Normothermic Ex Vivo Kidney Perfusion - a Novel Approach for the Storage, Assessment, and Repair of Renal Grafts

Normothermic Ex Vivo Kidney Perfusion - A Novel Approach for the Storage, Assessment, and Repair of Renal Grafts

Johann Moritz Kaths, MD

A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto

Normothermic Ex Vivo Kidney Perfusion - A Novel Approach for the Storage, Assessment, and Repair of Renal Grafts

Johann Moritz Kaths, MD

Master of Science

Institute of Medical Science University of Toronto

2016

Abstract

Kidney transplantation is the treatment of choice for patients with end-stage renal disease. A major problem is the worldwide severe graft shortage, which triggered interest in kidneys recovered from extended criteria donors (ECD) and donation after circulatory death (DCD).

Currently utilized techniques of hypothermic preservation induce injury, especially in ECD and

DCD grafts. This study investigates the feasibility and safety to preserve kidneys under normothermic, more physiologic conditions ex vivo. In a porcine model, kidneys were recovered, and preserved on ice or with “Normothermic Ex Vivo Kidney Perfusion (NEVKP)”. Kidneys preserved at normothermia providing physiologic flow, oxygen, and nutrition, demonstrated high metabolic activity and excellent graft viability during storage. Following autotransplantation, superior renal function with lower serum creatinine, higher creatinine clearance, and lower renal injury markers was demonstrated when compared to cold storage. This data suggests that

NEVKP offers superior storage and provides a platform for further graft assessment and repair.

Acknowledgments

Most grateful I am to my supervisors and mentors Dr. Lisa Robinson and Dr. Markus Selzner. Having given me the opportunity to commence this research project in Toronto turned out to be a unique experience and a very important step for my scientific and medical career as well as my personal life. Both being experts in their fields, they provided outstanding support, many helpful suggestions, and were always available to discuss novel approaches and find proper solutions. I am looking forward for many more collaboration with both of them in the future.

Furthermore, I want to thank my PAC members Dr. Darius Bagli and Dr. Craig Simmons, and the members of the “NEVKP-group” Dr. David Grant, Dr. Anand Ghanekar, and Dr. Istvan Mucsi for their professional advices in our biweekly meetings. Having been part of a group with such varying scientific and medical backgrounds was tremendously helpful for the success of this project here in Toronto. I thank Dr. Rohan John and Dr. Paul Yip for their great cooperative support in histological and biochemical analyses.

A very special thanks goes to Dr. Juan Echeverri. Having joined the “Toronto Organ Preservation Laboratory” by the end of 2014 was definitely key to this project. His tremendous support, generosity, and motivation to get this project going – especially while developing the transplant model – were amazing. His altruistic personality helped to push the NEVKP project to the same level of the preexisting liver perfusion model. In addition I thank Dr. Vinzent Spetzler and Dr. Nicolas Goldaracena, who enrolled me in the liver project at the beginning when I arrived in Toronto and helped translating the knowledge from the liver to the kidney perfusion setting.

Having established NEVKP and the heterotopic autotransplantation model, many more people helped to increase the number of experiments. Danny Cen and Yi-Min Chun joined many perfusions and transplants, staying long hours, and even doing overnight shifts. Dr. Ivan Linares joined the lab in August 2015 and since then participated in many kidney transplants, demonstrating great efforts to support the project further. Luke Dingwell and Aryn Wiebe were two more irreplaceable members of the kidney perfusion team, joining experiments, developing technical devices, and working on projects related to NEVKP. Sujani Ganesh and Kristine Louis were huge supporters of the NEVKP project. They joined experiments, performed further

iii analyses such as ELISAs, and performed most of the administrative work in our laboratory. IMS Master’s students Roberto Robiero, Chia Wei Teoh, Martin Sidler, and Michael Myunghyun made the courses and seminars fun and enjoyable. Fruitful discussions led to novel ideas and cooperation projects. Jessica Fairlie and Bernard Singh were incredibly helpful to organize meetings, and support in administrative issues.

Without the tireless support of the Animal Resources Centre, namely Guin Arciszewska, Walter Ingles, Shelley Belford, Shawna Lussier, and Tihomir Dryanovski, all the experiments would have never been performed in this way. The veterinarians Dr. Badru Moloo, Dr. Alyssa Goldstein, and Dr. Amanda Healy were always available to discuss improvements of research approaches.

Another very special thanks goes to Walter Propawa from Sorin Group, now LivaNova. His tireless effort supporting us with cardiopulmonary bypass technology and personal experience were amazing. Furthermore, I want to thank Andrew Dooley and Paul Parsons for their support. In addition, I want to thank John Houde from Braun, XVivo, Rieber as well as the John David and Signy Eaton Foundation.

Finally, I want to thank my parents and siblings for their encouragement and support throughtout the whole project. Lastly, all of my accomplishments to date would not have been feasible without the support of my girlfriend Vanessa Hissnauer. Having left Germany to join me for most of the time here in Toronto made my stay enjoyable, unique, and one of the most important periods for our joint future life.

Contributions

Data presented here was performed in collaboration with several individuals. My colleagues Dr. Juan Echeverri, Dr. Ivan Linares, Dr. Vinzent Spetzler, Dr. Nicolas Goldaracena, Danny Cen, Yi-Min Chun, Luke Dingwell, and Aryn Wiebe assisted with surgeries and animal care during postoperative follow up. Kristine Louis and Sujani Ganesh supported in isolation of erythrocytes, performance of ELISAs, and administrative procedures in our laboratory. Dr. Rohan John supported in evaluation of renal histology and Dr. Paul Yip and his team of the Core Laboratory performed some of the biochemical analyses.

Financially, this work was supported by the John David and Signy Eaton Foundation. Sorin Group (Milano, Italy), now LivaNova PLC (London, UK), provided cardiopulmonary bypass equipment, XVivo Perfusion Inc. (Goteborg, Sweden) provided STEENTM Solution, and Braun AG (Melsungen, Germany) provided syringe pumps.

Furthermore, the following student awards were won with this project and contributed financially:

- 2015 Faculty of Medicine GSEF Merit Scholarships for International Students from the Office of Graduate & Life Sciences - 2015 Institute of Medical Science International Student Fee Differential Award - 2015-16 Institute of Medical Science International Fee Subsidy Award - 2015-16 Institute of Medical Science Open Fellowship Award

Within the Multi Organ Transplant Program, the following award was won and supported the project financially:

- 2015-16 MOT Research Fellowship Competition

This work was performed as part of the Canadian National Research Program (CNTRP).

TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... III

CONTRIBUTIONS ...... V

LIST OF TABLES ...... X

LIST OF ABBREVIATIONS ...... XI

LIST OF FIGURES ...... XV

LIST OF APPENDICES ...... XIX

1 LITERATURE REVIEW ...... 1

1.1 Renal Transplantation ...... 2 1.1.1 Chronic Kidney Disease and Renal Transplantation ...... 2 1.1.2 Logistics and Surgical Procedure ...... 2 1.1.3 Postsurgical Aspects ...... 4 1.1.4 Historical Context of Renal Transplantation and Preservation ...... 7

1.2 Renal Graft Shortage ...... 10

1.3 Renal Graft Donor Sources ...... 12 1.3.1 Deceased Donation ...... 13 1.3.2 Living Donation ...... 15 1.3.3 Outcome by Donor Source ...... 16 1.3.4 Kidney Donor Profile Index – The New Allocation System ...... 18 1.3.5 Logistic Challenges in Kidney Donation ...... 19

1.4 Renal Ischemia Reperfusion Injury ...... 20 1.4.1 Endothelial Cells and Vascular Components of IRI ...... 20 1.4.2 Inflammation – Immune Response and Cells Enrolled in IRI ...... 21 1.4.3 Complement Cascade Activation in IRI ...... 23 1.4.4 Hypothermia in Renal IRI ...... 23

1.5 Renal Graft Preservation ...... 24 1.5.1 Principles of Static Cold Storage and Preservation Solutions ...... 24 1.5.2 Principles of Hypothermic Machine Perfusion ...... 28 1.5.3 Hypothermic Machine Perfusion vs. Static Cold Storage ...... 28 1.5.4 Subnormothermic Preservation ...... 30 1.5.5 Normothermic Preservation ...... 30

1.6 Importance of the topic ...... 33

2 HYPOTHESES AND AIMS ...... 34

3 NORMOTHERMIC EX VIVO KIDNEY PERFUSION FOR THE PRESERVATION OF KIDNEY GRAFTS PRIOR TO TRANSPLANTATION ...... 37

3.1 Introduction ...... 38

3.2 Materials and Methods ...... 40 3.2.1 Animals ...... 40 3.2.2 Organ Retrieval ...... 40 3.2.3 Normothermic Ex Vivo Kidney Perfusion (NEVKP) ...... 42

3.3 Results ...... 46

3.4 Discussion ...... 50

4 HETEROTOPIC RENAL AUTOTRANSPLANTATION IN A PORCINE MODEL: A STEP-BY-STEP PROTOCOL ...... 52

4.1 Introduction ...... 53

4.2 Materials and Methods ...... 55 4.2.1 Animals ...... 55 4.2.2 Kidney Graft Retrieval ...... 55 4.2.3 Kidney Graft Transplantation ...... 57 4.2.4 Postsurgical Follow Up ...... 61

4.3 Results ...... 62

4.4 Discussion ...... 65

vii

5 EIGHT HOUR CONTINUOUS NORMOTHERMIC EX VIVO KIDNEY PERFUSION IS A SAFE PRESERVATION TECHNIQUE FOR KIDNEY TRANSPLANTATION: A NEW OPPORTUNITY FOR THE STORAGE, ASSESSMENT, AND REPAIR OF KIDNEY GRAFTS ...... 68

5.1 Introduction ...... 70

5.2 Materials and Methods ...... 72 5.2.1 Study Design ...... 72 5.2.2 Animals ...... 72 5.2.3 Normothermic Ex Vivo Kidney Perfusion ...... 72 5.2.4 Static Cold Storage ...... 76 5.2.5 Kidney Retrieval and Transplantation ...... 76 5.2.6 Whole Blood, Serum, and Urine Measurements ...... 77 5.2.7 Histology ...... 78 5.2.8 Statistical Analysis ...... 78

5.3 Results ...... 79 5.3.1 Demographics ...... 79 5.3.2 Perfusion Characteristics during NEVKP ...... 79 5.3.3 NEVKP was associated with maintenance of physiologic biochemical parameters in the perfusate 81 5.3.4 NEVKP vs. SCS results in comparable graft function and injury after kidney transplantation ...... 83 5.3.5 NEVKP did not compromise animal survival ...... 85

5.4 Discussion ...... 86

6 GENERAL DISCUSSION AND CONCLUSION ...... 90

6.1 Normothermic Preservation of Ischemically Injured Renal Grafts ...... 92

6.2 Improving Organ Storage ...... 93

6.3 Improving Organ Assessment ...... 97

6.4 Improving Organ Repair ...... 98

6.5 Conclusion and Clinical Trial ...... 99

7 FUTURE DIRECTIONS ...... 100

viii

REFERENCES ...... 103

APPENDICES ...... 116

List of Tables

Chapter 1

Table 1.1 Allograft dysfunction classified by time and underlying causes.

Table 1.2 Grouping of grafts recovered in donation after circulatory death by the revised Maastricht Classification.

Table 1.3 Preservation solutions for static cold storage and hypothermic machine perfusion.

Chapter 3

Table 3.1 Kidney graft shortage in the US and Eurotransplant Region.

Chapter 5

Table 5.1 Composition of the perfusate solution.

Table 5.2 Blood gas analyses, osmolarity, and oncotic pressure at baseline.

Table 5.3 Histological findings.

Chapter Appendices

Table A.1 Reasons for kidney transplantation.

List of Abbreviations

AHR Adjusted Hazard Ratio

AOR Adjusted ODDs Ratio

AST Aspartate Aminotransferase

ATN Acute Tubular Necrosis

ATP Adenosintriphosphat

AMR Antibody-Mediated Rejection

BGA Blood Gas Analysis

BPH Benign Prostatic Hyperplasia

BUN Blood Urea Nitrogen

CAN Chronic Allograft Nephropathy

CNI Calcineurin Inhibitor

CPDA Citrate, Phosphate, Dextrose, Adenosine

CPR Cardiopulmonary Resuscitation

CKD Chronic Kidney Disease

CT Computed Tomography

CTS Collaborative Transplant Study

DBD Donation After Brain Death

DCD Donation After Circulatory Death / Donation After Cardiac Death

DD Deceased Donation

DGF Delayed Graft Function

DMS Data Management System dRO Double Reverse Osmosis

EAD Early Allograft Dysfunction

EC Euro-Collins

ECD Expanded Criteria Donor / Extended Criteria Donor

EGL Early Graft Loss

EMS Exsanguinous Metabolic Support

ESRD End-Stage Renal Disease

EVNP Ex Vivo Normothermic Perfusion

GFR Glomerular Filtration Rate

HBD Heart-Beating Donor

H&E Hematoxylin and Eosin

HES Hydroxyethyl Starch

HLA Human Leukocyte Antigen

HMP Hypothermic Machine Perfusion

HOC Hyperosmolar Citrate

HPF High Power Fields

HTK Histidine-Tryptophan-Ketoglutarate

xii

IGL-1 Institute George Lopez – 1

IVC Inferior Vena Cava

IRI Ischemia Reperfusion Injury

IRR Intrarenal Resistance

KDPI Kidney Donor Profile Index

KTx Kidney Transplantation

LD Living Donation

LDKT Live Donor Kidney Transplantation

LDH Lactate Dehydrogenase

MAP Mean Arterial Pressure

MMF Mycophenolate Mofetil

MPS Machine Perfusion Solution

MRI Magnetic Resonance Imaging

NDD Neurologic Determination of Death

NHBD Non Heart-Beating Donor

NP Normothermic Preservation

NEVKP Normothermic Ex Vivo Kidney Perfusion

OPTN Organ Procurement and Transplantation Network

OR Operating Room

PAS Periodic acid – Schiff stain

xiii

PEG Polyethylene Glycol

PNF Primary Non-Function

POD Postoperative Day

RCT Randomized Controlled Trial

ROS Reactive Oxygen Species

SCD Standard Criteria Donor

SCS Static Cold Storage

SD Standard Deviation

SLA Swine Leukocyte Antigen

SRTR Scientific Registry of Transplant Recipients

TBI Total Body Irradiation

UW University of Wisconsin

WHO World Health Organization

WI Warm Ischemia

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List of Figures

Chapter 1

Figure 1.1 Heterotopic kidney transplantation.

Figure 1.2 Historical timeline demonstrating developments in renal transplantation (top section) and renal preservation (lower section).

Figure 1.3 Kidney transplantation activities in 2013 worldwide.

Figure 1.4 “The need continues to grow”.

Figure 1.5 Renal grafts recovered and transplanted (in 1000) in the US by donor type.

Figure 1.6 Percentage of each kidney transplant type among all deceased-donor kidney-alone transplants.

Figure 1.7 Renal grafts recovered for transplantation but not transplanted, by DCD and ECD donor status.

Figure 1.8 Death-censored graft failure within 90 days among adult kidney transplant recipients.

Figure 1.9 Graft survival among adult kidney transplant recipients, 2008: deceased donors.

Figure 1.10 Graft survival among adult kidney transplant recipients, 2008: living donors.

Figure 1.11 Endothelial injury in renal IRI.

Figure 1.12 Immune response in renal IRI.

Chapter 3

Figure 3.1 Study Protocol.

Figure 3.2 Schematic Drawing of the Perfusion Circuit.

Figure 3.3 Mean arterial flow with standard deviation (mL/min).

Figure 3.4 Intrarenal resistance (IRR), mean and standard deviation (mmHg/mL/min).

Figure 3.5 Total urine volume, mean and standard deviation (mL).

Figure 3.6 Oxygen consumption, mean and standard deviation (mL/min/g).

Figure 3.7 pH venous, mean and standard deviation.

Figure 3.8 HCO3- venous, mean and standard deviation (mmol/L).

Figure 3.9 Venous sodium concentration, mean and standard deviation (mmol/L).

Figure 3.10 Venous potassium concentration, mean and standard deviation (mmol/L).

Figure 3.11 Venous aspartate aminotransferase, mean and standard deviation (AST; U/L).

Figure 3.12 Lactate, mean and standard deviation (mmol/L).

Figure 3.13 Osmolarity of the serum, mean and standard deviation (mosm/L).

Figure 3.14 Histology (H&E).

Figure 3.15 Histology (PAS).

Figure 3.16 Histology (TUNEL staining).

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Chapter 4

Figure 4.1 Study Protocol.

Figure 4.2 Serum creatinine values.

Figure 4.3 Serum BUN values.

Figure 4.4 Acid-base hemostasis.

Figure 4.5 Electrolyte levels.

Figure 4.6 Histology (H&E), 100X magnification.

Figure 4.7 Histology (H&E), 100X magnification.

Chapter 5

Figure 5.1 Schematic of the NEVKP Circuit.

Figure 5.2A Renal artery blood pressure during Normothermic Ex Vivo Kidney Perfusion.

Figure 5.2B Pressure in the renal vein during Normothermic Ex Vivo Kidney Perfusion.

Figure 5.2C Renal artery flow during Normothermic Ex Vivo Kidney Perfusion.

Figure 5.2D Intrarenal resistance during Normothermic Ex Vivo Kidney Perfusion.

Figure 5.3 Cumulative urine output during Normothermic Ex Vivo Kidney Perfusion.

Figure 5.4A pH of the perfusate during Normothermic Ex Vivo Kidney Perfusion.

Figure 5.4B HCO3- concentration in the perfusate during Normothermic Ex Vivo Kidney Perfusion.

Figure 5.5 Lactate levels in renal perfusate during Normothermic Ex Vivo Kidney Perfusion. xvii

Figure 5.6A Serum creatinine of the transplanted animals during 10 day postoperative follow-up for autologous kidney transplantation following SCS and NEVKP.

Figure 5.6B Serum BUN/urea during 10 day postoperative follow-up for autologous kidney transplantation following SCS and NEVKP.

Figure 5.6C Serum potassium during 10 day postoperative follow-up for autologous kidney transplantation following SCS and NEVKP.

Figure 5.6D 24-hour creatinine clearance during 10 day postoperative follow-up

Chapter Appendices

Figure A.2 Sorin SIII Heart-Lung-Machine, modular system.

Figure A3.1 Components of the customized perfusion circuit (1).

Figure A3.2 Components of the customized perfusion circuit (2).

Figure A4.1 Schematic drawing of customized, double-walled kidney perfusion chamber (1).

Figure A4.2 Schematic drawing of customized, double-walled kidney perfusion chamber (2).

Figure A.6 Renal graft perfused under normothermic conditions ex vivo.

Figure A7.1 Customized flooring to separate feces and food from urine in the metabolic cage.

Figure A7.2 Customized metabolic cage for 24-hour urine collection.

xviii

List of Appendices

A1 Reasons for kidney transplantation.

A2 Perfusion device.

A3 Perfusion circuit.

A4 Customized organ chamber.

A5 Red cell isolation protocol.

A6 Perfused renal graft.

A7 Metabolic cage.

A8 Portable perfusion device.

xix 1

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1 Literature Review

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1.1 Renal Transplantation

1.1.1 Chronic Kidney Disease and Renal Transplantation

Chronic kidney disease (CKD), also referred to as chronic renal disease, is a condition characterized by gradual loss of renal function over a period of time. As per definition, CKD is present when the glomerular filtration rate drops below 60 mL/min for more than 3 months or when the urine albumin-to-creatinine ratio is over 30 mg of albumin for each gram (g) of creatinine (30 mg/g). The two main causes for CKD are diabetes and high blood pressure. Currently, the approximate prevalence of CKD in the general American population is 14%, demonstrating an increased tendency throughout the last years. Progression of CKD finally leads to kidney failure, so called end-stage renal disease (ESRD). Either dialysis or kidney transplantation are then required to maintain vital life functions 1.

Kidney transplantation is currently the treatment of choice for end-stage renal disease (ESRD). Compared to dialysis, it results in lower rates of morbidity and mortality, improved quality of life, and reduced health care costs 2-4. Since January 1st 1988, 384,691 renal transplantations (> 400,000 since the first successful renal transplant) have been performed in the US; 251,620 were recovered in deceased donors, 133,071 in living donors. Annually, between 16,000 and 17,000 kidney transplants are performed in the US 5. However, a major limitation is the worldwide shortage of organs available for transplantation 6-9. The 117,733 organs transplanted in 2013 represent less than 10% of the global needs 6 (for detailed information see Chapter 1.2).

Indications for renal transplantation are diverse and can be divided by diagnostic category. Reasons for kidney transplantation are glomerular diseases, diabetes (type I and II), polycystic kidneys, hypertensive nephrosclerosis, renovascular and other vascular diseases, congenital, rare familial and metabolic disorders, tubular and interstitial diseases, neoplasm, retransplantation/ graft failure, and “other” indications (see Appendix A1) 10.

1.1.2 Logistics and Surgical Procedure

Logistic challenges for renal transplantation include graft recovery, graft preservation for transportation from the donor to the recipient hospital, and finally transplantation itself.

Surgical graft recovery techniques differ depending on the setting of donation. In deceased donation, usually multi organ retrievals are performed. In donation after brain death (DBD) (for detailed information see Chapter 1.3.1.1), following diagnosis of cessation of brain function 11,12, appropriate donor management 13, and medical check up, the potential donor is brought to the operating room for (multi) organ recovery. Single or dual organ perfusion, dissection in the cold or warm, and single or en bloc kidney removal are performed 13. In donation after circulatory death (DCD) (for detailed information see Chapter 1.3.1.2), logistics depend on the donor category. Donors of category I, II, and V are uncontrolled, donors of category III and IV are controlled 14,15. Protocols for DCD organ recovery vary with local practices 16,17. In controlled DCD, following withdrawal of life support, determination of death, and a defined stand-off period (most commonly 5 minutes), rapid retrieval operation is performed to recover organs with lowest warm ischemic injury as possible. In uncontrolled DCD, when cardiopulmonary resuscitation (CPR) fails, following declaration of death, and a defined stand- off period, rapid organ retrieval is performed 18. In living kidney donation (LD) (for detailed information see Chapter 1.3.2), following donor evaluation, nephrectomy can be performed using open (subcostal incision vs. intercostal mini-incision) or laparoscopic approaches (retroperitoneal vs. transperitoneal; hand-assisted vs. “pure” laparoscopic) 19-21.

