Evaluation of Persufflation and Cold Storage Preservation in Isolated Porcine Kidneys Using Novel Methods for Organ Quality Assessments

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Authors Min, Catherine Gyongeun

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EVALUATION OF PERSUFFLATION AND COLD STORAGE PRESERVATION IN ISOLATED PORCINE KIDNEYS USING NOVEL METHODS FOR ORGAN QUALITY ASSESSMENTS

Catherine G. Min

A Dissertation Submitted to the Faculty of the

GRADUATE INTERDISCIPLINARY STUDIES IN PHYSIOLOGICAL SCIENCES

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College THE UNIVERSITY OF ARIZONA 2018

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certifythat we have read the dissertation prepared by Catherine G. Min, titled "Evaluation of Persufflation and Cold Storage Preservation in Isolated Porcine Kidneys using Novel Methods for Organ Quality Assessments" and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy.

Defense Date: May 4, 2018

Jean-Philippe Galons

Thomas L. Pannabecker

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copies of the dissertation to the Graduate College.

I hereby certifythat I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

Dissertation Director: Klearchos K. Papas

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder.

SIGNED: Catherine G. Min

ACKNOWLEDGEMENTS

First, I would like to acknowledge the Papas lab that created the opportunity for such an exciting, interdisciplinary project. This project has been the greatest source of exposure to a variety of fields, professionals and collaborators, and pushed me to be independent, resourceful and persistent.

I would like to wholeheartedly thank my dissertation committee members. Thank you, Dr. Fregosi, for those many hours of conversations in your office about being a student, graduating, future jobs and everything in between. Thank you, Dr. Pannabecker, for the early thesis edits and educational blurbs on kidney physiology. Thank you, Dr. Harland, for training me on organ prep, driving to phoenix just for a research pancreas, bomb jokes about the PSF unit and stopping by my desk to discuss a variety of topics on organ transplantation. Your enthusiasm and willingness to teach was a huge motivation for me to keep going. Thank you Dr. Galons (JP!) for always making time to image pig kidneys, for always helping with the setup and cleanup of the circuit that would randomly explode, and for patiently helping me navigate through a project that had many moving pieces with invaluable mentorship. I thoroughly enjoyed having you all on my committee, and I am eternally grateful for your constant support.

I am thankful for the members of the Roy-Chaudhury lab, especially Diego and Aous for spending those precious hours trying to make rat kidney transplantation work. While that project didn’t work out, I had a lot of fun working with you all on the porcine allotransplants.

A special thanks to Dr. Taylor, Dr. Weegman (Brad) and Beth who have assisted with many technical and engineering parts of the project as well as manuscript editing.

Thank you Dr. Tempelman, Simon and Melissa for patiently guiding someone with no background in engineering through trouble shooting the persufflator.

To my wonderful family and friends from all stages of my life, I cannot thank you enough for your love and support. You were the best part of this experience.

DEDICATION This dissertation is dedicated to my parents who instilled the value of education, persistence, and the immigrant work ethic. I would not have been able to come this far without their worldly advice, support and love.

Table of Contents List of Figures ...... 7 List of Tables ...... 10 Abstract ...... 12 Chapter 1: Introduction...... 14 Chapter 2: Literature Review ...... 24 Section1: HISTORY OF ORGAN TRANSPLANTATION ...... 25 Section 2: DONOR TYPES ...... 27 Section 3: PATHOPHYSIOLOGY OF ORGAN DAMAGE...... 32 Section 4: ORGAN ASSESSMENT ...... 50 Section 5: ORGAN PRESERVATION ...... 60 Chapter 3: Methodology ...... 72 Chapter 4: Novel Methods for Real-Time Kidney Quality Assessment Prior to Transplantation ...... 96 Chapter 5: Quality Assessments of Persufflated and Cold Storage Preservation in Subnormothermic Isolated Porcine Kidney ...... 162 Chapter 6: Pilot Study Comparing the Effects of Persufflation and Hypothermic Machine Perfusion Using the LifePort ...... 220 Chapter 7: Pre-clinical Porcine Kidney Allotransplant ...... 239 Chapter 8: Summary and Interpretations of Results ...... 264 Complete Dissertation References ...... 276 Appendix A: Recent Developments of Persufflation for Organ Preservation ...... 310 Appendix B: Summary of Attempts at Rat Kidney Allotransplant ...... 337 Appendix C: Acute Ischemia Induced by High Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans ...... 342 Appendix D: In vitro Characterization of Neonatal, Juvenile, and Adult Porcine Islet Oxygen Demand, β-cell function, and Transcriptomes ...... 366

List of Figures FIGURE 1: DIAGRAM OF THE MAIN THEMES OF THIS THESIS...... 18

FIGURE 2: THESIS ORGANIZATION FLOW CHART...... 19

FIGURE 3: THESIS ORGANIZATION CHART WITH DESCRIPTIONS OF EACH CHAPTER AND APPENDIX...... 20

FIGURE 4: SUMMARY OF THE TYPES OF DECEASED DONORS AND THEIR CRITERIA ...... 29

FIGURE 5: SCHEMATIC FOR ORGAN DONATION, PRESERVATION AND TRANSPLANT...... 33

FIGURE 6: A SUMMARY OF THE MECHANISMS FOR CELLULAR CHANGES THAT ARE INDUCED BY ISCHEMIA IN

THE KIDNEY...... 37

FIGURE 7: A SUMMARY OF THE MECHANISMS FOR CELLULAR CHANGES THAT ARE INDUCED BY

REPERFUSION AFTER AN ISCHEMIC INSULT IN THE KIDNEY ...... 41

FIGURE 8: KIDNEY ANATOMY ILLUSTRATION...... 45

FIGURE 9: UNILATERAL HYDRONEPHROSIS IN DOMESTIC PIGS...... 75

FIGURE 10: BIFURCATED RENAL VEINS AND ABNORMAL FLUSHING...... 75

FIGURE 11: IMAGE OF THE GINER PERSUFFLATOR PROTOTYPE...... 76

FIGURE 12: SCHEMATIC OF POST-PRESERVATION KIDNEY REWARMING TIMELINE...... 78

FIGURE 13: SCHEMATIC OF THE WHOLE ORGAN OXYGEN CONSUMPTION RATE PERFUSION CIRCUIT...... 80

FIGURE 14: WHOLE ORGAN OXYGEN CONSUMPTION RATE SETUP...... 80

FIGURE 15: SCHEMATIC OF HYPOTHERMIC PERFUSION SYSTEM FOR DYNAMIC CONTRAST ENHANCED

MAGNETIC RESONANCE IMAGING ...... 82

FIGURE 16: MAGNETIC RESONANCE COMPATIBLE PERFUSION CIRCUIT...... 83

FIGURE 17: KIDNEY IMAGING AND TWO-COMPARTMENT MODEL...... 84

FIGURE 18: A COMPOSITE PANEL OF KIDNEY IMAGES TAKEN AT MULTIPLE TIMEPOINTS DURING DYNAMIC

CONTRAST ENHANCED MAGNETIC RESONANCE IMAGING...... 84

FIGURE 19: WHOLE ORGAN OXYGEN CONSUMPTION RATE IN CONTROL AND PRESERVED KIDNEYS...... 123

FIGURE 20: LACTATE LEVELS OF RENAL PERFUSATE...... 124

FIGURE 21: LACTATE PRODUCTION OF PRESERVED KIDNEYS DURING HYPOTHERMIC LIQUID PERFUSION...... 125

FIGURE 22: LIGHT MICROSCOPY EVALUATION OF HEMATOXYLIN AND EOSIN STAINED RENAL BIOPSIES. 125

FIGURE 23: TIMELINE AND THE WORK FLOW OF RNA SEQUENCING...... 128

FIGURE 24: THE MOST DOWNREGULATED AND UPREGULATED GENES IN STATIC COLD STORAGE KIDNEYS

AT T1 V T0...... 128

FIGURE 25: THE MOST DOWNREGULATED AND UPREGULATED GENES IN PERSUFFLATED KIDNEYS AT T1 V T0...... 129

FIGURE 26: THE MOST DOWNREGULATED AND UPREGULATED GENES IN PERSUFFLATED KIDNEYS

COMPARED TO STATIC COLD STORAGE KIDNEYS AT T1...... 130

FIGURE 27: A NETWORK MODEL DELINEATING THE RELATIONSHIP BETWEEN THE DOWNREGULATED

PROTEIN-PROTEIN INTERACTIONS IN STATIC COLD STORED KIDNEYS AT T1 COMPARED TO T0. ... 149

FIGURE 28: A NETWORK MODEL DELINEATING THE RELATIONSHIP BETWEEN THE UPREGULATED

PROTEIN-PROTEIN INTERACTIONS IN STATIC COLD STORED KIDNEYS AT T1 COMPARED TO T0. ... 151

FIGURE 29: A NETWORK MODEL DELINEATING THE RELATIONSHIP BETWEEN THE DOWNREGULATED

PROTEIN-PROTEIN INTERACTIONS IN PERSUFFLATED KIDNEYS AT T1 COMPARED TO T0...... 153

FIGURE 30: A NETWORK MODEL DELINEATING THE RELATIONSHIP BETWEEN THE UPREGULATED

PROTEIN-PROTEIN INTERACTIONS IN PERSUFFLATED KIDNEYS AT T1 COMPARED TO T0...... 155

FIGURE 31: A NETWORK MODEL DELINEATING THE RELATIONSHIP BETWEEN THE DOWNREGULATED

PROTEIN-PROTEIN INTERACTIONS BETWEEN STATIC COLD STORED KIDNEYS AND PERSUFFLATED

KIDNEYS AT T1...... 157

FIGURE 32: A NETWORK MODEL DELINEATING THE RELATIONSHIP BETWEEN THE UPREGULATED

PROTEIN-PROTEIN INTERACTIONS IN STATIC COLD STORED KIDNEYS AND PERSUFFLATED KIDNEYS

AT T1...... 160

FIGURE 33: HISTOLOGICAL ANALYSIS FROM HEMATOXYLIN AND EOSIN STAINED BIOPSIES ...... 195

FIGURE 34: LACTATE LEVELS OF RENAL PERFUSATE DURING THE REWARMING PERIOD...... 196

FIGURE 35: HISTOGRAM OF THE P-VALUE AND FREQUENCY...... 202

FIGURE 36: BLAND–ALTMAN PLOT (MA PLOT) THAT REPRESENTS DIFFERENCES BETWEEN

MEASUREMENTS TAKEN FROM PAIRED SAMPLES OF SCS AND PSF...... 203

FIGURE 37: THE MOST DOWNREGULATED AND UPREGULATED GENES IN PSF KIDNEYS COMPARED TO SCS

KIDNEYS...... 203

FIGURE 38: A TOPOLOGY MAP OF UPREGULATED GENES...... 204

FIGURE 39: A TOPOLOGY MAP OF DOWNREGULATED GENES...... 204

FIGURE 40: LIST OF UPREGULATED GENES...... 209

FIGURE 41: LIST OF DOWNREGULATED GENES...... 209

FIGURE 42: TIMELINE ILLUSTRATING THE DETAILS OF STUDY DESIGN...... 225

FIGURE 43: WHOLE ORGAN OXYGEN CONSUMPTION RATE OF ISOLATED KIDNEYS...... 230

FIGURE 44: FUNCTIONAL PARAMETERS MEASURED WITH DYNAMIC CONTRAST ENHANCED MAGNETIC

RESONANCE IMAGING...... 231

FIGURE 45: BLAND–ALTMAN PLOT (MA PLOT) THAT REPRESENTS DIFFERENCES BETWEEN

MEASUREMENTS TAKEN FROM PAIRED SAMPLES OF HMP AND PSF...... 231

FIGURE 46: COLLECTION P-VALUES PRODUCED BY ANALYZING EACH PROBE SET...... 232

FIGURE 47: LIGHT MICROSCOPY REPRESENTATION OF H&E-STAINED TISSUE SAMPLES...... 232

FIGURE 48: HISTOLOGICAL ANALYSIS OF RENAL BIOPSIES FROM HMP AND PSF TREATED KIDNEYS. .... 233

FIGURE 49: TIMELINE OF THE DOMINO TRANSPLANT USING A DONATION AFTER BRAIN DEATH MODEL. 249

FIGURE 50: TIMELINE OF THE PAIRED ALLOTRANSPLANT USING A DONATION AFTER CARDIAC DEATH

MODEL...... 250

FIGURE 51: PICTURES OF THE PERSUFFLATED KIDNEY WITH COMPLETE VASCULAR ANASTOMOSES FROM

THE DOMINO ALLOTRANSPLANT...... 251

FIGURE 52: H & E STAINING ON CONTROL AND POSTOPERATIVE DAY 1 KIDNEY BIOPSIES...... 254

FIGURE 53: LEVELS OF SERUM CREATININE AND BLOOD UREA NITROGEN (BUN) COLLECTED FROM THE

PERSUFFLATED KIDNEY RECIPIENT...... 254

FIGURE 54: H & E STAINING ON CONTROL AND POSTOPERATIVE DAY 7 KIDNEY BIOPSIES FROM THE

DOMINO ALLOTRANSPLANT...... 255

FIGURE 55: SERUM CREATININE AND BLOOD UREA NITROGEN OF STATIC COLD STORAGE AND

PERSUFFLATED KIDNEY RECIPIENTS...... 256

FIGURE 56: LIGHT MICROSCOPY IMAGES OF H&E-STAINED TISSUE SAMPLES FROM EXPLANTED STATIC

COLD STORAGE KIDNEYS...... 257

FIGURE 57: MICROSCOPY IMAGES OF H&E-STAINED TISSUE SAMPLES FROM EXPLANTED PERSUFFLATED

KIDNEYS...... 258

FIGURE 58: WOOCR MEASUREMENTS AT HYPOTHERMIC AND SUBNORMOTHERMIC TEMPERATURES FOR

STATIC COLD STORAGE AND PERSUFFLATED KIDNEYS...... 267

FIGURE 59: GFR MEASUREMENTS AT HYPOTHERMIC AND SUBNORMOTHERMIC TEMPERATURES FOR

STATIC COLD STORAGE AND PERSUFFLATED KIDNEYS...... 268

FIGURE 60: HISTOLOGICAL ANALYSIS AT HYPOTHERMIC AND SUBNORMOTHERMIC TEMPERATURES FOR

STATIC COLD STORAGE AND PERSUFFLATED KIDNEYS...... 269

FIGURE 61: EXPERIMENTAL TIMELINE FOR ORGAN PRESERVATION...... 338

FIGURE 62: EXPERIMENTAL TIMELINE FOR TRANSPLANT SURGERIES AND POST-SURGERY ASSESSMENTS...... 339

FIGURE 63: IMAGES OF THE RAT KIDNEY WITH COMPLETE VASCULAR ANASTOMOSES...... 340

List of Tables TABLE 1: DOWNREGULATED PATHWAYS AND BIOLOGICAL PROCESSES IN STATIC COLD STORAGE KIDNEYS

AT T1 COMPARED TO T0...... 131

TABLE 2: DOWNREGULATED GENES IN PRESERVED STATIC COLD STORAGE KIDNEYS AT T1 COMPARED TO T0...... 133

TABLE 3: UPREGULATED PATHWAYS AND BIOLOGICAL PROCESSES IN STATIC COLD STORAGE KIDNEYS AT

T1 COMPARED TO T0...... 134

TABLE 4: UPREGULATED GENES IN PRESERVED STATIC COLD STORAGE KIDNEYS AT T1 COMPARED TO T0...... 137

TABLE 5: DOWNREGULATED PATHWAYS AND BIOLOGICAL PROCESSES IN PERSUFFLATED KIDNEYS AT T1

COMPARED TO T0...... 137

TABLE 6: DOWNREGULATED GENES IN PERSUFFLATED KIDNEYS AT T1 COMPARED TO T0...... 138

TABLE 7: UPREGULATED PATHWAYS AND BIOLOGICAL PROCESSES IN PERSUFFLATED KIDNEYS AT T1

COMPARED TO T0...... 139

TABLE 8: UPREGULATED GENES IN PERSUFFLATED KIDNEYS AT T1 COMPARED TO T0...... 141

TABLE 9: DOWNREGULATED PATHWAYS AND BIOLOGICAL PROCESSES IN PERSUFFLATED KIDNEYS

COMPARED TO STATIC COLD STORAGE KIDNEYS AT T1...... 142

TABLE 10: DOWNREGULATED GENES IN PERSUFFLATED KIDNEYS COMPARED TO STATIC COLD STORAGE

KIDNEYS AT T1...... 143

TABLE 11: UPREGULATED PATHWAYS AND BIOLOGICAL PROCESSES IN PERSUFFLATED KIDNEYS

COMPARED TO STATIC COLD STORAGE KIDNEYS AT T1...... 145

TABLE 12: UPREGULATED GENES IN PERSUFFLATED KIDNEYS COMPARED TO STATIC COLD STORAGE

KIDNEYS AT T1...... 148

TABLE 13: HIGHLY CONNECTED, DOWNREGULATED PROTEINS IDENTIFIED IN STATIC COLD STORED

KIDNEYS AT T1 COMPARED TO T0...... 150

TABLE 14: PATHWAYS ENRICHED IN STATIC COLD STORAGE KIDNEY SAMPLES AT T1 COMPARED TO T0...... 150

TABLE 15: BIOLOGICAL PROCESSES ENRICHED IN STATIC COLD STORAGE KIDNEY SAMPLES AT T1

COMPARED TO T0...... 151

TABLE 16: HIGHLY CONNECTED, UPREGULATED PROTEINS IDENTIFIED IN STATIC COLD STORED KIDNEYS

AT T1 COMPARED TO T0...... 152

TABLE 17: PATHWAYS ENRICHED IN STATIC COLD STORED KIDNEY SAMPLES AT T1 COMPARED TO T0...... 152

TABLE 18: BIOLOGICAL PROCESSES ENRICHED IN STATIC COLD STORED KIDNEYS AT T1 COMPARED TO T0...... 153

TABLE 19: HIGHLY CONNECTED, DOWNREGULATED PROTEINS IDENTIFIED IN PERSUFFLATED KIDNEYS AT

T1 COMPARED TO T0...... 154

TABLE 20: PATHWAYS ENRICHED IN PERSUFFLATED KIDNEY SAMPLES AT T1 COMPARED TO T0...... 154

TABLE 21: BIOLOGICAL PROCESSES ENRICHED IN PERSUFFLATED KIDNEY SAMPLES AT T1 COMPARED TO T0...... 155 TABLE 22: HIGHLY CONNECTED, UPREGULATED PROTEINS IDENTIFIED IN PERSUFFLATED KIDNEYS AT T1

COMPARED TO T0...... 156

TABLE 23: PATHWAYS ENRICHED IN PERSUFFLATED KIDNEY SAMPLES AT T1 COMPARED TO T0...... 156

TABLE 24: BIOLOGICAL PROCESSES ENRICHED IN PERSUFFLATED KIDNEY SAMPLES AT T1 COMPARED TO T0...... 157

TABLE 25: HIGHLY CONNECTED, DOWNREGULATED PROTEINS IDENTIFIED IN PERSUFFLATED KIDNEYS

COMPARED TO STATIC COLD STORED KIDNEYS AT T1...... 158

TABLE 26: PATHWAYS ENRICHED IN PERSUFFLATED KIDNEYS COMPARED TO STATIC COLD STORAGE

KIDNEY SAMPLES AT T1...... 158

TABLE 27: BIOLOGICAL PROCESSES ENRICHED IN PERSUFFLATED KIDNEYS COMPARED TO STATIC COLD

STORAGE KIDNEY SAMPLES AT T1 ...... 159

TABLE 28: HIGHLY CONNECTED, UPREGULATED PROTEINS IDENTIFIED IN PERSUFFLATED KIDNEYS

COMPARED TO STATIC COLD STORED KIDNEYS AT T1...... 160

TABLE 29: PATHWAYS ENRICHED IN PERSUFFLATED KIDNEYS COMPARED TO STATIC COLD STORAGE

KIDNEY SAMPLES AT T1...... 160

TABLE 30: BIOLOGICAL PROCESSES ENRICHED IN PERSUFFLATED KIDNEY COMPARED TO STATIC COLD

STORAGE KIDNEY SAMPLES AT T1 ...... 161

TABLE 31: HIGHLY CONNECTED, UPREGULATED PROTEINS...... 205

TABLE 32: A CHART OF THE HUB GENES WITH THE MOST DEGREE AND BETWEENNESS FROM THE

NETWORK OF DOWNREGULATED PROTEINS...... 205

TABLE 33: PATHWAYS ENRICHED IN PSF COMPARED TO SCS KIDNEY SAMPLES...... 206

TABLE 34: PATHWAYS ENRICHED IN PSF COMPARED TO SCS KIDNEY SAMPLES...... 207

TABLE 35: IMMUNOSUPPRESSIVE DRUGS, ANTIBIOTICS AND ANTICOAGULANTS...... 252

TABLE 36: SUMMARY OF RAT AUTOTRANSPLANT...... 339

TABLE 37: SUMMARY OF RAT KIDNEY PROCUREMENT AND PRESERVATION...... 339

Abstract Organ shortage is a persistent, widespread problem that deprives hundreds of thousands of people from a better quality of life. While the improvements in preservation solutions, immunosuppressants and surgical techniques have steadily increased successful outcomes, the scarcity in organs continue to worsen with transplant patients on the waiting list dying every day. In order to overcome this dilemma, organs from many donor types are being used with varying degrees of risk factors and damage. Currently, there are no systematic, universal ways of quantifying organ quality in a way that can predict graft function once it’s been transplanted.

Although various preservation methods are being explored to extend the life of the organ, delayed graft function and complications after the transplant caused by ischemic damage during the procurement and preservation process has yet to be resolved.

We had two overarching goals for these experiments. The first was the development of noninvasive quality assessment methods that can characterize organs prior to transplantation. The second was to test anterograde persufflation as a method for organ preservation. Persufflation is a technique that delivers humidified, gaseous oxygen directly into the vasculature to ensure even and thorough oxygenation of the whole organ. In previous studies, it has been shown to extend the preservation time, replenish energy levels and improve the function of the organ.

For these experiments, whole organ oxygen consumption rate was used as a measure of tissue viability. Additionally, dynamic contrast enhanced magnetic resonance imaging was used as a tool to calculate glomerular filtration rate, the

12 standard measure of renal function. In conjunction with these assessment techniques, immunohistochemistry, biomarkers and genomic analyses were employed to further elucidate the effects of persufflation on isolated organs. Renal allotransplants were attempted in a pre-clinical porcine model to determine the physiological relevance of persufflation.

Overall, results from studies using isolated kidneys show higher oxygen consumption rate and higher GFR in the persufflated kidneys compared to the static cold storage kidneys, which were interpreted as increased metabolism and function.

Higher levels of lactate that are indicative of anaerobic metabolism were measured in the cold storage kidneys. on the other hand, histological analyses revealed no significant differences between the two treatment groups in terms of morphological changes Microarray data show an upregulation of genes and pathways involved in renal reparative actions, stress response and immune response in the persufflated kidneys. These results likely reflect a more metabolically active organ that is responding to the ischemic insult. The pig renal allotransplant model shows regeneration in the renal tubules of the persufflated kidney, while the cold storage counterpart shows diffuse necrosis. While it is premature to make any assumptions about the in vivo effects of persufflation from the transplant model, it provides a foundation for subsequent large animal model pre-clinical studies.

Chapter 1

Introduction

Overview and Thesis Organization

There is an increasing demand for not only more donor kidneys for transplant, but prognostic indicators that enable the assessment of kidney quality prior to transplantation. Pre-transplant kidney quality assessments are currently limited to biopsies, visual assessments, and vascular resistance measurements. These measurements are inefficient and do not reliably correlate with clinical transplant outcomes (Wang, et al. 2015, Polska, et al. 2001, El-Husseini, et al. 2007, Boddi,

Natucci and Ciani 2015, Pokorná, et al. 2000, Sung, et al. 2008). The use of expanded criteria donor (ECD) and donor after cardiac death (DCD) kidneys as a means to increase the donor pool of organs ironically increased the discard rate due to the risk aversion of using organs that fall outside the standard criteria (Cecka, et al. 2006, Hall, et al. 2015). Objective analyses and prospective studies that develop a reference marker for clinical outcomes are needed to reduce the number of discarded organs.

The use of organs outside the standards induced a paradigm shift; not only do these organs need optimal reconditioning during the preservation period, but also tools that can predict their transplant outcomes. The ability to characterize organ condition is especially important for ECD and DCD kidneys because they are more susceptible to delayed graft function, and recipients are at risk for longer hospital stays compared to kidneys from a standard donor (Saidi, et al. 2007, Ojo 2001).

Furthermore, there is a proclivity towards rejecting ECD and DCD kidneys due to the risk of poor clinical outcomes, even when they could be transplantable. Developing an objective, systemic way to assess organ quality from non-standard donors may

15 help determine graft outcomes, and prevent the discard of viable organs that can be used for transplant.

The chapters herein present studies that have been submitted for publications and unpublished studies that are in preparation for publication. Our present study has two aims of studying novel assessment techniques, and using persufflation (PSF, humidified oxygen gas perfusion) as an alternative, improved approach for preservation. The overall goal for this research is to establish better organ quality evaluation technology that can be used prior to transplant, and examining alternate preservation methods that will allow longer preservation times without compromising organ viability and function, and enabling organ reconditioning or repair.

Specific Aim 1: Develop whole organ oxygen consumption rate (WOOCR) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) as assessment methods to predict transplant outcomes ex vivo in a porcine model.

Specific Aim 2: Use the assessment methods established in Aim 1 to compare different methods of kidney preservation.

Aim 2a: Compare static cold storage (SCS, standard method for organ

preservation) and persufflation

Aim 2b: Compare hypothermic machine perfusion (HMP) using the LifePort

(state of the art method of kidney preservation) and persufflation

Specific Aim 3: Use the assessment methods established in Aim 1 to compare the effects of persufflation and static cold storage on kidneys at room temperature after the 24 hours of treatment.

Specific Aim 4: Translate the ex vivo assessment tools and preservation methods to an in vivo porcine model using pig allotransplant to evaluate renal function.

We report the feasibility of using whole organ oxygen consumption rate

(WOOCR) and measuring glomerular filtration rate (GFR) in isolated porcine kidneys to assess their viability and function. Additionally, our results confirm that oxygenating kidneys during the preservation period can be beneficial in maintaining their functional integrity as measured by WOOCR and GFR.

We believe using these assessment techniques will improve transplant outcomes and change the way kidneys are currently being allocated. This work has major implications, given that the number of donor organs that could be used for transplant can be increased through improved preservation, and quality assessment.

Ultimately, this can result in more lives being saved through transplantation, and better quality and longer life for transplant recipients.

Figure 1: Diagram of the main themes of this thesis. The overarching research goal is to expand the number of kidneys that can be transplanted. The studies presented in this thesis focus on developing novel assessment methods that can be used to evaluate the quality of donor kidneys, and persufflation as an improved preservation method prior to transplantation

Figure 2: Thesis organization flow chart. The bulk of this thesis covers the ground work and original studies that have been done to optimize organ assessment techniques and study the effects of persufflation as a method of organ preservation.

Figure 3: Thesis organization chart with descriptions of each chapter and appendix. The first three chapters cover the background information, objectives and methods of these studies. Chapters 4-7 cover the experiments that were conducted to address the aims and chapter 8 summarizes these experiments. Appendices include tangential, related studies for examining persufflation as a preservation technique and improving islet transplantation by evaluating their viability and function, and investigating alternative sources for islets.

Chapter 2 covers background information on the history of organ transplantation, organ damage, current assessment techniques and preservation methods. It includes the limitations and inadequacies of the status quo of preservation and organ allocation.

Chapter 3 describes in detail the methods that were used to study the research aims.

These methods include organ procurement and preservation, whole organ oxygen consumption rate, dynamic contrast enhanced imaging using gadolinium to calculate glomerular filtration rate, perfusate lactate measurements, histological analysis of renal biopsies, RNA sequencing and Agilent microarray analysis.

Chapter 4 includes a submitted manuscript that describes novel assessment techniques that can be used in isolated kidneys to evaluate graft quality. In this study, persufflated kidneys were compared to static cold stored kidneys by using WOOCR and GFR. Perfusate levels of lactate and renal biopsies were also used to evaluate the graft. The development of WOOCR and imaging to calculate GFR has important implications for it can quantitatively determine the viability of isolated kidneys in a noninvasive manner. Additionally, this study shows improved WOOCR and GFR in the persufflated kidneys, consequently strengthening the case for using persufflation as an alternative preservation method. This chapter also includes unpublished data from RNA sequencing analysis of the isolated kidneys.

Chapter 5 describes a pilot study that was performed to compare hypothermic machine perfusion to persufflation. Hypothermic machine perfusion is currently the state of the art preservation method to condition ECD and DCD kidneys prior to

21 transplant. It has shown to reduce the rates of delayed graft function in clinical trials, and many modifications of HMP is currently being investigated in kidneys and livers.

In this study, we aimed to directly compare HMP to PSF to fill the gap in the literature.

Our studies were inconclusive due to the malfunction of the LifePort, consequently not being able to gather data from an appropriate sample size with consistent experimental conditions. Overall, we did not see any significant differences in the kidneys evaluated after 24 hours of preservation with the LifePort and persufflator.

Chapter 6 includes a manuscript in preparation that uses WOOCR and GFR to assess persufflated kidneys and static cold stored kidneys at subnormothermic temperatures. Literature demonstrates that controlled rewarming kidneys prior to transplantation can improve subsequent organ quality upon reperfusion. In order to avoid exacerbating oxidative damage by the sudden rewarming and reoxygenation of the kidneys, we rewarmed them to subnormothermic temperatures to evaluate their viability and function with WOOCR and GFR. Similar to findings from Chapter 4, this study also shows improved WOOCR and GFR in persufflated kidneys.

Chapter 7 describes initial attempts at porcine allotransplants of kidneys preserved with persufflation or static cold storage. These studies investigate whether the differences in renal parameters that were observed in the ex vivo studies translate to functional differences in an in vivo model. Sorting out the logistics and creating a cross-functional team that can perform porcine kidney allotransplants is an invaluable step towards furthering pre-clinical studies.

Chapter 8 summarizes the studies presented in this dissertation, discusses the challenges, limitations and future directions.

Appendix A is an invited review on the current literature on the development and use of persufflation in the field of organ preservation and transplantation. It covers the key manuscripts published between 2014-2017 and summarizes their important findings, as well as the trends and trajectory of persufflation research.

Appendix B is a summary of attempts at rat kidney allotransplants. These endeavors were largely unsuccessful due to many technical difficulties that come with microsurgeries.

Appendix C is a published manuscript in which I am a co-author. This study covers the acute ischemic damage on islets in encapsulation devices. Islets are densely packed into biocompatible devices, which creates large diffusional distances for nutrients and stagnant hypoxic environments for the beta cells. These cells show changes in gene expression in terms of inflammatory cytokines, hypoxia-response signaling, nutrient transport and metabolism.

Appendix D is a published manuscript in which I’m a co-author. This study encompasses the characterization of neonatal, juvenile, and adult porcine islets that are being examined as an alternative source of islets for islet transplantation.

Chapter 2

Literature Review

Section1: HISTORY OF ORGAN TRANSPLANTATION

In the early 20th century, Alexis Carrel, a professor of surgery from Lyon,

France performed the first solid organ xenotransplants in human patients (Linden,

2009; Morris, 2004; Carpenter, Sayegh, & Charles, 2004). He used sheep and pig organs and connected their vessels to the human recipient brachial vessels. He then went on to develop hemostatic methods for renal vascular anastomoses and won the

Nobel Prize for medical research (Linden, 2009). There were other attempts at xenotransplants using rabbit, pig and monkey kidneys but these foreign grafts were received with severe host immune responses (Linden, 2009; Shayan, 2001). During the Second World War, immune rejection was studied due to the necessary composite tissue transplants for burn victims. During this time, it was discovered that immune responses are the reason behind allograft rejection (Shayan, 2001). After the war,

Hume and his colleagues notably performed a series of renal transplants without immune suppression in Boston. Some of these kidneys maintained renal function for weeks and months (Morris, 2004), and these case studies offer a plethora of information that helped characterize indicators for clinical and pathological immune rejection (Starzl, 2000). Shortly thereafter, Harrison, Murray and Merrill successfully reversed renal failure with transplantation surgery with a healthy donor’s kidney placed in his identical twin brother. (Shayan, 2001; Linden, 2009; Starzl, 2000;

Morris, 2004).

These sequences of transplant surgeries became the backdrop of realizing the need for immunosuppression for a successful transplantation with both graft function and host survival. In the mid-20th century, in tandem with the

25 allotransplants, total body irradiation was tested as a means for immunosuppression

(Starzl, 2000). Irradiation as a measure of controlling immunosuppression was too global, but this proof of concept experiment served as a platform for refining the development of targeted, pharmacological immunosuppressive agents. After a few failed attempts at renal transplantation in animal models with 6-mercaptopurine, this was replaced by azathioprine used in conjunction with corticosteroids (Morris,

2004). The efficacy of this cocktail of immunosuppressive drug was the gateway to proceeding with organ transplantation of hearts, lungs, livers and pancreas. While this increased renal graft survival rate to 40-50%, the adverse side effects of immunosuppression were not resolved until cyclosporine was discovered by a Swiss physician, Jean Borel in the late 20th century (Morris, 2004; Linden, 2009).

Cyclosporine is a calcineurin inhibitor that specifically targeted T-lymphocytes and depressed humoral and cellular immunity but without the telltale bone marrow depression of its predecessors. This drug was then challenged by tacrolimus, which had better potency and efficacy. While both drugs have harmful side effects such as nephrotoxicity, neurotoxicity and diabetogenicity, the utilization of the latter drug presented with less incidence of hypertension and hyperlipidemia for improved recipient health (Linden, 2009; Starzl, 2000).

The development of surgical techniques and immunosuppressive drugs paved the way for establishing organ transplantation as a preferred therapeutic option for end stage organ failure and other irreversible organ damage and disease. Since the first successful kidney allotransplant completed in 1954 by Dr. Joseph Murray at the

Peter Bent Brigham Hospital in Boston, there have been millions of organ

26 transplantations performed with increasing surgical technology advancement and improvement in graft outcomes. While this is a costly procedure with a significant recovery period, receiving a new organ reduces patient morbidity, improves life expectancy, and overall reduces the financial cost of maintenance for chronic disease

(Ojo, et al. 2001).

Despite advances in medicine and technology and increased awareness for the need for organ donation and transplantation, the number of patients going on the transplant waitlist has increased dramatically over the past few decades and quickly outpaced the available organs for transplant. As of October 2017, there were 127,377 active candidates on the waiting list that need lifesaving transplant surgeries while only 26,034 transplants were performed for heart, lung, liver, pancreas, kidney and intestines. For kidneys, there were a total of 14,776 transplant surgeries with 10,554 deceased donor transplants and 4,222 living donor transplants. 96,556 patients still remain on the waiting list for renal transplants (Organ Procurement and

Transplantation Network, 2017).

Section 2: DONOR TYPES

Organs are donated from both healthy living donors with no significant medical conditions, and deceased donors. Deceased donors are comprised of standard criteria donors (SCD), expanded criteria donors (ECD) and donors after cardiac death (DCD). SCDs who are also referred to as brain dead donors (DBD), are donors under the age of 50 who suffered irreversible loss of brain and brainstem function and permanent brain damage from trauma, cerebral vascular accidents and

27 other medical problems (Rao & Ojo, 2009; Wong, Tan, & Goh, 2017; Zambergs & Vyas,

2015). These patients are declared dead due to neurological criteria and have beating hearts when the organs are removed. The Organ Procurement and Transplantation

Network/United Network for Organ Sharing (OPTN/UNOS) defined ECDs as organs that come from donors who died from brain death but with certain number of risk factors. These are donors that are over the age of 60, or over the age of 50 with two or more comorbidities such as systemic hypertension, death from stroke and high creatinine levels (Rao & Ojo, 2009). DCD organs are from patients who show a very high probability of dying if taken off, or refuse life-sustaining treatment. These patients typically have suffered devastating and permanent brain injury or have end stage musculoskeletal disease and high spinal cord injury, but do not formally meet the clinical criteria to be declared brain dead. DCD is divided into two categories; controlled and uncontrolled. Controlled DCDs are kept alive on ventilators and other palliative and life sustaining measures until the proxy or family members consent to withdrawing care (Steinbrook, 2007). Once the physician that is not on the transplant team declares death by an irreversible cessation of circulatory and respiratory function, organs are procured (Bernat, et al., 2006). Uncontrolled DCDs describe patients who have suffered an unexpected cardiac arrest in conjunction with 30 minutes of failed resuscitation. These scenarios usually occur outside the hospital and cardiac compression with mechanical ventilation is used to preserve the organs until procurement (Bernat, et al., 2006; Ortega-Deballon, Hornby, & Shemie, 2015; Evrard,

2014).

Figure 4: Summary of the types of deceased donors and their criteria Organs from all donor types are exposed to ischemia, which is caused by the cessation of blood flow at the time of organ collection. Ischemia time is a complex variable that exists in organ transplantation that could be influenced by the type of donor, organ procurement, preservation, process of allocation, transport and the transplant surgery itself. Warm ischemia time (WIT) is the time that the organs are left at normothermic temperature without delivery of oxygen. In DCDs, this is experienced during the time of extubation and withdrawal of care until the initiation of cold perfusion. In DBDs it is from the aortic cross clamp until the initiation of cold perfusion (Halazun, Al-Mukhtar, Aldouri, Willis, & Ahmad, 2007). Once the donor is extubated, the clinical staff waits between 2-5 minutes to confirm the absences of circulation and then declares the death of the donor (Bernat, et al., 2006). Cold ischemia time (CIT) is the time the organs are left at hypothermic temperatures without delivery of oxygen. This is measured at the start of cold perfusion of the donor until the organ is transplanted into a recipient prior to reperfusion. The amount of

29 ischemia time influences the organ function and graft outcome. The effects of prolonged cold ischemia time on transplant outcomes remain controversial.

Stagnant organ supply and the ever-growing transplant waiting lists challenged the transplant community to find more sources for life saving organs. Over the past decade, the use of expanded criteria donor (ECD) and donor of cardiac death

(DCD) organs that would have not been considered in the past have substantially increased thereby alleviating excessive waiting times and organ shortages. The nature of the definition of ECD organs naturally leads to high levels of variability, which makes it difficult to objectively score and predict their risk for delayed graft function or graft failure. Out of the multifactorial characteristics of the ECD organs, donor age was the most significant factor that influenced graft survival (Pascual,

Zamora, & Pirsch, 2008). There are conflicting data on the graft survival with ECD kidneys. Over the years there have been retrospective studies comparing ECD and

SCD kidneys at numerous transplant centers across the U.S. and Europe. Many of these centers show a 77% greater risk for a worse outcome using ECD kidneys compared to the SCD counterpart. The transplants from 1996-2005 show at five years after transplant, approximately 53% of the ECD kidneys were showing renal function while 70% of the SCD kidneys had renal function (Andreoni, Brayman, Guidinger,

Sommers, & Sung, 2007). These kidneys also have a greater chance of host rejection, and a greater susceptibility to hypothermic and ischemic injuries, drug toxicity, and the effects of posttransplant hypertension (Ojo, et al. 2001, Port, et al. 2002). Despite this expected higher rate of graft failure, multiple studies have subsequently shown that kidney transplantation using ECDs is associated with a considerable reduction in

30 healthcare expenditure, morbidity and improvement in life expectancy compared with transplant candidates who remained on maintenance dialysis. The average increase of life expectancy of patients that received ECD organs was 5 years, and varied from 3-10 years compared to the life expectancy of those who were waitlisted

(Ojo, et al. 2001). Another study showed that the ECD organ recipients had a 27% lower chance of mortality compared to patients who were on the waitlist for greater than 3.7 years (Merion, et al., 2005).

When comparing SCD kidneys to DCD kidneys, several regional studies found that there is a difference in the incidence of delayed graft experienced by the patients

(Locke, et al., 2007; Summers, et al., 2015; Gentil, et al., 2016). There is some ambiguity and heterogeneity with the definition of delayed graft function with 18 different definitions and 10 diagnostic techniques used to identify it (Yarlagadda, et al., 2008), but it ranges from total anuria or slow recovery of function with needing postoperative dialysis at least once during the first 7 days post-transplant. Controlled

DCD kidney transplants done in the United Kingdom show a higher incidence of delayed graft function in DCD kidneys. However, there were no significant differences in graft survival and patient survival at 3 years after the transplantation. The DCD kidneys had a 4% chance and SCD kidneys had a 3% chance of primary non-function

(Summers, et al., 2015). With donors under the age of 50, both DCD and SCD kidneys had similar 5-year graft survival. DCD kidneys were shown to have higher rates of primary non-function compared to SCD kidneys, but lower than ECD kidneys. DCD kidneys that are from donors over the age of 50 showed an 80% increased risk of graft loss compared to kidneys acquired from younger DCDs. All DCD, SCD and ECD

31 kidneys had 38.7%, 19.5% and 30% chance of delayed graft function (Locke, et al.,

2007). When comparing initial hospitalization costs, SCD, ECD and DCD kidney recipients were charged on average $47,462, $70,030 and $72,789 with the latter two organs with higher incidence of delayed graft function, longer hospital stay and frequency of readmission, and longer time for serum creatinine to reach the ideal range for renal function (Saidi, et al., 2007). However, the occurrences of graft rejection and long-term patient survival between these groups are not significantly different. These results show that despite the poorer post-transplantation results of non SCD organs, several studies showed that receiving a kidney, albeit a marginal kidney is better than no kidney.

Section 3: PATHOPHYSIOLOGY OF ORGAN DAMAGE

All donated organs are subjected to a multi-step process that starts with the withdrawal of life support, flushing of the organs in the body cavity, organ procurement, preservation/transport and transplantation into the recipient. Kidneys from traditional and non-traditional donors are exposed to varying levels of hypoxia and ischemia during the cessation of blood flow at the time of organ collection. During organ preservation and transport, procured organs are inevitably exposed to a period of cold ischemia time (CIT), with longer CIT associated with delayed graft function

(Krishnan, et al., 2016; Peters, Shaver, Santiago-Delpin, Jones, & Blanton, 1995;

Kayler, Magliocca, Zendejas, Srinivas, & Schold, 2011). The combination of pre- transplant hypothermic damage, ischemic damage and post-transplant reperfusion damage creates a complex sequence of cellular events that can potentiate renal injury

32 after transplantation. The severity of these injuries found in kidneys has a crucial effect on graft recovery and clinical outcomes (Salvadori, Rosso, & Bertoni, 2015).

Figure 5: Schematic for organ donation, preservation and transplant. Once a patient meets the requirements to be an organ donor, they are taken off life support. The abdominal organs are flushed with cold preservation solution. After the organ is harvested, organ assessments can be run if a better evaluation of the organ is needed. Organ is then preserved in hypothermic conditions during its transport and then transplanted into the recipient.

HYPOTHERMIC DAMAGE: Hypothermia is widely used to reduce metabolic rate to diminish the demand for tissue oxygen and substrates. The lipid bilayers of cell membranes that modulate cell function and structural integrity transition into a gel like state, which causes the different lipid classes to separate (Hochachka, 1986). This process of solidification decreases membrane fluidity and causes abnormal transmembrane diffusion due to increased permeability of the cells (Fuller, Guibert, &

Rodriguez, 2010). While enzymatic activity shows a 1.5-2-fold decrease for every

10°C decrease in temperature and there is reduced cellular metabolism in procured

33 kidneys that are being stored at 4°C, these organs are still metabolically active (Belzer

& Southard, 1988). The metabolic rate of procured organs is reduced by approximately 90% but nonetheless they are slowly depleting the remaining ATP during the cold preservation period (Ratigan & McKay, 2016). The net result of cooling the whole organ is not completely understood because it does not affect all metabolic regulation equally (Taylor, 2007). The suppression of metabolic rate and metabolic regulation impedes energy costly mechanisms such as protein synthesis and ion motive adenosine triphosphatases (ATPases) that maintain cellular membrane potential. The reduction of adenosine triphosphate (ATP) synthesis accelerates the dissipation of ion gradients and switches the cells from aerobic to anaerobic glycolysis to produce energy, which is also an important proponent of ischemic injury.

ISCHEMIC INJURY: Ischemic injury denotes the damage that occurs from the cessation of blood supply that discontinues the delivery of oxygen and nutrients to the oxygen dependent tissue. This is typically experienced in the isolated organ during procurement and preservation period. The anoxic environment slows down mitochondrial activities of ATP synthesis and oxidative phosphorylation. The fall in oxygen levels induces a shift from aerobic to anaerobic glucose metabolism to meet the metabolic demands of the organ. Anaerobic metabolism does not produce sufficient levels of high energy phosphates and depletes cellular glycogen storage.

The accumulation of byproducts such as lactic acid, hydrogen ions and NADH inhibit glycolytic enzymes. The acidotic environment in the tissue created by glycolysis and the dysfunction of sodium-hydrogen exchanger (Na+/H+ exchanger) favors the

34 optimal pH of lysosomal protease activity, which is normally suppressed. Lysosomal disruption and instability can release catalytic enzymes for protein and lipid degradation that induces membrane component and subcellular structural changes

(Fuller, Guibert, & Rodriguez, 2010).

In parallel with alterations in membrane permeability, failure to maintain adequate ATP levels during ischemic stress destroys cell homeostasis and triggers the mis-localization of membrane proteins, loss of cytoskeletal integrity and cell polarity.

This inevitably leads to decreased sodium-potassium-ATPase (Na+/K+-ATPase) activity which is a crucial component for maintaining membrane potential and low levels of intracellular Sodium. This dysregulation depolarizes the cells and promotes potassium (K+) efflux, sodium (Na+) and chloride (Cl-) influx to the cell down their concentration gradients (Belzer & Southard, 1988; Rauen & de Groot, 2004). The buildup of solutes and residual intermediates of glycolysis increase the intercellular osmolarity, causing edema by drawing water into the cell through osmosis (Belzer &

Southard, 1988). Edema places hydrostatic pressure on the cellular membranes, stimulates stretch activated channels and disrupts the organelle membranes of mitochondria, endoplasmic reticulum and Golgi apparatus. Mitochondrion membrane permeability from edema can trigger apoptosis and necrosis. The influx of cations precedes membrane depolarization and opening of voltage dependent calcium (Ca2+) channels and a decrease in sodium-calcium exchanger (Na+/Ca2+ exchanger) activity, thus resulting in calcium influx into the cell. The sodium accumulation inside the cell cannot be pumped out with the Na+/K+ ATPase, which

35 eliminates the inward Na+ gradient and reverses the Na+/Ca2+ exchanger (de Groot &

Rauen, 2007; Fuller, Guibert, & Rodriguez, 2010).

This overall increase in cytosolic Ca2+ is one of the major mediators of ischemic injury through multiple cellular pathways. Under anoxic duress, the normally impermeable inner membrane opens its mitochondrial permeability transition pore

(mPTP), which is linked to mitochondrial dysfunction. It allows communication between the cytoplasm and mitochondrial matrix, permitting small molecular weight solutes to move freely across the membrane (Bernardi & Di Lisa, 2015). The accumulation of Ca2+ in the mitochondrial matrix through the mPTP can put the cellular ATP in contact with mitochondrial ATPase that further degrades ATP (de

Groot & Rauen, 2007; Kosieradzki & Rowinski, 2008). This adenine nucleotide catabolism creates the intracellular accumulation of hypoxanthine (Collard & Gelman,

2001). The change in mitochondrial permeability and mitochondrial uncoupling destroys the proton gradient and releases factors involved in necrosis and apoptosis.

Calpains are released into the cytoplasm, which are calcium-dependent, non- lysosomal cysteine proteases that are an essential component of necrosis.

Cytochrome c released from the inner mitochondrial membrane triggers the activation of Caspase-9, which promotes additional downstream caspase cascade that activities cell death (Slee, et al., 1999; Du, Fang, Li, Li, & Wang, 2000). Increased cytoplasmic Ca2+ activates endogenous hydrolases, phospholipases and proteases, which increase membrane phospholipid hydrolysis, protein damage and alters cellular cytoskeleton. This leads to an auto-digestion stage that causes phospholipid

36 mass and buildup of free fatty acids, lysophopholipids and un-esterified arachidonic acids (Van der Vusse, Reneman, & van Bilsen, 1997).

Figure 6: A summary of the mechanisms for cellular changes that are induced by ischemia in the kidney.(Kalogeris, Baines, Krenz, & Korthuis, 2012; Perico, Cattaneo, Sayegh, & Remuzzi, 2004)

REPERFUSION INJURY: the reinstitution of blood perfusion to the ischemic kidney stimulates a paradoxical sequence of events that enhances renal injury. Upon reperfusion, oxygen and pH levels are restored in the ischemic tissue that is experiencing mitochondrial damage, calcium overload, low pH and suppression of antioxidant activities. The normalization of extracellular pH creates a H+ gradient and pulls in more sodium into the cell through the Na+/H+ exchanger, which exacerbates the Na+/Ca+ exchanger that is working in reverse, which further worsens the cytosolic and mitochondrial Ca2+ overload. At the restored optimal pH, calpain that was

37 released from the mitochondria through the mTPT is activated and proceeds with proteolytic activities that destroy cellular structures and eventually prompt cell death.

This is a combination of inflammatory response and oxidative stress from the burst of reactive oxygen species (ROS) that are generated after reperfusion (Zweier,

Flaherty, & Weisfeldt, 1987). Free radicals have one or more unpaired electrons and are extremely reactive, and unstable with a very short half-life (Chatauret, Badet,

Barrou, & Hauet, 2014). They are typically generated as byproducts of cellular metabolism and controlled with endogenous scavenging mechanisms under normal conditions. Free radicals are generated in small amounts during hypoxia both from cellular metabolism, and the suppression of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase prime the tissue for further damage

(Chatauret, Badet, Barrou, & Hauet, 2014). These molecules can destroy membrane lipid, denature proteins and break DNA. Once normoxia is reinstated, the ROS generated during ischemic damage are converted into more potent forms and much more ROS is generated. Many studies show the reperfusion of organs with hypoxic solutions does not invoke the extent of free radical formation seen in organs reperfused with oxygenated solution (Korthuis, Smith, & Carden, 1989).

In the post ischemic tissue, the hypoxanthine created during ischemic exposure is metabolized to xanthine by xanthine oxidase (XO) which is mainly found in endothelial cells (Zweier, Flaherty, & Weisfeldt, 1987). XO needs molecular oxygen for its function so upon reperfusion, hypoxanthine is metabolized with O2. – and H2O2 produced as intermediate products (Yapca, Borekci, & Suleyman, 2013; Chambers, et

38 al., 1985). The Nox/Duox family of NADPH oxidases can be produced by phagocytic cells and plays an active role in converting oxygen to large quantities of O2. – and its derivatives (Dröge, 2002; Babior, Lambeth, & Nauseef, 2002). Mechanical stress and angiotensin 2 are also known to activate NADPH oxidases in the endothelium for O2. – production. The three isoforms of nitric oxide synthase (NOS) can be stimulated by elevated levels of H2O2, which produces nitric oxide (NO) from the catabolism of L- arginine (Granger & Kvietys, 2015). Typically, NO is deemed as protective against the ischemia reperfusion injury due to its antioxidant and anti-inflammatory properties.

However, large amounts of NO and NOS uncoupling paradoxically leads to further O2.

– production from NO, which mediates cellular constituent impairment (Fu & Li,

2017). O2. – will also react with free nitric oxide (NO) to form secondary reactive nitrogen species such as peroxynitrite (ONOO-), which is implicated in inflammation induced tissue injury (Zweier & Talukder, 2006; Jourd'heuil, et al., 2001). The acidotic and reducing state of the post ischemic tissue is conducive for ferritin and metalloproteins to release ferric and ferrous iron. The less potent O2. – and H2O2 go through conversion to a highly reactive oxidant, hydroxyl radical (•OH) which is primarily responsible for DNA damage (Ambrosio, Zweier, Jacobus, Weisfeldt, &

Flaherty, 1987). Monoamine oxidases located on the outer mitochondrial membrane can create H2O2 through degradation of amines.

The mitochondria play a crucial role in generating ROS. The electrons from the electron transport chain (ETC) that leak into the cytosol from the mitochondria during the ischemic phase reduce oxygen into O2. – (Zweier & Talukder, 2006). The cellular catabolism of carbohydrates, lipids and proteins yield substrates that are

39 used for the TCA cycle. The TCA cycle intermediates donate electrons and protons to create NADH and FADH2 to support the ETC. Four compartments comprise the ETC in the inner mitochondrial membrane to shuttle electrons. All compartments have the potential to transferring single electrons to oxygen to create O2. – that are kept at low levels of detection through antioxidant systems (Perrelli, Pagliaro, & Penna, 2011).

Upon reperfusion, there is amplified O2. – creation due to the damaged complexes from the original ischemic insult. The mTPT and inner membrane anion channel

(IMAC) are formed in an environment that favors redox reactions, leading to ROS production. This circular, self-amplifying mitochondrial damage experienced by ROS that subsequently results in the generation of more ROS release is named ROS- induced ROS release (RIRR) (Zorov, Juhaszova, & Sollott, 2014). The excessive production of ROS that outpaces the endogenous antioxidant activities can result in deleterious ischemia-reperfusion pathologies and cell death.

Figure 7: A summary of the mechanisms for cellular changes that are induced by reperfusion after an ischemic insult in the kidney(Kalogeris, Baines, Krenz, & Korthuis, 2012; Perico, Cattaneo, Sayegh, & Remuzzi, 2004)

INFLAMMATION: Oxygen radical production and resulting lipid peroxidation invokes changes in cytokines (IL-1, IL-6, TNF-a), chemokines (CC, CXC, CX3C) and their receptor expression that causes an inflammatory cascade (Thurman, 2007; Jang

& Rabb, 2009). Antigen independent, pro-inflammatory mediators promote the increase in adhesion molecule expression on the endothelial cell surface (selectins) that recruit leukocytes (Thurman, 2007; Jordan, Zhao, & Vinten-Johansen, 1999).

With reperfusion, the host cellular components of innate immunity are delivered to the damaged sites primed with integrins to tether these cells. Once there is a firm adhesion between the vascular wall and leukocytes via integrins interacting with intracellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1

(VCAM-1), transendothelial migration occurs. The leukocyte rolling, activation, adhesion and locomotion using selectins are reversible processes, but the migration is an all or nothing process (Muller, 2011). This excavation and infiltration are necessary components for vascular damage and increased permeability that occurs during the reperfusion phase (Bonventre & Yang, 2011; Zimmerman & Granger,

1994).

