The Impact of Nutritional Supplementation on Donor Kidneys During Oxygenated Ex Vivo Subnormothermic Preservation.

Evidence suggests that nutritional supplementation during normothermic ex vivo perfusion improves organ preservation. However, it is unclear whether the same benefit is observed during room temperature (subnormothermic) oxygenated perfusion. In this study, we tested the impact of providing complete nutrition during subnormothermic perfusion on kidney outcomes. Methods: Porcine kidneys were recovered after 30 min of cross clamping the renal artery in situ to simulate warm ischemic injury. After flushing with preservation solution, paired kidneys were cannulated and randomly assigned to perfusion with either (1) hemoglobin-carrier hemoglobin-based oxygen carrier or (2) hemoglobin-based oxygen carrier + total parenteral nutrition (TPN) for 12 h at 22 °C. To mimic reperfusion injury, all kidneys were reperfused with whole blood for an additional 4 h at 37 °C. Kidney function and damage were assessed. Results: Kidneys preserved with or without TPN performed equally well, showing similar renal function postreperfusion. Histological findings indicated similar levels of damage from apoptosis staining and acute tubular necrosis scores in both groups. Additionally, markers of renal damage (KIM-1) and inflammation (IL-6; high-mobility group box 1) were similar between the groups. Conclusions: Unlike other studies using normothermic oxygenated perfusion platforms, nutritional supplementation does not appear to provide any additional benefit during ex vivo kidney preservation over 12 h evaluated by whole blood-based reperfusion method at subnormothermic temperature. Further study should include a kidney autotransplant model to assess the role of TPN in vivo.

Comparison of Centrifugal and Pulsatile Perfusion to Preserve Donor Kidneys Using Ex Vivo Subnormothermic Perfusion.

Objective We have previously demonstrated benefits of kidney preservation utilizing an oxygenated subnormothermic ex vivo perfusion platform. Herein, we aim to compare pulsatile versus centrifugal (steady and uniform flow) perfusion with the goal of optimizing renal preservation with these devices. Materials and methods: Pig kidneys were procured following 30 min of warm ischemia by cross-clamping both renal arteries. Paired kidneys were cannulated and underwent either: oxygenated pulsatile or centrifugal perfusion using a hemoglobin oxygen carrier at room temperature with our ex vivo machine perfusion platform for 4 hr. Kidneys were reperfused with whole blood for 4 hr at 37° C. Renal function, pathology and evidence of inflammation were assessed post-perfusion. Results: Both pump systems performed equally well with organs exhibiting similar renal blood flow, and function post-reperfusion. Histologic evidence of renal damage using apoptosis staining and acute tubular necrosis scores was similar between groups. This was corroborated with urinary assessment of renal damage (NGAL 1) and inflammation (IL-6), as levels were similar between groups. Conclusion: In our porcine model with added warm ischemia simulating the effects of reperfusion after transplantation, pulsatile perfusion yielded similar renal protection compared with centrifugal perfusion kidney preservation. Both methods of perfusion can be used in ex vivo kidney perfusion systems.

Renal Protection Against Ischemia Reperfusion Injury: Hemoglobin-based Oxygen Carrier-201 Versus Blood as an Oxygen Carrier in Ex Vivo Subnormothermic Machine Perfusion.

BACKGROUND: The optimal method of oxygen delivery to donor kidneys during ex vivo machine perfusion has not been established. We have recently reported the beneficial effects of subnormothermic (22°C) blood perfusion in the preservation of porcine donation after circulatory death kidneys. Since using blood as a clinical perfusate has limitations, including matching availability and potential presence of pathogen, we sought to assess hemoglobin-based oxygen carrier (HBOC-201) in oxygen delivery to the kidney for renal protection. METHODS: Pig kidneys (n = 5) were procured after 30 minutes of warm in situ ischemia by cross-clamping the renal arteries. Organs were flushed with histidine tryptophan ketoglutarate solution and subjected to static cold storage or pulsatile perfusion with an RM3 pump at 22°C for 4 hours with HBOC-201 and blood. Thereafter, kidneys were reperfused with normothermic (37°C) oxygenated blood for 4 hours. Blood and urine were subjected to biochemical analysis. Total urine output, urinary protein, albumin/creatinine ratio, flow rate, resistance were measured. Acute tubular necrosis, apoptosis, urinary kidney damage markers, neutrophil gelatinase-associated lipocalin 1, and interleukin 6 were also assessed. RESULTS: HBOC-201 achieved tissues oxygen saturation equivalent to blood. Furthermore, upon reperfusion, HBOC-201 treated kidneys had similar renal blood flow and function compared with blood-treated kidneys. Histologically, HBOC-201 and blood-perfused kidneys had vastly reduced acute tubular necrosis scores and degrees of terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining versus kidneys treated with cold storage. Urinary damage markers and IL6 levels were similarly reduced by both blood and HBOC-201. CONCLUSIONS: HBOC-201 is an excellent alternative to blood as an oxygen-carrying molecule in an ex vivo subnormothermic machine perfusion platform in kidneys.

