(Taken directly from the application) Ischemia/reperfusion injury (IRI) is a major contributor to the organ damage that results in acute renal failure, one of the major causes of morbidity and mortality in hospitalized patients in the United States. We propose endothelial cell injury during the initiation phase of ARF (anoxia resulting from decreased vascular perfusion of the organ) occurs and is compounded thereafter by continued hypoxia in the cortical medullary region of the kidney. This results in further endothelial cell injury mediating epithelial cell injury and worsening organ dysfunction. Many factors are known to be involved in ischemia/reperfusion injury, but considerable data point to an important role for endothelial cells, as agents of injury in the kidney as well as other organs. Furthermore there is evidence that actin cytoskeletal alterations, occurring during cellular ATP depletion, mediate many of the structural and functional changes known to occur in ischemic cells. However, documentation of endothelial damage in the kidney is lacking. Therefore, we propose to test our hypothesis using an experimental model of ischemia/reperfusion injury in the mouse, and to determine the extent of initial and subsequent ischemia-induced endothelial injury and endothelial actin alterations. We will exploit recent advances in gene therapy and imaging technologies to image endothelial cell dynamics in live animals whose endothelial cells have been labeled with green fluorescent protein (GFP). We will also use this system to investigate the effect of ischemia/reperfusion on the dynamics of renal microvascular blood flow. Finally we will isolate GFP labeled microvascular endothelial cells and study the effects of ATP depletion on the actin cytoskeleton, actin depolymerizing factor (ADF) and expression of ADF using an adenoviral system to express a GFP-ADF chimera in cultured endothelial cells and in in vivo studies. These studies will provide a better understanding of the clinically important phenomenon of ischemia/reperfusion injury, and provide new methods for testing potential therapeutic approaches.
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