Neutrophils are one of the first types of leukocytes to be recruited to the site of injury. They use intracellular adhesion molecules (ICAM/CD54), selectins, platelet activating factor (PAF), platelet-endothelial cell adhesion molecule (PECAM-1) and many others to potentiate further injury to the tissue (Ioannou, Dalle Lucca, & Tsokos,

2011; Welbourn, et al., 1991). During a renal ischemia reperfusion injury, neutrophils regulate the adaptive immune response through recruiting IL-17, which activates natural killer T cells and interferon gamma (IFN-y) that subsequently activates macrophages. It produces a potent cytokine IL-8 which elicits further neutrophil infiltration (Jordan, Zhao, & Vinten-Johansen, 1999). Neutrophils contain myeloperoxidase, with which they can generate toxic ROS such as hypochlorous acid using the existing O2. – and H2O2 from ischemic cellular damage. Infiltration of neutrophils mediates the increase in microvascular permeability and accumulates at the vascular injury site (Hernandez, et al., 1987). They also contain proteinase elastase and metalloproteases that can degrade the extracellular matrix (Gidday, et al., 2005; Williams, 1996). Monocytes can migrate to the inflamed tissue and differentiate into macrophages and dendritic cells. Their migration is facilitated by chemokines and chemokine receptor signaling pathways, specifically CCR2 and

CX3CR1 (Li, et al., 2008). Macrophages elicit further tissue damage through ROS productions and cytokines (IL-1, TNF-a) that leads to inflammatory cascade.

Dendritic cells play a major role in linking the innate immune response to adaptive immune response by activating T cells via antigen presentation. It is also a major source of TNF during the early phases of ischemia reperfusion injury (Dong, et al.,

2007).

Toll like receptors (TLR) are pattern recognition receptors and are expressed primarily in antigen presenting cells such as macrophages, B cells and dendritic cells.

Products that result from ischemic injuries such as products of necrotic cells, heat shock proteins, and lipopolysaccharides act as endogenous ligands for TLRs (Boros &

Bromberg, 2006). When bound to these ligands, TLRs activate cytokines, kinases and factor NF-κB prototypical proinflammatory signaling pathway (Thurman, 2007; Jang

& Rabb, 2009). The complement system is another mediator for innate immune defense and inflammation. It differentiates between healthy host tissue and debris from the ischemic insult and plays a role in destroying invading foreign organisms and promotes phagocytic clearance of immune complexes. It not only regulates the innate immune response by mediating the migration of eosinophils and mast cells through upregulation of endothelial adhesion molecules, but it can also regulate adaptive immunity by enhancing B and T cell responses (Arumugam, Shiels,

Woodruff, Granger, & Taylor, 2004; Bonventre & Yang, 2011).

RENAL FUNCTION AND POST ISCHEMIC KIDNEYS: The renal system eliminates wastes and regulates homeostasis. It regulates electrolytes, acid-base balance, blood pressure, vitamin D activation and erythrocyte production. Kidneys are located at

T12 to L3 with the right kidney slightly lower than the left. It is attached to the ureter that leads to the bladder, and is protected by renal and adipose capsule. The three regions of the kidney are cortex, medulla and pelvis. Fluid is filtered through the glomerular capillaries into the renal tubules for tubular reabsorption and secretion of solutes and water. This process of glomerular filtration in the nephron forms urine to eliminate toxins and wastes while sodium, potassium and metabolites are conserved. Extracellular fluid homeostasis is maintained by altering urine composition. The nephron is the functional and structural unit and is comprised of glomerulus and tubule. Blood is carried to the glomeruli by the afferent arterioles and taken away by the efferent arterioles. The efferent arterioles carry blood to the vasa recta, and form the peritubular plexis, which are anastomotic capillaries in the cortex.

The filtrate moves through the proximal convoluted tubule (PCT). The PCT has a luminal brush border due to microvilli. The PCT precedes loop of Henle that has a thin descending limb and thick ascending limb. This connects to the distal convoluted tubule (DCT) which coalesces with collecting ducts. The collecting ducts pass through the cortex and medulla to empty urine into the renal pelvis.

Figure 8: Kidney anatomy illustration. This schematic shows the renal cortex and medulla, and the functional unit (nephron). Adapted from opentextbc.ca.

Renal blood flow is autoregulated at renal perfusion pressures greater than approximately 60–100 mm Hg. This is regulated by intrinsic and extrinsic factors. The renal system has intrarenal renin-angiotensin and autoregulatory mechanism to maintain consistent flow (Holechek, 2003). Myogenic mechanisms are controlled by baroreceptors that sense the vascular wall tension by using stretch receptors

(Loutzenhiser, Bidani, & Chilton, 2002). Pressure increases cause vasoconstriction of the afferent arterioles to prevent higher pressure exposure to the glomeruli, while pressure decreases cause vasodilation for consistent blood flow and glomerular filtration. Tubuloglomerular feedback uses the macula densa and juxtaglomerular cells to sense the sodium chloride (NaCl) levels in the distal nephrons. Decrease in

NaCl causes vasodilation to increase glomerular blood flow. Baroreceptors and

45 macula densa can activate the renin-angiotensin system to increase blood pressure.

Renin converts angiotensinogen to angiotensin I, which is converted to angiotensin II with angiotensin-converting enzyme from the lungs. Angiotensin II is a potent vasoconstrictor that can act on both the afferent and efferent arterioles of the nephron (Kobori, Nangaku, Navar, & Nishiyama, 2007; Levens, Peach, & Carey, 1981).

Glomerular and vascular endothelium produces vasodilatory factors such as prostaglandins to offset the effects of vasoconstrictors.

Oxygen supply to the kidney has heterogeneous patterns with most of the supply being shifted to the cortex instead of an even distribution between the cortex and medulla (Epstein, 1997). The proximal tubule uses gluconeogenesis and oxidative metabolism of fat as an energy source, while the ascending limb of loop of

Henle, distal convoluted tubules and collecting duct use glycolysis for energy. There is higher energy use in the proximal tubules because approximately 87% of electrolytes in the filtrate are reabsorbed in this segment of the nephron. Medullary oxygen tension is maintained at 10mmHg in canine kidneys even at high oxygen pressures (Baumgärtl , Leichtweiss, Lübbers, Weiss, & Huland, 1972). There is a corticomedullary gradient in oxygen availability due to the morphology and organization of vessels in the medulla. The tubules and vessels are looped to facilitate countercurrent exchange of solutes and water to modify urine concentration (Brezis,

Rosen, Silva, & Epstein, 1984). Because the oxygen diffuses from the artery to veins, the inner medullary portions are not oxygenated well (Levy, 1959). The medullary thick ascending limb of the loop of Henle consumes high levels of oxygen through

NaCl active transport to maintain the osmotic gradient, and blood flow is limited in

46 the medulla to prevent washout of this gradient. These factors combined leave the outer medulla vulnerable to ischemic injury.

Post ischemic kidneys display hemodynamic alterations. Adequate blood flow and hydrostatic pressure from the perfusion are necessary for proper renal filtration activity. A hallmark hypoperfusion of the outer medulla and congestion is present despite normalization of blood flow to the renal cortex (Yamamoto, Wilson, & Baumal,

1984). This perpetuates a vicious cycle of tubulointerstitial hypoxia that results in tubular cell damage and apoptosis, which activates interstitial fibrosis and inflammatory responses that ultimately impairs oxygen delivery to the cells, which further aggravates hypoxia. The vascular congestion of the outer medulla is a key player in ischemic renal failure, marked by inadequate renal perfusion (Mason,

Torhorst, & Welsch, Role of the medullary perfusion defect in the pathogenesis of ischemic renal failure, 1984; Mason, Joeris, Welsch, & Kriz W, 1989).

Tubular metabolism is altered by ischemia. The fall of ATP levels, calcium overload and the subsequent generation of ROS cause renal tubular injury through cell membrane destruction, DNA damage, cellular necrosis and apoptosis. ATP depletion disrupts the actin cytoskeleton and the microvillar structures turn into blebs in the extracellular space (Schrier, Wang, Poole, & Mitra, 2004). These blebs and cellular debris can end up in the tubular lumen, contributing to cast formation, local edema and tubular obstruction. Damage to the actin cytoskeleton disrupts the organization of tight junctions, which can cause tubular back leak, forcing the urinary filtrate from the tubules to the interstitium and absorbed into the peritubular capillaries (Sheridan & Bonventre, 2000). This exacerbates ischemic injury and

47 facilitates vacuole formation inside the cells, pyknotic nuclei undergoing and cells detaching from the basement membrane. The basolateral cytoskeleton damage during ischemia triggers the loss of Na+/K+-ATPase polarity and redistribution of integrins from the basement membrane to the apical membrane. This compromises sodium reabsorption and tubule epithelial integrity. Decreased tubular sodium reabsorption triggers the tubuloglomerular feedback which decreases glomerular filtration rate, which substantially reduces the need for energy-dependent tubular resorption (Schrier, Wang, Poole, & Mitra, 2004).

There are two waves of apoptosis that occur from ischemic injury. The first happens within 6-12 hours and it peaks at 3 days. Both intrinsic and extrinsic factors are activated, the former through Bcl-2 family, cytochrome c and caspase 9 and the latter through Fas, FADD and caspase 8. Crosstalk between these two pathways is facilitated by Bid. On the other hand, heat shock proteins inhibit apoptosis and reinstate denatured proteins to restore cytoskeletal integrity and Na+/K+-ATPase polarity to restore normal cellular function. Growth factors such as hepatocyte growth factor (HGF), IGF-1 and hepatocyte nuclear factor (Hnf-1B) play a role in the regeneration and proliferation of tubular repair after the ischemic injury (Devarajan,

2006; Faguer, et al., 2013). Heme oxygenase-1 (HO-1) is induced after cellular stress and has a protective role during ischemia reperfusion injury. It is expressed in the medulla and preserves medullary blood flow (Regner & Roman, 2012). While under normal circumstances NO has positive vasodilatory effects, NO produced during the propagation of ischemia reperfusion injury can exacerbate oxidative stress (Regner

& Roman, 2012).

Extensive studies show that the vascular endothelium is a source of inflammatory cascades, and the endothelial cells also experience actin cytoskeleton and cell polarity disruption. Endothelial edema and blebbing contributes to hypoperfusion of the renal vasculature with a disproportionate amount of reduction in the outer medulla. Vasoconstriction in small arterioles are seen in post ischemic kidneys due to the overexpression of endothelin-1, adenosine, thromboxane, prostaglandin, platelet activated factor and leukotrienes (Post, Kellum, Bellomo, &

Vincent, 2017; Devarajan, 2006). Increased sympathetic nervous system activation results in circulation of vasoconstrictors norepinephrine and stimulation of renin- angiotensin system. The reduction in a crucial vasodilatory factor production such as acetylcholine (ACh), nitric oxide (NO), and bradykinin (BK), and the damaged cells becoming refractory to the vasodilatory actions amplify vasoconstriction (Baylis,

Harton, & Engels, 1990; Post, Kellum, Bellomo, & Vincent, 2017). Vasoactive cytokines released with the inflammatory cascade activation obstruct the vasculature, and upregulation of chemokine receptors can lead to necrotizing crescentic glomerulonephritis (Ding, et al., 2006). Ischemic kidneys show abnormal vascular reactivity by responding to a reduction in perfusion pressure with vasoconstriction.

This is presented with hypersensitivity to sympathetic nervous system outputs and the loss of vasodilatory effects of ACh which inevitably leads to increased vascular resistance (Bilde, 1976; Kelleher, Robinette, & Conger, 1984).

Section 4: ORGAN ASSESSMENT

Organ quality assessments prior to transplantation are currently limited to biopsies, visual assessments by the procuring surgeons, and vascular resistance measurements, which do not necessarily correlate with clinical transplant outcomes.

Thus, there is a critical need for new and improved methods that can better preserve and/or recondition kidneys. This is especially crucial for organs from ECDs or DCDs that would have otherwise not been used, and it would decrease insensible discarding of organs from these groups that could be used. More importantly, the development and validation of real-time noninvasive whole organ quality assessments is crucial in excluding poor quality organs from transplantation.

RESISTIVE INDEX: Doppler ultrasound is a noninvasive, inexpensive method for imaging flow and evaluating blood circulation. High frequency ultrasonic waves emitted by the transducer are focused by using gel as a conductor. Echoes from the erythrocytes or other scatterers in the vasculature return to the receiver, which is used for spectral analysis (Nuffer, Rupasov, Bekal, Murtha, & Bhatt, 2017).

Ultrasound doppler measurement of renal resistive index is clinically used detect organ damage. It is typically measured at arcuate arteries (at the corticomedullary junction) or interlobar arteries of the kidneys. Resistive index is calculated by the difference between the peak systolic and end-diastolic blood velocities divided by the peak systolic velocity, and ranges from 0 to 1 (O'Neill, 2014; Tublin, Bude, & Platt,

2003). In healthy subjects, the renal resistive index averaged 0.60 (Tublin, Bude, &

Platt, 2003). It is used to assess renal allograft rejection, hydronephrosis, renal vein thrombosis, chronic kidney disease, and arterial stenosis (Boddi, Natucci, & Ciani,

2015). Increased renal resistive index is associated with tubulointerstitial fibrosis, atherosclerosis and primary hypertension in patients, indicating that vascular stiffening can influence resistive index changes (Viazzi, Leoncini, Derchi, &

Pontremoli, 2014). Several studies show the lack of change in resistive index with vasoconstrictor and vasodilators administration in human carotid arteries and retinal arteries (Loquet, et al. 1991, E. Polska, et al. 2001) and it is suggested that RI elevation is more likely to be present with a vascular or tubulointerstitial renal abnormalities, and is much less likely with glomerular damage (Platt, Ellis, Rubin,

DiPietro, & Sedman, 1990). Resistive index does not always imply vascular resistance because it does not consider for vascular compliance and capacitance that occurs in response to pressure changes (Tublin, Bude, & Platt, 2003). Furthermore, systemic pulse rate, heart rhythm, sex, age and body mass index have also shown to influence this measurement (Ponte, et al., 2013). A study that measured renal resistive index in the segmental arteries in 601 transplant patients 3 months after the surgeries found that patients with higher arterial resistance index presented with less creatinine clearance, required dialysis and had greater risk of mortality (Radermacher, et al.,

2003). Some studies found that it is not specific indicator for organ rejection posttransplant but rather an overarching marker for vascular damage (Seiler, et al.,

2012). Resistive indices as a diagnostic parameter is incredibly weak since some kidney diseases with considerable loss of renal function present with little or no change in renal vascular resistance (Platt J. , 1997).

BIOPSIES: Pre-transplant donor biopsies are commonly taken to assess the state of the organ before it is placed in a patient. This is especially practiced with

51 marginal donor organs that are projected to result in more graft function problems with higher risk of non-function. Hematoxylin and eosin (H&E), periodic acid–Schiff

(PAS), Masson's trichrome, Jones methenamine silver (JMS) are generally used for staining the needle or wedge biopsies. Out of the total deceased donor pool of kidneys,

37.3% of were not used because of biopsy findings. Interpretation of the biopsy results is the most common reason for discard (Kaiske, et al., 2014). This method was thought to be useful in assessing organ quality, but there have been heterogeneous results on its ability to accurately and reproducibly predict graft outcome. A single center study found that both needle and wedge biopsies can determine vascular damage, whereas interstitial fibrosis and glomerulosclerosis were over-scored in wedge biopsies (Mazzucco, Magnani, Fortunato, Todesco, & Monga, 2010). A Medline search for a correlation between implantation biopsies and outcome found that biopsies can give some insight on graft prognosis in the case of expanded criteria kidneys (El-Husseini, Sabry, Zahran, & Shoker, 2007). Glomerulosclerosis on the biopsy has previously been associated with lower graft function and survival.

However, out of the 14 retrospective studies that used glomerulosclerosis as a marker, it did not accurately predict graft failure in 7 of these studies (Wang,

Wetmore, Crary, & Kasiske, 2015; Naesens, 2016). Out of the deceased donor kidneys, approximately 19% of SCD and 75% of ECD kidneys are biopsied. Based on the biopsy findings 41% of the ECD kidneys were discarded despite no established correlation between the degree of glomerulosclerosis and the odds of delayed graft function

(Sung, et al., 2008). A multi-center study evaluating the relationship of acute tubular necrosis and graft function found no significant association between the pre-implant

52 biopsies and delayed graft function (Hall, et al., 2014; Malek, 2014). While there has been an increase in the number of donated ECD kidneys, their overall use dropped due to the perhaps inappropriate discard based on biopsy results. This method has limited value and it should be cautiously used in conjunction with other assessment methods for kidney evaluation.

BIOMARKERS: Biomarkers are biological markers that can be quantitatively measured accurately and reproducibly. They can be used to indicate disease progression, pathological development, pharmacological responses along with normal biological processes (Strimbu & Tavel, 2010). There aren’t many pre- transplant biomarkers that can successfully predict delayed graft function, primary non-function and the overall success of the transplantation. Tissue damage markers from renal effluent have been investigated to be used as a tool for organ assessment.

This pre-transplant method, not unlike renal resistive index and biopsy measurements, has mixed results in its prognostic capabilities. Biomarkers such as

Glutathione S-transferase (GST), alanine-aminopeptidase (AAP) and fatty acid binding protein were not found to correlate with long-term renal function (Gok, et al.,

2003). Conversely, other groups found GST, AAP, N-acetyl-β-d-glucosaminidase

(NAG), and heart-type fatty acid binding protein (H-FABP) to have predictive capabilities of delayed graft function but not for graft survival (Moers, et al., 2010).

Retrospective studies on biomarkers collected from the perfusate of machine perfused kidneys during the preservation period found that lactate dehydrogenase

(LDH), aspartate aminotransferase (AST), and AAP could not predict graft function

(Moers, et al., 2010), while others cite LDH and AST has having predictive properties

(Bhangoo, Hall, Reese, & Parikh, 2012). Other studies have shown that LDH and IL-18 were associated with primary non-function of the transplanted kidneys, but they lacked diagnostic accuracy (Hoogland, et al., 2013). There are however, some post- transplant biomarkers measured in the serum that reflects renal damage and function better than the standard serum creatinine. Plasma cystatin-C, AST, heart-type fatty acid–binding protein (H-FABP), neutrophil gelatinase–associated lipocalin (NGAL)

(Jochmans, Lerut, van Pelt, Monbaliu, & Pirenne, 2011; Coca, Yalavarthy, Concato, &

Parikh, 2008) and urinary kidney injury molecule-1 (KIM-1), NGAL, IL-18, and GST reflected renal injury and dysfunction and mortality risk (Coca, Yalavarthy, Concato,

& Parikh, 2008; Han, et al., 2008). Serum phosphorus levels traditionally have indicated chronic renal disease and cardiovascular disease, with hyperphosphatemia inducing vascular calcification and hemodynamic changes. There was significant association between recipient pre-transplant serum phosphorus and mortality post renal transplant, but this association existed only at very high levels of phosphorus

(Sampaio, et al., 2001). While biomarkers could be a valuable parameter for assessing renal damage, they lack the precision and accuracy to be used as a stand-alone method for predicting transplant outcomes.

OXYGEN CONSUMPTION RATE: Oxygen consumption and energy use is necessary to maintain homeostasis and fuel ion pumps, protein turnover, RNA and

DNA turnover, substrate cycling, signal transduction and many other biological processes (Rolfe & Brown, 1997). Oxygen consumption rate (OCR) in cells is used as a biochemical marker of metabolism, and several studies demonstrate its capability as a parameter for organ viability. OCR can discriminate between viable and dead

54 cells in preparations with mixed ratios of live and heat-treated cells, with OCR being linearly proportional to cell membrane integrity (Papas, Pisania, Wu, Weir, & Colton,

2007). OCR can also be used to detect real time changes in oxidative metabolism. In kidneys perfused with hypothermic solutions showed a decrease in oxygen consumption compared to the control kidneys, and this consumption rate was recovered upon rewarming the organs to normothermia(Levy, 1959) Real time change in OCR in rat hearts were demonstrated by measuring the consumption rate before and after potassium chloride induced arrest. OCR steadily decreased after cardiac arrest but recovered to the same level of oxygen uptake upon resuscitation

(Lochner, Arnold, & Muller-Ruchholtz, 1968). OCR measurements in porcine kidneys harvested from non-heart beating donors show that it can discern between healthy and unhealthy organs. In the same kidney, OCR was measured before and after it experienced formalin induced damage. OCR decreased from 187 nmol/min/g to 17 nmol/min/g, reflecting its ability to discern between healthy and damaged tissue

(Weegman, et al., 2010).

Several studies on islets demonstrated OCR’s prognostic capabilities in transplant outcomes. In a murine model of streptozotocin induced diabetes, purified human islets were transplanted in the renal subcapsule to normalize blood glucose.

Diabetes reversal was dependent on the OCR normalized to islet prep DNA, with 86% of mice with high OCR and 25% of mice with low OCR reaching diabetes reversal

(Papas, et al., 2007). Pigs implanted with embryos with higher OCR could carry piglets to term whereas pigs with low OCR embryos could not become pregnant (Sakagami, et al., 2015). In a small case study of 13 patients who received islet

55 allotransplantation, those who received islets with higher levels of OCR reached insulin independence within 45 days (Kizmann, et al., 2014). Similar to the allotransplants, OCR normalized to DNA for the number of viable islets was highly correlated to insulin independence 6-12 months after the autotransplant (Papas, et al., 2015). Clinical data show patients with glomerulonephritis with low glomerular filtration rate (GFR) presented with low OCR despite normal and increased perfusion to the kidneys (Cargill & Hickam, 1949). Rat studies show a correlation between OCR and GFR, linking higher oxygen consumption to better renal function after preservation using hypothermic machine perfusion (Bunegin, Tolstykh, Gelineau,

Cosimi, & Anderson, 2013). In bovine kidneys, increased warm ischemia time and ischemic insult decreased OCR, and this was associated with reduction in GFR and urine flow (Stubenitsky, et al., 2000). OCR has also been used in liver, composite tissue, and endothelium as a parameter for viability assessments (Yang, Jia, Acker,

Lung, & McGann, 2000; Zhang, Ohkohchi, Oikawa, Sasaki, & Satomi, 2000;

Steinlechner-Maran, et al., 1997; Schwitalla, Heres, Röhl, Pfau, & Kessling, 2001).

DYNAMIC CONTRAST IMAGING: Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has burgeoned as an assessment tool in many different diseases. One of its major benefits is that it is noninvasive, and it’s been studied in the detection, characterization and therapy monitoring of heart failure, cancers, and renal disease. It has several advantages over the more traditional tests in quantifying renal function such as endogenous creatinine and inulin clearance measurements, renal scintigraphy and contrast enhanced computed tomography. DCE-MRI doesn’t require multiple blood and urine sampling and does not have the risk of radiation and

56 nephrotoxicity from contrast agents. A diffusible tracer or contrast agent is administered into the blood or to the perfusate and T1-weighted images are taken over a time series. The transit of contrast agent in the tissue is denoted through the change in magnetic resonance (MR) signal intensities. Under the magnetic field, the protons’ axes from water molecules aligns and creates a magnetic vector along the axis of the scanner. Additional pulse sequences of radio waves are added, and when this is turned off, the magnetic vector returns to resting state and emits a signal. This is dependent on proton relaxation dynamics. MR instruments are sensitive to two different relaxation processes, the T1 (longitudinal relaxation time) and T2

(transverse relaxation time). Receiver coils are used to receive this signal, and these signals are plotted to form cross-sectional images over time.

There are variations in the length of acquiring and processing images before, during and after the contrast agent injection, conversion of the gadolinium signal intensity into the concentration in the organ compartments, and fitting these concentration curves over time (Chandarana & Lee, 2009). The movement of contrast agent and its kinetics are dependent on the perfusion flow rate, vascular permeability and the volume of the extravascular space in the tissue. The most commonly used contrast agent is gadopentetate dimeglumine (Gd-DTPA), which is gadolinium bound to a chelating agent to control toxicity. Images acquired from DCE-MRI can be quantitatively analyzed through parametric techniques. Kinetic parameters are estimated by using mathematical pharmacokinetic models designed for specific tissue type to fit the concentration curves of the contrast agent. The signal intensity- time curves acquired from images during the wash-in, plateau, and washout contrast

57 kinetics reflects tissue perfusion (Essig, et al., 2013). These models work under different assumptions and simplifications to understand the physiology of various tissue types (Khalifa, et al., 2014). There are many different acquisition techniques and models that can be used to interpret DCE-MRI data.

Compartmental models are commonly used with the assumption that the contrast agent is uniformly distributed at any given time and the flux movement of the contrast agent is proportional to its concentration. The arterial input function

(AIF) is one of the major factors for pharmacokinetic models in imaging. It typically refers to the large, major artery that carries the contrast agent into the organ or tissue.

The first pass AIF is characterized by a strong signal with short initial peak that diminishes over time as the blood or perfusate disperses the contrast agent to the branching vessels. The MR signal intensity reflects contrast agent concentration when this relationship is linear, but this is usually not the case because the signal can saturate if the contrast agent levels are too high. In a two-compartment model, the contrast agent uptake from the intravascular space to the extravascular space is used to study tissue perfusion. In the case of the kidney, the intravascular space is the blood vessels and the extravascular space is the renal tubules. Once the bolus of Gd-DTPA enters the renal artery, the cortex is visible followed by medullary enhancement. The tracer that enters the renal capillaries does not exit the kidney for approximately 90 seconds (Tofts, et al., 2008). Clinically relevant parameters as the renal volume, GFR and renal blood flow can be interpreted (Bokacheva, Rusinek, Zhang, & Lee, 2008).

The total filtration of the kidneys is measured by using the transfer constant (Ktrans)

58 and renal mass. Ktrans essentially measures the permeability of capillaries using contrast agent concentrations in the extravascular/extracellular space.

Many studies have demonstrated MRI’s ability to evaluate and measure renal function in animal and clinical studies. In rabbit kidneys, GFR values obtained from measuring plasma 51Cr-EDTA clearance rate and DCE-MRI were correlated (Annet, et al., 2004). Early clinical studies comparing MRI and scintigraphic procedures found that MR could accurately evaluate renal physiological parameters. They showed close correlation of the MR data of healthy kidneys to isotopic uptake in all patients.

However, MR functional results from kidneys with ischemic damage were overestimated (Laissy, et al., 1994). Tofts et al. found that renal parameters such as filtration rate, filtration fraction, blood volume and perfusion rates measured from human subjects using DCE-MRI were comparable to the values reported in the literature (Tofts, Cutajar, Mendichovszky, Peters, & Gordon, 2012). GFR measured with plasma clearance of iopromide was compared to single-kidney GFR measured using MRI, and high correlation was found when GFR was calculated 40-110 seconds after the aortic signal (Hackstein, Heckrodt, & Rau, 2003). In another similar study, the MRI derived GFR was 109.0 mL/min per 1.73 m2 ± 17.1 while the iopromide clearance rate derived GFR was 103.1 mL/min per 1.73 m2 ± 9.4 (Boss, et al., 2007).

In a few instances, MRI was used to evaluate graft function in kidney transplant recipients. A retrospective case study on the relationship between kidney volume and graft survival found kidney volume to recipient weight ratio less than 2 cm3/kg accompanied lower GFR (Saxena, Busque, Arjane, Myers, & Tan, 2004). Transplant patients with acute tubularnephritis did not show the classic biphasic pattern of

59 increased signal intensity in the cortex and medulla followed by a decreased signal in the medulla that is seen in healthy kidneys (Szolar, et al., 1997).

Section 5: ORGAN PRESERVATION

Organ preservation methods have evolved since the advent of transplantation research. The broad categories of preservation methods can be divided into static and dynamic methods. Further modifications of these methods have been explored with regards to changing the composition of preservation solutions, modifying the temperature of dynamic perfusion methods, pharmacological agent supplementation and the utilization of oxygen.

COLD STORAGE: Static cold storage is the most widely used preservation method due to its logistical simplicity and low cost. Procured organs are flushed with preservation solutions, placed in a sterile bag or container with the solution and immersed in ice. In the 1960s the Euro-Collins solution was developed to mimic the intracellular ionic composition using high potassium, and it enabled kidney preservation up to 36 hours (Guibert, et al., 2011; Southard & Belzer, 1995). High levels of glucose ended up being a contributing factor for lactate production and acidosis. In the 1980s the University of Wisconsin (UW) solution was developed with the assumption that interstitial spaces may not follow the stoichiometric rule of 3 Na+ out, 2 K+ in exchanger activity. UW has a high Na+ content along with biologically inert compounds such as lactobionate to offset edema (Guibert, et al., 2011). It is considered the gold standard and many studies from various organ types (pancreas, heart, kidney, liver, lungs, and intestines) demonstrated its superiority over the Euro-

Collins solution (Southard & Belzer, 1995; Swanson, Pasaoglu, Berkoff, Southard, &

Hegge, 1988; Olthoff, et al., 1990; Todo, et al., 1989; Ploeg, 1990). Since then, many other storage solutions with varying modification emerged. The HTK solution was originally developed for cardioplegia but now is used for preserving kidneys, livers and hearts. It has low concentrations of Na+, K+, and Mg+ and low viscosity that help in rapidly cooling organs. Renal grafts preserved with HTK in terms of initial non- function and graft survival were comparable to UW but superior to Euro–Collins (de

Boer, et al., 1999). In adult liver transplants, there were no significant differences between the two solutions with regards to graft function and patient survival

(Magnus, et al., 2006).

The overarching criteria for preservation solutions are to prevent cellular swelling, prevent intracellular acidosis and electrolyte imbalances, minimize ROS production and block enzymes that facilitate ROS production, contain agents that stimulate high-energy phosphate generation and physiological buffers to maintain pH

(Belzer & Southard, 1988; Moukarzel, Chalouhy, & Chemaly, 2015). Impermeant anions with large molecular mass and colloids such as hydroxylethyl starch manage the osmotic pressure to prevent cellular edema. ATP precursor such as adenosine is incorporated to facilitate energy production with organ reperfusion. Glutathione is used to reduce ROS and lipid peroxides to minimize reperfusion injury (Belzer &

Southard, 1988). They also contain electrolytes (Na+, K+, Ca+, Mg+), H+ ion buffers to regulate extracellular H+, metabolic inhibitors such as allopurinol that inhibits xanthine oxidase and antioxidants such as deferoxamine to reduce free radical injuries (Wille, de Groot, & Rauen, 2008). While the improvement in preservation

61 solutions enabled the successful storage of organs ex vivo, a certain percentage of transplanted organs experience delayed graft function and primary non-function. The use of these storage solutions in conjunction with other preservation technologies and pharmacological maneuvers may alleviate the dysfunction of organs once they are transplanted.

HYPOTHERMIC MACHINE PERFUSION: Hypothermic machine perfusion (HMP) is a preservation and storage method that cools down the whole organ with liquid perfusion. It is currently FDA approved for kidney preservation, but clinically not available for hearts and livers. It is largely based on evenly, rapidly reducing the core organ temperature to avoid ischemic and hypoxic injuries that are inevitable during organ procurement and storage (Fuller & Lee, 2007). Lower temperatures slow the physical and chemical processes and attenuate the cellular damage that occurs with energy depletion and metabolic uncoupling. Hypothermic perfusion can first and foremost deliver atmospheric oxygen and nutrients while removing the toxic metabolic byproducts that otherwise would remain in the organ. This allows for aerobically derived energy that can maintain ion gradients and pH control. It maintains vascular patency through pulsatile perfusion and facilitates the delivery of cryoprotective agents and pharmacological interventions that can improve organ quality through the machine that is already set up (Taylor, 2007). Furthermore, perfusion machines can measure pre-transplant parameters such as arterial pressure, renal flow and resistance of deceased donor kidneys that has the potential for prognostic significance (Polyak, Boykin, Arrington, Stubenbord, & Kinkhabwala,

1997).

Several studies demonstrate the benefits of using hypothermic machine perfusion over static cold storage. Perfused kidneys had lower renal vascular resistance, higher flow rate and maintained endothelial cell dependent vasodilation

(Hansen, D'Alessandro, & Southard, 1997). Rodent livers that were perfused had less superoxide formation and lipid peroxidation (Dutkowski, et al., 1999) and damaged rodent hearts that were perfused during storage had greater power recovery that traditionally stored hearts (Nickless, Rabinov, Richards, Conyers, & Rosenfeldt,

1998). Perfused hearts exhibited lower apoptotic myocyte death, higher myocardial oxygen consumption and graft function (Peltz, et al., 2005; Rosenbaum, et al., 2008).

Porcine studies have shown that HMP is effective if administered at the start of preservation, and its benefits are diminished if it is used after hours of cold storage

(Hosgood, Mohamed, Bagul, & Nicholson, 2011). In 2009 a large-scale international clinical study was done by assigning one deceased donor kidney to static cold storage and the other to hypothermic machine perfusion and each kidney was transplanted to a recipient. HMP significantly reduced the rates and duration of delayed graft function with no adverse events originating from this preservation method (Moers, et al., 2009). This benefit is particularly pronounced in ECD kidneys, with significantly lower delayed graft function, incidence of nonfunction and better 1-year graft survival in the HMP kidney recipients (Treckmann, et al., 2011; Gallinat, et al., 2012).

There are however no significant differences in the incidence of primary non- function, serum creatinine level and clearance and acute rejection (Jiao, et al., 2013;

Moers, et al., 2009).

Despite these studies, this method is not without its negative effects. Perfused canine kidneys presented with nuclear pyknosis, and dilation of endoplasmic reticulum membranes. These characteristics were found in all cell types, especially endothelial cells (Craig, Seibel, & Abouna, 1977).In isolated livers, LDH levels became significantly higher in HMP than in SCS after reperfusion and endothelial permeability increase was noted (Xu, Lee, Clemens, & Zhang, 2004). Paired studies using cold storage and pulsatile perfusion for renal allografts found severe endothelial and basement membrane damage with capillary congestion in the perfused kidneys

(Cerra, Raza, Andres, & Siegel, 1977), and these types of damages can be mitigated by using the lowest perfusion pressure possible (Grundmann, Raab, Meusel, Kirchhoff,

& Pichlmaier, 1975). HMP kidneys showed an increase in tubular cell dilatation and interstitial edema. They also had higher levels of lipid peroxidation which can induce inflammatory responses and mediate ischemia reperfusion injury (Hosgood, Yang,

Bagul, Mohamed, & Nicholson, 2010). Despite some of these limitations, HMP is currently the state of the art kidney preservation method that can expand the donor pool by conditioning marginal kidneys. It has potential to not only better maintain the organ quality but can measure a slew or renal parameters and biomarkers from the perfusate to help choose the organs that are predicted to have optimal clinical performance.

PERSUFFLATION: There is a huge interest in delivering oxygen to the organ to ameliorate the hypoxic and ischemic damage that occurs during the organ procurement and transport. It is one of the first metabolites to be exhausted during the ischemic state, and it is crucial for aerobic metabolism to synthesize sufficient ATP

64 levels to maintain homeostasis. Static cold storage depends on the passive diffusion of oxygen to the core of the organ, and it only penetrates a millimeter in from the organ surface when immersed in perfluorocarbon with 100% oxygen (Agrawal, et al.,

2010; Papas, et al., 2005). Hypothermic machine perfusion is known to ‘rescue’ marginal organs from delayed graft function but the exact mechanism in which it exerts its protective effects remains unclear. While some experiments state the sufficiency of atmospheric oxygen in maintaining liver sections during machine perfusion ('t Hart, et al., 2005), others have found that 100% oxygen in whole livers decreased oxidative damage (Lüer, Koetting, Efferz, & Minor, 2010; Minor, Klauke,

Vollmar, Isselhard, & Menger, 1997). Persufflation (PSF), the technique that delivers humidified, gaseous oxygen into the organ through the vasculature, may have its advantages over the other two preservation methods in terms of evenly oxygenating the whole organ during storage.

The concept of persufflation was serendipitously discovered when an isolated heart was left perfusing with defibrinated blood. Once all the blood was used, the perfusion circuit circulated oxygen through the heart and maintained rhythmic contractions for some time. In the literature both anterograde and retrograde persufflation has been used with the former using the arterial and latter using the venous system to deliver oxygen to the organ. The oxygen supply during persufflation can be connected directly to the arterial line for anterograde persufflation. For retrograde persufflation, oxygen is connected to the venous line in opposite flow of normal physiology, with needle pricks introduced to the anterior and posterior side of the kidney to allow the air to escape. Retrograde persufflation has been favored in

65 kidneys due to experiments showing higher mortality rates, decreased renal perfusion and uremia in the anterograde persufflated kidney transplants (Denecke,

Isselhard, Stelter, Berger, & Sachweh, 1971). Subsequent studies reported anterograde PSF kidneys presented with slower recovery of renal function after transplant, while the retrograde PSF kidneys had shorter recovery times to restore normal renal function (Isselhard, et al., 1972; Isselhard, et al., 1974). However, the reasons as to why anterograde persufflation presented with mixed results has not been studied extensively, and anterograde persufflation has been successfully used in hearts, pancreas and kidneys. One of the major concerns and a source of reluctance to use persufflation in the clinic are the fear of deleterious effects on the vascular endothelium from pressured gas and high levels of oxygen creating free radical damage. Several studies using hearts have shown that the normal hemodynamic flow, endothelium derived relaxation is preserved with equal amount of damage between

PSF and SCS hearts (Kuhn-Regnier, et al., 2004; Fischer, Funcke, Yotsumoto, Jeschkeit-

Schubbert, & Kuhn-Regnier, 2004).

Early studies of renal persufflation presents with mixed results. This might be attributed to many varying factors such as the persufflation technique, length of preservation, preservation solutions that were used, and in vivo versus ex vivo organ examinations. In the early 1970s, canine kidneys that were flushed and persufflated in vivo for 2-4 hours had less tubular damage and maintained renal function compared to kidneys that were flushed and then left without circulation for 2 hours

(Talbert, Riley, & Sabiston, 1961; Suszynski, et al., 2012). Following this study, PSF and SCS were compared in vivo. After 4 hours of the respective preservation methods,

66 the contralateral kidneys were removed and each animal was left with either a PSF or SCS kidney at the time of reperfusion. In this study, the anterograde PSF treated animals fared worse with over half of the group dying within the first 7 days

(Denecke, Isselhard, Stelter, Berger, & Sachweh, 1971). Isselhard and colleagues lead a series of experiments that compared anterograde and retrograde PSF, and coined the term presumably derived from the existing term insufflation. Compared to control kidneys, ischemic kidneys had a decrease in ATP levels by 50% in 30 minutes, while it took 48 hours in PSF kidneys for a similar loss (Isselhard, Denecke, Witte, Berger,

& Fischer, 1972). While anterograde PSF diminished the rapid depletion of ATP, post ischemic recovery and survival rates were better in canine recipients with the SCS kidneys (Isselhard, et al., 1973). In the case of retrograde PSF, it maintained higher levels of ATP than SCS kidneys in the tissue and the recovery time for inulin and PAH clearance, urea excretion, serum urea and creatinine levels were shorter with better renal function (Isselhard, et al., 1974).

Towards the late 1970s, retrograde PSF, perfusion and cold storage was tested in kidneys with 30 minutes of warm ischemia time. Kidneys treated with either PSF or HMP had renal function and higher urine production at 1-3 hours and 24 hours after the autotransplant, showing their potential for ameliorating ischemic injury extending organ preservation time (Fischer, Czerniak, Hauer, & Isselhard, 1978).

After these series of immediate kidney function observations, long-term function of canine autotransplants were used to demonstrate the benefits of oxygenating the organ through the vasculature. Kidneys from control, retrograde PSF, anterograde

PSF, flushed and immersed with oxygenated solution and flushed with oxygenated

67 solution with no further oxygenation after 30 minutes of warm ischemia time were assessed. There was a significant decrease in creatinine levels at day 14 and day 21 from both types of PSF compared to the other three groups of treatment (Ross &

Escott, 1979). Almost a decade later a massive autotransplant study was undertaken to study the effects of 60 minutes of WIT and then 24 hours of preservation with control, PSF, atmospheric air, nitrogen and helium. In tandem, 30 minutes of warm ischemia with 48 hours of preservation was studied with no PSF and PSF. 8 pricks

1mm deep were made with a needle on both anterior and posterior surfaces for the kidneys that would be insufflated to maintain even oxygen tension. Out of the five preservation methods in the 60-minute WIT group, PSF kidneys performed the best, followed by atmospheric air. Control, nitrogen and helium treated kidneys could not support life post-operatively for the full 3-month duration of the experiment.

Similarly, only 1 out of 5 no PSF kidney recipients survived long term while 4 out of 5 survived in the PSF group for kidneys with 30 minutes of WIT after 48 hours of storage (Rolles, Foreman, & Pegg, 1984). Interestingly enough, they did not see any treatment dependent effects on adenine nucleotide metabolism. The total adenine nucleotide, ATP and ADP content in PSF kidneys were comparable to kidneys perfused with atmospheric air and nitrogen (Rolles, Foreman, & Pegg, 1989).

To understand the mechanism behind retrograde PSF, rabbit kidneys with 60 minutes of warm ischemia time were stored with or without PSF. After the preservation period, ATP levels, cytochrome oxidase activity and morphology were examined. The ATP levels were higher in the treated kidneys, consistent with a previous study done with canine kidneys (Pegg, Foreman, Hunt, & Diaper, 1989). ATP

68 was also being synthesized and utilized during the hypothermic storage period, and the kidneys that were untreated had higher cellular edema and cytological injuries

(Pegg, Foreman, Hunt, & Diaper, 1989). The persufflated kidneys presented with a uniform mitochondrial configuration, especially in the proximal and distal tubules that are known to have high levels of metabolic activity while the cold storage kidney tubules displayed condensed mitochondria with dense matrixes and swelling (Pegg,

Foreman, Hunt, & Diaper, 1989). The presence of oxygen is the crucial ingredient for

ATP generation during hypothermia. Warm ischemia of 30 minutes reduced the total adenine nucleotides to 25% of a control kidney, and continued to deplete during cold storage. Persufflated kidneys preserved with UW solution could generate adenine nucleotides, even when preserved with UW that did not contain adenosine, implying that oxygen as the rate limiting step. Despite these differences, there were no statistically significant differences in functional outcomes between the two kidneys after heterotrophic autotransplant (Yin, et al., 1996).

Results from animal studies were deemed sufficient to venture into clinical trials. 10 pairs of donor kidneys were used for PSF and SCS preservation and transplanted into recipients. It is hard to make any wide sweeping claims about persufflation due to the small sample size, considerable variability between the donors, preservation time and recipient health. Out of the 10 PSF kidneys, 4 were scored as excellent, 2 were satisfactory, 2 had delayed graft function and the last 2 had to be removed due to venous thrombosis and acute tubular necrosis. Out of the

10 SCS kidneys, 5 were satisfactory, 4 had delayed graft function and 1 infarcted and had to be removed (Rolles, Foreman, & Pegg, 1989). Unfortunately, this study was not

69 followed up and in the mid- 2000 porcine studies were picked up to test retrograde

PSF after successful studies in porcine livers. Warm ischemia time of 60, 90 and 120 minutes was tested with retrograde PSF and SCS for 4 hours. These kidneys were autotransplanted into pigs and blood and urine samples were collected to assess renal function. All animals with 60 minutes of WIT survived while few died in the other groups from uremia. No benefits of retrograde PSF were seen in the 90 and 120- minute WIT groups. In the 60-minute group, oxygenated kidneys had significantly lower serum creatinine and urea levels compared to the cold storage counterpart, and the oxygen treatment did not induce increased lipid peroxidation (Treckmann, et al.,

2006). Follow this study Treckmann and colleagues compared 4 hours of SCS, HMP and retrograde PSF and found PSF was associated with better renal function and lower levels of lipidperoxidation markers (Treckmann, et al., 2006). PSF is also associated with higher renal vascular flow rates, enhanced urine output and less cellular debris and better preserved epithelial lining just after 90 minutes of exposure

(Minor, Efferz, & Lüer, 2012). A recent study compared used kidneys exposed to 20 minutes of warm ischemia time followed by 60 minutes of HMP and retrograde PSF in porcine kidneys. These kidneys were autotransplanted and observed for 2 hours before the animals were sacrificed. Investigators found no significant differences between the two groups in terms of urea levels and histology (Moláček, Opatrný,

Matějka, Baxa, & Třeška, 2016).

With the improvement in the quality preservation solutions and the discovery of hypothermic machine perfusion, organ preservation has advanced considerably since the first transplant surgery. Despite these advances, there is a shortage of

70 organs for transplant purposes, which fuels a demand for techniques that will minimize hypothermic, hypoxic damage and ischemia reperfusion injuries.

Persufflation can mitigate oxidative stress, maintain higher levels of energy and extend the preservation time. These are important factors that can overcome maintaining tissue quality and geographical barriers, especially since the use of marginal organs have increased to make up for the organ shortage. Numerous studies that show the benefits of persufflation using different types of organs and using diverse quality assessment assays make it clear that persufflation should be explored further to fully delineate its benefits.

In summary, there is a need for noninvasive organ assessment techniques that can accurately diagnose the quality of the organs and predict their functionality after transplantation. Our lab focuses on developing oxygen consumption rate as a standard for measuring cell and tissue viability, and we’ve adapted using imaging in isolated organs to measure functional parameters. We also have a strong interest in using oxygenation as a means to mitigate the inevitable hypoxic and ischemic damage that occurs during procurement and extends to the preservation periods.

Persufflation poses a unique advantage of utilizing and maximizing the use of sub-par organs by limiting the ischemic injury and even perhaps rescuing the organ from irreversible damage. Conditioning organs prior to allotransplant can moderate the level and intensity of ischemia-reperfusion injury they will experience after the transplant.

Chapter 3

Methodology

Methods

The work presented in this dissertation aimed to advance organ assessment by developing and optimizing a novel, multimodal system that can noninvasively evaluate organ quality and function. Developing a set of objective assessment tools that has the potential to predict clinical outcomes can change the way organs are discarded, allocated and transplanted into patients in a significant way. Based on the results found in the literature and by former students in our lab, we hypothesized that whole organ oxygen consumption rate (WOOCR) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to calculate glomerular filtration ate (GFR) can be used as an indicator of kidney health (Weegman, et al. 2010, Tofts, et al. 2012,

Buchs, et al. 2014, Martin, et al. 2008, Jones, et al. 2011). Additionally, we hypothesized that providing oxygen to the kidneys during hypothermic preservation will resuscitate and or improve organ quality, and WOOCR and GFR measurements will be able to detect differences between different preservation methods. In this chapter, the methods used to collect data for the presented studies are described in detail.

Organ Procurement and Preservation

This study was approved by the institutional animal care and use committee

(IACUC) of the University of Arizona, and all animal care procedures were in accordance with University policies. Domestic female juvenile pigs (35-50 kg) were sourced from a local farm in Wilcox, Arizona.

Heparinized porcine donors were euthanized, exsanguinated and flushed with

2 liters of room temperature, heparinized saline through the abdominal aorta.

Kidneys were procured after exposure to 30 minutes of warm ischemia time (WIT) inside the body cavity. Following procurement, kidneys were cannulated at the renal artery and vein using luer locks (Cole-Parmer). Subsequently, both kidneys were flushed with hanging IV bags of 400 mL of 4°C heparinized saline. Gravity was used instead of a pressure cuff or syringe during the ex vivo flush in order to avoid vascular damage. This was followed by a secondary flush with SPS-1 solutions (Organ

Recovery Systems) using a peristaltic pump at 20mL/min. The kidneys were preserved for 24 hours at 4°C in their respective conditions.

It is important to note that domestic pigs from local farms may have infections or diseases that can directly affect renal health, as shown in Figure 1. Additionally, approximately 30% of pigs have multiple renal arteries (Giraud , et al. 2011), and 88% of pigs have two tributaries forming the main renal vein (Gómez, Ballesteros and

Cortés 2017). Taking measurements from these kidneys without the proper length of renal vessels were not feasible (Figure 2) given that our systems were designed for one inlet and one outlet. Kidneys with abnormalities were excluded from these experiments.

Static cold storage

Cold storage of organs is typically described as ‘throwing them in an ice bucket’. It is the standard way of retaining kidney viability during transport for its simplicity and low cost. After the vascular flush, kidneys were kept in SPS-1 solution at 4°C during the 24 hours of preservation.

Figure 9: Unilateral hydronephrosis in domestic pigs. 6% of the pigs had hydronephrosis; a dilation of the renal pelvis due to obstruction of the urine outflow. The kidney on the right in A) is hydronephrotic with a cyst located in the center of the kidney. This condition was found in the right kidneys in our pig cohort. Some causes of this condition include urinary tract calculi, pyelonephritis and inflammation in the ureters or urethra. B) shows a fluid filled kidney with a dilated renal pelvis and minimal cortex tissue.

Figure 10: Bifurcated renal veins and abnormal flushing. Some of the porcine donors had bifurcated renal vessels. A) shows the drain of two renal veins during the vascular flush with saline. If the vessels were cut too close to the hilum, especially in the case of the renal arteries, the kidneys could not be flushed properly as illustrated in B).

Persufflation

The persufflator prototype (Giner, Inc., Newton, MA) delivered electrochemically generated, humidified, gaseous 40% oxygen mixed with atmospheric air. It maintained a manifold pressure of 40-50 mmHg to generate a flow rate of 60 mL/min. Tubing from the persufflator was attached to the renal artery, and leaks around the vessel were ligated to ensure that the entire kidney was adequately perfused. This tubing was then connected to a luer lock that was drilled into the inside of the lid of a custom-built organ container. A separate tubing was then connected to another luer lock on the outside of the lid and connected to the persufflator port.

Kidneys were kept in SPS-1 solution at 4°C during the 24 hours of preservation.

Figure 11: Image of the Giner persufflator prototype. This portable electrochemical oxygen concentrator mixes ambient air oxygen generated from water to get intermediate oxygen concentrations. Persufflation manifold delivery pressure and flow rate can be controlled at individual outlet ports. These ports can be modified to add filters for a semi-sterile delivery of oxygen to organs.

Hypothermic Machine Perfusion

The LifePort Kidney Transporter is a portable transport device that is mainly used for kidneys from non-standard donors (expanded criteria donors and donors of cardiac death). It is designed to liquid perfuse the kidney under hypothermic, aseptic conditions. The tubing of the organ cassette was properly connected to the pump, valves and sensors on the LifePort deck and filled with SPS-1 (Organ Recovery

Systems). The ice container was packed with water and ice to create a slush that would bring the temperature of the LifePort to 4-8°C. Once the LifePort was turned on, SPS-1 was circulated through the tubing and primed to verify that perfusate was contained within the tubing without bubbles or leaks. Instead of using the seal ring cannula that comes with the sterile cassette, the cannulated renal artery was directly connected to the infusion line of the LifePort. The seal ring cannula requires an aortic patch, and the kidneys that were used for these studies were too small for the aortic vascular tissue to cover the entire sealing ring. The cannulated kidney was placed on the cradle and checked for any torsion and occlusion of the renal artery. The mesh organ restraint then was draped over the kidney to keep it from drying out and to secure it in the cradle.

Kidney Rewarm

After the 24-hour preservation period, kidneys were rewarmed to subnormothermic temperatures (25°C) over a period of 30 minutes. Kidneys that were stored at 4°C in SPS-1 (Organ Recovery Systems) were placed on a liquid perfusion circuit that was gradually warmed using a water bath. The temperature was raised in a stepwise fashion from 4°C to 10°C, 15°C and 25°C. It took less than a minute

77 for the water bath to reach the set temperatures, and at each temperature, kidneys were perfused for 10 minutes to ensure even re-warming. Once we reached subnormothermic temperatures, kidneys were taken off this circuit for assessments.

Assessments included WOOCR, GFR, biomarker levels, histological analysis and

Agilent microarray analysis for kidney evaluation.

Figure 12: Schematic of post-preservation kidney rewarming timeline. Isolated porcine kidneys that were preserved in static cold storage or with persufflation were liquid perfused with SPS-1 solution after the 24-hour preservation period. The perfusion circuit used a water bath and heat exchanger to gradually warm the solution in a stepwise fashion from 10°C to 15°C to 25°C. Each temperature was held for 10 minutes and the kidney was warmed to subnormothermic temperatures over 30 minutes.

Whole Organ Oxygen Consumption Rate

Previous work from our lab on the oxygen consumption rate of islets have shown to be predictive of islet health prior to transplantation (K. K. Papas, M. D. Bellin, et al. 2015, K. K. Papas, C. K. Colton and R. A. Nelson, et al. 2007, K. K. Papas, C. K.

Colton and A. Qipo, et al. 2010). Preliminary data on whole organ oxygen consumption

78 rate (WOOCR) show that this method can be used to measure organ viability and differentiate between healthy pig kidneys and kidneys damaged with formalin

(Weegman, et al. 2010). For these reasons, WOOCR was used to measure kidney viability for pre-transplant assessments.

Flow-through fiber-optic oxygen sensors (Instech Laboratories, Inc.) were calibrated with 0%, 21% and 40% oxygen at 4-8°C prior to data collection. The sensors were connected directly to the hollow fiber oxygenator (Medtronic) on a SPS-

1 (Organ Recovery Systems) recirculating hypothermic perfusion circuit. Nitrogen, laboratory air and 40% oxygen were supplied to the oxygenator with Masterflex® peristaltic tubing (Cole-Parmer). After the calibration, the oxygen sensors were placed inline at the renal arterial cannula and one was placed distal to the venous cannula to measure the difference in oxygen partial pressure at the inlet and the outlet of the organ. The WOOCR (mol/min/g) of a kidney was calculated by using a mass balance equation where Q is the flow rate of perfusion (mL/min), α is oxygen solubility in aqueous solution at temperature (mol/mL/mmHg), and m is the mass of the kidney (g) (Weegman, et al. 2010):

푄 × (푝푂2 − 푝푂2 ) × 훼 푊푂푂퐶푅 = 퐴푟푡푒푟푖푎푙 푉푒푛표푢푠 푚

Figure 13: Schematic of the whole organ oxygen consumption rate perfusion circuit. schematic of the perfusion circuit where the organ is perfused with oxygenated preservation solution (SPS-1) in a recirculating bath maintained at 4°C. Perfusate enters the kidney through the inline flow cells containing an oxygen sensor at the arterial end to the second sensor at the venous end. The fluorometer that couples with oxygen sensors provides oxygen measurements.

Figure 14: Whole organ oxygen consumption rate setup. A depicts the custom built perfusion circuit with the peristaltic pump, flow meter and oxygenator that dispenses the perfusion fluid to the tubing that is directy connected to the oxygen sensor and arterial cannula. B shows the inline fiberoptic oxygen sensors at the renal artery (red) and the renal vein (blue).

Dynamic Contrast Imaging using Magnetic Resonance

In 2011, a group in the UK used magnetic resonance imaging (MRI) data from healthy human volunteers to demonstrate that kidney function can be measured with imaging. They used a two-compartment model to calculate glomerular filtration rate

(GFR) by looking at the change in concentration of the contrast agent over time in the intravascular and extravascular compartments. For kidneys, the intravascular compartment is primarily glomerular; the extravascular compartment is primarily tubular (Tofts, et al. 2012). In 2013 rodent kidneys that were treated with hypothermic machine perfusion and cold storage were used to test renal oxygen consumption rate as a potential way to measure organ viability. They found that oxygen consumption rate during hypothermic machine perfusion correlates with renal function and has the potential to predict better post-preservation glomerular filtration rate in ex vivo rat kidneys (Buchs, et al. 2014).