Subnormothermic Oxygenated Perfusion Optimally Preserves Donor Kidneys Ex Vivo.

INTRODUCTION: The current methods of preserving donor kidneys in nonoxygenated cold conditions minimally protect the kidney against ischemia-reperfusion injury (IRI), a major source of complications in clinical transplantation. However, preserving kidneys with oxygenated perfusion is not currently feasible due to the lack of an ideal perfusion mechanism that facilitates perfusion with blood at warm temperature. Here, we have designed an innovative renal pump circuit system that can perfuse blood or acellular oxygen carrier under flexible temperatures, pressures, and oxygenation. We have tested this apparatus to study optimal conditions of storage of our porcine model of donation after cardiac death (DCD) kidneys. METHODS: Porcine kidneys were retrieved after 30 minutes of cross-clamping renal pedicles in situ. Cessation of blood mimics postcardiac death in humans and simulates DCD warm ischemic injury. Procured kidneys were flushed and subjected to static cold storage (SCS) for 4 hours. For warm perfusion, kidneys were cannulated for pulsatile oxygenated perfusion with blood:PlasmaLyte for 4 hours at 15 °C, 22 °C, and 37 °C. To mimic posttransplant scenario, all kidneys were reperfused with blood for an additional 4 hours at 37 °C. RESULTS: Compared with all other groups, 22 °C perfusion resulted in significant reduction of acute tubular necrosis (ATN), apoptosis, kidney damage markers, Toll-like receptor signaling, and cytokine production. It was associated with maximal renal blood flow and urine output. Kidneys stored at 15 °C thrombosed within 2 hours under this condition. Martius Scarlet Blue staining confirmed that 22 °C was the optimal temperature to minimize hemorrhage and blood clots. CONCLUSION: Our novel study shows that oxygenated perfusion at near-room-temperature provides optimal donor kidney storage conditions.

CORM-401 Reduces Ischemia Reperfusion Injury in an Ex Vivo Renal Porcine Model of the Donation After Circulatory Death.

BACKGROUND: Carbon monoxide (CO) inhalation protects organ by reducing inflammation and cell death during transplantation processes in animal model. However, using CO in clinical transplantation is difficult due to its delivery in a controlled manner. A manganese-containing CO releasing molecules (CORM)-401 has recently been synthesized which can efficiently deliver 3 molar equivalents of CO. We report the ability of this anti-inflammatory CORM-401 to reduce ischemia reperfusion injury associated with prolonged cold storage of renal allografts obtained from donation after circulatory death in a porcine model of transplantation. METHODS: To stimulate donation after circulatory death condition, kidneys from large male Landrace pig were retrieved after 1 hour warm ischemia in situ by cross-clamping the renal pedicle. Procured kidneys, after a brief flushing with histidine-tryptophan-ketoglutarate solution were subjected to pulsatile perfusion at 4°C with University of Wisconsin solution for 4 hours and both kidneys were treated with either 200 µM CORM-401 or inactive CORM-401, respectively. Kidneys were then reperfused with normothermic isogeneic porcine blood through oxygenated pulsatile perfusion for 10 hours. Urine was collected, vascular flow was assessed during reperfusion and histopathology was assessed after 10 hours of reperfusion. RESULTS: We have found that CORM-401 administration reduced urinary protein excretion, attenuated kidney damage markers (kidney damage marker-1 and neutrophil gelatinase-associated lipocalin), and reduced ATN and dUTP nick end labeling staining in histopathologic sections. CORM-401 also prevented intrarenal hemorrhage and vascular clotting during reperfusion. Mechanistically, CORM-401 appeared to exert anti-inflammatory actions by suppressing Toll-like receptors 2, 4, and 6. CONCLUSIONS: Carbon monoxide releasing molecules-401 provides renal protection after cold storage of kidneys and provides a novel clinically relevant ex vivo organ preservation strategy.