Role of Myosin II in Ischemic Acute Renal Failure

肌球蛋白 II 在缺血性急性肾衰竭中的作用

• 批准号: 6686317
• 负责人: Timothy Alan Sutton
• 金额: $ 12.44万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-15 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6686317
• 关键词: MDCK cell; actins; acute renal failure; adenosine triphosphate; biological signal transduction; cell adhesion; cellular pathology; confocal scanning microscopy; guanine nucleotide binding protein; guanosinetriphosphatases; immunofluorescence technique; microfilaments; molecular pathology; myosin light chain kinase; myosins; phosphorylation; renal ischemia /hypoxia

DESCRIPTION (provided by applicant) The overall mortality rate for patients with acute renal failure (ARF) has remained 40-50% for the past 30 years. The associated annual cost of ARF is over $12 billion. Ischemia is the leading cause of ARF and alterations in the actin cytoskeleton of renal tubular cells during ischemia and recovery have important consequences for kidney function. The long-term goal of this project is to understand how myosin Il (MII) and the Rho GTPases interact to regulate protective and reparative alterations in the actin cytoskeleton of renal tubular cells during ischemia and recovery. The general strategy is to study ATP depletion as a reversible cell culture model of ischemia that has been shown to recapitulate alterations in the actin cytoskeleton observed from in vivo studies of ischemia. Mutant proteins will be used as molecular probes to conduct studies not possible in animals or humans. The proposed experiments will test the hypothesis that MII is an important distal effector of the Rho regulatory cascade and plays a critical role in regulating the actin cytoskeleton in renal tubular cells during ischemia and recovery. Grant funding and extended training in cell biology is a major factor in the PI's immediate goal of attaining research independence and the long-term career goal of becoming a successful and productive investigator in an academic institute. The specific aims of this present study are as follows: 1) Determine the effects of altering MII activation on the actin cytoskeleton and cell adhesion during cellular ATP depletion and recovery. Mutant forms of myosin Iight chain kinase (MLCK) will be expressed in MDCK cells to alter the activation state of myosin II. In all specific aims, immunofluorescence confocal microscopy will be utilized to examine actin and MII distribution as well as structures important for cell-matrix adhesion. Standard assays will be used to examine MII activation and cell adhesion. 2) Determine the effects on the actin cytoskeleton of altering MII activation while inversely altering Rho signaling during cellular ATP depletion and recovery. Mutant forms of MLCK and Rho proteins will be co-expressed in MDCK cells to alter cellular signaling. 3) Determine the effects on the actin cytoskeleton of altering Rho-kinase (ROCK) signaling while inversely altering Rho signaling during cellular ATP depletion and recovery. Mutant forms of ROCK and Rho proteins will be co-expressed in MDCK cells to alter cellular signaling. 4) Determine the effects on the actin cytoskeleton of altering MII activation while inversely altering ROCK signaling during cellular ATP depletion and recovery. Mutant forms of MLCK and ROCK proteins will be co-expressed in MDCK cells to alter cellular signaling.

描述（由申请人提供） 急性肾衰竭（ARF）患者的总死亡率已有 在过去的30年中，仍保持40-50％。相关的ARF年度成本是 超过120亿美元。缺血是ARF的主要原因和改变 缺血期间肾小管细胞的肌动蛋白细胞骨架和恢复 肾功能的重要后果。该项目的长期目标 是了解肌球蛋白IL（MII）和Rho GTPases如何相互作用以调节 肾脏肌动蛋白细胞骨架的保护性和修复性改变 缺血和恢复期间的管状细胞。一般策略是研究 ATP耗竭是一种可逆的缺血细胞培养模型 显示以概括从In观察到的肌动蛋白细胞骨架的改变 缺血的体内研究。突变蛋白将用作分子探针 在动物或人类中进行研究。提出的实验 将测试MII是RHO的重要远端效应子的假设 监管级联反应，在调节肌动蛋白中起关键作用 缺血和恢复期间肾小管细胞中的细胞骨架。 赠款资金和细胞生物学的扩展培训是 PI获得研究独立性和长期职业的直接目标 成为学术界成功和富有成效的研究员的目标 研究所。 本研究的具体目的如下：1）确定 改变MII激活对肌动蛋白细胞骨架和细胞粘附的影响 在细胞ATP耗竭和恢复期间。肌球蛋白链的突变形式 激酶（MLCK）将在MDCK细胞中表达，以改变 肌球蛋白II。在所有具体目的中，免疫荧光共聚焦显微镜将是 用于检查肌动蛋白和MII分布以及重要的结构 用于细胞 - 矩阵粘附。标准测定将用于检查MII 激活和细胞粘附。 2）确定对肌动蛋白的影响 改变MII激活的细胞骨架，而反向改变Rho信号传导 在细胞ATP耗竭和恢复期间。 MLCK和RHO的突变形式 蛋白质将在MDCK细胞中共表达以改变细胞信号传导。 3） 确定对改变Rho-激酶（岩石）的肌动蛋白细胞骨架的影响 信号传导，而在细胞ATP耗竭期间反向改变RHO信号传导 和恢复。岩石和Rho蛋白的突变形式将共表达 MDCK细胞改变细胞信号传导。 4）确定对肌动蛋白的影响 改变MII激活的细胞骨架，而反向改变岩石信号传导 在细胞ATP耗竭和恢复期间。 MLCK和岩石的突变形式 蛋白质将在MDCK细胞中共表达以改变细胞信号传导。