Ischemia-induced Actin Cytoskeletal Alterations

描述(由申请人提供)：继发于缺血和相关事件的急性肾功能衰竭，如败血症和横纹肌溶解，仍然是一个主要的健康问题，在过去的30年里，对此几乎没有什么治疗上的成果。肌动蛋白细胞骨架事件介导了许多细胞改变，进而导致器官功能障碍。尤其是近端肾小管上皮细胞微绒毛的破坏，导致肾小管梗阻、近端离子转运减少和近端肾小管细胞间溶质倒漏，在肾小球滤过减少和肾小管功能障碍中起重要作用。因此，这项资助的目的是进一步描述由肌动蛋白解聚因子(ADF)/cofilin、原肌球蛋白和肌球蛋白vs介导的近端小管细胞顶端肌动蛋白的变化。具体地说，我们认为，ATP的减少会导致ADF/cofilin的激活，而钙的增加会导致顶端结构域原肌球蛋白从F肌动蛋白细胞骨架上解离。这两个事件导致ADF药用F肌动蛋白的破坏，微绒毛完整性的丧失，通过起泡和内化而失去顶膜。肌球蛋白1既受ATP消耗的影响，又受钙离子的影响。我们认为，它在调节顶膜结构、内吞作用和离子通道功能方面的功能发生了改变。我们将使用多光子显微镜和腺病毒探针通过微穿孔技术在活体肾小管上皮细胞中追踪这些特定蛋白在生理条件下、缺血期间和再灌注期的动态变化。这些蛋白质的特定作用将通过其他已知调节其功能的蛋白质、定点突变和截短结构的使用来探讨。已经开发出了能够对体内观察到的影响进行定量分析的技术。综上所述，我们预计这些数据将极大地增强我们对体内整个过程的理解，因为利用特定的分子方法跟踪分化的近端肾小管细胞的能力以前是不可能的。

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.