CELL PATHOLOGY IN KIDNEY HYPOXIA ROLE OF BAX

描述(改编自《调查者摘要》)：本申请 阐述肾脏细胞死亡的潜在机制，以响应 缺氧。肾缺血是导致急性肾功能衰竭的常见原因 缺血性损伤是由低氧引起的。两人都观察到了损伤 在缺氧期和复氧期，以及一 造成这种损伤的机制可能是产生反应性的 氧气物种。本申请建议研究一种用于 这种损伤集中在Bax的易位上，Bax是一种促进死亡的 从胞浆到线粒体的蛋白质，线粒体进而诱导 将细胞色素c从线粒体释放到胞浆中。这个 主要研究人员显示，损伤的形态是细胞凋亡和 不需要分子氧就能发生损伤。校长 研究人员假设细胞死亡的形态和本质 在细胞色素c从线粒体释放到胞浆后 依赖于ATP的供应。如果ATP可用，则格式为 可能发生的死亡就是细胞凋亡。如果没有ATP，那么就会出现坏死 都会导致。这一假说是基于这样一种认识，即细胞凋亡 这是一个依赖于ATP的过程。线粒体细胞色素c的丢失 通过氧化损伤细胞产生三磷酸腺苷的能力 需要完整的电子传递链的磷酸化 在线粒体中。因此，如果细胞确实有现成的三磷酸腺苷供应 通过保存的糖酵解，细胞凋亡将是细胞的形式 死亡。因此，这一应用的关键要素是研究 Bax移位到线粒体，线粒体的变化 在线粒体膜外膜上诱导的通透性 Bax，细胞色素c释放到胞浆中，以及细胞损伤 随之而来。首席调查员提出了四个具体目标。第一 其中三个利用的是大鼠近端肾小管细胞的细胞培养模型。第一 目的：研究Bax在细胞色素c释放中的作用。 线粒体在低氧中的作用及其诱导机制 渗透率发生变化。这一具体目标将决定要求 Bax与细胞色素c泄漏的相互作用 与细胞死亡相关的特定蛋白质，以及是否改变了 线粒体内膜通透性也存在。第二个具体问题 AIM将确定Bax易位的分子机制 在缺氧过程中从胞浆到线粒体。重点将是 被置于磷酸化、核苷酸结合和 应激蛋白的关联性。BAX的缺失突变体将用于 绘制易位所需的蛋白质序列图。第三个具体目标 将研究抑制Bcl2基因表达的分子机制 缺氧时细胞色素c的释放。这一具体目标将决定 抑制细胞色素释放所需的Bcl-2序列， 与Bax的异二聚化对保护活性的要求 Bcl2与死亡途径相关蛋白的相关性研究 Bcl2对Bax周转的影响第四个具体目标将实现 将对培养细胞的观察扩展到肾小管的研究 显微解剖的大鼠肾和兔近端小管的肾脏。 此外，还将进行缺血再灌注损伤的研究。

DESCRIPTION (Adapted from Investigator's Abstract): This application addresses mechanisms underlying cell death in the kidney in response to hypoxia. Renal ischemia is a common cause for acute renal failure and such ischemia-induced injury is induced by hypoxia. Injury is observed both during the hypoxic phase and during the phase of reoxygenation, and one mechanism that may account for such injury is the generation of reactive oxygen species. The present application proposes to study a mechanism for such injury that is centered on the translocation of Bax, a death-promoting protein from the cytosol to the mitochondria which in turn induces the release of cytochrome c from the mitochondria into the cytosol. The principal investigator shows that the morphology of injury is apoptosis and does not require molecular oxygen for the damage to occur. The principal investigator hypothesizes that the morphology and the nature of cell death following the release of cytochrome c from the mitochondria into the cytosol is dependent upon a supply of ATP. If ATP is available, then the form of death that would occur is apoptosis. If ATP is not available, then necrosis would result. This hypothesis is based on the recognition that apoptosis is an ATP-dependent process. The loss of cytochrome c from the mitochondria impairs the ability of the cell to generate ATP through oxidative phosphorylation which necessitates integrity of the electron transport chain in the mitochondrion. Thus, if the cells do have a ready supply of ATP through preserved glycolysis, then apoptosis would be the form of cellular demise. Thus, the critical elements of this application are the study of translocation of Bax to the mitochondrion, the changes in mitochondrial permeability that are induced in the outer mitochondrial membrane induced by Bax, the release of cytochrome c into the cytosol and the cell injury that ensues. The principal investigator proposes four specific aims. The first three utilize a cell culture model of rat proximal tubular cells. The first specific aim will investigate the role of Bax in cytochrome c release from mitochondria during hypoxia as well as the mechanisms by which it induces the permeability change. This specific aim will determine the requirement of Bax for cytochrome c leak, the nature of the interaction of Bax with specific proteins related to cell death, and whether changes in the mitochondrial inner membrane permeability also occur. The second specific aim will determine the molecular mechanisms underlying Bax translocation from the cytosol to the mitochondria during hypoxia. Emphasis will be placed on the roles played by phosphorylation, nucleotide binding, and the association of the stress proteins. Deletion mutants of Bax will be used to map protein sequences required for translocation. The third specific aim will investigate molecular mechanisms that underlie Bcl-2 inhibition of cytochrome c release during hypoxia. This specific aim will determine the sequences of Bcl-2 that are required to inhibit cytochrome release, the requirement of heterodimerization with Bax for protective activity, the association of Bcl-2 with proteins related to death pathways, and the effects of Bcl-2 on Bax turnover. The fourth specific aim will perform studies to extend the observations made on cultured cells to tubules of the kidney in microdissected rat nephrons and rabbit proximal tubules. Additionally, studies will be conducted in ischemia reperfusion injury.