. Ginès et al., DOI 10.1007/978-1-60761-866-9, C Springer Science+Business Media, LLC 2011

561 562 Subject Index

Adrenal function and chronic (cont.) acute phase, 380 RAI diagnosis, 381 adverse outcome predispose, 380 steroid therapy, 382 anabolic processes, 380 stimulation test, 381 corticotropin-releasing hormone synthetic adrenocorticotropic (CRH), 378 hormone, 381 dystrophy, 380 with cirrhosis adrenal function effect optimization, 380 decompensations, 387 enzymatic inactivation, 378 hypothalamic-pituitary-adrenal axis factors, 378 activation, 387 high plasma cortisol levels, 378 non-SBP infections, 387 non-ACTH-mediated mechanism, RAI prevalence, 387 380 cirrhosis severe sepsis/septic shock response, 380 insufficiency stimulate corticotropin secretion advanced liver disease, 386 (ACTH), 378 APACHE III, 384 sustained activation, 379 controls, 384 vasopressin (ADH), 378 diagnostic testing, 386 insufficiency fungal infection, 386 adrenal blood flow reduction, 387 glucocorticoid treatment, 384 cholesterol precursor, 387 hemodynamic instability, 383 complications, 383 hospital mortality rates, 384 contributing factor, 387 hyperreninemic hypoaldosteronism endotoxin, 387 syndrome, 386 high-density lipoprotein (HDL), 387 infections, 384 immunosuppressive effects, 382 mineralocorticoids, 386 positive effects, 382 monitoring/treatment, 383 preexisting conditions, 387 natural course of, 386 RCT steroid administration, 382 relevant finding and study, 385 secondary infections, 382 retrospective comparative study, 384 vasoconstrictor drugs, 382 risk and benefits, 384 poor prognosis, 378 sequential organ failure assessment RAI, 378 (SOFA), 384 relative adrenal insufficiency incidence, severity and poor prognosis, 387 383 shock reversal rate and hospital serum cortisol levels, 379 survival, 385 steroid effect, 378 studies, 383 unsolved questions supplemental treatment effect, 384 corticotropin stimulation test, 388 treatment, 386 current consensus, 388 cortisol effects, 378 features, 388 adaptation and maintenance, 380 glucocorticoid secretion, 389 biological, 381 measuring total serum cortisol, 389 homeostatic mechanisms, 380 methodological problems, 388 immunosuppressive hormone, 380 physiological function, 388 inhibitory effect, 380 steroids, type/doses/duration, 389 metabolism aspects, 380 stress/replacement doses, 389 endothelial integrity, 378 Adrenomedullin in cirrhotic liver patients, hospital mortality, 378 313–314 hospital survival probability, 386 AKI, see Acute kidney injury (AKI) hydrocortisone administration Alanine aminotransferase (ALT), 48 effects, 385 Alcoholic and nonalcoholic liver disease, hypothalamic-pituitary-adrenal 12 axis, 379 Alcoholic steatohepatitis (ASH), 7 Subject Index 563

ALF, see Acute liver failure (ALF) LSECs, 345 Alpha-1 antitrypsin deficiency, 52 macrophages and lymphocytes, 347 ALT, see Alanine aminotransferase (ALT) MCP-1, 346 AMAs, see Antimitochondrial antibodies mitogenic, profibrogenic, and (AMAs) chemotactic agents, 347 Aminotransferases, 36 monoclonal antibody receptors, 347 Ammonium detoxification, 42 morphological components, 345 AMP-dependent protein kinase multitarget-based therapies, 352 (AMPK), 37 neovascularization, 345 central turntable, 38 pathological situations, 346 AMPK, see AMP-dependent protein kinase PDGF, 345 (AMPK) physiological conditions, 345 Angiogenesis and vascular growth, 19 PlGF, 346 cirrhotic rats vascular remodeling, 354 portal blood flow, 351 CLD, 343 preclinical investigations, 347 complex pathophysiological process, proinflammatory genes, 346 343 RTKs, 347 complications, 344 sinusoidal capillarization, 345 conductance vessels vascular Sunitinib treatment, 347 remodeling synergistic effect, 347 advanced liver disease, 353 therapeutic strategies, 345 characteristic features of, 353 VCAM-1, 346 promotes architectural changes, 355 VEGF-A, 346 remarkable features, 354 mechanisms, 344 remodeling processes, 353 NAFLD, 343 studies, 354 sinusoidal intrahepatic circulation, 344 unresponsiveness, 355 splanchnic angioarchitecture, vasorelaxation, 353 long-term structural changes in drugs action mechanism, 348–350 angiogenic process, 352 HCC, 343 hyperdynamic splanchnic hemodynamic abnormalities, 344 circulation, 353 hepatic angioarchitecture long-term neutralizing antibody, 353 structural changes in peritoneal circulation, 352 anatomical region, 352 persistent abnormalities, 352 angiogenesis, 345 PlGF deficiency, 353 antitumor and antiangiogenic agent, portosystemic shunting, 353 347 powerful inducer ascites, 353 approach validity, 352 preexisting venous channels, 353 beneficial effect Sunitinib, 347 signaling pathway activation, 353 beneficial treatment, 352 splanchnic area, 352 blood supply sources, 344 in vivo mesenteric assays, 352 colocalization, 346 Sunitinib treated cirrhotic rats COX2, 346 significance, 351 efficacy, 347 vascular tone modulation, 344 eNOS, 345 Antimitochondrial antibodies (AMAs), 60 fenestration, 345 APACHE, see Acute physiology and fibrogenic process, 346 chronic health evaluation HIF-1, 346 (APACHE) HSCs signaling pathway, 345, 351 Apical sodium bile acid transporter ICAM-1, 346 (ASBT), 35 inflammatory mediators, 346 Apolipoprotein B (Apo-B) synthesis, 37 intestinal mucosa, 344 APRI, see AST-to-platelet ratio index IRF-1, 346 (APRI) 564 Subject Index

APTT, see Activated partial thromboplastin proapoptotic factors, 510 time (APTT) proinflammatory cytokines, 510 Aquaporine 2 water channels (AQP2), 255 serum bilirubin and biliary acids, 508 Arachidonic acid-derived lipid mediators splanchnic and systemic COX pathway hemodynamics, 510 biosynthesis, 135–136 vasoactive systems, 510 eicosanoids, 138 in vitro and in vivo studies, 509 endothelial cells, 136 ASBT, see Apical sodium bile acid liver injury, 139 transporter (ASBT) NSAIDs, 137 Ascites management PGD synthase, 137 body fluid regulation disturbances, 411 cyclopentenone PGs, 139 in cirrhosis, pathogenesis of, 412 isoprostanes consequence, 411 cellular membranes, 139 diuretics side effects formation, 140 amiloride and eplerenone, 414 nonenzymatic lipid peroxidation chronological relationship, 414 products, 140 electrolyte disturbances, 414 oxidative stress injury, 140 hepatic encephalopathy, 414 5-LO pathway induced diagnosis, 414 biosynthesis, 141 muscle cramps, frequency and fibrosis, 144 intensity, 415 FLAP inhibitor, 142 natriuretic potency, 415 hepatic levels, 143 renal function tests, 414 lipid mediators, 142–143 therapy complications, 414 LT formation, 141 homeostatic activation, 411 platelet-activating factor, 142 large-volume ascites patients structural lipid components, 135 management recommendations, Arginine vasopressin (AVP), 255, 418 416 Arterial blood gas (ABG), 365 pharmacological therapy Arterial buffer system in liver ascites reduction, 412 adenosine role, 84 negative sodium balance, 412 dipyridamole level, 84 recommended sodium intake, 412 metabolic autoregulation, 83 recommendations, 418 oxygen demand, 83 renal function abnormalities, 411 portal flow, 83 splanchnic circulation, 411 sinusoidal microcirculation, 83 therapeutic paracentesis Artificial liver support (ALS), aspects, 415 physiological effects efficacy, 416 acute decompensation, 508 guidelines, 415 albumin dialysis in ACLF, 508 harmful effects, 415 circulatory dysfunction, 510 lower adverse events, 415 detoxifying capacity, 510 plasma volume expansion, 415 hepatocyte apoptosis, 510 shortened survival, 415 HVPG, 510 treatment strategy, 415 inflammatory response, 509 transjugular intrahepatic portosystemic malondialdehyde levels, 509 shunt (TIPS) MARS adverse effects, 417 and FPSA comparison, 511 bacterial peritonitis, 417 influence, 509 beneficial effect, 416 and Prometheus devices, 509 cardiopulmonary disease, 418 medical therapy, 509 cost, 417 monocyte chemoattractant protein, 510 discrepancies, 417 portal pressure change, 510 drawbacks, 416 Subject Index 565

