Molecular Regulation of Ischemic Renal Failure

描述（由申请人提供）： 缺血性肾损伤导致急性肾衰竭（ARF），这是一种与高死亡率相关的主要肾脏疾病。虽然ARF的发展涉及多种因素，并可能在几个阶段进行，但它最终是由肾小管细胞的亚致死性和致死性损伤引起的。本研究的长期目标是阐明肾小管细胞损伤的凋亡机制，并确定预防和治疗ARF的新策略。在上一个资助期间，我们和其他人已经表明肾小管细胞凋亡参与缺血性和肾毒性肾损伤。重要的是，这些研究已经证明了Bax/巴克介导的线粒体损伤的关键作用。我们进一步揭示了肾小管细胞凋亡过程中线粒体的显著形态学变化，即线粒体片段化。抑制线粒体断裂可防止线粒体损伤和细胞凋亡。尽管有这些发现，关键问题仍然存在：1）Bax是如何激活的？2)线粒体断裂是如何诱导和调节的？Endophilin B1（Endo-B1）最初是通过酵母双杂交筛选鉴定出的一种与β-淀粉样蛋白相互作用的蛋白质。我们的初步研究表明，在缺血性肾损伤早期，Endo- B1激活并移位到线粒体。重要的是，敲低Endo-B1抑制线粒体片段化和肾小管细胞凋亡。本课题的总体目标是阐明Endo-B1对Bax和线粒体形态学动力学的调控，并确定Endo-B1在缺血性肾损伤中肾小管细胞凋亡中的作用。我们推测，在细胞凋亡诱导，内皮B1与Bax相互作用，导致其易位到线粒体。在线粒体中，Endo-B1与分裂融合蛋白协同作用，使细胞器破碎，促进Bax/巴克寡聚化和病理性孔的形成，导致凋亡因子的释放。我们提出了三个具体的目的来验证这一假设：1）确定内皮素B1在Bax激活中的作用，并分析内皮素B1/Bax在肾细胞凋亡中的相互作用; 2）阐明内皮素B1在肾细胞损伤中线粒体断裂中的作用和调节;和3）使用新开发的基因敲除小鼠模型来确定Endo-B1在体内缺血性肾损伤中Bax活化和线粒体断裂中的作用该研究的完成有望显着推进对细胞凋亡过程中线粒体损伤机制的理解。此外，它可能导致缺血性肾衰竭的预防和治疗的新策略的发展。

DESCRIPTION (provided by applicant): Kidney injury by ischemia leads to acute renal failure (ARF), a major kidney disease associated with high mortality. While the development of ARF involves multiple factors and may proceed in several phases, it is ultimately precipitated by sublethal and lethal damage to renal tubular cells. The long-term goal of our research is to delineate the apoptotic mechanism of tubular cell injury and identify novel strategies for the prevention and treatment of ARF. During the last grant period, we and others have shown the involvement of tubular cell apoptosis in ischemic and nephrotoxic renal injury. Importantly, these studies have demonstrated a pivotal role for Bax/Bak- mediated mitochondrial damage. We have further revealed a striking morphological change of mitochondria during tubular cell apoptosis, i.e. mitochondrial fragmentation. Inhibition of mitochondrial fragmentation prevents mitochondrial damage and apoptosis. Despite these findings, critical questions remain: 1) how is Bax activated? 2) how is mitochondrial fragmentation induced and regulated? Endophilin B1 (Endo-B1) was originally identified as a Bax-interacting protein by yeast two hybrid screening. Our preliminary studies have demonstrated an early Endo- B1 activation and translocation to mitochondria during ischemic renal injury. Importantly, knockdown of Endo-B1 suppresses mitochondrial fragmentation and tubular cell apoptosis. The overall objective of this project is to elucidate the regulation of Bax and mitochondrial morphological dynamics by Endo-B1, and determine the role of Endo-B1 in tubular cell apoptosis during ischemic renal injury. We hypothesize that upon apoptosis induction, Endo-B1 interacts with Bax, leading to their translocation to mitochondria. In mitochondria, Endo-B1 collaborates with fission-fusion proteins to fragment the organelles, facilitating Bax/Bak oligomerization and formation of pathological pores, resulting in the release of apoptotic factors. We propose three Specific Aims to test this hypothesis: 1) determine the role of Endo-B1 in Bax activation and analyze Endo-B1/Bax interactions during renal cell apoptosis; 2) delineate the role and regulation of Endo-B1 in mitochondrial fragmentation during renal cell injury; and 3) use a newly developed knockout mouse model to determine the role of Endo-B1 in Bax activation and mitochondrial fragmentation during ischemic renal injury in vivo. Completion of the research is expected to significantly advance the mechanistic understanding of mitochondrial injury during apoptosis. In addition, it may lead to the development of novel strategies for the prevention and treatment of ischemic renal failure.