CELL PATHOLOGY IN KIDNEY HYPOXIA ROLE OF BAX

肾脏缺氧的细胞病理学 BAX 的作用

• 批准号: 6178087
• 负责人: POTHANA SAIKUMAR
• 金额: $ 15.04万
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178087
• 关键词: BCL2 gene /protein; animal tissue; apoptosis; cell death; cellular pathology; cytochrome c; cytoplasm; gene deletion mutation; intracellular membranes; intracellular transport; kidney cell; membrane permeability; mitochondria; mitochondrial membrane; necrosis; nucleotides; oxidative phosphorylation; protein sequence; renal ischemia /hypoxia; stress proteins; tissue /cell culture

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.

描述（改编自研究者摘要）：本申请 解决肾脏细胞死亡的潜在机制 缺氧。 肾缺血是急性肾功能衰竭等的常见原因 缺血性损伤是由缺氧引起的。 观察到双方均受伤 在缺氧阶段和复氧阶段，以及一 可能解释这种损伤的机制是反应性的产生 氧物种。 本申请提出研究一种机制 这种损伤以 Bax 的易位为中心，Bax 是一种促进死亡的物质。 蛋白质从细胞质转移到线粒体，进而诱导 细胞色素c从线粒体释放到细胞质中。 这 主要研究者表明损伤的形态是细胞凋亡 不需要分子氧就能发生损害。 校长 研究人员假设细胞死亡的形态和性质 细胞色素 c 从线粒体释放到细胞质中后 取决于 ATP 的供应。 如果 ATP 可用，则形式为 发生的死亡就是细胞凋亡。 如果 ATP 不可用，则坏死 会产生结果。 该假说基于以下认识：细胞凋亡 依赖于 ATP 的过程。 线粒体中细胞色素 c 的损失 损害细胞通过氧化产生 ATP 的能力 磷酸化需要电子传递链的完整性 在线粒体中。 因此，如果细胞确实有现成的 ATP 供应 通过保留的糖酵解，细胞凋亡将成为细胞的形式 灭亡。 因此，该应用的关键要素是研究 Bax 易位至线粒体，线粒体的变化 线粒体外膜诱导的通透性 Bax，细胞色素 c 释放到细胞质中并导致细胞损伤 随后发生。 主要研究者提出了四个具体目标。 第一个 三个利用大鼠近端肾小管细胞的细胞培养模型。 第一个 具体目标是研究 Bax 在细胞色素 c 释放中的作用 缺氧期间线粒体及其诱导机制 渗透率变化。 这个具体目标将决定要求 Bax 的细胞色素 c 泄漏，Bax 与细胞色素 c 的相互作用的性质 与细胞死亡相关的特定蛋白质，以及是否发生变化 线粒体内膜通透性也会发生。 第二具体 目的将确定 Bax 易位的分子机制 缺氧时从细胞质到线粒体。 重点将是 放在磷酸化、核苷酸结合和 应激蛋白的关联。 Bax 的缺失突变体将用于 绘制易位所需的蛋白质序列。 第三个具体目标 将研究 Bcl-2 抑制的分子机制 缺氧时细胞色素c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 异二聚化以实现保护活性的要求， Bcl-2 与死亡途径相关蛋白的关联 Bcl-2 对 Bax 周转的影响。 第四个具体目标将实现 将培养细胞的观察扩展到小管的研究 显微解剖的大鼠肾单位和兔近端小管中的肾脏。 此外，还将进行缺血再灌注损伤的研究。