guidelines, 418 by Pseudomonas aeruginosa, 396 hepatorenal syndrome, 417 quinolones use, 396 meta-analysis, 417 studies, 395 obstruction frequency, 417 consequences portosystemic shunt, 416 acute-onchronic liver failure, 397 procedure, 416 body’s systemic inflammatory rescue therapy, 417 response, 396 transjugular approach, 416 coagulopathy and treatment choice and efficacy thrombocytopenia, 397 adverse effects, 413 septic encephalopathy, 397 aldosterone antagonists, 412 nonspontaneous analysis, 413 CDAD, 405 diuretics, 412 child-pugh scores, 405 diuretic therapy, 413 MELD, 405 hyperkalemia, 413 respiratory tract infections, 404–405 hypovolemic hyponatremia, 413 urinary tract infections, 404–405 natriuretic potency, 413 SBP refractory ascites, 413 ascitic fluid analysis, 397–398 regime, 413 blood cultures, 398 spironolactone, 413 prophylaxis, 400–401, 403–404 theoretical advantages, 413 treatment, 398–400 ASH, see Alcoholic steatohepatitis (ASH) without/with antibiotic prophylaxis, Aspartate aminotransferase (AST), 48 rates of, 402 Association analysis spontaneous case–control design, 161 ascitic/serum fluid analyses, 398 large association studies, 161 asymptomatic disease, high rate, 397 one association study blood cultures, 398 procedure, 161 PMN, 398 AST, see Aspartate aminotransferase (AST) prompt diagnostic paracentesis, Astrocytes 397–398 glutamine Bacterial infections in liver cirrhosis accumulation, 172 ACLF, 221 generated, 172 associated with, 221 in hepatic encephalopathy (HE), 172 gram-negative organisms, 222 neuronal function, 172 gram-positive bacteremias, 222 neurosteroids synthesis, 172–173 risk factor for AST-to-platelet ratio index (APRI), 56 sepsis, 221 ATP-binding cassette (ABC), 40 screening and treatment, 221 Autoimmune hepatitis, 52 Bazett correction formula, 283 AV P, see Arginine vasopressin (AVP) BCRP, see Breast cancer-related protein (BCRP) Bile acids, 33 B bile salt export pump (BSEP), 34–35 Bacterial infections cholesterol in synthesis, 34 in cirrhosis, epidemiology conjugated, 34 common, 396 micelle formation and, 34 data, 396 production, 34 by Enterococcus faecium, 396 secondary, 34 by Escherichia coli, 396 signaling molecules, 35 GNB and GPB, 396 Biochemical tests by Klebsiella spp., 396 abnormal patterns methicillin-resistant Staphylococcus cholestasis, 52 aureus, 396 emerging tests, 52 multiresistant, 396 566 Subject Index

Biochemical tests (cont.) QT prolongation, 271–272 serum aminotransferase (AST and Cargo proteins, 35 ALT) levels, 52 Carnitine palmitoyltransferase-1 severity and reversibility of liver (CPT-1), 37 injury, 52 CDAD, see Clostridium difficile-associated synthetic function, 52 illness (CDAD) transport organic anions, 52 Child–Turcotte–Pugh (CTP) alkaline phosphatase, 49–50 scores, 71 aminotransferases, 48–49 and survival rates, 542 bilirubin, 51 system, 542 cholestatic pattern Cholestasis, 56–58 acute/chronic impairment in bile extrahepatic causes, 59 flow, 56 Cholestatic liver injury, 7–8 autoimmune diseases, 60 Chronic hepatitis B, 52 causes of, 57–58 Chronic liver disease (CLD), intensive care infiltrative disorders, 61 airway intrahepatic cholestasis, 60 aspiration risk, 544 GGTP, 50 catastrophic consequences, 544 hepatocellular pattern coagulopathy, 544 serum aminotransferase elevations, encephalopathy, 544 52 endotracheal intubation, 544 hyperbilirubinemia, 61–63 intra-abdominal pressure, 544 liver function tests, 48 percutaneous tracheostomy, 544 5nucleotidase (5 NT), 50 premature extubation and prolonged quantitative, 70 ventilation, 544 Biopsy, 66 prophylactic intubation, 544 indications and contraindications, re-intubation rates, 544 67–68 ultrasonography, 544 Bleeding time (BT), 291 AKI, 543 Body mass index (BMI), 364 APACHE, 542 Breast cancer-related protein (BCRP), 40 ascites and intra-abdominal pressure British Society of gastroenterology activation pathways, 546 (BSG), 457 albumin and vasopressor BSG, see British Society of analogues, 546 gastroenterology (BSG) CIPMN, 547 Budd–Chiari syndrome (BCS), 467 cirrhosis complications, 545 dysfunction, oncotic pressure and myocardial, 546 C effusions and ascites, worsening, Carbohydrate metabolism, 36–37 545 Cardiovascular abnormalities, 269 fat oxidation, 547 abnormal structure and histology, gluconeogenesis, 547 272–273 haemodynamics and effects, 546 histomorphological alterations, 273 hydrothorax, 546 blunted contractile response to stimuli long-term outcomes, 545 β -adrenergic stimulation, 270 malnutrition, 547 diastolic relaxation, 270–271 neuro-humoral responses, 545 systolic contractility, 271 parameters, 546 systolic responsiveness, 270 PEEP, 546 electrophysiological abnormalities PICD, 546 Child–Pugh score, 271 pressure fall, 546 chronotropic incompetence, 272 SIRS, 547 electromechanical dyssynchrony, splanchnic circulation, 546 272 Subject Index 567

breathing prognosis, 548 affect ventilation mechanics, 545 respiratory mechanics chronic obstructive pulmonary activation pathways, 546 disease, 545 albumin and vasopressor analogues, hepatopulmonary and 546 portopulmonary syndrome, 545 ascites accumulation, 545 pneumonia, 545 CIPMN, 547 respiratory infections and failure, cirrhosis complications, 545 545 dysfunction, oncotic pressure and cardiovascular myocardial, 546 accurate assessment, 548 effusions and ascites, worsening, arteriovenous shunts, portosystemic 545 and pulmonary, 549 fat oxidation, 547 cardiomyopathy characterization, gluconeogenesis, 547 548 haemodynamics and effects, 546 CO, 548 hydrothorax, 546 CVP, 549 long-term outcomes, 545 echocardiogram, 549 malnutrition, 547 electrophysiological abnormalities, neuro-humoral responses, 545 548 parameters, 546 hyperdynamic circulation, 548 PEEP, 546 IAH, 548 PICD, 546 initial resuscitation period, 548 pressure fall, 546 macro and microcirculations, 548 splanchnic circulation, 546 monitoring and support, 548 systemic inflammatory response parameters, 549 syndrome (SIRS), 547 reduced mortality, 549 specific pathologies, 543 serum lactate, 549 Chronic liver failure, transplantation issues Child–Turcotte–Pugh (CTP), 542 allocation complication encountered, 541 biochemical parameters, 527 dysfunction, sepsis and multi-organ, CTP scoring system, 526 542 etiology, 527 extensive variceal haemorrhage, 543 influential factor, 526 extra-hepatic and renal factors, 543 international normalized ratio (INR) HE, 541 of prothrombin, 527 HPS Mayo end-stage liver disease arterial hypoxaemia, 547 model, 527 diagnosis of, 547 models, 526 echocardiography, 547 mortality rate, 526 pulmonary/CT angiography, 547 National Organ Transplant Act pulmonary vasodilatation, 547 (NOTA), 526 qualitative assessment, 547 Organ Procurement and survival rates, 547 Transplantation Network infection risk, 542 (OPTN), 526 model of end-stage liver disease prediction tool, 527 (MELD), 542 priority/status, 526 mortality prediction power and score, transjugular shunts, 527 543 United Network for Organ Sharing pathologies, specific liver-related, (UNOS), 526 549–556 wait list and selection process, 526 PPH bioartificial liver support, 522 Doppler echocardiography, 548 critically ill liver patient, 533–534 prevalence of, 548 immunosuppression, 521 568 Subject Index

Chronic liver failure (cont.) genome-wide human haplotype, 156 liver transplant, referral and evaluation, global expansion, 156 522–525 Common disease–rare allele prediction models of, 527–533 hypothesis, 161 c-statistic measures, 527 extensive allelic, 161 prognostic variables, 527 heterogeneous population, 161 soothsayers, hepatologist and liver locus heterogeneity, 161 surgeon, 527 Complex diseases, 156–158 pretransplant evaluation, 524 Conductance vessels, vascular surgical technique, 521 remodeling in therapeutic approach, 522 advanced liver disease, 353 Chronic obstructive pulmonary characteristic features of, 353 disease, 545 promotes architectural changes, 355 Chronic viral hepatitis, 7 remarkable features, 354 Chronotropic incompetence, 272 remodeling processes, 353 Chylomicrons (CLMs), 20 unresponsiveness, 355 CIPMN, see Critical illness vasorelaxation, 353 polymyoneuropathy (CIPMN) Conjugated bile acids, 34 Cirrhosis Copper-transporting ATPases antifibrotic therapies, 95 (Cu-ATPases), 39 microvascular structure, 91 COX2, see Cyclooxygenase-2 (COX2) noninvasive tool, 99 CPT-1, see Carnitine progressive stages, 99 palmitoyltransferase-1 (CPT-1) Cirrhotic cardiomyopathy Critical illness polymyoneuropathy abnormal renal function, 273 (CIPMN), 547 β-adrenergic receptor system, 278 Critically ill liver patient, transplantation carbon monoxide, 281 issues cellular calcium kinetics, 279–280 futile therapy, 533 clinical consequences, 274–275 manifested condition, 533 endocannabinoids, 281–282 multiorgan failure, 533 liver transplantation, 277 prediction models in membrane physicochemical changes, aforementioned models, 534 278–279 APACHE, 534 myofilament proteins, 282 myocardial infarction, 534 NF-κB and cytokines, 282 organ system failure (OSF), 534 nitric oxide role of, 280–281 renal failure, 534 spontaneous bacterial peritonitis scoring systems, 534 (SBP), 276 sequential organ failure assessment TIPS insertion, responses and survival, (SOFA), 534 276–277 studies focused on, 534 treatment Cyclooxygenase-2 (COX2), 346 anti-aldosterone drug, 283 Cytokines β-blockers, 283 IL-6 cytokine family, 134 diuretics, 283 liver cell types, 132 non-pharmacological management, low-molecular-weight proteins, 133 283–284 TNF-α Clostridium difficile-associated illness alcoholic hepatitis, 134 (CDAD), 405 cellular and biological processes, Coagulation factors, 35 132–133 Common disease–common variant serum levels, 133 hypothesis, 156 signaling, 133–134 alleles, 156 toll-like receptors, 133 divergence of humans, 156 Cytomegalovirus (CMV) infections, 52 Subject Index 569

