MACHINE PERFUSION IN KIDNEY TRANSPLANTATION: CLINICAL APPLICATION & METABOLOMIC ANALYSIS By ALISON J GUY (MBChB, FRCS) A thesis submitted to the University of Birmingham for the degree of DOCTOR OF MEDICINE Department of Immunity and Infection University of Birmingham & Department of Renal Surgery University Hospitals Birmingham 30th March 2015 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Kidney Transplantation is the gold standard treatment for patients with end-stage renal failure. Most kidneys used for transplantation are from deceased donors and ensuring successful outcomes depends on many factors. One of these is organ storage. Hypothermic Machine Perfusion (HMP) of deceased donor organs has been shown to have several benefits. However, it has not been widely adopted and the underlying mechanism is poorly understood. The first section of this thesis examines the introduction of HMP into clinical practice. HMP outcomes were similar to those of standard storage techniques but with the additional benefit of increasing safe storage times. This was likely due to inherent benefits of the machine itself, improved recipient preparation and better peri- operative conditions. The second part of this study analysed HMP perfusate using metabolomics (Nuclear Magnetic Resonance) to identify potential predictors of graft outcome. Differences were identified in the metabolic profiles of perfusate from kidneys with immediate and delayed graft function. These may have a future role in viability assessment. Improved understanding of metabolism during storage may help target optimization strategies for deceased donor organs. 2 The final part of this study describes the development of a porcine model of transplantation to test future hypotheses. 3 ACKNOWLEDGEMENTS This work would not have been possible without the help and support of a number of people. I would first like to thank my supervisors Andrew Ready and Mark Cobbold for their invaluable guidance with this project. Also, other members of the renal research team who were a source of ideas and practical help – Nicholas Inston, Mel Field, Jay Nath and Damian McGrogan. The metabolomics work would not have been possible without the help, and patience, of Christian Ludwig and Daniel Tennant. Assistance with histology preparation and analysis was provided by Desley Neal and the UHB histology lab. I would also like to thank Peter DeMuylder and Gunther Vanwezer from Organ Recovery Systems who provided essential funding and shared their expertise in organ perfusion. Assistance with statistical analysis was provided by the UHB statistics department, mainly James Hodson, to whom I am very grateful. I would like to thank all of the clinical renal transplant team for co-operating with this research and providing their opinions and advice, along with Buta Basi who assisted with many practical arrangements. In addition, thanks to the staff at FA Gill Ltd who must have found my requests and arrangements for the perfusion of pig kidneys slightly unusual. 4 LIST OF CONTENTS CHAPTER ONE: INTRODUCTION 22 1.1 Chronic Kidney Disease (CKD) 23 1.1.1 CKD - Definitions & Staging 23 1.1.2 CKD - Prevalence, Incidence & Economic Burden 25 1.1.3 CKD - Aetiology/Pathology 30 1.1.4 CKD - Disease Progression 32 1.1.5 CKD - Management 33 1.2 Renal Transplantation (RT) 35 1.2.1 RT - Overview / Definitions 35 1.2.2 RT - Cold Ischaemic Time 37 1.2.3 RT - Outcomes 37 1.2.4 RT - The Organ Shortage & Marginal Organs 39 1.2.5 RT - Ischaemia-Reperfusion Injury 41 1.3 Organ Preservation 46 1.3.1 History of Organ Preservation 46 1.3.2 Preservation Fluids 48 1.3.3 Static Cold Storage 54 1.3.4 Hypothermic Machine Perfusion 55 1.3.5 HMP Mechanisms 60 1.3.6 HMP Viability Assessment 61 1.3.7 HMP Pharmacological Manipulation 64 1.3.8 HMP Clinical Outcomes 65 5 1.3.9 Other Preservation Methods 69 1.4 Overall Aims 74 CHAPTER TWO: ASSESSMENT OF CLINICAL UTILITY 76 2.1 Background 77 2.1.1 Aims 79 2.1.2 Setting 79 2.2 Methods 81 2.2.1 Patient Recruitment 81 2.2.2 Kidney Preservation 81 2.2.3 Data Collection & Outcome Measures 82 2.2.4 Statistical Analysis 83 2.3 Results 84 2.3.1 Donor & Recipient Demographics 84 2.3.2 Delayed Graft Function (DGF) 86 2.3.3 Cold Ischaemic Time and Timing of Surgery 87 2.3.4 HMP Parameters 90 2.3.5 Complications & Length of Stay 93 2.3.6 Post-Operative Creatinine 94 2.3.7 Histology 95 2.4 Discussion 97 2.4.1 Donor & Recipient Considerations 97 2.4.2 Use Of HMP & CIT 98 2.4.3 Resistance 100 6 2.4.4 Post-Operative