All imaging was done on a 3T Skyra scanner (Siemens, Erlangen, Germany).

Kidneys were secured in the organ cassette (LifePort) and cold-perfused (4-8°C) with

SPS-1 preservation solution (Organ Recovery Systems) using a MR compatible hypothermic perfusion circuit set up. The organ cassette was inserted in a standard

Siemens Head and Neck RF coil and secured in the coil for a snug fit using towels. 5mL of 2mM Multihance Gadolinium-DTPA solution (Bracco Diagnostics Inc.) was injected over 15 seconds into the cannulated artery after 20 seconds of precontrast, and continuous acquisition was recorded for 4 minutes.

Dynamic-contrast MRI was performed using a golden angle stack-of-star radial acquisition. (Feng, et al. 2016) Compressed Sensing reconstruction with total

81 variation applied in the temporal dimension was used to reconstruct images with a 1 second temporal resolution. (Pandey, et al. 2017, Jones, et al. 2011, Porschen, et al.

2016) The regions of interests are drawn on the reconstructed images (performed by CGM, AP), outlining the kidney margins, and these signals analyzed using a 2- compartment modified Toft’s model for pharmacokinetic modelling (Tofts, et al.

2012); a total of 90 sec post-contrast data was used to provide the maximum amount of data prior to excretion and leakage of contrast outside the ROI. (Jones, et al. 2011)

The arterial input function, the diffusive transport of gadolinium through intravascular and extravascular space was used to calculate Ktrans and glomerular filtration rate of the isolated kidneys.

Figure 15: Schematic of hypothermic perfusion system for dynamic contrast enhanced magnetic resonance imaging. This illustration shows the jacketed media reservoir, peristaltic

82 pump and water cooler are placed in the office area with the MRI viewing software that is separated from the magnet. 30 feet of insulated tubing is connected from the media reservoir to the heat exchanger, which has an injection port for contrast agent infusion. The outlet port of the heat exchanger is connected to the renal artery for cold perfusion of the kidney. A return line connected to a perfusion pump brings back the preservation solution from the organ container to the media reservoir for recirculation.

Figure 16: Magnetic resonance compatible perfusion circuit. This perfusion circuit is comprised of a jacketed medium reservoir that is connected to a water bath for temperature control. A peristaltic pump is used to perfuse SPS-1 from the media reservoir to a water jacketed heat exchanger that is connected to the kidney located in the magnet. A return line that is secured at the bottom of the organ container is connected to a second peristaltic pump to recirculate the media back into the media reservoir to prevent the overflow of the organ container. The tubing that goes to the kidney is insulated to maintain the desired temperature. A and B depict the cart that holds the majority of the perfusion circuit. This cart stays in the office/computer lab because they are not MR compatible. The insulated tubing is fed through a hole on the wall that separates the office and MR. C shows the heat

83 exchanger/oxygenator connected to the kidney container that is inside the coil. D is another view of the isolated kidney inside the magnet.

Figure 17: Kidney imaging and two-compartment model. A shows images of isolated kidney during imaging prior to compressed sensing reconstruction. B depicts the two-compartment model that uses the arterial input function, the transport of gadolinium through intravascular (largely glomeruli) and extravascular (largely tubules) space was used to calculate Ktrans and glomerular filtration rate. A region of interest (ROI) shown as the dotted line in B and yellow line in C was drawn around the parenchyma of reconstructed images for two-compartment analysis. The dotted box represents a traced region of interest or pixel in the kidney, which shows full kidney perfusion

Figure 18: A composite panel of kidney images taken at multiple timepoints during dynamic contrast enhanced magnetic resonance imaging. As the images progress the entrance of

84 gadolinium through the renal artery is visible, and this perfuses to the renal cortex and washes out over time.

Lactate measurement Organ function is critically linked to the available oxygen that can be used to fuel cellular metabolism. The interruption of blood flow and subsequent conditions of low oxygen tensions induces a shift in pathways that are used to produce cellular energy. Hypoxia and ischemia exposed cells use anaerobic metabolism of glucose for inefficient energy generation while creating large amounts of lactate in the process

(Jaeschke and Mitchell 1990). Lactate is a metabolite that is being studied as a biomarker in diagnosing and assessing the severity of systemic hypoperfusion and hypoxia (Fuller and Dellinger 2012, Watson and Heard 2010). Numerous candidate biomarkers are currently being studied because they can be measured from the perfusate or urine accurately and reproducibly. While there are no standalone biomarkers that can guide clinical practice, some correlate more with renal damage than others (Lo, Kaplan and Kirk 2014), and they have the potential to be used in profiling complex pathophysiological conditions of organs prior to transplantation.

At the end of the 24-hour preservation period, kidneys that were preserved using SCS or PSF were perfused with 500 mL of SPS-1 solution for 90 minutes at a flowrate of 80 mL/min. The perfusate was recirculated for this entire time period so that lactate collected from the kidney during the perfusion was retained within the

500 mL of solution in the perfusion circuit maintained at 4-8° C. 500µL of venous effluent was collected from the renal venous cannula immediately at the start of liquid perfusion, and the effluent lactate concentration was measured by using the YSI 2700 biochemistry analyzer (YSI Life Sciences), and normalized to the renal mass. The

85 concentration of the samples that were collected were converted to mmol/g using the following equation where the YSI reading of the venous effluent sample (mmol/L) is multiplied by the volume (v) of the sample, divided by the renal mass (m);

푚푚표푙 푚푚표푙 푣 = × 푔 퐿 푚 and these values were then converted to pmol/g. Due to the extremely low concentrations, values were converted to picomoles. Lactate concentration from the perfusate samples (pmol/g) taken over 90 minutes were graphed using Graphpad

Prism Version 6 software. The approximate net accumulation of lactate over 24 hours was calculated by using the area under the curve of the data points during the first 10 minutes of perfusion. The reasoning behind this is that in 10 minutes, the entire volume of the SPS-1 solution in the organ container passed through the kidney, and the amount of lactate collected in the 500 mL of the recirculating medium at the 10- minute mark can be interpreted as the approximation of the amount of lactate produced within the kidney over the 24-hour preservation period. This is with the assumption that the lactate produced and consumed during 10 minutes of perfusion is negligible.

Histology

Hematoxylin and eosin (H & E) staining is used for routine examination of a wide variety of tissue types. It enables the visualization of cytoplasmic, nuclear, and extracellular matrix (Atale, et al. 2014). Hematoxylin reacts with negatively charged, basophilic cell components, and stains nucleic acids a deep purple color. Eosin reacts

86 with positively charged, acidophilic components in the tissue stains proteins and the cytoplasm. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining (Fischer, et al. 2008). H &

E staining was used to evaluate renal morphology and structural changes induced during the preservation period.

Initially, needle biopsies were taken but the collected tissue did not contain the recommended ~10 glomeruli for a representative sample. The fixed samples were embedded using OCT (optimal cutting temperature), an inert support medium for cryosectioning. Unfortunately, the sectioned samples revealed questionable tissue integrity and they were deemed unsuitable for histopathological examination.

Paraffin embedding was recommended by the pathologist for better preservation of morphological details. Consequently, we started collecting wedge biopsies from the kidneys after the preservation period. They were fixed with 4% paraformaldehyde, dehydrated in a series of alcohol solutions, processed and paraffin wax embedded.

Tissue samples were sectioned at 5 microns with a microtome and stained with hematoxylin and eosin (Sigma-Aldrich). Slides were double blinded and the sections were assessed for tubular dilation, brush border integrity, tubular injury, interstitium edema, epithelial cell vacuolization and vascular damage. A veterinary pathologist scored the samples over 5 fields using a 4-point scale in which 0 indicates no abnormality; 1, mild changes; 2, moderate changes and 3, severe changes.

Genomic Analysis

In an effort to examine gene expression changes in our tissue of interest, we used RNA sequencing and Agilent microarrays. RNA sequencing is an approach for transcriptome profiling that uses high throughput, next generation sequencing to provide a precise measurement of levels of transcripts in the sample. The reads are either aligned to a reference genome or reference transcripts, or assembled de novo if the organism is lacking a reference genome (Wang, Gerstein and Snyder 2009,

Ozsolak and Milos 2001). Microarrays measure the expression levels of large numbers of genes with chips that are printed with thousands of spots containing a known DNA sequence or gene (probes). These types of hybridization-based approaches are high throughput and relatively inexpensive, but are limited to detecting transcripts that correspond to existing genomic sequences (Russo, Zegar and Giordano 2003, Heller 2002).

RNA Isolation

Wedge biopsies containing renal cortex and medulla were stored in RNAlater™ stabilization solution (ThermoFisher Scientific). After they were thawed, a tissue sample of approximately 1 cm in length and 1 mm in width was collected and placed in QIAzol Lysis Reagent (Qiagen Sciences). Tissue samples were homogenized in sterile tissue shredder tubes using the gentleMACS™ Dissociator (Miltenyi Biotec

GmbH). RNA was purified and isolated form the samples using an miRNeasy Mini Kit

(Qiagen Sciences) according to manufacturer instructions. The RNA integrity was checked on a NanoDrop™ 3300 Fluorospectrometer (ThermoFisher Scientific).

Samples with 260/280 ratios above 2 and 260/230 ratios above 1.8 were used for this study.

RNA Sequencing

RNA Samples were assessed for quality with an Advanced Analytics Fragment

Analyzer (High Sensitivity RNA Analysis Kit – Catalog # DNF-491 / User Guide DNF-

491-2014AUG13) and quantity with a Qubit RNA quantification kit (Qubit® RNA HS

Assay Kit – Catalog # Q32852).

Given satisfactory quality and quantity, samples were used for library builds with the Kapa Biosystems Stranded mRNA-Seq Kit (KapaBiosystems Stranded mRNA-

Seq Kit – Catalog # KK8420 / KapaBiosystems Stranded mRNA-Seq Kit TDS KR0960

– v3.15). Upon library build completion, samples were assessed for quality and average fragment size with the Advanced Analytics Fragment Analyzer (High

Sensitivity NGS Analysis Kit – Catalog # DNF-486 / User Guide DNF-486-

2014MAR10). Quantity was assessed with an Illumina Universal Adaptor-specific qPCR kit from Kapa Biosystems (Kapa Library Quantification kit for Illumina NGS –

Catalog # KK4824 / KAPA Library Quantification Technical Guide - AUG2014).

After final library QC was completed, samples were equimolar-pooled and clustered for sequencing on the HiSeq2500 machine. The sequencing run was performed using Illumina HiSeq Rapid SBS v2 chemistry (HiSeq Rapid SBS Kit v2 200 cycles - Catalog # FC-402-4021, HiSeq PE Rapid Cluster Kit v2 - Catalog # PE-402-

4002), and data was sent to UAGC Biocomputing Group for further analysis and transmission to the client. Reads were trimmed using Trimmomatic (Bolger, Lohse and Usadel 2014) to remove Illumina adapters and remove low quality sequence data

89 using the following parameters: PE_TruSeq_adapt_Index5UnivRC.fa:2:30:11:1: true, sliding window 4:15, trailing 3 and min length 20 (Bolger, Lohse and Usadel 2014).

Mapping of trimmed reads to the Ensembl version 82 Sus scrofa genome sequence was performed with Tophat 2.0.9 using Bowtie2 2.1.0 (Trapnell, et al. 2012) using default parameters. HTseq-count was used to calculate expression values for all genes in each sample. Differential expression analysis was carried out using DEseq to compare paired (pre- and post-treatment) kidney samples.

Genomics Data Analysis

Differentially expressed genes were analyzed using Kobas 3.0 and Network

Analyst (http://www.networkanalyst.ca/faces/home.xhtml) to identify gene networks and pathways. The International Molecular Exchange Consortium (IMEx), a literature-curated comprehensive data was used to evaluate first order protein to protein interactions. To facilitate this, we converted pig UniProt accessions to human accessions using Ensembl Biomart and deleted duplicates and unassigned transcript

IDs.

Microarray Analysis

In vitro transcription was carried out using Agilent Quick Amp labeling kit

(Agilent Technologies, Santa Clara, CA) in the presence of Cy3- and Cy5-CTP. Then,

825 ng (150 pmol) of labeled cRNA from individual tissues were purified separately, combined, and mixed with hybridization buffer before being applied on the microarray. The hybridization solution was prepared using an Agilent Gene expression hybridization kit (Agilent Technologies, Santa Clara, CA). An Agilent 4x44k

S. Scrofa (Pig) Oligo Microarray v2 (Agilent Technologies, Design # 026440) kit was

90 used for hybridization, performed in a hybridization oven G2545A (Agilent

Technologies, Palo Alto, CA). Hybridization and washing were performed according to the protocol described in the Agilent Oligonucleotide Microarray Hybridization manual (Agilent Technologies, Santa Clara, CA). After washing, microarrays were scanned using a Gene Pix 4000B Scanner (Molecular Devices, Sunnyvale, CA) with laser excitation at 532 and 635nm at the highest possible resolution (five pixels per micron), and saved as 16-bit grayscale TIFF images. Intensity values were extracted using GenePix Pro6.0 (Molecular Devices, Sunnyvale, CA), and the data for each array were normalized followed by ANOVA analysis using a Limma package in R version

R2.5.1 (http://www.r-project.org/).

Differentially expressed genes were analyzed using Network Analyst

(http://www.networkanalyst.ca/faces/home.xhtml) to identify gene networks and pathways. The International Molecular Exchange Consortium (IMEx), a literature- curated comprehensive data was used to evaluate first order protein to protein interactions. To facilitate this, we converted pig UniProt accessions to human accessions using Ensembl Biomart and deleted duplicates and unassigned transcript

IDs. Genes with a p < 0.101 and log2 fold change of ≥ 1 or ≤ 1 were considered to be differentially expressed.

Pre-clinical Allotransplantation

Domino Surgery- Organ Procurement

Domestic female pigs weighing 35-50 kg were used, after being obtained from a local farm in Wilcox, Arizona. Animal care procedures were in accordance with

91 university policies. The donor pig was pre-medicated with intramuscular Telazol, and intubated. General anesthesia was induced and maintained with isoflurane. Oxygen was administered and heparin was injected through the ear catheter. A midline incision was done to access the peritoneal cavity. The terminal abdominal aorta was cannulated and suprarenal aorta was cross-clamped. The ureter was dissected and the kidneys were separated by longitudinally splitting the aorta and inferior vena cava. The isolated kidney was flushed with 400 mL of heparinized saline and SPS-1.

The harvested kidney was stored at 4oC in SPS-1 for 24 hours. Once the kidney was procured, the donor pig was euthanized. For the first attempt at allotransplants, we decided to do a domino procedure with 2 donors and 2 recipients. The first donor kidney was harvested and cold stored for 24 hours and transplanted into the first recipient. The recipient went through bilateral native nephrectomy at the time of transplant. One of these kidneys was persufflated for 24 hours and transplanted into the second recipient, and bilateral native nephrectomy was performed on the second recipient. Because we first wanted to establish that the surgeries could be done successfully with survival of the pigs for 7 days, we opted out of using a donation after cardiac model to ensure that the kidneys would be exposed to a minimum level of warm ischemia.

Paired Surgery- Organ Procurement

Domestic female pigs weighing 35-50 kg were used from a local farm in

Wilcox, Arizona. Animal care procedures were in accordance with university policies.

The donor pig was anesthetized under isoflurane and heparin was circulated for 5 minutes to prevent thrombosis during the procurement. They were euthanized, and

92 flushed with 4 liters of room temperature, heparinized saline through the abdominal aorta. Kidneys were exposed to 30 minutes of warm ischemia time and cannulated at the renal artery and vein using sterile luer locks (Cole-Parmer). Isolated kidneys were flushed with 400 mL of 4°C heparinized saline using gravity followed by a secondary flush with SPS-1 solution (Organ Recovery System). Isolated kidneys were treated with either static cold storage (SCS) or persufflation (PSF) for 24 hours at 4°C in a container with SPS-1. The persufflator (Giner, Inc., Newton, MA) delivered electrochemically generated, humidified, 40% oxygen mixed with atmospheric air with a manifold pressure of 40 mmHg to generate a flow rate of 60 mL/min. Tubing from the persufflator was attached to the renal artery and leaks around the vessel were ligated to ensure maximum amount of gas perfusion. For the second allotransplant, we used a DCD model and the pair of kidneys from a single donor was treated, preserved and transplanted into two separate recipients as indicated in the figure below.

Renal Allotransplant

After the 24-hour preservation period, recipient pigs were intubated after intravenous anesthesia and maintained on a respirator with oxygen. The peritoneal cavity was accessed through a midline incision. Heparin was administered and the abdominal aorta, renal artery and vein were dissected and a nephrectomy was performed. The preserved kidney was placed at the original kidney site and vascular anastomoses were performed end-to-side renal vein to vena cava and end-to-end renal artery to the native renal artery. Reperfusion was established by release of the venous and arterial clamps. Ureteroureterostomy was performed and the native

93 contralateral kidney was removed. The muscle layer, subcutaneous layer and the skin were closed and the pig was extubated.

Immunosuppression and Anticoagulants

Recipient pigs received an immunosuppression regimen prior to and after the allotransplant to decrease chances of rejection and antibiotics to reduce chances of infection. The following drugs were administered:

o 2mg/kg Methotrexate, twice a week for 1-week pre- and post-transplant

o 15mg/kg Cyclosporine once a day for 1-week pre- and post-transplant

o 125mg/kg Methylprednisolone once day 3 days post-transplant

o 40,000 U/kg Penicillin, IM, once a day for 3 days pre-transplant

o 20 mg/kg amoxycillin, PO, once a day for 3-5 days post-transplant

o 0.15 mg/kg metoclopramide, IM [anti-emetic] once, day of transplant

o 50 U/kg heparin, intravenously (IV) via ear catheter once 1-day post-

transplant

o 5-10 mg Warfarin, daily starting 1-day post-transplant

Blood and Urine Collection

Blood samples (2-5 mL) were obtained by IV ear catheter for a standard blood workup with blood cell count, creatinine levels, blood urea nitrogen and electrolytes.

2-5 mL urine samples were obtained by either voluntary voiding, or collected with a syringe guided by ultrasound at the time of blood collection. Unless euthanasia was suggested by the university animal care veterinarians to protect the wellbeing of the

94 animal, the recipient pigs were kept for a maximum of 7 days. On day 7 pigs were euthanized and the transplanted organs were collected for assessments.

Statistics

The function and viability measurements of GFR, Ktrans, WOOCR and lactate levels were all made in matched pairs of kidneys. The differences between the treatment groups were analyzed using a Wilcoxson Signed Rank test, and a paired t- test was performed on transformed data to satisfy the prerequisite assumptions of normality, and area under the curve (AUC) was calculated for continuous data. All statistical analyses were performed using Graphpad Prism Version 6 software.

Statistical significance was set at P value less than 0.05. Unless otherwise indicated, data represent mean ± SEM values.

Chapter 4

Novel Methods for Real-Time Kidney Quality Assessment Prior to Transplantation

The presented work in this chapter is in preparation for publication. A summary of

the work and a contribution summary has been provided within this chapter

Study Summary

Before we could start this study, completion of building, testing and optimizing the circuit for whole organ oxygen consumption rate (WOOCR) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was necessary. We also had to proficiently and consistently perform the organ procurement and vessel ligation techniques. Once this groundwork was done we moved on to investigate the effects of persufflation (PSF) on kidney preservation.

The aim of this first chapter was to use WOOCR and DCE-MRI to characterize the viability and function of organs that were preserved using PSF or static cold storage (SCS). We used a donation after cardiac death (DCD) model and mimicked the conditions organs would experience in this type of donor. The kidneys were exposed to 30 minutes of warm ischemia time, which is within the “desirable” 60 minutes.

They were then stored at hypothermic temperatures for 24 hours which is typically experienced during organ matching and transport. Paired kidneys were stored with either SCS or PSF and assessed using WOOCR, DCE-MRI to measure glomerular filtration rate (GFR) which is a standard measurement of renal function, collected perfusate for lactate measurements and took wedge biopsies for histological analysis.

The PSF kidneys had significantly higher WOOCR and GFR. DCE-MRI images show more signal intensity from the contrast agent with a faster clearance rate. The perfusate of these kidneys had significantly decreased lactate levels. There were no significant differences in renal architecture and there was minimal damage in renal tubules and the vasculature. These findings indicate higher metabolism and filtration function with lower levels of anaerobic glycolysis in the PSF kidneys. There were no

97 significant morphological changes, or latent ischemic injury that was observed, which suggests that the lower GFR may be caused by hemodynamic responses to hypothermia, as opposed to tubular impairment. Additionally, the novel techniques of WOOCR and DCE-MRI to measure GFR in isolated kidneys successfully detected the differences between the two preservation methods.

Novel Methods for Non-invasive, Non-destructive, Real-Time Kidney

Quality Assessment Prior to Transplantation

Catherine G. Min MS 1,2, Jean-Philippe Galons PhD 3, Abhishek Pandey MS 4, Brad P.

Weegman PhD5,6, Leah V. Steyn PhD2, William G. Purvis MS2, Kate E. Smith MS 1,2,

Diana S. Molano BME 2, Nicholas D. Price BS 2, Michael J. Taylor PhD 6,7, Jana Jandova

PhD 8, Diego R. Martin MD, PhD 1,3,9, Robert C. Harland MD 2, Klearchos K. Papas PhD

1Physiology, University of Arizona, Tucson, AZ, United States; 2Surgery, University of

Arizona, Tucson, AZ, United States; 3Medical Imaging, University of Arizona, Tucson,

AZ, United States, 4Electrical and Computer Engineering, University of Arizona, Tucson,

AZ, United States; 5Department of Radiology, University of Minnesota, Minneapolis, MN,

United States; 6Sylvatica Biotech, Inc., N. Charleston, SC, United States; 7Mechanical

Engineering, Carnegie Mellon University, Pittsburgh, PA; 8School of Animal and

Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, 9Department of

Biomedical Engineering, University of Arizona

Correspondent: Klearchos K. Papas

Fax: (520) 626-3770

Phone: (520) 626-4494

Email: [email protected]

Sources of Support: Partial support received from the University of Arizona

“Improving Health Initiative”, Pilot Interdisciplinary Grant Program; PI: K.K. Papas

Abstract

There is a need to reduce the high numbers of kidney discard and maximally utilize kidneys from the existing deceased donor organ pool. Quantifying organ characteristics and improving their quality during the preservation period can prevent unnecessary discard of transplantable organs. In this study, quality assessment methods of whole organ oxygen consumption rate (WOOCR) and dynamic contrast imaging to calculate glomerular filtration rate (GFR) were used to noninvasively measure kidney viability and function. In tandem, persufflation was studied as an alternative preservation method that mitigates ischemic damage during the preservation period. Porcine kidneys were subjected to 30 minutes of warm ischemia to mimic a cardiac death donor. Following ischemia, kidneys were flushed and one group of kidneys was persufflated while the other was cold stored for 24 hours at 4°C. WOOCR, GFR, perfusate lactate concentration and biopsies were used to evaluate kidney quality after preservation. Persufflated kidneys had significantly higher WOOCR and GFR of 182 ± 33 nmol/min*g and 36.6 ± 12.1 ml/min while cold storage kidneys had 132 ± 21 nmol/min*g and 11.8 ± 4.25 ml/min. Cold stored kidneys contained more lactate and histological evaluation of renal biopsies revealed no significant differences in structural changes between the two groups. These results demonstrate that persufflation can improve viability and function of the kidney after ischemic stress by providing oxygen, and WOOCR and GFR can be measured in isolated kidneys as valuable pre-transplant assessments.

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Key Words: Kidney assessment, organ preservation, oxygen consumption rate, persufflation, magnetic resonance imaging

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Introduction

Despite medical advances and increased awareness surrounding organ donation and transplantation, the discrepancy between the number of available organs and the number of patients on the organ transplant waiting list continues to widen (1-2). To mitigate this discrepancy, organs from expanded criteria donors

(ECD) and donors after cardiac death (DCD) have been used to increase the available donor organ pool.

Ironically, with the steady rise in use of kidneys from nontraditional donors, an unfortunate side effect of higher discard rates of kidneys has been observed.

In the past 5 years, over 14,000 recovered kidneys were not used (3). Additionally, over 40% of ECD kidneys were discarded while over 10% of non ECD kidneys were not transplanted (4). Another notable trend is that biopsied kidneys for pre- transplant evaluation were also discarded at a higher rate at approximately 30% compared to 10% kidneys that were not biopsied (4). Given the lack of available organs for patients on the waiting list, measures need to be taken to reduce excessive discard of potentially transplantable kidneys.

There are a few approaches that can be taken to maximize the use of existing deceased donor organs. The first is by developing organ assessment techniques that are reliable, noninvasive with prognostic capabilities that can differentiate between healthy and unhealthy organs to screen for transplantable organs. The second is by using organ preservation methods that reduce damage and induce reparative responses during the preservation period for improved organ quality.

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Kidney quality assessments prior to transplantation are currently limited to biopsies, visual assessments, and vascular resistance measurements, which do not reliably correlate with clinical transplant outcomes (5-9). Oxygen consumption rate measurements and glomerular filtration rate calculation using medical imaging are two promising technologies for pre-transplant kidney assessments. Literature demonstrates oxygen consumption rate as a reliable method of measuring cell and tissue viability that can be used to predict transplant outcomes (10-14). Dynamic contrast enhanced magnetic resonance imaging is another non-invasive technique that enables modeling and calculation of intrarenal flow and contrast agent compartmentalization (15-18), which cannot be evaluated by simple vascular resistance measurements. Imaging can estimate physiological parameters such as regional filtration, perfusion, filtration fraction, and total glomerular filtration rate

(GFR), all of which are important determinants for kidney function (19-23).

In conjunction with kidney assessment, ischemic damage of kidneys that occurs during preservation can be mitigated by utilizing persufflation, a gas perfusion of humidified oxygen using the native renal vasculature. Persufflation has led to substantially prolonged preservation times and has enabled the reconditioning and utilization of deceased donor organs. It has benefited hearts

(24, 25), kidneys, (26-31), livers, (32-36) and pancreas (37-39) in a variety of animal models and in some clinical studies outside of the United States.

The aim of this study was to further investigate the feasibility of using

WOOCR and dynamic contrast enhanced imaging as assessment tools and

103 persufflation as an alternative method of preservation in an isolated DCD porcine kidney model.

Methods

Organ Procurement and Preservation

This study was approved by the institutional animal care and use committee

Heparinized porcine donors were euthanized, exsanguinated and flushed with 2 liters of room temperature, heparinized saline through the abdominal aorta.

Kidneys were procured after exposure to 30 minutes of warm ischemia time (WIT) inside the body cavity. Following procurement, kidneys were cannulated at the renal artery and vein using luer locks (Cole-Parmer). Subsequently, both kidneys were flushed with 400 mL of 4°C heparinized saline using gravity followed by a secondary flush with SPS-1 solutions (Organ Recovery Systems). The kidneys were preserved for 24 hours with either static cold storage (SCS) or persufflation.

The persufflator prototype (Giner, Inc., Newton, MA) delivered electrochemically generated, humidified, gaseous 40% oxygen mixed with atmospheric air (38), as summarized in Figure 6. It maintained a manifold pressure of 40-50 mmHg to generate a flow rate of 60 mL/min. Tubing from the persufflator was attached to the renal artery, and leaks around the vessel were ligated to ensure that the entire kidney was adequately perfused. Both kidneys were kept in SPS-1 solution at 4°C during the 24 hours of preservation.

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Whole Organ Oxygen Consumption Rate

The renal artery and renal vein were cannulated individually and connected to a custom built hypothermic perfusion system maintained at 4-8°C. SPS-1

(Organ Recovery Systems) was oxygenated with 40% oxygen using a hollow fiber oxygenator (Medtronic) and perfused through the artery throughout the measurements. Flow-through fiber-optic oxygen sensors (Instech Laboratories,

Inc.) were placed inline at the renal arterial cannula and one was placed distal to the venous cannula to measure the difference in oxygen partial pressure at the inlet and the outlet of the organ as shown in Figure 1A. Inline sensors were calibrated at 0%, 21% and 40% oxygen prior to data collection. The WOOCR

(mol/min/g) of a kidney was calculated by using a mass balance equation where

Q is the flow rate of perfusion (mL/min), α is oxygen solubility in aqueous solution at temperature (mol/mL/mmHg), and m is the mass of the kidney (g) (14):

푄 × (푝푂2 − 푝푂2 ) × 훼 푊푂푂퐶푅 = 퐴푟푡푒푟푖푎푙 푉푒푛표푢푠 푚

Dynamic Contrast Imaging using Magnetic Resonance

All imaging was done on a 3T Skyra scanner (Siemens, Erlangen,

Germany). Kidneys were secured in the organ cassette (LifePort) and cold- perfused (8°C) with SPS-1 preservation solution (Organ Recovery Systems) using a MR compatible hypothermic perfusion circuit set up in Figure 2A. The organ cassette was inserted in a standard Siemens Head and Neck RF coil. 5mL of 2mM

Multihance Gadolinium-DTPA solution (Bracco Diagnostics Inc.) was injected over

15 seconds into the cannulated artery after 20 seconds of precontrast, and continuous acquisition was recorded for 4 minutes.

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Dynamic-contrast MRI was performed using a golden angle stack-of-star radial acquisition (40). Compressed Sensing reconstruction with total variation applied in the temporal dimension was used to reconstruct images with a 1 second temporal resolution (21, 20). The regions of interests are drawn on the reconstructed images (performed by CGM, AP), outlining the kidney margins, and these signals analyzed using a 2-compartment modified Toft’s model for pharmacokinetic modelling (18); a total of 90 sec post-contrast data was used to provide the maximum amount of data prior to excretion and leakage of contrast outside the ROI (20). The arterial input function, the diffusive transport of gadolinium through intravascular and extravascular space was used to calculate

Ktrans and glomerular filtration rate of the isolated kidneys.

Lactate measurement

After 24 hours of preservation, kidneys that were preserved using SCS or

PSF were placed in an organ container with 500mL of fresh SPS-1 (Organ

Recovery Systems). Preserved kidneys were then liquid perfused through the renal arterial cannula on a custom built, recirculating hypothermic circuit

maintained at 4-8° C. 500µL of venous effluent was collected from the renal venous cannula immediately at the start of liquid perfusion, and the effluent lactate concentration was measured by using the YSI 2700 biochemistry analyzer (YSI

Life Sciences), and normalized to the renal mass. Lactate production rate was calculated by using the concentration (C) normalized to kidney weight (m), volume

(V) of the recirculating bath and preservation period (t):

퐶 푥 푉 푝푟표푑푢푐푡푖표푛 푟푎푡푒 = 푚푡

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Histology

Wedge biopsies were taken from the kidneys after the preservation period.

They were fixed with 4% paraformaldehyde, dehydrated, processed and paraffin wax embedded. Tissue samples were sectioned at 5 microns with a microtome and stained with hematoxylin and eosin (Sigma-Aldrich) to evaluate the glomeruli, renal tubules and the vessel wall morphology using light microscopy. Slides were double blinded and the sections were assessed for tubular dilation, brush border integrity, tubular injury, interstitium edema, epithelial cell vacuolization and vascular damage. A veterinary pathologist scored the samples over 5 fields using a 4-point scale in which 0 indicates no abnormality; 1, mild changes; 2, moderate changes and 3, severe changes.

Statistics

The function and viability measurements of GFR, Ktrans, WOOCR and lactate levels were all made in matched pairs of kidneys. The differences between the treatment groups were analyzed using a Wilcoxson Signed Rank test, and a paired t-test was performed on transformed data to satisfy the prerequisite assumptions of normality, and area under the curve (AUC) was calculated for continuous data. All statistical analyses were performed using Graphpad Prism

Version 6 software. Statistical significance was set at P value less than 0.05.

Unless otherwise indicated, data represent mean ± SEM values.

Results

Whole Organ Oxygen Consumption Rate

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WOOCR for PSF kidneys were significantly higher than cold storage. This was calculated in pairs of porcine kidneys (n=7 matched pairs) after 24 hours of preservation in their respective conditions as a measure of viability using the circuit depicted in Figure 1A. WOOCR for SCS kidneys was 132 ± 21 nmol/min*g, and

182 ± 33 nmol/min*g for persufflated kidneys (p < 0.05) as shown in Figure 1B, which indicates more oxygen utilization in the persufflated group.

Dynamic Contrast Enhanced Magnetic Resonance Imaging

Ktrans values and GFR for PSF kidneys were significantly higher than SCS kidneys. Organ function was measured in kidneys (n=5 matched pairs) post 24 hours of preservation period by using dynamic contrast enhanced magnetic resonance imaging with gadolinium as the contrast agent with the perfusion system shown in Figure 2A. Galolinium perfusion curves (Figure 2B) demonstrate more perfusion and faster clearance of the contrast agent from the renal cortex of

PSF kidneys vs. SCS kidneys as they have a steeper descending cortical slope (-

1.03 ± 0.3 vs. -0.39 ± 0.3, p<0.05) respectively. The volume transfer constant Ktrans, is measured through the 2-compartment model. It reflects the efflux rate of gadolinium from the vasculature, especially the glomeruli, into the tissue extravascular extracellular space, which in this case is largely the renal tubules.

Ktrans depends on the perfusate flow, vascular permeability, and the vascular surface area. On average, the SCS kidneys had a Ktrans value of 0.04 ± 0.01 mL/g/min, while persufflated kidneys had a significantly higher rate of 0.11 ± 0.02 mL/g/min (Figure 3A). The glomerular filtration rate is derived from Ktrans and the volume of the kidneys. Similar to the Ktrans data, the persufflated kidneys have a

108 significantly higher glomerular filtration rate of 36.6 ± 12.1 ml/min while the SCS kidneys averaged at 11.8 ± 4.25 ml/min (Figure 3B).

Perfusate Lactate Levels

PSF kidneys had significantly lower lactate levels than SCS kidneys.

Lactate concentration was measured from isolated kidneys (n=4 matched pairs) after the 24-hour preservation period by sampling the venous effluent during hypothermic liquid perfusion. The first pass concentration of lactate (Figure 4A) that was normalized to renal mass was significantly lower in PSF kidneys (11.5 ±

0.7 nmol/g) compared to SCS kidneys (20.82 ± 3.7 nmol/g) as shown in Figure

4B.

Histology

Histological evaluation revealed no significant differences in renal morphology or damage between treatment groups. Both exhibited mild tubular and glomerular space dilation. They also exhibited epithelial cell vacuolation and scattered pyknotic nuclei, which are early signs of degeneration and apoptosis.

Neither group exhibited notable damage to the renal vasculature (Figure 5).

Discussion

The development of improved organ preservation is crucial as it will not only better maintain the viability of the organ, but also preserve its functional integrity, which is essential for successful transplant outcomes. For this reason, there is a resurgence of clinical interest in alternative organ preservation methods such as hypothermic machine perfusion that allow longer preservation times without compromising the health of the organ, and enable organ reconditioning and repair

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(41). Achieving these goals is an important gateway to increase the use of organs from the already available, but widely dismissed, pool of marginal organs that are often discarded without objective assessment of their function. Results from this study demonstrate that persufflation improves kidney viability and function versus

SCS. Furthermore, our results demonstrate that it is feasible to conduct noninvasive real-time WOOCR measurements while using magnetic resonance imaging to measure GFR to assess the viability and function in excised kidneys.

In our study, we demonstrated that the oxygen consumption rate of porcine kidneys can be measured ex vivo at 4ºC using a perfusion circuit with an oxygenator and inline oxygen sensors. Oxygen consumption rate is correlated with cell and tissue viability (10-12). This is strongly evidenced in studies that assess islet quality, and is a compelling predictor of islet transplant outcome. We applied this approach in the context of assessing whole organs prior to transplantation.

Weegman et al. established that this method can differentiate between healthy and damaged kidneys, with injured kidneys exhibiting an irreversible decrease in oxygen consumption rate (14). Lower consumption rate seen in the SCS kidneys may be explained by increased kidney damage attributed to the hypoxic environment during the preservation period. In early persufflation studies, kidneys preserved with retrograde persufflation had less ATP depletion and continual synthesis and utilization of ATP during hypothermic preservation compared to SCS kidneys (42, 43). This may explain the more metabolically active, viable tissue that is in the persufflation group.

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Magnetic resonance imaging using gadolinium is another valuable noninvasive tool that can be used to evaluate kidney function. This technique has been used in lab animals and humans in vivo to estimate dynamic elements of kidney physiology, including GFR (21, 19). For example, using these methods, a correlation has been shown between GFR measured with dynamic contrast enhanced imaging and the plasma clearance of chromium-51 labeled ethylenediamine tetraacetic acid (51Cr-EDTA), a labeled filtration marker (44, 15).

Ex vivo MR perfusion imaging of porcine kidneys shows a significantly higher Ktrans value which can relate to higher GFR values. We can infer from an increased GFR that more non-energetic, passive glomerular filtration clearance is present in the tubules. This suggests more energy is being consumed, which is corroborated by the increase observed in WOOCR and increased functionality in the persufflated kidneys.

Lactate measurements in perfused organs can also provide additional objective measure of organ health. Anaerobic metabolism created by an ischemic environment is characterized by an increase in tissue lactate levels, which correlates with pH changes (45, 46). Lactate is observed at higher levels in the static cold storage kidneys when compared to the persufflated kidneys, which may indicate less anaerobic metabolism and functional utilization of the provided exogenous oxygen.

Despite the significant differences observed in WOOCR and GFR, there were no gross structural changes or significant differences between the two treatment groups observed in the renal biopsies. Histological sections revealed a

111 negligible amount of tubular stress with minimal infarct, apoptosis, and inflammation. The glomeruli and renal vessels in the biopsies were intact with no noticeable changes or damage. This suggests that 40% oxygen persufflation can be safely used without the fear of hyperoxic vascular endothelial injury. More importantly, these results show that WOOCR and GFR measurements can detect changes that are not detectable through the standard biopsy that is used to allocate kidneys.

These results confirm that oxygenating kidneys during the preservation period can be beneficial in maintaining their functional integrity as measured by

WOOCR and GFR. Studies show increased ATP levels in persufflated organs and an extension of ischemic tolerance (32, 43, 47), all of which can positively influence graft outcomes. Further analysis will be necessary to understand the specific mechanisms by which oxygen supplementation leads to functional preservation.

Nonetheless, persufflation has the potential to rescue and resuscitate organs after warm and cold ischemic periods that are experienced throughout the process of organ procurement and preservation. Additionally, the results of this study provide a multimodal, noninvasive method to assess kidneys for their quality prior to transplantation. Future studies will evaluate the correlation between ex vivo assessments and transplant outcomes. Being able to evaluate organ viability and function is undoubtedly valuable, especially for the transplant of marginal organs whose use will be essential in decreasing the gap between available organs and waiting organ recipients.

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Disclosure

K.K.P. declares a financial interest in Giner, Inc. This interest has been properly disclosed to the University of Arizona Institutional Review Committee and is managed by a Financial Conflict of Interest Management Plan in accordance with its conflict of interest policies.

Acknowledgements

The authors would like to acknowledge the members of the Papas laboratory and collaborators, including Thomas Suszynski, J. Brett Stanton, Jennifer P. Kitzmann,

AnnaMaria Burachek, Alexandra Hoeger, Ivan Georgiev, Liberty Kirkeide, Dr.

David G. Besselsen, Dr. Ronald Lynch, Dr. Heddwen Brooks and Dr. Tun Jie for their support. We also would like to thank Simon Stone and Dr. Linda Tempelman for technical assistance with the persufflator.

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Figure Legends

Figure 1: Schematic of the whole organ oxygen consumption rate perfusion circuit and WOOCR measurements from SCS and persufflated kidneys. A is a schematic of the perfusion circuit where the organ is perfused with oxygenated preservation solution (SPS-1) in a recirculating bath maintained at 4°C. Perfusate enters the kidney through the inline flow cells containing an oxygen sensor at the arterial end to the second sensor at the venous end. The fluorometer that couples with oxygen sensors provides oxygen measurements. B shows the values for whole organ oxygen consumption rate (WOOCR). WOOCR was obtained with a hypothermic perfusion circuit in isolated DCD porcine kidneys preserved with SCS or PSF. WOOCR values were calculated with the oxygen partial pressure difference at the renal artery and vein, flow rate, and oxygen solubility normalized by the mass of each kidney (n=7, * indicates p<0.05).

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Figure 2: Schematic of hypothermic perfusion system for dynamic contrast enhanced magnetic resonance imaging and whole parenchymal perfusion curves. A shows the jacketed media reservoir, peristaltic pump and water cooler are placed in the office area with the MRI viewing software that is separated from the magnet. 30 ft. of insulated tubing is connected from the media reservoir to the heat exchanger, which has an injection port for contrast agent infusion. The outlet port of the heat exchanger is connected to the renal artery for cold perfusion of the kidney. A return line connected to a perfusion pump brings back the preservation solution from the organ container to the media reservoir for recirculation. B portrays perfusion curves obtained from DCE-MRI of isolated porcine kidneys preserved for 24 hours. Regions of interest around the whole kidneys were taken and graphed to show the average signal intensity over time in the renal cortex and medulla. The descending cortical slope represents the tracer clearance from the renal cortex, and is significantly deeper with oxygenation (n=4, p<0.05).

Figure 3: Panel of renal characteristic data obtained from DCE-MRI. Images were obtained from isolated kidneys using a MR compatible hypothermic perfusion system. A shows Ktrans, the transfer constant of the tracer moving from the intravascular space into the extracellular, extravascular space was measured with dynamic contrast imaging using gadolinium as the tracer (n=5, p<0.05). B demonstrates the values for estimated glomerular filtration rate in static cold storage and persufflated kidneys. It was calculated by using Ktrans and kidney volume (n=5, p<0.05).

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Figure 4: Lactate levels of renal perfusate. At the end of the 24-hour preservation period, each kidney was perfused with 500 mL of SPS-1 solution for 90 minutes at a flowrate of 80 mL/min. A shows the concentration of lactate in the first 500 uL sample of venous effluent at the start of the perfusion prior to recirculation (n=4, p= 0.05). B shows the first pass lactate concentrations in graph A normalized to each individual renal mass (n=4, p< 0.05).

Figure 5: Light microscopy images of hematoxylin and eosin stained tissue samples. Light microscopy images of hematoxylin and eosin stained tissue samples. Tissue biopsies were taken after the 24 hours of preservation period from

116 isolated DCD kidneys. A and B are images from persufflated kidneys and C and D are images from static cold storage. No significant differences were detected between the two treatment groups.

SCS PSF

Score 0.45 ± 0.13 0.3 ± 0.13

Table 1: Histological evaluation assessing renal injury in wedge biopsies. After 24 hours of preservation. Scores are reported in means ± SEM from four pairs of kidneys (n=4). Sections were stained with H&E and scored over 5 fields with 0 indicating no abnormalities, 1 mild changes, 2 moderate and 3 severe changes. Kidney sections were evaluated for tubular dilation, vacuolation of epithelial cells, loss of brush border, tubular casts and lymphoplasmacytic inflammation in the interstitium.

Figure 6: Experimental Design Illustration. Kidneys from a porcine DCD model were procured after 30 minutes of WIT and exposed to 24 hours of hypothermia during the preservation period. Kidneys were preserved using either SCS or PSF for 24 hours, and assessments were done after the preservation period.

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29. Primary organ function of warm ischaemically damaged porcine kidneys after retrograde oxygen persufflation. Treckmann, Jürgen W, et al. 7, July 1, 2006, Nephrology Dialysis Transplantation, Vol. 21, pp. 1803-1808. 30. Function and quality of kidneys after cold storage, machine perfusion, or retrograde oxygen persufflation: results from a porcine autotransplantation model. Treckmann, Jürgen, et al. 1, August 2009, Cryobiology, Vol. 59, pp. 19-23. 31. Hypothermic reconditioning by gaseous oxygen persufflation after cold storage of porcine kidneys. Minor, Thomas, Efferz, Patrik and Lüer, Bastian. 1, August 2012, Cryobiology, Vol. 65, pp. 41-44. 32. Retrograde oxygen persufflation preservation of human livers: a pilot study. Treckmann, Jürgen, et al. 3, February 28, 2008, Liver Transplantation, Vol. 14, pp. 358- 364. 33. Hypothermic reconditioning by gaseous oxygen improves survival after liver transplantation in the pig. Minor, Thomas, et al. 12, December 2011, American Journal of Transplantation, Vol. 11, pp. 2627-2634. 34. Oxygen persufflation as adjunct in liver preservation (OPAL): study protocol for a randomized controlled trial. Minor, Thomas, et al. 1, October 28, 2011, Trials, Vol. 12, p. 234. 35. Energetic recovery in porcine grafts by minimally invasive liver oxygenation. Minor, Thomas, et al. 2, December 2012, Journal of Surgical Research, Vol. 178, pp. e59-e63. 36. The effect of anterograde persufflation on energy charge and hepatocyte function in donation after cardiac death livers unsuitable for transplant. Khorsandi, Shirin Elizabeth, et al. 6, June 2014, Liver Transplantation, Vol. 20, pp. 698-704. 37. Portal Venous Oxygen Persufflation of the Donation after Cardiac Death pancreas in a rat model is superior to static cold storage and hypothermic machine perfusion. Reddy, Mettu S, et al. 6, June 2014, Transplant International, Vol. 27, pp. 634-639. 38. Persufflation improves pancreas preservation when compared with the two-layer method. Scott, William E I I I, et al. 6, July 1, 2010, Transplantation Proceedings, Vol. 42, pp. 2016–2019. 39. Pancreas oxygen persufflation increases ATP levels as shown by nuclear magnetic resonance. Scott, William E I I I, et al. 6, July 1, 2010, Transplantation Proceedings, Vol. 42, pp. 2011-2015. 40. XD-GRASP: Golden-Angle Radial MRI with Reconstruction of Extra Motion-State Dimensions Using Compressed Sensing. Feng, Li, et al. 2, February 2016, Magnetic Resonance in Medicine, Vol. 75, pp. 775-788. 41. Current state of hypothermic machine perfusion preservation of organs: The clinical perspective. Taylor, Michael J and Baicu, Simona C. 3S, July 2010, Cryobiology, Vol. 60, pp. S20-S35.

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42. The mechanism of action of retrograde oxygen persufflation in renal preservation. Pegg, D E, et al. 2, August 1989, Transplantation, Vol. 48, pp. 210-217. 43. Metabolism of canine kidneys in anaerobic ischemia and in aerobic ischemia by persufflation with gaseous oxygen. Isselhard, W, et al. 2, 1972, Pflügers Archiv European Journal of Physiology, Vol. 337, pp. 87-106. 44. Validation of plasma clearance of 51Cr‐EDTA in adult renal transplant recipients: comparison with inulin renal clearance. Medeiros, Flavia Silva Reis, et al. 3, s.l. : Transplant International, March 2009, Vol. 22, pp. 323-331. 323-331. 45. Principles of solid-organ preservation by cold storage. Belzer, F O and Southard, J H. 4, April 1988, Transplantation, Vol. 45, pp. 673-676. 46. Lactate and pH change in close correlation in the extracellular space of the rat brain during cortical spreading depression. Scheller, D, Kolb, J and Tegtmeier, F. 1, January 20, 1992, Neuroscience Letters, Vol. 135, pp. 83-86. 47. Function and metabolism of canine kidneys after aerobic ischemia by retrograde persufflation with gaseous oxygen. Isselhard, W, et al. 1, March 1974, Research in Experimental Medicine, Vol. 164, pp. 35-44.

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Chapter 4 Supplementary data

Supplementary data for this chapter include T0 measurements for whole organ oxygen consumption rate, histological analyses and RNA sequencing analyses.

Due to the nature of measuring WOOCR with liquid perfusion, these assessments were not done on the same kidneys at the day of procurement and after 24 hours of preservation. Hypothermic liquid perfusion is currently the state of the art method for preserving marginal kidneys, and exposing the kidneys to perfusion for an hour would be a significant variable in evaluating the effects of persufflation. Similarly, liquid perfusion would also affect renal morphology and interfere with observing the effects of persufflation on the structural changes of kidneys. Therefore, control samples from separate pigs were obtained for WOOCR and histology. Control kidneys used for WOOCR were exposed to 15-30 minutes of warm ischemia time (WIT), and controls used for histology were exposed to 15-20 minutes of WIT. Static cold storage

(SCS) and persufflated (PSF) kidneys were exposed to 30 minutes of WIT and 24 hours of cold ischemia time (CIT).

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T0 Whole organ oxygen consumption rate and histological analyses

Figure 19: Whole organ oxygen consumption rate in control and preserved kidneys. WOOCR was obtained with a hypothermic perfusion circuit in isolated DCD control porcine kidneys and kidneys preserved with SCS or PSF.

The control and SCS kidneys (control: 127.3 ± 29.78; PSF: 132.1 ± 31.78 nmol/min*g) have very similar WOOCR values while the PSF kidneys have a higher average of 182.3 ± 33.02 nmol/min*g. The SCS kidneys appear to have retained viability at a comparable level to that of control kidneys, despite being exposed to 24 hours of CIT. This may be due to the metabolic depression of tissue during hypothermic storage, and the flushing of kidneys with preservation solution may have had a prophylactic effect on further damage. Belzer UW preservation solution

(same basic composition as SPS-1) is widely used for organ preservation. It contains cell impermeant agents that prevent the cell swelling and agents that stimulate recovery of normal metabolism upon reperfusion. PSF kidneys on the other hand show a modest increase in WOOCR compared to control. This difference reflects the

123 protective effects contributed by oxygenation, albeit the low metabolic rate of the kidneys at 4°C. This improved viability from the time of organ procurement in a DCD model has profound implications, for it may be able to resuscitate organs from all types of donors with varying degrees of warm ischemia time. Studies in the literature denote the importance of timing of oxygenation, and it is suggested that the benefits of persufflation increase when it is administered soon after procurement (Porschen,

Srinivasan, Iwasaki, Afify, & Tolba, 2016).

Figure 20: Lactate levels of renal perfusate. At the end of the 24-hour preservation period, each kidney was perfused with 500 mL of SPS-1 solution for 90 minutes at a flowrate of 80 mL/min. The perfusate was recirculated for this entire time period so that lactate collected from the kidney during the perfusion was retained within the 500 mL of solution in the perfusion circuit. A shows the very first single pass collection of venous effluent at the start of the perfusion (n=4, p<0.05). Data points represent the concentration of perfusate sample normalized to each individual renal mass. B shows the average lactate levels in perfusate collected at the different time points during 90 minutes of liquid perfusion. The first pass collection is concentrated with the lactate that is accumulated overnight. After perfusion there is a drop in lactate due to the washout, and subsequently reaches a steady state. There is a gradual increase due to the lactate that is already in the recirculating medium from the initial flush and new production of lactate. C shows the approximate net accumulation of lactate over 24 hours. This was calculated by using the area under the curve of the data points during the first 10 minutes of perfusion, depicted by the dotted vertical line in B. In 10 minutes, the entire volume of the SPS-1 solution in the organ container passed through the kidney, and the amount of lactate collected in the 500 mL of the recirculating medium is an approximation of the amount of lactate produced within the kidney over the 24-hour preservation period (n=4, p=0.1).

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Figure 21: Lactate production of preserved kidneys during hypothermic liquid perfusion. Kidneys were perfused with 500 mL of SPS-1 solution for 90 minutes in a recirculating bath post-preservation. A shows the production of lactate during liquid perfusion. This was calculated by using the area under the curve of the data points that were subtracted by the base level of lactate that was present in the 500 mL SPS-1 from recirculation. B depicts the production rate of lactate (slope) during liquid perfusion in isolated porcine kidneys.

Figure 22: Light microscopy evaluation of hematoxylin and eosin stained renal biopsies. Tissue biopsies were taken after the 24 hours of preservation period from isolated DCD kidneys.

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Interestingly, the average morphological abnormalities are the highest in the control kidneys, followed by the SCS and PSF (0.73 ± 0.24; 0.45 ± 0.13; 0.3 ± 0.13) treated kidneys. This may largely be attributed to the fact that the kidneys in both treatment groups were collected at 4°C, compared to the control kidney biopsies, which were taken under normothermic conditions. Nonetheless, it is surprising that the additional 24 hours of CIT did not incur noticeable morphological changes in the treated kidneys compared to the control.

Traditionally in open surgery, the recommended WIT for kidneys is 30 minutes (Laven, et al., 2004). However, some studies have found both laparoscopic and open surgeries, 90 minutes of WIT resulted in initial renal dysfunction, but normalized within 2 weeks after the surgeries (Laven, et al., 2004). Longer CIT is correlated with higher incidence of delayed graft function (Simpkins, et al., 2007;

Salahudeen A. K., Cold ischemic injury of transplanted kidneys: new insights from experimental studies, 2004; Kayler, Yu, Cortes, Lubetzky, & Friedmann, 2017), but its influence on long term graft survival remain controversial (Debout , et al., 2015;

Ponticelli, 2015). The detrimental effects of both warm and cold ischemic exposure times manifest in the form of delayed graft function, which is a phenomenon that occurs from ischemia-reperfusion injury post-transplant. Considering we are using isolated kidneys for assessments without the sudden rewarming, blood reperfusion and oxygenation, our model won’t reproduce these types of injuries. It is important to note that while the differences have been observed in these samples, the average values fall under the range of no abnormalities to mild changes in renal morphology.

Furthermore, there are mix results in the renal biopsy’s prognostic qualities for renal

126 transplants (Sung, et al., 2008; Anglicheau , et al., 2008), and it seems these results do not necessarily correlate with the differences seen in our WOOCR and GFR measurements for SCS and PSF kidneys.

RNA Sequencing Analysis

Introduction

The organ quality assessment techniques that we developed and studied are global, systemic level of observations. WOOCR determines the parenchymal oxygen consumption that we can interpret as viability, and GFR is the standard estimation of renal function. While lactate levels and histology can detail the effects of anaerobic metabolism during preservation and study the morphological changes that occur during preservation, they do not detail the pathways or the mechanism by which different environments produce functional changes. Gene expression technologies provide a snapshot of transcriptional activity in different tissues. It has the power to provide insights into normal cellular processes and diseased state pathogenesis.

Furthermore, it can provide a comprehensive comparative analysis of different phenotypes from diseased and normal tissue. In order to understand the effects of persufflation, RNA sequencing and microarrays were used to quantitatively survey the global behavior of transcriptomes in isolated, preserved porcine kidneys.

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Figure 23: Timeline and the work flow of RNA sequencing. Wedge biopsies from static cold storage and persufflated kidneys were collected at immediately after 30 minutes of warm ischemia time (WIT) at T0, and a second set of samples were collected 24 hours after the preservation period at T1.