D Endoscopic retrograde DDAVP, see Deamino-8-D-arginine cholangiopancreatography vasopressin (DDAVP) (ERCP), 60 Deamino-8-D-arginine vasopressin Endoscopic ultrasonography, 61 (DDAVP), 466 Endoscopic variceal ligation (EVL), 553 Detoxification pathways Endothelial nitric oxide (eNOS), 345 alcohol, 41 Endothelium-derived hyperpolarizing ammonium, 42 factor (EDHF) bilirubin, 40–41 arachidonic acid metabolites, 312 DHPRs, see Dihydropyridine receptors gap junctions, 312 (DHPRs) hydrogen peroxide, 312 DIC, see Disseminated intravascular monovalent cation K+, 312 coagulation (DIC) Epithelial-to-mesenchymal transition Dihydropyridine receptors (DHPRs), 279 (EMT), 8 DILI, see Drug-induced liver contribution of, 9 injury (DILI) derived fibroblasts, 9 Dissect disease-causing genetic variants, markers of, 9 study designs Epsilon aminocaproic acid (EACA), 466 candidate–gene approaches ERCP, see Endoscopic retrograde association analysis, 161–162 cholangiopancreatography GWAS (ERCP) application of, 162 Ethanol-induced injury, 13 benefit and education of patients, ETP, see Endogenous thrombin potential 162 (ETP) contribution of, 162 EVL, see Endoscopic variceal ligation defined, 162 (EVL) liver disease and traits, 162 Extracellular fluid volume National Human Genome Research fluid homoeostasis, 240 Institute, 162 fluid retention, 240 Disseminated intravascular coagulation plasma and interstitial space dynamics (DIC), 457 balance between, 241 Distal splenorenal shunt (DSRS), 484 blood stream transport, 244 DLST, see Drug lymphocyte stimulation capillarisation, 242 test (DLST) contractile elements, 247 Drug-induced liver injury (DILI) drainage from peritoneal cavity, 246 diagnosis of, 63 exchange of material, 241 Hy’s rule, 64 hepatic and gastrointestinal liver biopsy, 64 circulation, 243 LMT and DLST, 64 hydrostatic/oncotic equilibration, prognosis of patients with, 64 244 scales used, 63 hydrostatic pressure, 242 Drug lymphocyte stimulation test (DLST), intestinal lymph-blood protein ratio, 64 241–242 DSRS, see Distal splenorenal kinetic studies, 242–243 shunt (DSRS) microcirculation, 241 microvascular fluid, 246 pressure gradient, 241 E protein-free fluid, 244 EACA, see Epsilon aminocaproic acid spontaneous bacterial peritonitis (EACA) (SBP), 245 EBL, see Endoscopic band ligation (EBL) sympathetic and parasympathetic Electromechanical dyssynchrony, 272 nervous system, 246–247 Endogenous thrombin potential (ETP), 295 transperitoneal dynamics, 242 Endoscopic band ligation (EBL), 480 570 Subject Index

Extracellular fluid volume (cont.) F transperitoneal Starling forces in Farnesoid X receptor (FXR), 35 cirrhosis, 246 FasL-and TRAIL-mediated apoptosis, 7 transvascular transport in Fas-mediated apoptosis, 7 capillaries, 245 Fatty acids, peroxisomes and microsomes, two-pore and three-pore 37 models, 243 Fenestrations, 20 vasodilatation in splanchnic FFP, see Fresh frozen plasma (FFP) capillaries, 246 Fibroblast growth factors (FGFs) signaling, renal dysfunction, 241 112 Extracellular matrix (ECM) Fibroblast-specific protein 1 (FSP-1), 9 components, 92 Fibrogenesis, 17–18 composition, 92 Fibrosis heterogeneity, 93 noninvasive tests, 68–69 injured liver, 93 Forced vital capacity (FVC), 362 molecules, 93 Fresh frozen plasma (FFP), 461 Extracorporeal artificial liver support FSP-1, see Fibroblast-specific protein 1 (ALS) systems (FSP-1) ACLF, 502 FVC, see Forced vital capacity (FVC) action mechanisms and outcomes FXR, see Farnesoid X receptor (FXR) summary of, 504–505 ALF, 502 appealing therapeutic approaches, 501 G CLF, 502 Gamma glutamyl transpeptidase clinical efficacy of, 501–515 (GGTP), 50 clinically applied BAL system GCDCA, see Glycine-conjugated characteristics, 506–507 chenodeoxycholic acid dialysis procedures, 501 (GCDCA) difference, 502 GCS, see Glasgow coma scale (GCS) future prospects for, 508 Genome-wide associations studies (GWAS) liver failure application of, 162 manifestations, 502 benefit and education of patients, 162 prevention and management, 501 contribution of, 162 liver supportive strategies, 502 defined, 162 multiorgan dysfunction, 502 liver disease and traits, 162 pathophysiological approach National Human Genome Research and types Institute auxiliary transplantation, 503 web site, 162 bioartificial devices, 503 GFR, see Glomerular filtration rate (GFR) biological devices, 503 GGTP, see Gamma glutamyl transpeptidase clinical application, 503 (GGTP) detoxification process, 508 Glasgow coma scale (GCS), 554 dialyzer, 505 Glomerular filtration rate (GFR), 252, 430 infections and disturbances, 503 bilateral Goldblatt kidneys, 254 MARS, 503 filtration fraction, 254 necrosis and apoptosis death, 502 Gluconeogenesis, 37 nonbiological devices, 503 Glucose-1-phosphouridyltransferase, 36 noxious agent, duration Glutamine synthesis, 42 and severity, 502 Glycerol-3-phosphate acyltransferase theoretical approaches, 503 (GPAT), 37–38 toxic substance accumulation, 503 Glycine-conjugated chenodeoxycholic acid pathophysiological effects of, 508–511 (GCDCA), 7 pros and cons of, 505 TRAIL-induced apoptosis, 8 Glycolysis pathway, 36 Subject Index 571

GNB, see Gram-negative bacteria (GNB) TEG parameters, 458 GPAT, see Glycerol-3-phosphate trigger DIC, 458 acyltransferase (GPAT) variceal rebleeding, 458 GPB, see Gram-positive bacteria (GPB) invasive procedures of G-Protein-coupled receptor, 35 axiomatic, 458 Gram-negative bacteria (GNB), 396 bleeding risk factor, 456 Gram-positive bacteria (GPB), 396 BSG, 457 Granulomatous liver disease, 60 central venous access, 457 Gut ischemia–reperfusion injury, 79 clotting parameters, 457 GWAS, see Genome-wide associations DIC, 457 studies (GWAS) elective surgery, 456 findings and studies, 458 hepatic puncture site, 456 H INR thresholds, 457 HAART, see Highly active antiretroviral liver biopsy, 456 therapy (HAART) mortality and morbidity, 456 Haemostasis in CLD, abnormalities paracentesis/thoracentesis, 457 correction plugged liver biopsy, 457 bleeding risk assessment PT and platelet count, 456 clot formation and lysis, 456 stipulated range, 457 CP score, 456 ultrasound guidance, 456 drawbacks, 455 liver cirrhosis patients clinical epistaxis, 455 haemorrhage, 454 hyperfibrinolysis, 455 pro and anti-coagulative clotting impairment degree, 455 factors, 454 measuring coagulant activities, 455 pro and anti-thrombotic prognostic indexes, 455 processes, 454 reflect/predict coagulation, 455 splenic sequestration, 454 TEG, 456 therapy of, 460–467 trigger factor, 456 thrombosis risk and anticoagulation blood coagulation, 453 clinical use assessment cirrhosis and peritoneal infection acenocoumarol, 468 heparin-like effect in patient, 459 anticoagulant effect, 469 fibrinolysis, 455 antithrombotic prophylaxis, 468 genetic defects, 455 BCS, 467 infection and sepsis, coagulation cholestatic disease, 468 antibiotic therapy, 458 confluent fibrosis, 467 anti-Xa activity, 458 disadvantage, 469 coagulation derangement, 460 guidelines, 469 consumptive coagulopathy, 458 hypothesized, 467 endotoxaemia, 458 intracranial haemorrhage, 469 endotoxins and inflammation, 458 mesenteric infarction, 467 glycosaminoglycans, 460 paradoxical, 467 HVPG, 459 parameters and tailoring, 469 intestinal decontamination, 459 pneumatic compression, 469 neutralase, 460 PNH, 467 NO production, 458 prothrombotic disorders, 467 patients rifaximin, 460 pulmonary embolism, 468 plasma endotoxin level, reduction, retrospective studies, 468 460 serum albumin level, 468 portal haemodynamics, 459 underwent anticoagulation therapy, prevention, 459 468 protamine sulphate infusion, 460 veins recanalization, 467 protamine treatment, 460 572 Subject Index

