RHO GTPASES AND ACTIN FUNCTION IN RENAL ISCHEMIA

缺血性急性肾衰竭是发病率和死亡率的主要原因。 肾近端小管的细胞特别容易受到缺血性损伤，并且这种损伤的特征在于正常细胞结构的破坏，随之而来的是表面蛋白的极化分布的丧失。 这种细胞结构和功能的破坏与肌动蛋白细胞骨架组分的快速和可逆的重新分布相关。 我们认为这种肌动蛋白丝的异常分布是由于rho家族GTPases失活所致。Rho蛋白是调节细胞形态和运动性的小GTP酶的ras超家族的分支，其在控制肌动蛋白功能中的作用已在成纤维细胞中被广泛研究，但在上皮细胞中的特征较少。 我们提出了三个具体的目标，以测试参与的rho蛋白及其效应物在肌动蛋白重组的细胞培养模型缺血，使用ATP耗竭诱导的底物耗竭和抗霉素A治疗。 我们将使用显微注射和转染引入显性活性和显性负性突变体的Rho，Rac和Cdc 42到LLC-PK细胞中，以测试通过这些蛋白质对肌动蛋白细胞骨架响应ATP消耗或恢复的阻断失活或激活途径的影响。 我们将通过测量与GTP酶结合的GTP：GDP的比率，并使用GFP标记的蛋白质分析GTP酶的细胞定位，来测量在ATP耗尽和恢复的条件下rho家族GTP酶的活化状态。 肌球蛋白II的胞浆异构体在极化上皮细胞的肌动蛋白细胞骨架的功能中起重要作用，并且是Rho调节的重要靶点。 我们将检查肌球蛋白的本地化和轻链磷酸化在控制细胞和ATP耗竭，和ezrin调节，我们将确定肌球蛋白活性，Rho激酶和肌球蛋白轻链激酶（MLCK）的调节剂的影响，使用显性负性和组成型活性突变体。 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以及生长因子已知有益作用的机制提供基础。 这将为开发更好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.