Following graft preservation and transportation (for details see Chapter 1.5), renal transplantation is performed hetero- or orthotopic to the left or the right side using open approaches (midline vs. upper quadrant vs. lower quadrant incision). Typically, arterial and venous vessels are anastomosed end-to-side to the iliac vessels, and the ureter is connected to the urinary bladder using the Lich-Gregoir technique (ureteroneocystostomy) 22 (Figure 1.1); technical alternatives for vessel and uretheral anastomoses do exist. Surgical complications are of vascular (bleeding, thrombosis, stenosis), ureteral (leak, stricture, reflux), or wound healing (infection, dehiscence) nature. For living donors, complications are very few.

Figure 1.1: Heterotopic kidney transplantation. Artery and vein are anastomosed end-to-side to the iliac vessels, and the ureter is connected to the urinary bladder using the Lich-Gregoir technique. Image from http://tvasurg.ca. Reproduced with permission. © The Toronto Video Atlas of Liver, Pancreas and Transplant Surgery 2016ʼ.

Perioperative mortality is about 0.03%, perioperative morbidity 1-2%. Typical complications are bleeding with need of blood transfusion, bowel injury, bowel obstruction, hernia, deep vein thrombosis/pulmonary embolism, reoperation, infection, and interruption of work/life activities 23.

1.1.3 Postsurgical Aspects

Following renal transplantation, recipients depend on lifetime immunosuppression to prevent acute rejection and graft loss. Over time, as risks of acute rejection decrease, levels of immunosuppression can be lowered to reduce the risk of infection and malignancy. The most common immunosuppressants used in renal allotransplantation are combinations of corticosteroids, azathioprine, mycophenolate mofetil (MMF), mycophenolate sodium (myfortic), cyclosporine, tacrolimus, everolimus, rapamycin (sirolimus), and betalacept 24,25. Combination of immunosuppressive drugs aims to increase effectiveness, while reducing morbidity and mortality, which are associated with each class of medication. Despite decreased rates of acute rejection during recent time and improved one-year allograft survival time, long-term renal allograft survival rates lack improvement 26,27.

Allograft dysfunction is the most common complication following renal transplantation, which ultimately can lead to graft loss. Allograft dysfunction is classified by time (immediate, early,

5 and late period) (Table 1.1). “Immediate allograft dysfunction”: Immediately post transplantation, the requirement for dialysis in the recipient within 7 days is defined as delayed graft function (DGF) 28. In donation after brain death (DBD), DGF rates of up to 50% are reported 29,30. The incidence is higher in extended criteria donors (ECD; definition see chapter 1.3.1.1.2) and donation after circulatory death (DCD) 31. In general, less than 5% of renal allografts with DGF will never function, which would then be defined as primary non-function (PNF). Principal underlying causes can be of prerenal (hypotension, volume depletion) or postrenal (urinary bladder dysfunction, undiagnosed benign prostatic hyperplasia (BPH), ureteral obstruction) nature, or due to intrinsic renal disease (postischemic acute tubular necrosis (ATN)/reperfusion injury, hyperacute and antibody-mediated rejection (AMR), thrombosis). “Early allograft dysfunction” (EAD): EAD post transplantation occurs within 1 to 12 weeks. Again, causes can be divided into prerenal (hypotension, volume depletion), postrenal (urinary bladder dysfunction, BPH, lymphoceles), and intrinsic renal disease (acute rejection, cyclosporine or FK nephrotoxicity, infection, recurrence of primary disease). “Late acute allograft dysfunction” (prerenal: volume depletion, renal artery stenosis; postrenal: urinary tract obstruction and lymphocele; intrinsic renal: cyclosporine or FK nephrotoxicity, acute rejection, recurrence of the primary disease, de novo renal disease such as ATN) and “late chronic allograft dysfunction” (chronic allograft nephropathy, calcineurin inhibitor nephrotoxicity, hypertensive nephrosclerosis, viral infections, and recurrent or de novo renal disease) occur more than three months after renal transplantation. Development of chronic allograft nephropathy (CAN) remains the most common cause for allograft loss 32.

Underlying causes for allograft dysfunction

Prerenal Postrenal Intrinsic renal disease

Immediate allograft Hypotension, Urinary bladder Postischemic ATN, dysfunction (day 0-7) volume dysfunction, hyperacute and antibody- depletion undiagnosed BPH, mediated rejection (AMR), ureteral obstruction thrombosis

Early allograft Hypotension, Urinary bladder Acute rejection, CsA or FK dysfunction (week 1-12) volume dysfunction, BPH, nephrotoxicity, infection, depletion lymphoceles recurrence of primary disease

Late acute allograft Volume Urinary tract CsA or FK nephrotoxicity, dysfunction (>3 depletion, obstruction, acute rejection, recurrence of months) renal artery lymphocele primary disease, de novo stenosis renal disease such as ATN

Late chronic allograft Chronic allograft dysfunction (>3 nephropathy, calcineurin months) inhibitor nephrotoxicity, hypertensive nephrosclerosis, viral infections, recurrent or de novo renal disease

Table 1.1: Allograft dysfunction classified by time and underlying causes. BPH, benign prostatic hyperplasia; ATN, acute tubular necrosis; AMR, antibody-mediated rejection; CsA, cyclosporine.

One-year-graft survival for kidneys recovered in deceased donation in the US are 91% for white and Hispanic Americans, 89% for African Americans, and 91% in Europe. Five- and 10- year graft survival rates were considerably higher for European (77% and 56%, respectively) than for US populations (whites 71% and 46%, Hispanic 73% and 48%, African American 62% and 34%). Identifying factors explaining long-term survival differences between Europe and the US and reasons for racial differences is currently of high priority 33. One-year graft survival rates in living donor kidney transplantation are approximately 95% 5.

1.1.4 Historical Context of Renal Transplantation and Preservation

Austrian-German surgeon Erwin Payr and French surgeon Alexis Carrel were pioneers in the field of organ transplantation and developed the first workable methods of vascular suturing 34,35. In 1902, Carrel described the end-to-end vascular suture technique, which is still used in transplantation 36. In the following years, he published several advances in transplantation at the University of Chicago in close collaboration with Charles Guthrie 37. These techniques still remain the basis of modern organ transplantation. In 1912, Alexis Carrel received the Nobel Prize for his work in the field of transplantation.

First reports about experimental renal transplantation in large animal models and xenotransplantation were reported in the early 1900s. Emerich Ullmann described retrieval and transplantation of kidneys in a canine model in 1902 38. In 1906, Mathieu Jaboulay transplanted a porcine kidney into the left elbow of a female patient, which is referred to as the first human kidney (xeno-) transplant 39. During the following years, goat to human, monkey to human, and lamb to human kidney transplantations were performed without functional success 40,41.

In the 1930s, the first human kidney allografts were transplanted by Yuri Voronoy in the Ukraine without significant long-term renal function. Following 15 years without remarkable progress, important immunological advances were made in 1943 and 1944 42,43 and first successful renal transplants were reported in 1945 at the Peter Bent Brigham Hospital in Boston and in 1950 in Chicago by Richard Lawler using an intra-abdominal approach (1895 – 1982) 44. In the 1950s and 60s, the “French Transplant Club” performed several renal transplants using an extraperitoneal approach (“Küss procedure”). Although the concept of immune related rejection was well-established at this point 45, all nine patients rejected their grafts. In 1951, Billingham and Medawar published their landmark paper on immune tolerance 46. During the following years, several achievements were made including first long-term success 47 and living donations 48.

An important milestone happened at the Peter Bent Brigham Hospital in 1954. Joseph E. Murray, performed a long-term successful renal transplant between the identical twins Richard Herrick and Ronald Herrick 49. Following this event, extensive progress in renal allotransplantation occurred. Initially, sublethal body irradiation (TBI) was used for immunosuppression 50. First real and important progress in immunosuppression was made in the 1960s. 6 mercaptopurine 51,

8 corticosteroids 52, azathioprine 53, and later cyclosporine 54 became available and improved postoperative outcomes significantly. By 1965, one year survival rates of allograft kidney transplantations reached 65% in deceased donation and 80% in living donation 55. Figure 1.2 provides an overview about milestones in renal transplantation.

Graft preservation has become important since the development of transplant programs with national and international graft exchange schemes, requiring transportation of organs across long distances. In organ transplantation, primarily surface cooling was tested to reduce the renal grafts temperature 56. Due to slow achievement of low graft temperatures, continuous cold perfusion soon became the norm in kidney transplantation. Following preclinical work 57, the first successful human hypothermic machine perfusion kidney transplant was performed in 1968 58. Throughout the 1970s, HMP was mainly used for preservation. However, when improved and cheaper solutions for static cold storage (SCS) became available 59 and the definition of donation after brain death – which replaced donation after circulatory death – was implemented 11,60, renal grafts were rather stored statically on ice then perfused. Solutions such as University of Wisconsin (UW) solution and Histidine-tryptophan-ketoglutarate solution (HTK) have become the gold standard in abdominal organ transplantation. However, since the early 2000s, again interest in HMP has increased. This has been mainly due to a significant change in the donor profile with recovery of grafts of lower quality (ECD and DCD). In addition, some interest has been grown in subnormothermic and normothermic kidney preservation.

Figure 1.2: Historical timeline demonstrating developments in renal transplantation (top section) and renal preservation (lower section). HLA, human leukocyte antigen; FK, FK506 Tacrolimus; MMF, mycophenolate mofetil; DCD, donation after circulatory death; ECD, extended criteria donor; UW, University of Wisconsin solution; HTK, Histidine-tryptophan-ketoglutarate solution; Belzer MPS, Belzer machine perfusion solution; KTx, kidney transplant; DBD, donation after brain death. Figure composed by review of primary literature; citations to be found in Chapter 1.1.4 and 1.5.

1.2 Renal Graft Shortage

Organ transplantation has become a successful therapeutic option in clinical practice during the last decades. Since the first successful abdominal 61-63 and thoracic transplants 64,65, various medical innovations have improved the patient’s posttransplant outcome and survival. However, the limited number of organs available for transplantation represents a severe problem worldwide 6,7,66. In 2013, approximately 117,733 solid organ transplants were reported worldwide. Of those, 78,952 (67.1%) were renal transplants (Figure 1.3). Although an increase of 2.6% over 2012 was reported, the number of transplants performed reflects less than 10% of the global need 6.

Figure 1.3. Kidney transplantation activities in 2013 worldwide. Adapted from the Global Observatory on Donation & Transplantation, World Health Organization 6.

Data from the “Organ Procurement and Transplantation Network” (OPTN) from the United States demonstrates that “the need continues to grow”. While 15,029 patients had been waiting on the transplant list in 1988, 12,618 organ transplants had been performed. In contrast, last year 122,071 patients had been waiting on the list, while 30,973 organ transplants had been performed

67. The gap between patients on the waiting list and organs transplanted continues to grow (Figure 1.4).

140000

120000 Pa'ents Wai'ng at Year End Deceased Donor Organs Transplanted

100000 Deceased Donors Recovered

80000

60000

40000

20000

1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 Year

Figure 1.4. “The need continues to grow”. US American data demonstrating the increase in patients waiting for a transplant by the end of each year. Data retrieved from the “Organ Procurement and Transplantation Network” 67.

In the United States, as per today 100,434 patients are waiting for a kidney transplant 67. The median waiting time for an adult kidney transplant in the US increased from 3 years in 2003 to more than 4.5 years in 2009 7. To overcome the renal graft shortage and shorten the recipients’ waiting time, several strategies have been explored. One of the most promising in deceased organ donation is to utilize grafts recovered in extended criteria donors (ECD) and donation after circulatory death (DCD) and the combination of both (Chapter 1.3). Innovative strategies have also been described to enhance the utility of living donor kidney transplantation 68.

1.3 Renal Graft Donor Sources

The first long-term successful kidney transplantation performed in 1954 was a living donor kidney transplantation between identical twins 49. Following development of transplant programs with national and international graft exchange schemes, during the 1960s and 1970s, donation after circulatory death was the main donor source for renal transplantation. This changed, when mechanical ventilation could sustain respiratory functions 69. To determine death, a test for cessation of brain function was implemented. “Irreversible coma” or “brain death” were defined as a “new criterion for death” by the Ad Hoc Committee of the Harvard Medical School in 1968 11. Publication of the criteria for the diagnosis of brain death 70 resulted in increased use of organs for transplant following “donation after brain death” with improved outcomes when compared to DCD. Since its implementation, DBD represents the main donor source for organ transplantation in the Western World.

However, the increasing organ shortage required a change in the donors’ profile. Donation after circulatory death regained more interest again in the 1990s in the Netherlands 71 and was approved by the WHO in 2011 72. Since the 1990s, also extended criteria donor grafts (ECD), which are grafts of lower quality, are increasingly used in kidney transplantation to reduce the recipients waiting list 73,74.

As per today, deceased donation (DD) represents the main donor source for organ transplantation. 16,291 kidneys were recovered and transplanted in the US in 2015; 11,216 (68.8%) of these grafts were recovered in deceased donors and transplanted, while 5,075 (31.2%) were recovered in living donors (Figure 1.5) 67.

16 All Donor Types Deceased Donor 14 Living Donor

Renal Gra)s Transplanted in the US by Donor Type (in 1000) 0

1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 Year

Figure 1.5. Renal grafts recovered and transplanted (in 1000) in the US by donor type. Figure based on data obtained from OPTN 67.

1.3.1 Deceased Donation

Renal grafts recovered from deceased donors are mainly standard criteria donor grafts (SCD = non-ECD) donated after brain death (about 70%). The remaining 30% are recovered from extended criteria donors in DBD or donation after circulatory death (DCD, non-ECD), with 15% each. A minority is recovered from DCD with grafts fulfilling ECD criteria (DCD/ECD) (Figure 1.6) 7.

Figure 1.6. Percentage of each kidney transplant type among all deceased-donor kidney-alone transplants. Figure adapted from OPTN/SRTR 2013 Annual Data Report: Kidney 7.

1.3.1.1 Donation after Brain Death

1.3.1.1.1 Standard Criteria Donor

Solid organs for transplantation in the Western world are typically recovered from deceased donors with brain dead (DBD), also referred to as neurologic determination of death (NDD) donors. Standard criteria donor (SCD = non-ECD) grafts fulfill the criteria of “high-quality” organ grafts, which are recovered in young individuals with no history of medical illnesses. The typical donor of this kind would be a healthy 30-year old man with no history of drug abuse for whom the cause of death was a traffic accident 75. Discard rates following retrieval are currently about 10% for SCD kidneys, with increasing numbers over the past 12 years (Figure 1.6) 7.

1.3.1.1.2 Extended Criteria Donor

To increase the number of available donors, during the last decades extended criteria donor (ECD), also referred to as expanded criteria donor grafts have been used for transplantation. The term “expanded criteria donor” was introduced in 1997 76. By definition, ECD grafts are recovered in donors aged ≥60 or aged 50 to 59 years fulfilling two of the three following criteria: (1) cerebrovascular accident as cause of death, (2) terminal serum creatinine > 1.5 mg/dL (> 130 µmol/L), or (3) history of systemic hypertension 77. In 2013, approximately 45% of ECD grafts recovered were actually not used for transplantation (Figure 1.7).

Figure 1.7. Renal grafts recovered for transplantation but not transplanted, by DCD and ECD donor status. Figure adapted from OPTN/SRTR 2013 Annual Data Report: Kidney 7.

1.3.1.2 Donation After Circulatory Death (DCD)

As an additional donor source, donation after circulatory death (DCD) regained interest in the 1990s in the Netherlands 14. Since approval by the WHO in 2011 72, it is current clinical practice in 10 of 27 countries of the European Union, North America, South America, Australia, and Japan 78-80. DCD donors are classified according to the revised Maastricht Classification. Donors of category III and IV are considered controlled donors, those of category I, II, and V are considered uncontrolled donors (Table 1.2) 14,15.

Categories of Donation after Circulatory Death

I Dead on arrival Uncontrolled II Unsuccessful resuscitation (CPR)

III Awaiting cardiac arrest Controlled IV Cardiac arrest after brain-stem death

V Cardiac arrest in a hospital patient Uncontrolled (added in 2000)

Table 1.2. Grouping of grafts recovered in donation after circulatory death by the revised Maastricht Classification 14,15.

Donation after circulatory death is rated as one of the most promising sources to increase the donor pool 18. In the US, DCD kidney transplantation accounted for about 15% in 2013. In the Netherlands and the UK, controlled DCD has nearly doubled the number of deceased organ donations 81. In Europe, also increased numbers of grafts recovered in uncontrolled DCD are utilized demonstrating promising results 82. More recently, DCD grafts fulfilling ECD criteria were used for transplantation. However, concerns about posttransplant outcomes result in low amounts of DCD/ECD grafts transplanted (Figure 1.5), as about 51% were discarded in North America 83. In general, graft discard rates of about 20% following recovery are reported for DCD grafts (Figure 1.6) 7.

1.3.2 Living Donation

An additional donor source for kidney transplantation is living donation (LD). Living donor transplantations have become frequently used procedures demonstrating optimal results for

16 recipients 84,85 and donors with low risk of complication. For the success of living donor kidney transplantation (LDKT), the safety of the kidney donor is an important aspect. Several studies demonstrate the very low risk for individuals donating renal grafts (preserved GFR, no increased risk of hypertension, excellent quality of life, low rates of ESRD) 86-89. In current clinical practice, worldwide over 27,000 kidney donations are completed yearly 90. However, living donation rates have not increased and in fact decreased since 2002 (Figure 1.4) 7.

1.3.3 Outcome by Donor Source

Current graft failure rates (death censored) within 90 days among adult kidney transplant recipients are presented in Figure 1.8, sorted by donor. For living donor grafts, lowest failure rates are reported (<1%), followed by standard criteria donor grafts. Higher graft failure rates are reported for DCD and ECD donor grafts. Overall failure rates for deceased donor grafts are ≤3% 7. Throughout the last years, short-term graft survival rates constantly improved.

Figure 1.8. Death-censored graft failure within 90 days among adult kidney transplant recipients. Limited to kidney-alone recipients. Retransplant, graft failure, or return to dialysis within the first 90 days post transplant. Figure adapted from OPTN/SRTR 2013 Annual Data Report: Kidney 7.

1.3.3.1 Deceased Donation

The 5-year graft survival rates for deceased donation are presented in Figure 1.9. Overall 5- year graft survival rates were reported to be about 75%. Highest 5-year graft survival rates are reported for SCD (non-ECD) grafts recovered in DBD (>75%), second highest for non-

ECD/DCD grafts (about 74%). Second lowest and lowest 5-year graft survival rates were reported for ECD/DBD (about 64%) and ECD/DCD (about 54%) 7.

Figure 1.9. Graft survival among adult kidney transplant recipients, 2008: deceased donors. Graft survival estimated using Kaplan-Meier methods. Figure adapted from OPTN/SRTR 2013 Annual Data Report: Kidney 7.

For DCD kidney transplantation, randomized controlled clinical trials and systematic reviews demonstrated comparable long-term graft and survival outcomes when compared to DBD 81. However, rates of primary non-function (PNF), delayed graft function (DGF), and early graft loss (EGL) are increased 80,91-94. In particular prolonged cold ischemia times have shown to negatively impact on outcomes in DCD transplantation 81,95,96.

1.3.3.2 Living Donation

The 5-year graft survival rates for living donation kidney transplantation, classified by donor age, are presented in Figure 1.10 7. In general, longer short- and long-term graft survival rates are achieved with LD when compared to deceased donor kidney transplantation 84. Reasons for this are superior graft quality and shorter waiting times for LDKT recipients. Increased waiting times on dialysis prior to transplantation negatively affect the post transplant graft survival 97. Furthermore, pre-emptive transplantation enhances survival and facilitates better HLA-matching 98. In addition, optimal timing of surgery with retrieval and transplant performed during daytime and reduction of detrimental hypothermic preservation times can be reduced to a minimum 99.

Figure 1.10. Graft survival among adult kidney transplant recipients, 2008; living donors. Graft survival estimated using Kaplan-Meier methods. Figure adapted from OPTN/SRTR 2013 Annual Data Report: Kidney 7.

1.3.4 Kidney Donor Profile Index – The New Allocation System

Major parts of the statistical data in Chapter 1.3 are derived from the OPTN/SRTR 2013 Annual Data Report for the kidney, which is the most recent US data report published in January 2015 7. The 2013 and 2014 Annual Data Kidney Reports will be the last to depict the deceased donor waiting list and times under the “old” allocation system, which was based on the above- described categories of deceased donors: standard criteria donor (SCD), extended or expanded criteria donor (ECD), and donation after circulatory death (DCD).

In December 2014, a new kidney allocation system was implemented in the US. This system characterizes donors on a percent scale (0%-100%) using the kidney donor profile index (KDPI). The KDPI includes 10 donor factors to grade the graft: age, height, race/ethnicity, history of hypertension and diabetes, cause of death, serum creatinine level, hepatitis C status, and DCD. Lower scores are associated with increased, higher scores with decreased graft longevity 67. The 20% of deceased donor kidneys with greatest posttransplant longevity will be allocated to the 20% of candidates with best-expected posttransplant survival. Priority will also be given to candidates awaiting multi-organ transplants (as with the old allocation system), zero HLA mismatches, and highly sensitized candidates. Most recent ECDs will be in the 85th percentile or above. The reason for the change of renal graft allocation were the increasing

19 number of candidates on the waiting list and the high graft discard rates 7. Future statistic reports will therefore demonstrate data by KDPI rather than SCD, ECD, and DCD.

1.3.5 Logistic Challenges in Kidney Donation

Logistic challenges in deceased and living kidney donation include graft recovery and preservation to facilitate the transportation to the recipient hospital. During graft recovery, organs are flushed and preserved cold with specifically designed solutions to reduce cell metabolism and prevent injury. However, metabolic activity is not completely arrested and continues at a reduced level. Thus, interruption from blood flow, oxygen-, and nutrition supply induces a period of (cold) ischemia. Subsequent transplantation with graft reperfusion leads to a pathophysiologic phenomenon termed ischemia reperfusion injury (IRI). In DCD kidney transplantation, in addition to cold ischemia, also significant times of warm ischemia do occur. The following chapter describes IRI and its specific relevance in kidney transplantation.

