William Hebrard

Benefits of active oxygenation during hypothermic machine perfusion of kidneys in a preclinical model of deceased after cardiac death donors

BACKGROUND Deceased after cardiac death donors (DCDs) represent a valuable source of organs; however, preventing poor outcome is difficult, even with the use of machine perfusion (MP). It is of paramount importance to improve this method. We proposed to evaluate the benefits of active oxygenation during kidney graft hypothermic MP using a novel perfusion machine: Kidney Assist (KA). METHODS We used a pig model of DCD transplantation in Large White pigs. Cold preservation was performed by conventional non-oxygenated MP (KAnoO2) or oxygenated MP (KA). RESULTS In the first 2 wk post-transplant, KA grafts displayed a lower serum creatinine peak and a faster return to normal levels compared with KAnoO2 animals, translating into a smaller area under the curve. Urinary neutrophil gelatinase-associated lipocalin levels and serum aspartate amino transferase levels were lower in KA animals compared with the non-oxygenated group. These correlated with better chronic function. Longer follow-up of the animals (3 mo) permitted evaluation of chronic outcome lesions. Interstitial fibrosis was reduced in the KA group, and these kidneys also displayed significantly lower levels of vimentin staining. Further histologic investigation also showed a trend toward decreased chronic inflammation in kidneys preserved with oxygen. CONCLUSIONS This new MP system is efficient in preserving DCD kidneys, greatly enhancing the capacity of the graft to withstand preservation stress and improving outcome. Oxygen delivery during preservation is thus valuable for highly damaged organs and offers an important therapeutic tool for transplant teams faced with decreased quality of donor organs.

Direct Thrombin Inhibitor Prevents Delayed Graft Function in a Porcine Model of Renal Transplantation

Background. Kidney transplantations from donors after cardiac arrest (DCA) are characterized by an increase in the occurrence of delayed graft function and primary nonfunction. In this study, Melagatran, a selective reversible direct thrombin inhibitor was used to limit renal injury in a DCA pig kidney transplantation model. Methods. We used a porcine model of DCA to study the effects of treatment with Melagatran in the peri-conservation period. Thromboelastography was used to check Melagatran antithrombin effect on in vitro clot formation. Reverse-transcriptase polymerase chain reaction was used to analyze the peripheral immune cells activation status. Renal function and morphologic study were performed at days 1 and 7. Finally, we analyzed the mechanisms of Melagatran protection on kidney microvasculature primary endothelial cells. Results. Prolongation of coagulation time (Ex-Tem) was observed 10 min after injection; however, Melagatran did not modulate increases of thrombin-antithrombin complexes following reperfusion. Melagatran significant treatment lowered the proinflammatory status of circulating immune cells. Animals survival was increased in Melagatran-treated groups (9 of 10 in Melagatran groups vs. 4 of 10 in controls at day 7). Renal injury and inflammation were also significantly reduced in treated groups. We also demonstrated a direct protective effect of Melagatran against endothelial cell activation and inflammation in vitro. Conclusion. Direct thrombin inhibitor administration in the periconservation period improved graft outcome and reduced renal injury in a model of DCA.

A p38 mitogen-activated protein kinase inhibitor protects against renal damage in a non-heart-beating donor model

Ischemia-reperfusion injury is one of the central nonimmunologic processes involved in renal allograft dysfunction. Kidneys from non-heart beating donors (NHBD) exhibit higher rates of delayed graft function (DGF) than those from other donors. Primary nonfunction and DGF are the main barriers to the use of kidneys from NHBD. Using a pig model of NHBD transplantation, we studied the effect of FR167653 (a p38 MAP kinase inhibitor) on the recovery and reparation of kidneys exposed to both warm (WI: 1 h) and cold ischemia (24 h). Our results demonstrate that the addition of FR167653 increases the kinetics of proximal tubule cell regeneration after 60 min of WI. Hypoxia-inducible factor and vascular endothelial growth factor expression was also more important in FR167653-treated kidneys compared with those in nontreated groups. Also, expression of peripheral-type benzodiazepine receptor, involved in tissue repair, was increased in the FR167653-treated groups. At 3 mo, the protective effects of FR167653 were accompanied by a reduction of long-term inflammation process and tubulointerstitial fibrosis development associated with a limitation of ischemia-induced remodeling. This study suggests that such treatment may be useful in protocols aimed at improving the quality of renal transplants from NHBD. In addition, the beneficial role of FR167653 in limiting early injury is associated with secondary reduction in development of tubular atrophy and interstitial fibrosis which are together the hallmark of failing renal transplants. The more efficient effect was observed when FR167653 was added in combination before WI, during cold storage and reperfusion.

Analysis of perfusates during hypothermic machine perfusion by NMR spectroscopy: a potential tool for predicting kidney graft outcome.