Figure 24: The most downregulated and upregulated genes in static cold storage kidneys at T1 v T0. Genes that were downregulated are involved in vessel formation, enzyme activity, potassium handling, signaling for cell growth regulation and differentiation, proliferation suppression, extracellular matrix degradation, immune response, inflammation and wound healing. Genes that were upregulated are involved in signaling pathways involved in cell proliferation, survival, migration, ATP generation, immune response, vascular and epithelial

128 development, cell adhesion, anticoagulation and potassium and sodium-mediated fluid transport.

Figure 25: The most downregulated and upregulated genes in persufflated kidneys at T1 v T0. Genes that were upregulated are involved in fiber assembly, cell structural component regulation, cyclase activity, proliferation and organization of vascular smooth muscle, processes involved in apoptosis, cell transformation, and cytoskeleton maintenance. Genes that were downregulated are involved in immune system activity, cell signaling, cell migration, autophagy regulation, angiogenesis, pH regulation, cell structure and actin cytoskeleton regulation.

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Figure 26: The most downregulated and upregulated genes in persufflated kidneys compared to static cold storage kidneys at T1. Genes that were upregulated are involved in regulation of extracellular matrix, regulation of angiogenesis, stabilization of endothelial cell junctions, maintenance of hemostasis, sodium ion permeability and activation of cell proliferation, differentiation and migration pathways. Genes that were downregulated are involved in potassium ion permeability, chromatin remodeling, differentiation of cells in the distal renal tubular epithelium, cellular response to mitogenic signals, transcription factors regulating cell cycle, cell proliferation and identities on the anterior-posterior axis and cytokine activity.

KOBAS analysis of DE genes obtained from RNA Sequencing SCS kidneys at T1 compared to T0

Downregulated KEGG Pathways Input Background Corrected P-Value Calcium signaling pathway 16 179 0.0001 MAPK signaling pathway 19 257 0.0002 Wnt signaling pathway 13 142 0.0007 ErbB signaling pathway 10 90 0.001 FoxO signaling pathway 12 135 0.002 HIF-1 signaling pathway 10 105 0.003 Tight junction 11 139 0.005 cAMP signaling pathway 13 201 0.009 mTOR signaling pathway 11 155 0.01 Regulation of actin cytoskeleton 13 219 0.02 PI3K-Akt signaling pathway 16 343 0.04 Vascular smooth muscle contraction 8 123 0.05 Downregulated Biological Processes Input Background Corrected P-Value cytoskeleton organization 56 1041 1.5e-07 cell junction organization 24 246 3.2e-07 cation transmembrane transport 42 678 3.6e-07 tissue development 76 1729 7.9e-07 actin cytoskeleton 32 453 1.3e-06 regulation of signal transduction 103 2680 1.3e-06 cell migration 57 1180 2.8e-06 vasculature development 35 595 1.5e-05 phosphorylation 85 2194 1.5e-05 cell differentiation 121 3524 1.9e-05 ion homeostasis 37 659 1.9e-05 response to oxygen-containing compound 63 1475 2.5e-05 wound healing 31 541 8.4e-05 angiogenesis 25 417 0.0003 developmental growth 30 566 0.0004 growth 41 954 0.001

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Table 1: Downregulated pathways and biological processes in static cold storage kidneys at T1 compared to T0. KEGG pathways and biological processes were obtained by using KOBAS3.0 to analyze the differentially expressed genes list acquired through RNA Sequencing.

Downregulated Genes Vascular Development Wound Healing ADIPOR2 adiponectin receptor 2 TFPI tissue factor pathway inhibitor PIK3CB phosphatidylinositol-4,5- PIK3CB phosphatidylinositol-4,5- bisphosphate 3-kinase bisphosphate 3-kinase catalytic catalytic subunit beta subunit beta CALCRL calcitonin receptor like ADIPOR2 adiponectin receptor 2 receptor MEF2C myocyte enhancer factor 2C ARFGEF1 ADP ribosylation factor guanine nucleotide exchange factor 1 TGFB2 transforming growth factor LMAN1 lectin, mannose binding 1 beta 2 MAPK1 mitogen-activated protein P2RX5 purinergic receptor P2X 5 kinase 1 MIB1 mindbomb E3 ubiquitin CAPN3 calpain 3 protein ligase 1 FLT1 fms related tyrosine kinase 1 TGFB2 transforming growth factor beta 2 CAV1 caveolin 1 MAPK1 mitogen-activated protein kinase 1 WNT2 Wnt family member 2 F9 coagulation factor IX MEOX2 mesenchyme homeobox 2 CAV1 caveolin 1 EFNB2 ephrin B2 ENO3 enolase 3 PTPRB protein tyrosine phosphatase, VWF von Willebrand factor receptor type B RAMP1 receptor activity modifying PRKAR2A protein kinase cAMP-dependent type protein 1 II regulatory subunit alpha MYO18B myosin XVIIIB PLG plasminogen DAB2IP DAB2 interacting protein ITPR2 inositol 1,4,5-trisphosphate receptor type 2 EGF epidermal growth factor F13A1 coagulation factor XIII A chain SLC12A6 solute carrier family 12 F10 coagulation factor X member 6 CDH13 cadherin 13 POSTN periostin ACKR3 atypical chemokine receptor 3 CD36 CD36 molecule COL8A1 collagen type VIII alpha 1 chain MERTK MER proto-oncogene, tyrosine kinase SFRP2 secreted frizzled related PRKCA protein kinase C alpha protein 2 CYSLTR2 cysteinyl leukotriene receptor PDPN podoplanin 2 PDE3B phosphodiesterase 3B P2RY1 purinergic receptor P2Y1 PRKCA protein kinase C alpha TM4SF4 transmembrane 4 L six family member 4 PDPN podoplanin ANO6 anoctamin 6

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SGCB sarcoglycan beta ABAT 4-aminobutyrate aminotransferase FZD5 frizzled class receptor 5 PPARA peroxisome proliferator activated receptor alpha ESM1 endothelial cell specific HBD hemoglobin subunit delta molecule 1 CDH2 cadherin 2 HBB hemoglobin subunit beta S1PR1 sphingosine-1-phosphate PECAM1 platelet and endothelial cell adhesion receptor 1 molecule 1 PTEN phosphatase and tensin homolog HEG1 heart development protein Ion Homeostasis with EGF like domains 1 PECAM1 platelet and endothelial cell ABCC2 ATP binding cassette subfamily C adhesion molecule 1 member 2 GJA5 gap junction protein alpha 5 PIK3CB phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit beta ATP2B4 ATPase plasma membrane Ca2+ transporting 4 Actin Cytoskeleton PYGM glycogen phosphorylase, muscle associated

ACTN2 actinin alpha 2 ATP2B1 ATPase plasma membrane Ca2+ transporting 1

MYBPC2 myosin binding protein C, fast CA12 carbonic anhydrase 12 type

SORBS1 sorbin and SH3 domain ATP12A ATPase H+/K+ transporting non- containing 1 gastric alpha2 subunit

DAAM1 dishevelled associated SLC4A4 solute carrier family 4 member 4 activator of morphogenesis 1

TNNC2 troponin C2, fast skeletal type P2RX5 purinergic receptor P2X 5

MYOM1 myomesin 1 CAPN3 calpain 3

TNNT1 troponin T1, slow skeletal type SLC8A3 solute carrier family 8 member A3

COBL cordon-bleu WH2 repeat SLC30A4 solute carrier family 30 member 4 protein

TNNC1 troponin C1, slow skeletal and CAV1 caveolin 1 cardiac type

MYO18B myosin XVIIIB CLCN3 chloride voltage-gated channel 3

TMOD1 tropomodulin 1 SLC9A2 solute carrier family 9 member A2

RDX radixin ITPR2 inositol 1,4,5-trisphosphate receptor type 2

ABL2 ABL proto-oncogene 2, non- CD36 CD36 molecule receptor tyrosine kinase

MYH15 myosin heavy chain 15 SLC31A1 solute carrier family 31 member 1

ZNF185 zinc finger protein 185 with SLC39A8 solute carrier family 39 member 8 LIM domain

DST dystonin SYPL2 synaptophysin like 2

PDLIM3 PDZ and LIM domain 3 CASQ1 calsequestrin 1

SHROOM4 shroom family member 4 ABL2 ABL proto-oncogene 2, non-receptor tyrosine kinase

PPP1R9A protein phosphatase 1 XPR1 xenotropic and polytropic retrovirus regulatory subunit 9A receptor 1

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EPB41 erythrocyte membrane KLHL3 kelch like family member 3 protein band 4.1

TNNI1 troponin I1, slow skeletal type JPH2 junctophilin 2

ANKRD23 ankyrin repeat domain 23 AKAP6 A-kinase anchoring protein 6

TMOD4 tropomodulin 4 CACNA2D calcium voltage-gated channel 1 auxiliary subunit alpha2delta 1

LMOD3 leiomodin 3 MCU mitochondrial calcium uniporter

ABLIM2 actin binding LIM protein CNNM4 cyclin and CBS domain divalent metal family member 2 cation transport mediator 4

MYRIP myosin VIIA and Rab SLC30A7 solute carrier family 30 member 7 interacting protein

CDH2 cadherin 2 CXCL11 C-X-C motif chemokine ligand 11

LMOD2 leiomodin 2 P2RY1 purinergic receptor P2Y1

ABLIM3 actin binding LIM protein S1PR1 sphingosine-1-phosphate receptor 1 family member 3

MYO18A myosin XVIIIA ATP6V0A ATPase H+ transporting V0 subunit 2 a2

SVIL supervillin TRDN triadin

DMD dystrophin GJA5 gap junction protein alpha 5

ATPase Activity Glycogen Metabolism

ABCC2 ATP binding cassette PYGM glycogen phosphorylase, muscle subfamily C member 2 associated

ATP9A ATPase phospholipid GSK3B glycogen synthase kinase 3 beta transporting 9A

ATP11B ATPase phospholipid SORBS1 sorbin and SH3 domain containing 1 transporting 11B

ATP2B4 ATPase plasma membrane GBE1 1,4-alpha-glucan branching enzyme 1 Ca2+ transporting 4

ATP2B1 ATPase plasma membrane AGL amylo-alpha-1, 6-glucosidase, 4- Ca2+ transporting 1 alpha-glucanotransferase

ATP12A ATPase H+/K+ transporting INSR insulin receptor non-gastric alpha2 subunit

PCYOX1 prenylcysteine oxidase 1 PPP1R3B protein phosphatase 1 regulatory subunit 3B

ATP8A1 ATPase phospholipid PPP1R2 protein phosphatase 1 regulatory transporting 8A1 inhibitor subunit 2

ABCC4 ATP binding cassette PPP1CC protein phosphatase 1 catalytic subfamily C member 4 subunit gamma

ABCA5 ATP binding cassette subfamily A member 5 ATP6V0A ATPase H+ transporting V0

2 subunit a2

Table 2: Downregulated genes in preserved static cold storage kidneys at T1 compared to T0.

Upregulated KEGG Pathways Input Background Corrected P-Value Allograft rejection 8 36 8.2e-06 Cell adhesion molecules (CAMs) 11 143 0.0003 Phagosome 10 152 0.002

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MAPK signaling pathway 12 257 0.006 Endocytosis 11 264 0.02

Upregulated Biological Processes MHC protein complex 8 23 6.3e-07 cell differentiation 84 3524 2.5e-06 nitrogen compound metabolic process 131 6701 1.05e-05 response to stress 80 3656 0.0001 response to cytokine 27 799 0.0005 microtubule cytoskeleton 32 1064 0.0007 anatomical structure morphogenesis 57 2521 0.001 regulation of cell differentiation 39 1486 0.001 cytokine-mediated signaling pathway 20 542 0.002 defense response 38 1528 0.004 phosphorylation 49 2194 0.005

Table 3: Upregulated pathways and biological processes in static cold storage kidneys at T1 compared to T0. KEGG pathways and biological processes were obtained by using KOBAS3.0 to analyze the differentially expressed genes list acquired through RNA Sequencing.

Upregulated Genes Cytoskeleton Defense/Cytokine Response

SARM1 sterile alpha and TIR motif containing 1 SARM1 sterile alpha and TIR motif containing 1

TTC19 tetratricopeptide repeat domain 19 TNFRSF1A TNF receptor superfamily member 1A

CUL7 cullin 7 UNC13D unc-13 homolog D

CAPN6 calpain 6 ACIN1 apoptotic chromatin condensation inducer 1

CDC45 cell division cycle 45 NUP93 nucleoporin 93

TRIP4 thyroid hormone receptor interactor 4 RIPK2 receptor interacting serine/threonine kinase 2

CCDC61 coiled-coil domain containing 61 IL27RA interleukin 27 receptor subunit alpha

CCDC114 coiled-coil domain containing 114 CCL2 C-C motif chemokine ligand 2

PHF1 PHD finger protein 1 TCIRG1 T-cell immune regulator 1, ATPase H+ transporting V0 subunit a3

IFT57 intraflagellar transport 57 TRIM62 tripartite motif containing 62

WRAP73 WD repeat containing, antisense to NMI N-myc and STAT interactor TP73

UPF3B UPF3B, regulator of nonsense mediated C19orf66 chromosome 19 open reading frame mRNA decay 66

FAM110A family with sequence similarity 110 AKIRIN2 akirin 2 member A

TUBGCP6 tubulin gamma complex associated TLR4 toll like receptor 4 protein 6

NPHP4 nephrocystin 4 FLOT1 flotillin 1

TPGS2 tubulin polyglutamylase complex IFI44L interferon induced protein 44 like subunit 2

134

BICDL1 BICD family like cargo adaptor 1 HERC5 HECT and RLD domain containing E3 ubiquitin protein ligase 5

KIF12 kinesin family member 12 AHSG alpha 2-HS glycoprotein

FLOT1 flotillin 1 PTGES prostaglandin E synthase

CEP131 centrosomal protein 131 ZYX zyxin

NCKAP5L NCK associated protein 5 like NFKBID NFKB inhibitor delta

KIFC2 kinesin family member C2 BATF2 basic leucine zipper ATF-like transcription factor 2

E4F1 E4F transcription factor 1 MAP4K2 mitogen-activated protein kinase kinase kinase kinase 2

KIF5C kinesin family member 5C LGALS9 galectin 9

UMOD uromodulin UMOD uromodulin

CCDC151 coiled-coil domain containing 151 FOS Fos proto-oncogene, AP-1 transcription factor subunit

DAXX death domain associated protein PER1 period circadian regulator 1

TTLL3 tubulin tyrosine ligase like 3 USP18 ubiquitin specific peptidase 18

HLA-DRB1 major histocompatibility complex, class II, DR beta 1

Cell Differentiation and Proliferation HLA-DRB5 major histocompatibility complex, class II, DR beta 5

TNS2 tensin 2 HLA-C major histocompatibility complex, class I, C

FLOT1 flotillin 1 HLA-E major histocompatibility complex, class I, E

ATAT1 alpha tubulin acetyltransferase 1 HLA-G major histocompatibility complex, class I, G

IFT57 intraflagellar transport 57 HLA-F major histocompatibility complex, class I, F

AKIRIN2 akirin 2 HLA-A major histocompatibility complex, class I, A

ATOH8 atonal bHLH transcription factor 8 LAT linker for activation of T-cells

HLA-B major histocompatibility complex, class IRF9 interferon regulatory factor 9 I, B

DPH1 diphthamide biosynthesis 1 HLA-B major histocompatibility complex, class I, B

CEP131 centrosomal protein 131 CLIP3 CAP-Gly domain containing linker protein 3

DDX39B DExD-box helicase 39B ALDH1A2 aldehyde dehydrogenase 1 family member A2

TRIM62 tripartite motif containing 62 LRRC4B leucine rich repeat containing 4B

MED25 mediator complex subunit 25 IL15RA interleukin 15 receptor subunit alpha

HLA-B major histocompatibility complex, class ADAMTS7 ADAM metallopeptidase with I, B thrombospondin type 1 motif 7

TFAP2B transcription factor AP-2 beta CLCF1 cardiotrophin like cytokine factor 1

SRSF6 serine and arginine rich splicing factor DAXX death domain associated protein 6

DAXX death domain associated protein CPNE1 copine 1

PTGES prostaglandin E synthase

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TCIRG1 T-cell immune regulator 1, ATPase H+ Nitrogen Compound Metabolic Process transporting V0 subunit a3

IL15RA interleukin 15 receptor subunit alpha HOXA11 homeobox A11

DAXX death domain associated protein TEAD3 TEA domain transcription factor 3

HSF4 heat shock transcription factor 4 DKK3 dickkopf WNT signaling pathway inhibitor 3

GABBR1 gamma-aminobutyric acid type B PHF21B PHD finger protein 21B receptor subunit 1

RAMP2 receptor activity modifying protein 2 TLE2 transducin like enhancer of split 2

GABBR1 gamma-aminobutyric acid type B ME1 malic enzyme 1 receptor subunit 1

CLK1 CDC like kinase 1 MLH1 mutL homolog 1

TLR4 toll like receptor 4 ACTL6B actin like 6B

ALDH1A2 aldehyde dehydrogenase 1 family FUS FUS RNA binding protein member A2

GDI1 GDP dissociation inhibitor 1 CDC45 cell division cycle 45

NFKBID NFKB inhibitor delta CREM cAMP responsive element modulator

TBX2 T-box 2 TRIP4 thyroid hormone receptor interactor 4

DAXX death domain associated protein SNRNP70 small nuclear ribonucleoprotein U1 subunit 70

ENPP1 ectonucleotide PBX4 PBX homeobox 4 pyrophosphatase/phosphodiesterase 1

DAXX death domain associated protein HOXA5 homeobox A5

UNC13D unc-13 homolog D ENG endoglin

ATAT1 alpha tubulin acetyltransferase 1 HOXB6 homeobox B6

HLA-G major histocompatibility complex, class FOXM1 forkhead box M1 I, G

ATAT1 alpha tubulin acetyltransferase 1 PHF1 PHD finger protein 1

HLA-G major histocompatibility complex, class APBB3 amyloid beta precursor protein I, G binding family B member 3

CUL7 cullin 7 SMAD5 SMAD family member 5

ADRA1D adrenoceptor alpha 1D PDGFRB platelet derived growth factor receptor beta

HLA-B major histocompatibility complex, class IFT57 intraflagellar transport 57 I, B

DDX39B DExD-box helicase 39B LOXL3 lysyl oxidase like 3

GABBR1 gamma-aminobutyric acid type B DUSP1 dual specificity phosphatase 1 receptor subunit 1

FOS Fos proto-oncogene, AP-1 transcription TBX2 T-box 2 factor subunit

CDC25B cell division cycle 25B MAPK8IP1 mitogen-activated protein kinase 8 interacting protein 1

ADRA1B adrenoceptor alpha 1B UPF3B UPF3B, regulator of nonsense mediated mRNA decay

P3H2 prolyl 3-hydroxylase 2 HOXD3 homeobox D3

YWHAH tyrosine 3- HOXD9 homeobox D9 monooxygenase/tryptophan 5- monooxygenase activation protein eta

DDX39B DExD-box helicase 39B RRP8 ribosomal RNA processing 8

136

KCTD1 potassium channel tetramerization domain containing 1

SPAG8 sperm associated antigen 8

ASCC1 activating signal cointegrator 1 complex subunit 1

SKAP1 src kinase associated phosphoprotein 1

Table 4: Upregulated genes in preserved static cold storage kidneys at T1 compared to T0.

Persufflated kidneys at T1 compared to T0

Downregulated KEGG Pathways Input Background P-Value Glutathione metabolism 5 51 3.8e-05 Drug metabolism - cytochrome P450 5 68 0.0001 Downregulated Biological Processes Input Background P-Value epithelial cell differentiation 16 550 0.0004 keratinocyte proliferation 5 37 0.001 epithelium development 21 1045 0.002 cell adhesion 25 1422 0.002 glutathione metabolic process 5 52 0.005 multicellular organismal water homeostasis 5 59 0.007 cell migration 19 1180 0.02 lysosomal membrane 8 272 0.03 epithelial cell proliferation 9 343 0.03 leukocyte migration 9 356 0.03 cytokine activity 7 222 0.04 metalloendopeptidase activity 5 111 0.04 peptidase activity 12 615 0.04 NF-kappaB-inducing kinase activity 2 6 0.04

Table 5: Downregulated pathways and biological processes in persufflated kidneys at T1 compared to T0. KEGG pathways and biological processes were obtained by using KOBAS3.0 to analyze the differentially expressed genes list acquired through RNA Sequencing

Downregulated Genes Glutathione Metabolism Leukocyte Migration

GGT6 gamma-glutamyltransferase 6 ITGA3 integrin subunit alpha 3

GSTA3 glutathione S-transferase alpha 3 TBX21 T-box 21

GSTA5 glutathione S-transferase alpha 5 MMP9 matrix metallopeptidase 9

GSTA1 glutathione S-transferase alpha 1 GATA3 GATA binding protein 3

GSTA2 glutathione S-transferase alpha 2 ZAP70 zeta chain of T-cell receptor associated protein kinase 70

CSF3R colony stimulating factor 3 receptor

Epithelial Cell Differentiation TNFSF18 TNF superfamily member 18

137

CYP26B1 cytochrome P450 family 26 subfamily DOK2 docking protein 2 B member 1

TP63 tumor protein p63 CXCL11 C-X-C motif chemokine ligand 11

TGM1 transglutaminase 1

UPK1A uroplakin 1A Cytokine Activity

GATA3 GATA binding protein 3 ITGA3 integrin subunit alpha 3

PPL periplakin FAT2 FAT atypical cadherin 2

ST14 suppression of tumorigenicity 14 MMP9 matrix metallopeptidase 9

CERS3 ceramide synthase 3 FERMT1 fermitin family member 1

TMEM79 transmembrane protein 79 GATA3 GATA binding protein 3

HEG1 heart development protein with EGF EFNB2 ephrin B2 like domains 1

PSAPL1 prosaposin like 1 S100A2 S100 calcium binding protein A2

SLC9A4 solute carrier family 9 member A4

ZNF703 zinc finger protein 703 Cell Adhesion

KRT14 keratin 14 CYP26B1 cytochrome P450 family 26 subfamily B member 1

GSTA1 glutathione S-transferase alpha 1 CDH1 cadherin 1

GSTA2 glutathione S-transferase alpha 2 GATA3 GATA binding protein 3

ZAP70 zeta chain of T-cell receptor associated protein kinase 70

Epithelial Cell Proliferation TNFSF18 TNF superfamily member 18

TP63 tumor protein p63 EFNB2 ephrin B2

TGM1 transglutaminase 1 SCGB1A1 secretoglobin family 1A member 1

FERMT1 fermitin family member 1 KIF26B kinesin family member 26B

GATA3 GATA binding protein 3 LMO1 LIM domain only 1

EFNB2 ephrin B2 B4GALNT2 beta-1,4-N-acetyl-galactosaminyl transferase 2

SDR16C5 short chain dehydrogenase/reductase ZNF703 zinc finger protein 703 family 16C member 5

ZNF703 zinc finger protein 703

PLXNB3 plexin B3 Lysosome

SERPINB5 serpin family B member 5 SPHK2 sphingosine kinase 2

TLR8 toll like receptor 8

Water Homeostasis KIAA1324 KIAA1324

CYP26B1 cytochrome P450 family 26 subfamily TSPAN1 tetraspanin 1 B member 1

TP63 tumor protein p63 CD68 CD68 molecule

AVPR2 arginine vasopressin receptor 2 TMEM8A transmembrane protein 8A

TMEM79 transmembrane protein 79 SLC2A8 solute carrier family 2 member 8

AQP2 aquaporin 2 TMEM79 transmembrane protein 79

Table 6: Downregulated genes in persufflated kidneys at T1 compared to T0.

138

Upregulated KEGG Pathway Input Background Corrected P- Value Protein digestion and absorption 15 90 2.5e-12 ECM-receptor interaction 11 83 3.5e-08 Focal adhesion 15 206 6.03-08 cGMP-PKG signaling pathway 13 173 4.3e-07 Vascular smooth muscle contraction 10 123 7.4e-06 PI3K-Akt signaling pathway 15 343 1.6e-05 Platelet activation 7 125 0.002 Renin secretion 5 64 0.003 Gap junction 4 88 0.04 Hedgehog signaling pathway 3 46 0.04 Cell adhesion molecules (CAMs) 5 143 0.04 cAMP signaling pathway 6 201 0.04 Upregulated Biological Processes Input Background Corrected P- Value Anatomical structure development 129 5323 1.7e-29 Cell differentiation 80 3524 5.07e-14 Cell development 50 1875 2.1e-10 Blood vessel morphogenesis 25 489 3.9e-10 Organ morphogenesis 34 968 9.6e-10 Angiogenesis 21 417 1.9e-08 Extracellular matrix disassembly 11 87 5.3e-08 Cell-matrix adhesion 14 194 2.01e-07 Cell motility 34 1291 6.7e-07 Growth factor binding 11 126 1.2e-06 Response to wounding 22 650 3.8e-06 Cell-substrate adherens junction 17 392 3.9e-06 Renal system development 14 278 8.4e-06 Wound healing 18 541 5.01e-05 Renal system vasculature morphogenesis 3 7 0.001

Table 7: Upregulated pathways and biological processes in persufflated kidneys at T1 compared to T0. KEGG pathways and biological processes were obtained by using KOBAS3.0 to analyze the differentially expressed genes list acquired through RNA Sequencing

Upregulated Genes Angiogenesis Differentiation

THSD7A thrombospondin type 1 domain HGF hepatocyte growth factor containing 7A

DCN decorin EPHA3 EPH receptor A3

139

HGF hepatocyte growth factor GLI2 GLI family zinc finger 2

NOTCH3 notch 3 ITGA8 integrin subunit alpha 8

MCAM melanoma cell adhesion molecule NRP1 neuropilin 1

FAP fibroblast activation protein alpha ECM2 extracellular matrix protein 2

NRP1 neuropilin 1 LGI1 leucine rich glioma inactivated 1

SPARC secreted protein acidic and cysteine FN1 fibronectin 1 rich

FN1 fibronectin 1 PALLD palladin, cytoskeletal associated protein

PDGFRA platelet derived growth factor MAP1B microtubule associated protein 1B receptor alpha

NOV nephroblastoma overexpressed MATN2 matrilin 2

COL8A1 collagen type VIII alpha 1 chain LAMC1 laminin subunit gamma 1

EDNRA endothelin receptor type A FRMD6 FERM domain containing 6

ANGPT1 angiopoietin 1 CDH11 cadherin 11

PLXDC1 plexin domain containing 1 CNTN4 contactin 4

ROBO1 roundabout guidance receptor 1 VLDLR very low density lipoprotein receptor

SEMA3E semaphorin 3E NTRK2 neurotrophic receptor tyrosine kinase 2

CSPG4 chondroitin sulfate proteoglycan 4 NCAM1 neural cell adhesion molecule 1

CCBE1 collagen and calcium binding EGF NTNG1 netrin G1 domains 1

THBS2 thrombospondin 2 ANTXR1 anthrax toxin receptor 1

COL15A collagen type XV alpha 1 chain ROBO1 roundabout guidance receptor 1

SEMA3E semaphorin 3E

Growth Factor Response ROBO2 roundabout guidance receptor 2

LTBP1 latent transforming growth factor SLIT1 slit guidance ligand 1 beta binding protein 1

LTBP2 latent transforming growth factor LAMA2 laminin subunit alpha 2 beta binding protein 2

PXDN peroxidasin

PDGFRA platelet derived growth factor Extracellular Matrix Organization receptor alpha

NTRK2 neurotrophic receptor tyrosine DCN decorin kinase 2

COL1A2 collagen type I alpha 2 chain VCAN versican

IGFBP6 insulin like growth factor binding ELN elastin protein 6

COL3A1 collagen type III alpha 1 chain COL11A1 collagen type XI alpha 1 chain

A2M alpha-2-macroglobulin ITGA8 integrin subunit alpha 8

FAP fibroblast activation protein alpha

Renal System Development COL5A3 collagen type V alpha 3 chain

DCN decorin ADAMTS2 ADAM metallopeptidase with thrombospondin type 1 motif 2

140

GLI2 GLI family zinc finger 2 NID2 nidogen 2

NOTCH3 notch 3 ECM2 extracellular matrix protein 2

ITGA8 integrin subunit alpha 8 COL12A1 collagen type XII alpha 1 chain

NRP1 neuropilin 1 LAMA4 laminin subunit alpha 4

TBX18 T-box 18 LOX lysyl oxidase

NID1 nidogen 1 SPARC secreted protein acidic and cysteine rich

ALDH1A aldehyde dehydrogenase 1 family FN1 fibronectin 1

2 member A2

PDGFRA platelet derived growth factor NID1 nidogen 1 receptor alpha

AGTR1 angiotensin II receptor type 1 LTBP2 latent transforming growth factor beta binding protein 2

EDNRA endothelin receptor type A RECK reversion inducing cysteine rich protein with kazal motifs

ANGPT1 angiopoietin 1 PXDN peroxidasin

FBN1 fibrillin 1 GFAP glial fibrillary acidic protein

ROBO2 roundabout guidance receptor 2 LAMC1 laminin subunit gamma 1

ITGA11 integrin subunit alpha 11

Wound Healing FBN2 fibrillin 2

MYLK myosin light chain kinase FBLN5 fibulin 5

FAP fibroblast activation protein alpha COL8A1 collagen type VIII alpha 1 chain

LOX lysyl oxidase ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif 5

SPARC secreted protein acidic and cysteine MMP16 matrix metallopeptidase 16 rich

FN1 fibronectin 1 COL6A3 collagen type VI alpha 3 chain

PDGFRA platelet derived growth factor EGFLAM EGF like, fibronectin type III and receptor alpha laminin G domains

ADRA2A adrenoceptor alpha 2A COL1A2 collagen type I alpha 2 chain

CSRP1 cysteine and glycine rich protein 1 FBN1 fibrillin 1

COL1A2 collagen type I alpha 2 chain COL3A1 collagen type III alpha 1 chain

SCARA5 scavenger receptor class A member A2M alpha-2-macroglobulin 5

PRKG1 protein kinase, cGMP-dependent, COL14A1 collagen type XIV alpha 1 chain type I

GNG2 G protein subunit gamma 2 COL4A5 collagen type IV alpha 5 chain

ADRA2B adrenoceptor alpha 2B LAMA2 laminin subunit alpha 2

COL13A1 collagen type XIII alpha 1 chain

COL4A6 collagen type IV alpha 6 chain

ADAMTSL ADAMTS like 2

COL5A2 collagen type V alpha 2 chain

Table 8: Upregulated genes in persufflated kidneys at T1 compared to T0.

141

Persufflated kidneys compared to static cold storage kidneys at T1

Downregulated KEGG Pathways Input Backgroun Corrected P- d Value Carbohydrate digestion and absorption 5 48 0.002 Starch and sucrose metabolism 5 57 0.003 NF-kappa B signaling pathway 5 93 0.01 Downregulated Biological Processes Input Backgroun Corrected P- d Value amylase activity 5 6 2.8e-06 metabolic process 82 10669 0.002 transcription, DNA-templated 39 3661 0.002 cellular macromolecule biosynthetic process 47 4840 0.002 gene expression 48 5206 0.004 animal organ development 31 3095 0.02 regulation of lymphocyte activation 8 371 0.03 positive regulation of leukocyte mediated 4 81 0.04 immunity lymphocyte differentiation 7 300 0.04

Table 9: Downregulated pathways and biological processes in persufflated kidneys compared to static cold storage kidneys at T1. KEGG pathways and biological processes were obtained by using KOBAS3.0 to analyze the differentially expressed genes list acquired through RNA Sequencing.

Downregulated Genes Gene Regulation/Transcription Amylase Activity

BCL3 B-cell CLL/lymphoma 3 AMY1B amylase, alpha 1B (salivary)

PHRF1 PHD and ring finger domains 1 AMY1C amylase, alpha 1C (salivary)

ACTL6B actin like 6B AMY1A amylase, alpha 1A (salivary)

CNOT3 CCR4-NOT transcription complex AMY2B amylase, alpha 2B (pancreatic) subunit 3

FLT3LG fms related tyrosine kinase 3 ligand AMY2A amylase, alpha 2A (pancreatic)

HSF4 heat shock transcription factor 4

AXIN1 axin 1 Cellular Macromolecule Biosynthetic Process

CAPN15 calpain 15 PNKP polynucleotide kinase 3'-phosphatase

MED25 mediator complex subunit 25 NEURL1 neuralized E3 ubiquitin protein ligase 1

HOXA3 homeobox A3 MRPS2 mitochondrial ribosomal protein S2

GATA3 GATA binding protein 3 NUDT14 nudix hydrolase 14

HOXB6 homeobox B6 CCR7 C-C motif chemokine receptor 7

FOXM1 forkhead box M1 METTL17 methyltransferase like 17

HOXB3 homeobox B3 LARGE2 xylosyl- and glucuronyltransferase 2

TBX2 T-box 2 ALAS2 5'-aminolevulinate synthase 2

142

SRSF6 serine and arginine rich splicing factor 6

TRAF2 TNF receptor associated factor 2 Lymphocyte Differentiation/Activation

HOXD9 homeobox D9 FLT3LG fms related tyrosine kinase 3 ligand

TRAF7 TNF receptor associated factor 7 GATA3 GATA binding protein 3

TFAP2A transcription factor AP-2 alpha ZAP70 zeta chain of T-cell receptor associated protein kinase 70

PELP1 proline, glutamate and leucine rich CCR7 C-C motif chemokine receptor 7 protein 1

RNF25 ring finger protein 25 TRAF2 TNF receptor associated factor 2

H2AFZ H2A histone family member Z NFKBID NFKB inhibitor delta

ZSCAN21 zinc finger and SCAN domain containing TBC1D10C TBC1 domain family member 10C 21

TSC22D4 TSC22 domain family member 4 CLCF1 cardiotrophin like cytokine factor 1

CDT1 chromatin licensing and DNA replication factor 1

NFKBID NFKB inhibitor delta Organ Development

FOXI1 forkhead box I1 NPHP4 nephrocystin 4

HOXB2 homeobox B2 LTB lymphotoxin beta

PIDD1 p53-induced death domain protein 1 ZAP70 zeta chain associated protein kinase 70

ZNF354B zinc finger protein 354B CCNF cyclin F

IRAK1 interleukin 1 receptor associated kinase 1 LTB lymphotoxin beta

HMX2 H6 family homeobox 2 RNF207 ring finger protein 207

ZNF777 zinc finger protein 777 TRIM45 tripartite motif containing 45

ZNF251 zinc finger protein 251 CALB1 calbindin 1

ZNF511 zinc finger protein 511 CLDN4 claudin 4

DDX39B DExD-box helicase 39B ZC4H2 zinc finger C4H2-type containing

ZNF358 zinc finger protein 358 CLCF1 cardiotrophin like cytokine factor 1

ZNF579 zinc finger protein 579

Table 10: Downregulated genes in persufflated kidneys compared to static cold storage kidneys at T1.

Upregulated KEGG Pathways Inpu Backgroun Corrected P- t d Value Focal adhesion 22 206 6.5e-12 Cell adhesion molecules (CAMs) 17 143 6.2e-10 ECM-receptor interaction 13 83 5.9e-09 PI3K-Akt signaling pathway 22 343 3.3e-08 Protein digestion and absorption 12 90 1.2e-07 Ras signaling pathway 12 231 0.0005 cGMP-PKG signaling pathway 10 173 0.0009 Rap1 signaling pathway 11 216 0.001 Vascular smooth muscle contraction 8 123 0.002

143

MAPK signaling pathway 11 257 0.004 Calcium signaling pathway 9 179 0.004 Cytokine-cytokine receptor interaction 11 265 0.005 Complement and coagulation cascades 5 79 0.02 Leukocyte transendothelial migration 6 120 0.02 AMPK signaling pathway 6 125 0.03 Platelet activation 6 125 0.03 Gap junction 5 88 0.03 FoxO signaling pathway 6 135 0.04 Tight junction 6 139 0.04 Wnt signaling pathway 6 142 0.04 HIF-1 signaling pathway 5 105 0.05 Upregulated Biological Processes Inpu Backgroun Corrected P- t d Value extracellular matrix 54 422 9.9e-33 anatomical structure development 169 5323 9.9e-33 cell adhesion 86 1422 2.8e-31 angiogenesis 40 417 3.4e-20 cell differentiation 112 3524 3.3e-19 response to growth factor 39 627 6.7e-14 epithelial cell migration 22 219 1.9e-11 wound healing 31 541 2.9e-10 endothelial cell migration 17 152 1.4e-09 epithelial cell proliferation 24 343 1.5e-09 response to oxygen-containing compound 51 1475 3.3e-09 regulation of body fluid levels 27 508 2.2e-08 cell-cell junction organization 18 216 2.4e-08 hemostasis 21 361 3.2e-07 extracellular matrix disassembly 11 87 7.3e-07 endothelial cell proliferation 12 113 9.8e-07 leukocyte migration 20 356 1.1e-06 response to stress 82 3656 1.9e-06 ion transport 42 1415 5.2e-06 response to hypoxia 17 293 5.9e-06 regulation of protein metabolic process 60 2449 8.1e-06 adherens junction organization 10 116 5.1e-05 regeneration 12 182 7.4e-05 metabolic process 173 10669 0.0001 immune system process 55 2411 0.0002 response to nitrogen compound 28 904 0.0002 cell growth 19 490 0.0002

144

regulation of apoptotic process 36 1386 0.0004 regulation of programmed cell death 36 1398 0.0005 renal system development 13 278 0.0007 inflammatory response 21 647 0.0009 defense response 35 1528 0.004 ion homeostasis 18 659 0.01

Table 11: Upregulated pathways and biological processes in persufflated kidneys compared to static cold storage kidneys at T1. KEGG pathways and biological processes were obtained by using KOBAS3.0 to analyze the differentially expressed genes list acquired through RNA Sequencing.

Upregulated Genes Angiogenesis Inflammatory Response

THSD7A thrombospondin type 1 domain CFH complement factor H containing 7A

CX3CL1 C-X3-C motif chemokine ligand 1 SLC7A2 solute carrier family 7 member 2

HGF hepatocyte growth factor CX3CL1 C-X3-C motif chemokine ligand 1

ADGRA2 adhesion G protein-coupled receptor A2 HGF hepatocyte growth factor

FLT4 fms related tyrosine kinase 4 CALCRL calcitonin receptor like receptor

CALCRL calcitonin receptor like receptor NFATC3 nuclear factor of activated T-cells 3

TGFB2 transforming growth factor beta 2 P2RX7 purinergic receptor P2X 7

ANGPT4 angiopoietin 4 ACVR1 activin A receptor type 1

FLT1 fms related tyrosine kinase 1 FN1 fibronectin 1

CAV1 caveolin 1 TEK TEK receptor tyrosine kinase

MEOX2 mesenchyme homeobox 2 PLP1 proteolipid protein 1

SEMA5A semaphorin 5A PTGIS prostaglandin I2 synthase

SPARC secreted protein acidic and cysteine rich SERPINF1 serpin family F member 1

ACVR1 activin A receptor type 1 IL33 interleukin 33

FN1 fibronectin 1 NTRK2 neurotrophic receptor tyrosine kinase 2

EPAS1 endothelial PAS domain protein 1 ADAMTS12 ADAM metallopeptidase with thrombospondin type 1 motif 12

NRP2 neuropilin 2 GJA1 gap junction protein alpha 1

CTGF connective tissue growth factor JAM3 junctional adhesion molecule 3

TEK TEK receptor tyrosine kinase SELP selectin P

PTGIS prostaglandin I2 synthase F8 coagulation factor VIII

EFNB2 ephrin B2 TRIL TLR4 interactor with leucine rich repeats

PTPRB protein tyrosine phosphatase, receptor type B

KDR kinase insert domain receptor Ion Homeostasis

SERPINF1 serpin family F member 1 ATP2B4 ATPase plasma membrane Ca2+ transporting 4

EPHB2 EPH receptor B2 P2RX7 purinergic receptor P2X 7

145

PDGFRA platelet derived growth factor receptor ESR1 estrogen receptor 1 alpha

SLC12A6 solute carrier family 12 member 6 CAV1 caveolin 1

CDH13 cadherin 13 EPAS1 endothelial PAS domain protein 1

HSPG2 heparan sulfate proteoglycan 2 ADRA1A adrenoceptor alpha 1A

COL8A1 collagen type VIII alpha 1 chain KDR kinase insert domain receptor

RSPO3 R-spondin 3 PDGFRA platelet derived growth factor receptor alpha

PRKCA protein kinase C alpha SCN7A sodium voltage-gated channel alpha subunit 7

EPHB1 EPH receptor B1 TRPC6 transient receptor potential cation channel subfamily C member 6

TGFBR2 transforming growth factor beta ITPR1 inositol 1,4,5-trisphosphate receptor 2 receptor type1

JAM3 junctional adhesion molecule 3 AKAP6 A-kinase anchoring protein 6

ROBO1 roundabout guidance receptor 1 GJA1 gap junction protein alpha 1

S1PR1 sphingosine-1-phosphate receptor 1 CACNA2D1 calcium voltage-gated channel auxiliary subunit alpha2delta 1

MMRN2 multimerin 2 STC1 stanniocalcin 1

THBS2 thrombospondin 2 SCARA5 scavenger receptor class A member 5

COL15A1 collagen type XV alpha 1 chain S1PR1 sphingosine-1-phosphate receptor 1

P2RX2 purinergic receptor P2X 2

Endothelial/Epithelial Cell Proliferation

IGF1 insulin like growth factor 1 Endothelial/Epithelial Cell Migration

FLT4 fms related tyrosine kinase 4 ADGRA2 adhesion G protein-coupled receptor A2

TGFBR3 transforming growth factor beta FLT4 fms related tyrosine kinase 4 receptor 3

ESR1 estrogen receptor 1 TGFB2 transforming growth factor beta 2

TGFB2 transforming growth factor beta 2 ANGPT4 angiopoietin 4

CAV2 caveolin 2 CORO1C coronin 1C

CAV1 caveolin 1 SEMA5A semaphorin 5A

SEMA5A semaphorin 5A SPARC secreted protein acidic and cysteine rich

SPARC secreted protein acidic and cysteine rich NRP2 neuropilin 2

NRP2 neuropilin 2 TEK TEK receptor tyrosine kinase

TEK TEK receptor tyrosine kinase EFNB2 ephrin B2

EFNB2 ephrin B2 STAT5A signal transducer and activator of transcription 5A

STAT5A signal transducer and activator of KDR kinase insert domain receptor transcription 5A

KDR kinase insert domain receptor SERPINF1 serpin family F member 1

FOXP2 forkhead box P2 CDH13 cadherin 13

SERPINF1 serpin family F member 1 PTPRR protein tyrosine phosphatase, receptor type R

146

LAMC1 laminin subunit gamma 1 PRKCA protein kinase C alpha

CDH13 cadherin 13 STC1 stanniocalcin 1

COL8A1 collagen type VIII alpha 1 chain TGFBR2 transforming growth factor beta receptor 2

GJA1 gap junction protein alpha 1 AMOTL1 angiomotin like 1

PTPRK protein tyrosine phosphatase, receptor ZEB2 zinc finger E-box binding type K homeobox 2

PRKCA protein kinase C alpha ROBO1 roundabout guidance receptor 1

FZD7 frizzled class receptor 7 MMRN2 multimerin 2

ROBO1 roundabout guidance receptor 1

Negative Regulation of Apoptosis

Positive Regulation of Apoptosis CX3CL1 C-X3-C motif chemokine ligand 1

P2RX7 purinergic receptor P2X 7 IGF1 insulin like growth factor 1

MAP3K20 mitogen-activated protein kinase HGF hepatocyte growth factor [ kinase kinase 20

TGFB2 transforming growth factor beta 2 FLT4 fms related tyrosine kinase 4

SRPX sushi repeat containing protein, X- ANGPT4 angiopoietin 4 linked

CAV1 caveolin 1 CAV1 caveolin 1

ZBTB16 zinc finger and BTB domain containing SEMA5A semaphorin 5A 16

RASGRF2 Ras protein specific guanine nucleotide ACVR1 activin A receptor type 1 releasing factor 2

CTGF connective tissue growth factor FN1 fibronectin 1

TGFB3 transforming growth factor beta 3 FHL2 four and a half LIM domains 2

PTGIS prostaglandin I2 synthase TGFB3 transforming growth factor beta 3

ARHGEF9 Cdc42 guanine nucleotide exchange TEK TEK receptor tyrosine kinase factor 9

NTRK3 neurotrophic receptor tyrosine kinase 3 KDR kinase insert domain receptor

KALRN kalirin RhoGEF kinase NTRK2 neurotrophic receptor tyrosine kinase 2

ROBO1 roundabout guidance receptor 1 MERTK MER proto-oncogene, tyrosine kinase

AKAP13 A-kinase anchoring protein 13 PRKCA protein kinase C alpha

UNC5C unc-5 netrin receptor C MPZ myelin protein zero

ITGA1 integrin subunit alpha 1 AR androgen receptor

PDE3A phosphodiesterase 3A

Actin Cytoskeleton Regulation

MYLK myosin light chain kinase Response to Hypoxia

SORBS1 sorbin and SH3 domain containing 1 CX3CL1 C-X3-C motif chemokine ligand 1

CORO1C coronin 1C IGF1 insulin like growth factor 1

FHL2 four and a half LIM domains 2 TGFBR3 transforming growth factor beta receptor 3

TEK TEK receptor tyrosine kinase TGFB2 transforming growth factor beta 2

DST dystonin ANGPT4 angiopoietin 4

PGM5 phosphoglucomutase 5 FLT1 fms related tyrosine kinase 1

147

PDLIM3 PDZ and LIM domain 3 CAV1 caveolin 1

KALRN kalirin RhoGEF kinase EPAS1 endothelial PAS domain protein 1

ACTG2 actin, gamma 2, smooth muscle, enteric ECE1 endothelin converting enzyme 1

GABARAP GABA type A receptor-associated TGFB3 transforming growth factor beta 3 protein

CDH2 cadherin 2 TEK TEK receptor tyrosine kinase

AKAP13 A-kinase anchoring protein 13 PTGIS prostaglandin I2 synthase

SYNPO2 synaptopodin 2 TRPC6 transient receptor potential cation channel subfamily C member 6

PEAK1 pseudopodium enriched atypical kinase ITPR1 inositol 1,4,5-trisphosphate 1 receptor type 1

SVIL supervillin STC1 stanniocalcin 1

ACACA acetyl-CoA carboxylase alpha PENK proenkephalin

P2RX2 purinergic receptor P2X 2

Table 12: Upregulated genes in persufflated kidneys compared to static cold storage kidneys at T1.

148

Network Analyst analyses of differentially expressed genes from RNA Sequencing

SCS kidneys at T1 compared to T0

Figure 27: A network model delineating the relationship between the downregulated protein-protein interactions in static cold stored kidneys at T1 compared to T0. This figure visualizes signaling networks based on differentially expressed gene products. The map was drawn using a Fruchterman-Reingold force-directed layout with NetworkAnalyst. Each circle represents a protein, and each line an interaction between two proteins. Bigger and darker spots are the genes that are the most connected genes upregulated in the samples. The light- yellow dots are genes that are influenced by the downregulated genes in the biopsies.

Gene Name Degree Betweenness UBC 289 3685143 AR 276 875367.6 ATXN1 251 805937.8 GSK3B 243 719300.1 MCC 231 545594.2 MAPK1 187 522329.2 PPP1CC 185 540410.8 CAV1 118 308805.3 PRKCA 113 277615.5 EPB41 113 246261.4

149

Table 13: Highly connected, downregulated proteins identified in static cold stored kidneys at T1 compared to T0. The top most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

Downregulated Pathway (SCS T0 v T1) Total Hits P.Value MAPK signaling pathway 265 157 2.9E-20

ErbB signaling pathway 87 67 1.01E-17

Cell cycle 124 81 3.3E-14

T cell receptor signaling pathway 98 68 4.6E-14

Focal adhesion 200 115 9.4E-14

RIG-I-like receptor signaling pathway 49 41 1.5E-13

Apoptosis 83 59 4.4E-13

Adherens junction 70 51 3.8E-12

B cell receptor signaling pathway 75 53 1.02E-11

VEGF signaling pathway 76 51 4.9E-10

Table 14: Pathways enriched in static cold storage kidney samples at T1 compared to T0. A list of downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between T0 and T1.

Downregulated Pathway (SCS T0 v T1) Total Expected Hits P.Value transmembrane receptor protein tyrosine kinase 782 199 329 8.6E-26 signaling pathway negative regulation of apoptotic process 679 172 294 1.8E-25 epidermal growth factor receptor signaling pathway 167 42.4 100 2.8E-21 wound healing 700 178 281 1.2E-18 negative regulation of cell cycle 520 132 222 1.6E-18 programmed cell death 2160 548 714 1.6E-18 cellular response to stress 1620 413 558 7.9E-18 cell cycle arrest 428 109 188 2.2E-17 regulation of body fluid levels 680 173 270 3.3E-17 positive regulation of signal transduction 998 253 368 5.8E-17 establishment of protein localization 1460 370 504 6.9E-17

150

regulation of MAPK cascade 559 142 230 7.2E-17 cell-cell junction organization 186 47.2 100 9.8E-17 response to hypoxia 245 62.2 118 8.6E-15 cellular component disassembly involved in execution 78 19.8 52 1.9E-14 phase of apoptosis regulation of I-kappaB kinase/NF-kappaB cascade 210 53.3 104 3.4E-14

Table 15: Biological processes enriched in static cold storage kidney samples at T1 compared to T0. A list of downregulated Gene Ontology (GO) biological processes (BP) from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. BP are cellular physiological processes or signal transduction. It is a process that has more than one distinct steps or sets of molecular events with a defined beginning and an end. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ between T0 and T1.

Figure 28: A network model delineating the relationship between the upregulated protein- protein interactions in static cold stored kidneys at T1 compared to T0. This figure visualizes signaling networks based on differentially expressed gene products. The map was drawn using a Fruchterman-Reingold force-directed layout with NetworkAnalyst. Each circle represents a protein, and each line an interaction between two proteins. Bigger and darker spots are the genes that are the most connected genes upregulated in the samples. The light- yellow dots are genes that are influenced by the upregulated genes in the biopsies.

Gene Name Degree Betweenness PAN2 341 781951.1 HLA-B 281 639191.3 FOS 214 521645.9 SRRM2 186 327715.6

151

TNFRSF1A 167 376120.6 UBC 165 2208882 MLH1 140 311009 YWHAH 130 289273.2 NXF1 102 159628.1 SNRNP70 101 187734.1

Table 16: Highly connected, upregulated proteins identified in static cold stored kidneys at T1 compared to T0. The top most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

Upregulated Pathway (SCS T0 v T1) Total Hits P.Value Cell cycle 124 86 4.3E-22 Focal adhesion 200 106 2.7E-14 MAPK signaling pathway 265 130 6.9E-14 Toll-like receptor signaling pathway 97 60 2.4E-12 Adherens junction 70 47 8.7E-12 RIG-I-like receptor signaling pathway 49 36 4.4E-11 T cell receptor signaling pathway 98 58 7.4E-11 NOD-like receptor signaling pathway 49 35 2.9E-10 ErbB signaling pathway 87 51 1.7E-09 Regulation of actin cytoskeleton 182 86 1.5E-08

Table 17: Pathways enriched in static cold stored kidney samples at T1 compared to T0. A list of upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between T0 and T1.

Upregulated Pathway (SCS T0 v T1) Total Expected Hits P.Value regulation of programmed cell death 1550 325 502 4.4E-29 cellular response to stress 1620 340 509 7.1E-26 cytokine-mediated signaling pathway 374 78.3 166 4.4E-25 cellular protein complex disassembly 160 33.5 93 6.4E-25 negative regulation of cell cycle 520 109 207 1.3E-23 cell cycle arrest 428 89.6 179 2.4E-23 programmed cell death 2160 451 622 1.3E-21 cell cycle 1860 389 548 4.9E-21 innate immune response 638 134 231 6.6E-20

152

negative regulation of apoptotic process 679 142 241 1.9E-19 negative regulation of programmed cell death 691 145 242 1.1E-18 signal transduction in response to DNA damage 129 27 71 1.5E-17 regulation of DNA metabolic process 235 49.2 106 4.7E-17 stress-activated protein kinase signaling cascade 257 53.8 111 4.1E-16 I-kappaB kinase/NF-kappaB cascade 246 51.5 107 7.7E-16 cell proliferation 1900 398 532 2.4E-15

Table 18: Biological processes enriched in static cold stored kidneys at T1 compared to T0. A list of upregulated Gene Ontology (GO) biological processes (BP) from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. BP are cellular physiological processes or signal transduction. It is a process that has more than one distinct steps or sets of molecular events with a defined beginning and an end. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ between T0 and T1.

PSF kidneys at T1 compared to T0

Figure 29: A network model delineating the relationship between the downregulated protein-protein interactions in persufflated kidneys at T1 compared to T0. This figure visualizes signaling networks based on differentially expressed gene products. The map was drawn using a Fruchterman-Reingold force-directed layout with NetworkAnalyst. Each circle represents a protein, and each line an interaction between two proteins. Bigger and darker spots are the genes that are the most connected genes upregulated in the samples. The light- yellow dots are genes that are influenced by the downregulated genes in the biopsies.

Gene Name Degree Betweenness IKBKE 366 445938.6

153

CDH1 130 181698.5 TP63 129 156857 IRAK1 92 105128.9 TFAP2A 82 100668.6 UBC 67 397115.6 ZAP70 53 63851.86 USF2 45 59447.43 SH3GL3 44 57314.79 KRT14 44 50968.03

Table 19: Highly connected, downregulated proteins identified in persufflated kidneys at T1 compared to T0. The top most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

Downregulated Pathway (PSF T0 v T1) Total Hits P.Value RIG-I-like receptor signaling pathway 49 30 8.3E-15 Cell cycle 124 50 6.9E-14 Toll-like receptor signaling pathway 97 42 4.2E-13 Adherens junction 70 33 8.6E-12 Apoptosis 83 35 1.01E-10 ErbB signaling pathway 87 36 1.04E-10 B cell receptor signaling pathway 75 32 4.5E-10 Focal adhesion 200 60 5.4E-10 NOD-like receptor signaling pathway 49 24 2.4E-09 Regulation of actin cytoskeleton 182 51 1.4E-07

Table 20: Pathways enriched in persufflated kidney samples at T1 compared to T0. A list of downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between T0 and T1.