1.4 Renal Ischemia Reperfusion Injury

Ischemic kidney injury often occurs in multiple organ failure and sepsis. Ischemia reperfusion injury (IRI) originates from an impaired supply of renal cells with oxygen and nutrition, and reduced waste product removal. Pathophysiological key components are hemodynamic alterations, inflammation, and endothelial and epithelial cell injury. Subsequent repair mechanisms can restore epithelial integrity; however, maladaptive repair can lead to death of tubular epithelial cells by apoptosis and necrosis (acute tubular necrosis, ATN), ultimately resulting in kidney failure 100. Renal IRI is unavoidable in kidney transplantation and is clinically associated with delayed graft function, acute allograft rejection, chronic rejection, and reduced graft survival and reduced long-term renal function 101. Besides cold ischemia, transplantation IRI includes severe amounts of warm ischemia during the prerecovery phase (blood pressure fluctuation in HBD and near to complete cessation of blood flow in DCD) and rewarming during suturing anastomoses. Reperfusion represents the final step in IRI. The following sections first describe renal IRI in general, followed by a section emphasizing its specific relevance in hypothermic renal graft preservation.

1.4.1 Endothelial Cells and Vascular Components of IRI

Endothelial cells and vascular components play a pivotal role in IRI injury. Endothelial cells are capable to regulate vascular tone, leukocyte function, and smooth muscle response 102. In renal ischemia, a disproportionally high reduction of blood flow occurs in the outer medulla. Affected/injured endothelium leads to vasoconstriction of small arterioles due to increased tissue levels of endothelin-1, angiotensin II, thromboxane A2, prostaglandin H2, leukotrienes C4 and D4, and adenosine, and sympathetic nerve stimulation. Furthermore, reduced vasodilation in response to acetylcholine, bradykinin, and nitric oxide occurs 103. Vasoactive cytokines (TNF-α, IL-1β, IL-6, IL-12, IL-15, IL-18, IL-32, endothelin) further augment the constriction of arterioles 104. Endothelial-leukocyte interaction and coagulation leads to further ischemia, especially in the outer medulla. Tubulo-glomerular feedback seems to reinforce pre-glomerular vasoconstriction 105. Local edema further compromises local blood flow. Ischemia further leads to increased expression of cellular adhesion molecules (CAMs) on endothelial cells such as ICAM-1 and subsequent expression of counterreceptors on leukocytes. Activation and transmigration of leukocytes, production of cytokines, and inflammation result in disruption of glycocalyx,

21 cytoskeleton, and endothelial cell-cell contacts, increasing permeability and subsequent loss of fluid into the interstitium (see Figure 1.11) 106.

Figure 1.11. Endothelial injury in renal IRI. (A) Normal epithelium and endothelium (glycocalyx coated). (B) IRI induces: Endothelial cell swelling; disruption of glycocalyx and endothelial monolayer; upregulation of ICAMs, VCAMs, selectins, enhanced leukocyte- endothelium interaction; formation of microthrombi; leukocyte migration; expansion of interstitial compartment with enhanced numers of inflammatory cells and interstitial edema. Figure adapted from 100. Copyrights received.

1.4.2 Inflammation – Immune Response and Cells Enrolled in IRI

The innate immune response is responsible for the early response in renal IRI and acts in a non- antigen-specific way, compromising neutrophils, monocytes/macrophages, dentritic cells (DCs), natural killer (NK) cells, and NKT cells. The adaptive immune response acts in an antigen-

22 specific way and compromises DC maturation and antigen presentation, T cell proliferation and activation, and T to B cell interactions 100.

Tubular epithelium in IRI induces generation of proinflammatory and chemotactic cytokines (TNF- α, IL-1β, IL-6, IL-8, TGF- β, RANTES, epithelial neutrophil-activating protein 78 (ENA- 78), which activate inflammatory cells. Furthermore, Toll-like receptors (TLRs), complement and complement receptors, and costimulatory molecules (T lymphocyte regulation) are expressed 104. Proximal tubular epithelium furthermore expresses MHC II, presenting antigen to T cells and express costimulatory factors. Neutrophils attach to the activated endothelium early after reperfusion (30 minutes) and produce proteases, myeloperoxidase, reactive oxygen species (ROS), and cytokines which cause increased permeability and reduced cell integrity 107. Monocytes infiltrate injured renal tissue and differentiate into macrophages and DCs (within 1 hour after reperfusion). M1 type macrophages produce reactive oxygen and nitrogen intermediates and inflammatory cytokines; M2 macrophages are generally believed to support repair systems 100. Infiltrating T lymphocytes can induce further injury (such as CD4+ cells) but also support repair mechanisms (such as T regulatory cells (Tregs) (Figure 1.12) 108.

Figure 1.12. Immune response in renal IRI. Release of proinflammatory cytokines and chemokines by injured tubular epithelium, resulting in recruitment of immune cells. Epithelial cells also express CAMs, TLRs, and T cell costimulatory molecules, which leads to activation of immune cells. Neutrophils, macrophages, and NKT cells cause direct injury to tubular epithelial cells. DCs are involved in both (innate and adaptive) immune responses. Adapted from 100. Copyrights received.

1.4.3 Complement Cascade Activation in IRI

Activation of the complement cascade plays an important role in the development of IRI. In particular, activation occurs through the classical, lectin, alternative pathways (APs), or the coagulation activation pathway 109. In renal IRI, many studies indicate that complementary activation occurs through the AP, or the lectin pathway 110. Complement induces expression of CAMs and promotes maturation of DCs, resulting in activation of T cell responses 100.

1.4.4 Hypothermia in Renal IRI

During hypothermic organ preservation, the graft is disconnected from its blood, oxygen, and nutrition supply for transportation to the recipient hospital. The principle of hypothermic storage is to reduce the detrimental effects of IRI by slowing down the cell metabolism. The recommended temperature for cold storage is 4°C. Approaching 0°C can result in organ freezing and subsequent necrosis. Temperatures higher than 4°C will lead to increased metabolic activity with ATP depletion, accumulation of lactic acid, and mitochondrial damage causing parenchymal and endothelial injury 111. Clinical hypothermia slows down the cell metabolism 12-fold 111; however, residual ongoing metabolism results in continuing energy consumption. ATP is depleted to ADP, which accumulates in the cells. Following exhaustion of oxygen supply, metabolism changes from aerobic to anaerobic and lactate and protons are produced 112. Due to loss of energy substrate, membrane Na+/K+-ion transport stops, passive inflow of sodium into the cell occurs, and subsequent osmotic water redistribution causes cell swelling. Changes in pH, loss of phospholipid membrane integrity, stress protein response and cytoskeletal changes (due to hypothermia) finally result in cell death through apoptosis or necrosis 112. Although hypothermia is traditionally required for the storage of organs until transplantation, it is also the catalyst for further damage. Especially prolonged cold ischemic time reduce the grafts chance to fully recover to normal function following transplantation.

1.5 Renal Graft Preservation

Since the development of transplant programs with national and international graft exchange schemes, renal graft preservation has become important in the field of organ transplantation. For this research study, it is useful to divide renal graft preservation techniques by the temperature applied and its mechanical nature (perfusion vs. static storage).

1.5.1 Principles of Static Cold Storage and Preservation Solutions

Static cold storage (SCS) became the predominant preservation technique in organ transplantation when the definition of brain death was implemented 11,60. DBD replaced donation after circulatory death and became the main donor source in the Western World. DBD organs, which are of higher quality then those recovered from DCD, demonstrated good outcomes following static storage. HMP was replaced as utilized solutions improved and SCS in general became cheaper than HMP 59.

Hypothermia is a simple method to reduce the organs metabolic rate and protect against warm ischemia. Primarily, surface cooling was tested and demonstrated effectiveness in renal autotransplantation in animals 56. Due to slow achievement of low temperatures in the graft, the organs vascular system was soon used for faster cooling. Following usage of cooled autologous blood and continuous cold perfusion, soon better preservation solutions were developed that allowed rapid flush-out of blood in situ and subsequent static storage on ice until transplantation 59.

SCS preservation solutions limit ATP consumption, inhibit cellular enzymatic activity, and reduce cellular degradation by phospholipid hydrolysis 113 (Chapter 1.4). Of particular interest are the impermeant molecules (e.g. glucose, sucrose, mannitol, citrate, colloids), which prevent the movement of water and subsequent electrolytes over the cell membrane. They remain in the vascular space and/or the interstitium to be effective. Furthermore, buffers preventing changes in pH and subsequent protein degradation (e.g. bicarbonates, histidine, phosphates, lactobionate), free radical scavengers that counteract reactive oxygen species (ROS) (e.g. glutathione, lactobionate, tryptophan), and nutrients/nutrient precursors that serve for ongoing metabolism and reperfusion are added to preservation solutions. Table 1.3 demonstrates an overview of solutions used today 114.

Preservation solutions for static cold storage* and hypothermic machine perfusion Celsior EC HOC HTK IGL-1 UW Belzer MPS Colloids (mM) HES - - - - - 0.25 0.25 PEG - - - - 0.03 - - Impermeants (mM) Citrate* - - 80 - - - - Gluconate ------85 Glucose - 195 - - - - 10 Histidine* 30 - - 198 - - - Lactobionate* 80 - - - 100 100 - Mannitol* 60 - 185 38 - - 30 Raffinose - - - - 30 30 - Ribose ------5 Buffers (mM) HEPES ------10 K2HPO4 - 15 - - - - - KH2PO4 - 43 - - 25 25 25 NaHCO3 - 10 10 - - - - Electrolytes (mM) Calcium 0.25 - - 0.0015 - - 0.5 Chloride 42 15 - 32 20 20 1 Magnesium 13 - 40 4 5 5 5 Potassium 15 115 84 9 25 120 25 Sodium 100 10 84 15 120 30 100 ROS scavenger (mM) Allopurinol - - - - 1 1 - Glutathione 3 - - - 3 3 - Tryptophan - - - 2 - - - Nutrients (mM) Adenine ------5 Adenosine - - - - 5 5 - Glutamate 20 ------Ketoglutarate - - - 1 - - - Osmolality (mOsm) 255 406 400 310 320 320 300

Table 1.3: *Citrate, histidine and lactobionate also act as buffers. Histidine, lactobionate and mannitol also act as ROS scavengers. EC, Euro-Collins; HEPES, 4-(2-hydroxylethyl)-1- piperazine ethanesulfonic acid; HES, hydroxyethyl starch; HOC, hyperosmolar citrate; HTK, Histidine-tryptophan-ketoglutarate; IGL-1, Institute-George Lopez-1; MPS, machine perfusion solution; PEG, polyethylene glycol; UW, University of Wisconsin solution. Derived from 114.

1.5.1.1 Euro-Collins

Euro-Collins (EC) was developed in 1969 for static organ preservation and is the precursor of University of Wisconsin solution. It has a high content of potassium and a low content of sodium. Glucose acts as impermeant and it has a high osmolality (Table 1.2). Clinical practice at the time of EC development found that liver grafts were preserved well for up to 8 hours following flush 115.

1.5.1.2 UW

University of Wisconsin solution (UW) (Viaspan®, Bristol-Meyers Squibb; SPS-1®, Organ Recovery Systems; Belzer UW Cold Storage Solution®, Bridge to Life) was originally developed for pancreas preservation by Belzer and Southard in 1987 116. UW represents the most common cold preservation solution in abdominal organ preservation. Its composition (potassium-rich, sodium-depleted, osmotically active fluid) reflects the intracellular milieu. Hydroxyethyl starch (HES) represents the colloid in UW and permits more effective flushing. Lactobinate and raffinose reduce cellular swelling (Table 1.2). Several disadvantages are described for UW. Due to high viscosity (caused by the osmotically active substances), flushing is challenging. Adenosine crystals can form, so a pore filter is required. Hyperkalemic cardiac arrest, ischemic-type biliary lesions in liver preservation, and microcirculatory disturbances are described risks 117.

1.5.1.3 HTK

Histidine-tryptophan-ketoglutarate (HTK) (Custodiol®, Dr. Franz Köhler Chemie GmbH) was originally developed as a cardioplegic solution by HJ Bretschneider but has also been tested for the storage of kidney, liver, and pancreas 118,119. HTK is a crystalloid with low potassium content and an osmolarity slightly higher than the intracellular space. The amino acid histidine serves as an impermeant molecule (assisted by mannitol) and buffer, tryptophan serves as membrane stabilizer (free radical scavenger), and ketoglutarate as an energy substrate (Table 1.2). As HTK does not contain a colloid, it is less viscous than UW. Furthermore, it is cheaper and does not require a filter 117.

1.5.1.4 Celsior

Celsior (Celsior®, Genzyme) was initially developed for the storage of cardiac allografts in the early 1990s 120. It has also proven to be useful for preservation of kidney, liver, and pancreas in abdominal organ transplantation 121,122. Celsior has high sodium and low potassium content (extracellular-type solution). Lactobionate (high molecular weight and negative charge) serves as impermeant, histidine as buffer, and reduced glutathione as scavenger. Furthermore, mannitol has impermeant and free radical scavenger function (Table 1.2). Due to its composition, it has a low viscosity.

1.5.1.5 IGL-1

Institute Georges Lopez-1 Solution (IGL-1®, Institute George Lopez) has a similar composition in terms of impermeants and buffers like UW. Instead of HES, it contains polyethylene glycol (PEG) as an impermeant (Table 1.2). It demonstrated potential for the preservation of kidney, liver, pancreas, and small bowel but is not standard of care in clinical practice today 114.

1.5.1.6 Current Clinical Practice in Static Cold Kidney Preservation

Overall, static cold storage with UW solution is the most common clinical practice in deceased donor kidney transplantation 117. A most recent systematic review and meta-analysis investigated 15 trials (including prospective studies with 3584 deceased donor renal allografts) with delayed graft function (DGF) as the primary outcome. Euro-Collins was found to have greater DGF when compared to UW in two randomized controlled trials (RCTs) and also when compared to HTK in two RCTs. UW was associated with equal risk of DGF when compared to Celsior in three RCTs and in 2 RCTs when compared to HTK. Both, UW and HTK had lower DGF rates than Euro- Collins 123. A single-center retrospective experience recently published investigated the difference in outcome for renal grafts preserved with UW or HTK. Despite similar 2-year patient survival, rejection, renal function, and graft survival, HTK was associated with greater early graft loss (<6 months) due to primary non-function. As DGF and early graft loss only occurred in marginal grafts, the authors concluded that HTK might be inferior in marginal donors 124. Segev et al. analyzed the UNOS deceased donor kidney transplantation database and found an increased risk of death-censored graft loss for HTK preserved grafts vs. UW in kidneys transplanted between 07/2004 and 02/2008 125. In two other single-center retrospective reviews, UW vs.

Celsior was compared for renal graft preservation. Here, no significant differences were found for 12-month postoperative serum creatinine levels, 5-year graft survival rates 126. Similar incidence of DGF and acute rejection, and 3-year graft and patient survival were reported 127. Further studies comparing UW and HTK demonstrated those to be equal efficacious for clinical use with minor but organ-specific qualities and different complications 128. In a recent review Parsons and Guarrera conclude that UW is the most popular static preservation solution in abdominal organ preservation. HTK and Celsior demonstrate equal outcomes in most studies, especially when organs are exposed to short cold ischemic times 117.

1.5.2 Principles of Hypothermic Machine Perfusion

Hypothermic machine perfusion (HMP) for preservation of renal grafts prior to transplantation was developed by Dr. Folkert Belzer in the 1960s. Following the first successful human kidney HMP in 1967 58 at the University of Wisconsin, HMP was the predominant storage technique in the 1970s in the US. Although SCS became the gold standard since the 1980s, HMP attracts more interest again, as the deceased donor profile changed and more grafts today are recovered from extended criteria donors and donation after circulatory death. Data from the international Collaborative Transplant Study (CTS) indicates that from 1990-2005 SCS was utilized for 97- 98% of all renal grafts, with HMP making up the remaining 2-3% 114. In the US, in 2000 about 10% of renal grafts had been preserved with HMP; since 2010 over 40% are being preserved with HMP 99. The predominant solution used for HMP is Belzer MPS® UW Machine Perfusion Solution (Bridge to Life). It contains HES as colloid, has a sodium concentration of 100 mEq/L and a potassium concentration of 25 mEq/L (Table 1.2). The LifePort Kidney Transporter from Organ Recovery Systems is the most frequently used device for HMP. It provides a sealed, sterile environment where the perfusate is pumped though the kidney at a low pulsatile pressure of 30 mmHg at cold temperatures (4 °C). It is portable and allows continuous perfusion from organ recovery till transplantation. During preservation, data such as temperature, flow rate, vascular resistance, and pressure are recorded every 10 seconds 129.

1.5.3 Hypothermic Machine Perfusion vs. Static Cold Storage

Renal grafts of lower quality in particular are susceptible to preservation-induced injury that can lead to delayed graft function and primary non-function, which in turn affect long-term outcomes of transplant recipients. Several randomized controlled trials, meta-analyses, and systematic

29 reviews have been conducted to investigate whether HMP preserves deceased donor kidneys, in particular those of lower quality, better than SCS.

A meta-analysis from 2003 investigating 16 prospectively conducted studies comparing HMP and SCS between 1971 and 2001 demonstrated a relative risk of DGF of 0.80 (0.67 – 0.96) for HMP vs. SCS. No effect on 1-year graft survival was detected 130,131.

In a more recently conducted randomized controlled trial in the Eurotransplant region, 336 paired kidneys recovered from all deceased donor types (standard criteria donors, extended criteria donors, and controlled donation after circulatory death) compared HMP and SCS. DGF occurred in 20.8% vs. 26.5% of the HMP vs. SCS preserved kidneys; further analyses revealed a reduced risk of DGF for HMP (adjusted odds ratio 0.57 (0.36 – 0.88) 132. Significantly less functional DGF and PNF occurred in the HMP group. HMP demonstrated improved graft survival at year one (94% vs. 90%) and year 3 (91% vs. 87%) after transplantation 133. For ECD kidneys, trials indicate more significant beneficial effects for HMP when compared to SCS. In an international, randomized controlled trial 91 ECD kidneys were preserved with HMP and compared to their contralateral pair that underwent SCS. 22% kidneys had DGF in the HMP group, 29.7% in the SCS group; HMP significantly reduced the risk of DGF compared to CS (OR 0.46, p=0.047). Furthermore, incidence of nonfunction was lower (12% vs. 3%, p=0.04) and one-year graft survival significantly higher (92.3% vs. 80.2%, p=0.02) in HMP vs. SCS grafts 134. 3-year graft survival was maintained for ECD kidneys (86% vs. 76%, AHR 0.38 (0.18-080) 133.

Data for kidneys recovered in DCD is controversial. Some studies suggest beneficial effects for HMP vs. SCS, others not. In a separate randomized extension of the aforementioned trial 132, 82 category III DCD kidney pairs were analyzed. The risk of DGF was reduced in HMP vs. SCS (53.7% vs. 69.5%, p=0.007; AOR 0.43 (0.20-0.89). PNF was similar in both groups, and HMP did not increase 1- or 3-year graft survival 135. In contrast, a parallel RCT from the UK analyzed 45 Maastricht III DCD kidney pairs and did not reveal differences in DGF between HMP and SCS (58% vs. 56%; p=0.99). PNF and graft survival were similar in both groups 136.

Several meta-analyses and systematic reviews have recently been published. Analyses of data from all types of donors revealed a reduced risk of DGF for HMP vs. SCS in two meta-analyses (AORs of 0.83 (0.72-0.96) and 0.81 (0.71-0.98) 137,138. For DCD kidneys, two meta-analyses also demonstrated a protective effect of HMP vs. SCS (AORs 0.56 (0.36-0.86) and 0.64 (0.43-0.95)

139,140. For ECD grafts, one meta-analysis demonstrated reduced DGF risk (AOR 0.59 (0.54- 0.66) 141.

1.5.4 Subnormothermic Preservation

In the late 1990s and early 2000s, Brasile, Stubenitsky, Kootstra et al. investigated subnormothermic preservation of renal grafts. Exsanguinous Metabolic Support (EMS) is based on subnormothermic acellular, low pressure, pulsatile ex vivo kidney perfusion 142,143. Following 30 minutes of warm ischemia and 24 hours of SCS, renal grafts were either re-implanted directly or first perfused for 3 hours with EMS and then transplanted. 24 hours after transplant, serum creatinine levels and peak serum creatinine were significantly lower in the subnormothermic preserved kidneys 144. In another study, following 30 minutes of warm ischemia, renal grafts were subjected to various durations of cold storage and subnormothermic perfusion with EMS. The results demonstrated that reduction of SCS and prolongation of subnormothermic perfusion led to significantly improved renal graft function with decreased serum creatinine levels after graft autotransplantation 145. Further publications support the beneficial findings of subnormothermic perfusion on the EMS device 146,147.

1.5.5 Normothermic Preservation

Early in history of organ preservation and transplantation, pioneers in the field investigated machine perfusion. Already in the first half of the twentieth century, Carrel perfused organs with normothermic, oxygenated serum and demonstrated gross viability for several days 148.

1.5.5.1 Experimental Experience in Animal Models

First experiments investigating normothermic preservation techniques using heparinized whole blood were conducted in canine models in the late 1970s and the early 1980s by Kootstra et al. Aiming for prolongation of preservation times, a combination of hypothermic machine perfusion (8°C) and normothermic ex vivo perfusion (38°C) was performed for up to 144 hours in HBD kidneys. Following renal autotransplantation, grafts that underwent a phase of 3 or 4 hours of normothermic ex vivo perfusion during the preservation period demonstrated significantly lower serum creatinine values and improved animal survival when compared to continuously hypothermic perfused grafts 149,150. These beneficial effects of intermediate normothermic

31 perfusion have also been demonstrated for renal grafts that were subjected to 30 minutes of warm ischemia 151.

Nicholson et al. investigated normothermic perfusion (NP) for renal grafts using erythrocyte- based solutions in porcine models. They demonstrated beneficial results for the application of a short period of normothermic perfusion at the end of the preservation period when compared to hypothermic preservation. The post-preservation assessment was performed during reperfusion on a separate ex vivo perfusion system using whole blood perfusion for 3 hours as a model for transplantation 152,153. In one study, this group investigated a short additional period of normothermic perfusion in a porcine autotransplantation model. Renal grafts were subjected to 30 minutes of warm ischemia and 22 hours of HMP, or 20 hours of HMP with an additional period of normothermic perfusion for 2 hours. No significant differences were found in graft survival or kidney function, but lower levels of lipid peroxidation were measured in the NP group 60 min after transplantation. This study demonstrated the feasibility of a short NP time of 2 hours in a porcine model of renal transplantation 154.

