CELL PATHOLOGY IN KIDNEY HYPOXIA. ROLE OF BAX

描述（由申请人提供）：小管上皮的丧失有助于缺血性急性肾功能衰竭的发展。我们的长期目标是阐明参与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耗尽引起的细胞损伤以及再氧化/ATP补充过程中发生的细胞凋亡中起主要作用。在缺氧时，Bax从细胞质转移到线粒体并形成低聚物。连同伴生的Bak低聚物。Bax渗透线粒体释放细胞色素c （Cyt.c）。Cyt.c促进一种蛋白质复合物的形成，即凋亡体，这是caspase激活和随后的凋亡执行所必需的。抗凋亡Bcl-2的表达不能阻止Bax的易位，但可以消除Bax和Bak的寡聚，从而导致Cyt.c的渗漏。体内实验显示肾缺血再灌注过程中Bax和Bcl-2的相互调节。基于这些发现，我们提出了四个具体目标：目的1假设Bax依赖的细胞色素c释放是一个两步过程，包括易位和BH3-only蛋白诱导的寡聚化。在这方面的研究将使用基因沉默，体外重建，以及em -免疫组织化学和FRET技术来检查线粒体外膜渗透的机制。目的2假设Bax和Bak形成的孔结构含有其他线粒体外膜蛋白。这些实验将使用免疫沉淀，2D-PAGE和基于质谱的肽序列分析。目的3假设凋亡途径的顺序阶段对ATP的不同需求。caspase的激活和抑制机制将通过免疫印迹、体外重组、基因表达和诱变来研究。目的4假设Bax表达调节肾小管损伤。Bax启动子在缺血和再灌注期间的转录调控将通过迁移迁移实验和体外足迹实验进行研究。在体内将Bax基因沉默，试图阻断Bax的调控，从而防止缺血肾细胞损伤。

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.