Junctional complex dysregulation during ischemic injury

缺血性肾组织损伤是许多肾脏疾病患者死亡率和发病率的重要因素。缺血性损伤的一个主要后果是上皮细胞极性的丧失，通过肌动蛋白细胞骨架和细胞-细胞连接复合物（粘附连接和紧密连接）的大规模破坏。连接复合物对于维持正常肾功能所需的极化离子运输系统、受体和酶分布是必要的。atp耗竭可作为肾缺血的体外模型。在这里，我们建议分析在atp耗竭过程中调节紧密连接的机制，并确定这些调节机制如何保护细胞免受损伤或加速恢复。实验将检查粘附连接（钙粘蛋白介导的细胞-细胞粘附）如何调节紧密连接的组装和功能。我们假设ATP耗竭导致紧密连接解体，这是粘附连接解体的结果。通过在上皮细胞中操纵钙粘蛋白连接（表达突变的钙粘蛋白）来检测缺血时钙粘蛋白细胞粘附分子连接，并分析对紧密连接组装和细胞极性的影响。通过粘附连接的信号传导过程也可能在atp耗竭期间中断。本项目要验证的一个主要假设是，在肾缺血期间，上皮细胞中的rho家族gtpase功能被抑制，导致连接复合物的破坏。Rho家族gtpase （Rho， Rac和Cdc42）是Ras基因超家族的成员，并已被证明调节肌动蛋白细胞骨架组装。初步证据表明，rho家族gtpase控制上皮细胞中细胞-细胞连接复合物的组装。这些信号系统的激活也可以保护细胞免受缺血性损伤。我们的研究将为缺血期间被破坏的关键调控机制提供新的和基本的见解。

Ischemic renal tissue damage is a significant factor in the mortality and morbidity of patients suffering from numerous renal disorders. A primary consequence of ischemic injury is the loss of epithelial cell polarity through large-scale disruption of the actin cytoskeleton and cell-cell junctional complexes (adherens junctions and tight junctions). Junctional complexes are necessary for maintenance of polarized ion transport systems, receptors and enzyme distributions required for proper kidney function. ATP-depletion serves as an in vitro model for renal ischemia. Here we propose to analyze mechanisms that regulate tight junctions during ATP-depletion, and determine how these regulator mechanisms may protect cells from damage or accelerate recovery. Experiments will examine how adherens junctions (cadherin-mediated cell-cell adhesion) regulate tight junction assembly and function. We hypothesize that ATP- depletion causes tight junction disassembly as a consequence of adherens junction disassembly. Cadherin cell adhesion molecule junction during ischemia will be examined by manipulating cadherin junction (expressing mutant cadherins) in epithelial cells, and effects on tight junction assembly and on cell polarity will be assayed. Signaling processes leading through the adherens junction may also be disrupted during ATP-depletion. A major hypothesis to be tested in this project is that Rho-family GTPases functions in epithelial cells are inhibited during renal ischemia leading to disruption of junctional complexes. Rho-family GTPases (Rho, Rac and Cdc42) are members of the Ras gene superfamily, and have been shown to regulate actin cytoskeleton assembly. Preliminary evidence suggests that Rho-family GTPases control cell-cell junctional complex assembly in epithelial cells. Activation of these signaling systems may also protect cells from ischemic injury. Our studies will provide new and fundamental insight into critical regulatory mechanisms that are disrupted during ischemia.