Downregulated Biological Process (PSF T0 v T1) Total Expected Hits P.Value regulation of programmed cell death 1550 142 271 5.5E-28 regulation of apoptotic process 1540 141 267 2.6E-27 innate immune response 638 58.5 144 1.9E-25 regulation of defense response 519 47.6 124 2.8E-24 cell proliferation 1900 175 301 7.9E-24 I-kappaB kinase/NF-kappaB cascade 246 22.6 75 1.2E-21 regulation of immune system process 1190 109 208 3.8E-21

154

signal transduction in response to DNA damage 129 11.8 51 1.2E-20 protein polyubiquitination 177 16.2 59 2.3E-19 regulation of I-kappaB kinase/NF-kappaB cascade 210 19.3 65 2.7E-19 response to wounding 1310 120 217 3.8E-19 protein ubiquitination 658 60.4 131 3.6E-18 leukocyte activation 707 64.9 136 1.7E-17 cellular response to stress 1620 149 247 4.9E-17 cytokine production 576 52.9 115 3.9E-16 lymphocyte activation 601 55.1 117 1.5E-15

Table 21: Biological processes enriched in persufflated kidney samples at T1 compared to T0. A list of downregulated Gene Ontology (GO) biological processes (BP) from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. BP are cellular physiological processes or signal transduction. It is a process that has more than one distinct steps or sets of molecular events with a defined beginning and an end. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ between T0 and T1.

Figure 30: A network model delineating the relationship between the upregulated protein- protein interactions in persufflated kidneys at T1 compared to T0. This figure visualizes signaling networks based on differentially expressed gene products. The map was drawn using a Fruchterman-Reingold force-directed layout with NetworkAnalyst. Each circle represents a protein, and each line an interaction between two proteins. Bigger and darker spots are the genes that are the most connected genes upregulated in the samples. The light- yellow dots are genes that are influenced by the upregulated genes in the biopsies.

Gene Name Degree Betweenness

155

FN1 689 1176546 BARD1 247 406326.7 A2M 102 166324.3 UBC 87 627307.3 FLNC 77 132022.5 FHL2 75 117763.6 PGR 73 110489 MAP4 50 87026.77 MAP1B 45 57540.96 GPRASP1 40 72536.03

Table 22: Highly connected, upregulated proteins identified in persufflated kidneys at T1 compared to T0. The top most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

Upregulated Pathway (PSF T0 v T1) Total Hits P.Value ECM-receptor interaction 84 46 4.0E-14 Regulation of actin cytoskeleton 182 71 1.9E-11 Adherens junction 70 34 6.1E-09 Cell cycle 124 47 1.6E-07 Gap junction 89 37 2.2E-07 Chemokine signaling pathway 189 57 3.7E-05 MAPK signaling pathway 265 74 5.0E-05 TGF-beta signaling pathway 84 30 0.0001 ErbB signaling pathway 87 29 0.0005 Vascular smooth muscle contraction 109 34 0.0007

Table 23: Pathways enriched in persufflated kidney samples at T1 compared to T0. A list of upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between T0 and T1.

Upregulated Biological Process (PSF T0 v T1) Total Expected Hits P.Value wound healing 700 94.3 184 1.4E-20 regulation of body fluid levels 680 91.6 172 1.8E-17 hemostasis 570 76.8 151 2.4E-17 cell proliferation 1900 256 367 1.3E-14 regulation of cell cycle 886 119 200 1.5E-14

156

response to wounding 1310 177 271 2.7E-14 epidermal growth factor receptor signaling pathway 167 22.5 61 3.8E-14 cell morphogenesis involved in differentiation 827 111 188 4.9E-14 regulation of programmed cell death 1550 209 305 3.9E-13 cytoskeleton organization 980 132 211 4.2E-13 regulation of cell adhesion 294 39.6 85 1.9E-12 negative regulation of cellular component organization 370 49.8 100 2.0E-12 regulation of apoptotic process 1540 207 299 2.0E-12 cell-matrix adhesion 159 21.4 56 2.4E-12 positive regulation of MAPK cascade 368 49.6 99 3.4E-12 transforming growth factor beta receptor signaling pathway 221 29.8 69 5.0E-12

Table 24: Biological processes enriched in persufflated kidney samples at T1 compared to T0. A list of upregulated Gene Ontology (GO) biological processes (BP) from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. BP are cellular physiological processes or signal transduction. It is a process that has more than one distinct steps or sets of molecular events with a defined beginning and an end. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ between T0 and T1.

PSF kidneys compared to SCS kidneys at T1

Figure 31: A network model delineating the relationship between the downregulated protein-protein interactions between static cold stored kidneys and persufflated kidneys at T1. This figure visualizes signaling networks based on differentially expressed gene products. The map was drawn using a Fruchterman-Reingold force-directed layout with NetworkAnalyst. Each circle represents a protein, and each line an interaction between two proteins. Bigger and darker spots are the genes that are the most connected genes

157 upregulated in the samples. The light-yellow dots are genes that are influenced by the downregulated genes in the biopsies.

Gene Name Degree Betweenness TRAF2 241 295238.6 SNRNP70 101 131217 IRAK1 92 97467.65 TFAP2A 82 101826.1 SNRPA 68 68056.17 DDX39B 66 82269.94 AXIN1 64 65641.6 SAT1 62 88925.72 UBC 54 432425.7 ZAP70 53 70627.5

Table 25: Highly connected, downregulated proteins identified in persufflated kidneys compared to static cold stored kidneys at T1. The top most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

Downregulated Pathway (SCS v PSF T1) Total Expected Hits P.Value Cell cycle 124 16.1 59 1.7E-21 T cell receptor signaling pathway 98 12.8 46 1.1E-16 Toll-like receptor signaling pathway 97 12.6 43 1.6E-14 NOD-like receptor signaling pathway 49 6.38 25 1.4E-10 RIG-I-like receptor signaling pathway 49 6.38 25 1.4E-10 Apoptosis 83 10.8 34 1.4E-10 MAPK signaling pathway 265 34.5 68 7.6E-09 Wnt signaling pathway 144 18.7 44 1.7E-08 Adherens junction 70 9.11 26 2.3E-07 B cell receptor signaling pathway 75 9.76 26 1.1E-06

Table 26: Pathways enriched in persufflated kidneys compared to static cold storage kidney samples at T1. Pathways enriched in persufflated kidneys compared to static cold storage kidney samples at T1. A list of downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between the two treatment groups at T1.

158

Downregulated Biological Process (SCS v PSF T1) Total Expected Hits P.Value regulation of apoptotic process 1540 139 277 3.9E-32 regulation of programmed cell death 1550 141 279 4.8E-32 cellular response to stress 1620 147 271 1.2E-25 programmed cell death 2160 195 326 1.6E-23 positive regulation of NF-kappaB transcription factor 115 10.4 50 1.1E-22 activity negative regulation of cell cycle 520 47.1 120 1.4E-22 protein ubiquitination 658 59.6 135 2.3E-20 apoptotic signaling pathway 261 23.7 72 2.1E-18 regulation of nucleobase-containing compound 4540 411 553 2.5E-18 metabolic process stress-activated protein kinase signaling cascade 257 23.3 71 3.3E-18 regulation of immune response 727 65.9 139 4.2E-18 innate immune response 638 57.8 127 4.6E-18 negative regulation of signal transduction 790 71.6 145 3.2E-17 positive regulation of immune response 487 44.1 104 3.9E-17 positive regulation of immune system process 739 67 138 4.5E-17 cell proliferation 1900 172 276 4.7E-17

Table 27: Biological processes enriched in persufflated kidneys compared to static cold storage kidney samples at T1. A list of downregulated Gene Ontology (GO) biological processes (BP) from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. BP are cellular physiological processes or signal transduction. It is a process that has more than one distinct steps or sets of molecular events with a defined beginning and an end. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ between the two treatment groups at T1.

159

Figure 32: A network model delineating the relationship between the upregulated protein- protein interactions in static cold stored kidneys and persufflated kidneys at T1. This figure visualizes signaling networks based on differentially expressed gene products. The map was drawn using a Fruchterman-Reingold force-directed layout with NetworkAnalyst. Each circle represents a protein, and each line an interaction between two proteins. Bigger and darker spots are the genes that are the most connected genes upregulated in the samples. The light- yellow dots are genes that are influenced by the upregulated genes in the biopsies.

Gene Name Degree Betweenness ESR1 799 1718332 FN1 689 1369446 AR 276 517679.6 GJA1 126 176046.5 CAV1 118 273413.4 UBC 116 880260.3 PRKCA 113 226225.7 ZBTB16 111 229654.1 EPAS1 101 152383 GABARAP 87 185527.5

Table 28: Highly connected, upregulated proteins identified in persufflated kidneys compared to static cold stored kidneys at T1. The top most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

Upregulated Pathway (SCS v PSF T1) Total Expected Hits P.Value Focal adhesion 200 51.7 136 1.92E-37 ECM-receptor interaction 84 21.7 53 4.66E-13 ErbB signaling pathway 87 22.5 53 3.54E-12 Adherens junction 70 18.1 45 1.10E-11 Regulation of actin cytoskeleton 182 47.1 88 2.40E-11 MAPK signaling pathway 265 68.5 115 1.29E-10 Cell cycle 124 32.1 64 4.73E-10 mTOR signaling pathway 45 11.6 29 5.14E-08 T cell receptor signaling pathway 98 25.3 50 6.43E-08 TGF-beta signaling pathway 84 21.7 44 1.50E-07

Table 29: Pathways enriched in persufflated kidneys compared to static cold storage kidney samples at T1. A list of upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed

160 using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between the two treatment groups at T1.

Upregulated Biological Processes (SCS v PSF T1) Total Expected Hits P.Value wound healing 700 138 276 2.3E-35 hemostasis 570 112 231 1.1E-31 coagulation 568 112 230 1.7E-31 regulation of body fluid levels 680 134 261 3.4E-31 regulation of programmed cell death 1550 305 467 1.4E-25 regulation of apoptotic process 1540 302 461 4.1E-25 negative regulation of programmed cell death 691 136 245 1.7E-23 cell morphogenesis involved in differentiation 827 163 280 2.8E-23 regulation of phosphorylation 1070 210 340 4.9E-23 negative regulation of apoptotic process 679 134 239 1.9E-22 cell proliferation 1900 374 533 1.8E-21 cell-substrate adhesion 241 47.4 112 2.3E-21 epidermal growth factor receptor signaling pathway 167 32.8 86 2.7E-20 regulation of cell migration 456 89.7 170 5.0E-19 regulation of cell differentiation 1290 254 379 8.7E-19 angiogenesis 426 83.8 160 2.5E-18 negative regulation of cell proliferation 585 115 201 1.1E-17

Table 30: Biological processes enriched in persufflated kidney compared to static cold storage kidney samples at T1. A list of upregulated Gene Ontology (GO) biological processes (BP) from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. BP are cellular physiological processes or signal transduction. It is a process that has more than one distinct steps or sets of molecular events with a defined beginning and an end. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ between the two treatment groups at T1.

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Chapter 5

Quality Assessments of Persufflated and Cold Storage Preservation in Subnormothermic Isolated Porcine Kidney

The presented work in this chapter is in preparation for publication. A summary of

the work and a contribution summary has been provided within this chapter.

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Study Summary

The aim of this chapter was to preserve pre-damaged kidneys using persufflation (PSF) and static cold storage (SCS) and assess the organs at subnormothermic temperature. Hypothermic temperatures are not conducive to accurately assess organs, and a period of compulsory normothermic condition is ideal for quantifying organ function prior to transplantation. While normothermic perfusion is most biologically relevant, it stipulates challenging, near-physiologic conditions. Furthermore, studies have found that quality assessments performed at subnormothermic temperatures can predict renal function after transplant.

Additionally, the preservation solution we were using could be safely warmed to subnormothermic temperatures without compromising the integrity of the solution components, so we opted to assess organs at 25°C.

Porcine kidneys were procured post euthanasia to mimic a DCD model. After the isolated kidneys were exposed to 30 minutes of warm ischemia time (WIT), they were flushed with SPS-1 and preserved at 4°C for 24 hours in their respective conditions. After the preservation period, the organs were gradually rewarmed for

30 minutes to subnormothermic temperatures. Whole organ oxygen consumption rate (WOOCR) and glomerular filtration rate (GFR) were measured at 25°C to assess renal viability and function. Renal perfusate and biopsies were collected post preservation to evaluate renal damage using molecular analyses, biomarkers and histology.

Results show significantly higher WOOCR and GFR in the persufflated kidney compared to that of the static cold storage control. Lactate and glutathione-s-

163 transferase levels were higher in the SCS kidneys when measured after 90 minutes of subnormothermic perfusion. On average, hematoxylin and eosin stained renal biopsies exhibit more renal damage such as tubular epithelial necrosis, nephritis, tubulitis and multifocal, subcortical early infarct.

Persufflated kidneys trended towards having more viable, oxygen consuming tissue and increased filtration activity and less indication of renal damage post preservation. Persufflation is a promising tool that has the potential to find a niche as a preservation method, especially since organs with a wide range of cold and warm ischemic damage are being used for transplantation.

164

Quality Assessments of Persufflated and Cold Storage Preservation in

Subnormothermic Isolated Porcine Kidneys

Catherine G. Min MS 1,2, Jean-Philippe Galons PhD 3, Abhishek Pandey MS 4, Brad P.

Weegman PhD5,6, Leah V. Steyn PhD2, William G. Purvis MS2, Kate E. Smith MS 1,2,

Diana S. Molano BME 2, Nicholas D. Price BS 2, Michael J. Taylor PhD 6,7, Jana Jandova

PhD 8, Whitney M. Zoll, DVM, DACVP 9, Diego R. Martin MD, PhD 1,3,10, Robert C.

Harland MD 2, Klearchos K. Papas PhD 2.

1Physiology, University of Arizona, Tucson, AZ, United States; 2Surgery, University of

Arizona, Tucson, AZ, United States; 3Medical Imaging, University of Arizona, Tucson,

AZ, United States, 4Electrical and Computer Engineering, University of Arizona, Tucson,

AZ, United States; 5Department of Radiology, University of Minnesota, Minneapolis, MN,

United States; 6Sylvatica Biotech, Inc., N. Charleston, SC, United States; 7Mechanical

Engineering, Carnegie Mellon University, Pittsburgh, PA; 8School of Animal and

Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, 9Arizona

Veterinary Diagnostic Laboratory, University of Arizona, Tucson, AZ; 10Department of

Biomedical Engineering, University of Arizona

Correspondent: Klearchos K. Papas

Fax: (520) 626-3770

Phone: (520) 626-4494

Email: [email protected]

165

Abstract

Alternative organ preservation methods are actively being pursued to extend preservation times and minimize tissue damage. These approaches may enable the use of organs that are currently being discarded, especially those that are donated after cardiac death. Preventing energy depletion of the organ by providing gaseous oxygen through the vasculature (persufflation) has the potential to prevent ischemic damage both during and post preservation. In the present study, kidneys from a donation after cardiac death (DCD) porcine model were preserved via static cold storage (SCS) or persufflation (PSF) at 4°C for 24 hours. After the preservation period, the kidneys were gradually rewarmed to subnormothermic temperatures using liquid perfusion. Whole organ oxygen consumption rate

(WOOCR) and glomerular filtration rate (GFR) were measured at 25°C to assess renal viability and function. Renal perfusate and biopsies were collected post preservation to evaluate renal damage using biomarkers, and histology. Results show significantly higher WOOCR and GFR for PSF kidneys compared to SCS kidneys. Accumulated lactate levels were significantly higher in the SCS kidneys.

Hematoxylin and eosin stained renal biopsies indicate more renal damage in SCS kidneys than PSF kidneys. PSF kidneys trend towards having more viable, metabolically active tissue and increased filtration activity, with less indication of renal damage post preservation. PSF is a promising tool that has the potential to minimize tissue damage and preserve renal function, especially since organs with a wide range of cold and warm ischemic damage are being used for transplantation.

166

Introduction

It is widely recognized that inevitable ischemic stress and its metabolic sequelae during standard kidney procurement and preservation by static cold storage (SCS) cause tissue injury (1-5) This is further exacerbated during the reperfusion phase that is accompanied by the abrupt reoxygenation after transplantation (6). Consequently, alternative approaches for kidney preservation are being studied to minimize ischemic injury and extend preservation time to efficiently use all available organs from the current deceased donor pool. These improvements will be particularly useful for enabling the use of kidneys that are being discarded, especially organs from expanded criteria donors (ECD) and donors of cardiac death (DCD).

Currently, hypothermic machine perfusion (HMP) is the preferred method of preserving ECD and DCD kidneys, and transplant centers around the world have been adopting this technique. Experimental and clinical studies have demonstrated that HMP reduces the incidence of delayed graft function and results in better graft viability compared to the standard SCS method (7-12). Economic evaluation established the short and long-term cost effectiveness of using MP over

SCS, with reduced costs related to hospital stay, dialysis treatment, and complications from delayed graft function (13,14). While HMP has the benefits of maintaining vascular patency and clearing metabolic waste products, it is not clear whether it can provide adequate oxygen to the tissue. Hypothermia results in reduced oxygen utilization; at 20°C oxygen consumption is only 16% of the normothermic value (15). Still, throughout the period of hypothermic metabolic

167 suppression, energy consumption remains very much present, and the lack of oxygen leads to a quick depletion of the remaining adenosine triphosphate (ATP) levels.

Preventing complete energy exhaustion of the organ by providing gaseous oxygen via the native vasculature using persufflation (PSF) has the potential to prevent ischemic damage during preservation. Providing a steady supply of exogenous oxygen has been shown to replenish ATP (16-19) and is capable of mitigating ischemia induced cellular morphological changes (20-22). Previous studies have found PSF to improve graft function in kidneys, livers, and hearts after transplant (23-26). While there are concerns about endothelial damage from vascular exposure to hyperoxia, hearts and livers preserved by PSF showed no topographical signs of damage (27-29). Harnessing the benefits of PSF and MP has the potential to synergistically provide oxygen needed for cellular function and clear out toxic byproducts from metabolism during the preservation period, which can subsequently lead to improved graft outcomes.

Hypothermic PSF can help restore ATP levels after the initial ischemic insult, and buys time for the organ to regain homeostasis. Extending the time for ischemic tolerance may circumvent the abrupt reoxygenation and subsequent reperfusion injury of the organ during reperfusion after transplant. While hypothermia has protective effects during preservation, it is not conducive to organ function assessments as most metabolic activities are repressed. A period of perfusion at higher temperatures is necessary to accurately characterize the organ. A growing body of evidence suggests that subnormothermic MP applied

168 during the whole or partial preservation period is superior to HMP or SCS alone

(30-32). Subnormothermic perfusion enabled graft assessment prior to transplant, and in tandem, conditioned the organs for normothermic perfusion that occurs at the time of transplant. Allotransplant studies have found that controlled rewarming reduced cellular enzyme loss, decreased lipid peroxidation (33) with minimal injury and improved energy content (34). At hypothermic temperatures, the correlation between renal vascular resistance and clearance were weak, and an improved predictive discrimination was seen with stronger correlation between the two parameters at subnormothermic temperatures (35).

In the present study, the effectiveness of PSF as a method of kidney preservation was evaluated in kidneys exposed to warm ischemia to mimic donation after cardiac death. Damaged kidneys from porcine donors were preserved by PSF or SCS in a pairwise fashion. Gradual rewarming to subnormothermic temperatures using liquid perfusion was performed for organ flushing and quality assessments.

Materials and Methods

Organ Procurement and Preservation

Domestic female pigs weighing 35-50 kg were provided by a local farm in

Wilcox, Arizona. Animal care procedures were in accordance with university institutional animal care and use committee policies. Pigs were anesthetized under isoflurane, and heparin at 300 U/Kg was circulated for 5 minutes as outlined in

Figure 1. Subsequently, they were euthanized, exsanguinated, and flushed with 2 liters of room temperature, heparinized saline through the abdominal aorta.

169

Kidneys were exposed to 30 minutes of warm ischemia inside the body cavity prior to procurement. After the procurement, kidneys were cannulated at the renal artery and vein using luer locks (Cole-Parmer, Vernon Hills, IL) and flushed with 400mL of 4°C heparinized saline using gravity, followed by a secondary flush with SPS-1 solutions (Organ Recovery Systems, Istaca, IL). The kidneys were then preserved for 24 hours with either SCS, current standard clinical procedure, or PSF. The persufflator (Giner, Inc., Newton, MA) delivered electrochemically generated, humidified, 40% oxygen mixed with atmospheric air with a manifold pressure of

40-50 mmHg and a flow rate of 60 mL/min. Tubing from the persufflator was attached to the renal artery, and leaks around the vessel were tied off with surgical silk to ensure that all of the gas entered the kidney. Both kidneys were kept in SPS-

1 solution at 4°C during the 24 hours of preservation.

Following this phase, the organs were systematically re-warmed to 25°C over the course of 30 minutes via liquid perfusion. SPS-1 perfusion solution was used because it was specifically designed for organ protection at hypothermic and subnormothermic temperatures. During rewarming, the water bath that was connected to the insulated circuit was raised from 4°C to 10°C, equilibrated for 10 minutes, and then stepped up to 15°C. The system was then again equilibrated for

10 minutes and warmed to 24°C, and equilibrated for another 10 minutes prior to quality assessments, illustrated in Figure 2.

Whole Organ Oxygen Consumption Rate

The renal artery and renal vein were cannulated individually and connected to a custom-built, temperature controlled machine perfusion system as shown in

170

Figure 3. This was maintained at 21-23°C using a water bath and water jacketed media reservoir with insulated tubing. SPS-1 (Organ Recovery Systems) was oxygenated with 40% oxygen using a hollow fiber oxygenator (Medtronic,

Minneapolis, MN) and perfused through the artery throughout the measurements.

Calibrated flow-through fiber-optic oxygen sensors (Instech, Plymouth Meeting,

PA) were placed proximal to the renal arterial cannula, and additionally distal to the venous cannula to measure the difference in oxygen pressure across the kidneys (36). The WOOCR (mol/min/g) was calculated by using a mass balance equation where Q is the flow rate of perfusion (mL/min), α is oxygen solubility in aqueous solution at temperature (mol/mL/mmHg), and m is the mass of the kidney

(g):

푄 × (푝푂2 − 푝푂2 ) × 훼 푊푂푂퐶푅 = 퐴푟푡푒푟푖푎푙 푉푒푛표푢푠 푚

Dynamic Contrast Imaging using Magnetic Resonance

All imaging was conducted on a 3T Skyra scanner (Siemens, Munich,

Germany). Kidneys were secured in the organ cassette (LifePort, Woodland, WA) and perfused with room temperature SPS-1 preservation solution (Organ

Recovery Systems) using a MR compatible subnormothermic perfusion circuit.

The organ cassette was secured in a standard Siemens Head and Neck RF coil.

After 15-20 minutes of perfusion, 4mM Multihance Gadolinium-DTPA solution

(Bracco Diagnostics Inc., Milan, Italy) was injected to cannulated artery after 20 seconds of pre-contrast and continuous acquisition was conducted for 4 minutes.

Dynamic-contrast MRI was performed using Siemens golden angle stack of star

171 radial acquisition (radialVibe). Compressed Sensing reconstruction with total variation applied in temporal dimension was used to reconstruct images with a 1 second temporal resolution (37,38). A Modified Look Locker Inversion recovery method (MOLLI) was used to generate T1 map pre-and post-injection of

Gadolinium-DTPA solution (Bracco Diagnostics Inc.). Manual segmentation was done on CS reconstructed images using only 21 lines of k-space (2 sec temporal resolution). The arterial input function (AIF) curve was generated using the signal from an ROI placed on renal artery and the kidney signal was obtained by segmentation of the kidney parenchyma. Data were continuously collected for total of 4 minutes but fitted over a 90 second acquisition window from the arrival of

Gadolinium. The arterial input function, the diffusive transport of gadolinium through intravascular and extravascular space was used to calculate Ktrans and glomerular filtration rate of the isolated kidneys (39,40).

Lactate measurement

After 24 hours of preservation, kidneys that were stored with or without oxygen were rewarmed on a recirculating liquid perfusion circuit to 25°C over 30 minutes. After the rewarming, kidneys were placed in 500 mL of fresh SPS-1 solution. This circuit was maintained at 22-25°C with a flowrate of 80 mL/minute.

Kidneys were perfused through the renal artery for 60 minutes, and 500 uL of venous effluent was collected every 10 minutes. The effluent lactate level was measured using the YSI 2700 biochemistry analyzer in mmol/L (YSI Life Sciences,

Yellow Springs, OH). Lactate concentration in each 500 uL samples were converted to mmol/g using the following equation where the YSI reading of the

172 venous effluent sample (mmol/L) is multiplied by the volume (v) of the sample, divided by the renal mass (m);

푚푚표푙 푚푚표푙 푣 = × 푔 퐿 푚 and these values were then converted to umol/g. The converted lactate values were plotted from 0 to 60 minutes. The area under the curve of this graph reflects the accumulation of lactate over the hour of perfusion.

Histology

Wedge biopsies were taken from the kidneys after the preservation period.

They were fixed with 4% paraformaldehyde overnight, dehydrated with ethanol, processed and paraffin wax embedded. Biopsies were sectioned at 5 microns and stained with hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO) to evaluate gross renal morphology using light microscopy. The sections were assessed for tubular, glomerular and vascular damage at 10x, 20x and 40x magnifications. A veterinary pathologist estimated the amount of damage per biopsied tissue section on the double blinded slides using a scoring system of 0-3. 0 indicates no abnormalities; 1, mild changes; 2, moderate changes and 3, severe changes.

RNA Isolation

Wedge biopsies containing renal cortex and medulla in QIAzol Lysis

Reagent (Qiagen Sciences) were homogenized in sterile tissue shredder tubes using the gentleMACS™ Dissociator (Miltenyi Biotec GmbH). RNA was purified and isolated form the tissue samples using an miRNeasy Mini Kit (Qiagen

Sciences) according to manufacturer instructions. The RNA integrity was checked

173 on a NanoDrop™ 3300 Fluorospectrometer (ThermoFisher Scientific). Samples with 260/280 ratios above 2 and 260/230 ratios above 1.8 were used for this study.

Microarray Analysis

In vitro transcription was carried out using Agilent Quick Amp labeling kit

(Agilent Technologies, Santa Clara, CA) in the presence of Cy3- and Cy5-CTP.

Then, 825 ng (150 pmol) of labeled cRNA from individual tissues were purified separately, combined, and mixed with hybridization buffer before being applied on the microarray. The hybridization solution was prepared using an Agilent Gene expression hybridization kit (Agilent Technologies, Santa Clara, CA). An Agilent

4x44k S. Scrofa (Pig) Oligo Microarray v2 (Agilent Technologies, Design # 026440) kit was used for hybridization, performed in a hybridization oven G2545A (Agilent

Technologies, Palo Alto, CA). Hybridization and washing were performed according to the protocol described in the Agilent Oligonucleotide Microarray

Hybridization manual (Agilent Technologies, Santa Clara, CA). After washing, microarrays were scanned using a Gene Pix 4000B Scanner (Molecular Devices,

Sunnyvale, CA) with laser excitation at 532 and 635nm at the highest possible resolution (five pixels per micron), and saved as 16-bit grayscale TIFF images.

Intensity values were extracted using GenePix Pro6.0 (Molecular Devices,

Sunnyvale, CA), and the data for each array were normalized followed by ANOVA analysis using a Limma package in R version R2.5.1 (http://www.r-project.org/).

Data Analysis

Differentially expressed genes were analyzed using Network Analyst

(http://www.networkanalyst.ca/faces/home.xhtml) to identify gene networks and

174 pathways. The International Molecular Exchange Consortium (IMEx), a literature- curated comprehensive data was used to evaluate first order protein to protein interactions. To facilitate this, we converted pig UniProt accessions to human accessions using Ensembl Biomart and deleted duplicates and unassigned transcript IDs. Genes with a p < 0.101 and log2 fold change of ≥ 1 or ≤ 1 were considered to be differentially expressed.

Statistics

The function and viability measurements of GFR, Ktrans, WOOCR, lactate, and histological analyses were all conducted in a pairwise manner. The differences between the treatment groups were analyzed by transforming the data and using a paired t-test on Graphpad Prism version 6 software. P < 0.05 was considered significant. Unless otherwise indicated, data represent mean ± SEM values.

Results

Organ oxygen consumption rate was measured in paired kidneys after 24 hours of preservation and rewarming to subnormothermic temperatures. Kidneys preserved by PSF had significantly higher WOOCR values than kidneys preserved by SCS, shown in Figure 4 (SCS: 225.5 ± 43.7 nmol/min*g; PSF: 281.8 ± 32.1 nmol/min*g).

The volume transfer constant Ktrans was measured to reflect the filtration rate at which the gadolinium was moving from the intravascular space to the extravascular space, i.e. the glomeruli to the tubules. On average, the SCS kidneys had a Ktrans value of 0.04 ± 0.01 mL/g/min while persufflated kidneys had a higher rate of 0.15 ± 0.05 mL/g/min as shown in Figure 5A. The GFR derived from Ktrans

175 and the volume of the whole kidney shows that the persufflated kidneys had a significantly higher rate of 22.0 ± 6.62 mL/min while the static cold storage kidneys averaged at 6.33 ± 2.16 mL/min (Figure 5B).

Renal perfusate was collected after rewarming and an hour of subnormothermic perfusion for lactate measurements. First pass collection of lactate prior to recirculation in Figure 6A show significantly higher levels in the

SCS kidneys (14.5 ± 1.9 umol/g, 6.3 ± 1.2 umol/g). The collection of venous effluent after 60 minutes on the subnormothermic perfusion circuit in Figure 6B first shows high levels of accumulated lactate followed by a decrease and subsequent gradual increase from the lactate that is already in the recirculating medium from the initial flush of residual lactate that remained in the kidneys from the rewarming and new production of lactate during the perfusion period. Total lactate levels in rewarmed kidneys during reperfusion shown in Figure 6C were calculated using the area under the curve, and show lactate levels of 387.4 ± 61.44 umol*min/g in SCS kidneys and significantly lower levels of 247.6 ± 23.76 umol*min/g in PSF kidneys.

On average, there was a slightly higher prevalence of tubular epithelial necrosis, nephritis, tubulitis, and multifocal, subcortical early infarct with pyknotic nuclei noted in the SCS kidneys (Table 1, Figure 7). There were no notable differences or blatant injury to the glomeruli and the renal vasculature in both groups.

Agilent microarrays with approximately 44,000 probes were used to identify the global changes in gene expression in SCS and PSF kidneys after 24 hours of

176 cold preservation and 30 minutes of rewarming to subnormothermic temperatures.

Global gene expression changes show 268 genes were differentially expressed, with 256 upregulated and 12 downregulated genes. The most altered genes by fold change are detailed in Figure 8.

The differences between SCS and PSF groups in genomic response to the preservation methods and rewarming were investigated through NetworkAnalyst to reveal biologically informative hub genes, KEGG pathways and biological processes. The most prominent hub genes associated with the differentially expressed gene list are summarized in Table 2. Canonical pathways and biological processes that are significantly associated with the differentially expressed genes and their associate nodes show an enhancement in metabolism, cell cycle, regulation of cell structure, immune response and response to damage, summarized in Table 3 and Table 4.

Discussion

There is a surge in the use of donor kidneys that fall outside of standard criteria due to organ scarcity (41-43). These kidneys are linked to a higher incidence of delayed graft function, longer time to reach optimal renal function, longer length of hospital stays, and more frequent readmissions (44). Delayed graft function has been associated with higher risk of graft loss, acute rejection, and decreased long term survival (2, 45-47). Because ischemia reperfusion injury is a crucial determining factor in acute kidney injury and delayed graft function, there has been a renewed interest in oxygenating kidneys. In the present study, we used

177 persufflation as the method of delivering oxygen to attenuate the effects of ischemic damage.

Here we demonstrate that at subnormothermic temperatures, there are viability and functional differences between kidneys that have been preserved with and without oxygen. Higher WOOCR seen in PSF kidneys were reflected by the most predominant changes seen global gene expression, corroborated by an enrichment in the TCA cycle pathway and glucose metabolism. Our studies show higher levels of lactate in the SCS kidneys, which reflects more anaerobic glycolytic metabolism and reduced ability to recycle and consume lactate by the cortex.

ATP depletion disrupts the actin cytoskeleton, which contributes to cast formation, local edema and tubular obstruction (48). The outer medulla has a natural proclivity to ischemic injury, and the proximal tubule and thick ascending limb of the loop of Henle in this area are most susceptible to anoxic conditions (48).

While the thick ascending limb contains more glycolytic machinery for energy synthesis (49,50), complete energy depletion will inevitably lead to cellular damage. Indeed, our biopsies show more tubular epithelial necrosis, nephritis, tubulitis, and multifocal, subcortical early infarct in the cold storage kidneys after they’ve been rewarmed. Moreover, the SCS group displayed a significant drop in

GFR, which is in theoretical agreement with the notion that tubulointerstitial changes can be used as an index of functional impairment, and tubular damage has a correlation to renal function (51,52).

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However, no assumptions can be made about the long-term consequences renal function in these SCS kidneys. Since tubular cells have proliferative and reparative capabilities after an injury (53,54), the morphological changes that are shown in the biopsies could be reversed if these organs were transplanted. Renal ischemic injury activates cell cycling, and the damage results in regulation of cell death and cell proliferation (55), and restoration of original tissue architecture can be achieved through regeneration with the renal progenitor system (56). In this study, Heat shock proteins (HSP) and hepatic nuclear factor (Hnf) were upregulated in PSF kidneys, and network analysis revealed them to be critical signaling nodes, both of which can have protective and ameliorative effects on sublethally damaged tissue by driving nephrogenesis, tubulogenesis and epithelial homeostasis through the regulation of epithelial-mesenchyme-epithelial cycling

(57,59). In addition, there is an upregulation of autophagy, which degrades damaged protein to maintain intracellular homeostasis and plays a role in maintaining tubular cell integrity under ischemic conditions (60).

We speculate that the supplementary oxygen prevents energy depletion, which enables renal cells to partake in stress responses, wound healing, and activates intrinsic dedifferentiating and proliferating capabilities to adapt to the loss of adjacent cells and accelerate renal repair. In rodent studies, tubular regeneration showed a peak at day 7 (61). It is doubtful that the differences in renal function between the treatment groups reflect regenerative activities of the renal tissue. The higher glomerular filtration rates seen in the PSF kidneys likely insinuate more filtration due to less damage to the nephrons during prolonged 24

179 hours of preservation as opposed to repaired tubules. While these results are encouraging, specific genes of interest need to be validated using quantitative real- time PCR.

Anoxic and hypothermic injuries are serious variables that lead to graft dysfunction and poor prognosis after transplantation. There is great potential for using WOOCR and GFR measurements calculated through imaging to evaluate and characterize renal health and function without rewarming the kidneys to normothermic temperatures. Our study shows that the deleterious effects of ischemia can be mitigated by supplying oxygen to the organ with PSF. It is plausible to establish a niche for PSF as a preservation method considering that immediate PSF after kidney procurement may reestablish optimal ATP levels and prevent irreversible, lethal damage caused by ischemia. During a time when substantial number of kidneys are being discarded without clear justifications

(62,63) and transplant centers are replete with risk aversion, developing a methodical protocol for kidney assessments and enhancing organ quality during preservation provides a much-needed opportunity to increase the use of organs and number of transplants.

Disclosure

K.K.P. served as Consultant/Chair, SAB Giner Inc and as a co-inventor on pending patents related to persufflation with University of Minnesota and Giner

Inc., and was the PI or co-PI on the NIH/NIDDK grants that partially funded the work. He is no longer affiliated with Giner Inc.

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Acknowledgements

The authors would like to acknowledge the members of the Papas laboratory and collaborators, including Thomas Suszynski, J. Brett Stanton,

Jennifer P. Kitzmann, AnnaMaria Burachek, Alexandra Hoeger, Ivan Georgiev,

Liberty Kirkeide, Dr. David G. Besselsen, Dr. Ronald Lynch, Dr. Heddwen Brooks and Dr. Tun Jie for their support. We also would like to thank Simon Stone and Dr.

Linda Tempelman for technical assistance with the persufflator, the Schott

Foundation, and the Carol Olson Memorial Diabetes Research Fund for the early development of the persufflation work.

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Figures and Legends

Figure 1: Experimental timeline of the isolated kidney preservation study. Kidneys from DCD porcine donor is preserved for 24 hours in cold storage or PSF. After the preservation period, they were rewarmed for 30 minutes using liquid perfusion for an even rewarming of the kidneys to subnormothermic temperatures. Under liquid perfusion at 25C, GFR and WOOCR were measured and samples were collected for biomarkers, histology and molecular analyses.

Figure 2: Schematic of post-preservation kidney rewarming timeline. Isolated porcine kidneys were liquid perfused with SPS-1 solution after the 24-hour preservation period. The perfusion circuit used a water bath and heat exchanger to gradually warm the solution in a stepwise fashion from 10°C to 15°C to 25°C. Each temperature was held for 10 minutes and the kidney was warmed to subnormothermic temperatures over 30 minutes.

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Figure 3: Schematic of whole organ oxygen consumption rate circuit. The organ is perfused with oxygenated preservation solution in a recirculating bath. The temperatures were controlled with the water bath using water jacketed media reservoir and insulated tubing. In this circuit, the perfusate enters the kidney through the cannula connected to the inline oxygen sensor at the arterial end to the other sensor at the venous end. The fluorometer that couples with oxygen sensors provide oxygen measurements.

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Figure 4: Whole organ oxygen consumption rate in SCS and PSF kidneys after preservation and rewarming. * indicates p<0.05 vs. SCS. WOOCR values were calculated using the perfusion flow rate, PO2 difference across the kidney and the oxygen solubility constant, which are normalized by the kidney mass. There was a significant increase in consumption rate in the persufflated kidneys (n=4, p<0.05).

Figure 5: Ktrans and glomerular filtration rate in SCS and PSF kidneys after preservation and rewarming. (A) Transfer constant of the tracer moving from the intravascular space into the extracellular, extravascular space measured through dynamic contrast imaging with gadolinium as the tracer (n=6). (B) * indicates p<0.05 vs. SCS. Values for estimated glomerular filtration rate in static cold storage

184 and persufflated kidneys. It was measured with dynamic contrast imaging and calculated by using the two-compartment model (n=6).

Figure 6: Lactate levels of renal perfusate. After 24-hour preservation period, static cold storage and persufflated kidneys were rewarmed to subnormothermic temperatures. After the rewarming, kidneys were perfused with fresh 500 mL of SPS-1 solution at a flowrate of 80 mL/min in a recirculating system for 60 minutes. At each timepoint, 500 uL of perfusate was collected from the renal vein. A shows first pass lactate concentration of the effluent sample prior to any recirculation (n=4, p < 0.5). B graphs the average lactate levels in the venous effluent collected at the different time points during liquid perfusion. C shows the approximate net production of lactate during the perfusion period acquired by calculating the area under the curve of B (n=4, p < 0.5).

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Figure 7: Light microscopy images of H&E-stained tissue samples. A and B are images from persufflated kidneys and C and D are images from static cold storage kidneys after preservation and rewarming. Arrows indicate damage in the renal tubules.

SCS PSF

Scores 1.3 ± 0.1 1.2 ± 0.07

Table 1: Histological evaluation assessing renal injury in wedge biopsies after 24 hours of preservation. Scores are reported in means ± SEM from four pairs of kidneys (n=4). Sections were stained with H&E and scored over 5 fields with 0 indicating no abnormalities, 1 mild changes, 2 moderate and 3 severe changes. Kidney sections were evaluated for tubular dilation, vacuolation of epithelial cells, loss of brush border, tubular casts and lymphoplasmacytic inflammation in the interstitium.

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M o s t A l t e r e d G e n e s b y F o l d C h a n g e

T M E D 1 0 T R I A P 1 C O X 6 C P R D X 3 T U B G C P 3 V D A C 2 L Y P L A 1 P T C H 1 P E G 1 0 T L K 1 P G K 1 A K R 1 A 1 G S T Z 1 A S S 1 P D Z K 1 C E S 1 I T P A I D H 2 P S M D 4 I D H 2 A S S 1 E E F 1 A 1 T M B I M 6 L G M N

- 2 0 2 4

L o g 2 F o l d C h a n g e

Figure 8: Summary of differential expression of microarray data. The most upregulated and downregulated genes in persufflated kidneys compared to static cold storage kidneys as determined by fold change.

Gene Name Degree Betweenness Upregulated Nodes HSP90AB1 565 1249938.17 HNF1A 294 723897.95 EEF1A1 286 475053.88 HNRNPA1 237 411212.21 EIF4A3 220 367022.87 EWSR1 213 433737.19 ACTB 198 394177.64 PPP2CA 159 267415 PABPC1 152 205357.24 HSPD1 143 208154.82 Downregulated Nodes VDAC2 55 4245.1 TMED10 40 3280.5 TUBGCP3 13 1143.4 TRIAP1 5 426

Table 2: Highly connected, upregulated proteins identified in Study B. The top n most highly connected proteins, along with their degree and betweeness is shown.

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Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

KEGG Pathways Total Hits P.Value FDR Cell cycle 124 73 2.89E-16 4.50E-14 Regulation of actin cytoskeleton 182 90 2.01E-13 1.09E-11 Chemokine signaling pathway 189 79 1.19E-07 1.29E-06 Apoptosis 83 35 0.000343 0.00146 Citrate cycle (TCA cycle) 30 16 0.000711 0.00275 Regulation of autophagy 8 5 0.0262 0.0707

Table 3: Pathways enriched in PSF compared to SCS kidney samples. A list of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between SCS and PSF kidneys.

Biological Processes Total Hits P.Value FDR Cellular macromolecule catabolic process 849 341 6.38E-48 1.74E-45 Signal transduction in response to DNA damage 129 81 1.83E-27 6.51E-26 Regulation of cell cycle 886 293 6.89E-24 1.49E-22 Cellular response to stress 1620 460 5.16E-21 8.64E-20 Regulation of apoptotic process 1540 438 1.10E-20 1.69E-19 Regulation of programmed cell death 1550 442 1.20E-20 1.82E-19 Hemostasis 570 179 1.37E-12 1.08E-11 Wound healing 700 207 1.36E-11 9.64E-11 Innate immune response 638 192 1.47E-11 1.03E-10

Table 4: Biological processes in PSF compared to SCS kidney samples. A list of altered cellular processes from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between SCS and PSF kidneys.

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Chapter 5 Supplementary Data

Supplementary data for this chapter include T0 measurements for histological analyses, lactate data collected during the organ rewarming phase and Agilent

Microarray analyses. Initially, RNA Sequencing was used to profile the transcriptome of preserved kidneys, but this proved to be an expensive method with a long wait time for the completion of sequencing. For these reasons, microarrays were used for subnormothermic studies. While this method of genomic evaluation is not new, decades have passed since its first application, and technological advancement enabled reliable data acquisition with low cost of service. The expression of thousands of known genes can be evaluated simultaneously, and custom arrays can be designed for specific species and panels of genes of interest. We chose gene panels for apoptosis, cell cycle and proliferation, extracellular matrix and cell adhesion, inflammation, replication and repair and gene regulation.

T0 Histological Analysis

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Figure 33: histological analysis from hematoxylin and eosin stained biopsies. DCD model porcine kidneys preserved for 24 hours in static cold storage or persufflation were compared to control kidneys with no cold ischemia time.

While the differences did not reach significance, on average SCS kidneys have the most abnormalities, followed by the persufflated kidneys and the control group.

This data suggests that the level of damage that was incurred during the cold preservation and rewarming to subnormothermic temperatures were similar in low or high oxygen conditions, and the renal morphology was retained at the level of kidneys exposed to a short amount of warm ischemia time and no cold ischemia time.

It may be the case that 30 minutes of rewarming is not enough time to incur significant damage to the renal tissue, and the liquid perfusion provides some level of oxygenation due to its exposure to atmospheric air. Additionally, the kidneys were rewarmed to subnormothermic temperatures that suppress normal metabolic activity and the requirement for high oxygen demands (Matsuno, et al., 2014). Studies have shown that cold preservation causes necrosis in human tubular cells, and rewarming to normothermic temperatures is associated with apoptosis. It is speculated that the rewarming activates apoptotic processes in damaged tubular cells

(Salahudeen, Joshi and Jenkins 2001). More differences in the structural integrity of renal tissue between the two treatment groups may have been observed at normothermic temperatures in which case, oxygenating the blood substitute or blood during rewarming becomes critical (Hoyer, et al., 2016; Brockmann , et al., 2009;

Schön , et al., 2001; Vogel, Brockmann, & Friend, 2010).

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Additional Lactate Data Analysis

Figure 34: Lactate levels of renal perfusate during the rewarming period. After 24-hour preservation period, static cold storage and persufflated kidneys were rewarmed using its vasculature on a perfusion circuit that was temperature controlled with a water bath. Kidneys were perfused with 500 mL of SPS-1 solution at a flowrate of 80 mL/min. The temperature of the preservation solution was incrementally increased from 4°C to 25°C over 30 minutes. The perfusate was recirculated for this entire time period so that lactate collected from the kidney during the perfusion was retained within the 500 mL of solution in the perfusion circuit. 500 uL of perfusate was collected every 5 minutes from the renal vein. A shows the very first single pass collection of venous effluent at the start of the perfusion prior to recirculation (n=4, p<0.05). Lactate concentration in the perfusate samples were normalized to renal mass. B shows the approximate net accumulation of lactate over 24 hours. This was calculated by using the area under the curve of the data points during the first 5 minutes of perfusion. In 5 minutes, almost the entire volume of the SPS-1 solution in the organ container passed through the kidney, and the amount of lactate collected in the 500 mL of the recirculating medium is an approximation of the amount of lactate produced within the kidney over the 24-hour preservation period. C shows the average lactate levels in perfusate collected at the different time points during liquid perfusion. C shows the approximate net production of lactate during the rewarming phase.

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On average, there is a higher level of lactate in the SCS kidney perfusate. The initial first pass of lactate that was collected after the preservation period show significant difference between the two treatment groups (SCS: 19.8 ± 4.72; PSF: 7.49

±1.15 pmol/g). However, there were no significant differences in the accumulation of lactate during the preservation period (SCS: 66.8 ± 15.7; PSF: 30.9 ±3.97 pmol/min*g), nor the net lactate level in the perfusate at the end of the rewarming phase (SCS: 251

± 57.1; PSF: 154 ± 22.2 pmol/min*g). Lactate levels can be interpreted as increased lactate production from anaerobic or aerobic glycolysis, and a reduction in lactate clearance. Acidic environments are known to increase the metabolism of lactate in the kidneys, unlike the liver where a drop in pH would depress lactate removal. In

SCS kidneys, this ability may be compromised, or the production of lactate exceeds the rate at which it can be metabolized.

Agilent Microarray Analysis

Introduction

A common complication experienced by transplant patients comes from delayed graft function (DGF). Fostering improvement of donor organ management and the advancement of diagnostic and therapeutic modalities can greatly reduce the incidence of DGF. Assessing the effects of DGF can be convoluted because there are so many variations of its definition. When DGF is defined as needing at least one dialysis session within the first week of transplant, it occurred in 27% recipients that received organs from cardiac death donors. Of these patients, 30% incompletely recovered from DGF (Lee, 2017). Incompletely recovered patients had lower renal function, and

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DGF was associated with increased risk for patient death (Lee, 2017), and a significant reduction of short- and long-term graft survival (Ojo, Wolfe, Held, Port, & Schmouder,

1997). Other studies have found that when DGF is defined by the time it takes for creatinine clearance rate to reach the normal physiological range, DGF lasting for more than 6 days reduced graft survival while less than 6 days did not impact graft survival (Giral-Classe, 1998). Furthermore, DGF is associated with an increased likelihood of acute rejection episodes after transplantation (Boom, 2000). Despite these conflicting results and a lack of universal definition for DGF, it is clear that minimizing the odds of having this symptom will reduce complications that can arise from renal transplants.

Risk factors for DGF include donor age (Ojo, Wolfe, Held, Port, & Schmouder,

1997; Mikhalski, 2008; Moreso, 1999), organs from expanded criteria donors

(Maathuis, 2007; O'callaghan, 2013) and longer cold ischemia time (Mikhalski, 2008;

Salahudeen A. K., 2004; Shoskes D. A., 1998), with a 23% increase in DGF risk for every 6 hours of cold ischemia (Ojo, Wolfe, Held, Port, & Schmouder, 1997). Each minute of warm ischemia is associated with a 6% increased risk of acute renal failure

(Patel, 2011) and increased risk of patient mortality. Additionally, graft failure associated with longer ischemia time persisted after stratification by donor types

(Tennankore, 2016). Organs are exposed to a mix of warm and cold ischemia during procurement, followed by preservation during transport. Most organs are preserved using static cold storage (SCS), during which they experience hours of cold ischemia.

Furthermore, the duration of exposure to ischemia may have synergistic effects once the organ is transplanted to the recipient due to ischemia reperfusion injury. The

198 effects of ischemia experienced in the deceased donor setting are typically reversible

(Shoskes D. A., 1996). Under ischemic stress, oxidative metabolism is not possible, and the tissue reverts to anaerobic metabolism.

Due to the low yield of adenosine triphosphate (ATP) from this energy generating mechanism, ATP is quickly depleted and ion pumps that rely on energy for active transport cease to function. One of the most energy intensive actions in the cell is the sodium potassium pump (Na+/K+-ATPase) that maintains sodium and water homeostasis. Consequently, the loss of Na+/K+-ATPase activity causes mitochondrial and nuclear swelling. Disruption of the Na+/K+-ATPase slows down active calcium efflux, which results in calcium accumulation in the cytosol and the subsequent dissipation of mitochondrial membrane potential by opening the mitochondrial permeability transition pore. Elevation of calcium activates calpains, which degrade cytoskeletal proteins, mitochondrial proteins, and the endoplasmic reticulum membranes (Huttenlocher, 1997). Hypoxanthine, potentially the most damaging degradation product of renal ischemia, accumulates in the cell. It is created by the ischemic environment that converts ATP to AMP and xanthine dehydrogenase to xanthine oxidase (Granger, 1988). Xanthine oxidase catalyzes the oxidation of hypoxanthine to produce free radicals, which can irreversibly damage the purines needed to synthesize ATP.

Kidneys can recover from ischemic injury through the ability of non-injured cells to proliferate and the capacity of damaged cells to regain function. Post-ischemic kidneys are characterized by increased vascular resistance, decreased glomerular hydraulic pressure and permeability (Mason, 1987). The congested tubules in the

199 outer medullary area increase vascular resistance by putting back pressure on the vasculature, coupled with the damaged endothelium inducing inflammatory responses and vasoactive molecules that can cause vasospasms (Sutton, 2002).

The most commonly injured cells are the epithelial cells of the proximal tubules. The S3 segment of the proximal tubules is located in the outermedullary area.

This area is highly metabolically active with limited capability for anaerobic metabolism, and severe depletion in ATP after ischemic insult can cause cell death.

Cells comprising the thick ascending limb of the loop of Henle are better equipped with glycolytic machinery, so despite being in the same region, fewer cells are lost during ischemia (Nangaku, 2006).

Given the extent to which ischemia can damage renal function and the subsequent impact on clinical outcomes, solutions to minimize ischemic exposure are actively being developed. One such approach is oxygenating organs through persufflation (PSF). To study the mechanism by which PSF may be changing cellular activity during the preservation period, tissue biopsies were taken for genomic analysis. Genomic studies are increasingly being used to garner a mechanistic understanding of disease states, pathology, and normal physiology. In this study, we hypothesized that providing damaged tissue with oxygen after 30 minutes of warm ischemia time will lessen the degree of injury and activate more of the wound healing, stress response pathways to start repairing the reversible damage that occurred during the warm and cold ischemic period. Microarrays were implemented as they provide a snapshot of global gene expression in a given tissue. Samples that were taken after 24 hours of preservation using SCS and PSF were compared using SCS as

200 the control. We used NetworkAnalyst to analyze the pathways and derive meaning from a list of differentially expressed genes. It is a program that uses innovative visual analytics that uses interactive features that can group genes by networks, patterns, functions and connections (Xia J. B., 2014; Xia J. G., 2015).

Methods

Data Analysis

Differentially expressed genes were analyzed using Network Analyst

IDs. Genes with a p < 0.101 and log2 fold change of ≥ 1 or ≤ 1 were considered to be differentially expressed.

Results

Out of approximately 44,882 transcripts that were analyzed, we did not have any differentially expressed genes with a p < 0.05. However, the distribution of the genes and p-values shown on Figure 2 was promising with the peak at 0, especially despite the observed variability in expression changes between each porcine donor in Figure 3. From this study, 180 genes with p < 0.101 were analyzed to explore the trends that existed between our treatment groups. The genes with the highest fold changes are depicted in Figure 4, the interactions between differentially expressed proteins for these transcripts are shown in Figures 5 & 6. Since highly connected

201 proteins (“hubs”) typically control these networks, Tables 1 & 2 indicate the most connected proteins for up and down regulated networks, respectively. Enriched

KEGG Pathways for the upregulated and downregulated RNAs are listed in Table 3 and Table 4.

Figure 35: Histogram of the p-value and frequency. Individual dots indicate genes with A being the average log intensity of the gene expression (x-axis) and M being the binary log of intensity ratio (y-axis). This graph shows a right shift on the histogram with many probe sets (transcripts) that are not significantly different (n=4).

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Figure 36: Bland–Altman plot (MA plot) that represents differences between measurements taken from paired samples of SCS and PSF. Each graph shows mRNA expression levels for each sample, with individual dots representing individual mRNAs identified. For each plot, the x-axis is the average log intensity of mRNA expression and the y-axis is the binary log of intensity ratio.

Figure 37: The most downregulated and upregulated genes in PSF kidneys compared to SCS kidneys. These genes are related to protein trafficking, regulation of cell cycle, mitochondrial apoptotic pathways, degradation of proteins, metabolism and energy production.

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Figure 38: A topology map of upregulated genes. This figure visualizes signaling networks based on differentially expressed gene products. Each circle represents a protein, and each line an interaction between two proteins Bigger and darker spots are the genes that are the most connected genes upregulated in the samples. The light-yellow dots are genes that are influenced by the upregulated genes in the biopsies.

Figure 39: A topology map of downregulated genes. This figure visualizes signaling networks based on differentially expressed gene products. Each circle represents a protein, and each line an interaction between two proteins Bigger and darker spots are the genes that are the most connected genes upregulated in the samples. The light-yellow dots are genes that are influenced by the upregulated genes in the biopsies.

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Gene Name Degree Betweenness HSP90AB1 565 1249938.2 HNF1A 294 723898 EEF1A1 286 475053.9 HNRNPA1 237 411212.2 EIF4A3 220 367022.9 EWSR1 213 433737.2 ACTB 198 394177.6 PPP2CA 159 267415 PABPC1 152 205357.2 HSPD1 143 208154.8 PSMD4 133 204852.8 RPLP0 124 118914.3 RPL3 112 104006.4 KDM1A 100 195518.1

Table 31: Highly connected, upregulated proteins. The top n most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

Gene Name Degree Betweenness VDAC2 55 4245.1 TMED10 40 3280.5 TUBGCP3 13 1143.4 TRIAP1 5 426 UBC 4 1780.6 TRAF6 2 479.2 CDK2 2 479.2 ATF2 2 479.2 ELAVL1 2 318

Table 32: A chart of the hub genes with the most degree and betweenness from the network of downregulated proteins. The top n most highly connected proteins, along with their degree and betweeness is shown. Degree represents the number of connections a protein has to other proteins in the network. Betweenness is the number of shortest path going through the gene products and indicates its capability of transferring communication between the genes in the module. High betweeness indicate more biologically informative nodes in a module.