Haemostasis in CLD (cont.) optimal levels, 464 transplantation, 455 pharmacological approaches, 464 vitamin K deficiency, 454 PLT production, 465 Haemostasis in CLD, therapies rheological studies, 464 antifibrinolytics splenectomy, 465 aprotinin, 466 splenic embolization, 465 blood transfusion-free thrombin production, 464 hepatectomy, 466 thrombocytosis, 465 EACA, 466 TPO receptor, 465 endoscopic treatment, 467 prothrombin complex hepatectomy/transplantation, 466 PCC, 462 intraoperative, 466 thrombin–antithrombin, 462 nafamostat mesilate efficacy, 466 thrombotic complications, 462 perioperative mortality, 466 recombinant activated factor VII serine protease, 466 acenocoumarol, 463 tranexamic acid, 466 coagulation disorders treatment, 463 antithrombin III infusion endoscopic and medical therapy, 464 blood loss/decrease mortality, 467 haemophilia patients, 463 fibrinogen concentration, 467 hepatectomy, 463 cryoprecipitate intracranial pressure contents, 462 monitoring, 463 hypofibrinogenemia, 463 laparoscopic liver biopsy, 463 recommended, 462 prothrombotic effects, 464 desmopressin rebleeding and mortality effect, 464 antidiuretic hormone, 466 thrombin generation, 463 DDAVP, 466 thromboembolic complications, 463 endothelial storage sites, 466 test abnormalities, 460 hepatic resection, 466 vitamin K FFP and transfusion requirements acute liver failure, 460 clotting factors, 461 cholestasis/malabsorption, 460 controlled trial, 462 coagulation factors, 460 crystalloid infusion, 461 subclinical deficiency, 461 crystalloids effect, 461 HALT-C model, see Hepatitis C Antiviral FFP infusion, 461 Long-term Treatment against half-life of, 461 Hepatitis C (HALT-C) model intracerebral pressure, 461 HapMap, see Human haplotype map neurological procedures, 461 (HapMap) plasma-based blood products, 461 HCV, see Hepatitis C virus (HCV) replacement therapy, 461 HDL, see High-density lipoprotein (HDL) transfusion strategy, Heart and central arterial tree, dynamic restrictive/liberal, 462 coupling transjugular biopsy, 462 arterial pulse wave velocity, 249–250 venous pressure gradient, 461 fast Fourier analysis, 250 life-threatening bleeding, 460 latent and manifest cardiac peptic ulcers, 460 dysfunction, 250 platelet transfusions and structural and functional alterations, 249 thrombocytopaenia treatment Hemochromatosis, 52 amelioration, 464 Hemojuvelin (HJV), 39 antiviral therapy, 465 Hemostasis abnormalities in CLD chemotherapy, 465 characterized defect, 289 chronic hepatitis, 465 coagulation pathophysiology eltrombopag, 465 cellular/humoral process, 292 haematocrit levels, 464 complex tissue factor, 292 Subject Index 573

plasmatic factors activation, 292 endothelial cell dysfunction, pro-and anti-coagulant factors, 293 293 procoagulant factors, 292 ETP, 295 TFPI, 292 exogenous triggers, 295 tissue factor activation, 292 exposure phospholipids, 295 fibrinolysis hemophilia and allied disorder, 294 fibrin deposition, 298 hemorrhagic procedures, 294 future efforts, 300 hemorrhagic risk, 296 global assays, 299 INR, 294 global test, 299 LRP, 293 hyper-or hypo-fibrinolysis, 298 mature coagulation factors, 293 integrated operating system, 298 multiligand receptor, 293 pathway, 299 plasma operating drivers, 295 plasmatic fibrinolytic capacity, 299 platelet test, 295 plasmin, generation and postribosomal carboxylation, 293 perturbation, 298 prophylactic replacement, 294 pro-and anti-fibrinolytic factors, 299 protein C and antithrombin, 293 procarboxypeptidase synthesis, 298 proteolytic inactivation, 293 schematic representation, 299 PT test, 294 special attention, 299 relative characterization, 293 TAFI, 298–299 synthetic capacity, 293 tPA, 298 threshold value, 296 uncertainty reasons, 298 thrombin generation, 295–296 uPA, 298 thrombocytopenia, 296 hemorrhagic disease epitome, 289 thromboelastography, 296 hypercoagulability thrombomodulin, 294–295 congenital defects, 297 in vivo operating, 294 factor levels, 298 literature and clinical practice, 290 gain-offunction mutations, 297 platelet adhesion injury and genetic risk factor, 297 aggregation, 290 heparin/oral anticoagulants, 298 primary hemostasis median ratio, 297 activated platelets, 290 plasma from 134 investigation, 297 ADP, 290 potent drivers, 298 antidiuretic hormone, 291 prolonged traditional tests, 297 BT prolongation, 291 PT-INR, 296 coagulation activation, 291 pulmonary embolism, 297 desmopressin infusion, 291 ratio index, 297 platelet adhesion study, 291 resistance result, 298 splenic and hepatic sequestration, retrospective study, 296 291 thrombin generation, 297 surrogate tests, 292 typical features, 298 thrombin formation, 291 VTE, 296 thrombocytopenia/ hypocoagulability thrombocytopathy, 291 apparent paradox, 294 TXA2, 290 application of, 295 vessel wall and platelets interaction, APTT, 294 290 arbitrary cutoff values, 294 VWF, 290 balance of, 295 tests and treating, 289 characterized condition, 294 thrombin generation coagulation, concomitant deficiency, 294 schematic representation, 292 congenital deficiencies, 294 Henle’s loop and distal tubules conventional tests, 296 aldosterone sensitivity, 255 574 Subject Index

Henle’s loop (cont.) treatment 11-beta-hydroxy-steroid antifibrotic therapies, 101 dehydrogenase, 255 anti-inflammatory activity, 100 endogenous glucocorticoids, 255 cirrhosis, 100 renal calcium/ polyvalent cation, 255 HSC activation, 99 sodium reabsorption, 255 leukemia, 101 Hepatic angioarchitecture, long-term NASH and HCV, 100–101 structural changes in stellate cells, 101 anatomical region, 352 Hepatic hydrothorax (HH), 366 angiogenesis, 345 Hepatic microvascular subunit (HMS), 324 angiogenic factors, 346 Hepatic stellate cell (HSC), 345 antitumor and antiangiogenic apoptosis, 99 agent, 347 HCV receptors, 97 blood supply sources, 344 indirect activators, 96 colocalization, 346 leptin, 100–101 COX2, 346 liver transplantation, 101 eNOS, 345 lymphocytes, 94 fenestration, 345 pathways, 94 fibrogenic process, 346 perisinusoidal cell, 92 HIF-1, 346 transcription factors, 95 HSCs signaling pathway, 345, 351 Hepatic stellate cells (HSCs), 4, 11, 325 inflammatory mediators, 346 biological actions associated with, 18 intestinal mucosa, 344 embryonic origin of, 15 IRF-1, 346 in hepatic physiology and LSECs, 345 pathophysiology macrophages and lymphocytes, 347 angiogenesis, 19 MCP-1, 346 contraction, 19 mitogenic, profibrogenic, and fibrogenesis, 17–18 chemotactic agents, 347 liver-specific pericytes, 17 monoclonal antibody receptors, 347 matrix degradation, 18–19 multitarget-based therapies, 352 modulation of inflammation, 19 neovascularization, 345 normal extracellular matrix Hepatic encephalopathy (HE), 171, 554 homeostasis, 16–17 Hepatic fibrosis, chronic liver disease retinoid storage and metabolism, biochemical and structural features, 92 15–16 bone marrow-derived cells, 95 and stem cell niche, 19 CD4/CD8 ratio, 93 physiological function of, 15 clinical aspects sinusoidal microcirculatory network, 14 cirrhosis, 99 in space of Disse, 14 histological staging systems, 98 Hepatic stem cells imaging methods, 97–98 activation liver biopsy, 97 Hedgehog (Hh) signaling, 114–115 noninvasive tests, 97 nonparenchymal cells, 115–116 disease-specific patterns, 96–97 oval cell proliferation, 114 ECM molecules, 93 progenitor cell markers, 115 extracellular release, 93 signaling pathways, 113–114 HSC, 92 in adult liver immune system, 93 endodermal stem cells, 112 resolution iron-induced liver injury, 112 antifibrotic therapies, 95 oval cells activation, 112–113 apoptosis, 96 salt and organic anion matrix remodeling, 96 transporters, 113 TIMPs, 95–96 and cancer Subject Index 575