Creatinine 101 2.4.5 Biopsy-Proven Acute Rejection Rates 101 2.4.6 Limitations 101 2.4.7 Conclusion 103 CHAPTER THREE: ANALYSIS OF HMP PERFUSATE 105 3.1 Background 106 3.1.1 Metabolomics 107 3.1.2 Metabolomic Investigations 110 3.1.3 Principles of Nuclear Magnetic Resonance (NMR) 111 3.1.4 NMR in Disease Studies 112 3.1.5 NMR in Transplantation 112 3.1.6 Key Metabolic Pathways 114 3.1.6.1 Glycolysis 115 3.1.6.2 The Citric Acid Cycle 117 3.1.6.3 Oxidative Phosphorylation 118 3.1.6.4 The Pentose Phosphate Pathway 119 3.1.6.5 The Urea Cycle 120 3.1.6.6 Fatty Acid Beta-Oxidation 121 3.1.6.7 Gluconeogenesis 122 3.2 Aims 125 3.3 Methods 125 3.3.1 Ethics & Sponsorship 125 3.3.2 Patient Recruitment 125 7 3.3.3 Kidney Preservation 126 3.3.4 Sample Collection 126 3.3.5 Sample Processing 127 3.3.6 Spectral Acquisition 127 3.3.7 Spectral Analysis 128 3.3.8 Statistical Analysis 133 3.4 Results 134 3.4.1 Identified Metabolites 136 3.4.2 Metabolites in KPS-1® 137 3.4.3 New Metabolites in Perfusate 138 3.4.4 Graft Function & Metabolomic Profile 140 3.4.4.1 Glucose 140 3.4.4.1.1 Correlation of Glucose Measurements 141 3.4.4.2 Inosine 143 3.4.4.3 Leucine 143 3.4.4.4 Gluconate 144 3.4.4.5 Other Metabolites 145 3.4.5 ROCS for Prediction of Delayed Graft Function 150 3.5 Discussion 152 3.5.1 Constituents of KPS-1® 154 3.5.1.1 Adenine & Ribose 155 3.5.1.2 Gluconate & Mannitol 156 3.5.1.3 Glutathione 156 3.5.1.4 Glucose 157 8 3.5.2 New Metabolites in Perfusate 158 3.5.2.1 Leucine 158 3.5.2.2 Inosine 159 3.5.2.3 Other Metabolites 159 3.5.3 Prediction of Delayed Graft Function 160 3.5.4 Limitations 161 3.5.5 Conclusion 162 CHAPTER FOUR: DEVELOPMENT & ASSESSMENT OF A PORCINE MODEL 163 4.1 Background 164 4.2 Aims 165 4.3 Methods 166 4.3.1 Sourcing of Porcine Kidneys 166 4.3.2 Porcine Kidney Recovery 166 4.3.3 Porcine Kidney Flush 166 4.3.4 HMP Technique 167 4.3.5 Sample Collection 167 4.3.6 Modifications 168 4.3.7 Preparation for NMR 170 4.3.7.1 Sample Processing 170 4.3.7.2 Spectral Acquisition 171 4.3.7.3 Spectral Analysis 171 4.3.7.4 Statistical Analysis of Porcine Data 173 4.3.8 Histological Preparation for Light Microscopy 174 9 4.3.9 Histological Preparation for Electron Microscopy 174 4.3.10 Comparison of Porcine & Human HMP Kidneys 179 4.3.11 Statistical Analysis of Porcine & Human Kidneys 180 4.4 Results 181 4.4.1 Ischaemic Times 181 4.4.2 Porcine HMP Parameters 181 4.4.3 Porcine NMR Analysis 184 4.4.4 Porcine Histology 190 4.4.4.1 Light Microscopy 190 4.4.4.2 Electron Microscopy 190 4.4.4.2.1 Endothelial Cells 190 4.4.4.2.2 Epithelial Cells 192 4.4.4.2.3 Glomerular Basement Membrane/Endothelium 194 4.4.4.2.4 Arteriolar Smooth Muscle 196 4.4.4.2.5 Tubules 198 4.4.4.2.6 Peritubular Capillaries 200 4.4.5 Comparison of Porcine and Human HMP Kidneys 202 4.4.5.1 Ischaemic Times 202 4.4.5.2 HMP Parameters 202 4.4.5.3 Metabolomics 204 4.5 Discussion 209 4.5.1 The Porcine Model 209 4.5.2 Porcine Ischaemic Times 211 4.5.3 Porcine HMP Parameters 211 10 4.5.4 Porcine NMR Analysis 211 4.5.5 Porcine Histology 214 4.5.6 Comparison of Porcine & Human HMP Kidneys 215 4.5.7 Limitations 218 4.5.8 Conclusions 219 CHAPTER 5: CONCLUDING REMARKS 221 CHAPTER 6: REFERENCES 228 CHAPTER 7: APPENDICES 263 7.1 Research Protocol 264 7.2 Ethics Favourable Opinion Letter 287 7.3 Cut-Off Values for ROC Curves 291 7.4 Metabolite Concentrations Measured in Kidney Perfusate of 292 SCS & HMP Porcine Kidneys (All Metabolites) 7.5 Box and Whisker Plot to Represent Concentrations of Metabolites 294 in HMP & SCS Porcine Kidney Perfusate (Non-Significant Metabolites) CHAPTER 8: PUBLICATIONS AND PRESENTATIONS 296 Declaration 299 Transplantation article 300 11 LIST OF TABLES Table 1.1: Staging System for CKD – National Kidney Foundation Table 1.2: Revised Staging System for CKD - KDIGO Table 1.3: Grading System for Albuminuria – KDIGO Table 1.4: Classification of Causes of CKD Based on Presence or Absence of Systemic Disease and Location Within the Kidney of Pathological- Anatomical Findings Table 1.5: Percentage Distribution of Primary Renal Diagnosis by Age (2012) Table 1.6: Constituents of KPS-1® Table 1.7: Proposed Advantages and Disadvantages of Normothermic Preservation Table 2.1: Donor and recipient demographics Table 2.2: Delayed Graft Function by Donor Type and Storage Group Table 2.3: In-patient Complications Following Deceased Donor Renal Transplantation Table 2.4: Histology of Post-transplant Kidney Biopsies Table 3.1: Chemical Shifts References Used for Metabolite Quantification Table