UC Davis Medical Center Ex Vivo Normothermic Preservation in Deceased Donor Renal Transplantation Richard Perez, MD Transplant Surgery UC Davis Medical Center

Deceased Donor Kidney Transplantation: A Model of Ischemia-Reperfusion Injury Potential Deceased Donor Hypotension, shock, acute inflammation, kidney injury Brain death or DCD Ex vivo Organ preservation – Hypothermia and hypoxia Recipient Vascular anastomosis with re-perfusion injury

Disadvantages of hypothermic preservation Ongoing low level metabolism/tissue breakdown Loss of energy stores Predictions of ultimate viability and function limited in highest risk marginal donors Not metabolically active so intervention for reconditioning/repair is limited Many high risk kidneys discarded

Ex vivo normothermic perfusion for organ preservation/pre-conditioning Maintain body temperature Oxygenation Support aerobic metabolism Normal physiologic function Advantages Restore ATP Able to assess organ function Minimize cold ischemia injury Regeneration and repair initiated?

Can ex vivo normothermic perfusion preservation be used to “rescue” discarded kidneys deemed “not transplantable” (2500/year)?

Hosgood / Nicholson, Transplantation 2011

Normothermic Perfusion System Pressure Display Pressure probe Medtronic Console Urine Collection Flow probe Heat Exchanger, 370C Venous Reservoir Kidneys were put on our system of EVNP for 3 hours. The perfusate was an Packed Red Blood Cells (PRBC) oxygenated and supplemented with parenteral nutrition and insulin. Exogenous creatinine was added to measure blood creat levels and clearance. One kidney was given Furosemide (10 mg) at the beginning to induce diuresis and assess acute kidney injury. Hemodynamic pump parameters and urine output were monitored. Blood and urine samples were collected at the start and every 30 minutes and analyzed for pH, oxygen, electrolytes, creatinine, and lactate. Wedge biopsies were collected pre & post perfusion & stained with alizarin red to measure calcium oxylate tubular deposition. Oxygenator Centrifugal Pump

UC Davis Pre-Clinical Ex Vivo Normothermic Renal Perfusion April 2013 - First sheep kidney perfused Jan 2014 - First human kidney perfused Jan 2014 - Nov 2015 - 53 human kidneys perfused Donor ages: 1 day – 78 years old Perfusion times: 1 - 12 hours

I Palma, Y Smolins, J Woloszyn, et al. UC Davis Medical Center Ex Vivo Normothermic Perfusion to Assess and Repair Kidney Ischemia-Reperfusion Injury I Palma, Y Smolins, J Woloszyn, et al. UC Davis Medical Center Academic Surgical Congress 2015

Study Design Deceased donors (n=4) presumed suitable for transplantation Met criteria for discard Paired kidneys (n=8) entered into study Whole blood EVNP to simulate clinical allograft reperfusion (n=4) Packed RBC EVNP to test optimized normothermic preservation (n=4)

Donor Characteristics Pair 1 Pair 2 Pair 3 Pair 4 Donation Type ECD DCD Reason for being discarded Biopsy Results Age (yrs.) 67 58 54 64 Cause of Death Anoxia CVA KDPI 99 79 75 94 Cold Ischemia Time (hrs) 40.33 40.67 52.82 53.15 56.08 56.28 60.37 60.88

Human Kidney Pre-Perfusion 1 hour 2 hours 3 hours 10A- WB 10B- PRBCs They appear to have normal appearance in color. 10B- PRBCs

Improved Hemodynamics with Optimized Normothermic Perfusion These are the flows for the human kidneys 9 and 10 using 2 Way ANOVA analysis. There was no statistical significance but as you can see the flows were higher in the PRBCs throughout the 3 hour perfusion. Academic Surgical Congress 2015

Ex vivo normothermic perfusion increases urine production Academic Surgical Congress 2015

Decreased injury biomarkers with optimized normothermic perfusion These are the Urine NGAL for the human kidneys 9 and 10 using 2 Way ANOVA analysis. There was statistical significance (p= 0.0090). The PRBCs had a lower level of urine NGAL which is an acute kidney injury biomarker. It is also secreted by white cells so we ran KIM1 to verify the NGAL results. This was measured by a Triage machine.

UC Davis Medical Center Ex vivo Normothermic Perfusion for Assessment of High Risk Deceased Donor Kidneys for Transplantation S Kabagambe, I Palma, Y Smolin, T Boyer, IP Palma, J Sageshima, Csanthanakrishnan, C Troppmann, J McVicar, R Perez UC Davis Medical Center Cutting Edge of Transplantation 2016

What criteria can we use to determine if an organ is transplantable? Retrospective review of locally discarded kidneys Study period: June 2014 – Oct 2015 Kidneys provisionally accepted for transplant but turned down due to biopsy and/or hypothermic perfusion parameters Placed on EVNP research protocol Assessment of gross appearance, blood flow, and urine output

n=6 Globally pink and well perfused High blood flow Good urine output Probably transplantable with Low Risk n=2 Pink, well perfused but patchy High blood flow Low urine output Possibly transplantable with Moderate Risk n=1 Pink and patchy Low blood flow No urine output Deemed not transplantable

Donor & pump Characteristics Moderate Risk ( n=2) Low Risk (n=6) P value Age (years) 51.00 ± 13.00 56.17 ± 3.48 0.76 KDPI (%) 73.50 ± 20.50 80.33 ± 5.35 0.80 Terminal Creatinine 2.44 ± 1.89 1.42 ± 0.27 0.68 Cold time (hrs) 50.93 ± 12.96 44.00 ± 4.90 0.69 Term hypo flow (ml/min) 73.50 ± 7.50 73.33 ± 4.13 0.99 Terminal hypo RRI 0.43 ± 0.05 0.39 ± 0.04 0.64 Terminal EVNP flow 250.0 ± 30.00 353.3 ± 33.83 0.09 Terminal EVNP RRI 0.29 ± 0.03 0.21 ± 0.03 0.15 Urine output (ml/hr) 2.25 ± 1.75 125.8 ± 43.14 0.04

Prospective clinical study of ex vivo normothermic perfusion in high risk deceased donor renal transplantation Purpose Demonstrate safety and feasibility of EVNP Comparison of standard of care hypothermic machine perfusion versus hypothermic perfusion + short additional period (2-4 hrs) of ex vivo normothermic perfusion prior to transplantation Establish a normothermic platform for further studies

Clinical trial of ex vivo normothermic perfusion in renal transplantation Inclusion criteria: Paired kidneys from donors with increased risk for DGF Donors with acute renal injury (Creat > 2.0) Donation after circulatory death Prolonged cold ischemia time (>36hrs) Donors with KDPI >70%

Clinical trial of ex vivo normothermic perfusion in renal transplantation Clinical protocol Standard hypothermic machine perfusion preservation to paired kidneys from high risk donors Control kidney transplanted after cold machine perfusion Study kidney to have additional 2-4 hours of normothermic perfusion just prior to implantation of organ

Ex Vivo Normothermic Perfusion: Future Directions Routine assessment of high risk/marginal organs Normothermic perfusion as a means to intervene and optimize organ function pre-transplant Pharmacologic Gene therapy Stem cells Development of “Organ Repair Centers”

Participating OPOs One Legacy Sierra Donor Services Donor Network of Arizona LifeShare Gift of Life Philadelphia Gift of Hope Illinois Life Link of Florida Life Share Oklahoma Life Center Northwest LifeNet Health Midwest Transplant Network Southwest Transplant