Red blood cells as oxygen carrier during normothermic machine perfusion of kidney grafts: Friend or foe?

Renal ex vivo normothermic machine perfusion (NMP) is under development as an assessment tool for high-risk kidney grafts and as a means of achieving more physiologically accurate organ preservation. On-going hemolysis has been reported during NMP, as this technique relies on red blood cells for oxygen delivery. In this study, we confirm the occurrence of progressive hemolysis during 6-hour kidney NMP. NMP-associated erythrostasis in the glomeruli and in peri-glomerular vascular networks points to an interaction between the red blood cells and the graft. Continuous hemolysis resulted in prooxidative changes in the perfusate, which could be quenched by addition of fresh frozen plasma. In a cell-based system, this hemolysis induced redox stress and exhibited toxic effects at high concentrations. These findings highlight the need for a more refined oxygen carrier in the context of renal NMP.

Metabolic needs of the kidney graft undergoing normothermic machine perfusion.

Normothermic machine perfusion (NMP) is emerging as a novel preservation strategy. During NMP, the organ is maintained in a metabolically active state that may not only provide superior organ preservation, but that also facilitates viability testing before transplantation, and ex situ resuscitation of marginal kidney grafts. Although the prevailing perfusion protocols for renal NMP are refined from initial pioneering studies concerning short periods of NMP, it could be argued that these protocols are not optimally tailored to address the putatively compromised metabolic plasticity of marginal donor grafts (i.e., in the context of viability testing and/or preservation), or to meet the metabolic prerequisites associated with prolonged perfusions and the required anabolic state in the context of organ regeneration. Herein, we provide a theoretical framework for the metabolic requirements for renal NMP. Aspects are discussed along the lines of carbohydrates, fatty acids, amino acids, and micronutrients required for optimal NMP of an isolated kidney. In addition, considerations for monitoring aspects of metabolic status during NMP are discussed.

Extracellular Vesicles Released During Normothermic Machine Perfusion Are Associated With Human Donor Kidney Characteristics.

BACKGROUND: Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. The characterization of EVs released by kidney grafts during normothermic machine perfusion (NMP) may present a promising avenue to assess graft status before transplantation. METHODS: We phenotyped and determined the concentrations of EVs in the perfusate of 8 discarded expanded-criteria donor human kidneys during 6 h of NMP. Perfusate samples were taken at 0/60/180/360 min and examined with nanoparticle tracking analysis and imaging flow cytometry (IFCM). Using IFCM, EVs were identified by their expression of common EV markers CD9, CD63, and CD81 (tetraspanins) in combination with either platelet endothelial cell adhesion molecule (CD31), pan-leukocyte protein (CD45), or carboxyfluorescein succiminidyl ester (CFSE) fluorescence. RESULTS: Nanoparticle tracking analysis measurements revealed the release of nanoparticles <400 nm into the perfusate during NMP. With IFCM, tetraspanin protein signatures of the released nanoparticles were characterized, and the majority (~75%) of CFSE+ EVs were found to be CD81+, whereas ~16% were CD9+ and ~8% CD63+. Correlation analysis of concentrations of identified EV subsets with crude donor characteristics and NMP viability characteristics revealed significant correlations with cold ischemia time, donor age, and renal flow. CONCLUSIONS: Our findings demonstrate that discarded expanded-criteria donor kidney grafts release distinct EV subsets during NMP. Because these subsets correlate with well-established indicators of transplant outcome, EVs might represent new potential candidates for assessment of kidney graft quality.

Complement Is Activated During Normothermic Machine Perfusion of Porcine and Human Discarded Kidneys.

Background: The gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP. Methods: NMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition. Results: Complement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death. Conclusion: Normothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation.