MITOCHONDRIAL CALPAIN MEDIATED RENAL CELL DEATH

描述(申请人提供)：该项目的长期目标是阐明在缺血/再灌流和毒物诱导的急性肾功能衰竭(ARF)中导致线粒体功能障碍的事件，并确定防止线粒体功能障碍和减少ARF的治疗方法。在许多ARF和肾毒性模型中，线粒体功能障碍和钙稳态的破坏在肾细胞损伤和死亡中的作用已被证明。钙激活的中性半胱氨酸蛋白酶在肾近端小管细胞(RPTC)缺氧/复氧和中毒所致的损伤和死亡中的重要性已被应用于钙激活的中性半胱氨酸蛋白酶抑制剂。尤其是两种不同的钙蛋白酶抑制剂，不仅可以阻断缺氧/复氧RPTC的死亡，还可以阻断线粒体功能障碍，促进复氧过程中呼吸的恢复。这些结果有力地支持了钙调蛋白在线粒体功能障碍中的关键作用。上述实验表明，Calain抑制剂对RPTC线粒体功能具有保护作用，提示线粒体中可能含有Calain。在使用分离的肾皮质线粒体(RCM)进行的一些不同的初步实验中，我们获得了新的线粒体钙蛋白的额外证据，该酶与线粒体功能障碍有关。这些数据导致假设线粒体钙摄取导致线粒体钙蛋白的激活，从而导致线粒体功能障碍，最终导致RPTC死亡和ARF。这一应用的具体目的是：特定目的I：鉴定和鉴定线粒体钙蛋白酶，并检测其在分离的RCM和RPTC中的调控。特定目的II：阐明线粒体Calain介导的RPTC和RCM线粒体功能障碍的机制，并确定线粒体Calain的蛋白靶点。具体目的III：确定目前已描述的Calain抑制剂对线粒体Calain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Calain特异性抑制剂，并确定其在RPTC和分离的RCM中的有效性。具体目的IV：在线粒体功能障碍和ARF的体内模型中，确定当前和/或开发的钙蛋白酶抑制剂的有效性。完成这些特定的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的抑制剂。最终，这些研究可能会导致治疗药物的开发，从而改善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.