biliary cell markers, 116 arterial–venous pathways, 368 dysplastic foci, 119 atelectasis, 372 immunophenotyping, 116 cadaveric/living donor, 370 identification and isolation diagnostic criteria, 368 cell-specific markers, 116 echocardiography, 369 phenotypic expression, 117–118 embolotherapy, 369 Hepatic venous pressure gradient (HVPG), hospitalization mortality, 370 99, 510 hyperdynamic circulatory, 369 Hepatitis C Antiviral Long-term Treatment medical treatments, 370 against Hepatitis C (HALT-C) NO effect, 370 model, 56 orthodeoxia, 369 Hepatitis C virus (HCV), 56 paradoxical, 372 Hepatocellular cancer (HCC), 92, 101, 343 portal hypertension triad, 368 biliary cell markers, 116 post-LT mortality, 370 pathogenesis, 114 pulmonary angiography, 369 stem cell-like cells, 115 screening for, 369 tumor specimens, 115 TIPS, 370 Hepatocytes, 4 transthoracic/transesophageal, 369 apoptosis, 6 true dilatation, 368 ALF, 7 See also Pulmonary ASH, 7 vasculopathies-dilemma Bax and Bak activation, 7 Hepatorenal syndrome (HRS) cholestatic liver injury, 7–8 nephropathy for c-Jun N-terminal kinases (JNKs) arterial blood pressure, 257 activation, 7 arterial hypovolaemia, 256 death receptor activated signaling autoregulation, 257 cascades, 7 GFR, 256–257 endoplasmic reticulum (ER) hepatorenal reflex, 256 stress, 7 opioid antagonists, 258 fibrogenesis progression, 8 RBF, 256 lysosomal permeabilization, 7 renal sympathetic nervous nonalcoholic steatohepatitis, 7 activity, 256 as hepatic effector cells, 5 types of, 256 injury, 5 vasoconstrictor and vasopressin, 257 in physiological conditions V2 receptor antagonists, 258 ammonia inactivation, 6 Hepcidine expression, 39 bile secretion, 6 HH, see Hepatic hydrothorax (HH) exogenous and endogenous HIF-1, see Hypoxia-inducible factor-1 metabolism/inactivation, 6 (HIF-1) glucose blood levels regulation, 5 HIFs, see Hypoxia-inducible factors (HIFs) homeostasis, 6 High-density lipoprotein (HDL), 387 lipid metabolism, 6 Highly active antiretroviral therapy plasma proteins, 6 (HAART), 525 as source of myofibroblasts HMG-CoA reductase (HMGR), 38 EMT, 8–9 HMS, see Hepatic microvascular subunit surface domains, 5 (HMS) zone 1 and 2 of Rappaport’s acinus, 6 Hodgkin’s lymphoma, 60 Hepatopulmonary syndrome (HPS), HPS, see Hepatopulmonary syndrome 362, 369 (HPS) altered ventilation–perfusion, 368 HSCs, see Hepatic stellate cells (HSCs) alveolar-capillary level, 368 Human genome anatomic arteriovenous structure and variation communications, 369 alternative splicing, 158 576 Subject Index

Human genome (cont.) fluid restriction environmental exposures, 158 clinical experience, 420 genetic polymorphisms, 158 efficacy, 420 genomic sequence, 158 homeostatic activation, 411 SNP, 158–159 hypervolemic hyponatremia, Human genome project (HGP), 156 pathogenesis of, 419 Human genomics hypo and hyper-volemic, 419 genetic information hyponatremia and hepatorenal benefits/risks of, 163 syndrome, 412 impact in medicine, 164 identification and treatment, 419 legal and social ramifications, 163 neurological complications, 418, 420 public health agencies, 164 predisposing factor, 419 Human haplotype map (HapMap), 162 prognosis marker, 418 ethnic groups, 163 renal function abnormalities, 411 Han Chinese, 163 sodium and fluid loss, 419 Japanese, 163 sodium chloride administration US, 163 disadvantage, 420 Yoruba of Nigeria, 163 short-lived effect, 420 genes and genetic variants, splanchnic circulation, 411 identification of, 162–163 Hypoxia-inducible factor-2α (HIF2 α) GWAS, 163 immunohistochemistry, 5 haplotype blocks with SNPs, 163 Hypoxia-inducible factor-1 (HIF-1), 346 HVPG, see Hepatic venous pressure Hypoxia-inducible factors (HIFs), 22 gradient (HVPG) Hyperbilirubinemia causes of, 62 hepatocyte/bile duct injury, 61 I multidrug resistance associated protein ICAM-1, see Intercellular adhesion (MRP2) activity, 61 molecule-1 (ICAM-1) serum bilirubin level, 63 Immune dysfunction, pathophysiology Hyponatremia management adaptive immune response advantages, 419 memory T lymphocytes, 224 albumin administration, 420 albumin role antidiuretic hormone, 418 endotoxin-inactivating rate, 226 AVP antagonists-vaptans endotoxin-induced IL-1 secretion of clinical and analytical macrophages, 226 surveillance, 422 LAL assay, 226 drug administration gut–liver axis interruption, 422 bacterial DNA, 224 efficacy and safety, 424 bacterial translocation rates, 224 investigation protocols, 422 endotoxemia, 224 marked and dose-dependent, 421 Kupffer cells, 224 osmotic demyelination immunopathology models in ACLF syndrome, 422 compensatory anti-inflammatory pharmacodynamic actions, 422 response syndrome (CARS), 226 renal failure, 422 hypothetical model, 227 side effect, 422 immunopathology, 227 treatment and normalization, 421 pro-and anti-inflammatory body fluid regulation disturbances, 411 cytokines, 227 cirrhosis hypervolemic hyponatremia SIRS, 226 management recommendations, innate immunity 423 aspects of, 223 disproportionate water retention, 418 consequences, 222 endotoxin, 223 Subject Index 577

macrophage opsonophagocytic oxidative stress injury, 140 dysfunction, 223 Isoproterenol-stimulated systolic malnutrition correlates, 223 velocity, 271 multiple cytokine expression abnormalities, 223 K neutrophils component, 222 Kupffer cells (KCs), 4, 325 NK cells, 223 activation, 10 toll-like receptors (TLRs) in acute and chronic liver injury, 10 antimicrobial products, 225 acetaminophen-mediated, 13 cytokines, 225 alcoholic and nonalcoholic liver defined, 225 disease, 11–12 generation of reactive oxygen chitotriosidase expression in, 12 species, 225 endotoxin-mediated injury, 11 peripheral blood mononuclear cells ethanol-induced, 13 (PBMCs), 225 hepatic fibrosis, 11 Immune-related proteins, 35 host defense, 11–12 Immunofunctions of liver, 44 infection, role in, 12 Indocyanine green (ICG) method, 79 nitric oxide (NO) production, 11 Inflammatory mediators role, liver failure ROS release, 11 arachidonic acid-derived lipid mediators ischemia–reperfusion and liver COX pathway, 135–139 transplantation, 13 cyclopentenone PGs, 139 liver cancer, 14 isoprostanes, 139–141 mediators secretion, 10 5-LO pathway, 141–144 oxidative damage and microcirculation, cytokines 9–10 IL-6 cytokine family, 134 physiological and pathophysiological low-molecular-weight proteins, 133 characteristics, 10 TNF-α, 132–134 portal hypertension, 14 inflammatory response, 131 as resident liver macrophages, 9 reactive oxygen species free radicals, 134 L mitochondria and detoxification Lactate dehydrogenase (LDH), 56 reactions, 134 ALT–LDH index, 56 INR, see International normalized ratio half-life of, 56 (INR) serum levels, 56 Intercellular adhesion molecule-1 Leishmania, 12 (ICAM-1), 346 Leucocyte migration test (LMT), 64 Interleukin (IL)-8, 19 Lipid metabolism, 37 International normalized ratio (INR), Lipoprotein receptor-related protein 56, 527 (LRP), 293 Intrahepatic cholestasis “Lipostats,” 38 autoimmune diseases with, 60 Liposynthesis, 37 causes of, 57–58 Listeriosis model, 12 evaluating patients with, 60 Liver failure effects on brain function extrahepatic causes, 61 astrocytes Injury Iron homeostasis, 39 Alzheimer type II, 171 Ischemia–reperfusion and liver glutamine, 172 transplantation, 13 in HE, 172 Ischemic hepatitis, 52 neuronal function, 172 Isoprostanes neurosteroids synthesis, 172–173 cellular membranes, 139 brain atrophy nonenzymatic lipid peroxidation neuroimaging techniques, 175 products, 140 prevalence, 175 brain edema 578 Subject Index

Liver failure effects (cont.) toxins production, 180 factors, 173 antibiotics, 180 intracranial hypertension, 174 effects, 180 magnetic resonance, 173 lactulose, 180 neuronal function, 174 neomycin and rifaxim antibiotics, pathogenetic mechanisms, 174 181 energy impairment prebiotics, 180 cerebral blood flow, 173 probiotics, 180 excessive glutamatergic therapies, 181 activation, 173 worsen neurological function, 175 fulminant hepatic failure, 173 Liver-related pathologies in HE, 173 AKI interaction between ammonia, 174 ATN, 550 metabolic encephalopathies, 173 haematuria and proteinuria, 550 neuronal function, 173 isotonic sodium bicarbonate, 550 tricarboxylic acid cycle, 173 meticulous assessment, 550 neurotransmission disturbances RIFLE classification, 550 excitatory glutamatergic, 171 serological testing, 550 inhibitory GABAergic, 171 serum creatinine (SCr) neuroimaging techniques, 171 concentration, 549 neurological manifestations, 171 spontaneous bacterial peritonitis Liver failure induced HE, mechanisms (SBP), 550 ammonia toxicity uropathy, 550 arterial ammonia level, 175 hepatic encephalopathy (HE) effects, 175 detoxification, 554 protein tyrosin nitration, 176 Glasgow coma scale (GCS), 554 RNA oxidation, 176–177 gut distension, 555 trafficking and metabolism, 176 hyperammonaemia, 556 circulatory dysfunction hypokalaemia, 555 cirrhosis and organic hypo-osmolar states, 555 nephropathies, 178 inflammatory cytokines, 554 renal and cerebral circulation, 177 portosystemic shunts, 554 vascular autoregulation, 177 West Haven criteria, 554 inflammation hepatorenal syndrome in HE, 177 cellular metabolism, 551 inflammatory mediators, 177 dysfunction, congestion and neuroinflammation, 177 microvascular, 551 proinflammatory cytokines, 177 ischaemia centric concepts, 550 renal function, 177 placebocontrolled clinical trial, 551 new therapies terlipressin, 551 ammonia generation, 181 TIPSS, 551 aquaretic drugs, 182 vasopressors and albumin, uses, 551 L-ornithine–L-aspartate, 181 RRT Molecular Adsorbents Recirculating antibiotic doses, 551 System (MARS), 182 anticoagulation of, 552 nutritional measures chronic condition severity, 551 branched-chain amino acids, 180 citrate anticoagulation, 552 calorie-to-nitrogen ratio, 178–180 clotting pathways, 552 isonitrogenous levels, 178 intermittent regimens outcomes, 551 protracted nitrogen restriction, 178 pharmaco-kinetics and vegetable-based diets, benefits, 180 dynamics, 551 portosystemic shunting, 175 pro-and anticoagulant, 552 precipitating factors, 178 prostacyclin, 552 Subject Index 579