Data described above highlights the potential of adding normothermic ex vivo preservation periods to commonly utilized hypothermic storage. This approaches aim to prolong graft storage times or repair grafts of lower quality (ECD, DCD) that were exposed to various times of hypothermia. However, currently no studies are available that investigated the potential to replace hypothermia using more physiologic storage at normothermic temperatures. Thus, the aim of the study described in this manuscript was to develop a preservation technique that facilitates prolonged, continuous normothermic perfusion of grafts ex vivo prior to transplantation replacing hypothermic storage.

1.5.5.2 Clinical Experience

Preclinical investigations resulted in the first in man renal transplantation following one additional hour of normothermic ex vivo kidney perfusion in 2011. The kidneys of a 62-year-old extended criteria donor, who died of an intracranial haemorrhage and had undergone cardiopulmonary resuscitation for a 30-minute cardiac arrest, were recovered. Following 11 hours of static cold storage, the left kidney was perfused at a temperature of 33.9°C for 35 minutes ex vivo prior to transplantation. The right kidney instead underwent static cold storage for 14 hours only. Following graft transplantation, the normothermic preserved kidney

32 demonstrated slow graft function, but the recipient remained dialysis independent. In contrast, the recipient who received the static cold stored kidney had a delayed graft function for 26 days and a higher serum creatinine at 3 months after transplantation 155.

Nicholson and Hosgood published the first clinical study investigating normothermic ex vivo kidney perfusion in 2013. Eighteen kidneys from ECD donors underwent a period of 63 ± 16 minutes normothermic perfusion with a plasma free red-cell based solution at a temperature of 34.6 °C immediately before transplantation. The outcome of these kidneys was compared to a control group of 47 ECD renal grafts that underwent static cold storage only. Following graft transplantation, the normothermically preserved grafts demonstrated a significantly lower rate of DGF when compared to the control group (p = 0.014) 156.

1.6 Importance of the topic

Kidney transplantation remains the treatment of choice for patients with end-stage renal disease. Currently, there is no therapy that provides outcomes comparable to transplanted renal grafts. However, a major limitation in kidney transplantation worldwide is the shortage of donor grafts resulting in long waiting lists. The waiting time for a patient can be up to 8 years in Ontario, Canada. Therefore, during the past decades the donor pool has been enlarged by accepting grafts of lower quality from extended criteria donors and donation after circulatory death. Unfortunately, these grafts often have delayed graft function following transplantation, with initial dialysis requirement and decreased long-term function.

The decreased outcome of extended criteria and donation after circulatory death grafts is linked to their poor tolerance to hypothermic storage as the current preservation technique. Static cold storage with preservation solutions developed in the 1980s remains the gold standard in kidney transplantation. It occurs that this kind of preservation is incapable of fully protecting ECD and DCD kidney grafts in renal transplantation. Therefore, several approaches are being explored to prevent ischemia reperfusion injury in renal grafts, including changes in donor management, preconditioning of the kidney, improvement of preservation solutions, post conditioning and regenerative techniques following transplantation.

A novel strategy to improve kidney preservation, allow assessment prior to transplantation, and repair grafts of lower quality during storage is normothermic ex vivo kidney perfusion. More physiologic conditions at normothermic temperatures providing oxygen and nutrition result in excellent graft viability during preservation. This novel technique reduces ischemia reperfusion injury, as it is capable of replacing hypothermia. Assessment of kidney function and injury during preservation facilitates decision making of whether a graft is transplantable or not. Furthermore, due to the organs’ high metabolic activity, administration of repair strategies is feasible.

Overall, normothermic ex vivo kidney perfusion will help to improve graft outcome in kidney transplantation, facilitate the usage of more marginal grafts based on improved assessment and repair strategies. Finally, normothermic ex vivo kidney perfusion will help solving the organ shortage problem and reduce the recipients’ waiting list.

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2 Hypotheses and Aims

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In the setting of organ transplantation, current research focuses on ischemia reperfusion injury and alternative organ preservation techniques. Despite the current gold standard static cold storage, hypothermic machine perfusion is increasingly used. Recent data support its superiority when compared to SCS, especially in ECD and DCD grafts. However, both techniques are based on the principle of slowing down the organ’s metabolic activity by application of hypothermic temperatures. In contrast, the goal of this study was to establish a model of NEVKP that could serve as an alternative preservation technique for renal grafts. The hypothesis of this study is that NEVKP provides a more physiologic and protective environment for superior graft storage. Due to the grafts high metabolic activity, NEVKP furthermore provides a platform for assessment and repair strategies during preservation. This study contained five main research aims:

1) To develop the technique of Normothermic Ex Vivo Kidney Perfusion in a porcine model.

2) To establish a porcine model of renal transplantation.

3) To investigate NEVKP in a renal transplantation model of heart-beating donation and compare the postoperative outcome to SCS.

4) To investigate NEVKP in a renal transplantation model of ischemically injured grafts and compare the postoperative outcome to SCS.

5) To translate the findings to a clinical safety trial at Toronto General Hospital.

The first aim of this study was to develop the novel preservation technique named Normothermic Ex Vivo Kidney Perfusion in a porcine model. Therefore, an S3 heart-lung-machine provided by Sorin Group was utilized. Based on neonatal cardiopulmonary bypass technology, a customized perfusion circuit, adapted to the needs of perfusing a kidney ex situ, was constructed. To provide a physiologic environment, an organ chamber was built. Furthermore, based on measurements in healthy pigs, a perfusion solution was developed and proper perfusion circuit characteristics were elaborated. The second aim was to develop a large animal model of renal transplantation. 30 kg Yorkshire pigs were used for graft retrieval and transplantation purposes. The third aim of the study was to investigate the feasibility and safety of NEVKP in a model of heart-beating donation. The postoperative outcome of grafts preserved with NEVKP vs. SCS was compared.

The fourth aim was then to investigate the potential of NEVKP to improve renal graft function in renal grafts that were subjected to warm ischemia. The final aim will then be to – based on the acquired data in the large animal model – translate the findings into a clinical safety trial at Toronto General Hospital.

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3 Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts Prior to Transplantation

______

This chapter is modified from the following:

Kaths J.M., Spetzler V.N., Goldaracena N., Echeverri J., Louis K.S., Foltys D.B., Strempel M., Yip P., John R., Mucsi I., Ghanekar A., Bagli D., Robinson L.A., Selzner M. (2015). Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts Prior to Transplantation. The Journal of Visualized Experiments (JoVE).

A visualized version of this study can be viewed at the following: www.jove.com/video/52909/normothermic-ex-vivo-kidney-perfusion-for-preservation-kidney- grafts

3.1 Introduction

Kidneys are the most frequently transplanted solid organs. For patients suffering from end-stage renal disease, kidney transplantation offers better life expectancy, and improved quality of life when compared with dialysis 2,157-159. The persisting organ shortage represents a severe problem in the field of transplant medicine (Table 1) 160.

United States* Eurotransplant region**

Patients on kidney transplant waiting list 100 867 (July 10 757 (December, 2013) 2014)

Deceased donor Kidneys transplanted in 11 163 2 959 2014

Median waiting time to deceased donor Up to 5 years* Up to 4 years** kidney transplant (in years)

Table 3.1. Kidney Graft Shortage in the US and Eurotransplant Region.

The outcome of kidney transplantation is negatively affected by the waiting time, with poorer outcome for patients subjected to prolonged dialysis 161. This has triggered interest in marginal kidney grafts as an additional donor source, such as kidneys from older donors, donors with multiple comorbidities (extended criteria donors (ECD), and kidneys donated after cardiac death (DCD). Marginal donor kidneys that would have been declined in the past are now considered for transplantation 162.

A major obstacle for the use of marginal kidney grafts is the preservation technique of cold anoxic storage. Currently, kidney grafts are stored statically on ice or perfused at 4°C without oxygen. The cold anoxic preservation technique is associated with ongoing graft injury during kidney preservation and does not allow graft assessment because of the lack of metabolism and urine production. In particular, marginal kidney grafts tolerate cold storage poorly, resulting in significant kidney injury, and high rates of delayed graft function 96,163. Delayed graft function is a prognostic factor for poor long-term graft function.

Extracorporeal kidney perfusion represents an alternative method for the preservation, assessment and repair of organs. In a porcine model, beneficial results were presented for

39 kidneys perfused ex vivo under normothermic conditions 154,164. The first clinical trial performed in 2013 demonstrated a lower rate of delayed graft function when kidneys retrieved from extended criteria donors were perfused for one hour immediately before transplant 156.

This paper presents a model of normothermic ex vivo kidney perfusion (NEVKP). The goal of this study is to reduce the applied cold ischemia time to a minimum and extend the period of NEVKP. NEVKP is an alternative preservation method that aims to reduce the damage that can be caused by cold storage techniques.

3.2 Materials and Methods

A schematic overview of the study protocol is presented in Figure 1.

Figure 3.1. Study Protocol. This study protocol of normothermic ex vivo kidney perfusion is based on a porcine model. After surgical dissection of the vessels of the kidney graft and flushing with 500 ml of histidine-tryptophan-ketoglutarate (HTK), the graft can be retrieved. After cold storage (SCS) for 3 hrs, the kidney graft is perfused normothermically ex vivo (NEVKP) for multiple hours until the designated transplantation.

All animals received humane care in compliance with the ‘‘Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research and the ‘‘Guide for the Care of Laboratory Animals’’ published by the National Institutes of Health, Ontario, Canada. The Animal Care Committee of the Toronto General Research Institute approved all studies.

3.2.1 Animals

Use male Yorkshire pigs (27 - 33 kg) in this protocol.

3.2.2 Organ Retrieval

3.2.2.1 Preoperative Procedure

1. House the male Yorkshire pigs in a research facility for at least one week to reduce their stress level. Fast the pigs for a minimum of six hours before induction of the anesthesia.

2. Initiate anesthetization of the pig by an intramuscular injection of ketamine (25 mg/kg), atropine (0.04 mg/kg), and midazolam (0.15 mg/kg). Subsequently, transport the animal from the housing facility to the operating room (OR).

3. Place the pig in a supine position on the OR table. Let it breathe 2 L of oxygen with 5% of isoflurane spontaneously. After relaxation, expose the vocal cords with a laryngoscope and spray them with 2% lidocaine to prevent a spasm caused by intubation. After

intubation with a 6.5 mm tube, block the cuff with 3 - 5 mL of air. Note: Capnometry reveals the correct position of the tracheal tube.

4. Lower the isoflurane gas to 2.5%. Set the ventilator to 14 - 16 breaths/min and the tidal volume to 10 - 15 mL/kg body weight. Monitor the pig closely. Heart rate and oxygen saturation are recorded by pulse oxymetry.

5. Under sterile conditions, introduce an 8.5 Fr. x 10 cm catheter into the jugular vein in Seldinger technique. Therefore, use a needle to puncture the venous vessel. After introducing a wire, replace the needle with a catheter. Eliminate the wire and fix the catheter to the skin. Administer 200 ml of Ringer’s lactate solution per hour throughout the surgery.

3.2.2.2 Surgical Procedure

1. Following disinfection and coverage of the surgical field, perform a midline incision from xyphoid to pubic symphysis. To ameliorate the exposure, extend the surgical approach with a left lateral incision. Cover the large and small bowels with a towel and position them to the left side for optimal access to the right kidney.

2. Separate the inferior vena cava (IVC) from the abdominal aorta. Ligate aortic branches from the back of the aorta.

3. After complete aortic dissection to the back, pass a ligature around the aorta cranial to the renal branches. Additionally, place two ligatures cranial of the iliac bifurcation. Place a tie around the left renal artery.

4. Free the right kidney from its adherent tissue. Dissect the renal vein, artery, and ureter.

5. Open the diaphragm and administer 1,000 IU heparin per kg donor weight into the heart. For a DCD model, inject 40 mval of KCl intracardially 3 min after systemic heparinization to induce cardiac arrest. The cardiac arrest is valued as the starting point of warm ischemia.

6. Meanwhile, for collection of blood, connect the lines of CPDA bags (citrate, phosphate, dextrose, adenosine) to the catheter introduced to the left upper jugular vein. Perform a

soft spin (1,500 x g without brake). Remove plasma and buffy coat under sterile condition (biosafety cabinet class II) and store the erythrocytes for transfusion.

7. Cannulate the aorta with an organ flush line above the iliac bifurcation. Tie the ligatures at the aorta and the left renal artery.

8. Flush the kidney with histidine-tryptophan-ketoglutarate (HTK) solution with a pressure

of 100 cm H2O. Clamp the thoracic cava and collect the blood via the jugular catheter. Cut the abdominal cava below the renal vein to secure an optimal flush of the kidney.

9. After a complete flush of the right kidney, retrieve the graft with a segment of the aorta. Cut the renal vein and leave the ureter long.

3.2.2.3 Back Table Preparation of the Kidney Graft for the Perfusion

1. Free the kidney from adherent tissue. Close the cranial part of the aorta with a tie and cannulate the lower part with a 1/4” x 3/8” reducer. Tie off smaller arterial branches coming from the aorta.

2. Cannulate the renal vein with a 1/4” x 3/8” reducer directly. Intubate the ureter with an 8 Fr. feeding tube.

3. Place the kidney on ice until the start of the NEVKP.

3.2.3 Normothermic Ex Vivo Kidney Perfusion (NEVKP)

3.2.3.1 Preparation of the Perfusion Circuit

The perfusion circuit consists of neonatal cardiopulmonary bypass equipment (Figure 2).

Figure 3.2. Schematic Drawing of the Perfusion Circuit. The circuit consists of neonatal cardiopulmonary bypass technology. The perfusion solution is collected in the venous reservoir. A centrifugal pump propels the solution into the oxygenator, where it is enriched with oxygen and warmed to 37 °C. After passing the arterial bubble filter, the perfusate is driven with a pressure of 60 – 80 mmHg through the renal artery into the kidney. The venous outflow (0 - 3 mmHg) leads the perfusate back into the venous reservoir. Syringe and infusion pumps secure the supply with additional compounds. The urine is collected throughout the perfusion. Perfusion circuit characteristics are recorded continuously. Hourly venous and arterial blood gas samples, and kidney injury markers are analyzed.

1. Connect the custom-made perfusion circuit to the kidney perfusion device.

2. Connect the tubing to the venous reservoir and oxygenator. Connect the arterial line tubing to the arterial outflow of the oxygenator and position the bubble filter in its holder. Connect the purge line. Connect the venous line tubing to the inlet of the venous reservoir.

3. For assessment during the perfusion, plug the temperature probe into the arterial outlet, connect the flow meter and the bubble sensor to the arterial line tubing, and connect the pressure lines. Connect the level sensor.

4. Connect the venous and arterial sample lines to the venous and arterial sample ports.

5. Position the organ chamber on a stand and introduce the venous and arterial tubing through the prepared holes. Fix the tubing to the table and chamber firmly.

6. Insert the suction tubing into the roller pump and position one end into the chamber to collect the fluids.

7. Connect the oxygen tubing to the gas tank containing 95% O2 / 5% CO2 and the oxygenator. Connect the heating unit tubing to the oxygenator and the organ chamber.

8. Use tubing clamps to close the venous and arterial outflow lines. Apply another tubing clamp to the outflow of the venous reservoir.

3.2.3.2 Preparation of Perfusion Solution, Additional Supplements, and Priming the Circuit

1. Use one infusion pump to replace the produced urine with Ringer’s lactate.

2. Use one syringe pump to administer nutrition (glucose 0.5 mL/hr, amino acids 0.5 mL/hr) and insulin (5 IU/hr) into the venous reservoir. Utilize a second syringe pump to infuse a vasodilator (verapamil, 0.25 mg/hr) directly into the arterial line.

3. Fill the venous reservoir with the perfusion solution. Therefore, pour Ringer’s Lactate (175 mL), STEEN solution (200 mL), dRO (27 mL), heparin (1,000 IU), sodium bicarbonate to adjust the pH, and calcium gluconate into the venous reservoir. Finally, add washed erythrocytes (125 mL).

4. Switch on the heart lung machine (HLM). Activate the pressure, temperature, level, and bubble sensor panels. Activate the Data Management System (DMS) to record the data throughout the perfusion. Activate the heating unit to warm the perfusion solution and the

organ chamber to 37 °C. Open the O2/CO2 supply.

5. Open the tubing clamp behind the venous reservoir and free the centrifugal pump head from air completely. Start the centrifugal pump at 1,000 rpm and allow the solution to be propelled throughout the circuit. Clamp the tubing bypassing the arterial filter and release air from the arterial filter.

6. Zero the pressure lines. Activate the syringe and infusion pumps.

3.2.3.3 Kidney Graft Perfusion

1. Remove the kidney from the ice, and position the kidney on bedding in the organ chamber. Place the urine catheter into the urine collector. After ensuring that the venous and the arterial tubing are free of air, plug the connectors to the tubing.

2. Close the shortcut between the arterial and venous tubing lines. Set the arterial pressure to 75 mmHg by regulating the speed of the centrifugal pump.

3. Record pressures, arterial flow, temperature, and presence of bubbles continuously with the DMS. Observe the values carefully throughout the perfusion. During the perfusion, blood leaking into the chamber is collected via the suction tubing back into the venous reservoir.

4. Record the quantity of urine produced. Collect venous blood and urine samples hourly. Monitor the perfusion by taking venous and arterial blood gas samples and aspartate aminotransferase (AST), and lactate analysis.

5. At the end of perfusion, disconnect the tubing from the renal artery and vein, flush the graft with cold HTK, and store it on ice in a sterile organ bag until transplantation.

3.3 Results

In the following the results of six experiments using a model of heart-beating kidney retrieval are presented. After in situ flush and kidney retrieval, the grafts were stored on ice for 3 hours (SCS) while the erythrocytes were prepared. For the clinical setting, this simulates the time required for the retrieval and the back table preparation. NEVKP was performed for 10 hr.

To maintain physiological conditions and simulate an in vivo surrounding for the kidney, the organ chamber should be heated and sealed. Perfusion and urine replacement solution should represent physiological values for blood gas analysis, oncotic pressure, and osmolarity. Normal values (baseline values) obtained from Yorkshire pigs in situ, are located in each figure description, respectively (Figures 3 - 13). The aim of NEVKP is to ensure that the graft is supplied with sufficient oxygen and nutrition. As ischemia causes vasoconstriction, thus increasing intrarenal resistance, achieving a constant flow with a stable pressure is a good indicator for adequate oxygenation. After the target graft temperature of 37 °C is reached via rewarming of the organ after SCS, flow values and intrarenal resistance remain stable with a constant physiological pressure of around 60-80 mmHg throughout the whole perfusion (Figures 3 and 4). The quantity of urine production depends mainly on the composition of the perfusion solution (Figure 5). Hourly measurements of venous and arterial pO2 reveal the metabolic activity of the kidney. The oxygen consumption was calculated using the equation ((pO2art – 144 pO2ven) x flow / weight) (Figure 6) .

Figure 3.3. Mean arterial flow with standard deviation (L/min). Throughout the perfusion the flow remains in a physiological range. Porcine physiological values, measured in situ: mean art. flow: 170 ± 57 mlLmin (range 83 – 325 mlLmin). Figure 3.4. Intrarenal resistance (IRR), mean and standard deviation (mmHg/mL/min). The mean arterial pressure (MAP) remains

47 constant between 60 and 80 mmHg. The intrarenal resistance is below 0.5 mmHg/mL/min constantly.

Figure 3.5. Total urine volume, mean and standard deviation (mL). The total urine volume mainly depends on the composition of the perfusion solution. The higher the oncotic pressure and the osmolarity, the lower the urine production. Figure 3.6. Oxygen consumption, mean and standard deviation (mL/min/g).

During the perfusion pH, HCO3-, and electrolytes are stable without requiring interventions (Figures 3.7- 3.10). Real-time AST and lactate measurements serve to monitor cellular damage. No increase of parameters of cell injury is detected during the NEVKP period (Figures 3.11 and 3.12). The osmolarity of the perfusion solution is stable (Figure 3.13). Histological assessment reveals minor changes (Figure 3.14 – 3.16).

Figure 3.7. pH venous, mean and standard deviation. The pH remains constant in a physiological range without administration of bicarbonate. Porcine physiological values, measured in situ: pH 7.46 ± 0.06 (range 7.34 – 7.63). Figure 3.8. HCO3- venous, mean and standard deviation (mmol/L). The HCO3- remains in a physiological range without administration of bicarbonates. Porcine physiological values, measured in situ: HCO3- 30.3 ± 2.4 mmol/L (range 21.6 – 35.8 mmol/L).

Figure 3.9. Venous sodium concentration, mean and standard deviation (mmol/L). The sodium remains in a physiological range. Porcine physiological values, measured in situ: 137.1 ± 3.8 mmol/L (range 118.7 – 140.9 mmol/L). Figure 3.10. Venous potassium concentration, mean and standard deviation (mmol/L). The potassium remains constant in a physiological range. Porcine physiological values, measured in situ: 3.85 ± 0.46 mmol/L (range 3.5 – 5.36 mmol/L).

Figure 3.11. Venous aspartate aminotransferase, mean and standard deviation (AST; U/L). In the ex vivo normothermic kidney perfusion, AST demonstrates a cell injury marker. AST values are low throughout perfusion. Figure 3.12: Lactate, mean and standard deviation (mmol/L). In the ex vivo normothermic kidney perfusion, lactate represents a cell injury marker. The values are stable throughout the perfusion.

Figure 3.13: Osmolarity of the serum, mean and standard deviation (mosm/L). A constant osmolarity in the perfusion solution secures low but constant urine production. Porcine physiological values, measured in situ: 282 ± 1.7 mosm/L (range 279 – 283 mosm/L).

Figure 3.14: Histology (H&E). 50x / 200x magnification of corticomedullary junction showing mild tubular vacuolization. No signs of necrosis. Figure 3.15: Histology (PAS). 50x / 200x magnification of corticomedullary junction showing mild tubular vacuolization. No signs of necrosis.

Figure 3.16: Histology (TUNEL staining). 25x / 200x magnification. Very occasionally nuclei are stained demonstrating very low rates of apoptosis.

3.4 Discussion

This study demonstrates that NEVKP with an erythrocyte-based solution can be performed with excellent results for a prolonged period of time in a porcine model. During the 10 hrs ex vivo perfusion the kidneys demonstrated stable perfusion parameters, active renal metabolism, homeostasis, and minimal renal injury.

Urine production and kidney injury depend on the composition of the perfusion solution. It is important to keep oncotic pressure and osmolarity of the perfusate within a physiologic range. In particular, a low oncotic pressure will result in an unphysiologically high urine production with significant kidney edema and increasing markers of kidney injury. STEEN solution containing albumin is chosen in this model to regulate the oncotic pressure and to simulate physiologic conditions for the kidney. Sodium bicarbonate and calcium gluconate are added to the system to achieve physiological values of pH, HCO3-, sodium, potassium, calcium, and chloride. The selection and dosage of the vasodilator is important to secure sufficient blood flow and oxygen supply.

