The Role of Caspases in Warm and Cold Ischemia

描述(由申请人提供)： 移植肾功能延迟(DGF)独立地预测肾移植后1年和5年存活率的降低，并有助于慢性移植物肾病(CAN)的发展。DGF是由冷缺血和热缺血引起的。冷缺血发生在搏动性机器灌流和肾脏静态冷藏过程中。这笔拨款将集中在冷缺血和热缺血的病理生理学，特别是半胱氨酸天冬氨酸蛋白酶的作用。Caspase-3是细胞凋亡和坏死性死亡的主要介质。Caspase-1将促炎细胞因子IL-1β和IL-18转化为其活性形式。总体假设是，在冷和热缺血中，半胱氨酸天冬氨酸氨基转移酶介导近端肾小管损伤。在冷缺血模型中，将评估caspase-3在近端肾小管细胞凋亡和严重刷状缘损伤中的作用。在静止性和搏动性冷缺血过程中，将测定caspase-3、7、9(线粒体凋亡途径)和8(死亡受体途径)的基因表达、蛋白和活性。将进行近端小管中活化的caspase-3的免疫组织化学检查。半胱氨酸氨基转移酶抑制对肾脏形态的影响将被评估。这些实验结果将有助于理解冷缺血损伤和器官保存。Caspase-1介导的IL-18的产生介导了缺血性ARF。IL-18是干扰素-γ的有效诱导剂，而干扰素-伽马又是MHC表达的强大诱导剂。在热缺血模型中，将确定Caspase-1和IL-18介导的肾脏干扰素伽马表达增加和近端小管MHC表达增加。将评估干扰素γ和MHC I、II类基因在肾脏中的表达。将评估caspase-1、IL-18和干扰素γ抑制对MHC表达的影响。MHC表达降低与移植肾免疫原性降低有关，并对DGF对移植肾功能的短期和长期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