variceal haemorrhage (VH) comorbidities, 525 antibiotic therapies, 553 eligibility, 523 CP and MELD, mortality prediction extrahepatic malignancy, 525 scores, 552 highly active antiretroviral therapy endoscopic sclerotherapy, 553 (HAART), 525 EVL, 553 human immunodeficiency virus failure prediction, 554 (HIV), 525 gastroesophageal junction, 553 laparotomy, 525 prophylaxis, 552 medically and ethically Sengstaken Blakemore tube justifiable, 526 (SBT), 553 MELD/CTP scores, 523 somatostatin analogues, 553 neoadjuvant chemoradiation, 525 ultrasonography, 553 thrombectomy, 525 Liver sinusoidal endothelial cells (LSECs), evaluation 4, 19, 345 anatomy and hepatocellular fenestrations, 20 carcinoma, 523 in immune response, 23 cardiopulmonary and leucocytes and cancer cells, 22–23 psychosocial, 523 and oxygen tension, 22 protocol, 523 as scavenger endothelium, 20 indications as source of biologically active CTP systems, 522 mediators, 21–22 diseases and conditions, 522 Liver support system, clinical efficacy MELD score, 522 evaluation prothrombin time ratio, 522 different trials, endpoints and sclerosing cholangitis, 522 protocols, 515 LMT, see Leucocyte migration test (LMT) single-center experience, 515 LRP, see Lipoprotein receptor-related future ideal requirements of protein (LRP) adequate time frame, 515 LSECs, see Liver sinusoidal endothelial bioartificial devices, 515 cells (LSECs) liver failure alterations, 515 L-Type calcium channel currents, 279 preferably survival, 516 nonbiological artificial devices in ACLF, 512 M coagulation disturbances, 514 Magnetic resonance cohort study, 513 cholangiopancreatography cost-effectiveness MARS (MRCP), 60 treatment, 513 Mammalian target of rapamycin CPT scores, 511 (mTOR), 37 encephalopathy, 513 MARS, see Molecular adsorbent glomerular filtration rate, 513 recirculation system (MARS) hemodynamic profile, 514 Matrix metalloproteinases (MMPs), 18 hepatic coma, 513 HSC proliferation, 97 hepatorenal syndrome, 511 pathways, 96 mortality rate, 513 type I collagen, 96 risk, 514 MCP-1, see Monocyte chemoattractant therapeutic measure, 514 protein-1 (MCP-1); Monocyte vasoconstrictors and plasma chemotactic protein-1 (MCP-1) expansion, 513 Mendelian diseases, 156, 158 Liver transplant, referral and evaluation single gene, 157 contraindications Mesenchymal stem cells (MSCs), 113 cholangiocarcinoma and portal vein MHC, see Myosin heavy chains (MHC) thrombosis, 525 Microcirculation hepatic perfusion in sepsis and shock 580 Subject Index

Microcirculation (cont. ) N treatment, effect, 337–338 Nadolol use, 283 HMS, 324–325 NADRPS, see Naranjo Adverse Drug HSCs, 325 Reactions Probability Scale immunoelectron microscopy and (NADRPS) biochemical studies, 325 NAFLD, see Nonalcoholic fatty liver Kupffer cells, 325 disease (NAFLD) nitric oxide synthase (eNOS), 325 Naranjo Adverse Drug Reactions noxious stimuli, 325 Probability Scale (NADRPS), 63 pathophysiology National Human Genome Research arteriovenous shunts in sepsis, role, Institute 336–337 web site, 162 in cirrhosis, 327–328 National Organ Transplant Act CO activates guanylate (NOTA), 526 cyclase, 329 Natural killer (NK) cells, 4 cyclooxygenase pathway (TXA2), Neoglucogenesis, 36 role, 333 Neurohumoral regulation endothelin-1 and nitric oxide, role, calcitonin gene-related peptide 334–336 (CGRP), 251 failure in steatosis, 329 circulating vasodilators, heme oxygenase (HO), 329 overproduction, 250 hepatic failure, 327 collecting ducts ischemia and, 328–329 AQP2, 255 MAPK signaling pathway, role, AV P, 255 332–333 cAMP, 255 mean arterial pressure (MAP), 326 V2 receptors, 255 mitogen-activated protein kinase water transport, 255 (MAPK), 328 effective arterial filling, 251 multiple organ failure, 326 endogenous vasoconstrictors, 250 normal coagulation cascade, glomerular filtration rate (GFR), 252 role, 336 bilateral Goldblatt kidneys, 254 red blood cell (RBC), role, 336 filtration fraction, 254 in sepsis, 326, 329–331 Henle’s loop and distal tubules TGF-β signaling, role, aldosterone sensitivity, 255 331–332 11-beta-hydroxy-steroid TNF-α, 328 dehydrogenase, 255 regulation of, 334 endogenous glucocorticoids, 255 TLR signaling pathway, renal calcium/ polyvalent role, 332 cation, 255 MMPs, see Matrix metalloproteinase sodium reabsorption, 255 (MMPs) kidney function Model of end-stage liver disease (MELD) renal dysfunction, 252 scoring system, 72, 543 nitric oxide, 251 Molecular adsorbent recirculation system parasympathetic and sympathetic (MARS), 503 dysfunction, 251 Monocyte chemoattractant protein-1 proximal tubules (MCP-1), 19 animal models of cirrhosis, 254 Monocyte chemotactic protein-1 reabsorption fraction, 254–255 (MCP-1), 346 renal blood flow (RBF), 252 mTOR, see Mammalian target of angiotensin II, 254 rapamycin (mTOR) beta-adrenoceptors, 253 Muscle disease, 56 calcium/polyvalent cation Myosin heavy chains (MHC), 282 receptors, 253 Subject Index 581

early pre-ascitic cirrhosis, 253 development rate, 479 endothelin 1, 253 experimental models, 479 renal perfusion pressure, 253 multicenter study, 479 renin angiotensin aldosterone system timolol/placebo, 479 (RAAS), 250–251 PBC, see Primary biliary cirrhosis (PBC) sinusoidal and portal hypertension, 250 PCC, see Prothrombin complex sympathetic nervous system, 250 concentrates (PCC) Neurological manifestations, 171 PDGF, see Platelet-derived growth factor Neurotransmission disturbances in HE (PDGF) excitatory glutamatergic, 171 PEEP, see Positive end expiratory pressure inhibitory GABAergic, 171 (PEEP) neuroimaging techniques, 171 PEPCK, see Phosphoenolpyruvat Neutrophils carboxykinase (PEPCK) phagocyte bacterial antigens, 43 Peroxisome proliferator-activated receptors studies, 222 (PPARs), 38 Nonalcoholic fatty liver disease (NAFLD), PFIC, see Progressive familial intrahepatic 52, 343 cholestasis (PFIC) Nonalcoholic steatohepatitis, 7 PFT, see Pulmonary function test (PFT) Nonparenchymal liver cells, 4 PHG, see Portal hypertensive gastropathy Nonsteroidal anti-inflammatory drugs (PHG) (NSAIDs), 137 Phosphoenolpyruvat carboxykinase NOTA, see National Organ Transplant Act (PEPCK), 38 (NOTA) PIGF, see Placental growth factor (PlGF) Notch pathway, 22 Pit cells, 4 Noxious compounds, 40 Placental growth factor (PlGF), 346 NTCP, see Na+-Taurocholate Plasma volume, distribution and regulation cotransporting polypeptide splanchnic and peripheral vasodilatation (NTCP) animal studies, 248 arterial vasodilation theory, 248 O plasma atrial natriuretic peptide, 248 OPTN, see Organ Procurement and plasma renin activity (PRA), 248 Transplantation Network transition of fluid, 247 (OPTN) vascular and arterial Organ Procurement and Transplantation compliance, 248 Network (OPTN), 526 vasopressor systems, 248–249 Organ system failure (OSF), 534 Platelet-derived growth factor Orthotopic liver transplantation (OLT) (PDGF), 18, 345 fulminant hepatic failure, 110 PMN, see Polymorphonuclear (PMN) PNH, see Paroxysmal nocturnal P haemoglobinuria (PNH) Parenchymal liver cells, see Hepatocytes Polymorphonuclear (PMN), 398 Paroxysmal nocturnal haemoglobinuria Polytetrafluoroethylene (PTFE), 485 (PNH), 467 POPH, see Portopulmonary hypertension Partial pressure of carbon dioxide (POPH) (PaCO2), 365 Portal hypertensive gastropathy (PHG), 491 Partial pressure of oxygen (PaO2), 365 Portal vein ligation (PVL), 307 Partial thromboplastin time (PTT), 64 Portopulmonary hypertension (POPH), 372 Patients selection for prophylaxis autoimmune liver disease, 372 high-risk varices patients classification of, 373 clinical decompensation, 480 diagnosis of, 372 follow-up endoscopy, 480 diastolic or systolic heart failure, 372 progression rate, 479 endothelin receptor, 373 patients without varices heart catheterization, 372 beta-adrenergic blocker, 479 582 Subject Index