The technique of normothermic ex vivo kidney perfusion has several limitations. Ex vivo perfusion is not associated with hormonal support of the kidney, which could negatively impact longer perfusion periods. In addition, the new technology, at this point in time, is associated with increased costs. Future improvements might simplify the technology and reduce the costs. The development of a portable kidney perfusion device might allow to completely avoiding cold kidney storage in the future.

The severe and persisting organ shortage leads to an increased use of marginal organs (ECD or DCD kidney grafts) 162. Currently, organ preservation is based on static cold storage or hypothermic machine perfusion. As a prolonged cold ischemic time has a significant impact on the outcome of kidney function of standard criteria 165 and marginal grafts 96,163, new preservation techniques minimizing cold storage are of particular interest 166-169.

A major obstacle to use marginal grafts more extensively is the inability to assess the quality and viability of organs prior to transplantation. Currently, only clinical parameters such as donor age, donor related diseases, and warm ischemia time of the grafts are used for the decision of whether

51 an organ is accepted or declined for transplantation. By preserving the graft under normothermic conditions, graft assessment based on perfusion characteristics and data is possible. Real-time parameters such as renal vascular flow, pressure, intrarenal resistance, urine production, oxygen consumption, and kidney injury parameters (such as AST and lactate) are supposed to be useful parameters to assess the viability of the graft.

In addition, the active metabolism during NEVKP allows the application of repair strategies to improve marginal kidney grafts prior to transplantation. For example, inhibition of proinflammatory pathways, immunomodulation, gene transfer, as well as stem cell administration could be future techniques to modify kidney grafts during the preservation time and improve recipient outcome.

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4 Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

______

This chapter is modified from the following:

Kaths J.M., Echeverri J., Goldaracena N., Louis K.S., Yip P., John R., Mucsi I., Ghanekar A., Bagli D., Selzner M., Robinson L.A. (2016). Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol. The Journal of Visualized Experiments (JoVE).

A visualized version of this study can be viewed at the following: http://www.jove.com/video/53765/heterotopic-renal-autotransplantation-porcine-model-step-step

4.1 Introduction

Kidney transplantation is the treatment of choice for patients with end-stage renal disease, due to associated lower rates of morbidity and mortality when compared to dialysis 2,3,159. Despite major improvements in patient outcomes following kidney transplantation, graft shortage still poses a severe challenge worldwide. The number of patients waiting for a kidney transplant by far exceeds the number of organs available 66,170,171. To increase the number of kidneys available for transplantation and to reduce patient waiting times, further sources of kidney grafts are needed.

Commonly, standard criteria donor (SCD) and extended criteria donor (ECD) kidney grafts from donation after brain death (DBD) as well as kidneys recovered from live donors (LDKT) are utilized. Since the 1990s, an increasing number of kidney grafts have been recovered in a donation after circulatory death (DCD) scenario, to further expand the donor pool 18,78. However, DCD and ECD kidney grafts demonstrate acceptable but decreased outcomes after transplantation, depending on different factors, such as donor age, warm and cold ischemia times, and the preservation technique used 96,132,172. Thus, additional research is required to improve the outcome of patients receiving marginal kidney grafts and to further increase the donor pool.

The porcine model of renal transplantation is well established and provides a clinical important scenario to investigate innovative approaches for the improvement of marginal kidney graft outcomes. In contrast to rodent and canine kidneys, which are unilobular, porcine and human kidneys are multilobular and are anatomically similar, particularly in regard to the arterial, venous, and urinary collecting systems 173,174. In addition, porcine and human kidneys demonstrate similarities in the pathophysiology of ischemia reperfusion injury (IRI), biochemistry, and immunological parameters 175. Thus, porcine renal transplantation is well- suited to investigate new organ preservation methods for marginal kidney grafts 154,176,177, model human IRI 165, study immunological pathways and allograft tolerance 178, provide surgical training 179-181, test new pharmacological therapies 182, implement new medical devices, and study new immunological mechanisms in xenotransplantation 183-185.

The renal porcine and human transplantation settings are not completely analogous. This article focuses on important technical details that will facilitate successful establishment of a renal

54 autotransplantation model. Species-adapted pre- and postoperative housing, administration of anesthesia with close monitoring, and matched surgical techniques are described in the protocol and demonstrated in the video. Resection of the contralateral native kidney provides the opportunity to assess the function of the transplanted kidney. The placement of venous and urinary catheters and the use of metabolic cages allow more in-depth assessment. For studies aimed at investigating alternative graft preservation methods and mechanisms of IRI, autotransplantation models are superior to allotransplantation models, as they avoid the complications and confounding bias associated with rejection and use of immunosuppressive medications.

4.2 Materials and Methods

All animals received humane care in compliance with the ‘‘Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research and the ‘‘Guide for the Care of Laboratory Animals’’ published by the National Institutes of Health. The Animal Care Committee of the Toronto General Research Institute, Ontario, Canada, approved all studies.

Note: A schematic overview of the study protocol is presented in Figure 1.

Figure 4.1. Study Protocol.

4.2.1 Animals

1. Use male Yorkshire pigs (30 kg) in this protocol.

4.2.2 Kidney Graft Retrieval

4.2.2.1 Preoperative Procedure

1. House the male Yorkshire pigs in a research facility for at least one week to acclimatize them. Use intramuscular injection of a third-generation cephalosporin, such as ceftiofur, for 3 days to reduce the potential risk of infections with Streptococcus suis and Salmonella. Fast the pigs for a minimum of 6 hr before induction of anesthesia to prevent aspiration.

2. Initiate anesthetization of the pig by an intramuscular injection of ketamine (20 mg/kg), atropine (0.04 mg/kg), and midazolam (0.3 mg/kg). Subsequently, transport the animal from the housing facility to the operating room (OR).

3. Place the pig in a supine position on the OR table. Allow the pig to breathe 2 L of oxygen with 5% of isoflurane spontaneously. Expose the vocal cords with a laryngoscope and spray them with 2% lidocaine topical solution to prevent intubation-induced

laryngospasm. After intubation with a 6.5 mm tube, block the cuff with 3-5 ml of air. Note: Capnometry confirms the correct position of the tracheal tube.

4. Decrease the isoflurane gas to 2.5%. Set the ventilator to 14-16 breaths/min and the tidal volume to 10-15 ml/kg body weight. Monitor the pig closely. Heart rate and oxygen saturation are recorded by pulse oxymetry. Confirm proper anesthetization by reduced heart rate (below 150 beats/min) and blood pressure (below systolic values of 100 mmHg) as well as absence of porcine movements (no usage of muscle relaxants).

5. Under sterile conditions, introduce an 9.5 Fr. single lumen permanent catheter into the internal jugular vein using Seldinger technique 186. Briefly, use a needle to puncture the vein. After introducing the guide-wire, replace the needle with the peel-away introducer, followed by replacement of the wire with the vascular catheter. Fix the catheter to the skin using a 3-0 silk or non—absorbable monofilament suture.

6. Administer 500 mg of metronidazole, 1 g of cefazolin, and 20 mg of pantoprazole. Administer 200 mL of lactated Ringer's solution with 5% dextrose (D5W) and 1 mL of fentanyl citrate per hour intravenously throughout the surgery. Apply veterinary ophthalmic ointment on eyes to prevent dryness while under anesthesia.

4.2.2.2 Surgical Procedure

1. Following sterile disinfection and coverage of the surgical field, perform a midline incision of 25 cm in length. Insert a retractor. Cover large and small bowels with a towel and position them to the left side for optimal access to the right kidney.

2. Free the ureter and the right kidney itself from any adherent tissue using the cautery.

3. Dissect the right renal vein and artery using the cautery until their origin from the inferior vena cava and the aorta, respectively, are free. To avoid arterial vasospasm, administration of 30-65 mg of papaverine should be considered.

4. After complete renal dissection, tie (silk, 3-0) and cut the ureter distally. Prepare a bowl of ice and a sterile organ bag.

5. First, clamp the renal artery close to the aorta and second, clamp the renal vein close to the vena cava using vessel clamps. Next, resect the kidney graft and immediately cannulate the renal artery with a renal artery cannula. Use 500 ml of ice-cold histidine- tryptophan-ketoglutarate (HTK) solution to flush out the blood. Store the kidney on ice until transplantation.

6. In situ, close the remaining renal artery with a ligature (silk, 2-0) and the renal vein with a running suture (prolene, 6-0).

7. After checking the dissected area for bleeding, close the abdominal wall with a running suture (monofil, 1) and the skin with a 3-0 silk or non—absorbable monofilament suture.

4.2.2.3 Postoperative Procedure

1. Fix the venous catheter subcutaneously with a suture (silk, 3-0) and tunnel it to the pig's back to prevent unwanted manipulation. After placing the pig prone, suture (silk, 3-0) the catheter firmly to the skin.

2. Wean the pig from the ventilator and let it recover in its housing area after extubation. Administer Ringer's lactate intravenously for volume expansion and administer 0.3 mg buprenorphine for analgesia. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.

4.2.3 Kidney Graft Transplantation

4.2.3.1 Preoperative Procedure

1. Anesthetize the pig using intravenous injection of propofol (1-2 mg/kg body weight) followed by a continuous infusion of propofol at a rate of 50-100 mg/hr. Re-intubate the pig as described in step 2.1.3 and 2.1.4 and set the isoflurane gas to 3-4%.

2. Administer 1 g of cefazolin and 20 mg of pantoprazole i.v. During surgery, use the same anesthetic protocol as described in 2.1.4.

3. Following sterile disinfection, make a cut of 4 cm next to the trachea. Dissect the tissue to expose the carotid artery. Pass over-holt forceps and a silk tie (2-0) around the artery. Use the Seldinger technique to introduce a plastic catheter to continuously measure the

arterial pressure throughout the surgery. Alternatively, non-invasive blood pressure measurement techniques can be utilized.

4.2.3.2 Surgical Procedure

1. After sterile disinfection, reopen the abdominal cavity by cutting the stitches of the skin and fascia sutures, reintroduce the surgical retractor to expose the abdominal cavity, and reposition the bowel to the left side to allow better access to the infrarenal vessels.

2. Transplant the preserved kidney graft end-to-side to the infrarenal vena cava and aorta. Therefore, dissect vena cava and aorta over 5-8 cm above the iliac bifurcation using pickups and cautery. If possible, do not disturb the lymphatic vessels; if not possible, close them with 5-0 prolene sutures.

3. After completing the dissection, check for bleeding and remove remaining tissue from the vessels. Ensure that complete clamping of the vena cava and aorta with a Satinsky clamp is feasible.

4. Next, resect the contralateral (left) kidney. To do so, position the bowel to the right; dissect the ureter, the kidney itself, the renal vein, and the renal artery from adherent tissue. Tie the ureter and blood vessels and resect the kidney. Check for bleeding.

5. Reposition the bowel to the left to expose the infrarenal aorta and vena cava. Inject heparin (100 IU/ kg body weight) and wait for at least two minutes.

6. Venous Anastomosis:

a. Use a Satinsky clamp to completely clamp the vena cava and make a slit incision that matches the size of the opening of the renal vein, using an 11 blade. Pott scissors can be used to further extend the slit.

b. After wrapping the kidney into a cloth containing sterile ice, remove it from the ice and position it into the surgical field. Use two double-armed 6-0 prolene sutures to perform a cranial and a caudal corner stitch.

c. Approximate the kidney, tie the upper corner and perform a running suture using 6-0 prolene, starting with the back wall. After having finished 2/3, use the other end of the tie to complete the suture at the front side. After tying the cranial stitches, tie the stitches at the caudal corner.

d. Position a bulldog clamp on the renal vein and open the Satinsky clamp. Check the anastomosis for bleeding.

7. Arterial Anastomosis:

a. Use the Satinsky clamp again to completely clamp the aorta. Use an 11 blade to make a slit incision, matching the opening of the renal artery. Use a 4.0 mm round punch to secure a clean opening.

b. Use one 6-0 prolene suture to perform the arterial anastomosis, starting at the recipient side. Ensure that the arterial endothelium is included in each suture to prevent a dissection. Meanwhile, start a continuous drip of 10 mL norepinephrine (16 mg/250 mL) diluted in 500 ml of Ringer's lactate and titrate to keep the systolic pressure above 100 mmHg.

c. Inject verapamil intra-arterially before completion of the arterial anastomosis and administer papaverine topically to the outside of the vessel to prevent vasospasm.

d. Position a bulldog clamp on the renal artery and open the Satinksy clamp. Check the anastomoses for bleeding.

e. Unwrap the kidney from the cloth and remove the ice. Open the venous bulldog clamp first, followed by the arterial bulldog clamp. After reperfusion, urine production should start immediately.

f. Use cloth to secure a favorable position for the transplanted graft and maintain a homogenous reperfusion.

8. Ureteral Anastomosis:

a. Use Pott scissors to open the ureter from the graft and the recipient over a longitudinal length of 0.5 cm.

b. Use two 6.0 polyester, poly (p-dioxanone) sutures for the side-to-side ureteral anastomosis. Perform a corner stich at each side, then run the back wall in a continuous manner first, followed by the front wall.

c. After checking for bleeding, remove the cloth and wrap some of the small bowel around the kidney to hold it in position. Close the abdominal wall with two monofil 1 sutures. Close the skin with 3-0 silk or non—absorbable monofilament suture.

d. Maintain the systolic pressure above 100 mmHg continuously by carefully titrating the norepinephrine infusion until the pig has been placed into prone position.

4.2.3.3 Postoperative Procedure

1. After abdominal closure as mentioned above, keep the pig warm using a heating pad and heat-circulating blanket. Remove the arterial line, close the puncture hole in the artery with a 6-0 prolene stich and close the incision site.

2. Turn the pig onto prone position, stop the norepinephrine drip and wean the pig from the ventilator. Allow the pig to recover in its housing area and monitor it closely to ensure its smooth recovery from the procedure. Take blood gas samples each hr via the implanted jugular catheter. Provide Ringer's lactate to substitute volume and administer 0.3 mg buprenorphine for analgesia.

3. Following extubation, monitor the pig closely until it is able to drink spontaneously. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.

4.2.4 Postsurgical Follow Up

1. Administer 0.3 mg buprenorphine i.v. every 8 hr for at least 2 days post surgery or longer if needed. Routinely administer a single prophylactic dose of antibiotic during surgery. In the event of signs of infection, administer cefazolin 1 g i.v. twice per day and metronidazole i.v. once per day until clinical improvement occurs. Administer Ringer's lactate until the pig drinks sufficient water. 1,000 IU heparin can be used to lock the catheter to prevent clotting.

2. Collect venous blood samples via the jugular catheter and urinary samples to assess the pig's clinical condition and renal function.

3. For euthanasia, induce anesthesia of the pig with propofol i.v. (5-10 mL) and maintain it with isoflurane 5%. Intubate the pig as described above. After relaparatomy and renal tissue sample collection, induce cardiac arrest by intravenous injection of 40 mval KCl.

4.3 Results

In the following, the results of renal autotransplantation experiments (n = 4) are demonstrated. After the initial graft retrieval, the pigs recovered in their housing area. Meanwhile, the kidney grafts were stored on ice for a mean time of 7 hr 35 min (± 18 min). After reinduction of anesthesia and repeat laparotomy, the contralateral kidneys were resected and the cold-stored grafts transplanted heterotopically as described. After weaning from the ventilator, pigs were recovered from surgery and followed up for 10 days (see Figure 4.1). Daily (1-4 post-operative day; pod) or every second day (6-10 pod) blood samples were collected to perform blood gas analyses; to assess the renal function, serum creatinine and blood urea nitrogen (BUN) values were estimated. For comparison, the results of one allotransplanted kidney graft are presented. For immunosuppression, this pig received cyclosporine 100 mg p.o. and cortisone 250 mg i.v. b.i.d. The surgical technique used was the same as in the autotransplant protocol; no warm ischemia time was applied.

All pigs were in good clinical condition during the follow up period. The serum creatinine and BUN values revealed the highest increase at day one after surgery (Crea 2.8 ± 0.7 mg/dL, BUN 25.3 ± 7 mg/dL) and decreased until pod 10 (Crea 1.7 ± 0.4 mg/dL, BUN 10.7 ± 4 mg/dL) close to the initial baseline values. The allotransplanted kidney graft demonstrated higher creatinine and BUN values after good initial graft function, when compared to the autografts, most likely due to rejection (Figure 4.2 and 4.3). Acid-base hemostasis (Figure 4.4) and electrolyte levels (Figure 4.5) were stable without intervention. Histological examination showed preserved tubulointerstitium in the autotransplanted kidney (Figure 4.6), and diffuse interstitial inflammation, tubulitis, and glomerulitis in the allotransplanted kidney (Figure 4.7).

Figure 4.2: Serum creatinine values. Serum creatinine values (mean and standard deviation) for baseline and 10 days after surgery. Figure 4.3: Serum BUN values. Serum BUN values (mean and standard deviation) for baseline and 10 days after surgery.

Figure 4.4: Acid-base hemostasis. Acid-base hemostasis (mean and standard deviation) for baseline and 10 days after surgery.

Figure 4.5: Electrolyte levels. Electrolyte levels (mean and standard deviation) for baseline and 10 days after surgery.

Figure 4.6: Histology (H&E), 100x magnification. Normal tubulointerstitium in the autotransplanted kidney 10 days after surgery. Figure 4.7: Histology (H&E), 100x magnification. Extensive interstitial inflammation, tubulitis, and glomerulitis, consistent with rejection, in the allotransplanted kidney 10 days after surgery.

4.4 Discussion

The model of porcine kidney transplantation provides a unique opportunity to further the field of human transplantation due to similarities in surgical aspects, physiology, biochemistry, and immunology 175.

Depending on the purpose of the experimental study, the model of renal autotransplantation has several advantages compared to the allotransplantation model. Although several groups report good renal graft function after allotransplantation 187, immunosuppression in pigs is challenging, especially in renal transplantation. Preoperative blood sample analyses to ensure compatibility for swine leukocyte antigen (SLA) are feasible, but expensive and impractical 175. Postoperatively, proposed immunosuppressive agents such as tacrolimus and cyclosporine (calcineurin inhibitors, CNI) are administered orally or i.v. 187. Oral administration is impractical, as pigs usually refuse to swallow oral medication. Furthermore, intestinal obstructions might obviate sufficient absorption of immunosuppressive medications and maintenance of therapeutic drug levels. The continuous infusion of CNI’s i.v. in active animals is technically demanding. I.v. bolus administration leads to high peak values, which cause toxicity. Thus, for the investigation of new preservation techniques, the model of renal autotransplantation has several advantages. In the representative results of the allotransplantated kidney graft demonstrated above, a delayed and increased peak of creatinine and BUN indicate rejection, which was demonstrated by histological assessment.

The porcine model of autotransplantation has previously been used to investigate new preservation techniques 165,175,188. However, the reported postoperative serum creatinine and BUN values of autotransplanted pigs in a heart-beating scenario vary considerably depending on the experimental system 181,189. The heart-beating donor protocol we present here results in a low postoperative serum creatinine peak of 2.8 mg/dL (± 0.7) and BUN peak of 25.3 mg/dL (± 7.4). These results are comparable with the low peak values presented by Hanto and colleagues 187 and Snoeijs and colleagues 190.

To ensure a successful outcome after renal transplantation in a porcine autotransplantation model, we have identified several key technical factors that minimize the rate of certain complications. The use of histidine-tryptophan-ketoglutarate solution (HTK) reduces the risk of

66 vasospasm due to its lower content of potassium when compared to University of Wisconsin (UW) solution. To further decrease the risk of vasospasm at the point of reperfusion, verapamil can be injected into the renal artery, and papaverine can be administered topically during retrieval and after reperfusion. In addition, a continuous drip of norepinephrine titrated to maintain the systolic blood pressure above 100 mmHg ensures a homogeneous reperfusion. It is useful to maintain this blood pressure at least until the pig is positioned prone. Furthermore, the positioning of the transplanted graft is important to prevent kinking of the newly anastomosed blood vessels. Therefore, it is helpful to resect the contralateral left kidney prior to sewing the anastomoses of the graft to avoid extensive mechanical manipulation. After finishing the ureteral anastomosis, wrapping small intestine around the transplanted graft secures its position after closure of the abdominal wall. Complications such as bowel obstructions due to kinking of the intestine are rarely observed but can lead to severe complications, including ileus, bowel perforation, and death. Overall, accurate surgical technique, attentive anesthesia and close monitoring during follow up ensure good clinical outcome and graft function.

Arterial and venous anastomoses can be performed using different techniques. Orthotopic placement of the graft allows end-to-end anastomoses of the renal artery and vein. In the case of heterotopic transplantation, the graft can be positioned in the contralateral renal fossa for end-to- end anastomoses, onto the iliac vessels, or the distal aorta directly. Heterotopic transplantation with anastomoses to aorta and cava directly in end-to-side technique are preferred in this model as it can reduce the risk of thrombosis and vasospasm 191. Anatomical variations with very early venous bifurcations might lead to the need of sewing two separate venous anastomoses. If the artery or vein are relatively short, the graft can be turned 180° to gain length of the vessels. Ureteral side-to-side anastomosis can achieve good experimental results without complicating strictures or urinary leak.

In general, the porcine model of renal transplantation offers advantages compared to other animal models. As described above, certain similarities exist between the porcine and the human setting, which allows relatively fast translation of new techniques into clinical practice. The technique of transplantation is technically easier compared to rodent models. In addition, by placement of venous catheters, peripheral blood samples can be collected easily and processed for further investigation. The collection of urine allows further assessment of kidney injury and function. To collect urine samples, a percutaneous catheter can be inserted into the urinary

67 bladder. To avoid manipulation by the pig, the distal end should be tunneled subcutaneously to the back of the animal. Another option for urine collection is the use of metabolic cages, which allow prolonged collection periods to estimate the creatinine clearance and concentration of additional biomarkers in the urine. Sonography, CT scans, and MRI images are possible. Donation after circulatory death protocols can be mimicked by applying warm ischemia prior to retrieval. Furthermore, pigs are relatively easy to handle if castrated to limit their aggressive behavior.

Disadvantages include the high costs of animal purchase, housing, surgical and other medical equipment, and manpower. These factors mean that it is not feasible to include large numbers of animals in each study group. Furthermore, compared to rodent models, a limited number of references are available in the literature for pig normative biological data. As an alternative for the assessment of new developed techniques, such as novel preservation methods, other groups have described the normothermic ex vivo reperfusion as an alternative to renal transplantation 192,193. This technique is easier to perform and less expensive. However, standardized kidney graft transplantation provides a model more similar to the clinical practice and allows longer follow up periods. Therefore, it serves for a more realistic graft assessment.

