RHO GTPASES AND ACTIN FUNCTION IN RENAL ISCHEMIA

缺血性急性肾功能衰竭是发病率和死亡率的主要原因。肾脏近端小管的细胞特别容易受到缺血性损伤，这种损伤的特征是正常细胞结构的破坏，从而导致表面蛋白极化分布的丧失。这种细胞结构和功能的破坏与肌动蛋白细胞骨架成分的快速和可逆的重新分布相关。我们认为这种肌动蛋白细丝的异常分布是Rho家族GTP酶失活的结果。Rho蛋白是调节细胞形态和运动的ras超家族的一个分支，其在成纤维细胞中控制肌动蛋白功能的作用已被广泛研究，但在上皮细胞中的特征较少。我们提出了三个特定的目标来测试Rho蛋白及其效应物在缺血细胞培养模型中参与肌动蛋白重组的作用，该模型使用底物耗竭和抗霉素A处理引起的ATP耗竭。我们将利用显微注射和转染法将Rho、Rac和CDc42的显性活性突变体和显性负性突变体导入LLC-PK细胞，以检测通过这些蛋白阻断失活或激活的通路对肌动蛋白细胞骨架对ATP耗竭或恢复的影响。我们将通过测量与GTP酶结合的GTP/GDP的比率，并使用GFP标记的蛋白来分析GTP酶的细胞定位，来测量Rho家族GTP酶在ATP耗竭和恢复条件下的激活状态。肌球蛋白II的胞浆亚型在极化上皮细胞肌动蛋白细胞骨架的功能中起重要作用，是Rho调控的重要靶点。我们将研究肌球蛋白在对照细胞中的定位和轻链磷酸化，以及对ATP耗竭和Ezrin调节的响应，并将使用显性负性和结构性活性突变体来确定肌球蛋白活性调节剂Rho-Kinase和Myosin Light Chain Kinase(MLCK)的作用。从拟议的研究中获得的数据将为理解细胞骨架对缺血的异常反应的细胞机制提供基础，并为生长因子的已知有益影响提供机制。这将为开发改进的治疗方法来管理人类缺血性急性肾功能衰竭提供基础。

Ischemic acute renal failure is a major cause of morbidity and mortality. Cells of the kidney proximal tubule are particularly vulnerable to ischemic injury, and this injury is characterized by breakdown of normal cellular architecture with consequent loss of polarized distribution of surface proteins. This disruption of cell structure and function correlates with rapid and reversible redistribution of components of the actin cytoskeleton. We propose that this abnormal distribution of actin filaments results from inactivation of rho family GTPases. Rho proteins are the branch of the ras superfamily of small GTPases that regulate cell morphology and motility, and their role in controlling actin function has been extensively studied in fibroblasts, but is less well characterized in epithelial cells. We propose three specific aims to test the involvement of rho proteins and their effectors in actin reorganization in a cell culture model of ischemia, using ATP depletion induced by substrate depletion and antimycin A treatment. We will use microinjection and transfection to introduce dominant active and dominant negative mutants of Rho, Rac and Cdc42 into LLC-PK cells to test the effect of blocking inactivation or activation of pathways through these proteins on the actin cytoskeletal response to ATP depletion or recovery. We will measure the activation state of rho family GTPases under conditions of ATP depletion and recovery by measuring the ratio of GTP:GDP bound to the GTPase, and analysing cellular localization of the GTPase using GFP-tagged proteins. Cytoplasmic isoforms of myosin II play an important role in function of the actin cytoskeleton in polarized epithelia, and are an important target of Rho regulation. We will examine myosin localization and light chain phosphorylation in control cells and in response to ATP depletion, and ezrin regulation, and we will determine the effect of regulators of myosin activity, Rho-kinase and myosin light chain kinase (MLCK), using dominant negative and constitutively active mutants. The data derived from the proposed studies will provide a basis for understanding the cellular mechanisms that underlie the abnormal response of the cytoskeleton to ischemia, and a mechanism for the known beneficial effects of growth factors. This will provide a basis for the development of improved therapeutic approaches to the management of human ischemic acute renal failure.