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预计将显着推进对细胞凋亡期间线粒体损伤机制的了解。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。 公共卫生相关性： VA相关性：包括脱水、低血压、败血性休克、创伤和动脉夹闭手术在内的广泛临床状况导致流向肾脏的血流减少。当肾脏的血液供应不足时，肾组织变得缺血，导致细胞损伤、组织损伤和肾衰竭1 -4。在美国，每年诊断出超过200，000例急性肾衰竭（ARF），导致数十亿美元的直接医疗费用。老龄化人群，反映了退伍军人，是非常容易受到ARF条件下缺血和肾毒性5 -7。来自奥斯汀自动化中心患者档案的数据显示，超过3%接受住院治疗的VA患者发生ARF（1999年359，608例患者中有11，187例）。急性肾衰竭也是战场上军人伤亡的常见并发症。特别是缺血性ARF与超过50%的高死亡率相关。本申请旨在鉴定介导缺血性ARF的关键分子因子。虽然是机械的，它可能会导致新的战略，预防和治疗这种毁灭性的疾病，大大有助于改善退伍军人的健康。参考文献：1. Bonventre JV，温伯格JM：缺血性急性肾衰竭病理生理学的最新进展。J Am Soc Nephrol 2003，14：2199-2210 2. Devarajan P：缺血性急性肾损伤机制的更新。J Am Soc Nephrol 2006，17：1503-1520 3. Schrier RW，Wang W，Poole B，Mitra A：急性肾功能衰竭：定义、诊断、发病机制和治疗。J Clin Invest 2004，114：5-14 4. Molitoris BA：缺血性急性肾功能衰竭的过渡治疗。J Am Soc Nephrol 2003，14：265-267 5.施密特R，坎特利LG：衰老对肾脏修复的影响。美国肾脏生理学杂志2008，294：F1265-1272 6。Xue JL，Daniels F，星星RA，Kimmel PL，Eggers PW，Molitoris BA，Himmelfarb J，柯林斯AJ：1992 - 2001年医疗保险受益人急性肾功能衰竭的发病率和死亡率。J Am Soc Nephrol 2006，17：1135-1142 7. Zhou XJ，Rakheja D，Yu X，Saxena R，Vaziri ND，Silva FG：The aging kidney.肾脏国际2008，74：710-720

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. PUBLIC HEALTH RELEVANCE: VA RELEVANCE: A broad range of clinical conditions including dehydration, hypotension, septic shock, trauma and operative arterial clamping leads to the reduction of blood flow to the kidneys. When blood supply to the kidneys is inadequate, renal tissues become ischemic, resulting in cell injury, tissue damage, and renal failure1-4. In the United States, over 200,000 cases of acute renal failure (ARF) are diagnosed each year, resulting in a direct medical expense in billions of dollars. Aging population, mirrored by veterans, is highly susceptible to ARF under conditions of ischemia and nephrotoxicity5-7. Data from the Austin Automation Center Patient Files show that over 3% of VA patients receiving in-patient care develop ARF (11,187 out of 359,608 patients in year 1999). ARF is also a common complication among military casualties in battle- fields3. Particularly, ischemic ARF is associated with high mortality, over 50%. This application aims at the identification of the key molecular factors that mediate ischemic ARF. While being mechanistic, it may lead to novel strategies for the prevention and treatment of this devastating disease, contributing significantly to the improvement of veterans' health. References: 1. Bonventre JV, Weinberg JM: Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol 2003, 14:2199-2210 2. Devarajan P: Update on mechanisms of ischemic acute kidney injury. J Am Soc Nephrol 2006, 17:1503-1520 3. Schrier RW, Wang W, Poole B, Mitra A: Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. J Clin Invest 2004, 114:5-14 4. Molitoris BA: Transitioning to therapy in ischemic acute renal failure. J Am Soc Nephrol 2003, 14:265-267 5. Schmitt R, Cantley LG: The impact of aging on kidney repair. Am J Physiol Renal Physiol 2008, 294:F1265-1272 6. Xue JL, Daniels F, Star RA, Kimmel PL, Eggers PW, Molitoris BA, Himmelfarb J, Collins AJ: Incidence and mortality of acute renal failure in Medicare beneficiaries, 1992 to 2001. J Am Soc Nephrol 2006, 17:1135-1142 7. Zhou XJ, Rakheja D, Yu X, Saxena R, Vaziri ND, Silva FG: The aging kidney. Kidney Int 2008, 74:710-720