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.
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