Comparison of competing ex vivo preservation techniques and investigation of the mechanisms of graft recovery during ex-vivo perfusion in marginal kidney grafts

肾移植是治疗终末期肾病的首选方法。供需之间的巨大差距导致更频繁地使用来自延长标准的器官和循环性死亡后的捐赠。这些边缘移植物的一个主要问题是由于对缺血和再灌注损伤的易感性增加而导致移植物功能延迟，从而导致长期功能下降。因此，移植界一直致力于研究改善这些移植物功能的方法。体外正常肾灌注和氧合低温灌注是实现这一目标的有希望的选择。在猪循环死亡后的捐赠模型中，研究表明，边缘移植物经常温离体肾灌注而不是传统的冷藏，可以改善移植物的功能。此外，其他动物研究也有证据表明，氧合低温灌注优于冷藏。本研究项目的目的是研究哪种方法提供了肾脏保存的最佳选择，以及哪种机制参与了灌注移植物在静态冷藏后循环死亡后移植功能的改善。我们假设为移植物提供氧气可以防止无氧代谢，从而导致参与缺血再灌注的介质减少。然而，其他新的途径也可能牵涉其中。因此，我们将通过微阵列分析开始对基因表达进行公正的检查，然后使用定量聚合酶链反应验证结果，然后将这些结果与适当的蛋白质表达研究相关联，例如通过酶联免疫吸附试验和Western blots。此外，可以识别与移植物功能改善相关的潜在生物标志物，然后这些标志物可用于评估移植前患者进行离体灌注的边缘移植物的功能和健康状况。最后，我们将探讨缺氧低温灌注和恒温离体肾灌注联合是否能更好地保存边缘移植物。本研究结果将支持氧合低温灌注和常温离体肾灌注向人体研究的转化，由于认为不适合移植的边缘移植物可在移植前进行氧合低温灌注和常温离体肾灌注，以恢复、改善和评估移植物的功能，因此可以采取个性化的方法。

Kidney transplantation represents the treatment of choice for end-stage renal disease. The enormous gap between supply and demand has lead to the more frequent use of organs from extended criteria and donation after circulatory death. A major problem with these marginal grafts is delayed graft function due to an increased susceptibility to ischemia and reperfusion injury, which leads to decreased long-term function. Therefore, the transplant community has focused its efforts on investigating methods for improving the function of these grafts. Normothermic ex-vivo kidney perfusion and oxygenated hypothermic perfusion are promising options to achieve that. In a porcine model of donation after circulatory death, studies have shown that marginal grafts subjected to normothermic ex-vivo kidney perfusion instead of the traditional cold storage demonstrate improved function of the grafts. Also, there is evidence from other animal studies that oxygenated hypothermic perfusion is superior to cold storage. The objective of the present research project is to investigate which method offers the best option for kidney preservation and which mechanisms are involved in the improved donation after circulatory death graft function in perfused grafts over static cold storage. We hypothesize that providing oxygen to the grafts prevents anaerobic metabolism, which results in a reduction in mediators involved in ischemia-reperfusion. However, other novel pathways may also be implicated. Therefore, we will initiate an unbiased examination of gene expression through microarray analysis, to then validate the results using quantitative polymerase chain reaction and then correlate these results with appropriate protein expression studies such as through enzyme-linked immunosorbent assays and Western blots. Also, potential biomarkers that correlate with improved graft function can be identified and these markers may then be utilized to assess the function and health of marginal grafts undergoing ex-vivo perfusion prior to transplant in patients. Finally, we will investigate whether a combination of oxygenated hypothermic perfusion and normothermic ex-vivo kidney perfusion offers a better preservation method in marginal grafts. The results of this study will support the translation of oxygenated hypothermic perfusion and normothermic ex-vivo kidney perfusion to human studies, and a personalized approach could be adopted as marginal grafts deemed unfit for transplant may be subjected to oxygenated hypothermic perfusion and normothermic ex-vivo kidney perfusion for restoration, improvement and assessment of graft function prior to transplantation.