MEMBRANE PATHOLOGY IN RENAL CELL INJURY

肾细胞损伤中的膜病理学

• 批准号: 3235901
• 负责人: MANJERI A VENKATACHALAM
• 金额: $ 26.08万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235901
• 关键词: adenosine triphosphate; alanine; animal tissue; atomic absorption spectrometry; bioenergetics; brush border membrane; calcium flux; cell death; cellular pathology; electron microscopy; epithelium; fatty acid metabolism; fluorescent dye /probe; glycine; kidney cell; kidney disorder; kidney pharmacology; lipid metabolism; membrane lipids; membrane reconstitution /synthesis; membrane structure; mitochondrial membrane; phospholipids; radiotracer; renal ischemia /hypoxia

Research proposed in this application aims to unravel the complex mechanisms which underlie the molecular pathogenesis of lethal membrane damage during cell injury due to depletion of adenosine triphosphate (ATP). The investigations will use a comprehensive approach covering the metabolic, lipid biochemical and structural aspects of cellular pathology caused by energy depletion. The major thrust of the effort will be to study how catabolism of membrane lipids relates to membrane damage. The experimental design hinges on the use of inhibitory interventions which retard specific aspects of the injury process. A restricted set of small amino acids (glycine, alanine and their analogs) powerfully inhibit the cellular degeneration which occurs during energy deprivation in a manner unrelated to their metabolism (FASEB J, in press Dec 1990). Use of these protective amino acids in conjunction with techniques to "clamp" cytosolic Ca++ at fixed levels has led to the development of experimental models appropriate for critical examination of the role played by Ca++ dependent and Ca++ independent mechanisms of cell damage. One mechanism, triggered by Ca++, damages the mitochondria and the plasma membrane, and is accompanied by polyphosphoinositide hydrolysis. The other is independent of calcium, primarily damages nonmitochondrial intracellular membranes, and appears to be lethal in an unknown fashion. Prevention of intracellular Ca++ increase inhibits the first process, and protective amino acids inhibit the second. The amino acids also have an overriding protective role and retard cell death by either mechanism. Selective or simultaneous manipulations of these two factors were found to specifically and uniquely alter the structural patterns of injury. Using these well-established protocols, we will address the roles played by phospholipid hydrolysis, unesterified fatty acid release, and fatty acid cytotoxicity in the injury process. Firstly, we will ask if the protective amino acids cause decreased hydrolysis of phospholipids and decreased release of unesterified fatty acids and thereby ameliorate membrane damage. Secondly, we will also investigate whether the protective amino acids promote the action of fatty acid binding proteins to bind and thereby segregate fatty acids away from membranes and other important proteins. Thirdly, since our experiments indicate that Ca++ dependent and amino acid responsive mechanisms of injury are associated with clearly identified cellular compartments, we will determine the structural and biochemical basis for such effects. These investigations will target the catabolism of phospholipids which are enriched in organelles such as mitochondria and the brush border membrane. In related experiments, we will also use fluorescent probes to label specific membrane compartments in living cells, and trace the sequence of membrane disintegration during injury, with emphasis on how inhibitory interventions modulate these events.

本应用程序中提出的研究旨在揭开复合物 致命膜分子发病机理的基础的机制 三磷酸腺苷（ATP）导致细胞损伤期间的损伤。 调查将采用涵盖该方法的全面方法 细胞病理的代谢，脂质生化和结构方面 由能量耗尽引起。 这项努力的主要力量将是 研究膜脂质的分解代谢与膜损伤如何关系。 这 实验设计取决于使用抑制性干预措施 延迟伤害过程的特定方面。 一组限制的小型 氨基酸（甘氨酸，丙氨酸及其类似物）有力抑制 细胞变性以某种方式发生在能量剥夺期间 与它们的新陈代谢无关（Faseb J，在1990年12月出版社）。 使用这些 保护性氨基酸与“夹具”胞质的技术结合 固定水平的Ca ++导致了实验模型的发展 适合对Ca ++依赖的作用进行批判性检查 和CA ++独立的细胞损伤机制。 一种机制，触发 由Ca ++，损坏线粒体和质膜，是 伴有多磷酸肌醇水解。 另一个是独立的 钙，主要损害非蒙蒙膜软骨内膜和 似乎以未知的方式致命。 预防细胞内 Ca ++增加抑制了第一个过程和保护性氨基酸 抑制第二个。 氨基酸还具有压倒性的保护性 两种机制的角色和延迟细胞死亡。 选择性或同时 发现这两个因素的操纵是特定而独特的 改变损伤的结构模式。 使用这些已建立的 协议，我们将解决磷脂水解扮演的角色， 损伤中未酯化的脂肪酸释放和脂肪酸细胞毒性 过程。 首先，我们将询问保护性氨基酸是否引起 磷脂的水解减少，未酯化的释放减少 脂肪酸，从而改善膜损伤。 其次，我们也会 研究保护性氨基酸是否促进脂肪的作用 酸结合蛋白结合，从而将脂肪酸从 膜和其他重要蛋白质。 第三，由于我们的实验 表明损伤的CA ++依赖性和氨基酸反应性机制 与清晰识别的细胞室有关，我们将 确定此类作用的结构和生化基础。 这些 研究将针对磷脂的分解代谢 富含线粒体和刷边膜等细胞器。 在相关实验中，我们还将使用荧光探针标记 活细胞中的特定膜室，并追踪 受伤期间的膜崩解，重点是抑制 干预措施调节这些事件。