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Pathway Total Hits P-value Endocytosis 101 48 5.07E-07 Fc gamma R-mediated phagocytosis 97 41 0.0001 Regulation of autophagy 8 5 0.03 Cell cycle 124 73 3.4E-16 ErbB signaling pathway 87 45 4.9E-08 MAPK signaling pathway 265 103 1.4E-07 mTOR signaling pathway 45 25 9.6E-06 p53 signaling pathway 68 33 1.8E-05 Apoptosis 83 35 0.0004 TGF-beta signaling pathway 84 33 0.002 VEGF signaling pathway 76 30 0.003 Regulation of actin cytoskeleton 182 90 2.4E-13 Focal adhesion 200 91 6.7E-11 Adherens junction 70 38 1.1E-07 Tight junction 118 51 7.6E-06 Wnt signaling pathway 144 51 0.003 Gap junction 89 34 0.003 ECM-receptor interaction 84 28 0.05 Chemokine signaling pathway 189 79 1.3E-07 B cell receptor signaling pathway 75 37 3.4E-06 T cell receptor signaling pathway 98 44 9.9E-06 RIG-I-like receptor signaling pathway 49 25 6.6E-05 NOD-like receptor signaling pathway 49 25 6.6E-05 Adipocytokine signaling pathway 63 30 6.8E-05 Leukocyte transendothelial migration 108 42 0.0008 Fc epsilon RI signaling pathway 75 29 0.005 Natural killer cell mediated cytotoxicity 138 44 0.04 Toll-like receptor signaling pathway 97 32 0.04 Citrate cycle (TCA cycle) 30 16 0.0007 Glycolysis / Gluconeogenesis 65 23 0.04

Table 33: Pathways enriched in PSF compared to SCS kidney samples. A list of upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Total: total number of gene products in pathway, Hits: number of gene products in pathway that significantly differ in abundance between SCS and PSF kidneys.

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Pathway Total Hits P.Value Protein processing in endoplasmic reticulum 129 7 0.0007 Notch signaling pathway 47 3 0.02 p53 signaling pathway 68 3 0.05

Table 34: Pathways enriched in PSF compared to SCS kidney samples. A list of downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the differentially expressed genes identified by microarrays were analyzed using NetworkAnalyst. Differentially expressed gene products in specified pathway are represented by hits, while the total number represents the number of all genes in the pathway.

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Regulation of Apoptosis Mitochondrial Matrix KDM1A Lysine demethylase 1A MIPEP Mitochondrial intermediate peptidase HSP90AB1 Heat shock protein 90 alpha family CS Citrate synthase class B member 1 LGMN Legumain OAT Ornithine aminotransferase RTN4 Reticulon 4 COASY Coenzyme A synthase IVNS1ABP Influenza virus NS1A binding protein BCKDHB Branched chain keto acid dehydrogenase E1 subunit beta TCP1 T-complex 1 HADHA Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha ATF4 Activating transcription factor 4 ACO2 Aconitase 2 TMBIM6 Transmembrane BAX inhibitor motif IDH3B Isocitrate dehydrogenase 3 (NAD (+)) containing 6 beta HSPD1 Heat shock protein family D (Hsp60) NDUFS2 NADH:ubiquinone oxidoreductase member 1 core subunit S2 ARL6IP5 ADP ribosylation factor like GTPase NAXE NAD(P)HX epimerase 6 interacting protein 5 ITGB1 Integrin subunit beta 1 NUDT13 Nudix hydrolase 13 ARF4 ADP ribosylation factor 4 IDH2 Isocitrate dehydrogenase LAMP1 Lysosomal associated membrane protein 1 TXNIP Thioredoxin interacting protein TCA Cycle/ATP Generation CS Citrate Synthase Cell Cycle ACO2 Aconitase 2 ANAPC5 Anaphase promoting complex IDH3B Isocitrate dehydrogenase 3 (NAD(+)) subunit 5 beta GNAI2 G protein subunit alpha i2 PCK1 Phosphoenolpyruvate Carboxykinase 1 ITGB1 Integrin subunit beta 1 NDUFS2 NADH:ubiquinone oxidoreductase core subunit S2 KIF3B Kinesin family member 3B PC Pyruvate carboxylase

PPP2CA Protein phosphatase 2 catalytic GPD1 Glycerol-3-phosphate dehydrogenase subunit alpha 1

PRKAG1 Protein kinase AMP-activated non- PRKAG1 Protein kinase AMP-activated non- catalytic subunit gamma 1 catalytic subunit gamma 1 PSMB4 Proteasome subunit beta 4 PSMD4 Proteasome 26S subunit, non- Glycolysis/ Gluconeogenesis ATPase 4 RAE1 Ribonucleic acid export 1 PYGL Glycogen Phosphorylase L SRSF5 Serine and arginine rich splicing PGK1 Phosphoglycerate kinase 1 factor 5 TXNIP Thioredoxin interacting protein TPI1 Triosephosphate isomerase 1 PPP2CA Protein phosphatase 2 catalytic subunit alpha Growth AKR1A1 Aldo-Keto Reductase Family 1 Member A1 SEC61A1 Sec61 translocon alpha 1 subunit PCK1 Phosphoenolpyruvate Carboxykinase 1 CD320 CD320 molecule FBP1 Fructose-bisphosphatase 1 CD81 CD81 molecule GNAS GNAS complex locus Cytoskeleton

ITGB1 Integrin subunit beta 1 ACTB Actin beta

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LGMN Legumain TUBGCP3 Tubulin gamma complex associated protein 3 RTN4 Reticulon 4 TPM1 Tropomyosin 1 TKT Transketolase KEAP1 Kelch like ECH associated protein 1

DENND1A DENN domain-containing protein 1A KIF3B Kinesin family member 3B

Figure 40: List of upregulated genes. Identified genes with a fold change of greater than 1 in PSF kidneys compared to SCS kidneys after 24 hours of preservation.

Apoptosis Mitochondrial Gene TRIAP1 TP53 regulated inhibitor of COX6C Cytochrome C Oxidase apoptosis 1 PEG10 Paternally expressed 10 PRDX3 Peroxiredoxin 3 VDAC2 Voltage dependent anion channel 2

Cell Cycle/Development Cytoskeleton TLK1 Tousled like kinase 1 COL27A1 Collagen type XXVII alpha 1

PTCH1 Patched 1 TUBGCP Tubulin gamma complex associated 3 protein 3 KLF2 Kruppel like factor 2

Miscellaneous

TMED10 Transmembrane p24 trafficking protein 10 LYPLA1 Lysophospholipase I

Figure 41: List of downregulated genes. Identified genes with a fold change of greater than 1 in PSF kidneys compared to SCS kidneys after 24 hours of preservation.

Discussion

Prolonged ischemia experienced by isolated organs can compromise tissue integrity and function, and can lead to worse clinical outcomes. Some of these effects can be mitigated by providing oxygen, which consequently aids in producing more

ATP to avoid complete energy depletion.

In the present study, PSF was used to deliver oxygen to kidneys during preservation, and transcriptome analysis was used to evaluate its effects compared to SCS. Ischemic injury can cause morphological changes in the renal tubules, and if severe enough, cause subsequent cell death from necrosis and apoptosis. Kidneys

209 have the capacity to repair itself through dedifferentiation and migration of viable cells to restore the damaged epithelium and renal function (Thadhani, Pascual, &

Bonventre, 1996). Progenitor cells exist in adult kidneys which have the potential to differentiate into glomerular and tubular epithelial cells, especially upon ischemia reperfusion injury (Cantaluppi, et al., 2012). In this discussion, we highlight the enriched pathways and how they relate to ischemic damage in the kidneys.

There is an enrichment in autophagy in the PSF kidneys after preservation and rewarming. Autophagy is a process of self-digestion, and degradation of protein aggregates and damaged organelles. These cytoplasmic components are sequestered into autophagosomes and delivered to lysosomes for degradation (Mariño, Niso-

Santano, Baehrecke, & Kroemer, 2014). It is a process observed in renal proximal tubular epithelial cells in response to ischemia, and we saw an increased fold change in Legumain (LGMN) that is required for lysosomal protein degradation (Jinq-May,

Dando, Stevens, Fortunato, & Barrett, 1998). It is typically induced prior to the activation of apoptotic pathways during ischemic damage. When autophagy is impaired, kidneys were sensitized to ischemic injury, which suggests its role in maintaining tubular cell integrity under ischemic conditions (Liu, et al., 2012). In mice with induced renal damage, autophagy inhibition resulted in more renal damage, functional loss and apoptosis (Jiang, et al., 2012; Jiang, Liu, Luo, & Dong, 2010). It is primarily a protective cellular process, but if unregulated it can have lethal effects

(Mizushima, Levine, Cuervo, & Klionsky, 2008).

We observed an upregulation of the mammalian target of rapamycin (mTOR) pathway that is known to regulate autophagy and plays a role in mediating cell size

210 and proliferation (Yu, et al., 2010; Inoki, 2014). There are two types, mTORC1 and mTORC2. mTORC1 induces autophagy in response to nutrient starvation, environmental stresses, and reduced growth factor signaling, while mTORC2 regulates autophagy in skeletal muscle through the Akt-FoxO3 pathway in response to fasting (Jung, Ro, Cao, Otto, & Kim, 2010). mTORC2 is activated by renal injury, and mTOR inhibition is associated with delayed recovery (Lieberthal & Levine, 2009), with prolonged delayed graft function in rapamycin (mTOR inhibitor) treated patients (Grigoryev, Liu, Cheadle, Barnes, & Rabb, 2006).

PSF kidneys exhibited an increase in transmembrane BAX Inhibitor Motif

Containing 6 (TMBIM6) expression, which has a role in suppressing apoptosis and autophagy (Yadav, et al., 2015). Conversely, P53 Regulated Inhibitor of Apoptosis 1

(TRIAP1) expression was decreased in PSF kidneys. The genes involved in the p53 pathway that initiates cell cycle arrest, cellular senescence and apoptosis (Harris &

Levine, 2005) are both upregulated and downregulated in PSF treated kidneys, which indicates a complex interaction of signal transduction pathways, positive and negative autoregulatory feedback loops that act on p53 and its downstream targets.

In rodent models of acute kidney injury, necrosis and apoptosis are both found in the damaged nephrons. The former is primarily found in proximal tubules with the latter in the distal tubules. Renal injury activates cell cycling, and the damage results in cell death and cell proliferation (Price, Safirstein, & Megyesi, 2009), and restoration of original tissue architecture can be achieved through regeneration with the renal progenitor system (Romagnani, Lasagni, & Remuzzi, 2013). Hepatocyte nuclear factor-1β (Hnf-1β) is a transcription factor that drives nephrogenesis, tubulogenesis

211 and epithelial homeostasis through the regulation of epithelial-mesenchyme- epithelial cycling (Faguer, et al., 2013). It functions either as a homodimer or as a heterodimer with Hnf-1α to regulate gene expression (Kanda, et al., 2016). In mice,

Hnf-1 is increased within 1-24 hours of hypoxia and was required for proper differentiation of renal proximal tubule cells (Pontoglio, 1996; Faguer, et al., 2013).

In our studies, we found an increased fold change of Hnf-1α, which was also an important ‘hub’ gene that interacts with many networks.

Renal cells that are typically quiescent enter mitogenesis and differentiation after an injury. They express proliferating cell nuclear antigen (PCNA) along with immediate early genes such as c-Fos that guide transition into the cell cycle (Witzgall,

Brown, Schwarz, & Bonventre, 1994). An enrichment in cell cycle pathways was observed in the PSF kidney compared to the SCS kidney, consistent with the activation of cell death and proliferation pathways that can contribute to both injury and protection and recovery of the organ seen in experimental models (Witzgall, Brown,

Schwarz, & Bonventre, 1994). In patients with tubular necrosis, more progenitor cells and better proliferative activity of tubular cells were associated with less renal failure and mortality (Ye, et al., 2011). However, studies have shown that the proliferative response combined with cellular damage can exacerbate the injury. Inhibiting the cell cycle progression reduced renal injury and resulted in higher survival rates in the rodent model (Pabla, et al., 2015).

Mitogen-activated protein kinases (MAPK) are a superfamily of heterogeneous kinases that are comprised of extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 kinases (Cowan & Storey,

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2003). They form ubiquitous pathways involved in a broad range of cellular processes including cell growth, proliferation, and death (Cowan & Storey, 2003). MAPK can be activated by ischemic injury and inflammatory cytokines, and activation of p38 can attenuate ischemic effects and preserve renal function during ischemic preconditioning (Park, Chen, & Bonventre, 2001). Further study will be needed to determine whether the activation and enrichment of this pathway in PSF kidneys is actually involved in the mechanisms behind ischemia induced cell damage and death, or if it is a byproduct of cell death.

The Raf-MAPK-ERK pathway that is upregulated in the PSF kidneys is a key downstream effector of Ras which is a key downstream effector of the epidermal growth factor receptor (EGFR) (Roberts & Der, 1997). The epidermal growth factor receptor (EGFR) family, also known as ErbB receptors (ErbBs), can contribute to cell proliferation, migration, and epithelial to mesenchymal transdifferentiation

(Norman, et al., 1987; Zhuang, Dang, & Schnellmann, 2004; Zeng, Singh, & Harris,

2009) that are important in renal repair after an insult. Increased EGFR phosphorylation is seen in many types of ischemic insult (Hise, et al., 2001; Homma, et al., 1995). EGFR deletion in the proximal tubules showed persistent proximal tubule dilation and cast formation with a delay in renal cell proliferation (Chen, Chen,

& Harris, 2012) while administering EGF accelerated both structural and functional recovery (Harris R. C., 1997). Sustained activation of EGFR is also involved in the development of renal fibrosis with transforming growth factor-b (TGF-β) (Tang, Liu,

& Zhuang, 2013; Tang J. L., 2013). TGF-βpromotes the structure of cytoskeleton of the proximal epithelium in culture (Humes, et al., 1993) and its expression is elevated at

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12 hours and 24 hours after ischemic injury (Basile, Rovak, Martin, & Hammerman,

1996). It also is a key mediator in tubular epithelial-myofibroblast transdifferentiation that is a cause an accumulation of extracellular matrix proteins that can lead to tubulointerstitial fibrosis (Fan, et al., 1999; Bonventre, 2003).

Heat shock proteins (HSP) are involved in post-translational folding, stability, and protein maturation, and are essential in mediating cell cycle progression. Additionally, they play a cytoprotective protein chaperone that participates in translocating proteins to the mitochondria (Budas, Churchill,

Disatnik, Sun, & Mochly-Rosen, 2010). They are stimulated by hyperthermia and environmental stressors, and elevations in HSP is seen after an ischemic event

(Knowlton, Brecher, & Apstein, 1991; Donnelly, Sievers, Vissern, Welch, & Wolfe,

1992). HSP90 is upregulated in the PSF organs, and acts as a hub gene within its network. It has proliferative capacities by forming a complex with AKT of the phosphatidylinositol 3′-kinase (PI3)/Akt pathway that is activated in proliferation and cell survival (Basso, et al., 2002). The inhibition of HSP90 is associated with tumor suppression (Solit, Basso, Olshen, Scher, & Rosen, 2003) and in cancer cell lines, inhibition of HSP90 and MAPK decreased cell proliferation and migration. It also increases endothelial nitric oxide synthase (eNOS) activation in hypoxic coronary endothelial cells through PI3-AKT (Chen & Meyrick, 2004). HSP are also involved in the regulation of apoptotic cell death, and mediates protection from cell death in ischemic preconditioning (Kennedy, Jäger, Mosser, & Samali, 2014; Hutter, et al.,

1996).

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Hypoxic conditions during organ procurement and preservation can activate the hypoxia inducible factor (HIF) system to increase vascular endothelial growth factor (VEGF) expression. It induces proliferation and migration of vascular endothelial cells that are involved in both developmental and pathological angiogenesis (Olsson, Dimberg, Kreuger, & Claesson-Welsh, 2006). VEGF induction can be stimulated by other factors such as reactive oxygen species, nitric oxide inhibition, growth factors, and inflammatory response mediators, and its pathways are enriched in the PSF kidneys. VEGF expression was found in the glomeruli and tubules with the receptors mainly in the endothelial cells of renal vasculature. Its production was needed for maintenance of the glomerular filtration barrier

(Eremina, Baelde, & Quaggin, 2007), vascular stabilization, and blocking VEGF receptor attenuated renal fibrogenesis (Lin, et al., 2011). Altered VEGF expression was seen in kidneys with peritubular capillary damage (Choi, et al., 2000), and it is an important angiogenic factor for vascular maintenance after kidney injury (Tögel,

Zhang, Hu, & Westenfelder, 2009). Interestingly enough in the context of renal ischemia, VEGF mRNA or protein levels were not increased following ischemia or ischemia and reperfusion (Kanellis, Mudge, Fraser, Katerelos, & Power, 2000) and in some cases, were repressed when compared to control values of VEGF expression

(Basile, Fredrich, Chelladurai, Leonard, & Parrish, 2008; Bonventre & Yang, 2011).

Renal ischemia causes the loss of surface membrane proteins and the redistribution of apical and basolateral cell components of the tubular cells. We see enriched pathways relevant to tissue remodeling and cell structure in the PSF treated kidneys. Cytoskeletal and structural changes cause instability of the surface

215 membrane and form blebs that contain detached microvilli from the apical cell surface (Sharfuddin & Molitoris, 2011). ATP depletion leads to apical actin depolymerization and redistribution of the actin. Tropomyosin that binds to actin for stabilization is dissociated from the microfilament during ischemia. Disruption of actin cytoskeleton has detrimental effects on tight junctions and adherens junctions that facilitate paracellular transport, morphology, and polarity. Disruptions of tight junctions lead to lateral diffusion of surface membrane components (Molitoris, Falk,

& Dahl, 1989) and back leak of glomerular filtrate to the interstitium (Molitoris &

Marrs, The role of cell adhesion molecules in ischemic acute renal failure, 1999). Our pathway analysis shows enriched genes associated with tight junction and adherens junction regulations in PSF kidneys, which may be a response to the structural damage incurred by ischemia during preservation. The basolateral Na+/K+-ATPase redistribution to apical membrane is a major source of sodium and water moving back to the tubular lumen (Sharfuddin & Molitoris, 2011). Na+/K+-ATPase is redistributed as soon as 10 minutes post ischemia. Re-establishment of cell polarity occurred 24 hours to several days after the ischemic insult ranging from 15-50 minutes in a dose dependent manner (Spiegel, Wilson, & Molitoris, 1989).

After the ischemic insult, surviving tubular epithelial cells and epithelial progenitor or stem cells are the main cell types to contribute to epithelial regeneration and renewal (Humphrey, et al., 2008). Adhesion molecules and cytokines regulate epithelial cell migration, proliferation and differentiation.

Adhesion molecules allow the cell to move forward and facilitate cell-matrix and cell- cell interactions. Loss of adhesion molecules, specifically integrins, was associated

216 with decreased polarity. Leukocyte endothelial cell adhesion molecules also plays a role in epithelial cell spreading through generating cytokines and chemokines that can influence renal repair (Bonventre, 2003).

Sterile inflammation occurs with the innate immune response to intracellular contents released by damaged and necrotic cells (Chen & Nunez, 2010). These factors can be passively released from cellular injury or leaked out due to cell structural damage. They can also be actively released, and immune cells can recognize altered cell surface structures from the injury (Thurman, 2007). Toll like receptors (TLRs) located on cell surfaces, NOD-like receptors (NLRs) located in the cytoplasm and RIG-

I-like receptors (RLRs) can be activated by cellular injury (Kawai & Akira, 2009). This induces pro-inflammatory cytokines, chemokines, and recruits leukocytes to the injury site. Inflammation is crucial for wound repair, but unchecked inflammation can result in creating more reactive oxygen species, vascular permeability, fibrosis, and tissue destruction.

The oxygenated kidneys, after a period of rewarming, show upregulation of pathways that are involved in tissue remodeling. The necessary elements of cell structural modifications can be inferred form the upregulation of cell cycling and regulation of actin cytoskeleton, tight junctions, and adherens junctions. MAPK signaling is difficult to interpret because it interacts with many different signaling cascades, but the increase in MAPK, TGF, mTOR and EGFR combined with cell structural regulation might indicate the beginning stages cell proliferation and migration which are necessary for renal tubular repair. Signs of the innate immune response are present in PSF kidneys with augmented signaling pathways such as RIG-

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I-like receptor, NOD-like receptor and Toll-like receptor signaling pathways.

Leukocyte transendothelial migration pathways are also upregulated. This is not surprising as cellular damage from 30 minutes of warm ischemia followed by 24 hours of hypothermia can elicit sterile inflammation. Studies have found a reduction in chemotactic migration under low pH and low oxygen conditions (Rotstein, Fiegel,

Simmons, & Knighton, 1988; Turner, 1999).

While ischemic conditions can induce inflammation, inflamed tissue is prone to hypoxia due to metabolic demands of cells and a drop in available metabolic substrates (Eltzschig & Carmeliet, 2011). The augmented inflammatory activity may be explained by the PSF tissue having more oxygen availability to support the energy requirements needed to elicit this response. There was an upregulation of the TCA cycle, glycolysis and gluconeogenesis pathways, which indicates both aerobic and anaerobic metabolism in the PSF kidneys. Gluconeogenesis removes glucose precursors in the kidney (Newsholme & Gevers, 1987), which may explain the lower levels of lactate in PSF treated kidneys seen in Experiment 1 and Experiment 2. HSP and Hnf was found to be upregulated in PSF kidneys, and network analysis revealed them to be critical signaling nodes, both of which can have protective and ameliorative effects on sublethally damaged tissue.

218 reperfusion. In rodent studies, tubular regeneration showed a peak at day 7

(Nonclercq, et al., 1992). In our studies, it is doubtful that the mix of warm and cold ischemia during organ procurement and preservation induced regenerative activities of the renal tissue that is reflected on the renal function after 24 hours. The higher glomerular filtration rates seen in the PSF kidneys from Experiment 1 and

Experiment 2 likely insinuates more filtration due to less damage during prolonged

24 hours of preservation as opposed to repaired tubules. While these results are encouraging, specific genes of interest need to be validated using quantitative real- time PCR.

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Chapter 6

Pilot Study Comparing the Effects of Persufflation and Hypothermic Machine Perfusion Using the LifePort

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Introduction

The prevailing method of organ preservation is static cold storage after a flush with preservation solution. The improvement in the content of these solutions has helped in minimizing organ edema and acidosis during the period between procurement and transplant. Furthermore, it is favored due to its simplicity and low cost. However, there has been a rise of using organs outside the standard criteria for deceased donors, subsequently driving the development of alternative ways to preserve organs with the goals of extending preservation periods, rehabilitating the damaged tissue and priming the organs for transplant.

The original, extracorporeal machine perfusion was discovered in 1935 with the idea of providing oxygenated fluid to the organs using a pulsatile circulation device (Monbaliu, Liu, Vekemans, & Pirenne, 2009). This was further developed and readopted when Belzer found that plasma perfusate can be successfully used to perfuse canine kidneys (Belzer, Park, & Vetto, 1964). This was discovered at a time when continuous perfusion with whole blood was possible only for a short period of time. Moreover, blood perfusion of isolated organs presented with an increase in perfusion pressure within 30 minutes of perfusion (Belzer, Park, & Vetto, 1964;

Belzer, Belzer, Ashby, Huang, & Dunphy, 1968; Belzer, Ashby, & Dunphy, 1967). The first documented clinical case of using HMP resulted in preserving an organ that was exposed to 25 minutes of warm ischemia time for 17 hours (Belzer F. O., Ashby ,

Gulyassy, & Powell, 1968). However, subsequent studies of machine perfused, non- heart beating donor kidneys were not successful and resulted in poor, nonfunctioning grafts (Belzer, Reed, Pryor, Kountz, & Dunphy, 1970). Around this time, a seminal

221 study using canine kidneys showed comparative effects of cold storage and perfusion, with cold storage being more successful (Collins, Bravo-Shugarman, & Terasaki,

1969). Kidneys were cooled either by surface cooling using oxygenated saline followed by hyperbaric oxygen for storage, or by perfusing them with 100-150 mL of cold solution and preserved in ice saline for the rest of the storage period. Results from this study show that kidney storage could be maintained for up to 12 hours with simple cooling, and this could be extended for up to 16 hours in conjunction with hypertension drugs such as mannitol and phenoxybenzamine. (Collins, Bravo-

Shugarman, & Terasaki, 1969). Moreover, earlier studies could not firmly establish any advantage of HMP over simple cold storage in organs that were not damaged with prolonged periods of warm ischemia time (van der Vliet, Vroemen, Cohen,

Lansbergen, & Kootstra, 1983). However, other groups found that when the warm ischemia time exceeded 30 minutes, kidneys preserved by HMP recovered their normal function whereas the cold storage kidneys did not (Johnson, Anderson,

Morley, Taylor, & Swinney, 1972). This was supported by a clinical trial that demonstrated HMP’s ability to reduce delayed graft function and primary nonfunction in non-heart beating donor organs compared to those that were cold stored (Daemen, et al. 1997).

The current state of the art method is of preservation is hypothermic machine perfusion (HMP) using the LifePort. More recent clinical studies cite the reduction in delayed graft function and less complications in expanded criteria and cardiac death donor organs (Moers, et al., 2009; O'callaghan, Morgan, Knight, & Morris, 2013;

Schold, et al., 2005; Wight J. P., Chilcott, Holmes, & Brewer, 2003), with reduced costs

222 and hospital stay (Wight J. , Chilcott, Holmes, & Brewer, 2003; Polyak, et al., 2000).

The parameters of long-term graft survival, patient survival and incidence of primary non-function however, were similar in cold storage and machine perfusion groups

(O'callaghan, Morgan, Knight, & Morris, 2013). A study published this year on donation after cardiac death kidneys exhibited higher urine volumes, lower creatinine levels and higher 1 and 3-year graft survival rates (Zhong, et al., 2017).

In parallel with the development of superior preservation techniques, the advancement and validation of minimally invasive methods that can measure organ viability and function ex vivo is imperative to pre-transplant characterization and organ allocation. In this study, we hypothesized that using persufflation to supplement whole kidneys with hypothermic, humidified oxygen gas will be a valid method that can mitigate the effects of ischemia during cold preservation. We opted for whole organ oxygen consumption rate (WOOCR) to be used in quantitating kidney viability, and glomerular filtration rate (GFR) using imaging. Dynamic contrast enhanced imaging using magnetic resonance (DCE-MRI) is a non-invasive technique that has been widely transitioning from giving a depiction of morphology to being studied to measure organ functions in vivo (P. S. Tofts, M. Cutajar, et al. 2012, Zhang, et al. 2014, J. B. Buchs, et al. 2014, Baumann and Rudin 2000, Annet, Hermoye, et al.

2004, Notohamiprodjo, Reiser and Sourbron 2010). Imaging has important capabilities of assessing vital physiological parameters such as vascular permeability, perfusion, filtration fraction and total GFR, all of which are essential information that can be used to anticipate graft function and longevity once they are transplanted.

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In the first study presented in Chapter 4, we found significant differences between the standard, static cold stored kidneys and persufflated (PSF) kidneys in terms of viability and function. In this separate pilot study, we compared the effects of hypothermic machine perfusion using the LifePort, and persufflation. The benefits of HMP are that they clear out toxin buildup from metabolic byproducts, maintain vascular patency while PSF can deliver more oxygen, maintain adequate ATP levels, extend the preservation periods and improve graft function (Taylor and Baicu 2010,

T. M. Suszynski, M. D. Rizzari and W. E. Scott, et al. 2012). Despite studies that span a few decades for both PSF and HMP, there are few studies that compare them directly with each other. Experimental evidence is needed to further elucidate both the benefits or disadvantages of these two preservation methods. This study aims to understand the effects of 24 hours of preservation with these two methods in pre- damaged, isolated porcine kidneys.

Methods

Organ Procurement

Domestic female pigs weighing 35-50 kg were used from a local farm in

Wilcox, Arizona. Animal care procedures were in accordance with university policies.

Pigs were anesthetized under isoflurane and heparinized with 300 U/kg for 5 minutes. Subsequently they were euthanized with pentobarbital, exsanguinated and flushed with 2 liters of room temperature, heparinized saline. Kidneys were exposed to 30 minutes of warm ischemia time inside the body cavity. After the procurement, they were promptly placed on ice and cannulated at the renal artery and vein using

224 luer locks (Cole-Parmer) and flushed with 400 mL of 4°C heparinized saline using gravity followed by a secondary flush with SPS-1 solutions (Organ Recovery System).

The kidneys were treated for 24 hours with either or persufflation (PSF) or hypothermic machine perfusion (HMP) as shown in Figure 42.

Figure 42: Timeline illustrating the details of study design. Kidneys from a porcine DCD model were procured after 30 minutes of warm ischemia time and exposed to 24 hours of cold ischemia time during the preservation period. Kidneys were preserved using either the LifePort or persufflator for HMP and PSF for 24 hours, and assessments were done after the preservation period.

Separate kidneys were harvested from porcine donors to be used as controls.

These kidneys were exposed to 15-30 minutes of warm ischemia time and were used to measure oxygen consumption rate and histological analyses.

Organ Preservation

The persufflator (Giner, Inc., Newton, MA) delivered electrochemically generated, humidified, 40% oxygen mixed with atmospheric air with a manifold pressure of 40-50 mmHg and gas flow of 60 mL/min. Tubing from the persufflator was attached to the port on the lid of the organ container. Separate tubing was connected to another port on the inside of the lid, and this was connected to the renal

225 artery. Leaks around the vessel were ligated and the kidney was stored in an icebox during the preservation period.

The LifePort (Organ Recovery Systems) was prepared by putting ice and water to the ice bin to create a slush. Once the temperature read 4-8°C, the LifePort cassette was connected to the pump deck that measures pressure and resistance. The cassette was filled with cold SPS-1 (Organ Recovery Systems) for perfusion. These organ cradles and cassettes typically use cannula for aortic patches, but we used a cannula that could be directly connected to the renal artery to avoid the complications that came with using an aortic patch. This cannula was then connected to the infusion line after they were cleared of air bubbles and primed for perfusion. Kidneys were hypothermically perfused for 16-24 hours.

Whole Organ Oxygen Consumption Rate

The renal artery and renal vein were cannulated individually and connected to a custom-built machine perfusion system. This was maintained at 21-23°C using a water bath and water jacketed media reservoir with insulated tubing. SPS-1 (Organ

Recovery Systems) was oxygenated with 40% oxygen using a hollow fiber oxygenator

(Medtronic) and perfused through the artery throughout the measurements.

Calibrated flow-through fiber-optic oxygen sensors (Instech) were placed proximal to the renal arterial cannula and one was placed distal to the venous cannula to measure the difference oxygen pressure across the kidneys. The WOOCR

(mol/min/g) was calculated by using a mass balance equation where Q is the flow rate of perfusion (mL/min), α is oxygen solubility in aqueous solution at temperature

(mol/mL/mmHg), and m is the mass of the kidney (g):

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푄 ∗ (푝푂2 퐴푟푡푒푟푖푎푙 − 푝푂2 푉푒푛표푢푠) ∗ 푎 푊푂푂퐶푅 = 푚

Dynamic Contrast Imaging using Magnetic Resonance

All imaging was done on a 3T Skyra scanner (Siemens). Kidneys were secured in the organ cassette (LifePort) and perfused at 75 mL/min with cold SPS-1 preservation solution (Organ Recovery Systems) using a MR compatible perfusion circuit. The organ cassette was secured in a standard Siemens Head and Neck RF coil.

After positioning the kidneys, 4mM Multihance Gadolinium-DTPA solution (Bracco

Diagnostics Inc.) was injected to a port in the heat exchanger connected to the renal artery after 20 seconds of pre-contrast. Dynamic-contrast MRI was performed using

Siemens golden angle stack of star radial acquisition (radialVibe). A Modified Look

Locker Inversion recovery method (MOLLI) was used to generate T1 map pre-and post-injection of Gadolinium-DTPA solution (Bracco Diagnostics Inc.). Compressed

Sensing reconstruction with total variation applied in temporal dimension was used to reconstruct images with a 1 second temporal resolution, and manual segmentation was done on these images using only 21 lines of k-space (2 sec temporal resolution).

The arterial input function (AIF) curve was generated using the signal from an ROI placed on the first appearance of gadolinium in the renal artery and the kidney signal was obtained by segmentation of the whole kidney. Imaging was done for a total of 4 minutes but fitted over a 90 second acquisition window from the arrival of

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Molecular Analysis

Microarrays can give a snapshot of the transcriptome at the time of the biopsy.

Biopsies were stored in RNAlater (Qiagen Sciences, Gaithersburg, MD) overnight at

4C and then stored in the -80C. RNA was isolated form the cortical tissue samples using miRNeasy Mini Kit (Qiagen Sciences). The RNA integrity was checked by

NanoDrop™ 3300 Fluorospectrometer (ThermoFisher Scientific). RNA from 4 pairs of tissue samples were used. The RNA was reverse-transcribed into cDNA for in-vitro transcription for aRNA synthesis using Quick Amp Labeling Kits (Agilent

Technologies). These samples were prepared for dye-coupling and labeled with Cy3

(green) or Cy5 (red). The samples were mixed with hybridization solution that was prepared using Agilent Gene expression hybridization kit (Agilent Technologies), and applied to the microarray. Agilent 4x44k S. Scrofa (Pig) Oligo Microarray v2 (Agilent

Technologies, Design # 026440) was used for hybridization performed in the hybridization oven G2545A (Agilent Technologies, Palo Alto, CA). Fluorescence intensities were measured by scanning microarrays with the GenePix 4000B Scanner

(Molecular Devices) with laser excitation at 532 and 635 nm. Measurements of differential expression were obtained from the relative abundance of hybridized mRNA.

Histology

Wedge biopsies were taken from the kidneys after the preservation period.

They were fixed with 4% paraformaldehyde overnight, dehydrated, processed with

Leica TP1020 tissue processor (Leica Biosystems) and paraffin wax embedded.

Biopsies were sectioned at 5 microns using a microtome and stained with

228 hematoxylin and eosin (H&E) (Sigma-Aldrich) to evaluate gross renal morphology.

The sections were double blinded and assessed for tubular, glomerular and vascular damage using light microscopy. A veterinary pathologist used a scoring system ranging from 0-3; 0 = abnormalities, 1 = mild, 2 = moderate and 3 = severe changes.

HMP and PSF biopsies were compared to control samples with warm ischemia time of 15-30 minutes and no cold ischemia time.

Statistics

Unless otherwise indicated, data represent mean ± SEM values. The function and viability measurements of GFR, Ktrans, WOOCR, and histological analyses were all from paired kidneys. The differences between PSF and HMP groups were analyzed by using Wilcoxon Signed Rank test on Graphpad Prism version 6 software. P < 0.05 was considered significant.

Results

Two animals from this study were excluded due to the malfunction of the

LifePort. We did not see any significant differences in any of the parameters we measured. The average WOOCR for control, HMP and PSF kidneys in Figure 43 were

127.3 ± 29.8, 117.9 ± 32.1 nmol/min*g and 127.9 ± 23.6 nmol/min*g. The Ktrans shown in Figure 44A (HMP: 0.08 ± 0.07 ml/g/min; PSF: 0.11 ± 0.04ml/g/min) and GFR values shown in Figure 44B (HMP: 10 ± 8.54 ml/min; PSF: 10.67 ± 2.6 ml/min) were low in both treatment groups. Agilent microarray results also do not show any differential gene expressions that were significant. The variability between porcine donors in terms of gene expression and fold changes are illustrated in each panel of

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Figure 45. The p-value histogram shows a frequency distribution of the genes, with minimal genes falling in the category of p < 0.05 (Figure 46). There were very minimal morphological differences scored between the control and treatment groups

(Control: 0.73 ± 0.24; HMP: 1.24 ± 0.25; PSF: 1.06 ± 0.25) as shown in Figure 47 &

48.

Figure 43: Whole organ oxygen consumption rate of isolated kidneys. Control kidneys and kidneys preserved with HMP or PSF were evaluated after 24 hours of preservation. WOOCR was measured under hypothermic liquid perfusion using flow through oxygen sensors at the arterial and venous cannulas to calculate the pO2 difference across the whole organ (n=4).

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Figure 44: Functional parameters measured with dynamic contrast enhanced magnetic resonance imaging. Images were obtained from isolated kidneys using a custom-built MR compatible hypothermic perfusion system. (A) shows Ktrans, the transfer constant of the tracer moving from the renal vasculature to renal tubules (n=3). (B) shows the calculated values for glomerular filtration rate by using Ktrans and kidney volume (n=3).

Figure 45: Bland–Altman plot (MA plot) that represents differences between measurements taken from paired samples of HMP and PSF. Each hybridization (hyb) shows gene expression of each animal used (n=4). Individual dots indicate genes with A being the average log intensity of the gene expression (x-axis) and M being the binary log of intensity ratio (y-axis).

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Figure 46: Collection p-values produced by analyzing each probe set. This histogram shows there are many probe sets that are not significantly different (n=4). A B

C D

Figure 47: Light microscopy representation of H&E-stained tissue samples. Top two images (A, B) are from persufflated kidneys and the bottom two images (C, D) are from hypothermic

232 machine perfusion kidneys. There are minimal tubulitis, ectasia, and apoptosis. The only notable difference between the two groups were that the HMP tissue showed more dilation and space between the tubules.

Figure 48: Histological analysis of renal biopsies from HMP and PSF treated kidneys. The percentage of vascular and tubular ischemic damage over the whole corticomedullary surface was measured semiquantitatively using H&E sections on double blinded slides (n=4).

Discussion

In our study, none of the parameters we measured were significantly different between HMP and PSF kidneys. It is difficult to conclude much from this data set considering the variability in the LifePort function during the preservation period and the small sample size of the experiment. The WOOCR and GFR data show similar levels of metabolism and renal function between the two experimental groups. The

WOOCR values for control and PSF groups are extremely close, with HMP values being lower by approximately 10 nmol/min*g. This implies that the viability of the HMP and PSF kidneys are similar to that of organs with only warm ischemic exposure without the 24 hours of cold ischemic exposure during preservation. Additionally,

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HMP kidneys were able to retain viability despite interrupted perfusion with different perfusion times, implying that continues HMP may not be necessary for organ reconditioning during preservation. HMP kidneys show a wider range of GFR (0-27 mL/min) compared to the PSF kidneys that ranged from 6-15 mL/min. This may be explained by the wide range in perfusion time of the HMP group spanning from 16-

24 hours, with a static cold period ranging from 0-8 hours for each kidney. Microarray analysis show that for each pig there were genes that changed significantly for the individual pair of kidneys from the same animal. However, there were no genes that were significantly upregulated or downregulated across all 4 pig donors. The biopsies collected for microarrays were only from the outer cortical area to minimize the variability that comes with the collection of different ratios of cortex and medulla between the kidneys. However, the corticomedullary area is more susceptible to low oxygen conditions (Fry, Edwards, Sgouralis, & Layton, 2014), and due to our area specific biopsy, we did not capture the full picture of gene expression changes in all segments of the kidney. From the histology data, we can infer that PSF and HMP did not grossly influence renal architecture, nor did we see substantial damages to the nephron and renal vasculature. It is likely that these damages may not fully manifest under hypothermic temperatures.

A recent study comparing retrograde PSF and HMP found no differences in urea levels nor in histopathological analyses of the graft biopsies. In this experiment, kidneys were exposed to 20 minutes of warm ischemia time and preserved in their respective treatments for 2 hours ex vivo and autotransplanted. Urea was collected for 2 hours and then the preserved organs were explanted (Kalenski, et al., 2016).

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Conversely, A porcine study comparing static cold storage, HMP and retrograde PSF showed inferior results for HMP. These organs were exposed to 60 minutes of warm ischemia time and preserved for 4 hours and stored on ice for 10 minutes. They were autotransplanted and renal function was observed for 7 days. The PSF transplants recovered normal renal function at 7 days while the cold storage and HMP transplanted animals had elevated creatinine and urea levels. Lactate was significantly higher in the cold storage group compared to the other two groups.

Histology showed a near-normal morphology in the PSF kidneys, and cold storage kidneys showed almost no pathological signs. HMP kidneys on the other hand exhibited more lipid peroxidation and protein precipitates inside the tubules

(Treckmann, et al., 2009). The higher levels of lipid peroxidation are consistently seen in other studies, and suggest free radical induced vascular endothelium damage.

However, this marked increase did not translate to tubule cell function and was not associated with an increased inflammation (Hosgood, Yang, et al. 2010).

Treckmann’s study may have seen more benefits of oxygen after the longer warm ischemia time. These results were analogous to an animal study comparing the effects of HMP between standard organs with no warm ischemia time, and organs from donation after cardiac death model that were subjected to 30 minutes warm ischemia. A noticeable improvement in renal function was observed in HMP of standard organs, but these effects did not carry over in the pre-damaged kidneys

(Nicholson, Hosgood, Metcalfe, Waller, & Brook, 2004). It is difficult to extrapolate from these studies because the preservation period was 2-4 hours, compared to our extended 24-hour period. In a study that compared the functions of PSF and HMP

235 directly, rodent livers with 30 minutes of WIT and 18 hours of cold, retrograde PSF or HMP were assessed after rewarming. Livers stored and rewarmed with oxygen had improved bile production and enhanced recovery in energy charge compared to HMP livers (Stegemann, Hirner, Rauen, & Minor, 2009). Interestingly, in kidneys from a heart beating donor that had a 19-hour cold storage period and subsequent 2-hour

HMP had less lactate dehydrogenase (LDH) and lipid peroxidation than kidneys with

21-hour HMP. No significant differences in renal resistance and creatinine clearance rates were detected, indicating that a shorter period of perfusion right before reperfusion is comparable to HMP during the whole preservation period (Gallinat, et al., 2012). It is unclear whether HMP is necessary during the entire duration of storage, if it has benefits for organs from all donor types, and if additional oxygen supplementation is needed.

A combination of PSF and HMP have been investigated, either through oxygenated HMP or HMP followed by PSF. Kidneys with minimal warm ischemia were stored with HMP or oxygenated HMP for 21 hours and then autotransplanted. The oxygenated HMP group had lower vascular resistance but elevated free radical mediated injury, and creatinine clearance was better in the HMP group (Gallinat, Paul, et al. 2012). Conversely, kidneys exposed to 30 minutes of warm ischemia and preserved with HMP, 21% oxygen HMP and 100% oxygen HMP for 21 hours show significantly increased blood perfusion and creatinine clearance and less biomarkers of cell damage and tubular injury in the order of 100% oxygen 21% oxygen and standard HMP (Hoyer, et al., 2014). The benefits of oxygenation with HMP was seen long term, with less interstitial fibrosis and chronic inflammation at 3-month after

236 transplantation (Thuillier, et al., 2013). In kidneys preserved with 21 hours of HMP followed by 90 minutes of retrograde PSF, a two-fold increase creatinine and urea clearances were detected. The retrograde PSF group also led to higher renal vascular flow rates, enhanced urine output better structural integrity (Minor, Efferz and Lüer

2012). These studies suggest the benefits of adding oxygenation to machine perfusion, especially for organs that come from a donation after cardiac model with a certain amount of ischemic exposure.

One of the major difficulties and limitations of our pilot study was the malfunction of the LifePort. The time it took to set up the LifePort was substantial, and we experienced many difficulties with this prototype from leaky cassettes to bubble errors that would interrupt the perfusion. The perfusion time for these organs ranged from 16-24 hours because the LifePort would sporadically stop functioning overnight. The technical errors we experienced were different for each experiment, and using this perfusion device proved to be a complicated to implement with a lot of troubleshooting for its successful application. With that said, our lab has an older model of the LifePort and these technical problems may not persist if the most updated machine is used. Perhaps the most promising information we gathered from this experiment is that the characteristics of kidneys treated with persufflation were comparable to the kidneys that were treated with the current state of the art method of preservation.

Further studies are needed to comprehensively compare PSF and HMP for organ preservation, and to identify the range of ischemic times in which they would be most efficacious. While we found little advantages or differences in PSF and HMP

237 due to blatant limitations to this pilot study, it laid some groundwork for future studies that compares PSF, HMP and a combination of both. Ultimately, the full potential of PSF and HMP on ischemically damaged kidneys will need to be assessed by a large study using equipment that is in perfect operating conditions.

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Chapter 7

Pre-clinical Porcine Kidney Allotransplant

239

Introduction

Organ transplantation is a heterogenous, multidisciplinary field that strives to meet the current demand for organs by increasing organ accessibility and improving transplant outcomes. Many new strategies are being adopted to advance the science behind organ transplant and implement new policies that can synergistically improve the number and quality of organs. These endeavors can be generally divided into three categories that are pertinent to changing policies, increasing the number of live donations and increasing the deceased donor pool of organs.

The first category is changing the policies for organ donation and allocation to increase the number of registered donors. One option is the opt-out donor policy that presumes everyone by default is a consenting donor until they choose to opt out.

Additionally, interventions aimed at increasing donor registrations in the US and worldwide are utilizing online registries to facilitate enrollment (Kennedy, et al.,

2015). In 2013, the Organ Procurement and Transplantation Network in the United

States approved a new deceased donor kidney allocation policy using the kidney donor profile index (KDPI). KDPI is a scoring system that combines a variety of donor factors into a single number that predicts the postoperative graft survival and function. Instead of using the time on the waiting list as the main determinant of receiving an organ, the OPTN created a new system that would allocate the top donor kidneys to transplant candidates that are projected to have the best graft survival

(Hippen, Thistlethwaite, & Ross, 2011). The donor factors include age, weight, height, race, history of hypertension, diabetes and serum creatinine (Organ Procurement &

Transplantation Network, n.d.). Lower KDPI scores are associated with longer

240 estimated function, while higher KDPI scores are associated with shorter estimated function (Israni, et al., 2014). KDPI of 0-20% on average function for 11 years, 21-

85% function for 9 years without any clinical significance between these scores on this wide range, and any score over 85% function for approximately 5.5 years (Organ

Procurement & Transplantation Network, n.d.; Gupta, Chen, & Kaplan, 2014).

The second category is focused on increasing the number of live donations.

These discussions are more controversial as they center on crafting a legal system to give monetary benefits and financial incentives for healthy donors to donate a kidney.

Creating a market that uses commercial transactions for organ exchange raises ethical and legal questions, especially that of taking advantage of people that are financially vulnerable and the potential for coercion into selling one’s organs (Gill, et al., 2014; Delmonico, et al., 2002). On a more technical aspect, current surgical techniques are being polished to not only optimize the surgical process, but also to reduce the risk donors would absorb by donating their organs. New surgical techniques include single-port nephrectomies (Leeser, Wysock, Gimenez, Kapur, &

Del Pizzo, 2011; Raman, Bensalah, Bagrodia, Stern, & Cadeddu, 2007) and transvaginal extraction of the kidney (Gurluler, Berber, Cakir, & Gurkan, 2014) to reduce morbidity, decrease hospital stay and to expedite the recovery process so that the donors can resume their normal daily functions.

The third category is increasing the organ pool is by enabling the utilization of the organs that are currently being discarded, essentially capitalizing on the already existing pool of organs. Manipulations on the deceased donor organs can occur during three therapeutic windows of before, during and after the ischemic event. Ischemic

241 preconditioning and postconditioning have been investigated to mitigate the adverse effects of oxygen starvation and subsequent reoxygenation of the organ in many pre- clinical and clinical studies.

On the donor level, regional normothermic circulation and ischemic preconditioning are being tested in situ. Maintaining a level of physiological blood flow to the abdominal organs using extra-corporeal membrane oxygenation (ECMO) at body temperature has been used to alleviate warm ischemia the organs experience at withdrawal of care and organ procurement (Oniscu, et al., 2014). This procedure was applied without noticeable short term aversive effects, and it managed to lower the ischemic time and increase the use of donation after cardiac death (DCD) organs

(Magliocca, et al., 2005; Gravel, et al., 2004; Hoogland, Snoeijs, & van Heurn, 2010;

Zhao, et al., 2003). Ischemic preconditioning is another approach that primes the organs to the prolonged cessation of blood supply by exposing them to repeated, transient periods of ischemia. This method manipulates the tissue’s inherent adaptation to stress and strengthens these defense mechanisms by building resistance to protect the organ from the subsequent, inevitable ischemia reperfusion injury. This concept was first introduced and tested in the myocardium. Brief, repetitive 5-minute occlusions followed by a 40-minute occlusion time resulted in a smaller infarct size in canine hearts compared to hearts without preconditioning

(Murry, Jennings, & Reimer, 1986). This method reduced neutrophil adherence in coronary vessels, attenuated endothelial-dependent relaxation and reduced apoptosis (Thourani, et al., 1999; Nakamura, et al., 2000). Along with its success in the heart, it has been studied in the liver, kidneys, intestines and brain (Clavien, 2000;

242

Zimmerman, et al., 2011; Ambros, Herrero-Fresneda, Borau, & Boira, 2007; Barone, et al., 1998; Hotter, et al., 1996). Literature demonstrates the benefits of preconditioning, especially through their mechanisms involving elevated heat shock proteins (Marber, Latchman, Walker, & Yellon, 1993; Kume, et al., 1996), adenosine and nitric oxide production for vasodilation (Jaeschke, 2003; Peralta, et al., 1998;

Peralta, et al., 1997; Nakano, Liu, Heusch, Downey, & Cohen, 2000), protein kinase C

(PKC) (Ping, et al., 1997; Mitchell, et al., 1995; Speechly-Dick, Mocanu, & Yellon, 1994) and endothelin (Maczewski & Beresewicz, 2000; Wang, Gallagher, Downey, & Cohen,

1996). Unfortunately, this is not a feasible technique that can be used on donors.

On the recipient level, the application of ischemic postconditioning can limit ischemia reperfusion injury. While organ preconditioning elicits protective mechanisms that lessen the degree of damage incurred after the transplant surgery, it is not a feasible technique that can be used on human donors. Conversely, ischemic postconditioning exposes the organ to a series of short bursts of ischemia at the onset of reperfusion in the recipient (Vinten-Johansen, et al., 2005). These organs phenotypically express less apoptosis, smaller infarct size and more functional vascular physiology with less tissue edema and lipid peroxidation (Vinten-Johansen

& Shi, 2011; Zhao, Sapolsky, & Steinberg, 2006; Vinten-Johansen, et al., 2005; Zhao, et al., 2003). In a clinical study involving patients submitted for coronary angioplasty, one group was subjected to the standard procedure and the other was postconditioned. After stenting, the postconditioning group was exposed to 1 minute reflow followed by 4 episodes of 1-minute perfusion and 1-minute occlusion of the angioplasty balloon (Staat, et al., 2005). Postconditioned patients did not experience

243 any adverse side effects and presented with less infarct size and more blood flow

(Staat, et al., 2005; Laskey, 2005). Postconditioning and preconditioning counteract ischemia reperfusion injuries through multiple mechanisms, and many of these are similar (Vinten-Johansen, et al., 2005). Postconditioning seem to have specific permutations for the length of time of ischemia, the duration and number of ischemic bursts, and the duration of reperfusion (Manintveld, et al., 2007; Ovize, et al., 2010).

Ischemic postconditioning activates ATP-sensitive K+ channels and reperfusion injury salvage kinase (RISK) pathways to prevent the opening of mitochondrial permeability transition pore, preserves mitochondrial function, produces nitric oxide and prevents apoptosis (Lim, Davidson, Hausenloy, & Yellon, 2007; Penna, Mancardi,

Raimondo, Geuna, & Pagliaro, 2008; Hausenloy & Yellon, 2009). Furthermore, pharmacological interventions can be taken within minutes of reperfusion to elicit protective effects that are comparable to ischemic postconditioning. Drugs such as adenosine, statins and anesthetics that activate the RISK pathway which subsequently inhibits the mitochondrial permeability transition pore opening can mimic postconditioning and its protective effects (Meybohm, et al., 2011; Zang, Sun,

& Yu, 2007; Heusch, 2015).

Protective measures to minimize ischemic injury can be taken before organ procurement and after transplantation, but most importantly, during the preservation period. Organ preservation research on isolated organs has expanded considerably since the end of the 20th century. Isolated organs provide a unique opportunity to try a variety of techniques that can enhance preservation during the time it takes to allocate and transport the organs. Cold storage has been improved

244 through the evolution of preservation solutions and refining the composition of these solutions for different organ types and donor types (Belzer and Southard 1988,

Southard and Belzer 1995). Machine perfusion research made a comeback with the wide use of expanded criteria donor and donor after cardiac death organs (Taylor and

Baicu 2010). Hypothermic machine perfusion (HMP) reduces delayed graft function, decrease length of hospital stay and enables the use of these nontraditional organs that fall short of standard organs (C. Moers, et al. 2009, J. M. O'callaghan 2013, Taylor and Baicu 2010, Maathuis, et al. 2007). Because hypothermia is known to cause tissue damage during preservation, temperature has been modified to study subnormothermic and normothermic machine perfusion. These methods, not unlike

HMP, maintained better organ function with minimal injury compared to static cold storage (Bruinsma B. G., 2014; Bruinsma B. G., 2013; Mahboub, et al., 2015; Nicholson

& Hosgood, 2013; Valero, et al., 2000). Alternatively, pharmacological drugs that can prevent thrombosis (Jespersen, 2016), increase vasodilation (Feuerstein & Ruffolo,

1996), and reduce immune and inflammatory response mediated by ischemic tissue damage were successfully tested in isolated organs (Lewis, Köhl, Ma, Devarajan, &

Köhl, 2008; Reilly, Schiller, & Bulkley, 1991; Davis, 2010; Castellano, 2010). In the same line of supplementing the organs with exogenous pharmacological agents, oxygenation of organs has been a topic of interest given that it is theoretically the most straightforward way to mitigate the damage incurred by an anoxic environment during organ preservation. Hyperbaric oxygenation combined with hypothermic storage yielded positive results in various tissue types (Manax, Block, Eyal, Lyons, &

Lillehei, 1965; Rudolf & Mandel, 1967; Yamada, Taguchi, Hirata, Suita, & Yagi, 1995;

245

Bayrakci, 2008). In kidneys with ischemia reperfusion injury, it improved vasodilatory responses, resulted in less reduction of glomerular filtration rate

(Rubinstein, et al., 2008), and preserved kidneys for 24 hours (Ladaga, et al., 1966).

