CELL PATHOLOGY IN KIDNEY HYPOXIA. ROLE OF BAX

描述(由申请者提供)：肾小管上皮细胞的缺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与缺氧细胞损伤有关的分子机制，并开发保护策略。我们已经证明，Bax是Bcl2家族中的一个促凋亡成员，在缺氧/ATP耗竭引起的细胞损伤以及在复氧/ATP再耗尽过程中发展的细胞凋亡中发挥着重要作用。在缺氧过程中，Bax从胞浆移位到线粒体，形成寡聚体。以及相关的BAK低聚物。Bax使线粒体通透性，释放细胞色素c(Cyt.c)。Cyt.c促进caspase激活和随后的凋亡执行所需的蛋白质复合体--凋亡体的形成。抗凋亡基因Bcl2的表达不能阻止Bax易位，但可以阻止Bax和Bak的寡聚化，从而导致Cyt.c的泄漏。体内实验表明，在缺血再灌流过程中，肾脏中Bax和Bcl2的表达相互调节。基于这些发现，我们建议追求四个特定的目标：目的1假设Bax依赖的细胞色素c的释放是一个两步过程，涉及转位，随后是BH3蛋白诱导的寡聚。这一目标的研究将利用基因沉默、体外重组以及EM免疫组织化学和FRET技术来研究线粒体外膜通透的机制。目的2假设Bax和Bak形成的孔结构中含有其他线粒体外膜蛋白。这些实验将使用免疫沉淀、2D-PAGE和基于质谱学的多肽序列分析。目的3假设细胞凋亡途径的不同阶段对ATP的不同需求。通过免疫印迹、体外重组、基因表达和诱变等方法研究caspase的激活和抑制机制。目的4假设Bax基因表达调控肾小管损伤。将使用迁移率改变分析和体外足迹法来研究Bax启动子在缺血和再灌注期间的转录调控。在体内，Bax的基因沉默将被尝试阻止Bax的调节，从而防止缺血肾脏的细胞损伤。

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.