In conclusion, the porcine model of heterotopic renal autotransplantation provides a clinical important scenario to investigate innovative novel approaches for the improvement of kidney graft outcomes. In particular, this protocol features important technical details that will facilitate successful establishment of a renal autotransplantation model and allows the rapid translation of new findings to clinical trials.

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5 Eight Hour Continuous Normothermic Ex Vivo Kidney Perfusion is a Safe Preservation Technique for Kidney Transplantation: A New Opportunity for the Storage, Assessment, and Repair of Kidney Grafts

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This chapter is modified from the following:

Eight Hour Continuous Normothermic Ex Vivo Kidney Perfusion is a Safe Preservation Technique for Kidney Transplantation: A New Opportunity for the Storage, Assessment, and Repair of Kidney Grafts. Transplantation. In press.

J. Moritz Kaths1, 2, 3, Juan Echeverri1,4, Nicolas Goldaracena1, Kristine S. Louis1, Yi-Min Chun1, Ivan Linares1, 3, Aryn Wiebe1, Daniel Foltys5, Paul M. Yip6, Rohan John6, Istvan Mucsi7, Anand Ghanekar1, Darius Bagli3, 8, 9, David R. Grant1, Lisa A. Robinson*2, 3, 10, Markus Selzner*1, 3

(*Both authors share senior authorship).

1 Multi Organ Transplant Program, Department of Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada 2 Division of Nephrology, The Hospital for Sick Children, Toronto, Ontario, Canada 3 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada 4 Programa de Doctorat en Medicina de la Universitat Autònoma de Barcelona, Barcelona, Spain 5 Department of General, Visceral, and Transplant Surgery, University Medical Center Mainz, Mainz, Germany 6 Laboratory Medicine & Pathobiology, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada 7 Multi Organ Transplant Program, Department of Medicine, University of Toronto, Toronto, Ontario, Canada 8 Departments of Surgery (Urology) & Physiology, The Hospital for Sick Children, Toronto, Ontario, Canada 9 Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada 10 Transplant and Regenerative Medicine Centre, The Hospital for Sick Children, Toronto, Ontario, Canada

Corresponding Author:

Markus Selzner, MD Associate Professor of Surgery, University of Toronto General Surgery & Multi-Organ Transplant Program Toronto General Hospital, University Health Network 585 University Avenue, 11 PMB 178 Toronto, ON M5G 2N2 t.416-340-4800 ext. 5884 f.416-340-5321 email: [email protected]

Lisa A. Robinson, MD, FRCP(C) Division of Nephrology The Hospital for Sick Children 555 University Avenue, Room 5265 Toronto, Ontario M5G 1X8 Phone: 416-813-7654 ext. 206289 Fax: 416-813-6271 e-mail: [email protected]

5.1 Introduction

Kidney transplantation is the treatment of choice for patients with end-stage renal disease as it results in lower morbidity and mortality rates when compared to dialysis 2-4. However, the limited number of deceased donor grafts available represents a severe problem worldwide 7,66,194. Deceased standard criteria donors (SCD) represent the main source for kidney transplantation. The organ shortage has triggered interest in expanding the pool of available kidneys by using renal grafts from donors with higher age, hypertension, increased terminal serum creatinine levels of >1.5 mg/dL, or death from cerebrovascular accident (extended criteria donors; ECD) 75 to increase the number of available donors. Donation after circulatory death (DCD) represents another large potential donor pool and has been practiced around the world since the late 1990s 18. However, the poorer outcome of ECD and DCD grafts has been a consistent concern. Higher rates of primary non-function (PNF), delayed graft function (DGF), and increased rates of early graft loss (EGL) have been reported 94,96.

Current renal graft preservation techniques prior to transplantation include static cold storage (SCS) and hypothermic machine perfusion (HMP). The principle of hypothermic techniques is based on the reduction of the tissue’s metabolic activity to reduce tissue injury. Unfortunately, ECD and DCD grafts tolerate cold anoxic storage only poorly and the detrimental impact of hypothermia has been demonstrated in several studies 96,195. Even in SCD heart-beating donor (HBD) kidney transplantation DGF rates of up to 20-50% have been reported in particular after prolonged hypothermic preservation 29,30,196. DGF in HBD kidney transplantation is known to result in decreased long-term outcomes and increased health care costs 197,198. Solid evidence supports the concept that even modest lengthening of cold ischemia times may worsen outcome in kidney transplantation of HBD grafts 199. In a recently published study, Debout et al. investigated the relationship between cold ischemia time and post transplant outcomes in 3839 adult recipients of a first heart-beating deceased donor kidney transplant between 2000 and 2011. The results demonstrated a significant increase in the risk of graft failure and mortality for each additional hour of cold ischemia 200.

Awareness of detrimental effects of hypothermia has resulted in the exploration of alternative storage techniques. A novel approach to solid organ preservation is normothermic ex vivo machine perfusion with avoidance of prolonged cold storage. Cypel and colleagues have

71 demonstrated favorable outcomes in experimental models and clinical practice using normothermic ex vivo lung perfusion 201. Others noted similar benefits using subnormothermic ex vivo liver perfusion in experimental models 167,202-205. In renal transplantation, Brasile, Stubenitsky, Kootstra et al. investigated acellular, low pulsatile pressure perfusion at subnormothermic temperatures of 32 °C demonstrating promising results in canine models in the early 2000s 144,145,147. Recently, Hosgood and colleagues demonstrated improved outcomes for ECD kidney grafts by an additional application of one hour of normothermic ex vivo kidney perfusion prior to transplantation in clinical practice 156.

We report here a novel technique of continuous, pressure-controlled, erythrocyte-based normothermic ex vivo kidney perfusion (NEVKP) 206. Our ultimate goal is to entirely replace cold storage with NEVKP as the preservation method. The aim of our study was to determine feasibility and safety of replacing cold storage with normothermic ex vivo kidney perfusion in a pig model of SCD HBD kidney transplantation.

5.2 Materials and Methods

5.2.1 Study Design

Heart-beating donor kidney retrieval was performed and the grafts were either stored for 8 h in cold histidine-tryptophan-ketoglutarate (HTK) solution, or preserved using 8 h of NEVKP (n=5 in each group). Then, kidney autotransplantation was performed in both groups with 10 d of follow-up. Perfusion characteristics, graft injury, and graft function after transplantation were determined. The study was approved by the Animal Care Committee of the Toronto General Research Institute, Ontario, Canada.

5.2.2 Animals

Male Yorkshire pigs (30 kg) were used and housed in species-adapted pens to acclimatize for one week. Water and food were provided ad libitum. All animals received humane care in compliance with the ‘‘Principles of Laboratory Animal Care’’ formulated by the National Society for Medical Research and the ‘‘Guide for the Care of Laboratory Animals’’ published by the National Institutes of Health.

5.2.3 Normothermic Ex Vivo Kidney Perfusion

A neonatal cardiopulmonary bypass circuit was used for the ex vivo kidney perfusion at 37°C. The customized perfusion circuit consisted of a venous reservoir and an oxygenator (D100 neonatal venous reservoir and oxygenator; Sorin Group Inc., Markham, Canada), an arterial filter (D130 neonatal arterial filter; Sorin Group Inc.), P.h.i.s.i.o coated PVC tubing (Sorin Group Inc.), and a customized double-walled organ chamber. The centrifugal pump of an S3 heart-lung- machine (Sorin Group Inc.) was used to perfuse the oxygenated perfusion solution through the circuit and the kidney graft. Perfusion circuit parameters such as temperature, arterial and venous pressure, and arterial flow were recorded continuously with the Data Management System (Sorin Group Inc.). Bubble and reservoir level sensors were used to prevent air embolism (Figure 5.1).

Figure 5.1: Schematic of the NEVKP Circuit. The circuit consists of neonatal cardiopulmonary bypass technology. The perfusion solution is collected in the venous reservoir. A centrifugal pump propels the solution into the oxygenator, where it is enriched with oxygen and warmed to 37°C. After passing the arterial filter, the perfusate is driven with a pressure of 65 mmHg through the renal artery into the graft located in the customized double-walled kidney chamber. The venous outflow (0 – 3 mmHg) leads the perfusate back into the venous reservoir. Syringe and infusion pumps secure the supply with additional compounds. The urine is collected throughout the perfusion. Control panel and Data Management System (DMS) indicate and record perfusion parameters continuously.

Prior to graft retrieval, whole blood was collected from a separate donor animal and passed through a leukocyte filter. Following centrifugation, the isolated erythrocytes were washed in sterile saline solution to avoid contamination with leukocytes or plasma. Heart-lung machine and perfusion circuit were prepared and primed with a physiologic perfusion solution just prior to initiation of the retrieval procedure. The composition of the perfusion solution is displayed in Table 5.1. It consisted of leukocyte-depleted washed erythrocytes (125 mL), mixed with STEEN

SolutionTM (150 mL; XVIVO Perfusion AB, Goteborg, Sweden). In addition, Ringer’s lactate (200 mL), double reverse osmosis (DRO) filtered water (27 mL), sodium bicarbonate (pH dependent; Hospira, Montréal, Canada), calcium gluconate (1.8 mL; Pharmaceutical Partners of Canada Inc., Richmond Hill, Canada), and heparin (1000 IU; LEO Pharma, Thornhill, Canada) were added resulting in physiologic blood gas values, oncotic pressure, and osmolarity (Table 5.2).

Composition of the perfusate solution Ingredient Amount / Rate Stock solution Ringer’s lactate 200 mL STEEN solutionTM 150 mL Erythrocytes (leukocyte-depleted) 125 mL Double reverse osmosis (DRO) filtered water 27 mL Sodium bicarbonate 8 mL Calcium gluconate 1.8 mL Heparin 1000 IU Continuous administration Ringer’s lactate Replacement of produced urine and evaporation Amino acids and glucose (intravenous) Target glucose concentration: 5 – 15 mmol/L Insulin (intravenous) 5 IE/h Verapamil (intraarterial) 0.25 mg/h

Table 5.1: Ingredients in perfusate solution and amount or rate administered.

Blood gas analyses, osmolarity, and oncotic pressure at baseline Physiologic values for Values for NEVKP set Yorkshire pigs at up at baseline baseline (n = 20) (n = 5) Venous blood gas analysis pH 7.46 ± 0.06 7.37 ± 0.02 pCO2 43.5 ± 7.3 mmHg 36.1 ± 5.5 mmHg pO2 47.5 ± 7.3 mmHg 633 ± 21 mmHg - HCO3 30.3 ± 2.4 mmol/L 20.2 ± 2.8 mmol/L Hb 104 ± 10 g/L 105 ± 14 g/L

O2 Sat - 99.9 % Na+ 137 ± 3.9 mmol/L 142 ± 0.8 mmol/L K+ 3.9 ± 0.5 mmol/L 3.5 ± 0.1 mmol/L Ca2+ 1.25 ± 0.10 mmol/L 1.36 ± 0.15 mmol/L Cl- 101 ± 1.9 mmol/L 108 ± 2 mmol/L Glucose 4.7 ± 2.5 mmol/L 4 ± 0.4 mmol/L Lactate 0.94 ± 0.19 mmol/L 10.38 ± 0.76 mmol/L Osmolarity 282 ± 2 mosmol/L 286 ± 4 mosmol/L (n = 6) (n = 5) Oncotic pressure 14 ± 0.8 mmHg 11 ± 0.9 mmHg (n = 6) (n = 5)

Table 5.2: Blood gas analysis, osmolarity, and oncotic pressure measured at baseline in Yorkshire pigs and at the start of normothermic ex vivo kidney perfusion.

In the NEVKP group, kidney grafts were retrieved and flushed with 300-500 mL Ringer’s lactate solution. Meanwhile, arterial (1.6”, Sorin Group Inc.) and venous cannulas (1/4” x 1/8”, Sorin Group Inc.) were fixed with 2-0 silk ties (Covidien, Mississauga, Canada). Immediately after the flush, grafts were connected to the perfusion circuit for preservation under normothermic conditions. Perfusion was started with a mean arterial pressure of 70 mmHg; following graft adaption to the system, a physiologic arterial pressure of 65 mmHg was targeted (Figure 5.2). After the first hour of perfusion, the pressure was stable without the need to adapt the speed of

76 the centrifugal pump. Throughout the perfusion, verapamil (0.25 mg/h; Sandoz Canada Inc., Toronto, Canada), amino acids mixed with glucose solution (1 mL/h, adapted to a target glucose concentration of 5 – 15 mmol/L; Travasol 10%, Dextrose 50%, Baxter, Mississauga, Canada), and insulin (Novorapid 5 IE/h, Novo Nordisk Canada Inc., Mississauga, Canada) were added continuously. The urine was continuously replaced with Ringer’s lactate (Table 5.1). Blood gas parameters, concentration of lactate, and potential cell injury markers aspartate aminotransferase (AST) 207 and lactate dehydrogenase (LDH) 208 were measured in the perfusate hourly 144,209. Perfusate samples were collected hourly and frozen at -80°C after centrifugation for further assessment. Following NEVKP, the grafts were flushed with 4°C cold histidine-tryptophan- ketoglutarate (HTK; Metapharm Inc., Bratford, Canada) and kept cold for the duration of the vascular anastomosis. A detailed visualized description of the NEVKP technique has recently been published by our group 206.

5.2.4 Static Cold Storage

In the control group, kidney grafts were retrieved and flushed with 300-500 mL of 4 °C cold HTK solution with a pressure of 100 cmH2O. In a sterile organ bag (CardioMed Supplies Inc., Lindsay, Canada), grafts were submerged in preservation solution and placed on ice until autotransplantation.

5.2.5 Kidney Retrieval and Transplantation

A porcine model of heterotopic renal autotransplantation was chosen to further investigate the technique of normothermic ex vivo kidney perfusion. Anesthesia was administered as an intramuscular injection of ketamine (20 mg/kg; Bimeda-MTC Animal Health Inc., Cambridge, Canada), atropine (0.04 mg/kg; Rafter 8 Products, Calgary, Canada), and midazolam (0.3 mg/kg; Pharmaceutical Partners of Canada Inc., Richmond Hill, Canada). After intubation, anesthesia was continued with inhaled isoflurane (2.5%; Pharmaceutical Partners of Canada Inc., Richmond Hill, Canada). For administration of fluids and medication, a permanent venous catheter (9.5 French; Cook Medical Company, Bloomington, US) was placed into the right internal jugular vein using the Seldinger technique. After a midline incision, dissection of the right kidney and its adherent structures was performed, the renal artery and vein were clamped, and the graft was resected. Immediately, the renal artery was cannulated with a 1.6” cannula (Sorin Group Inc., Italy) and kidneys were either flushed with Ringer’s lactate and placed on pump (NEVKP; study

77 group), or flushed with 4 °C cold HTK solution and placed on ice (SCS; control group) for preservation, respectively. Following abdominal closure, the pigs recovered from surgery. After graft preservation time (8h), pigs were re-anaesthetized with propofol (Pharmascience Inc., Montréal, Canada) and re-intubated for the process of autotransplantation. Following repeat laparotomy and resection of the contralateral (left) kidney, grafts were removed from pump (study group) and flushed with 4°C cold HTK, or removed from ice directly (control group) for sewing of the anastomoses. Following autotransplantation of the graft to the infrarenal vena cava (end-to-side) and aorta (end-to-side), the donor ureter was anastomosed side-to-side to the recipient ureter. Shortly before reperfusion of the graft, 500 mg of solu-medrol (Pfizer Canada Inc.) was administered intravenously; norepinephrine (Baxter Corporation) was administered to ensure a systolic pressure, measured non-invasively, of at least 80 mmHg. After abdominal closure, the pigs recovered from surgery and were followed for ten days for further assessment. Peri- and postoperatively, pigs received antibacterial prophylaxis (Ceftiofur 3 mg/kg i.m. OD for 3 d pre-operatively, Pfizer Canada Inc., Calgary, Canada; metronidazole 500 mg i.v. OD for 10 d postoperatively, Baxter Corporation; and cefazolin 1 g i.v. for 10 d postoperatively, Pharmaceutical Partners of Canada Inc.). Pantozole (20 mg bid on day of surgery; Sandoz Canada Inc.) was given to prevent gastrointestinal bleeding; buprenorphine (0.3 mg; RB Pharmaceuticals LTD, Mississauga, Canada) was administered every 8 hours following surgical procedure for at least 2 postoperative days. The decision to administer further analgesia was based on clinical behavior of the pigs but was rarely necessary. Throughout retrieval and transplant procedure and recovery in the evening, animals received in total 3 L of intravenous fluids. During the initial postoperative course, about 200 mL of intravenous fluids were given in the morning and the evening when sampling the animals until full recovery. Animals were also permitted to drink ad libitum. A visualized description of the heterotopic renal autotransplantation technique has recently been published by our group (Kaths et al., The Journal of Visualized Experiments, in press).

5.2.6 Whole Blood, Serum, and Urine Measurements

Perfusate and whole blood were sampled for blood gas analysis (RAPIDPoint 500 Systems, Siemens AG, Berlin, Germany) hourly during NEVKP, or at baseline and each morning during follow up of the transplanted pigs, respectively. Serum samples were collected for analysis of AST and LDH (Vitros DT60 II, Johnson & Johnson, Markham, Canada) during NEVKP and for

78 measurement of creatinine and blood urea nitrogen (BUN) Piccolo Xpress, Union City, Canada) for baseline and each morning during follow up. 24h urine collection was performed using a metabolic cage to investigate the creatinine clearance before transplantation (day 0), and on postoperative day 10. Further serum and urine analyses were performed in the core lab using Abbott Architect Chemistry Analyzer using the manufacturer’s reagents (Abbott Laboratories, Abbott Park, IL, USA).

5.2.7 Histology

Ten days after transplantation, the abdomen was opened under anesthesia, a wedge of renal tissue retrieved and placed into 10% neutral buffered formalin for histology and immunohistochemistry analyses. Fixed kidney tissue was paraffin-embedded, sectioned and stained. 3-µm periodic acid-Schiff (PAS) stained sections were used to score tubular injury, edema, fibrosis, and interstitial inflammation on a scale of 0 to 3 as previously described by us and others blinded to the experimental group. Histopathologic changes representing tubular injury including brush border loss, tubular dilatation, epithelial vacuolation, thinning and sloughing, and luminal debris were scored in 10 high power fields (HPF) and averaged to assess overall tubular injury 206 210 211. Interstitial inflammation was scored in 10 low power fields and averaged. TUNEL staining was performed according to standard protocol. Because of the low rate of TUNEL-positive cells, the total number of positive cells were manually counted in 25 HPF and averaged.

5.2.8 Statistical Analysis

Statistical analysis was performed using SPSS software version 23.0 (IBM, Armonk, NY, USA). Differences in mortality were calculated with Fisher’s exact test (2-sided). Variables were tested for normal distribution using Kolmogorov-Smirnov Test / Shapiro-Wilk Test. Students t-test was used to compare differences of continuous values between the two groups. A paired t-test was used to calculate differences between paired continuous values. To compare ordinal, non- parametric data the Mann Whitney U test was used. Significance was defined as p < 0.05.

5.3 Results

5.3.1 Demographics

The weights of the pigs were not different between both groups (NEVKP 30.4 ± 1.9 kg vs. SCS 32.7 ± 1.9 kg, p = 0.092). Preservation and anastomoses times in NEVKP groups vs. SCS group were similar with 452.4 ± 22.7 vs. 461.0 ± 20.7 minutes (p = 0.549) and 33.4 ± 3.8 vs. 36.2 ± 8.8 minutes (p = 0.533), respectively.

5.3.2 Perfusion Characteristics during NEVKP

Normothermic ex vivo kidney perfusion was initiated with an arterial pressure set to 70 mmHg. After rewarming of the graft, the pressure was adjusted to 65 mmHg throughout the whole perfusion; this is identical to the mean systemic pressure measured invasively in anesthetized healthy pigs (Figure 5.2A). Venous pressure was maintained at around 2 mmHg by height regulation of the venous reservoir (Figure 5.2B). An initial renal artery blood flow rate of 114 ± 18 mL/min was measured. After rewarming of the renal graft flow rates of around 200 mL/min were attained. The achieved flow rates were above the mean values of anesthetized healthy pigs, which were measured in situ using flow probes (Figure 5.2C) as described previously by our group 212,213. Intrarenal resistance (IRR) was calculated by dividing the arterial pressure by the arterial flow. Baseline IRR at the start of the NEVKP was 0.63 ± 0.1 (mmHg/mL/min) which decreased during NEVKP below the mean values that were measured in situ in 30 pigs. Comparison between baseline IRR and IRR at last hour of perfusion demonstrated a significant decrease (p = 0.003) (Figure 5.2D). The urine output during NEVKP is displayed in Figure 5.3.

A NEVKP (n=5) B NEVKP (n=5) 80 10 75 8 70

65 6

60 4 55

50 2 Renal Artery Pressure (mmHg) Pressure Renal Vein (mmHg) 45 0 1 2 3 4 5 6 7 0 Normothermic Ex Vivo Kidney Perfusion (Hours) 0 1 2 3 4 5 6 7 Normothermic Ex Vivo Kidney Perfusion (Hours) C NEVKP = 5 D NEVKP (n=5) 350 0,8

300 0,6 250

200 0,4 150

100 0,2

Flow Renal Artery (mL/min) 50

0 Intrarenal Resistance (mmHg/mL/min) 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Normothermic Ex Vivo Kidney Perfusion (Hours) Normothermic Ex Vivo Kidney Perfusion (Hours)

Figure 5.2A: Renal artery blood pressure during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD in mmHg. Dashed line and grey area represent mean systemic blood pressure and SD measured invasively in situ in 30 anesthetized pigs by placing a catheter into the carotid artery. Figure 5.2B: Pressure in the renal vein during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD in mmHg. Figure 5.2C: Renal artery flow during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD in mL/min. Dashed line and grey area represent mean flow rate with SD measured in situ in 30 anesthetized pigs; upper and lower lines represent maximal and minimal renal artery flow rates in these pigs. The measurements were performed in control pigs following laparotomy and minimal dissection of the right renal artery with a flow probe. Figure 5.2D: Intrarenal resistance during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD in mmHg/mL/min. Dashed line and grey area represent mean IRR with SD based on measurements performed in situ in 30 anesthetized pigs.