Portopulmonary hypertension (cont.) survival benefit models hemodynamic problem, 372 complex statistics, 533 medical treatments, 374 donor factors, 532 MPAP and PVR, 373 donor organs scarcity, 532 pathophysiology of, 372 iterations and validations, 533 prognostic factors, 374 posttransplant mortality, 532 transthoracic echocardiography, 372 simulated model, 533 vasoconstriction result, 372 Primary biliary cirrhosis (PBC), 60, 528 vasomodulating therapies, 373 Primary sclerosing cholangitis (PSC), 60 Positive end expiratory pressure Progressive familial intrahepatic cholestasis (PEEP), 546 (PFIC), 61 Postprandial hyperemia, 80–81 Propranolol use, 283 functional adaptation, 81 Protein metabolism, 35 Potential therapeutic strategies pathways antibiotic therapy, 228 autophagic–lysosomal, 36 end-stage liver diseases, 230 ubiquitin–proteasome-related, 36 granulocyte colony-stimulating Prothrombin complex concentrates factor, 230 (PCC), 462 gut permeability, 228 Prothrombin time (PT), 294 liver cirrhosis, 228 PSC, see Primary sclerosing cholangitis Molecular Adsorbent Recirculating (PSC) System (MARS), 229–230 PT, see Prothrombin time (PT) pro-and anti-inflammatory PTFE, see Polytetrafluoroethylene (PTFE) cytokines, 229 Pulmonary alterations, chronic liver failure scavenge endotoxin, 230 arterial oxygenation and pulmonary TLRs, 230 function testing PPARs, see Peroxisome ABG, 365 proliferator-activated receptors DLCO, 365 (PPARs) hypoxemia, 365 Prediction models of chronic liver failure, obstructive diseases, 365 transplantation issues PaCO2, 365 Child–Turcotte–Pugh (CTP) PaO2, 365 ceiling effect, 529 PFT, 365 complications, 528 radiographs, 365 flaws, 528 screening method, 365 floor effect, 529 splanchnic vasodilatation, 365 heterogeneity, 529 ascites effects scoring system, 528 arterial oxygenation, 363 Mayo primary biliary cirrhosis (PBC) dyspnea, 363 natural history model FVC, 362 identified variables, 528 lung atelectasis degree, 363 listing criteria, 528 massive/tense, 363 optimal time, 528 and obstructive sleep apnea, 363 predictors, clinically and TLC, 362 statistically, 528 dyspnea, 362 MELD model and variants encephalopathy effects advantages, 530 fatigue, 363 anticoagulation effect, 530 hyperventilation, 363 clinicians calculator for, 529 metabolic consequence, 363 delta-MELD, 531 pneumonitis, 363 d-MELD, 531 progesterone receptors, 363 mathematical formula, 529 hepatic dysfunction, 361 MELDNA, 530 hepatic hydrothorax (HH) Subject Index 583

bacterial peritonitis, 368 efficacy, 484 diaphragmatic fenestrations, 366 pharmacological treatment, 484 mortality, 367 drug therapy negative inspiratory pleural efficacy, 483 pressure, 366 meta-analyses, 483 pleural effusion, 366 mortality, 483 positive cytology, 366 optimal pharmacological therapy, repeat thoracenteses, 367 483 SBE, 367 endoscopic therapy thoracentesis, diagnostic and endoscopic band ligation, 484 therapeutic, 366 sclerotherapy injection, 484 thoracoscopic pleurodesis, 367 high risk, 483 TIPS, 367 mortality rate, 483 unilateral fluid, 366 rebleeding prevention tips hepatopulmonary syndrome, diagnostic DSRS, 484 and prognostic criteria, 366 encephalopathy, 484 moderate right-sided hepatic PTFE, 485 hydrothorax, 367 reintervention rate, 485 overnight oximetry (OvOx) risk indicators, 483 patterns, 364 treatments, 483 pathophysiology, 362 Regulatory T (Treg) cells expression, 43 portopulmonary hypertension, 362 Relative or functional adrenal insufficiency pulmonary circulation, effects of, 362 (RAI), 378 pulmonary vasculopathies–dilemma, Renal blood flow (RBF), 252 368–374 angiotensin II, 254 sleep-disordered breathing effects beta-adrenoceptors, 253 atrial fibrillation, 365 calcium/polyvalent cation BMI, 364 receptors, 253 disease sleep abnormalities, 364 early pre-ascitic cirrhosis, 253 endothelial dysfunction, 363 endothelin 1, 253 nocturnal hypoxemia, 363 renal perfusion pressure, 253 oxygen saturation, 363 Renal failure management vasculopathies, 362 ascites reabsorption, 429 Pulmonary function test (PFT), 365 ATN, 430 Pulmonary vasculopathies-dilemma cirrhosis HRS, pathogenesis diagnostic and prognostic criteria, 373 adrenal insufficiency, 440 hypothesized hypertension, 368 angiotensin-II powerful liver disease Kaplan–Meier survival effects, 437 curves, 371 antidiuretic hormone, 433 pathophysiological perspective, 368 arterial accentuation, 435 pulmonary vascular dilatation, 370 arterial hipovolemia, 433 vasoactive factors, 368 arterial vasodilation, 436 VEGF, 368 blood flows, 437 See also Portopulmonary hypertension characterized cardiomyopathy, 440 (POPH) complex mechanism, 436 contributory mechanism, 440 cortisol synthesis, 440 R electrophysiological, 440 RAI, see Relative or functional adrenal extrasplanchnic vascular insufficiency (RAI) territories, 434 RARs, see Retinoic acid receptors (RARs) glomerular filtration rate, 435 Recurrent bleeding prevention heart rate, 439 controlled trials, 483 hepatic encephalopathy, 437 drug and endoscopic therapy 584 Subject Index

Renal failure management (cont.) HRS multiorgan failure, 439 hepatic venous pressure gradient middle cerebral artery resistive (HVPG), 438 index, 438 hepatorenal syndrome, 439 nephrotoxic antibiotics, 430 hydrocortisone, 440 nonsteroidal anti-inﬂammatory hyperdynamic circulation, 433 drugs, 430 hyponatremia, 434 peripheral arterial vasodilation infection resolution, 438 hypothesis, 434 inotropic and chronotropic peripheral vasodilation hypothesis, 436 functions, 439 type 1 HRS treatment, 441–443 intrahepatic hemodynamics, 438 type 2 HRS treatment, 443–445 juxtaglomerular apparatus, 437 Renal replacement therapy (RRT), 551 nonsplanchnic vasoconstriction, 437 Retinoic acid receptors (RARs), 16 peripheral arterial vasodilation Roussel Uclaf Causality Assessment hypothesis, 433 Method (RUCAM), 63 progression setting, 435 RRT, see Renal replacement therapy (RRT) renal vasoconstriction, 435 RUCAM, see Roussel Uclaf Causality resistive index, 437 Assessment Method (RUCAM) sympathetic nervous systems, 433 Ryanodine-release receptor (RyR), 280 systemic and hepatic hemodynamics, 435 systolic and diastolic responses, 440 S Sarcoidosis, 60–61 TIPS, 440 2+ treatments, 440 Sarcoplasmic reticulum Ca -ATPase urinary excretion, 436 (SERCA2), 280 vascular effect, 434 SBE, see Spontaneous bacterial empyema vasoactive systems, 437, 439 (SBE) vasoconstrictor systems, 437 SBP, see Spontaneous bacterial peritonitis diagnosis of (SBP) aminoglycosides, 431 SBT, see Sengstaken Blakemore tube circulatory dysfunction, 432 (SBT) echostructure, 432 Secondary bile acids, 34 extrarenal ﬂuid losses, 431 Sengstaken Blakemore tube (SBT), 553 SERCA2, see Sarcoplasmic reticulum HRS types, 432 2+ intravenous albumin, 431 Ca -ATPase (SERCA2) mechanism, 432 Serum aminotransferase elevations, 52–55 nonazotemic cirrhosis, 433 Serum glutamic oxaloacetic transaminase oliguria, 430 (SGOT), 48 osmolality ratio, 430 Serum glutamic pyruvic transaminase polymorphonuclear leukocytes, 432 (SGPT), 48 reduced GFR, 430 SGOT, see Serum glutamic oxaloacetic refractory ascites, 433 transaminase (SGOT) SBP, 432 SGPT, see Serum glutamic pyruvic septic shock, 431 transaminase (SGPT) signs and symptoms, 432 Single-gene diseases, 156 ultrasonography, 432 Sinusoidal endothelial cells (SECs), 85 urinary tract infection, 432 concentration–effect curve, 87 diuretics urine volume, 429 endothelial NOS (eNOS) isoform, 87 GFR, 430 factors regulating, 86 glomerulonephritis, 430 NO, role in, 86–87 hemorrhagic shock, 430 vasoconstrictors and vasodilators, 86 α hepatorenal syndrome diagnostic Smooth muscle isoform of -actin α criteria, 431 ( -SMA), 15 immunohistochemistry for, 16 Subject Index 585