Synthetic oxygen carriers such as perfluorocarbon have been used in the Two Layer

Method (TLM) for cold storage where the organ is placed between two layers of oxygen carriers. While this was beneficial in a small animal model (Maluf, et al., 2006;

Marada, Zacharovova, & Saudek, 2010), TLM was inadequate to fully oxygenate large porcine kidneys (Hosgood, Mohamed, & Nicholson, 2011) and pancreas (Scott, et al.,

2010). Persufflation has been a resurfacing technique that bubbles humidified, gaseous oxygen directly to the organ. While it took a backseat due to the development and dominance of cold solutions and machine perfusion, it is regaining interest for its ability to replenish adenosine triphosphate (ATP) levels, extend preservation time and improve graft function after warm ischemic injury (Kuhn-Regnier, et al., 2000;

Koetting, Lüer, Efferz, Paul, & Minor, 2011; Stegemann & Minor, 2009; Rolles,

Foreman, & Pegg, 1984; Pegg, Foreman, Hunt, & Diaper, 1989; Isselhard, et al., 1972).

It has been wildly successful in pre-clinical liver preservation, especially with the addition of antioxidants (Minor, Saad, et al. 1998, Minor, Klauke and Isselhard 1996,

J. W. Treckmann, et al. 2006).

In our study, we tested the effects of anterograde persufflation for its simplicity, ability to restore ATP levels in the kidneys and its ability to extend the preservation period. Our ex vivo experiments with the porcine model had two main goals. The first goal was to establish whole organ oxygen consumption rate (WOOCR) as a measure of viability and quantifying GFR using dynamic contrast enhanced

246 imaging as a measure of function. These two techniques were the main methods used for non-invasively characterizing organ quality that can potentially predict graft outcomes. We wanted to make headway in organ assessments because currently there is no universal way to systemically characterize organ quality to predict its function in vivo. Developing a protocol for evaluating organs and a range of values that can predict organ function would be an astronomical advancement in efficiently utilizing the existing organs from all types of donors. The second goal was to determine if anterograde persufflation would be a suitable preservation method that can mitigate ischemic and hypothermic damage while conditioning the organ for transplant. We found that WOOCR and imaging are feasible tools for evaluating isolated organs and can differentiate between the two treatment methods in hypothermic and subnormothermic temperatures. While we saw statistically relevant differences in isolated kidneys, it is never a certainty that these differences will have any biological significance in an in vivo model. To investigate whether these differences lent themselves to any biological and functional changes, we proceeded to pre-clinical model of renal allotransplants.

We first attempted renal allotransplants in a rodent model using Lewis rats.

Our goal was to do paired studies by transplanting static cold stored and persufflated kidneys from one donor into two rodent recipients. Unfortunately, all 5 rodents died during practice surgeries and we were not able to complete a successful transplant.

These rodent recipients were all under isoflurane for 4-5 hours with the completion of the venous anastomoses and partial arterial anastomoses when they died. Due to the complications and technical challenges that came with performing

247 microsurgeries, we moved to a porcine pre-clinical model for renal allotransplants.

We established a team with a transplant surgeon, transplant nephrologist and a veterinarian surgeon to attempt a controlled study of comparing cold storage and persufflated kidneys in vivo.

Methods

Domino Surgery- Organ Procurement

Domestic female pigs weighing 35-50 kg were used from a local farm in

Wilcox, Arizona. Animal care procedures were in accordance with university policies.

The donor pig was pre-medicated with intramuscular Telazol, and intubated. General anesthesia was induced and maintained with isoflurane. Oxygen was administered and heparin was injected through the ear catheter. A midline incision was done to access the peritoneal cavity. The terminal abdominal aorta was cannulated and suprarenal aorta was cross-clamped. The ureter was dissected and the kidneys were separated by longitudinally splitting the aorta and inferior vena cava. The isolated kidney was flushed with 400 mL of heparinized saline and SPS-1. The harvested kidney was stored at 4°C in SPS-1 for 24 hours. Once the kidney was procured, the donor pig was euthanized. For the first attempt at allotransplants, we decided to do a domino procedure with 2 donors and 2 recipients, summarized in Figure 49. The first donor kidney was harvested and cold stored for 24 hours and transplanted into the first recipient. The recipient went through bilateral native nephrectomy at the time of transplant. One of these kidneys was persufflated for 24 hours and transplanted into the second recipient, and bilateral native nephrectomy was performed on the

248 second recipient. Because we first wanted to establish that the surgeries could be done successfully with survival of the pigs for 7 days, we opted out of using a donation after cardiac model to ensure that the kidneys would be exposed to a minimum level of warm ischemia.

Figure 49: timeline of the domino transplant using a donation after brain death model. This model was used to facilitate establishing the transplant model, and to test the feasibility and logistics of doing an allotransplant. Kidney procured from porcine donor 1 was preserved using static cold storage (SCS) for 24 hours, and transplanted into recipient 1. Native kidneys from recipient 1 was procured and one was preserved using persufflation (PSF) for 24 hours. The PSF kidney was transplanted into recipient 2. Post-operative follow-up lasted 7 days. Blood and urine samples were collected at days 1, 3 and 7 with the necropsy done on day 7.

Paired Surgery- Organ Procurement

Domestic female pigs weighing 35-50 kg were used from a local farm in

Wilcox, Arizona. Animal care procedures were in accordance with university policies.

The donor pig was anesthetized under isoflurane and heparin was circulated for 5 minutes to prevent thrombosis during the procurement. They were euthanized, and flushed with 4 liters of room temperature, heparinized saline through the abdominal aorta. Kidneys were exposed to 30 minutes of warm ischemia time and cannulated at the renal artery and vein using sterile luer locks (Cole-Parmer). Isolated kidneys were

249 flushed with 400 mL of 4°C heparinized saline using gravity followed by a secondary flush with SPS-1 solution (Organ Recovery System). Isolated kidneys were treated with either static cold storage (SCS) or persufflation (PSF) for 24 hours at 4°C in a container with SPS-1. The persufflator (Giner, Inc., Newton, MA) delivered electrochemically generated, humidified, 40% oxygen mixed with atmospheric air with a manifold pressure of 40 mmHg to generate a flow rate of 60 mL/min. Tubing from the persufflator was attached to the renal artery and leaks around the vessel were ligated to ensure maximum amount of gas perfusion. For the second allotransplant, we used a DCD model and the pair of kidneys from a single donor was treated, preserved and transplanted into two separate recipients as indicated in the figure below.

Figure 50: timeline of the paired allotransplant using a donation after cardiac death model. A pair of kidneys from the porcine donor were preserved using static cold storage (SCS) or persufflation (PSF). After 24 hours of preservation, kidneys were transplanted into their respective recipients. Post-operative follow-up lasted 7 days. Blood and urine samples were collected at days 1, 3 and 7 with the necropsy done on day 7.

Renal Allotransplant

After the 24-hour preservation period, recipient pigs were intubated after intravenous anesthesia and maintained on a respirator with oxygen. The peritoneal

250 cavity was accessed through a midline incision. Heparin was administered and the abdominal aorta, renal artery and vein were dissected and a nephrectomy was performed. The preserved kidney was placed at the original kidney site and vascular anastomoses were performed end-to-side renal vein to vena cava and end-to-end renal artery to the native renal artery. Reperfusion was established by release of the venous and arterial clamps, shown in Figure 51. Ureteroureterostomy was performed and the native contralateral kidney was removed. The muscle layer, subcutaneous layer and the skin were closed and the pig was extubated.

Figure 51: Pictures of the persufflated kidney with complete vascular anastomoses from the domino allotransplant. A) shows the kidney before perfusion and B) shows the kidney within a minute of unclamping and blood reperfusion. Immunosuppression and Anticoagulants

Recipient pigs received an immunosuppression regimen and antibiotics prior to and after the allotransplant to decrease chances of rejection and infection.

Anticoagulants were given to prevent thrombosis during and after the surgery to minimize secondary complications. The following drugs were administered:

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Drug Name Dose Frequency Duration

Methotrexate 2mg/kg Twice a week 1-week pre- and post-transplant

Cyclosporine 15mg/kg Once a day 1-week pre- and post-transplant

Methylprednisolone 125mg/kg Once a day 3 days post-transplant

Penicillin 40,000 U/kg Once a day 3 days pre-transplant

Amoxycillin 20 mg/kg Once a day 3-5 days post-transplant

Metoclopramide 0.15 mg/kg Once Day of transplant

Heparin 50 U/kg Once 1-day post-transplant

Warfarin 5-10 mg Daily 1-week post-transplant

Table 35: Immunosuppressive drugs, antibiotics and anticoagulants.

Blood and Urine Collection

Blood samples were obtained by IV ear catheter for a standard blood workup with blood cell count, creatinine levels, blood urea nitrogen and electrolytes. 2-5 mL urine samples were obtained by either voluntary voiding, or collected with a syringe guided by ultrasound at the time of blood collection. Unless euthanasia was suggested by the university animal care veterinarians to protect the wellbeing of the animal, the recipient pigs were kept for a maximum of 7 days. On day 7 pigs were euthanized and the transplanted organs were collected for assessments

Histology

Wedge biopsies were taken from the kidneys after the preservation period.

They were fixed with 4% paraformaldehyde overnight, dehydrated, processed and paraffin wax embedded. Biopsies were sectioned at 5 microns and stained with hematoxylin and eosin (Sigma-Aldrich). The veterinary pathologist evaluated gross

252 renal morphology and hypoxic and ischemia reperfusion damage using light microscopy.

Results

Domino Allotransplant

The SCS kidney recipient from the domino surgery was euthanized the day after surgery. The ureteroureterostomy had come undone and per our IACUC protocol we could not perform another major surgery to reconnect the ureters.

Histological analysis showed minimal tubular damage in the explanted SCS kidney. It presented with protein in the glomerular space and tubules (Figure 52). The PSF kidney recipient on the other hand survived for the full 7 days. On the day of the transplant surgery, it had normal creatinine levels at 1.1 mg/dL and a slightly lower

BUN of 7.9 mg/dL. On postoperative days 1, 3, and 7, serum creatinine levels were

5.6, 4.1 and 1.9 mg/dL (Figure 53A) while BUN was 58.8, 45.0 and 16.3 mg/dL

(Figure 53B). By day 7, both parameters were within normal range of 1.0-2.7 mg/dL for creatinine and 10-30 mg/dL for BUN. The PSF recipient started producing urine at postoperative day 2 with no physical or clinical signs of discomfort. As delineated in Figure 7, The PSF kidney showed signs of tubular damage with some proximal tubules lacking the brush border that functions to increase surface area of the tubular epithelial cells to facilitate their electrolyte resorption. In contrast, reparative actions through flattened epithelium with visible squamous were also present, indicating renal injury repair (Figure 54).

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Figure 52: H & E staining on control and postoperative day 1 kidney biopsies. The top panel (A, B, C) show different magnifications of control kidneys while the bottom panel (D, E, F) show sections from an explanted kidney on the 1st day after transplantation from the domino allotransplant. Arrows indicate proteinaceous casts in the tubules and glomerular space (n=1).

Figure 53: Levels of serum creatinine and blood urea nitrogen (BUN) collected from the persufflated kidney recipient. Blood was collected at postoperative days 1, 3 and 7 (n=1). A) shows a gradual decrease in serum creatinine levels, with the arrow indicating serum creatinine in the normal range of 1.0-2.7 mg/dL. B) shows a gradual decrease in BUN levels, with the arrow indicating BUN in the normal range of 10-30 mg/dL.

254

Figure 54: H & E staining on control and postoperative day 7 kidney biopsies from the domino allotransplant. The top panel (A, B, C) show different magnifications of control kidneys while the bottom panel (D, E, F) show sections from an explanted kidney on the 7th day after transplantation (n=1).

Paired Allotransplant

The SCS kidney recipient from the paired allotransplant did not produce urine for 3 days after the surgery and the incision site was edematous and warm to touch.

Ultrasound showed an empty bladder and per recommendation of the university animal care veterinarians, we euthanized the pig on postoperative day 4. The PSF kidney recipient was noted to experience discomfort in her hind legs since postoperative day 1. She stood on her toes or remained immobile most of the time.

She started producing urine but she refused to move and did not eat or drink few days after the surgery. Per recommendation of the university animal care veterinarians, we euthanized the pig on postoperative day 3.

On the day of the transplant surgery, SCS recipient had creatinine level of 0.6 mg/dL and BUN of 7.1 and the PSF recipient had 0.8 mg/dL and 10.7 mg/dL. On

255 postoperative days 1 and 3, serum creatinine levels were 7.0 mg/dL and 16.6 mg/dL for the SCS recipient and 5.3 mg/dL 11.0 mg/dL for the PSF recipient (Figure 55A).

BUN levels were 58.8 mg/dL and 120.6 mg/dL for the SCS recipient and 54.8 mg/dL and 122.6 mg/dL for the PSF recipient at day 1 and 3 (Figure 55B).

Histological analysis for the SCS recipient in Figure 56 display diffuse hemorrhage and necrosis with widespread pyknotic nuclei. It also had mineralized tubules and inflammation. These observations were consistent with vascular compromise and hypoxia. The PSF kidney shown in Figure 57 in appearance had slight, mild hemorrhage with focal inflammation. The tissue showed some proteinaceous cast and regeneration of renal tubules. The pathologist stated that he would have expected normal renal function in a week had the pig survived the full 7 days.

Figure 55: serum creatinine and blood urea nitrogen of static cold storage and persufflated kidney recipients. Blood samples were collected at the day of transplant (day 0) and postoperative day 1, 3 of the static cold storage and persufflated kidney recipients of the paired allotransplant (n=1). A) reflects a gradual increase in serum creatinine and B) reflects a gradual increase in BUN.

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Figure 56: Light microscopy images of H&E-stained tissue samples from explanted static cold storage kidneys. Samples were collected at postoperative day 4 of the paired allotransplant. Top two images (A, B) are taken at 5x, bottom left (C) at 10x and bottom right (D) at 20x (n=1).

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Figure 57: microscopy images of H&E-stained tissue samples from explanted persufflated kidneys. Samples were collected at postoperative day 3 of the paired allotransplant. Top two images (A, B) are taken at 5x, bottom left (C) at 10x and bottom right (D) at 20x (n=1).

Discussion

It is difficult to make conclusions from this pilot study due to the multiple variables present in these series of allotransplants. On a logistics level, we have learned that these surgeries are feasible, and that we have a team in place comprised of experts in organ quality assessments, transplant surgery and nephrology that can complete these studies. While the premature euthanasia of the domino allotransplant

SCS kidney recipient was disappointing, the survival of the PSF kidney recipient from the domino surgery is promising. The presence of proteins in the glomeruli of the SCS kidney suggests abnormal filtration across the glomerular basement membrane.

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Proteinuria may have been observed had the recipient survived through the full 7 days of the experiment. Several studies demonstrated a correlation between glomerular proteinuria and renal failure, and it is an indication of interstitial inflammation (Burton, 1996; Abbate, Zoja, & Remuzzi, 2006). The DCD model was not implemented for this surgery and the kidney it started functioning after 24 hours of preservation with normal creatinine levels by day 7. This may largely be attributed to the lack of 30 minutes of warm ischemia time, which is associated with adverse long- term graft survival after kidney transplantation (K. K. Tennankore, et al. 2016).

In the paired allotransplant, serum creatinine levels were higher in the SCS kidney recipient for postoperative day 1 and 3, and it increased by a greater degree than the PSF kidney. Minimal differences were observed in the BUN levels between the SCS and PSF kidney recipients. The beginning stages of tubular regeneration observed in the PSF kidneys suggest that given this recipient survived for another week or so, we may have observed the creatinine and BUN levels stabilizing back to the normal range, barring any other complications.

The SCS kidney recipient did not produce urine at 3 days after transplant, and biopsies show hemorrhage and wide spread tubular necrosis and inflammation.

Recent clinical study demonstrated that different cell death pathways are present during the different stages of kidney transplant. It found that necrosis typically occurs during extended cold storage in renal allografts, and apoptosis occurs soon after reperfusion (Salahudeen, Huang, Joshi, Moore, & Jenkins, 2003). These processes are crucial in tubular repair and function that can determine the functional outcomes of transplanted kidneys.

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Acute tubular necrosis is frequently seen in injured kidneys from extended cold storage (Salahudeen, Joshi and Jenkins, Apoptosis versus necrosis during cold storage and rewarming of human renal proximal tubular cells 2001). Necrosis causes cytosolic constituents to leak into the extracellular space and provoke an inflammatory response at the time of reperfusion (Proskuryakov, Konoplyannikov, &

Gabai, 2003). Cell and interstitial swelling, calcium overload, reactive oxygen species

(ROS) generation, mitochondrial swelling, lactic acidosis, altered lysosomal and plasma membrane rupture experienced from ischemic injury contribute to necrotic cell death (Golstein & Kroemer, 2007). With reperfusion, there is a robust burst of

ROS that overwhelms the antioxidant mechanisms such as glutathione and superoxide dismutase that are typically repressed under cold storage (Shoskes and

Halloran 1996). Furthermore, chemokines and cytokines released from the injured cells and increased expression of adhesion molecules induce an immune response that further exacerbates and potentiates renal damage (Shoskes and Halloran 1996).

The damaged epithelium can obstruct tubules, and the interactions of the endothelium and leukocytes can obstruct the capillary beds to restrict blood flow and cause vascular congestion (Egidi & Giannese, 2017).

Acute tubular necrosis seen in renal sections can manifest in the form of delayed graft function (DGF) in the recipient, and is associated with acute cellular rejection (ACR) or antibody-mediated rejection (AMR) (Egidi & Giannese, 2017). The more functional definition of DGF is based on the inability of the graft to decrease serum creatinine level by 10% for 3 consecutive days during the first week after surgery (Sharif & Borrows, 2013). This is seen in the SCS kidney recipient with

260 creatinine increasing from 7 mg/dL to 16.6 mg/dL between postoperative day 1 through 3. However, this is not unexpected since DGF affects 25 - 50% of deceased donor kidney recipients, and this increases risk for acute rejection episodes and chronic renal dysfunction (Miglinas, Supranaviciene, Mateikaite, Skebas, & Kubiliene,

2013).

In conjunction with acute tubular necrosis and delayed graft function, there are surgical complications that can compromise graft perfusion and function.

Bleeding is the most common surgical complication, and postoperative surgical-site hemorrhage (SSH) can occur within the first few days after kidney transplantation

(Marietta, Facchini, Pedrazzi, Busani, & Torelli, 2006). Vascular anastomoses and dilation of vessels on the renal hilum at the onset of reperfusion can be a source of hemorrhage (Hachem, et al., 2017). Additionally, hematoma in the kidney graft area is usually caused by bleeding from tiny vessels that are constricted during the surgical procedure that start to bleed during reperfusion (Pawlicki, Cierpka, Krol, & Ziaja,

2011). Thrombosis in the renal vasculature is another potential complication that can result in graft loss. It is typically caused by torsion of the vessels, narrow anastomosis, injury to the vascular endothelium and compression by hematomas (Humar & Matas,

2005). Vascular injury and the altered endothelium lose regulatory function vascular tone and vasopermeability. Moreover, the activation of coagulation pathways by the damaged endothelium and the loss of nitric oxide production can cause impairment of renal perfusion, continued renal hypoxia (Sutton, Fisher and Molitoris 2002).

The results from this pilot study shows lower creatinine levels and less renal damage in the PSF kidney recipient, and a potential benefit to using PSF over SCS in

261 ischemically damaged kidneys. the limitations to this study are without a doubt, the number of animals used and euthanasia of the animals for reasons other than graft failure. The behavioral signs of hindlimb discomfort from the PSF kidney recipient may be caused by distal aorta clamping, which could be a source of nerve ischemia.

The protocol for deciding on animal euthanasia need to be a point of discussion as we had dissent amongst the veterinarians with regards to pain management, graft function and animal wellbeing. A technical improvement that can be made in the future is using more heparin to prevent thrombosis. It is difficult to gauge how the infarction happened in the cold storage kidney of the paired allotransplants, and it is premature to say that it was because of the preservation method. To rule out thrombosis in future studies, it would be prudent to increase anticoagulant doses within the safe range of use.

In conclusion, more transplants using this pre-clinical model should be completed to fully understand the potential and effects of PSF on graft function.

Future studies should also measure WOOCR and GFR before transplant so that these values can be correlated with renal function in vivo. If PSF is beneficial in an in vivo model, it can provide a relatively simple method of mitigating ischemic damage that occurs at all stages of organ procurement, allocation and transplantation. It is also more time and cost effective than HMP as it does not require extensive troubleshooting or expensive ancillary accessories. In conclusion, PSF has the potential to treat a wide range of ischemic exposure from a variety of donor types with numerous applications. Furthermore, it can diminish the risk of delayed graft function secondary to ischemic damage, and mitigate the ongoing problem we have

262 with organ shortage. Nonetheless, more robust preclinical studies need to be performed to confirm the efficacy and efficiency of PSF.

263

Chapter 8

Summary and Interpretations of Results

264

The presented experiments contribute to developing novel methods for organ quality assessments, and testing anterograde persufflation (PSF) as an alternative preservation technique to the traditional static cold storage (SCS) and hypothermic machine perfusion. Moreover, these findings may explain the effects of oxygenating kidneys during the preservation period, and how that might translate to renal function after an allotransplant setting.

Comparing kidneys that were persufflated (PSF) or cold stored (SCS) at hypothermic temperatures: In these experiments, we assessed and compared the effects of PSF to SCS, which is the standard method for organ preservation. We used the techniques developed in house to measure whole organ oxygen consumption rate

(WOOCR) and glomerular filtration rate (GFR) by using dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) in isolated kidneys. In these studies, we could confirm the technical feasibility of these quality assessment tools. These methods were used to discern the differences between the PSF and SCS kidneys, and demonstrated that PSF treated kidneys had significantly higher WOOCR and GFR.

Lactate levels indicated more anaerobic metabolism in the SCS kidneys and histological analyses showed no significant differences in morphology and the extent of damage between the two treatment groups. Increase in oxygen consumption rate in the PSF group may be attributed to cellular machinery that experienced a lesser degree of ischemic damage from oxygenation during the 24 hours of hypothermic preservation. Furthermore, the PSF kidneys had WOOCR levels higher than control organs without any cold ischemia time. This can be extrapolated as the exogenous oxygen improving organ viability during the storage period. Increased GFR may be

265 explained by maintenance of vascular patency and less damaged tubules from the gas perfusion of oxygen. From this data, we inferred that the PSF kidneys were more metabolically active with more passive filtration function than SCS kidneys due to less damage sustained by the renal tissue during preservation.

Comparing kidneys that were persufflated or cold stored at subnormothermic temperatures: In these set of experiments, we preserved the kidneys with either PSF or SCS. After the preservation period, they were rewarmed to subnormothermic temperatures and assessed for WOOCR and GFR. Assessing the organs at a higher temperature was necessary since hypothermic temperatures are not conducive to properly gauging physiological parameters. Similar results were obtained with significantly increased WOOCR and GFR in the PSF kidneys. When we compared hypothermic WOOCR to subnormothermic WOOCR, we see an approximate 2-fold increase from PSF kidneys (132.01 ± 21.77 nmol/min*g to 281.8

± 32.1 nmol/min*g) and approximately 1.7-fold increase in SCS kidneys (132.01 ±

21.77 nmol/min*g to 225.5 ± 43.7 nmol/min*g), summarized in Figure 58.

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Figure 58: WOOCR measurements at hypothermic and subnormothermic temperatures for static cold storage and persufflated kidneys.

The GFR values on the other hand, decreased with rewarmed temperature for both treatment groups as shown in Figure 59. This is an interesting phenomenon since we expected the filtration activity to increase with vasodilation in the renal vasculature at subnormothermic temperatures. We speculate that these changes may be related to renal hemodynamics and not enough hydrostatic pressure from the perfusion during imaging. The renal perfusion pressure can influence sodium excretion and urine flow (Khraibi, Haas, & Knox, 1989; Hall, et al., 1977), and in future studies our circuit should be modified to control for pressure as opposed to only controlling for flow rates. Moreover, agonal vasospasms occur in ischemically damaged allografts during isolated perfusion, and they can influence the renal filtration rate and result in inadequate perfusion (Pryor, Keaveny, Reed, & Belzer,

1971; D'Alessandro, 1989). Another factor to consider is using solutions that have a

267 similar viscosity to blood. The research grade SPS-1 solutions that were used for these experiments were sticky and viscous, which is an indisputable element that can influence the passive filtration rate of gadolinium. These factors can influence GFR and give measurements that are not physiologically relevant.

Figure 59: GFR measurements at hypothermic and subnormothermic temperatures for static cold storage and persufflated kidneys.

268

Figure 60: Histological analysis at hypothermic and subnormothermic temperatures for static cold storage and persufflated kidneys.

One study that performed histological evaluation on kidneys with varying degrees of warm ischemia time (15 min to 120 min) after 3 hours of reperfusion show an average range from mild to moderate injury (Hosgood, Shah, Patel, & Nicholson,

2015). These conditions without a doubt are very different from our samples with 30 minutes of WIT, 24 hours of CIT and no reperfusion, but our scoring system revealed an average score of minimal to mild abnormalities in renal tubules and vasculature.

This suggests that longer warm ischemia times and normothermic reperfusion may have a bigger impact on histological changes than an extended exposure to cold ischemia time. Comparatively, while there are no significant differences between the two treatment groups, we saw an overall increase in morphological abnormalities with rewarming (Figure 60).

269

There is extensive literature on the detrimental effects of cold ischemia time during preservation (Belzer & Southard, 1998; Southard & Belzer, 1995), and its negative impact on graft function (Ojo, Wolfe, et al. 1997, A. K. Salahudeen 2004). It is difficult to gauge how the level of damage observed on biopsies correlate with renal functional changes as there are no standard method or scoring system for ischemic damage. It is a subjective, semi-quantitative measure that can provide insight on renal morphology and abnormalities, but cannot be a standalone clinical predictor of graft health and survival outcomes. In our studies we saw lower GFRs in the rewarmed kidneys and concordant increase in renal tubular damage in the subnormothermic temperatures. Interestingly, while there are significant differences in GFR between the rewarmed SCS and PSF kidneys, these differences are not reflected in structural changes.

Comparing kidneys that preserved with persufflation or hypothermic machine perfusion (HMP): In this pilot study, we preserved the kidneys with either

PSF or HMP. After the preservation period, they were assessed for WOOCR, GFR, histological analysis and global gene expression. There were no significant differences between the two groups in all parameters. Undeniably, the biggest limitation for this study were inconsistent HMP times for kidneys that were preserved on the LifePort, and the small sample size. Given the limitations of this study, we were unable to make strict comparisons between the groups and find the results largely inconclusive. However, it is interesting to note that despite the HMP kidneys being exposed to a mix of perfusion and static cold storage, their viability and functional measurements were comparable to PSF kidneys. Further studies that

270 determine the duration in which maximum levels of organ reconditioning and repair can be achieved with HMP and PSF would be insightful on the management of organ preservation. For example, it would be useful to know if a couple of hours of HMP or

PSF would have similar effects as using these techniques for 12-24 hours to streamline the preservation process to be as clinically and pragmatically effective as possible.

Genomic analyses: In these experiments, biopsies that were collected from the first two studies were analyzed using microarrays. There were no differentially expressed genes that had a p < 0.05, so genes with p < 0.101 were analyzed, all of which were from samples collected at subnormothermic temperatures. This was perhaps the most insight we had to the pathways that were involved in the ways persufflation affects kidneys during preservation period. We identified many enriched KEGG pathways that were pertinent to autophagy and apoptosis, which is consistent with ischemic damage (Price, Safirstein and Megyesi 2009). Shortly after the injury, tubular cells are known to enter the cell cycle. Upregulation of these pathways may indicate proliferation of the formerly quiescent tubular cells in response to cell stress and death (J. V. Bonventre 2003). This is seen in combination with enrichment in inflammatory pathways that are most likely activated from the dying cells (Chen C. J., 2007), and tissue remodeling through the upregulation of Actin

Beta (ACTB), noncanonical Wnt signaling pathway and the cellular junctions that are important in maintaining cell polarity and structure (Hartsock & Nelson, 2008;

Widelitz, 2005; Reya & Clevers, 2005). The most interesting and critical signaling genes revealed by NetworkAnalyst were HSP and Hnf that can exert protective effects

271 on damaged tissue (S. Faguer, et al. 2013, G. R.-R. Budas 2010). These results likely reflect the effects of oxygenation and subsequent prevention of complete energy depletion that allows for the organs to afford energy expenditure on these responses stimulated by ischemia. The variability we saw between the pigs is an innate quality that cannot be controlled, but technical aspects of collecting samples can be streamlined. In future studies, sections from the cortex and medulla should be dissected by a pathologist to ensure that similar amounts of cortical and medullary areas are being represented per sample, per kidney. As mentioned earlier, genes of interest identified from this experiment need to be validated using quantitative real- time PCR.

Paired renal allotransplant: in this study, we were able to compare the effects of PSF and SCS in a pre-clinical porcine model. While we have n=1 per group, the preliminary data we collected showed remarkable differences between the two treatment groups. The SCS recipient presented with delayed graft function with no urine production for 3 days. The explanted kidney showed severe signs of widespread hemorrhage, necrosis, diffuse inflammation that indicated acute kidney injury. The

PSF recipient on the other hand started producing urine at day 2. Explanted kidney in appearance had diffuse loss of brush border cells, flattened epithelium and mild inflammation indicative of ischemia reperfusion injury and tubular regeneration.

This project is in its primitive stages, and it’s too early to make any conclusions about the effects of PSF in vivo. Both recipients did not make it to the full 7 days of the study, and the PSF recipient was euthanized for reasons other than its renal function. Its hind limb discomfort may have been caused by surgical complications such as nerve

272 ischemia or thrombosis. Future studies need to measure WOOCR and GFR sterilely before the transplant and correlate these values with creatinine clearance and GFR measured by DCE-MRI in vivo.

Undoubtedly, the most significant findings of these studies are that alternative methods for organ assessments can be achieved. Oxygen consumption rate of cells and tissue have been measured to study cells (K. K. Papas, C. K. Colton, et al. 2007, K.

K. Papas, M. D. Bellin, et al. 2015) tumors (Dewhirst, Secomb, Ong, Hsu, & Gross , 1994;

Chen, Cairns, Papandreou, Koong, & Denko, 2009; Gatenby & Gillies, 2004), composite tissue (Avgoustiniatos & Colton, 1997; Bakhach, 2009), and this can be applied to whole organs with WOOCR. It is a method that can estimate viability (B. P. Weegman,

V. A. Kirchner, et al. 2010) with the understanding that cellular function is dependent on continuous, efficient usage of oxygen for energy generation that can fuel normal physiological events (Pegg, 1989; Jain, Langham, & Wehrli, 2010). Comparatively, another existing technology, such as dynamic contrast imaging can be applied to quantitatively estimate an important renal functional parameter, GFR (Stevens,

Coresh, Greene, & Levey, 2006). Specifically, it has been established that magnetic resonance imaging (MRI) can measure GFR in patients without the risk of radiation and nephrotoxicity from contrast agents (Annet, Hermoye, et al. 2004), and it’s been studied in kidney donors before and after nephrectomy to evaluate filtration fraction of the kidneys (Cutajar, et al., 2015). These approaches can be implemented, rather than reinventing the wheel for measuring organ quality.

273

Another important finding is the potential for anterograde persufflation.

Similar to oxygen consumption rate and imaging techniques, persufflation is not a new method for organ preservation. However, the increasing use of organs outside the standard donor and the increasing number of patients that require new organs created a renewed interest in this technique. Ischemic damage is one of the major source for dysfunction of the graft after transplantation (Collard and Gelman 2001,

Bonverte and Yang 2011, Perico, Cattaneo, et al., Delayed graft function in kidney transplantation 2004). Some of these effects can be mitigated through providing oxygen to the tissue immediately after the procurement. Aside from the physiological benefits of supplementing the organ with oxygen such as maintenance of ATP levels, extended preservation periods and improvement of primary organ function (T. M.

Suszynski, M. D. Rizzari and W. E. Scott, et al. 2012), it has logistical benefits.

Persufflators are technically less complicated to use and less expensive than using hypothermic machine perfusion that require considerable effort in the setup and maintenance of organ perfusion. Furthermore, there are less extensive regulatory processes involved in implementing medical intervention on isolated organs compared to donors before death, or recipients after transplantation.

The next step for this work is completing porcine renal allotransplants to 1) determine whether WOOCR and GFR acquired through imaging correlates to transplant outcomes and define an acceptable ex vivo WOOCR and GFR value that can be used for prognostic parameters and 2) determine whether the differences seen between the SCS and PSF kidneys translate to functional differences in transplant recipients. While we were able to complete the ex-vivo studies, the critical next step

274 is to determine whether these assessment tools and alternative preservation methods are translatable in pre-clinical models.

Overall, these studies show the feasibility of using WOOCR and DCE-MRI to measure GFR in isolated, preserved porcine kidneys to characterize and quantify organ quality prior to transplant. PSF may represent a useful tool for a safe and improved preservation with restored organ viability and function. Ultimately, the technology described in these studies can be used to resuscitate marginal organs, improve graft outcomes and expand the donor organ pool to enable more lifesaving transplants for patients.

275

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Appendix A

Recent Developments of Persufflation for Organ Preservation

The presented work in this chapter is published in Current Opinion in Organ

Transplantation

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Recent developments in persufflation for organ preservation

Running title: Persufflation for organ preservation

Min, Catherine G.a,b; Papas, Klearchos K.b

aDepartment of Physiology, bDepartment of Surgery, Institute for Cellular

Transplantation, University of Arizona, Tucson, Arizona, USA

Published in: Current Opinion in Organ Transplantation

Issue: Volume 23(3), June 2018, p 330-335

Corresponding Author: Klearchos K. Papas, PhD

Department of Surgery, Institute for Cellular Transplantation

University of Arizona, Medical Research Building

1656 E Mabel Street, Room 122,

Tucson, AZ 85724, USA.

Tel: +1 520 626 4494 fax: +1 520 626 3770 e-mail: [email protected]

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Abstract

Purpose of review: To summarize current literature and recent findings on the potential of humidified oxygenated gas perfusion (persufflation) as an alternative method for improved organ preservation.

Recent findings: Although there are some conflicting data, the majority of the evidence suggests that persufflation, by enhancing oxygenation, can improve preservation and even rescue organs, including organs with prior exposure to warm ischemia. In some cases, persufflation produced better results than hypothermic machine perfusion. The timing of persufflation is of importance; benefits of persufflation appear to increase as the timing of its administration post procurement decreases. This may be particularly true for tissues that are more sensitive to ischemia, such as the pancreas prior to islet isolation. Combining oxygen persufflation with nitric oxide and addition of pulsatile flow may provide further benefits and amplify its effects on improving transplant outcomes.

Summary: Persufflation is a promising, relatively simple, preservation technique that enables improved oxygenation, which provides protection and improvement in the graft condition during preservation and prior to transplantation. More detailed studies are needed to optimize persufflation and evaluate its short and long-term effects in vivo.

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Keywords: organ preservation, organ quality, oxygenation, persufflation, resuscitation

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INTRODUCTION

Organ transplantation is increasingly used for a variety of organ failure and end stage organ diseases, and the demand for donated organs that qualify for transplantation far exceeds the supply [1-3]. The scarcity in organs prompted the use of expanded criteria donors (ECD) and donors of cardiac death (DCD). These organs are more susceptible to stresses associated with procurement and preservation, in particular, long static cold storage (SCS) times, as well as ischemia and ischemia reperfusion injury and would benefit from improved organ preservation techniques [4-7]. A variety of techniques such as hypothermic and normothermic machine perfusion are currently being explored as alternatives to

SCS and have begun to demonstrate promising preclinical and clinical results [8-

17]. Recent studies started incorporating exogenous enhanced oxygen concentration (higher than that found in air - for example, 40 or 95% oxygen) to machine perfusion (OMP) in attempt to manage the tissue injury that occurs with hypoxia and anoxia [18,19]. Enhanced oxygen delivery to organs has been a topic of significant interest, given that it is theoretically the most straightforward way to mitigate the damage incurred by hypoxic and anoxic environments during organ procurement and preservation.

To that end, persufflation, a technique that delivers humidified, gaseous oxygen directly to the organ via its vasculature, which was serendipitously discovered in 1902 [20], is resurfacing for its simplicity, cost-effectiveness, and its superior ability to effectively deliver oxygen to preserved organs. Although it took a backseat because of the development and the dominance of SCS and machine

314 perfusion, it is regaining interest, given reports on its ability to replenish and maintain tissue ATP levels, to decrease oxidative stress and lipid peroxidation and to extend preservation time (e.g. up to 24 h for pancreas and 14 h for hearts [21-

23]) and improve graft function after warm ischemic injury [24-29]. Persufflation has been shown to benefit hearts, livers, kidneys, pancreas and intestines, and these studies are summarized in detail elsewhere [30].

One of the major concerns and a source of reluctance to use persufflation in the clinic is the fear of gas emboli. Reperfusing preserved organs using Krebs solution can be used to clear the gas in the vascular system [21], and no evidence of embolism was cited in hearts preserved using free flow of gaseous oxygen [31].

Another hindrance in the use of this technique is the fear of deleterious effects on the vascular endothelium from pressured gas perfusing high levels of oxygen creating free radical damage. Several studies using hearts have shown that the normal hemodynamic flow and endothelium-derived relaxation are preserved with equal amount of damage between persufflated and SCS hearts [32,33]. In this review, we summarize the recently published studies that focus on persufflation as an option for improved preservation.

ANTEROGRADE VERSUS RETROGRADE PERSUFFLATION

The oxygen gas supply during persufflation can be connected directly to the arterial line for anterograde persufflation (A-PSF). For retrograde persufflation (R-

PSF), oxygen is connected to the venous line in opposite flow of normal physiology, with small perforations introduced to the anterior and posterior side of

315 the organ to allow the gas to escape. A series of studies performed by Isselhard et al. [24,34,35] reported A-PSF kidneys presented with slower recovery of renal function after transplant, whereas the R-PSF kidneys had shorter recovery times to restore normal renal function. In these studies, A-PSF required higher pressures and R-PSF about a two-fold lower pressure to maintain ATP levels [24,34,35]. Use of lower perfusion pressures was recommended, so as to minimize the concerns of damaging the endothelium and resultant vasospasms and vascular homeostasis [36,37]. Some investigators believed bypassing the glomeruli and arterial vasculature using R-PSF could result in less glomerular damage. However, there is a lack of published studies on the effects of persufflation on glomerular tissue morphology [38]. A-PSF has been successfully used in hearts, pancreas and kidneys [22,23,27,39] and the reasons as to why there are conflicting results for A-PSF are not fully elucidated, but they may relate to differences in the quality of organ flushing prior to persufflation, pressures used during persufflation and potentially the level of oxygen used (e.g. 95 or 100 versus 40%). The current persufflation literature primarily tested R-PSF, with the exception of split liver studies that used A-PSF.

LIVER

Significant benefits of persufflation were demonstrated in ischemically damaged rat livers [40-42], followed by pre-clinical allotransplants that showed persufflations beneficial effects of extending ischemic tolerance and resuscitating

DCD organs [43,44]. The encouraging results from these experiments lead to a

316 pilot clinical study [45]. Organs that were used for this pilot study were rejected by multiple transplant centers. These livers were donated by 37-64-year-old donors with warm ischemic damage greater than 20 min and less than 20% steatosis.

Results demonstrated patient survival in all five recipients with good hepatic function after 2 years posttransplant. Liver biopsies taken before and after persufflation revealed increased ATP levels after persufflation, and postoperative biopsies showed undamaged vascular structures [45]. A subsequent proof of concept study was established to test a hypothermic reconditioning protocol for isolated human liver persufflation for 2 h prior to transplant, with the end points of transaminase serum level for 3 days posttransplant to evaluate liver injury, graft and recipient survival hepatic perfusion and graft function, and results are pending

[46].

Recently, Khorsandi et al. [47] published two studies on persufflated human livers. Clinically rejected livers with mild steatosis and moderate/severe steatosis were split with one lobe oxygenated with A-PSF for 2 h and the other serving as an internal cold storage control. They did not find any differences in apoptosis and necrosis between the two groups. With regards to the adenylate levels and energy charge changes in A-PSF livers and control, ATP levels increased in the mild steatosis group whereas it decreased in the moderate/severe steatosis lobes.

Similarly, energy charge significantly increased in the mild and significantly decreased in moderate/severe steatosis group, demonstrating that A-PSF may not exert its beneficial effects on the latter group of livers with more damage.

Subsequently, isolated hepatocyte analyses of cell mitochondrial

317 dehydrogenase activity and cell attachment to characterize function and viability were completed in only the mild steatosis group. Quantification of these activities revealed improvement in both function and viability in the A-PSF liver sections [47].

In a subsequent small cohort study by this group, split livers preserved with A-PSF and cold storage were used to study the mitochondrial composition of Complex 1 that controls the switch from oxidative phosphorylation to glycolysis. They found no changes in the Complex 1 subunit levels with A-PSF treatment, and it was fully assembled with no degradation of associated subunits in livers from donors of brain death, donors of cardiac death with a wide range of warm ischemic times.

However, this study was limited in the small sample size and the lack of functional measurements of Complex 1 [48].

KIDNEY

The early studies for kidney persufflation research were mostly spearheaded by scientists in Germany in the 70s [24,34,35,38], which were followed by rather sporadic bursts of research throughout the past few decades.

There are limited published clinical studies using PSF as a method of kidney preservation. The first documented case is Flatmark's study in which human kidneys were accidentally persufflated during hypothermic machine perfusion [49].

They found that although the renal pressure went up during PSF, this normalized after reperfusion and kidneys treated with up to 2 h of PSF exhibited immediate function after transplantation [49]. It would not be until after another decade that

PSF was further clinically tested. Ten pairs of donor kidneys were used for R-PSF

318 and SCS preservation and transplanted into recipients. Although it is hard to make any wide sweeping claims about persufflation because of the small sample size, the considerable variability between donors, preservation time and recipient health, the authors state that this technically simple method has the potential to improve initial graft function and increase the overall pool of usable organs [50].

In a more recent study, Kalenski et al. [51*] directly compared cold storage,

R-PSF and OMP in isolated DCD porcine kidneys to control kidneys with no ischemic damage after 24 h of preservation. R-PSF and OMP kidneys had higher urine output with lower protein concentrations. Sodium excretion and creatinine clearance in R-PSF and OMP were comparable to nondamaged controls indicating the maintenance of renal function through these preservation methods. OMP had higher intrarenal resistance and lower oxygen consumption rate than R-PSF.

Similar lipid peroxidation levels were observed in R-PSF and controls with higher levels in OMP, and lower oxidative stress was seen in R-PSF compared with OMP kidneys. These results show R-PSF's capacity to ameliorate oxidative damage with the maintenance of these parameters to be similar with the non-DCD controls

[51[black small square]]. In contrast, Molacek et al. published a study comparing

R-PSF to hypothermic liquid perfusion in DCD kidneys exposed to 20 min of warm ischemia time (WIT). After a total of 2 h of preservation, kidneys were assessed for 2 h immediately after autotransplantation and restoration of blood perfusion.

Venous urea and histopathological analyses were used to assess graft quality, which revealed no significant differences between the two groups [52]. This may be anticipated, given that this study was limited in evaluating substantial ischemic

319 damage and the effects of R-PSF because of the short time period (2 h) in which the kidneys were preserved. It is not clear whether the animals were under anesthesia during the urine collection, and using blood urea and histology for the evaluation of renal function is inadequate to fully understand the effects of R-PSF and hypothermic liquid perfusion. Further examination of multiple parameters is needed to clarify the effects of R-PSF posttransplant. These recent studies show conflicting results, but these may be attributed to the differences in ischemic exposure during the preservation periods used in their respective treatments as well as the difference in characterizing the viability and function of the kidneys in an ex-vivo versus an in-vivo model.

HEART

Although PSF was first discovered using hearts [20,39,53], PSF research has been dominated by livers and kidneys since its discovery. The most recent published study used a DCD model of pig hearts to compare the utility of cold storage, hypothermic OMP and R-PSF [54]. All organs were treated for 2 h in their treatment groups, and authors reported seven out of 11 successfully reanimated in the cold storage group, two out of six hearts successfully reanimated in the hypothermic OMP group, all six hearts successfully reanimated in the R-PSF group for up to 5 h [54]. This study was preliminary in nature and it appears that results included those from hearts that were utilized in the process of technique development with protocol amendments introduced during the course of the study.

For example, some of the hearts of the OMP group and all of the PSF group hearts

320 benefitted from the use of dialysis filters during near-normothermic blood reperfusion to eliminate lactate buildup and maintain pH homeostasis. The R-PSF group was given additional insulin, corticosteroid and antibiotic in the initial flush solution unlike the hearts in the OMP and cold storage groups [54]. Given the above considerations, concrete conclusions cannot be drawn from this proof-of- concept study using R-PSF to reanimate hearts procured after approximately 20 min of warm ischemia. Nonetheless, the results of this early study suggest that R-

PSF may be a promising approach to resuscitate DCD hearts.

PANCREAS

Pancreas persufflation is being studied in the context of improving islet isolations for allotransplantation. Previous studies have shown A-PSF to increase

ATP levels and maintain it for up to 24 h of preservation in human and porcine pancreata [23,55]. Improved islet viability and yield after persufflation have also been reported relative to SCS even after 24 h of persufflation [56-58].

Reddy et al. [59] studied a DCD model to test the effects of cold storage,

HMP and R-PSF in rat pancreatic islets. Islets were isolated from pancreata preserved in these conditions for 6 h. Results show significantly higher islet yields in the R-PSF group compared with cold storage and HMP. They did not find any significant differences between islet viability and function measured through staining and glucose stimulation indices. The authors note that the damaged islets from cold storage and HMP may be filtered out during the islet purification process to yield comparable viability and function as the R-PSF islets. Furthermore, rat

321 pancreata are small in size relative to porcine and human [60-62] and do not experience the same degree of oxygen limitations during SCS or HMP and this may have mitigated some of the more clinically relevant positive effects of PSF in this study [58]. Nevertheless, improved islet yield alone is an important achievement, especially when humans typically need a dose of islets that require multiple pancreatic isolations for successful islet transplant to achieve insulin independence [63-66]. More recently, Kelly et al. demonstrated that pancreas preservation by persufflation rather than SCS prior to islet isolation reduced inflammatory responses and promoted metabolic pathways in human islets.

Additionally, persufflation improved [beta]-cell function (stimulated insulin secretion) and these effects persisted for at least 2 days in culture after islet isolation. They also demonstrated that these positive effects were diminished the longer the human pancreas remained in SCS post procurement and prior to the onset of persufflation, emphasizing the importance of initiating persufflation as early as possible after procurement [67,68].

MODIFIED PERSUFFLATION

As the renewed interest in the safety and efficacy of persufflation is being pursued, it is evolving through alterations and shifting paradigms of organ preservation. Recently published studies have brought in the addition of nitric oxide and pulsatile flow [69,70]. Although nitric oxide can be a source of free-radical damage, it has been shown to protect against ischemia reperfusion injury by limiting oxidative damage and leukocyte adhesion [71,72]. The physiological shear stress exerted from pulsatile flow may improve circulation [73], and promote anti-

322 inflammatory pathways in the liver [74]. The following studies have added these two factors to refine persufflation for better preserved organs.

Kageyama et al. used DCD rat livers exposed to 30 and 60 min of WIT. The effects of 3 h of cold storage and R-PSF supplemented with nitric oxide (R-PSF-

NO) were compared after transplantation. The 30-min WIT R-PSF-NO group had significantly lower hepatocellular damage with less expression of inflammatory cytokines than the cold storage livers with near normal morphology after transplantation. In the 60-min WIT experiment, cold stored liver recipients did not survive past 24 h of the surgery whereas 71% of R-PSF-NO group survived. The cold stored livers had significantly higher expression of endothelin-1 that can exacerbate ischemia reperfusion injury [69]. The notable finding of this study is the potential of R-PSF-NO to successfully treat the prolonged oxidative damage that occurred over 60 min of WIT to improve recipient survival. Similarly, Porschen et al. tested the effects of R-PSF-NO during the first hour of a 24-h cold preservation period and the last hour of a 24-h cold preservation period. Results show significantly lower aspartate aminotransferase, lactate dehydrogenase and malondialdehyde levels, which are indicative of liver damage and lipid peroxidation in livers persufflated during the first hour of preservation period [70]. These studies suggest the timing of oxygen provision is important in rescuing the organ from harmful effects of prolonged ischemic preservation periods, and R-PSF-NO can be used to recondition livers with up to 60 min of prolonged warm ischemia.

Additionally, Luer et al. [75] studied the influence of pulsatile gas flow in the context of persufflation using isolated DCD rat livers. After 18 h of pulsatile and

323 nonpulsatile R-PSF, ALT and AST levels were significantly lower in the pulsatile

R-PSF after reperfusion with higher bile flow and ATP levels. Furthermore, pulsatile R-PSF livers presented with less vascular resistance upon reperfusion with better maintained NO levels. In these studies, persufflation resulted in improved liver viability and function compared with cold storage counterparts, signifying the advantages of providing oxygen to DCD organs during preservation

[75]. They also demonstrated that there is still room for improvement in the PSF technique by modification of the gas content and introducing a more physiological, pulsatile flow to progress towards enhancing its beneficial effects.

CONCLUSION

Improved preservation methods, especially ones enhancing oxygenation are actively pursued to improve organ preservation. Persufflation is currently investigated to a limited degree as a simple, cost-effective and efficient method for oxygenating organs during preservation. Most of the recent literature demonstrated favorable outcomes and the superiority of persufflation over the traditional cold storage in DCD organs and some studies have begun to compare it with machine perfusion. More detailed and properly powered prospective studies will be needed to compare the different modalities and to further validate, evaluate and establish parameters such as retrograde versus anterograde persufflation, concentration of oxygen used, perfusion pressure, flow rate and preservation periods for the well tolerated, standard use of persufflation. These parameters are expected to be organ and tissue specific and extrapolations from one organ to

324 another should not be made. Future application of persufflation in conjunction with other dynamic preservation methods offers a promising strategy for preservation.

It has the potential to increase the quality and quantity of cells and solid organs for transplant, and warrants further investigation and optimization.

KEY POINTS

• There is a trend of superior organ viability and quality in persufflated organs

than cold storage organs as demonstrated by functional assessments,

biomarker levels and genomic profiles

• Persufflation appears to be a simple, cost-effective preservation method

that should be further investigated

• The benefits of anterograde and retrograde persufflation need to be further

delineated

• Timing of persufflation is of importance, with its beneficial effects most

pronounced when it is applied shortly after procurement, especially for DCD

organs

325

Acknowledgements

Early work on the development and establishment of persufflation for the pancreas was supported in part by funding from the Schott Foundation and the Carol Olson

Memorial Diabetes Research Fund.

Financial support and sponsorship

Funding source: This work was supported by the National Institutes of Health grant

5R44DK070400.

Conflicts of interest

K.K.P. served as Consultant/Chair, SAB Giner Inc and as a co-inventor on pending patents related to persufflation with University of Minnesota and Giner Inc., and was the PI or co-PI on the NIH/NIDDK grants that partially funded the work. He is no longer affiliated with Giner Inc.

326

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

[*] of special interest

[*][*] of outstanding interest

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336

Appendix B

Summary of Attempts at Rat Kidney Allotransplant

337

Aim: to translate the ex vivo assessment tools and preservation methods to an in vivo model using pig allotransplant to evaluate renal function.

Figure 61: Experimental timeline for organ preservation. Heparinized rats are euthanized and flushed with room temperature heparinized saline through the abdominal aorta. Kidneys are procured after 30 minutes of warm ischemia time (WIT), which is the 30-minute duration between the euthanasia and placing the organs on ice. Following procurement, kidneys are cannulated at the renal artery and vein using cannulas. The kidneys are treated for 24 hours with either static cold storage (SCS) or persufflation(PSF). Both kidneys are kept in SPS-1 solution at 4°C during the 24 hours.

Samples Assessments

Blood Creatinine, BUN, Albumin

Urine Urinalysis

Biopsy * Histology

338

Figure 62: Experimental timeline for transplant surgeries and post-surgery assessments. Rat renal allotransplants will be performed with kidneys that have been preserved for 24 hours with either persufflation or cold storage. The recipient rats will be monitored for 7 days post- transplant for renal function. Rat urine output, urine and serum creatinine and urinary physiological markers will be measured daily until sacrifice. At day 7 the allografts will be harvested and histological examination will follow to assess ischemia reperfusion injury and any additional damage.

Results

Total time Complete Anastomosis Rat under Vein Artery Ureter Reperfusion anesthesia 1 5.5 hr Yes Yes No No, arterial anastomosis completed post mortem 4 3 hr 40 min Yes No No No 5 4.5 hr Yes Yes Attempted No, arterial anastomosis completed post mortem 6 4 hr Yes Partial No No 7 3 hr 50 min Yes No No No

Table 36: Summary of rat autotransplant.Rat kidneys were isolated and repositioned for transplant in the same animal for practicing surgical techniques. End-to-side and end-to-end vascular anastomoses were done, and an end-to-end ureter anastomosis was attempted. Rats did not survive the surgeries.

Rat Tail Vein Kidney Vessel Cannulation Catheter Procurement 2 No Yes No 3 No Yes Yes

Table 37: Summary of rat kidney procurement and preservation. These were a series of unsuccessful attempts at kidney procurement, flushing and preservation using persufflation. Due to the unsuccessful placement of catheters, no heparin was administered to prevent thrombosis.

339

Figure 63: Images of the rat kidney with complete vascular anastomoses. A end-to-side arterial anastomosis and B depicts end-to-end venous anastomosis.

Methods

In order to practice techniques involving organ isolation, dissection and anastomoses, we took the kidney from the same rat and transplanted distally from its original site. Unfortunately, none of the rats survived the surgery and often died before the complete anastomoses of the renal artery.

Male Lewis rats (300-400g) were used for this experiment. The donor rat anesthesia was induced and maintained with continuous inhalation of oxygen and isoflurane. After heparin (50 IU/Kg) injection, hair was removed from the surgical site using clippers followed by a surgical scrub alternating between betadine and alcohol. A midline incision was made to access peritoneal cavity. The abdominal aorta, inferior vena cava, renal artery, renal vein, ureter and the kidney was blunt dissected. The inferior vena cava and abdominal aorta were clamped beneath the diaphragm by using hemostatic forceps. The renal vein and artery were respectively clamped with vascular clips next to abdominal aorta and IVC. Both renal vessels were incised as near as possible to the left kidney. Stay sutures were placed in the opposed ends of the vessels opposite to each other and 3-4 stitches were placed on each side. The vein was anastomosed first, followed by the arterial anastomosis.

340

Discussion

Rat kidney transplants presented with profound technical difficulties that come with microsurgeries. We wanted to establish a protocol and iron out the logistics of preforming rat kidney transplants with no prior experience in this type of surgery. The biggest hurdle we faced was the rats dying during the surgeries at the completion of venous anastomosis and sometimes through partial arterial anastomosis. This may be caused by too much respiratory depression during the long hours of surgery, tracheal secretions that blocked airways and not enough heat from the heating pad to help maintain their body temperature. We decided to proceed with using pigs as a pre-clinical model. Porcine work to evaluate different types of preservation methods are well established in the literature. Pig kidneys are close in structural features to human kidneys, and the processes of anesthesia and surgical procedures are also similar to human conditions, which makes them a model of choice for preclinical studies.