NEVKP (n=5) 80 70 60 50 40 30 20 10 Cumula&ve Urine Produc&on (mL) 0 0 1 2 3 4 5 6 7 Normothermic Ex Vivo Kidney Perfusion (Hours)

Figure 5.3: Cumulative urine output during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD in mL.

5.3.3 NEVKP was associated with maintenance of physiologic biochemical parameters in the perfusate

Blood gas analyses were performed at baseline (Table 5.1) and then hourly during normothermic ex vivo perfusion. Measures of acid-base homeostasis, including pH and bicarbonate concentration, remained stable during preservation time and were physiologic when compared to basal values observed in 20 healthy control pigs (Figure 5.4A and 5.4B).

A NEVKP (n=5) 7,65

7,6

7,55

7,5 pH 7,45

7,4

7,35

7,3 BL 1 2 3 4 5 6 7 Normothermic Ex Vivo Kidney Perfusion (Hours)

B NEVKP (n=5) 40 35 30 25 20 15 HCO3- (mmol/L) 10 5 0 BL 1 2 3 4 5 6 7 Normothermic Ex Vivo Kidney Perfusion (Hours)

Figure 5.4A: pH of the perfusate during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD. Dashed line and grey area represent mean serum pH with SD measured in situ in 20 anesthetized pigs; upper and lower lines represent maximal and minimal pH values in these pigs. Figure 5.4B: HCO3- concentration in the perfusate during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD in mmol/L. Dashed line and grey area represent mean serum HCO3- with SD measured in situ in 20 anesthetized pigs; upper and lower lines represent maximal and minimal HCO3- values in these pigs.

Tissue injury during NEVKP was assessed by hourly measurement of AST and LDH levels in the perfusate. AST and LDH were below the detectable analyzer measurement limits of 4 U/L and 100 U/L, respectively. Lactate levels were measured in the perfusate as an indicator of graft

83 injury during ex vivo perfusion. Perfusate lactate levels decreased from baseline until the last hour of NEVKP (10.38 ± 0.76 vs. 1.22 ± 0.26 mmol/L, p = 0.0001) (Figure 5.5).

NEVKP (n=5) 12

4 Lactate (mmol/L)

0 BL 1 2 3 4 5 6 7 Normothermic Ex Vivo Kidney Perfusion (Hours)

Figure 5.5: Lactate levels in renal perfusate during Normothermic Ex Vivo Kidney Perfusion. Values presented as mean ± SD in mmol/L.

5.3.4 NEVKP vs. SCS results in comparable graft function and injury after kidney transplantation

During the 10-day postoperative follow-up, daily comparison of serum creatinine and BUN demonstrated a trend to lower values in NEVKP perfused grafts compared to cold storage kidneys (Figure 5.6A and 5.6B). Peak creatinine and BUN levels in the NEVKP vs. SCS group after transplantation were 2.0 ± 0.5 mg/dL vs. 2.7 ± 0.7 mg/dL (p = 0.114) and 19 ± 3.5 mg/dL vs. 24 ± 6.7 mg/dL (p = 0.159), respectively. Interestingly, 10 days after transplantation, creatinine (1.1 ± 0.2 at day 10 vs. 1.0 ± 0.2 mg/dL at baseline; p = 0.49) and BUN (8 ± 1 at day 10 vs. 8 ± 3 mg/dL at baseline; p = 0.59) levels in the NEVKP group were comparable to their baseline values. In contrast, SCS preserved kidneys had significantly higher creatinine (1.5 ± 0.4 vs. 0.9 ± 0.1 mg/dL; p = 0.01) and BUN (10 ± 3 vs. 6 ± 1 mg/dL; p = 0.03) values on day 10 after transplant when compared to baseline. Serum potassium values during follow up and estimation of 24-hour creatinine clearance on postoperative day 10 were similar to baseline values (Figure 5.6C and 5.6D). No hematuria was observed.

A Auto-KTx HBD SCS8h (n=5) Auto-KTx HBD NEVKP8h (n=5) B Auto-KTx HBD SCS8h (n=5) Auto-KTx HBD NEVKP8h (n=5)

3,5 35 300 12,0 3,0 30 250 10,0

2,5 mol/L) 25 μ

200 8,0 mol/L) 2,0 20 μ 150 6,0 1,5 15 100 1,0 10 4,0 Serum BUN (mg/dL) 50 Serum Urea ( Serum Crea)nine (mg/dL) 2,0 0,5 Serum Crea)nine ( 5

0,0 0 0 0,0 BL 1h 2h 10h 1pod 2pod 3pod 4pod 5pod 6pod 7pod 8pod 9pod 10pod BL 1h 2h 10h 1pod 2pod 3pod 4pod 5pod 6pod 7pod 8pod 9pod 10pod Postopera)ve Follow Up (Hours/Days) Postopera)ve Follow Up (Hours/Days)

C Auto-KTx HBD SCS8h (n=5) Auto-KTx HBD NEVKP8h (n=5) D Auto-KTx HBD 8h SCS (n=5) Auto-KTx HBD 8h NEVKP (n=5) 7,0 120,0

100,0 6,0

80,0 5,0 60,0 4,0 K+ (mmol/L) 40,0

3,0 20,0 Crea)nine Clearance (mL/min)

2,0 0,0 BL 1h 2h 3h 1pod 2pod 3pod 4pod 5pod 6pod 7pod 8pod 9pod 10pod 0 pod 10 pod Postopera)ve Follow Up (Hours/Days) Postopera)ve Follow Up (Days)

Figure 5.6A: Serum creatinine of the transplanted animals during 10 day postoperative follow-up for autologous kidney transplantation following SCS and NEVKP. Values presented as mean ± SD in mg/dL and µmol/L. Figure 5.6B: Serum BUN/urea during 10 day postoperative follow-up for autologous kidney transplantation following SCS and NEVKP. Values presented as mean ± SD in mg/dL and µmol/L. Figure 5.6C: Serum potassium during 10 day postoperative follow-up for autologous kidney transplantation following SCS and NEVKP. Values presented as mean ± SD in mmol/L. Figure 5.6D: 24-hour creatinine clearance during 10 day postoperative follow-up. Values presented as mean ± SD in mL/min.

Kidney histology was assessed 10 days after transplantation. Between NEVKP vs. SCS preserved kidneys, there were no significant differences for tubular injury (0.5 (0.5 – 1.5) vs. 0.5 (0 – 1.5), p = 0.690), interstitial inflammation (1.0 (0.5 – 2.0) vs. 1.0 (0.5 – 2.0), p = 0.841), edema (0 (0 – 0) vs. 0 (0 – 1), p = 0.690), or fibrosis (0 (0 – 0) vs. 0 (0 – 1), p = 0.310) (Table 5.3). In all cases, the majority of glomeruli were mildly shrunken. TUNEL staining showed extremely low levels of apoptotic cells with no differences between NEVKP and SCS preserved kidneys (data not shown).

Histological Findings NEVKP SCS p-value Tubular injury 0.5 (0.5 – 1.5) 0.5 (0 - 1.5) 0.690 Inflammation 1.0 (0.5 – 2.0) 1.0 (0.5 – 2.0) 0.841 Edema 0 (0 – 0) 0 (0 – 1.0) 0.690 Fibrosis 0 (0 – 0) 0 (0 – 1.0) 0.310

Table 5.3: Histological findings ten days after transplantation assessed by H&E / PAS staining. Biopsies were scored on a scale of 0-3 for tubular injury, inflammation, edema, and fibrosis. Data represent median (range) of 30 fields.

5.3.5 NEVKP did not compromise animal survival

Four pigs out of 5 survived until day 10 in the group in which the grafts were preserved with NEVKP. On day 5 one pig in the NEVKP group developed an ileus and had to be sacrificed with a well-functioning renal graft. All pigs in the hypothermic preserved group survived. There was no significant difference in animal survival between both groups (p = 1.000).

5.4 Discussion

This is the first demonstration that NEVKP can be performed for at least 8 hours in HBD grafts under physiologic conditions, resulting in excellent early outcomes after kidney transplantation. During NEVKP physiologic perfusion parameters were maintained, high ex vivo metabolic activity, low intrarenal resistance, and no markers of tissue injury were seen. Further assessment after renal graft autotransplantation demonstrated comparable kidney function for NEVKP when compared to cold storage control during 10 days follow up.

Our findings are in keeping with those of Brasile, Stubenitsky, and Kootstra who investigated ex vivo kidney perfusion using a canine model of subnormothermic (32°C), acellular, low pulsatile pressure (MAP 35 mmHg) perfusion in the early 2000s. Following application of 30 minutes of renal warm ischemia, grafts were stored statically on ice (4 °C), perfused at subnormothermic temperatures (32 °C), or subjected to a combination of both preservation techniques. Following renal autotransplantation, grafts preserved with subnormothermic perfusion demonstrated lower serum creatinine values, while grafts stored statically on ice had a significantly reduced renal function. Only prolonged rather than short subnormothermic perfusion following SCS was capable to fully recover renal function 145. These findings demonstrated (1) the detrimental effects of cold ischemic preservation, (2) the option of replacing SCS continuously with perfusion at subnormothermic temperatures, and (3) the potential of prolonged subnormothermic perfusion to repair grafts following SCS. Some key differences exist between our experimental set-up and that of Brasile et al. We utilized healthy porcine HBD grafts in our study whereas Brasile used canine DCD kidneys. Furthermore, Brasile et al. used a different perfusion set up with an acellular, low pulsatile pressure perfusion at a subnormothermic temperature of 32 °C 144. Despite these promising results, this system has never been reported to have been successfully translated to clinical trials for the preservation of human kidney grafts and subsequent transplantation 214.

Hosgood et al. previously investigated shorter periods of normothermic ex vivo kidney perfusion in a porcine model of renal autotransplantation. Kidneys were subjected to 30 minutes of warm ischemia and preserved with either 22 hours of hypothermic machine perfusion (HMP) or 20 hours of HMP followed by 2 hours of normothermic preservation (NP) using autologous blood. There was no significant difference in graft survival or kidney function, but lower levels of lipid

87 peroxidation were measured in the NP group 60 min after transplantation 154. In a first clinical trial in 2013, ex vivo normothermic perfusion (EVNP) was investigated in ECD kidney grafts. Eighteen kidneys from extended criteria donors underwent one hour of EVNP immediately prior to transplantation. Comparing the outcome of these kidneys to 47 ECD kidneys that underwent SCS demonstrated a significant reduction in DGF (5.6% vs. 36.2%, respectively) 156. Although Hosgood and Nicholson demonstrated benefits of short term ex vivo perfusion in marginal grafts, the effects of continuous perfusion and avoidance of cold storage were not assessed. In addition, possible negative effects of normothermic ex vivo perfusion on good quality HBD grafts were not investigated.

For renal grafts recovered from ECD or DCD the susceptibility for additional injury caused by hypothermia has been well described 93,96,192,195. In addition, prolonged cold ischemia is also known to have detrimental effects on healthy HBD grafts 199,200. Delpech et al. demonstrated the detrimental impact of 24 hours cold storage in kidneys retrieved in a porcine heart-beating model. Three days, 7 days, and 3 month after autotransplantation, the creatinine clearance was still reduced and significantly lower when compared to baseline. Even kidneys in which the renal hilum was clamped in situ for 60 min without undergoing transplantation afterwards demonstrated better function and nearly returned to baseline after 3 month. As expected, the combination of 30 minutes of warm ischemia, 24 hours of cold storage, and autotransplantation demonstrated the lowest values of creatinine clearance 165.

In our study we demonstrated that NEVKP can replace hypothermic storage techniques and therefore offers the potential to avoid harmful effects of cold storage. We aimed to provide the most physiologic conditions for the renal graft during the ex vivo perfusion period. Thus, the arterial and venous pressures, the chosen temperature, and the composition of the perfusion solution were based on physiologic values obtained in healthy pigs (Table 5.1 and 5.2, Figures 5.2) 212,213. Leukocyte-depleted washed erythrocytes were used to provide an immunologically protected environment 215. No bicarbonate had to be given throughout the perfusion as the kidneys demonstrated physiologic function in keeping the acid-base hemostasis stable. Verapamil was chosen for vasodilation as it allowed stable perfusions of 10 hours and more in our recently conducted study 206. Furthermore, the perioperative application of verapamil demonstrated improved renal graft function after transplantation 216 and reduced the incidence of acute tubular necrosis in a Cochrane Database systematic review 217. Hourly, potential marker of

88 renal graft injury (AST and LDH) were assessed 144 207,209 and demonstrated to be below analyzer range. The initial high values of lactate can be explained with the high content of Ringer’s lactate in the perfusate solution. Biopsies were only taken at the end of the study period so as to avoid bleeding and to not compromise the animal outcome. In a former set of experiments, biopsies were taken immediately following 10 hours of NEVKP of good quality HBD grafts. These biopsy specimens demonstrated minimal changes and no necrosis 206.

Normothermic ex vivo kidney perfusion can offer several advantages in comparison to static cold storage or hypothermic machine perfusion. First, as described above, negative effects of cold storage can be avoided. Second, during the normothermic ex vivo perfusion period grafts are metabolically active and the function of the graft can be assessed. Third, graft repair strategies could be applied during the period of normothermic perfused preservation.

We chose a pig model of heart-beating donation with a short preservation time to investigate the safety of continuous NEVKP. By keeping the storage time short and avoiding the deleterious effects of brain death we created a control group that would be expected to have excellent graft function, thus maximizing the probability that any potential deleterious effect of normothermic perfusion would be detected. The use of an autotransplantation model excluded rejection as a confounding factor and allowed us to evaluate exclusively the effects of storage and reperfusion. Clinical trials in the future will likely include acceptable kidney grafts for transplantation and demonstrating that the new technology is not detrimental to good quality grafts provides reassurance about the safety of this technique for future clinical trials. This information will also facilitate clinical trials of new therapeutic strategies such as stem cell therapy, gene transfer, or microRNA administration to modify grafts of which some are more likely to target standard criteria donor rather than ECD or DCD grafts.

Our study has several limitations. Our model did not include kidneys recovered in donation after brain death or kidneys with severe preservation injury. Thus, possible protection against deleterious effects of brain death or preservation injury was not investigated. In addition, in the absence of severe preservation injury, we did not assess mechanisms of graft injury such as cytokine release, infiltration of inflammatory cells, or ATP depletion. Furthermore, keeping the preservation time to less than 8 hours may be impossible in organ procurement regions where prolonged storage periods are sometimes required. To address the question of safety, we on

89 purpose chose a short preservation time with minimal injury in the control group. This might explain that there was no significant difference in peak serum creatinine and BUN in between both groups. Future studies will focus on grafts with severe kidney injury, such as kidneys recovered after circulatory death.

In conclusion, we demonstrated that normothermic ex vivo kidney perfusion can be performed safely in good quality heart-beating donor kidney grafts without causing graft injury. NEVKP maintains a physiologic environment during the preservation period, preserving excellent kidney function ex vivo. Future studies will evaluate the effects of NEVKP for preservation of ECD and DCD grafts.

______

6 General Discussion and Conclusion

______

The structure of this thesis in a “multiple paper format” allows specific discussion sections for each chapter that is derived from published data (see Chapter 3.5, 4.5, 5.5). In order to avoid repetition, this general unifying discussion focuses on the main aspects this research study addresses: Using Normothermic Ex Vivo Kidney Perfusion for improvement of storage, assessment, and repair of renal grafts prior to transplantation, and its translation to a clinical trial.

The importance of this study results from the main challenge in transplantation, which is the organ graft shortage. Thus, effort focuses on exploring strategies to increase the donor pool. Kidney transplantation from ABO- or HLA-incompatible living donation, paired kidney donation, and organ recovery from extended criteria donors and donation after circulatory death is performed. Among the most promising strategies to increase the donor pool are ECD and DCD kidney transplantation, which currently represent about 30% (15% each) of all deceased kidney transplants in the US 7,78. However, reduced outcomes following ECD and DCD kidney transplantation are found, largely caused by the organs’ increased sensitivity to ischemia reperfusion injury. Different approaches including donor management, graft-preconditioning, improvement of preservation solutions, postconditioning, and regenerative therapies are investigated to reduce IRI 218.

Altering organ preservation represents an auspicious approach to improve ECD and DCD graft outcome. Hypothermia has been proven to be detrimental for prolonged storage periods, especially for grafts with lower quality. For organ systems such as lung and liver, several studies demonstrated beneficial effects of normothermic preservation with superior outcomes when compared to cold storage 166,219. Current data about normothermic kidney preservation is limited 156.

Therefore, the main goal of this study was to investigate the possibility of normothermic kidney preservation in a porcine model. In particular, aims were to develop NEVKP in a porcine model (Chapter 3), to establish a porcine transplant model (Chapter 4), and to investigate the feasibility and safety of NEVKP in this transplant model (Chapter 5). Furthermore, the potential to use NEVKP for the improvement of ischemically injured kidneys was assessed (Chapter 6.1). Finally, the findings will be translated to a clinical safety trial.

6.1 Normothermic Preservation of Ischemically Injured Renal Grafts

Recent data from our group demonstrated the superiority of continuous pressure-controlled NEVKP over cold storage in grafts that were injured by warm ischemia (unpublished data). We compared the outcome of ischemically injured grafts preserved with NEVKP or SCS in a porcine model kidney autotransplantation. After 30 minutes of warm ischemia, right kidneys were removed and preserved with 8h NEVKP or in 4°C histidine-tryptophan-ketoglutarate solution (SCS). Throughout NEVKP, electrolytes and pH values were maintained. Intrarenal resistance decreased over the course of perfusion (0h 1.6±0.51mmHg/mL/h vs. 7h 0.34±0.05mmHg/mL/h, p=0.005). Perfusate lactate concentration also decreased (0h 10.5±0.8mmol/L vs. 7h 1.4±0.3mmol/L, p<0.001). Cellular injury markers LDH and AST were persistently low (LDH<100U/L, below analyzer range; AST 0h 15.6±9.3U/L vs. 7h 24.8±14.6U/L, p=0.298). Following autotransplantation, renal grafts preserved with NEVKP demonstrated lower serum creatinine on day 1–7 (p<0.05) and lower peak values (NEVKP 5.5±1.7mg/dL vs. SCS 11.1±2.1mg/dL, p=0.002). The creatinine clearance on day 4 was increased in NEVKP preserved kidneys (NEVKP 39±6.4mL/min vs. SCS 18±10.6mL/min, p=0.012). Serum NGAL at day 3 was lower in the NEVKP group (1267±372 vs. 2697±1145ng/mL, p=0.029). In conclusion, continuous pressure-controlled NEVKP improves function of transplanted kidneys that underwent warm ischemic injury prior to transplantation.

6.2 Improving Organ Storage

The current gold standard in kidney transplantation – static cold storage – has been developed in the 1980s. SCS has been demonstrated to be an appropriate storage technique for organs of high quality (standard criteria donor organs), which were mainly used in the 1980s and 1990s. However, since then the donor profile has changed. Increasing amounts of kidneys transplanted are recovered from ECD and DCD, which subsequently led to changes in graft preservation, too. Prolonged cold ischemia times are proven to be detrimental for SCD, and especially ECD and DCD kidneys. Thus, since the 2000s an increased portion of kidneys are being preserved with hypothermic machine perfusion; today more than 40% are preserved on pump in the US 99.

Further investigations aim to improve hypothermic machine perfusion. Traditionally, SCS and HMP are performed at ice-cold temperatures (4 °C) without oxygen and nutrition supply. Experimental studies conducted in a porcine DCD model compared the conventional HMP technique with active oxygenation during HMP. Following transplantation, renal grafts preserved with active oxygenation demonstrated lower serum creatinine peak, faster return to normal levels, and lower urinary NGAL and serum AST levels. In addition, chronic function was improved with reduced interstitial fibrosis and lower levels of vimentin staining 177. Oxygenation in HMP kidney preservation might lead to increased production of ATP during hypothermic phase. Energy consumption during hypothermic storage still occurs as cell metabolism is continuing at a reduced level. Providing oxygen might facilitate increased rates of aerobic metabolism with superior cell function and less production of toxic metabolites 220.

Further experimental data from another group investigated kidney perfusion at subnormothermic temperatures (20 °C). In a porcine DCD model, subnormothermic perfusion resulted in increased creatinine clearance when compared to oxygenated HMP and SCS. As higher temperature results in higher metabolic demands, the preservation set up in the subnormothermic group was adapted. Active oxygenation, increased perfusion flow, and altered perfusion solution were utilized 221. Underlying mechanisms for beneficial results provided by subnormothermic perfusion are not completely understood. Some studies indicated that endothelial shear stress 222 and control of acidosis 223 might be of importance. Increased temperatures during perfusion might improve the environment for these processes 221.

Brasile, Stubenitsky, and Kootstra conducted preservation experiments with perfusions at temperatures just below normal successfully in the early 2000s. Their Exsanguinous Metabolic Support (EMS) is based on subnormothermic (32 °C), acellular, low pressure, pulsatile ex vivo kidney perfusion 142,143. In canine DCD kidney transplant models, subsequent preservation with EMS following cold storage or replacement of hypothermic preservation techniques demonstrated significantly improved kidney graft function with decreased serum creatinine levels during follow up 146,147. However, having been developed nearly 20 years ago, EMS has not been brought to clinical trials yet. Potential explanations are of economic and regulatory nature.

Most recently, Hosgood and Nicholson demonstrated the first clinical experience of ex vivo normothermic kidney perfusion using an erythrocyte-based solution. Following the first in man perfusion demonstrating positive results 155, they published the first clinical trial in 2013. ECD kidneys treated with one hour of normothermic kidney perfusion ex vivo following cold storage directly before transplantation reduced the rate of DGF significantly 156.

The approach of NEVKP is significantly different to former investigated normothermic techniques. Instead of adding a period of one hour of normothermic perfusion 155,156, the aim is to replace hypothermic preservation. This study, for the first time, demonstrates the feasibility and safety to perfuse porcine high-quality renal grafts save for at least ten hours (Chapter 3) 206. The perfusion characteristics’ set up for NEVKP was developed based on measurements performed in healthy pigs. Mean arterial pressure for the perfusion was calculated by invasive measurement of arterial pressures in 30 anesthetized pigs; normal ranges of flow were assessed by in situ flow measurement in 30 healthy pigs 213. The perfusate solution was created to aim for the most physiologic composition possible. Therefore, blood gas analyses were performed in 20 healthy pigs; furthermore, osmolarity and oncotic pressure were investigated in 6 pigs. Using Ringer’s lactate, Steen solution, washed erythrocytes, sodium bicarbonate, calcium gluconate, heparin, and DRO water resulted in a perfusate similar to a pigs blood baseline values. Key components for the solution are washed erythrocytes (leukocyte-depleted) and Steen solution. Washed erythrocytes serve as oxygen carrier; furthermore, leukocyte-depletion allows graft perfusion in an immunologically protected environment, potentially reducing inflammation 215. Steen solution, providing appropriate amounts of albumin, secures optimal oncotic pressure during

95 perfusion to minimize cell swelling. Dextran 40 protects and coats endothelium from further excessive leukocyte interactions 224.

