CELL PATHOLOGY IN KIDNEY HYPOXIA. ROLE OF BAX

肾脏缺氧的细胞病理学。

• 批准号: 6878602
• 负责人: POTHANA SAIKUMAR
• 金额: $ 31.3万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6878602
• 关键词: BCL2 gene /protein; Bax gene /protein; apoptosis; cellular pathology; cysteine endopeptidases; cytochrome c; electron microscopy; enzyme activity; fluorescence resonance energy transfer; gel mobility shift assay; gene induction /repression; genetic transcription; immunocytochemistry; immunoprecipitation; intracellular transport; laboratory rat; mass spectrometry; membrane permeability; mitochondrial membrane; protein protein interaction; protein sequence; protein structure function; renal ischemia /hypoxia; site directed mutagenesis; tissue /cell culture; two dimensional gel electrophoresis; western blottings

DESCRIPTION (provided by applicant): Loss of tubular epithelium contributes to the development of ischemic acute renal failure. Our long term objective is to elucidate the molecular mechanisms that are involved in hypoxic cell injury and to develop protective strategies. We have shown that Bax, a pro-apoptotic member of the Bcl-2 family, plays a major role in cell injury caused by hypoxia/ATP depletion, and in the apoptosis that develops during reoxygenation/ATP repletion. During hypoxia, Bax translocates from cytosol to mitochondria and forms oligomers. Together with associated Bak oligomers. Bax permeabilizes mitochondria to release cytochrome c (Cyt.c). Cyt.c facilitates the formation of a protein complex, the apoptosome, required for caspase activation and subsequent apoptotic execution. Expression of anti-apoptotic Bcl-2 does not prevent Bax translocation, but abrogates Bax and Bak oligomerization, and thereby Cyt.c leakage. Experiments in vivo showed reciprocal regulation of Bax and Bcl-2 in the kidney during reperfusion after ischemia. Based on these findings, we propose to pursue four specific aims: Aim 1 hypothesizes that Bax dependent cytochrome c release is a two step process involving translocation followed by oligomerization induced by a BH3-only protein. Studies in this aim will use gene silencing, in vitro reconstitution, as well as EM-Immuno histochemistry and FRET techniques to examine the mechanisms of mitochondrial outer membrane permeabilization. Aim 2 hypothesizes that pore structures formed by Bax and Bak contain other mitochondrial outer membrane proteins. These experiments will use immunoprecipitation, 2D-PAGE and mass spectrometry based peptide sequence analysis. Aim 3 hypothesizes differential ATP requirements for sequential stages of the apoptotic pathway. Mechanisms of caspase activation and its inhibition will be studied by immunoblotting, in vitro reconstitution, gene expression and mutagenesis. Aim 4 hypothesizes that Bax expression regulates tubular injury in the kidney. Transcriptional regulation of Bax promoter during ischemia and reperfusion will be studied using mobility shift assays, and in vitro foot-printing. In vivo gene silencing of Bax will be attempted in efforts to block up regulation of Bax and thereby prevent cell injury in the ischemic kidney.

描述（由申请人提供）：肾小管上皮的丧失导致缺血性急性肾衰竭的发生。我们的长期目标是阐明缺氧细胞损伤的分子机制并制定保护策略。我们已经证明，Bax 是 Bcl-2 家族的促凋亡成员，在缺氧/ATP 耗竭引起的细胞损伤以及复氧/ATP 补充过程中发生的细胞凋亡中发挥着重要作用。在缺氧期间，Bax 从细胞质转移到线粒体并形成寡聚体。与相关的 Bak 低聚物一起。 Bax 透化线粒体以释放细胞色素 c (Cyt.c)。 Cyt.c 促进蛋白质复合物（凋亡体）的形成，这是 caspase 激活和随后的细胞凋亡执行所需的。抗凋亡 Bcl-2 的表达不会阻止 Bax 易位，但会消除 Bax 和 Bak 寡聚化，从而消除 Cyt.c 渗漏。体内实验显示缺血后再灌注期间肾脏中 Bax 和 Bcl-2 的相互调节。基于这些发现，我们建议追求四个具体目标： 目标 1 假设 Bax 依赖性细胞色素 c 释放是一个两步过程，涉及易位，然后是仅 BH3 蛋白诱导的寡聚化。该目标的研究将使用基因沉默、体外重建以及电磁免疫组织化学和 FRET 技术来检查线粒体外膜透化的机制。目标 2 假设 Bax 和 Bak 形成的孔结构含有其他线粒体外膜蛋白。这些实验将使用基于免疫沉淀、2D-PAGE 和质谱的肽序列分析。目标 3 假设细胞凋亡途径的连续阶段对 ATP 的需求存在差异。将通过免疫印迹、体外重建、基因表达和诱变来研究半胱天冬酶激活及其抑制的机制。目标 4 假设 Bax 表达调节肾脏肾小管损伤。将使用迁移率变化测定和体外足迹来研究缺血和再灌注期间 Bax 启动子的转录调节。我们将尝试体内基因沉默 Bax，以阻止 Bax 的调节，从而防止缺血肾脏中的细胞损伤。