341

Appendix C

Acute Ischemia Induced by High Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans

342

Acute ischemia induced by high density culture increases cytokine expression

and diminishes the function and viability of highly purified human islets of

Langerhans.

Kate E. Smith, MS1,2, Amy C. Kelly, BS3, Catherine G. Min, MS1,2, Craig S. Weber, BS4, Fiona M. McCarthy, PhD3, Leah V. Steyn, PhD1, Vasudeo Badarinarayana, PhD5, J. Brett Stanton, BS1, Jennifer P. Kitzmann, MPH1, Peter Strop, PhD5, Angelika C. Gruessner, PhD6, Ronald M. Lynch, PhD4, Sean W. Limesand, PhD3, and Klearchos K. Papas, PhD1

1Department of Surgery, University of Arizona, Tucson AZ 2Department of Physiological

Sciences GIDP, University of Arizona, Tucson AZ 3School of Animal and Comparative

Biomedical Sciences, University of Arizona, Tucson AZ 4Department of Physiology,

University of Arizona, Tucson AZ 5Sanofi-Aventis Group, Tucson, AZ 6Department of

Surgery, SUNY Upstate Medical University, Syracuse, NY

Corresponding author:

Klearchos K. Papas, PhD

Department of Surgery

University of Arizona

Medical Research Building

1656 E. Mabel Street, Room 122

Tucson AZ 85724

Telephone: (520) 626-5853

Fax: (520) 626-3770

E-Mail: [email protected]

343

AUTHORSHIP

Kate E. Smith – Coordinated and contributed to physiological assessments and analyses, contributed directly to manuscript preparation and content, figures, and review. This author declares no conflicts of interest.

Amy C. Kelly – Coordinated and conducted RNAseq analysis and directly contributed to manuscript preparation and content, figures, and review. This author declares no conflicts of interest.

Catherine Min – Contributed to physiological assessments, analysis, and manuscript review. This author declares no conflicts of interest.

Craig S. Weber – Contributed to physiological assessments, analysis, and manuscript review. This author declares no conflicts of interest.

Fiona M. McCarthy – Contributed to RNAseq analysis and manuscript review. This author declares no conflicts of interest.

Leah V. Steyn – Contributed to physiological assessments and manuscript review. This author declares no conflicts of interest.

Vasudeo Badarinarayana – Contributed to RNAseq analysis and manuscript review. This author declares no conflicts of interest.

Peter Strop – Contributed to study design, manuscript review, and funding. This author declares no conflicts of interest.

Angelika C. Gruessner – Conducted statistical analysis and manuscript review. This author declares no conflicts of interest.

J. Brett Stanton – Performed fluorescence microscopy and quantification and contributed to manuscript review. This author declares no conflicts of interest.

Jennifer P. Kitzmann – Consulted on physiological assessments and manuscript review.

This author declares no conflicts of interest.

344

Ronald M. Lynch – Contributed to study design, consulted on physiological assessments, and manuscript review. This author declares no conflicts of interest.

Sean W. Limesand - Contributed to study design, manuscript preparation, content, statistics, and review. This author declares no conflicts of interest.

Klearchos K. Papas – Principal Investigator, contributed directly to study funding, conception, design, data analysis and presentation, manuscript preparation, and review.

Dr. Papas declares a financial interest in Giner, Inc. This interest has been properly disclosed to the University of Arizona Institutional Review Committee and is managed by a Financial Conflict of Interest Management Plan in accordance with its conflict of interest policies.

FUNDING

Support for this study was provided by the Sanofi-Aventis Group, the Heart, Lung, and

Blood Training Grant (T32HL007249), NIH/NIDDK Phase 1 SBIR Grant (Giner, Inc.) R43

DK100999, and the Juvenile Diabetes Research Foundation (JDRF Grant JDRF: 5-2013-

141) for their generous support.

345

ABBREVIATIONS

ARG1, Arginase 1, AP1, Activator protein 1, B2M, Beta-2 microglobulin, CCL2, C-C motif chemokine ligand 2, CCL20, C-C motif chemokine ligand 20, CCL3, C-C motif chemokine ligand 3, CCL3L3, C-C motif chemokine ligand 3-like 3, CPM, Counts per million, CSF2,

Colony stimulating factor 2, CSF3, Colony stimulating factor 3, CXCL1, C-X-C motif chemokine ligand 1, CXCL3, C-X-C motif chemokine ligand 3, CXCL5, C-X-C motif chemokine ligand 2, CXCL8, C-X-C motif chemokine ligand 8, DNA, deoxyribonucleic acid, FDA/PI, fluorescein diacetate/propidium iodide, FLT1, Fms related tyrosine kinase

1, FOS, Fos proto-oncogene, AP-1 transcription factor subunit, FPKM, Fragments per kilobase per million mapped reads, GO, Gene Ontology, GSIS, Glucose Stimulated Insulin

Secretion, HBA2, Hemoglobin subunit alpha 2, HBB, Hemoglobin subunit beta, HGF,

Hepatocyte growth factor, HIF1A, Hypoxia inducible factor 1 alpha subunit, HK1,

Hexokinase 1, HMOX1, Heme Oxygenase 1, HPRT1, Hypoxanthine

Phosphoribosyltransferase 1, ICAM1, Intercellular adhesion molecule 1, IE, Islet

Equivalents, IIDP, Integrated Islet Distribution Program, IL6, Interleukin 6, ITx, Islet transplantation, JUN, Jun proto-oncogene, AP-1 transcription factor subunit, KCNJ11, potassium voltage-gated channel subfamily J member 11, KCNJ3, potassium voltage- gated channel subfamily J member 3, OCR, Oxygen Consumption Rate, RNAseq, RNA

Sequencing, RPLP0, Ribosomal Protein Lateral Stalk Subunit P0, SC-βs, Stem Cell

Derived β-Cells, SELE, Selectin E, SLC2A1, Solute Carrier Family 2 Member 1,

SLC38A4, Solute Carrier Family 38 Member 4, T1D, type 1 diabetes, TNF, Tumor

Necrosis Factor, UPP1, uridine phosphorylase 1, VCAM1, Vascular cell adhesion molecule 1

346

ABSTRACT

Background: Encapsulation devices have the potential to enable cell based insulin replacement therapies (such as human islet or stem cell derived β–cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β–cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention.

Methods: Human islets were exposed in a pairwise model simulating high density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing (RNAseq) was conducted to assess transcriptome-wide changes in gene expression.

Results: Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (p<0.01). Insulin secretion was also diminished, with ischemic β–cells losing their insulin secretory response to stimulatory glucose levels (p<0.01). RNAseq revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism.

Conclusions: In order for cell based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to β–cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and β–cell function, and leads to increased inflammatory signaling.

347

INTRODUCTION

Islet transplantation (ITx), a form of cell based insulin replacement therapy, is an attractive approach for the treatment of uncontrolled or “brittle” type 1 diabetes (T1D). However, large scale application of ITx is limited by the need for lifelong immunosuppression and a shortage of human cadaveric pancreas donors, which is exacerbated by the need for islets from multiple pancreata per patient to achieve insulin independence1,2. Encapsulation

(immunoisolation) devices have the potential to address these critical limitations. They have demonstrated efficacy in protecting transplanted islets even in the absence of immunosuppression, and may allow for alternative cell based insulin replacement therapies for T1D, for example by enabling the use of stem-cell derived β-cells (SC-βs) which can be sourced on a virtually unlimited scale3-10.

However, encapsulating therapeutic β-cell doses into reasonably sized (postage stamp) immunoisolation devices creates a high density environment in which the availability of nutrients (especially glucose and oxygen) is limited, leading to ischemia7. This has ultimately hindered the clinical application of macro-encapsulation in β–cell replacement therapies. Insufficient oxygen is a particular concern for the insulin secreting β–cells within islets. β-cells lack sufficient levels of the enzyme lactate dehydrogenase α (LDHα) to generate ATP using anaerobic glycolysis, and overexpression of LDHα in β–cells diminishes their glucose responsiveness11,12. Moreover, once separated from their native vasculature, islets depend solely on diffusion for the delivery of oxygen and energetic substrates. Deprivation of these critical nutrients and the accumulation of toxic metabolites leads to impaired insulin secretion and eventual cell death13-19. These effects are even more pronounced in islets with large diameters, and clinical ITx outcomes are improved for patients receiving preparations comprised of smaller average diameter islets20-22.

Considering the recent surge of interest in immunoisolation devices and the benefits that their application can offer, it will be of critical importance to understand how these devices

348 impact β–cell physiology in order to facilitate successful clinical outcomes. Therefore, we investigated the viability, insulin secretion, and transcriptional adaptations of highly purified human islets with an emphasis on β–cell function, stress, and inflammation following acute ischemic exposure in high density “pellet” model. This model has been characterized previously in the context of islet shipping, and is used here to simulate and study the effects of the high density environments created by delivery of therapeutic β– cell doses in reasonably sized immunoisolation devices23,24.

349

MATERIALS AND METHODS

Islet Source and Maintenance. Human islets (n=11 independent preparations) were obtained from the Integrated Islet Distribution Program (IIDP), the University of Minnesota, the University of Arizona, the McGill University Health Centre, and the University of

California - San Francisco. Islets were cultured in oxygen permeable silicone rubber membrane GRex vessels (Wilson Wolf, St Paul, MN). Islet culture media consisted of

CMRL 1066 (Mediatech, Inc., Manassas, VA) supplemented with 0.5% Human Serum

Albumin (HSA, BioChemed Services, Winchester, VA), 1% heparin (SAGENT,

Schaumburg, IL), 1% L-Glutamine (Mediatech, Inc.), and 1% Penicillin/Streptomycin (GE

Healthcare Life Sciences, Logan, UT). Prior to experiments, islets were maintained at

25ºC and ambient pO2 supplemented with 5% CO2.

Induction of Pelletized Culture. Following standard culture, islets from each donor were divided into normoxic and ischemic conditions. Ischemia was modeled through high density, “pelletized” culture in which 10,000 islet equivalents (IE) as quantified by DNA were allowed to settle in the bottom of a 1.5 mL centrifuge tube25. This condition was selected since it mimics both traditional islet shipping conditions as well as the high density environments that can result from encapsulation. For the normoxic condition, an equal number of islets were cultured in a 10 cm2 GRex vessel (Wilson Wolf). Normoxic and ischemic islets were cultured for 12 hours at 37°C, ambient O2, and 5% CO2.

Islet Encapsulation. In order to test the effects of high density encapsulation on human islets and verify comparable effects to pelletized culture, 8,000 IE as quantified by DNA were loaded into 4.5µl (0.34cm2) TheraCyteTM (TheraCyteTM, Laguna Hills, CA) devices using a Hamilton syringe. Devices were then placed into oxygen permeable, 10 cm2 GRex vessel (Wilson Wolf) filled with culture media and maintained for 12 hours at 37°C, ambient

O2, and 5% CO2. Encapsulated islets were compared to matched pellets and controls prepared as described above.

350

Measurement of DNA. Islets were suspended in 1 mL of AT Buffer (1M solution of ammonium hydroxide in nanopure water, supplemented with 0.2% Triton X-100) and sonicated using a Sonic Dismembrator Model 500 (Fisher Scientific, Waltham, MA) for 30 seconds at 11% amplitude. DNA was assessed using a Quant-iTTM PicoGreen dsDNA kit

(Life Technologies, Carlsbad, CA) according to manufacturer instructions. 96 well plates were read using a SpectraMax M5 plate reader and SoftMax Pro software (Molecular

Devices, Sunnyvale, CA).

Measurement of Oxygen Consumption Rate Normalized to DNA (OCR/DNA). After

12 hours, islets and media were mixed to ensure homogenous sampling. OCR was conducted as previously described26. Briefly, islets were resuspended in Media 199

(Mediatech, Inc.) warmed to 37°C and divided evenly between three OCR chambers of known volumes, or conducted on whole devices as in the case of encapsulated islets

(Instech Laboratories, Inc., Plymouth Meeting, PA). Measurements of pO2 in each chamber over time were recorded using fiber optic sensors and NeoFox viewer software

(Ocean Optics, Inc., Dunedin, FL). The oxygen consumption rate in nanomoles of O2 per minute was then estimated from the slope of the decline in pO2 over time. This value was normalized to the DNA content of each chamber (as described above) to give a final measurement of OCR/DNA (nmol O2/min*mg DNA).

Measurement of % Viability by fluorescein diacetate/propidium iodide (FDA/PI) staining. From each condition, 100µl samples of islets were combined with 377.6µl of dithizone (Sigma-Aldrich, St. Louis, MO). To this, 1.39µl of fluorescein diacetate (Sigma-

Aldrich) and 21.05µl of propidium iodide (Sigma-Aldrich) were added for final concentrations of 0.067µM and 4.0µM respectively. Islets were incubated in the dark for

20 minutes, after which they were allowed to settle and 100µl of islets were placed on a slide for imaging. To determine the proportion of live vs. dead cells, islets were imaged

351 on a Zeiss Observer.Z1 (Zeiss, Oberkochen, Germany) using the 10X objective and an

AxioCam MRm camera with ZEN 2012 (blue addition) software (Zeiss).

β-cell Function. The Biorep Technologies Peri-4.2 Perifusion System (Biorep

Techonologies, Inc., Miami Lakes, FL) was used to measure dynamic glucose stimulated insulin secretion (GSIS). Triplicate measurements using 100 IE each underwent baseline stimulation with 2.8 mM glucose in oxygen-saturated (95% O2) Krebs-Ringer Bicarbonate buffer (KRB) for 20 minutes before and after a 40 minute stimulation period with 16.7 mM glucose. Perfusate was collected at a rate of 100 μl/min in a 96 well plate. Perfusate was tested for insulin content using an insulin ELISA (Mercodia, Winston-Salem, NC). ELISA plates were read using Softmax Pro software and a SpectraMax M5 plate reader

(Molecular Devices). Insulin content was normalized to DNA as described above.

RNA Library Preparation. Paired normoxic and ischemic islets from four donors were selected at random for high throughput RNA Sequencing (RNAseq). For RNA samples, islets were collected in microcentrifuge tubes and washed twice in 1X DPBS to remove serum. RNA was isolated using the RNeasy Mini Kit (Qaigen, Valencia, CA) according to manufactures instructions. RNA quality was assessed by Agilent Bioanalyzer (Agilent

Technologies, Santa Clara, CA), and a minimum RIN of 7.0 was required for inclusion in the study. Libraries were prepared using the Illumina TruSeq Stranded mRNA Sample

Preparation Kit (Illumina, San Diego, CA). mRNA was sequenced using Illumina HiSeq

2000 sequencer and quantified into transcripts using EA-Quintiles mRNAv8 pipeline.

RNAseq Analysis. Briefly, 50x50 base, paired-end reads were checked for quality by comparison to intergenic and ribosomal sequences. Sequencing reads were aligned to the human UCSC known gene transcriptome using RSEM v1.2.0 and transcript abundance was quantified. An average of 97.4 ± 0.4% of total reads mapped to reference genomes. The aligned reads for each gene were summarized as described in Li and

Dewey 201127. All genes and isoforms have been assigned a normalized coverage rate in

352 fragments per kilobase per million mapped reads (FPKM). Differential gene expression was performed using the edgeR R package scripts (bioconductor.org) using paired sample statistical procedures28,29. A paired-sample (generalized paired t-test) analysis was conducted to investigate the effects of ischemia while adjusting for baseline differences between patients. Genes with a p<0.05 following Benjamini-Hochberg correction were considered to be statistically significant.

Functional Analysis of Differentially Expressed Genes.

Significantly up or downregulated genes were submitted to KOBAS 2.0 in order to determine which canonical pathways were enriched by ischemia, as well as to determine associated Gene Ontology (GO) terms30. Significant enrichments were defined as a corrected p<0.05 following a Fisher’s Exact Test with Benjamini Hochberg correction in

KOBAS 2.0. To summarize GO enrichment and reduce redundant terms, ReviGO was used to cluster similar GO terms using small (0.5) SimRel similarity and the whole Uniprot as the database for categorical GO term sizes 31. Significant pathways were manually curated to remove irrelevant pathways containing redundant or non-specific gene findings.

Preferential association between the lists of up- and down-regulated genes was also evaluated.

RT qPCR Array. Results from RNAseq were compared to RTqPCR results for a single islet preparation. Purified islet total RNA was run on RT² Profiler™ PCR Array Human

Hypoxia Signaling Pathway plates (Qiagen) using iQ5 Real-Time PCR Detection System

(Bio-Rad Laboratories, Irvine, CA). Fold changes were determined according to manufactures instructions. Gene expression was normalized to housekeeping genes

RPLP0, B2M, and HPRT1.

Statistics. Statistical analysis of physiological parameters was conducted in SAS 9.4

(SAS Institute Inc., Cary, NC). OCR/DNA data were analyzed using Wilcoxon Signed

Rank Test. Matched FDA/PI and OCR/DNA data were analyzed using a Student’s t-test.

353

Perifusion data were analyzed using a generalized linear mixed model for repeated measurements. Fold changes from the RT qPCR array were correlated to fold changes from RNAseq using linear regression analysis in GraphPad Prism version 6.07 (GraphPad

Software, Inc., La Jolla, CA). Unless otherwise indicated, data represent mean ± standard deviation.

RESULTS

Donor Metrics. Individual donor characteristics and islet quality parameters (% viability measured by membrane integrity staining, purity, and cold ischemia time) as reported by the distributing isolation center are presented in Table 1.

Pellet Model Characterization and Comparison to Encapsulation. The viability of islets from n=2 human islet preparations was compared under matched normoxic, pelletized, and encapsulated conditions in order to confirm that the ischemic damage caused by the pellet condition is similar to what is caused in devices loaded at high densities7. Pelletized islets and encapsulated islets showed similar % reductions in OCR/DNA values versus control (57.7 ± 19.5% and 80.3 ± 21.1% respectively).

Islet Viability. Islet viability was determined in n=8 independent islet preparations.

Average OCR/DNA values for normoxic islets align closely with clinical averages observed for both islet auto and allotransplantation32,33. Islet OCR/DNA values are reduced following warm ischemic exposure (p<0.01, Fig. 1), which correlates to lower, although not entirely diminished, viability. In a sub-cohort of n=4 independent islet preparations, OCR/DNA results were compared to FDA/PI staining. Staining revealed a significant loss of viability for control vs. pelletized islets (% 87.3 ± 1.0% vs. 8.8 ± 5.5% respectively, p<0.01).

OCR/DNA measurements for matched samples were similarly reduced for control vs. pelletized islets (122.9 ± 53.3 and 53.4 ± 12.0 nmol O2/min*mg DNA respectively, p<0.05).

β-cell Function. GSIS was measured in a subset of control and ischemic islet pairs (n=4 independent preparations, Fig. 2). GSIS of ischemic islets is virtually absent compared to

354 control islets. Basal secretion was not different between control and ischemic islets (Fig.

3A). However, under stimulatory glucose concentrations (Fig. 3B), ischemic islets secrete less insulin than control islets (p<0.01). Normoxic islets show elevated (p<0.01) insulin secretion in response to stimulatory glucose concentrations while ischemic islets are unresponsive.

Global Gene Expression Changes Following Ischemia. An average of 19,932 ± 138 genes were detected with RNAseq. Of these, 657 genes were differentially expressed in ischemic versus control islets (p<0.05) (Fig. 4A). Genes most up-regulated include immune molecules interleukins 6 and 8 (IL6, IL8), chemokine 3 (CCL3), and chemokine 3

+ like (CXCL3). Genes most down-regulated include the inward rectifier K Channel Kir 3.1

(KCNJ3) and Na+ Coupled Neutral Amino Acid Transporter 4 (SLC38A4) (Fig. 4B).

Fold changes calculated from RNAseq correlated (p<0.0001, r2=0.75) to fold changes calculated by RT qPCR array (Supplemental 1). The high correlation between independent measures of gene expression corroborates findings from RNAseq model.

Functional analysis of differentially expressed genes.

Canonical pathways significantly associated with differentially expressed genes were identified (Table 2). Specific differentially expressed genes associated with inflammation and cytokine signaling, and also associated with pathways for the HIF-1α response, AP-1

(FOS) signaling, and ion transport are detailed in Fig. 5. Enriched GO terms were in agreement with pathway findings, showing enhancement of cytokine signaling and inflammation as well as downregulation of cellular ion transport (Table 3). Genes associated with hypoxia and nutrient deprivation (including SLC2A1, HK1, HMOX1,

ARG1, and UPP1) were also upregulated (p<0.05).

355

DISCUSSION

Ischemia remains a pervasive issue that directly influences the outcomes of cell based insulin replacement therapies such as ITx. In this study, we implement an ischemic model that has previously been used to study islet shipping, and captures the conditions seen in high density islet encapsulation, including a significant reduction of islet viability which is associated with oxygen and nutrient deprivation, and consistent with results from the transcriptome analysis19,23,24. Furthermore, we demonstrate that the viable population of islets following acute ischemia exhibits significant β–cell dysfunction corresponding to global alterations in gene expression. Inflammatory responses were evident in the gene signatures of islets following ischemia, with concurrent downregulation of genes involved in nutrient and ion transport. The persistence of β-cell dysfunction due to ischemic stress has been previously demonstrated, whether due to cellular reprogramming or ischemia- reperfusion injury, and the endurance of a concurrent pro-inflammatory signature is likely, given the present work19,34,35. Interestingly, kidney capsule islet transplants represent a commonly used in vivo correlate to our high-density in vitro model in which islets are transplanted as a “pellet” below the renal capsule36. Although this method induces less ischemic stress than what might be experienced in an immunoisolation device, when these islets are exposed to nerve growth factor (which has an anti-apoptotic effect), graft function is improved37-39. This supports our finding that even acute ischemia is detrimental to islet viability and function, and indicates that early mitigation of these factors is beneficial to transplant outcomes.

The differential gene expression profile caused by acute ischemia was predominantly increased inflammatory and stress response signaling. More specifically, there was increased expression of key inflammatory genes (including TNF and IL6), which are known to contribute to islet death in the peri-transplant period by the instant blood mediated inflammatory reaction40. The link between hypoxia and this pro-inflammatory

356 profile has been demonstrated previously in rat islets41. This increased inflammatory signaling may be important not only from an immunological standpoint, but also in the context of β-cell function. TNFα has previously been shown to inhibit glucose stimulated insulin secretion in β-cell lines as well as downstream insulin signaling in adipocytes42-44.

The potential damage to insulin signaling is compelling, as autocrine insulin signaling in

β–cells has been demonstrated to influence insulin expression45. Key genes involved in the coupling of glucose sensing to insulin secretion (for example KCNJ11, which encodes the major subunit of the ATP sensitive K+ channel) are also significantly downregulated.

Together with the effects of inflammatory factors, these changes may underlie the significant loss of β–cell function. By mitigating these damaging adaptations, loss of islets in the peri-transplant period may be minimized and ultimately help to facilitate lower curative doses of islets for diabetes reversal46.

Lifelong immunosuppression remains a critical barrier which limits the application of β– cell replacement therapies. Implantable macro-encapsulation devices are a possible solution and have been demonstrated to be allo-protective in animal models as well as in humans3,47. However, packing therapeutic β-cell doses into practically-sized devices results in a high density environment in which diffusion of oxygen and nutrients is severely limited. This study demonstrates that such high density environments are damaging to β- cell function, and may ultimately affect transplant outcomes5,48. Addressing the effects if ischemia by ensuring adequate nutrient delivery will be critical to the success of cell based insulin replacement therapies going forward. With respect to maintaining oxygenation, numerous advances have been made with regard to supplying oxygen to the pancreas during the preservation period as well as to isolated islets during culture49,50. Attempts have also been made to supply islets in encapsulation devices with oxygen, and this remains an active area of research although a permanent solution has yet to be achieved3.

357

ACKNOWLEDGEMENTS

The authors would like to acknowledge the members of the Papas laboratory and collaborators, including William Purivs, Nicholas Price, Ivan Georgiev, Anna Maria

Burachek, Alexandra Hoeger, Diana Molano, Jana Jandova, Kristina Rivera, Aikaterina

Assimacopoulos, and Breonna Smith for their support. We would like to thank our collaborators at the University of California – San Francisco, McGill University Health

Centre, and the University of Minnesota as well as the Integrated Islet Distribution

Program for supplying islets used in these studies, as well as Dr. Linda Tempelman and

Dr. Jan Burt for their advisement and mentorship. We would additionally like the ARCS

Foundation Phoenix Chapter for their generous support.

358

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Appendix D

In vitro Characterization of Neonatal, Juvenile, and Adult Porcine Islet Oxygen Demand, β-cell function, and Transcriptomes

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In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen

demand, β-cell function, and transcriptomes.

Kate E. Smith1, 2, William G. Purvis2, Melissa A. Davis3, Catherine G. Min1,2, Amanda M.

Cooksey3, Craig S. Weber4, Jana Jandova3, Nicholas D. Price2, Diana S. Molano2, J.

Brett Stanton2, Amy C. Kelly3, Leah V. Steyn2, Ronald M. Lynch4, Sean W. Limesand3,

Michael Alexander5, Jonathan R. T. Lakey5, Karen Seeberger6, Gregory S. Korbutt6,

Kate R. Mueller7, Bernhard J. Hering7, Fiona M. McCarthy3, Klearchos K. Papas2

1Department of Physiological Sciences, University of Arizona

2Department of Surgery, University of Arizona

3School of Animal and Comparative Biomedical Sciences, University of Arizona

4Department of Physiology, University of Arizona

5Department of Surgery, University of California – Irvine

6Department of Surgery, Alberta Diabetes Institute, University of Alberta

7Schulze Diabetes Institute, Department of Surgery, University of Minnesota

Corresponding author:

Klearchos K. Papas, PhD

Department of Surgery

University of Arizona

Medical Research Building

1656 E. Mabel Street, Room 122

Tucson, AZ 85724

Telephone: (520) 626-5853

Email: [email protected]

367

AUTHOR CONTRIBUTIONS

KES contributed to manuscript preparation, data analysis, and physiological assessments,

WGP contributed to physiological assessments and manuscript preparation, MAD contributed to physiological assessments and manuscript preparation, CGM contributed to physiological assessments and manuscript preparation, AMC contributed to data analysis and manuscript preparation, CSW contributed to physiological assessments and manuscript review, JJ contributed to data analysis and manuscript preparation, NDP contributed to physiological assessments and manuscript review, DSM contributed to physiological assessments and manuscript preparation, JBS contributed to physiological assessments and manuscript review, ACK contributed to data analysis and manuscript review, LVS contributed to physiological assessments and manuscript review, RML contributed to data analysis and manuscript preparation, SWL contributed to data analysis and manuscript preparation, MA contributed to JPI isolation and manuscript review, JRTL supplied JPI and contributed to manuscript preparation, KS isolated and shipped NPI preparations, GSK supplied NPI and contributed to manuscript preparation, KRM contributed to API isolations and manuscript review, BJH supplied API and contributed to manuscript preparation, FMM contributed to data analysis and manuscript preparation,

KKP is directly responsible for study design, data review and analysis, funding, and manuscript preparation.

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ABBREVIATIONS

APAF1 - apoptotic peptidase activating factor 1, API - adult porcine islets, APP - amyloid beta precursor protein, ATP - adenosine triphosphate , CACNA1E - calcium voltage gated channel subunit alpha 1 E, CASP3 - caspase 3, CDK2 - cyclin dependent kinase 2, CMAH

- cytidine monophosphate-N-acetylneuaminic acid hydroxylase, DAPI - 4',6-diamidino-2- phenylindole, DE - differentially expressed, DNA - deoxyribonucleic acid, DPBS -

Dulbecco’s phosphate-buffered saline, DTZ - dithizone, ECM - extracellular matrix, EdU -

5-ethynyl-2´-deoxyuridine, ELISA - enzyme linked immunosorbent assay, FDA -

Fluorescein diacetate, FN1 - fibronectin 1, FPKM – fragments per kilobase of transcript per million mapped reads, GGTA1 - α-1,3-galactosyltransferase , GSIS – glucose stimulated insulin secretion, HNF4A - hepatocyte nuclear factor 4 alpha, IE - islet equivalents, JPI - juvenile porcine islets, KEGG - Kyoto Encyclopedia of Genes and

Genomes, KRB - Krebs-Ringer Bicarbonate, LDHA - lactate dehydrogenase alpha , MYC

- MYC proto-oncogene BHLH transcription factor, NPI - neonatal porcine islets, OCR - oxygen consumption rate, OCT - optimal cutting temperature , PI - propidium iodide,

PTPRN - protein tyrosine phosphatase, receptor type N, RAB3A - RAS-associated protein

RAB3A, RNA - ribonucleic acid, RNAseq - RNA sequencing, SNAP25 - synaptosome associated protein 25, SP1 - specificity protein 1, TP53 - tumor protein P53, VAMP2 - vesicle associated membrane protein 2, VWF - won Willebrand factor

369

ABSTRACT

Background: There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted.

Methods: Neonatal porcine islets (NPI; 1-3 days), juvenile porcine islets (JPI; 18-21 days), and adult porcine islets (API; 2+ years) porcine islets were compared in vitro, including assessments of oxygen consumption rate, membrane integrity determined by FDA/PI staining, β-cell proliferation, dynamic glucose stimulated insulin secretion, and RNA sequencing.

Results: Oxygen consumption rate normalized to DNA was not significantly different between ages. Membrane integrity was age dependent, and API had the highest percentage of intact cells. API also had the highest glucose stimulated insulin secretion response during a dynamic insulin secretion assay and had 50-fold higher total insulin content compared to NPI and JPI. NPI and JPI had similar glucose-responsiveness, β-cell percentage, and β-cell proliferation rate. Transcriptome analysis was consistent with physiological assessments. API transcriptomes were enriched for cellular metabolic and insulin secretory pathways, while NPI exhibited higher expression of genes associated with proliferation.

Conclusions: The oxygen demand, membrane integrity, β-cell function and proliferation, and transcriptomes of islets from API, JPI, and NPI provide a comprehensive physiological comparison for future studies. These assessments will inform the optimal application of each age of porcine islet to expand the availability of islet transplantation.

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INTRODUCTION

A shortage of human donor pancreata limits the availability of islet transplantation (ITx) as a treatment for uncontrolled or “brittle” type 1 diabetes1,2. Due to this restriction, alternative islet sources are required to enable this course of treatment. Procuring islets from porcine donors is one such approach, and advances in gene editing have made the use of porcine organs and tissues an increasingly realistic and safe option3-11.

Porcine islets have established therapeutic potential in numerous pre-clinical and clinical studies, demonstrating diabetes reversal in non-human primates and long-term survival in human subjects3,12-21. However, within this cohort of studies, there is a range of porcine donor ages from which the islets were isolated, leading to debate as to the optimal donor age for clinical use. For instance, islets isolated from younger pigs (neonatal and juvenile) are more stable in vitro as compared to adult islets and have the capacity for proliferation, both of which are desirable traits for large-scale application22-24. Neonatal porcine islets have also specifically been shown to resist hypoxic damage, which may be a critical advantage given the significant impact of hypoxia on β-cell function25-27. However, islets isolated from adult pigs may be less immunogenic and are more immediately glucose responsive than islets from younger pigs23,28-30.

Despite the breadth of research detailing the specific merits of islets from individual age groups, comparatively few studies include and directly compare islets from multiple donor ages24,28,31-33. We directly compared neonatal (NPI), juvenile (JPI), and adult (API) porcine islets to guide their optimal translation in future applications. A comprehensive range of assays was used to evaluate relevant physiological traits including oxygen demand, β-cell function and proliferation, and whole transcriptome sequencing.

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MATERIALS & METHODS

Islet Isolation and Culture. Porcine islets were shipped to the University of Arizona from specified isolation centers in GRex vessels (Wilson Wolf Corporation, St. Paul, MN), This method has previously been demonstrated to maintain islet viability and function during shipment34. Neonatal Porcine Islets: NPI were isolated in the Korbutt laboratory and cultured as previously published22,35. Juvenile Porcine Islets: JPI were isolated by the

Lakey laboratory according to previously published protocols36,37. Adult Porcine Islets: API were isolated by the Hering laboratory using the automated method and tissue dissociating enzymes at low temperatures before being purified on continuous iodixanol density gradients as previously described38,39. Following receipt, islets were cultured at

37°C for 24 hours prior to assessment.

DNA Quantification. Replicate 100µl islet samples were collected in microcentrifuge tubes and suspended in 1 mL of AT Buffer (1M solution of ammonium hydroxide in nanopure water, supplemented with 0.2% Triton X-100). Islets were lysed by sonication for 30 seconds (Sonic Dismembrator Model 500, Fisher Scientific, Waltham, MA). A

Quant-iTTM PicoGreen dsDNA kit (Life Technologies, Carlsbad, CA) was used according to manufacturer instructions to measure the concentration of DNA in each sample.

Samples plates were read on a SpectraMax M5 plate reader using SoftMax Pro software

(Molecular Devices, Sunnyvale, CA).

Oxygen Consumption Rate (OCR) Measurement. OCR was measured as previously described40. Samples of approximately 3000 islet equivalents (IE) were suspended in serum free ME-199 media (Mediatech, Inc., Manassas, VA) and divided evenly between three 200µl water jacketed titanium chambers (Instech Laboratories, Inc., Plymouth

Meeting, PA). The chambers were sealed and pO2 was measured over time using fiber optic sensors and NeoFox Viewer software (Ocean Optics, Inc., Dunedin, FL). The change in pO2 over time was normalized to the DNA content of each chamber, measured as

372 described above, giving a final value in nmol O2/min*mg DNA. All OCR measurements were conducted at 37ºC.

Membrane Integrity. Samples of 50-100IE were collected in 100µl of media and combined with 377.6µl of dithizone (DTZ, Sigma-Aldrich, St. Louis, MO). Fluorescein diacetate (FDA, Sigma-Aldrich) and propidium iodide (PI, Sigma-Aldrich) were added for final concentrations of 0.067µM and 4.0µM respectively. Samples were then incubated in the dark for 20 minutes prior to imaging. Islets were imaged on a Keyence BZ-X710 fluorescence microscope using a 10x objective to determine the fraction of live vs. dead cells (Keyence, Osaka, Japan).

Dynamic Glucose Stimulated Insulin Secretion (GSIS). β-cell function was evaluated by measuring the levels of basal and glucose stimulated insulin secretion. Insulin secretion was measured using a Biorep Technologies Peri-4.2 Perifusion System (Biorep

Techonologies, Inc., Miami Lakes, FL). 100-300IE samples were loaded in triplicate into perifusion chambers and exposed to basal glucose (2.8mM) in Krebs-Ringer Bicarbonate

(KRB) buffer for 20 minutes, followed by stimulatory glucose (16.7mM) for 40 minutes.

Perfusate was continuously collected in 96 well plates at a rate of 100µl/minute. Insulin content of the perfusate was measured using an insulin enzyme-linked immunosorbent assay (ELISA, Mercodia, Winston-Salem, NC). ELISA plates were read using Softmax Pro software and a SpectraMax M5 plate reader (Molecular Devices). Insulin secretion rate was normalized to the DNA content of each perifusion chamber, measured as described above. All perifusion measurements were conducted in triplicate at 37ºC. Area under the curve was calculated beginning upon glucose stimulation and indicates total insulin secreted during this interval. The average basal secretion rate was multiplied by the stimulated time interval to yield basal secretion for that period. Stimulation index was calculated as a ratio of total secretion to basal secretion.

373

Insulin Content. 100-300IE were suspended in 500µl of insulin content buffer (100% ethanol supplemented with 21.4% ddH2O and 8.6% 11.6M hydrochloric acid). Islets were stored at -20ºC for 24 hours. Following this period, islets were centrifuged at 13,300x g for

15 minutes at 4ºC. Supernatant was collected and diluted in KRB buffer, and DNA was collected in 1mL of AT buffer. Supernatant was tested for insulin content using a colorimetric insulin ELISA (Alpco, Salem, NH) according to manufacturer instructions and read on a SpectraMax M5 plate reader using SoftMax Pro software (Molecular Devices).

Insulin content was normalized to sample DNA content, with DNA quantified as described above.

Histology. Islets (500IE) were fixed in 4% paraformaldehyde, then snap frozen in Optimal

Cutting Temperature compound (OCT, Sakura Finetek USA, Inc., Torrance, CA) and stored at -80°C. Embedded islets were sectioned at 10µm and ≥100-μm intervals with a

Microm HM 520 cryostat (Southeast Pathology Instrument Service, Charleston, SC) onto

Superfrost Plus microscope slides (Fisher Scientific, Pittsburg, PA). Insulin positive cells were immunostained using a guinea pig anti-porcine insulin polyclonal primary antibody

(1:500, Agilent Technologies, Santa Clara) and detected with donkey anti-guinea pig IgG conjugated to Alexa Fluor® 594 (1:500, Jackson ImmunoResearch Laboratories, Inc.,

West Grove, PA). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, 1µg/mL,

Sigma-Aldrich, St. Louis, MO). For cellular proliferation, islet samples were cultured with

10µM 5-ethynyl-2´-deoxyuridine (EdU, Molecular Probes, Eugene, OR) for 48 hours prior to embedding. Proliferating β-cells were stained for EdU (Click-iT EdU Alexa Fluor 488

HCS Assay) and co-stained with anti-insulin and DAPI as described above. Fluorescent images were visualized on the Leica DM5500 microscope system at 20x magnification and digitally captured with an ORCA-Flash4.0 LT Digital CMOS Camera C11440

(Hamamatsu Phototonics K.K, Japan) using HCImage Live software (Hamamatsu

Phototonics K.K, Japan). Morphometric analysis was performed with ImagePro 6.3

374 software (Media Cybernetics, Silver Spring, MD). The percentage of insulin positive cells was calculated as the number of cells positive for insulin divided by the total cell nuclei

(minimum 10,000 per isolation). The percentage of proliferating β-cells was calculated as the number of cells positive for both EdU and insulin divided by the total number of insulin positive cells

RNA Library Preparation. 2500IE samples were washed twice with 1X Dulbecco’s phosphate-buffered saline (DBPS) to remove serum, and RNA was then isolated using an

RNeasy Mini Kit (Qaigen, Valencia, CA) according to the manufactures instruction’s. 2 x

100 paired end reads were prepared using the Kapa Biosystems Stranded mRNA Kit

(Kapa Biosystems, Wilmington, MA). Sequencing was conducted on an Illumina HiSeq

2500 platform. Four samples were sequenced per lane.

RNA Sequencing (RNAseq) Analysis. Reads were trimmed using Trimmomatic to remove Illumina adapters and remove low quality sequence data using the following parameters: PE_TruSeq_adapt_Index5UnivRC.fa:2:30:11:1:true, sliding window 4:15, trailing 3 and min length 2041. Mapping and differential expression analyses were conducted using the Tuxedo RNAseq analysis pipeline42. Mapping of trimmed reads was performed with Tophat 2.0.9 using Bowtie2 2.1.0. Reads were mapped using both genome and transcriptome references (Sus scrofa, Ensembl version 82) and default parameters.

Transcripts were assembled and quantified using Cufflinks 2.2.1. The --max-bundle-frags option was set to 5,000,000 to allow for the inclusion of highly expressed transcripts such as insulin in the quantification and differential expression analyses. Cufflinks files were merged using Cuffmerge.

Functional Analysis of DE Genes. Analysis of significantly altered pathways was conducted using the Annotate + Identify function in the KOBAS 3.043. Significantly up or down regulated genes were submitted for Kyoto Encyclopedia of Genes and Genomes

(KEGG) Pathway results. Network analysis was conducted with NetworkAnalyst, using the

375

IMEx Interactome protein-protein interaction database and zero-order network options44,45. For input into NetworkAnalyst, porcine Ensembl gene IDs were converted to human orthologs with one to one homology type using BioMart (www.ensembl.org).

Statistics. For parameters excluding molecular profiling, statistical significance was determined in GraphPad Prism 6.07 (GraphPad Software, Inc., La Jolla, CA) using a

Kruskal-Wallis test with Dunn’s correction for multiple comparisons. For RNAseq, statistical analysis was performed using CuffDiff with the --max-bundle-frags option set to

5,000,000. Genes were considered differentially expressed if the if the Benjamini-

Hochberg corrected p-value was ≤0.05. Pathways in the KEGG database were deemed significantly enriched using KOBAS 3.0 if the corrected p-value was ≤0.05 as determined by Fisher’s Exact Test with Benjamini-Hochberg correction. Pathways were manually reviewed to exclude enriched pathways that only contained proteins which were identified in multiple other pathways or were disease specific.

RESULTS

Oxygen Consumption and Membrane Integrity. Oxygen demand was found to be similar across age groups (Fig. 1A). The percentage of cells with intact membranes increased with donor age, and was significantly higher for API as compared to NPI

(p<0.01, Fig. 1B).

Insulin secretion and β-cell composition. GSIS for API, JPI, and NPI are shown in Fig.

2A-C respectively. Insulin secretion rates from JPI and NPI were lower under both basal and stimulated conditions compared to insulin secretion from API, as were stimulation indices (Table 1). Relative to total secretion, a greater proportion of insulin was secreted under basal conditions from JPI and NPI versus API (Fig. 3A, p<0.01). The proportion of insulin secreted during the first phase was not different across age groups, while second phase secretion was lower for JPI and NPI compared to API (Fig. 3B & C, p<0.01). Islet

376 insulin content normalized to DNA and percentage of β-cells were significantly higher for

API versus either JPI or NPI (Fig. 4A & B). As shown in Fig. 2D-F, NPI and JPI secrete a higher proportion of their total insulin content than API.

β-cell Proliferation. The percentage of proliferating β-cells in API, JPI, and NPI is shown in Fig. 5. NPI have a higher percentage of proliferating β-cells than API (p<0.05). Although the sample size for JPI was too small to determine significance, their rate of β-cell proliferation was similar to NPI.

Functional Analysis of RNAseq. Due to the substantial similarities between JPI and NPI observed in other assessments, only API and NPI samples were compared using

RNAseq. Expression data from each sample is shown in Supplemental Table 1 for differentially expressed transcripts. Pathways enriched in NPI are shown in Table 2 and include extracellular matrix (ECM)-receptor interaction, signaling pathways regulating pluripotency of stem cells, and cell cycle. Pathways enriched in API are listed in Table 3.

In concordance with the results of physiological assessments, significantly enriched pathways include Protein Export and Insulin Secretion. Changes in expression of DE transcripts associated with insulin secretion and cell cycle KEGG pathways are shown in

Fig. 6. Network analysis results are summarized in Supplemental 2. Two subnetworks were derived for genes with significantly higher expression in NPI and five subnetworks were derived for genes with significantly higher expression in API. Within the subnetworks for genes enriched in NPI, nodes with the highest connectivity (>100 connections) included hepatocyte nuclear factor 4 alpha (HNF4A), MYC proto-oncogene BHLH transcription factor (MYC), specificity protein 1 (SP1), fibronectin 1 (FN1), and cyclin dependent kinase 2 (CDK2). Within the subnetwork for genes enriched in API, the most highly connected node was amyloid beta precursor protein (APP).

377

DISCUSSION

The implementation of new gene editing technologies such as CRISPR/Cas9, as well as the use of micro- and macro-encapsulation strategies to bypass the need for immunosuppression have made the use of porcine islets an increasingly pragmatic approach to address the shortage of available human donor tissue8,9,21,46. This study presents a comprehensive evaluation of relevant physiological characteristics of porcine islets from three donor ages under a unified set of protocols. Parameters such as oxygen demand were found to be similar across age groups, while membrane integrity, β-cell proliferation, the kinetics of insulin secretion, and transcriptomes were found to differ in an age dependent manner.

An enduring concern for islet survival post-transplantation, especially in encapsulated environments, is ischemic stress26,47-49. In developing adjacent technologies to address this issue, for example oxygen generating biomaterials, it is important to ensure that the oxygen demand of the transplanted tissue is met50. In the present study, no differences in basal oxygen demand were observed across donor ages. Although oxygen demand appears to be consistent across donor ages, NPI expressed a significantly higher number of transcripts for lactate dehydrogenase alpha (LDHA), which allows for adenosine triphosphate (ATP) production under anaerobic conditions. While this likely reflects the different cell populations that comprise the sequenced islets, it does support previously published results that NPI resist hypoxic damage, though further study will be required to understand whether this translates to improved survival in encapsulated environments, particularly when coupled with enhanced oxygenation25. Although oxygen consumption rate has previously been reported to correspond to insulin secretion, no such correlation was observed here51.

Membrane integrity of the islets evaluated was age dependent, with NPI having the highest proportion of compromised cells. This may be attributed to the cell turnover

378 associated with normal pancreatic development52. Importantly, although FDA/PI is still used as a standard measure of islet viability, it fails to predict diabetes reversal53.

OCR/DNA, which in this case did not vary by age, is a predictive measure of diabetes reversal, and therefore may be a more informative measure of islet quality than FDA/PI staining53.

β-cell proliferation, although still low, was higher on average for JPI and NPI. The rate of

API β-cell proliferation is similar to that previously reported for adult mice54. Likewise, pathway analysis showed an enrichment in cell-cycle and DNA replication associated transcripts. Network analysis also identified HNF4A, which is critical for β-cell expansion, as a key signaling node in NPI55.

Consistent with the existing literature, this study found substantially lower insulin content and number of β-cells within JPI and NPI compared to API28,31. Dynamic GSIS analysis also revealed nearly identical secretion kinetics for JPI and NPI. While API secreted a greater absolute amount of insulin, their secretion profile also revealed proportionally more insulin secreted during the second phase. This is consistent with higher expression of transcripts including calcium voltage gated channel subunit alpha 1 E (CACNA1E), which encodes a Cav2.3 calcium channel required for second phase insulin secretion, and protein tyrosine phosphatase, receptor type N (PTPRN), which is involved in insulin expression and vesicle accumulation56-58. Indeed, several transcripts including RAS- associated protein RAB3A (RAB3A), vesicle associated membrane protein 2 (VAMP2), and synaptosome associated protein 25 (SNAP25) involved in insulin vesicle cycling all have significantly lower expression in NPI59,60. These results are consistent with a previously published high throughput sequencing comparison of sorted adult and fetal human β-cells61. This would indicate that these findings cannot be attributed solely to differing islet cell compositions, and the insulin secretory machinery of JPI and NPI β-cells is likely still immature.

379

A previously published study compared the insulin secretion of API to those isolated from pigs aged 3 months or younger. Notably, while relative insulin content for the islets in

Mueller, et al. was slightly higher compared to API than was observed in the present study, the difference in peak insulin secretion rates was consistent with the findings presented here28. Although a more detailed evaluation will be needed, this indicates that it may not be efficient or economical to use islets from older (several months) animals, as function is not proportionally increased. Interestingly, while insulin secretion in vitro differs by at least an order of magnitude for JPI and NPI versus API, NHP studies have achieved diabetes reversal using NPI with a dose just two times higher than used for API15,16,19. In vitro maturation has also been shown to improve the glucose responsiveness of JPI, though further study will be needed to understand the capacity for islets from younger porcine donors to mature in vivo24.

Finally, a persistent impediment to the use of porcine islets is their xenoantigenicity.

Humans have pre-formed antibodies which can react to carbohydrate epitopes present on the surface of porcine cells. The most prominent of these is galactose-α-1,3-galactose, which contributes significantly to the hyperacute rejection of porcine tissues62,63. The use of islets from animals lacking these epitopes (α-1,3-galactosyltransferase [GGTA1] knockout) has been shown to improve graft survival and transplant outcomes19. The elimination of another carbohydrate xenoantigen N-glycolylneuraminic acid (Neu5Gc), synthesized by cytidine monophosphate-N-acetylneuaminic acid hydroxylase (CMAH), has also been demonstrated to reduce the binding of pre-formed human antibodies64.

CMAH and GGTA1 double knockout pigs do not appear to have altered insulin secretion65.

Consistent with previous studies, our data indicate a higher number of GGTA1 transcripts in NPI versus API, but also a higher number of CMAH transcripts29. However, von

Willebrand factor (VWF) has significantly higher expression in API, and has been linked with graft dysfunction due to platelet activation66. Given that GGTA1, CMAH, and VWF

380 knockout pigs have already been created, it is likely that immunologic factors will continue to be modified and may not preclude the use of any individual age in the future66,67.

However, when engineering optimal porcine islets for transplantation, age dependent xenoantigen expression should be taken into consideration so that appropriate genetic models are used.

This study presents a unified characterization of API, JPI, and NPI. In addition to providing benchmarks for parameters such as oxygen demand, this study presents the first high- throughput sequencing comparison of islets from different ages of porcine donors. The results of this study will guide the optimal application of each age of porcine islet, and aid in the eventual treatment of patients with type 1 diabetes.

ACKNOWLEDGEMENTS

We would like to acknowledge the members of the Papas Laboratory, including Anna

Maria Burachek, Alexandra Hoeger, Erik Rohner, and Ivan Georgiev. We would also like to thank the University of Arizona Genetics Core, especially Jonathan Galina-Mehlman, the T32HL007249 Training Grant, and the ARCS Foundation Phoenix Chapter for their support.

FUNDING

This research was supported by JDRF grant #2-SRA- 2014-289- Q-R. JPI supply was supported by JDRF grant #17-2013-288.

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TABLES

Parameter API (n=11) JPI (n=5) NPI (n=6) Basal Insulin Secretion 1.02 ± 0.12 0.19 ± 0.04* 0.14 ± 0.05** (pg insulin/ng DNA) Stimulated Insulin Secretion 7.23 ± 0.87 0.15 ± 0.06** 0.12 ± 0.05** (pg insulin/ng DNA) Stimulation Index 8.5 ± 1.2 1.8 ± 0.2** 1.8 ± 0.3** (Stimulated/Basal Secretion)

Table 1. Analysis of insulin secretion for API, JPI, and NPI. GSIS profiles were integrated to determine insulin secreted under basal and stimulated conditions, as well as stimulation indices for API, JPI, and NPI. Values represent mean ± SEM. * indicates p<0.05 vs. API. ** indicates p<0.01 vs. API.

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Input Background Corrected P- Pathway number number Value Axon guidance 60 134 3.0112E-07 Focal adhesion 63 147 3.0112E-07 ECM-receptor interaction 36 57 3.7726E-07 PI3K-Akt signaling pathway 84 248 2.3240E-06 Regulation of actin cytoskeleton 61 167 1.5111E-05 Rap1 signaling pathway 57 159 4.5152E-05 Hippo signaling pathway 42 103 7.5997E-05 Leukocyte transendothelial migration 39 95 0.0001 Protein digestion and absorption 29 63 0.0003 MAPK signaling pathway 58 192 0.0014 Bacterial invasion of epithelial cells 26 60 0.0017 Cell adhesion molecules (CAMs) 37 106 0.0024 Tight junction 36 104 0.0030 Cytokine-cytokine receptor interaction 54 186 0.0038 Adherens junction 24 58 0.0038 Fc gamma R-mediated phagocytosis 27 70 0.0039 Hippo signaling pathway -multiple 12 18 0.0049 species DNA replication 15 29 0.0072 Ras signaling pathway 50 178 0.0083 Cell cycle 31 93 0.0083 Gap junction 24 66 0.0114 Signaling pathways regulating 33 105 0.0120 pluripotency of stem cells Endocytosis 53 204 0.0218 Phospholipase D signaling pathway 32 107 0.0244 Pancreatic secretion 23 68 0.0253 Complement and coagulation 23 69 0.0286 cascades EGFR tyrosine kinase inhibitor 21 62 0.0348 resistance Proximal tubule bicarbonate 10 20 0.0427 reclamation TGF-beta signaling pathway 21 64 0.0434 Bile secretion 19 56 0.0460 Wnt signaling pathway 28 96 0.0460 Oxytocin signaling pathway 33 120 0.0489

Table 2. Pathways Enriched in NPI compared to API. DE genes identified by RNAseq which belong to the specified pathway are represented by the input number, while the background number represents the number of all genes in the pathway.

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Pathway Input number Background number Corrected P-Value Protein processing in 40 129 0.0019 endoplasmic reticulum Protein export 11 18 0.0322 Insulin secretion 20 59 0.0480 Metabolic pathways 168 982 0.0480

Table 3. Canonical Pathways Enriched in API compared to NPI. DE genes identified by RNAseq which belong to the specified pathway are represented by the input number, while the background number represents the number of all genes in the pathway.

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FIGURE LEGENDS

Figure 1. Oxygen Demand and Membrane Integrity by Donor Age. (A) Oxygen consumption rate normalized to DNA is shown for API (n=11), JPI (n=5), and NPI (n=6).

No significant differences between age groups are observed. The % cell membrane integrity of API (n=9), JPI (n=5), and NPI (n=6) is given in (B). ** indicates p<0.01 vs. API.

All plots shown represent the median, lower and upper quartiles, and minimum and maximum of each dataset.

Figure 2. Dynamic GSIS Profiles by Donor Age. (A) GSIS profile for API (n=11). (B) GSIS profile for JPI (n=5). Expanded axes are provided within the main graph area. (C) GSIS profile for NPI (n=6). Expanded axes are provided within the main graph area. Error bars indicate SEM. Figures D-F reflect A-C normalized to total insulin content/DNA (API n=8,

JPI n=5, NPI n=5).

Figure 3. Fractional Insulin Secretion by Donor Age. (A) Fraction of basal insulin secretion vs. donor age. ** indicates p<0.01 vs. API. (B) Fraction of first phase insulin secretion vs. donor age. No significant differences are observed between age groups. (C) Fraction of second phase insulin secretion vs. donor age. ** indicates p<0.01 vs. API. All plots shown represent the median, lower and upper quartiles, and minimum and maximum of each dataset. API n=8, JPI n=5, NPI n=5.

Figure 4. Insulin Content and β-cell Composition by Donor Age. (A) Total insulin content normalized to DNA is shown for API (n=8), JPI (n=5), and NPI (n=5). * indicates p<0.05 vs. API, ** indicates p<0.01 vs. API. Expanded axes are provided within the main graph area. (B) The percentage of insulin positive cells per islet for API (n=5), JPI (n=3), and NPI

394

(n=6) is shown. ** indicates p<0.01 vs. API. All plots shown represent the median, lower and upper quartiles, and minimum and maximum of each dataset.

Figure 5. β-Cell Proliferation by Donor Age. The percentage of proliferating β-cells

(defined as positive for insulin and EdU) is shown for API (n=3), JPI (n=2), and NPI (n=5).

* indicates p<0.05 vs. API.

Figure 6. Differentially Expressed Genes in Insulin Secretion and Cell Cycle Pathways.

Shown in (A) are DE genes associated with the KEGG insulin secretion pathway which are enriched in API, while (B) shows DE genes associated with the cell cycle which are enriched in NPI. Fold changes are NPI relative to API.

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