Ischemia-induced Actin Cytoskeletal Alterations

描述（由申请人提供）：继发于缺血和相关事件（如脓毒症和横纹肌溶解症）的急性肾衰竭仍然是一个主要的健康问题，在过去三十年中，在治疗方面几乎没有取得进展。肌动蛋白细胞骨架事件介导许多细胞改变，这些细胞改变反过来导致器官功能障碍。特别是，近端肾小管细胞微绒毛破坏，导致肾小管阻塞，近端离子转运减少和近端肾小管细胞之间的溶质回漏，已知在肾小球滤过减少和肾小管功能障碍中起主要作用。因此，本补助金的目的是进一步描绘近端小管细胞顶端肌动蛋白的改变介导的肌动蛋白解聚因子（ADF）/cofilin，原肌球蛋白和肌球蛋白Vs。具体而言，我们建议，减少ATP的结果ADF/cofilin激活和增加钙导致顶端结构域原肌球蛋白从F肌动蛋白细胞骨架解离。这两个事件然后导致ADF介导的F肌动蛋白破坏、微绒毛完整性的丧失、通过起泡和内化的顶端膜的丧失。肌球蛋白1受ATP耗竭和钙的影响。我们认为它在介导顶端膜结构、内吞作用和离子通道功能方面的功能被改变。我们将使用多光子显微镜和腺病毒探针通过微穿刺技术递送到体内肾小管上皮细胞，以在生理条件下，在缺血期间和再灌注期间体内追踪这些特异性蛋白。这些蛋白质的具体作用将使用已知调节其功能的其他蛋白质、定点诱变和使用截短的构建体来接近。已经开发了允许对体内观察到的效应进行定量分析的技术。总之，我们预计这些数据将大大提高我们的整体过程中，在体内的能力，以遵循一个分化的近端肾小管细胞利用特定的分子方法之前是不可能的理解。

DESCRIPTION (provided by applicant): Acute Renal Failure secondary to ischemia and associated events such as sepsis and rhabdomyolysis remains a major health problem, about which little has been accomplished therapeutically in the last three decades. Actin cytoskeletal events mediate many of the cellular alterations that in turn result in organ dysfunction. In particular, proximal tubular cell microvillar destruction, resulting in tubular obstruction, reduced proximal ion transport and backleak of solutes between proximal tubular cells, is known to play a major role in reduced glomerular filtration and tubular dysfunction. Therefore, the purpose of this grant is to further delineate proximal tubule cell apical actin alterations as mediated by actin depolymerizing factor (ADF)/cofilin, tropomyosins and myosin Vs. Specifically, we propose that a reduction in ATP results in ADF/cofilin activation and increases in calcium result in apical domain tropomyosin dissociation from the F actin cytoskeleton. These two events then lead to ADF medicated F actin destruction, loss of microvillar integrity, loss of apical membrane through blebbing and internalization. Myosin 1 is affected by both ATP depletion and calcium. We believe its function in mediating apical membrane structure, endocytosis and ion channel function is altered. We will use multi-photon microscopy and adenoviral probes delivered by micropuncture techniques to tubular epithelial cells in vivo to follow intravitally these specific proteins under physiologic conditions, during ischemia and during the reperfusion period. Specific roles for these proteins will be approached using additional proteins known to regulate their function, site directed mutagenesis and the use of truncated constructs. Techniques have been developed to allow for quantitative analysis of the effects observed in vivo. Taken together, we anticipate these data will greatly enhance our understanding of the overall process in vivo as-the ability to follow a differentiated proximal tubular cell utilizing specific molecular approaches has not been previously possible.