Background Machine perfusion use has been reported to promote graft outcome in case of donation after cardiac death. Our objective was to evaluate the potential for nuclear magnetic resonance (NMR) to predict graft outcome by analyzing perfusates during machine perfusion time. Method We used a renal autotransplantation model mimicking deceased after cardiac death donors with pigs. Organs were subjected to 60 min of warm ischemia before the hypothermic machine preservation during 22 hr. We studied the correlation between creatinemia after transplantation and the NMR data from perfusates. Results A metabonomic analysis allowed us to highlight the evolution of several metabolites during perfusion: the concentration of lactate, choline, or amino acids such as valine, glycine, or glutamate increased with time, whereas there was a diminution of total glutathione during this period. The changes in these biomarkers were less severe in the group with the better outcome. Statistical analysis revealed a strong association between the level of those metabolites during machine perfusion and function recovery (Spearman rank ≥0.89; P<0.05). Conclusion Multivariate analysis of lesion biomarkers during kidney perfusion using NMR data could be an interesting tool to assess graft quality, particularly because analyses times (2 hr total) are compatible with clinical application.

A new approach to the evaluation of liver graft function by nuclear magnetic resonance spectroscopy. A comparative study between Euro-Collins and University of Wisconsin solutions

Abstract The incidence of primary dysfunction or non-function of liver grafts still occurs at an unacceptable rate and the identification of new markers of graft viability and metabolic capacity is essential. Proton nuclear magnetic resonance (H NMR) spectroscopy has previously shown potential in the evaluation of renal allograft dysfunction after ischaemia reperfusion. The aim of this study was to compare liver graft function in a rat isolated perfused liver model after 24 hours of preservation in either Euro-Collins (EC) or University of Wisconsin (UW) solutions. Livers were reperfused for 90 min with a modified Krebs-Henseleit medium. Functional parameters measured were: pressure and resistances, bile and transaminase production. The production of lactate, pyruvate, citrate and succinate, β-hydroxybutyrate and aceto-acetate was measured by H NMR. There was a significant difference in both haemodynamics and bile production in favour of the UW group. The transaminases were similar in the two groups. Measurements of citrate, succinate and ketone bodies by H NMR were all higher in the UW group. These markers indicate better metabolic function of these grafts during reperfusion. In conclusion, discriminating different hepatic metabolic pathways is possible and easy by H NMR and can be used to assess both the preservation quality of liver grafts and their functional recovery.

Evaluation of renal medulla injury after cold preservation and transplantation: noninvasive determination of medullar damage by proton nuclear magnetic resonance spectroscopy of urine and plasma ☆

PRIMARY and early allograft dysfunction remains a challenge for the nephrologist. Usually, the assessment of renal graft dysfunction following transplantation is based on nonspecific measurement of renal function or an invasive method such as transplant biopsy. A rapid diagnosis is necessary because a delay in treating early rejection deteriorates the prognosis of the allograft function. In addition, the antirejection treatment based on a mere clinical diagnosis of rejection could improve delayed graft function caused by acute tubular necrosis or cyclosporine damage through possible side effects of antirejection treatment. In the present study, we wanted to assess whether proton nuclear magnetic resonance (HNMR) spectroscopy can detect ischemic damage and predict acute tubular necrosis after pig kidney preservation in two preservation solutions (Euro-Collins: EC and University of Wisconsin: UW).

Comparison of Euro-Collins and university of wisconsin solutions in the isolated perfused rat liver model: evaluation by proton nuclear magnetic resonance spectroscopy.

PRIMARY dysfunction or nonfunction of liver graft is still an important problem in liver transplantation despite surgical progress. The determination of new markers of graft viability and metabolic activities seems to be an important challenge to assess the early graft function. Proton nuclear magnetic resonance spectroscopy (NMRp) has previously demonstrated its interest in the evaluation of renal allograft dysfunction after ischemia reperfusion in isolated perfused pig kidney. For the kidney, NMRp study determines specific markers of function like TMAO and citrate. The aim of this study was to assess liver graft function in an isolated perfused rat liver model after 24 hours of preservation in Euro-Collins (EC) or University of Wisconsin (UW) solution.

Defining the optimal duration for normothermic regional perfusion in the kidney donor: A porcine preclinical study

Kidneys from donation after circulatory death (DCD) are highly sensitive to ischemia‐reperfusion injury and thus require careful reconditioning, such as normothermic regional perfusion (NRP). However, the optimal NRP protocol remains to be characterized. NRP was modeled in a DCD porcine model (30 minutes of cardiac arrest) for 2, 4, or 6 hours compared to a control group (No‐NRP); kidneys were machine‐preserved and allotransplanted. NRP appeared to permit recovery from warm ischemia, possibly due to an increased expression of HIF1α‐dependent survival pathway. At 2 hours, blood levels of ischemic injury biomarkers increased: creatinine, lactate/pyruvate ratio, LDH, AST, NGAL, KIM‐1, CD40 ligand, and soluble‐tissue‐factor. All these markers then decreased with time; however, AST, NGAL, and KIM‐1 increased again at 6 hours. Hemoglobin and platelets decreased at 6 hours, after which the procedure became difficult to maintain. Regarding inflammation, active tissue‐factor, cleaved PAR‐2 and MCP‐1 increased by 4‐6 hours, but not TNF‐α and iNOS. Compared to No‐NRP, NRP kidneys showed lower resistance during hypothermic machine perfusion (HMP), likely associated with pe‐NRP eNOS activation. Kidneys transplanted after 4 and 6 hours of NRP showed better function and outcome, compared to No‐NRP. In conclusion, our results confirm the mechanistic benefits of NRP and highlight 4 hours as its optimal duration, after which injury markers appear.