CASPASES IN RENAL TUBULAR EPITHELIAL CELL INJURY

胱天蛋白酶（ICE/Ced3蛋白酶）是最近鉴定的一组蛋白酶， 在细胞死亡中起关键作用的密切相关的半胱氨酸蛋白酶。我们在 体外和体内研究提供了强有力的证据， 半胱天冬酶在肾小管上皮细胞缺氧损伤中的作用体外 我们的研究表明，特异性的半胱天冬酶肽抑制剂， 防止肾近端小管上皮细胞的细胞死亡和DNA损伤 暴露于缺氧和氧化损伤。稳定转染的LLC-PK1细胞系 p35可以过度表达caspase-3抑制剂， 缺氧以及氧化损伤。探讨半胱天冬酶在肾脏疾病中的作用， 在体内的病理生理学中，了解哪些半胱天冬酶在 在肾脏。由于这些信息是未知的，我们用RT-PCR方法测定了 老鼠的肾皮质转录半胱天冬酶1，-2，-3，-6，-7，-8的基因 在已知的半胱天冬酶家族成员中是-9。我们还证明 分别通过Western和北方印迹分析， 在肾缺血/再灌注损伤中，存在活化和 caspase-3的上调和caspase-1、-2、-3-6和 -8个mRNA。检查刽子手的免疫组化定位 Caspase-3在肾缺血/再灌注损伤中的作用 大鼠重组caspase-3抗体，并证明了再分布， Caspase-3在肾小管上皮细胞中的胞浆核转位 损伤时的上皮细胞。基于这些研究，我们假设， 半胱天冬酶的亚细胞再分布和激活起着重要作用 在肾缺血/再灌注损伤中细胞死亡和DNA损伤中的作用， 抑制半胱天冬酶活化将保护或减轻缺血性损伤。 因此，本提案的目标是检查信令 半胱天冬酶的活化和亚细胞再分布的途径， 调节肾缺血性损伤中的DNA损伤和细胞死亡。基于 增强的mRNA表达、半胱天冬酶激活和半胱天冬酶上调 在缺氧和缺血/再灌注损伤过程中，我们将重点研究 执行者半胱天冬酶，半胱天冬酶-3，和起始者半胱天冬酶，半胱天冬酶-8， -9.虽然这些研究将集中在缺血性损伤，我们相信， 从这些研究中获得的信息将适用于未来的研究 这将检查半胱天冬酶在其他形式的急性肾衰竭中的作用。 该提案的具体目标是：I.检查胱天蛋白酶在以下方面的作用： 体外缺氧/复氧和体内缺血/再灌注损伤 调节它们的表达和/或活性。二.检查亚细胞 caspase-3在体外和体内的分布及转位机制 缺血/再灌注损伤的体内模型。三.研究的机制 半胱氨酸蛋白酶在缺氧/复氧和缺血/再灌注中的活化 损伤IIIA.为了检测胰腺癌细胞中caspase-3的活化， 缺氧和氧化损伤及非神经依赖性 途径：IIIB。探讨缺氧和氧化诱导的 线粒体PT在半胱天冬酶激活中的改变。四.识别 半胱天冬酶在核酸内切酶活化中的作用。这些研究将提供新的 深入了解我们的细胞和分子机制， 缺血性肾损伤以及其他形式的急性肾衰竭。

Caspases (ICE/Ced3 proteases) are a recently identified group of closely related cysteine proteases that play key roles in cell death. Our in vitro and in vivo studies provide strong evidence for an important role of caspases in hypoxic injury to renal tubular epithelial cells. In in vitro studies we have demonstrated that specific peptide inhibitors of caspases prevent cell death and DNA damage in renal proximal tubular epithelial cells exposed to hypoxic and oxidant injury. A stably transfected LLC-PK1 cell line developed to overexpress the caspase-3 inhibitor, p35 provided protection to hypoxic as well as oxidant injury. To delineate the role of caspases in renal pathophysiology in vivo it was essential to know which caspases are expressed in the kidney. Since this information was not known, we determined by RT-PCR that rat kidney cortex transcribes the genes for caspases 1, -2, -3, -6, -7, -8 and -9 out of the known family members of caspases. We have also demonstrated by Western and Northern blot analyses, respectively, that in rats subjected to ischemia/reperfusion injury to the kidney, there is both activation and upregulation of caspase-3 and enhanced expression of caspases -1, -2, -3 -6 and -8 mRNAs. To examine the immunohistochemical localization of the executioner caspace-3 in renal ischemia/reperfusion injury we developed a polyclonal antibody to rat recombinant caspase-3 and demonstrated redistribution and translocation of caspase-3 from the cytoplasm to the nuclei in renal tubular epithelial cells during injury. Based on these studies, we hypothesize that subcellular redistribution and activation of caspases plays an important role in cell death and DNA damage in renal ischemia/reperfusion injury and that the inhibition of caspase activation will protect or reduce the ischemic injury. Thus, the objectives of the present proposal are to examine the signaling pathway/s of activation and subcellular redistribution of caspases that regulate DNA damage and cell death in renal ischemic injury. Based on the enhanced mRNA expression, caspase activation and upregulation of caspases during hypoxic and ischemia/reperfusion injury, we will focus our studies on the executioner caspase, caspase-3, and the initiator caspases, caspase-8 and -9. While these studies will focus on ischemic injury, we believe the information derived from these studies will be applicable in future studies which will examine the role of caspases in other forms of acute renal failure. The specific aims of the proposal are: I.To examine the role of caspases in hypoxia/reoxygenation in vitro and ischemia/reperfusion injury in vivo by modulating their expression and/or activity. II. To examine the subcellular distribution and mechanisms of translocation of caspase-3 in in vitro and in vivo models of ischemia/reperfusion injury. III. To examine the mechanism of activation of caspases in hypoxia/reoxygenation and ischemia/reperfusion injury. IIIA. To examine mitochondrial-mediated activation of caspase-3 in hypoxic and oxidant injury and contribution by nonmitochondrial-dependent pathway: IIIB. To examine the relation between hypoxia- and oxidant-induced alterations in mitochondrial PT in activation of caspases. IV. To identify the role of caspases in endonuclease/s activation. These studies will provide new insights into our understanding of the cellular and molecular mechanisms of ischemic renal injury as well as other forms of acute renal failure.