The Role of Caspases in Warm and Cold Ischemia

描述（由申请人提供）： 移植肾功能延迟恢复（DGF）可独立预测1年和5年肾移植存活率降低，并促进慢性移植肾肾病（CAN）的发生。DGF由冷缺血和热缺血引起。冷缺血发生在搏动机灌注和静态冷藏肾脏。该基金将重点研究冷缺血和热缺血的病理生理学，特别是半胱天冬酶的作用。Caspase-3是凋亡和坏死细胞死亡的主要介质。Caspase-1将促炎细胞因子IL-1 β和IL-18转化为它们的活性形式。总的假设是，半胱天冬酶介导近端肾小管损伤在冷和热缺血。在冷缺血模型中，将评估半胱天冬酶-3对近端肾小管细胞凋亡以及严重刷状缘损伤的贡献。将在静态和脉动冷缺血期间测定半胱天冬酶-3，7，9（细胞凋亡的线粒体途径）和8（细胞凋亡的死亡受体途径）的基因表达、蛋白质和活性。将对近端小管中的活化半胱天冬酶-3进行免疫组织化学。将评估半胱天冬酶抑制对肾脏形态的影响。这些实验结果将有助于了解冷缺血损伤和器官保存。Caspase-1介导的IL-18的产生介导缺血性ARF。IL-18是干扰素γ的有效诱导剂，干扰素γ是MHC表达的强力诱导剂。将在热缺血模型中测定胱天蛋白酶-1和IL-18介导的肾中干扰素γ表达的增加和近端小管上MHC表达。将评估肾脏中的干扰素γ和MHC I类和II类mRNA表达。将评价半胱天冬酶-1、IL-18和干扰素γ抑制对MHC表达的影响。MHC表达减少与肾移植免疫原性降低有关，并对DGF对短期和长期移植肾功能的有害影响具有重要意义

DESCRIPTION (provided by applicant): Delayed graft function (DGF) independently predicts reduced 1- and 5- year kidney transplant survival and contributes to the development of chronic allograft nephropathy (CAN). DGF is caused by both cold and warm ischemia. Cold ischemia occurs during both pulsatile machine perfusion and static cold storage of kidneys. This grant will focus on the pathophysiology of cold and warm ischemia and specifically the role of caspases. Caspase-3 is a major mediator of apoptotic and necrotic cell death. Caspase-1 converts the proinflammatory cytokines IL-1beta and IL-18 to their active forms. The overall hypothesis is that caspases mediate proximal tubular damage in both cold and warm ischemia. In a model of cold ischemia the contribution of caspase-3 to proximal tubular apoptosis as well as severe brush border injury will be assessed. Gene expression, protein and activity of caspases-3, 7, 9 (mitochondrial pathway of apoptosis) and 8 (death receptor pathway of apoptosis) will be determined during both static and pulsatile cold ischemia. Immunohistochemistry for activated caspase-3 in proximal tubules will be performed. The effect of caspase inhibition on kidney morphology will be assessed. The findings of these experiments will contribute to the understanding of cold ischemia injury and organ preservation. Caspase-1-mediated production of IL-18 mediates ischemic ARF. IL-18 is a potent inducer of interferon gamma, which is a powerful inducer of MHC expression. Caspase-1 and IL-18-mediated increase in interferon gamma expression in the kidney and MHC expression on proximal tubules will be determined in a model of warm ischemia. Interferon gamma and MHC class I and II mRNA expression in the kidney will be assessed. The effect of caspase-1, IL-18 and interferon gamma inhibition on MHC expression will be evaluated. Reduced MHC expression is associated with decreased kidney graft immunogenicity and has important implications for the deleterious effect of DGF on both short and long-term allograft function