Having established a porcine renal autotransplantation model improves outcome assessment of grafts preserved with NEVKP and is an important step of this study. Renal autotransplantation appears to be more appropriate to investigate organ preservation techniques and ischemia reperfusion injury, excluding confounding factors of rejection that occur in allotransplant models. In Chapter 4, the model of porcine renal autotransplantation is described in detail and a visualized version can be viewed online. Although renal autotransplantation in large animal models has been described, this manuscript for the first time provides a visualized step-by-step protocol that highlights key differences to the clinical scenario. Important steps to achieve successful posttransplant results include: venous and arterial anastomoses in end-to-side technique to vena cava and aorta, rather than to iliac vessels to avoid arterial and venous thrombosis as well as vasospasm; using norepinephrine to achieve a systolic pressure of 80-100 mmHg ensuring homogenous renal graft reperfusion; topic application of papaverine to avoid vasospasm; uretheral end-to-side anastomosis to minimize the risk of postoperative obstructions; and proper positioning of the graft to prevent from vessel kinking 225. Actual transplantation provides a more realistic assessment of NEVKP preserved grafts than reperfusion models, which are frequently used by most other groups 154,193.

Chapter 5 finally demonstrates feasibility and safety of NEVKP in a HBD transplant model using high-quality, SCD grafts. Comparison with SCS demonstrated superior outcomes for NEVKP preserved kidneys 226. On purpose, this study was designed using rather short preservation times and non-injured renal grafts to indicate non-inferiority of NEVKP over SCS. Mean preservation times of renal grafts prior to transplantation in the US are described to be about 15 hours; however, depending on donation scenarios (living vs. deceased donor) and logistic aspects, preservation times vary between very few hours (below 3 hours) and more than 30 hours. The high quality of the grafts used in the study presented in Chapter 5 with short preservation times explain the mostly non-significant differences in between both groups.

In addition to its feasibility and safety in high-quality SCD grafts, NEVKP was investigated in renal grafts that were exposed to warm ischemia prior to recovery. NEVKP preserved grafts demonstrated to be significantly better with lower serum creatinine and higher creatinine

96 clearance following transplantation when compared to cold storage (Chapter 6.1) (unpublished data).

We propose several key advantages of our study design compared to hypothermic, subnormothermic, or short additional normothermic perfusion scenarios. Only prolonged, continuous normothermic preservation reduces cold ischemic injury to a minimum. Providing a physiologic environment ex vivo during preservation appears to be crucial – especially for ischemically injured grafts – to improve organ storage. Cold ischemic injury, as described in chapter 1.4, will always occur using hypothermic storage techniques, as cell metabolism is still ongoing, only on reduced levels. Using physiologic perfusate components, normothermic temperatures, species adapted perfusion pressures, delivering oxygen and nutrition, and providing an immunologically protected environment appear to be crucial 215.

6.3 Improving Organ Assessment

A major drawback in static cold kidney storage is the inability to assess the grafts quality during preservation. The basic principle of hypothermia is to reduce the organs metabolic activity to a minimum, which facilitates extracorporeal storage. However, this reduced metabolism makes it demanding to assess the renal function.

HMP to some extend allows renal graft assessment prior to transplantation. Several studies describe vascular intrarenal resistance (IRR) as a potential marker to predict graft outcome. In one study, IRR values were prospectively collected in 302 HMP preserved deceased donor kindeys of all types. An association was found between IRR and DGF and 1-year graft survival. IRR at the end of HMP was an independent risk factor for DGF (OR 21.12 [1.03-435.0], p=0.048) with however low predictive values, and an independent risk factor for 1-year graft failure (HR 12.33 [1.11-136.85], p=0.004) 227. A systematic review investigated HMP perfusate biomarkers. Results indicated a significant association of perfusate lactate dehydrogenase (LDH), gluthatione-S-transferase (GST), and aspartate transaminase (AST) with delayed graft function 228. A more recent study demonstrated the association of LDH and IL-18 with PNF and DGF 229. Although associations between perfusate biomarkers have been demonstrated, present viability tests are not reliable predictors of transplant outcome 230.

NEVKP represents a novel approach to improve assessment of grafts during preservation. The grafts metabolic activity potentially allows investigation of renal function. Functional parameters, such as diuresis, creatinine clearance, or inulin clearance as well as more specific kidney injury markers such as KIM-1 and NGAL could potentially improve outcome prediction. This study design, utilizing continuous, prolonged NEVKP, again provides superior potential over short normothermic perfusion. Longer assessment times might offer advantages for more in depth investigations (such as lactate decrease) and more accurate outcome prediction.

Improving assessment techniques during preservation is of particular importance to reduce currently high kidney discard rates of up to 52% for recovered ECD/DCD kidneys 83 and help increasing the kidney donor pool. Our current studies therefore focus on exploring the potential to use NEVKP for graft quality assessment prior to transplantation.

6.4 Improving Organ Repair

Another advantage originating from the graft’s high metabolic activity is the potential to repair injured kidneys. Novel techniques such as drug delivery 231, gene transfer 232, and application of stem cells 233 could help to improve grafts while being preserved.

In a porcine model of normothermic kidney perfusion, Hosgood et al. investigated the use of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitior. Assessment during 3 hours of reperfusion demonstrated improved renal function, reduced oxidative stress and neutrophil infiltration when compared to control 182. Brasile et al. used their subnormothermic, acellular perfusion model for immunocloacking. During 3 hours of warm perfusion, the renal vasculature was treated with a bioengineered interface consititing of a nano-barrier membrane. In a canine renal allotransplantation model, untreated control dogs demonstrated signs of renal rejection 6 days after transplantation. Treated dogs with modified renal vascular surfaces demonstrated first signs of rejection after 30 days 234. Elimination of immunosuppression during the initial phase following transplantation is of importance to reduce side effects caused by immunosuppressive drugs.

NEVKP as described in this study provides superior storage compared to SCS. Especially in warm ischemically injured grafts, continuous prolonged normothermic perfusion demonstrated significantly improved renal function following transplantation. Longer vs. shorter times of NEVKP demonstrated to improve graft outcome substantially (data not shown). Thus, NEVKP not only improves graft function by excluding cold ischemic injury; in addition, it seems to have a repair effect itself. The grafts high metabolic activity over a prolonged time also allows applying additional repair strategies. Future studies will have to address this approach.

6.5 Conclusion and Clinical Trial

In conclusion, this study demonstrates that NEVKP can be performed for a prolonged time to preserve renal grafts at normothermic temperatures ex vivo in a large animal model, which is the closest available to the human. Following development of a heterotopic porcine autotransplantation model, this study determined the feasibility and safety of NEVKP in a model of heart-beating donation and renal transplantation. Superior outcomes were demonstrated, in particular for grafts recovered following warm ischemia. Based on these findings, a clinical trial will be initiated soon in the Multi Organ Transplant Program at Toronto General Hospital.

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7 Future Directions

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A clinical trial based on the findings presented in this thesis will be initiated in mid/end of 2016. Therefore, our porcine NEVKP system was adapted to the needs of a human scenario. The primary goal of this trial will be to demonstrate the safety of NEVKP in a human scenario. In a follow up clinical trial, we will investigate its efficacy in human donation after circulatory death kidney transplantation.

Further experimental research should focus on the exploration of different timings of NEVKP and its combination with SCS and HMP. The question, whether a short period of NEVKP following cold storage is sufficient, or if prolonged continuous NEVKP with complete replacement of cold storage is superior should be investigated. In addition, it is unclear to which extend NEVKP can repair the damage hypothermia induces in renal grafts during hypothermic transportation. The question, whether a transportable normothermic perfusion device will be necessary needs further evaluation. Complete exclusion of hypothermia using normothermic flushes before and after NEVKP might be a future strategy.

To further understand the potential benefits of NEVKP over hypothermic storage, underlying mechanisms will need to be explored. Analyses of inflammatory cytokines (such as TNF-alpha, IL-1, and IL-6), more specific renal injury markers (such as NGAL, KIM-1, and IL-18), and oxidative stress markers (such as 8-isoprostane and malondialdehyde (MDA) – investigated during NEVKP and after transplantation - will help to further understand differences between hypothermic and normothermic preservation and facilitate to improve NEVKP.

Another important goal of normothermic graft storage is to improve assessment strategies during kidney preservation to judge whether a graft of lower quality is transplantable or not. Further studies will need to investigate biomarkers and determine cut off values to decide if an organ should be discarded or used for transplantation.

An auspicious advantage of NEVKP is the grafts increased metabolic activity during normothermic preservation. Therefore, the organs improved susceptibility to apply repair mechanisms are higher compared to hypothermic preservation. Administration of drugs to reduce ischemia reperfusion injury or post transplant immunogenic rejection, and utilizing gene transfer or adding stem cells to the perfusate might improve the grafts post transplant outcome. Drug

102 repurposing studies might help to identify potential reagents that can repair grafts of lower quality.

In the far future, the extracellular matrix of decellularized kidneys or even 3D printed structures could be recellularized with our device using autologous stem cells of the recipient. Bioengineered kidneys of optimal quality would solve the problems of the organ shortage and posttransplant immunosuppression would become redundant.

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Appendices

A1. Reasons for Kidney Transplantation.

Kidney Diagnosis Category Kidney Diagnosis

Glomerular Diseases Anti-GBM Chronic Glomerulonephritis: Unspecified Chronic Glomerulosclerosis: Unspecified Focal Glomerularsclerosis Idio/Post-Inf Crescentic Glomerulonephritis IGA Nephropathy Hemolytic Uremic Syndrome Membranous Glomerulonephritis Mesangio-Capillary 1 Glomerulonephritis Mesangio-Capillary 2 Glomerulonephritis Systemic Lupus Erythematosus Alport's Syndrome Amyloidosis Membranous Nephropathy Goodpasture's Syndrome Henoch-Schoenlein Purpura Sickle Cell Anemia Wegeners Granulomatosis

Diabetes Diabetes: Type I Insulin Dep/Juvenile Onset Diabetes: Type II Insulin Dep/Adult Onset Diabetes: Type I Non-insulin Dep/Juv Onset Diabetes: Type II Non-insulin Dep/Adult Onset

Polycystic Kidneys Polycystic Kidneys

Hypertensive Nephrosclerosis Hypertensive Nephrosclerosis

Renovascular And Other Vascular Chronic Nephrosclerosis: Unspecified Diseases Malignant Hypertension Polyarteritis Progressive Systemic Sclerosis Renal Artery Thrombosis Scleroderma

Congenital, Rare Familial, And Congenital Obstructive Uropathy Metabolic Disorders Cystinosis

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Fabry's Disease Hypoplasia/Dysplasia/Dysgenesis/Agenesis Medullary Cystic Disease Nephrophthisis Prune Belly Syndrome

Tubular And Interstitial Diseases Acquired Obstructive Nephropathy Analgesic Nephropathy Antibiotic-induced Nephritis Cancer Chemotherapy-Induced Nephritis Chronic Pyelonephritis/Reflex Nephropathy Gout Nephritis Nephrolithiasis Oxalate Nephropathy Radiation Nephritis Acute Tubular Necrosis Cortical Necrosis Cyclosporin Nephrotoxicity Heroin Nephrotoxicity Sarcoidosis Urolithiasis

Neoplasms Incidental Carcinoma Lymphoma Myeloma Renal Cell Carcinoma Wilms' Tumor

Retransplantation/Graft Failure Retransplant/Graft Failure

Other Other Rheumatoid Arthritis Other Familial Nephropathy

Table A.1: Reasons for kidney transplantation. Adapted from “Organ Procurement and Transplantation Network” 10.

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A2. Perfusion device.

For renal perfusion experiments in the porcine model, a third generation SIII Heart-Lung- Machine provided by Sorin Inc. was utilized (Figure A2; Soring Group Inc., Markham, ON, Canada). The modular system was initially configured and adapted to the needs to perform ex vivo kidney perfusion. A S3 Console with Standard Mast System served as a platform. A Sorin Centrifugal Blood Pump (SCP, part number 60-00-00) – consisting out of SCP Drive Unit (60- 01-00) and SCP Pump Control Panel (60-02-15) – was mounted on the left mast to propel the perfusate trough the perfusion circuit. An SCP Flow Sensor 3/8” (96-414-120) was used to analyze and record flow rates on the arterial site. Venous and arterial pressures were recorded with one pressure transducer each, temperature with Temperature Probes for direct measure in the oxygenator (45-03-10). A Bubble Detector (23-07-50) at a 3-joint-mast holder (23-26-96) recorded air in the arterial line; a Level Sensor (23-27-40) ensured sufficient volume of perfusate in the venous reservoir. A Heater-Cooler Unit (HU 35, Maquet Getinge Group, Rastatt, Germany) maintained a physiologic temperature of 37 °C of perfusate and the customized double-walled organ chamber. Measured data such as flow per minute (mL/min), venous and arterial pressures (mmHg), perfusate temperature (°C), bubble detection (yes/no), and level of venous reservoir were displayed on the individualized SIII System Panel. For safety reasons, a Sorin Roller Pump (10-60-00) was attached to transport leaked blood from the organ chamber back into the venous reservoir. The Perfusor® Space Infusion Pump System (B. Braun Medical Inc., Melsungen, Germany) was utilized for continuous administration of drugs; infusion pumps (Graseby 500, Smiths Medical Ltd., Markham, ON, Canada) for replacement of produced urine during organ perfusion. An oxygen tank (95% O2, 5% CO2) provided sufficient oxygen for perfusate and renal graft. The Data management system (DMS, Sorin Group Inc.) was installed on a notebook and connected to the SIII for continuous real-time recording of time, arterial and venous pressure, temperature, flow, urine output, and unforeseen changes or events.

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Figure A2: Sorin SIII Heart-Lung-Machine, modular system. Mounted devices: Sorin Centrifugal Blood Pump, SCP Flow Sensor 3/8”, Pressure Transducer, Bubble Detector, Level Sensor, Heater-Cooler Unit, SIII System Panel, Sorin Roller Pump, Perfusor® Space Infusion Pump System (2x), Infusion Pumps (3x), stand for positioning of the organ chamber.

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A3. Perfusion circuit.

A perfusion circuit (Figure A3.1 and A3.2), consisting of cardiopulmonary bypass technology, was customized to provide optimal conditions for ex vivo normothermic kidney perfusion. It was built under sterile conditions and provided by Sorin Group Inc. (Milano, Italy). A hard shell neonatal venous reservoir and oxygenator (kids D100) were utilized for pooling and oxygenation of the perfusate. The oxygenator was connected via tubing including a gas filter to the oxygen tank. A Revolution® Pump Head was used to propel the perfusate from the venous reservoir to the oxygenator and through the arterial P.h.i.s.i.o coated PVC tubing to the renal graft. An arterial filter (D130 neonatal arterial filter) was placed in the arterial line to prevent air embolism; a purge line was connected to the venous reservoir. Venous and arterial sample ports served for perfusate collection during the experiment. For vessel cannulation, a 1.6" arterial 1 cannula was used for the artery, and a ¼” – /8” connector for the vein. Extension lines and pressure isolators served to measure invasive blood pressure in arterial and venous line. A suction line was used to remove blood leaking into the organ chamber.

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Figure A3.1: Components of the customized perfusion circuit (1): Neonatal venous reservoir and oxygenator, arterial and venous tubing, arterial filter, Revolution® centrifugal pump head, sample ports, purge lines.

Figure A3.2: Components of the customized perfusion circuit (2): Suction line, extension lines and pressure isolators, various connectors, stopcocks, and syringes.

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A4. Customized organ chamber.

To provide a physiological environment for perfused grafts during perfusion, a customized chamber (Figure A4.1) was constructed. The chamber consisted out of stainless steel to be reusable and provide sterility. A double-walled design with heated water inlet and outlet ensures a temperature of 37 °C when connected to the heater cooler unit. Arterial inlet and venous outlet served to fix the vessel cannulas and perfusion circuit tubing to the chamber to avoid vessel lesions. The urinary outlet ensured continuous transportation of produced urine from the graft to a glass cylinder by gravity. An organ support bedding prevented organ damage. Adapted dimensions and a clear polycarbonate lid insert (Figure A4.2) served to prevent drying-out of the graft and allowed visual monitoring.

Figure A4.1: Schematic drawing of customized, double-walled kidney perfusion chamber (1). Arterial inlet, venous outlet, urinary outlet, blood outlet, heated water inlet and outlet, organ support bed, dimensions given in centimeter.

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Figure A4.2: Schematic drawing of customized, double-walled kidney perfusion chamber (2). The clear polycarbonate lid insert with ventilation holes is demonstrated.

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A5. Red cell isolation protocol.

Balancing and handling of units containing red blood cells must be performed inside a BSC.

1. Preparation

i) Sterilize a BSC with UV light and 70% ethanol.

ii) Sterilize scissors, syringes, needles, clamp, and ice bucket with 70% ethanol before use/placing inside the BSC.

iii) Cool the centrifuge to 4°C.

2. Procedure for Collecting and Leukoreducing Whole Blood

i) Cut off the needle of the quadruple blood-pack unit.

ii) Attach the end of the tubing to the tubing from the catheter in the animal.

iii) Once the desired volume of blood has been collected (in one or more blood-pack units), tie off and remove the bag(s).

iv) Hang the unit(s) and “crack” the tubing leading to the leukoreduction filter so that the whole blood will filter through. Note: do not apply pressure to the bag. It will take about 15-60 minutes to filter.

v) Tie off and remove the original collection bag(s).

vi) Place the remainder of the unit on ice and transport to a BSC.

3. Procedure for Isolating Red Blood Cells

i) If more than one blood-pack unit is being used, balance the volumes in swinging buckets by transferring blood from one unit to the other, using a syringe and 18G needle. If an odd number of units are being used, balance with a Transfer Pack (600 mL, 12/pack, Fenwal Inc., 4R2023) containing double reverse osmosis water.

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Note: The unit must be placed in the bucket as follows: from closest to the centre of the centrifuge to its diameter – SAG-M bag, Transfer Container, bag containing leukoreduced whole blood.

ii) Use at least 2 cloths per bucket to keep the bags in place.

Note: Ensure that the bags and/or tubing do not interfere with the range of motion of the buckets by using tape.

If using the SX4750 rotor (i.e. in Dr. Li Zhang’s lab in TMDT room 2-701), contents must be balanced within 2 mg. Point the “tabs” on the rotor upwards and place the units in positions 1 and 3.

If there is still an imbalance error (sometimes due to very heavy masses), use the SX4750A rotor in Dr. Gary Levy’s research lab in MBRC 2R414 Research 17.

If using the SX4750A rotor, contents must be balanced within 50 mg.

iii) Centrifuge at 1500xg for 15 minutes with no brake (setting: off) at ~4°C.

Note: It will take about 15 minutes to slow down to 0xg.

iv) Carefully remove the buckets from the centrifuge, bring to the BSC, and carefully remove the units from the buckets, ensuring that the separation of the red blood cells and plasma are not disturbed.

Note: While processing a unit, place any additional units on ice.

v) “Crack” the tubing leading to the Transfer Container. Press off the plasma until the buffy coat and some red blood cells can be seen entering the Transfer Container.

vi) Clamp, remove, and tie off the tubing leading to the Transfer Container.

4A. Procedure for Storing Leukoreduced Red Blood Cells for Future Use

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i) If the leukoreduced red blood cells will be used in future, “crack” the tubing leading to the bag containing SAG-M and apply pressure to transfer the red blood cells to the bag. Rock the SAG-M bag to ensure that the cells are in contact with the SAG-M.

ii) Cut off the Transfer Containers and any additional tubing, ensuring that enough tubing is left for future use.

iii) Place the unit at 4°C on a horizontal or vertical rotator or shaker at a low speed.

Note: The leukoreduced blood can be stored in SAG-M for up to 42 days at 4°C.

4B. Procedure for Washing Red Blood Cells for Immediate Perfusion

i) If the leukoreduced red blood cells will be used immediately for subnormothermic ex vivo liver perfusion (SNEVLP) or ex vivo kidney perfusion (EVKP), add 250 mL (for one unit) of 0.9% cold saline to each unit, using a 60 mL syringe and 18G needles.

ii) Remove the SAG-M bag. It can be tied off and saved for potential future use.

iii) Balance as in 3i-ii above.

iv) Centrifuge at 1500xg for 15 minutes with a slow brake (setting: 1 on the Avanti X- 15R) at ~4°C.

Note: It will take about 4-5 minutes to slow down to 0xg.

v) Carefully remove the buckets from the centrifuge, bring to the BSC, and carefully remove the units from the buckets, ensuring that the separation of the red blood cells and plasma are not disturbed.

Note: While processing a unit, place any additional units on ice.

vi) Press off the saline and any remaining plasma into a waste container until some red blood cells can be seen exiting.

vii) Ensure that the red blood cells are resuspended by gently rubbing the bag.

127 viii) Tie off the tubing and place the unit(s) on ice while transporting to the operating room for use.

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A6. Perfused renal graft.

Figure A6. Renal graft perfused under normothermic conditions ex vivo.

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A7. Metabolic cage.

For further assessment and urine collection, a metabolic cage was developed. A former pig truck for lifting of heavy animals was reconstructed: The flooring (Figure A7.1) was adapted to separate feces and food from the urine; an ABC plastic funnel system let the urine into a collection chamber. A customized drinking device was constructed to avoid spilling of water into the urine chamber (Figure A7.2).

Figure A7.1: Customized flooring to separate feces and food from urine in the metabolic cage.

Figure A7.2: Customized metabolic cage for 24-hour urine collection. Pig truck, adapted flooring, ABS plastic funnel, water feeding system with piglet nipple, permanent plastic lattice as fence.

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A8. Portable perfusion device.

In collaboration with the Biomedical Engineering Department of the University of Toronto, a prototype of a portable normothermic perfusion device was developed (BME489 Capstone Design Project). The full report is attached to the printed version of this thesis.

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Figures 1.11 and 1.12 are adapted from 100. Reproduced with permission.

Copyrights were received for the included manuscripts from The Journal of Visualized Experiments and Transplantation.