MITOCHONDRIAL CALPAIN MEDIATED RENAL CELL DEATH

描述（由申请人提供）：该项目的长期目标是阐明在缺血/再灌注和毒性诱导的急性肾功能衰竭（ARF）中导致线粒体功能障碍的事件，并确定一种预防线粒体功能障碍和减少ARF的治疗方法。线粒体功能障碍和ca2 +稳态破坏在肾细胞损伤和死亡中的作用已在许多ARF和肾毒性模型中得到证实。钙蛋白酶（Ca2+活化的中性半胱氨酸蛋白酶）在肾近端小管细胞（RPTC）损伤和死亡中由缺氧/再氧化和毒物产生的重要性已通过钙蛋白酶抑制剂得到证实。特别是两种不同类型的calpain抑制剂不仅阻断缺氧/再氧合RPTC死亡，而且阻断线粒体功能障碍，促进再氧合时呼吸恢复。这些结果有力地支持钙蛋白酶在线粒体功能障碍中的关键作用。上述实验显示calpain抑制剂对RPTC线粒体功能的保护作用，提示线粒体可能含有calpain。在使用分离的肾皮质线粒体（RCM）进行的一系列不同的初步实验中，我们已经获得了一种新的线粒体钙蛋白酶导致线粒体功能障碍的额外证据。这些数据导致了线粒体ca2 +摄取导致线粒体calpain激活的假设，这导致线粒体功能障碍，最终导致RPTC死亡和ARF。该应用程序的具体目的是：具体目的1：鉴定和表征线粒体钙蛋白酶，并检查其在分离的RCM和RPTC中的调节。具体目的二：阐明线粒体calpain介导的RPTC和离体RCM线粒体功能障碍的机制，鉴定线粒体calpain的线粒体蛋白靶点。具体目标III：确定目前描述的calpain抑制剂对线粒体calpain的有效性，并使用新的非天然氨基酸类似物开发新的线粒体calpain特异性抑制剂，并确定其在RPTC和分离的RCM中的有效性。特定目的IV：确定当前和/或开发的钙蛋白酶抑制剂在线粒体功能障碍和ARF的体内模型中的疗效。完成这些特定目标将大大增加我们对细胞损伤和死亡的基本理解，特别是介导线粒体功能障碍的事件。此外，我们将鉴定线粒体钙蛋白酶并开发新的钙蛋白酶抑制剂，包括那些线粒体钙蛋白酶特异性抑制剂。最终，这些研究可能会导致治疗药物的开发，以改善ARF患者的临床结果。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the events that cause mitochondrial dysfunction in ischemia/reperfusion- and toxicant-induced acute renal failure (ARF), and to identify a therapeutic approach that prevents the mitochondrial dysfunction and reduces ARF. The role of mitochondrial dysfunction and disruption of Ca 2+ homeostasis in renal cell injury and death has been demonstrated in numerous models of ARF and nephrotoxicity. The importance of calpains (Ca2+-activated neutral cysteine proteases) in renal proximal tubule cellular (RPTC) injury and death produced by hypoxia/reoxygenation and toxicants has been shown using calpain inhibitors. In particular, two dissimilar calpain inhibitors not only blocked hypoxia/reoxygenation RPTC death, but also blocked the mitochondrial dysfunction and promoted the recovery of respiration during reoxygenation. These results strongly support a key role for calpains in mitochondrial dysfunction. The above experiments showing calpain inhibitor protection of mitochondrial function in RPTC, suggest that mitochondria may contain a calpain. In a number of diverse preliminary experiments using isolated renal cortical mitochondria (RCM) we have obtained additional evidence of a novel mitochondrial calpain that is responsible for mitochondrial dysfunction. These data resulted in the hypothesis that mitochondrial Ca 2+- uptake leads to the activation of a mitochondrial calpain, which causes the mitochondrial dysfunction and ultimately results in RPTC death and ARF. The specific aims of this application are: Specific Aim I: Identify and characterize the mitochondrial calpain and examine its regulation in isolated RCM and RPTC. Specific Aim II: Elucidate the mechanism of mitochondrial calpain-mediated mitochondrial dysfunction in RPTC and isolated RCM, and identify the mitochondrial protein targets of mitochondrial calpain. Specific Aim III: Determine the effectiveness of currently described calpain inhibitors on mitochondrial calpain and develop new specific inhibitors of mitochondrial calpain using novel, non-natural amino acid analogues and determine their effectiveness in RPTC and isolated RCM. Specific Aim IV: Determine the efficacy of current and/or developed calpain inhibitors in an in vivo model of mitochondrial dysfunction and ARF. Completion of these Specific Aims will add significantly to our basic understanding of cell injury and death, particularly events mediating mitochondrial dysfunction. Further, we will identify a mitochondrial calpain and develop novel calpain inhibitors, including those that are mitochondrial calpain specific. Ultimately, these studies may lead to the development of therapeutic agents that improve clinical outcomes in patients with ARF.