Space of Disse representation, 4 local, 81 Speed limiting gluconeogenesis enzyme, 38 Spontaneous bacterial empyema Splanchnic and systemic hemodynamic (SBE), 367 abnormalities Spontaneous bacterial peritonitis circulation in liver cirrhosis, (SBP), 397, 550 development, 306 ascitic fluid analysis, 397–398 intestinal and splanchnic circulations blood cultures, 398 mild portal hypertension, 307 prophylaxis, 400 portal pressure, 308 in hospitalized patients with portal vein ligation (PVL), 307 gastrointestinal hemorrhage, systemic circulatory abnormalities, 401, 403 308–309 in patients with/without prior vasodilatation, 309 episodes of, 403–404 VEGF, 307–308 treatment nitric oxide (NO), discovery, 306 albumin, role of, 400 vasodilatation antibiotic therapies, 399–400 adrenomedullin, 313–314 principles, 398 carbon monoxide (CO), role, SREBP, see Sterol regulatory element 311–312 binding protein (SREBP) EDHF, 312–313 Star-shaped phagocytes, 14 endocannabinoids, role, 312 Stauffer syndrome, 60 endothelial cells, 309 Steatosis and steatohepatitis, 56 hydrogen sulfide (H2S), 313 Stem cells and chronic liver failure nitric oxide (NO), role, 309–311 adult hepatic progenitor/stem cells, 110 prostacyclin (PGI2), role, 312 cell-based therapy TNFα, 313 adult hepatic stem cells, 121 VEGF, 313 ES cells, 120–121 Splanchnic angioarchitecture, long-term exogenous recombinant factor structural changes in VII, 120 angiogenic process, 352 hepatocytes, 119 hyperdynamic splanchnic human leucocyte antigen circulation, 353 (HLA), 121 neutralizing antibody, 353 mesenchymal stem cells peritoneal circulation, 352 (MSCs), 121 persistent abnormalities, 352 multipotent tissue-specific, 111 PlGF deficiency, 353 phototherapy, 119–120 portosystemic collateral vessels, 353 signaling pathways, 111–112 portosystemic shunting, 353 donor organs, 110 powerful inducer ascites, 353 embryonic stem (ES) cells, 110 preexisting venous channels, 353 hepatic stem cells signaling pathway activation, 353 activation, 113–116 splanchnic area, 352 in adult liver, 112–113 in vivo mesenteric assays, 352 and cancer, 116–119 Splanchnic arterial vasodilatation, 307 identification and isolation, 116 Splanchnic circulation Steroid transporters (Ostα,Ostβ), 35 anatomy, 78 Sterol regulatory element binding protein blood flow, 80 (SREBP), 38 branching pattern, 79 Synthetic function test Fick principle, 80 albumin ICG method for, 79 globulin levels, 66 regulation half-life, 66 autoregulation, 82 prothrombin time extrinsic, 82–83 INR, 64 586 Subject Index

Synthetic function test (cont.) Type 1 HRS treatment international sensitivity index (ISI) extracorporeal albumin dialysis value, 65 (MARS) measurement, 64 dialysis process, 443 sepsis, 65 survival rate, 443 vitamin K deficiency, 65 veno-venous hemofiltration monitor, Systemic inflammatory response syndrome 443 (SIRS) liver transplantation acute functional renal impairment, cyclosporine/tacrolimus, 441 220–221 hemodialysis, 441 defined, 220 morbidity and mortality, 441 development of, 220–221 nephrotoxicity, 441 neurohormonal abnormalities, 441 transjugular intrahepatic portosystemic T shunt (TIPS) TAFI, see Thrombin-activatable fibrinolysis medical treatment, 443 inhibitor (TAFI) + novo hepatic encephalopathy, 443 Na -Taurocholate cotransporting pilot studies, 442 polypeptide (NTCP), 35 refractory ascites, 443 TEG, see Thromboelastography (TEG) vasoconstrictors and albumin TFPI, see Tissue factor pathway inhibitor dopamine/octreotide, 442 (TFPI) effective therapy, 441 T-helper (Th) cells expression, 43 efficacy of, 442 Thrombin-activatable fibrinolysis inhibitor maximal dose, 442 (TAFI), 298 retreatment, 442 Thrombocytopenia, 56 vasoconstrictor agent, 442 Thromboelastography (TEG), 552 Type 2 HRS treatment Thromboxane A2, 14 HRS, prevention of Thyroid disorders, 56 clinical settings, 444 TIPS placement, see Transjugular CP score, 445 portosystemic shunt (TIPS) hospital mortality rate, 444 placement inhibitor pentoxifylline, 445 Tissue factor pathway inhibitor tumor necrosis factor, 445 (TFPI), 292 transjugular intrahepatic portosystemic Tissue inhibitors of metalloproteinases shunt (TIPS) (TIMPs), 95 ascites control, 444 Tissue plasminogen activator (tPA), 298 complications, type TLC, see Total lung capacity (TLC) and rate, 444 Total lung capacity (TLC), 362 refractory/recidivant ascites, 443 Toxin-or drug-induced liver injury, 52 vasoconstrictors and albumin tPA, see Tissue plasminogen activator effect of, 444 (tPA) studies, 444 Transferrin receptor 2 (Tfr-2) proteins, 39 Transjugular intrahepatic portosystemic stent-shunt (TIPS), 276, 442 U Transjugular portosystemic shunt (TIPS) United Network for Organ Sharing placement, 72 (UNOS), 526 Tumor necrosis factor-α (TNF-α), 313 UNOS, see United Network for Organ alcoholic hepatitis, 134 Sharing (UNOS) cellular and biological processes, uPA, see Urokinase plasminogen activator 132–133 (uPA) serum levels, 133 Urea/ornithine cycle, 42 signaling, 133–134 Urokinase plasminogen activator toll-like receptors, 133 (uPA), 298 Subject Index 587

V meta-analysis random effects van den Bergh method, 51 model, 481 Variceal bleeding, treatment and prevention patients selection for prophylaxis, acute bleeding episode 479–480 blood spurting/oozing, 485 portal hypertensive gastropathy (PHG) clot adherent, 485 bleeding emergency endoscopy, 485 chronic bleeding, 491 gastrointestinal bleeding, 485 gastric perfusion condition, 491 ruptured esophageal, 485 Helicobacter pylori infection, 491 acute variceal bleeding treatment, mild and severe, 491 485–490 mosaic characteristic, 491 cirrhosis complications, 478 mucosa and submucosa, 491 cirrhosis varices, natural history prevention, 478 annual incidence, 478 prophylactic efficacy, 478 Child–Pugh class, 478 recurrent bleeding prevention, 483–485 collaterals development, 478 screening for drug therapy, 478 elastography measurements, 479 esophageal decrease, 478 endoscopically, 479 NIEC index, 478 noninvasive tests, 479 predictive factors, 478 prophylactic treatment, 479 risk, 479 Variceal haemorrhage (VH), 553 esophageal or gastric varices, 478 Vascular cell adhesion molecule-1 first bleeding prevention treatment (VCAM-1), 346 beneficial effects, 480 Vascular endothelial growth factor (VEGF), blood transfusion, 482 20, 307–308, 313, 368 breath shortness and hypotension, Vasodilatation 482 adrenomedullin clinical efficacy, 480 cirrhotic liver patients, 313–314 EBL advantage, 480–481 carbon monoxide (CO), role effectiveness and complications, 480 heme oxygenase (HO), isoforms, eradication rate, 480 311 guidelines, 482 zinc protoporphyrin (ZnPP), acute improved bleeding-related intraperitoneal injection, survival, 483 311–312 intrinsic alpha-adrenergic blocker decreased response effect, 482 and vasoconstriction, balance placebo-controlled study, 480 between, 314 prophylaxis, 480 EDHF side effects, 482 arachidonic acid metabolites, 312 therapy, pharmacology and gap junctions, 312 endoscopic, 482 hydrogen peroxide, 312 underpowered trials, 481 monovalent cation K+, 312 gastric varices bleeding endocannabinoids, role cyanoacrylate, 490 CB1 receptors, blockade, 312 glue injection, 490 endothelial cells, 309 intravascular obliteration, 491 hydrogen sulfide (H2S) ligation group, 490 intravenous bolus injection, 313 Linton-Nachlas tube, 490 molecules and factors, 310 mortality rate, 490 nitric oxide (NO), role obturation, 490 cofactors, 311 portal hypertension, 490 eNOS regulation, 311 TIPS and surgery, 491 NO synthases (NOSs), 309–311 liver function deterioration, 478 protein–protein interactions, 311 588 Subject Index

Vasodilatation (cont.) VEGF, see Vascular endothelial growth prostacyclin (PGI2), role factor (VEGF) adenylyl cyclase (AC), 312 Venous thromboembolism (VTE), 296 cyclic adenosine monophosphate Voltage-dependent calcium channels (cAMP), 312 (VDCC), 279 cyclooxygenase (COX), 312 Von Willebrand factor (VWF), 290, 454 TNFα, 313 VTE, see Venous thromboembolism (VTE) VEGF, 313 VCAM-1, see Vascular cell adhesion molecule-1 (VCAM-1) VDCC, see Voltage-dependent calcium W channels (VDCC) Wilson’s disease, 39, 52