Molecular Regulation of Ischemic Renal Failure

描述(由申请人提供)： 肾脏缺血损伤导致急性肾功能衰竭(ARF)，这是一种与高死亡率相关的主要肾脏疾病。虽然ARF的发展涉及多个因素，并可能分几个阶段进行，但它最终是由对肾小管细胞的亚致死性和致死性损伤所致。我们研究的长期目标是阐明肾小管细胞损伤的凋亡机制，并寻找防治ARF的新策略。在最后一次资助期间，我们和其他人已经表明肾小管细胞凋亡参与了缺血和肾毒性肾损伤。重要的是，这些研究已经证明了Bax/Bak介导的线粒体损伤的关键作用。我们进一步揭示了在肾小管上皮细胞凋亡过程中线粒体的显著形态变化，即线粒体碎裂。抑制线粒体碎裂可防止线粒体损伤和细胞凋亡。尽管有这些发现，关键的问题仍然存在：1)Bax是如何激活的？2)线粒体断裂是如何诱导和调控的？亲内蛋白B1(Endo-B1)最初被酵母双杂交筛选鉴定为Bax相互作用蛋白。我们的初步研究表明，在缺血性肾损伤过程中，Endo-B1早期激活并移位到线粒体。重要的是，敲除Endo-B1可以抑制线粒体碎裂和肾小管细胞的凋亡。本项目的总体目标是阐明Endo-B1对Bax和线粒体形态动力学的调节，并确定Endo-B1在肾缺血损伤时肾小管细胞凋亡中的作用。我们推测，在诱导细胞凋亡时，Endo-B1与Bax相互作用，导致它们移位到线粒体。在线粒体中，Endo-B1与裂变融合蛋白合作，使细胞器碎裂，促进Bax/Bak的寡聚和病理性孔洞的形成，导致凋亡因子的释放。我们提出了三个具体目标来验证这一假说：1)确定Endo-B1在Bax激活中的作用，并分析Endo-B1/Bax在肾细胞凋亡过程中的相互作用；2)描述Endo-B1在肾细胞损伤过程中线粒体碎裂中的作用和调控；3)使用新开发的基因敲除小鼠模型来确定Endo-B1在体内缺血肾损伤中对Bax激活和线粒体碎裂的作用。这项研究的完成有望极大地促进对线粒体在细胞凋亡过程中损伤的机制的理解。此外，它可能导致开发新的预防和治疗缺血性肾功能衰竭的策略。 公共卫生相关性： VA相关性：包括脱水、低血压、感染性休克、创伤和手术动脉阻断在内的多种临床情况都会导致流向肾脏的血流量减少。当肾脏的血液供应不足时，肾组织就会变得缺血，导致细胞损伤、组织损伤和肾功能衰竭。在美国，每年确诊的急性肾功能衰竭(ARF)病例超过20万例，造成数十亿美元的直接医疗费用。退伍军人反映的老龄化人口在缺血和肾毒性条件下极易发生ARF。来自奥斯汀自动化中心患者档案的数据显示，在接受住院治疗的VA患者中，超过3%的人患上了ARF(1999年359,608名患者中有11,187名)。急性呼吸窘迫综合征也是战场上军事伤亡的常见并发症3。特别是，缺血性ARF与高死亡率相关，超过50%。这一应用旨在确定介导缺血性ARF的关键分子因素。虽然是机械性的，但它可能会导致预防和治疗这种毁灭性疾病的新战略，为改善退伍军人的健康做出重大贡献。参考文献：1.Bonventre JV，Weinberg JM：缺血性急性肾功能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最新进展。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：过渡到治疗缺血性急性肾功能衰竭。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。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：1992-2001年间医疗保险受益人中急性肾衰竭的发生率和死亡率。中华肾脏病杂志，2006，17：1135-1142 7.周学军，Rakheja D，Yu X，Saxena R，Vaziri ND，Silva FG：肾脏老化.国际肾联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