The overall objective of the proposal is to understand the pathogenetic mechanisms involved in the ischemic renal failure with a central hypothesis that hypoxia leads to the production of nitric oxide which is followed by damage to the tubular cells with rise in intracellular calcium, activation of calpain, disruption of microvillar network, and ultimately a decrease in the glomerular filtration rate. To establish the relationship of these events leading to renal failure various in vitro and in vivo experiments are proposed under 3 specific aims.
In specific aim 1, the effect of nitric oxide synthase (NOS) and calpain (calcium dependent protease) inhibition on the progression of renal failure will be assessed. The inhibition will be carried out with various inhibitors of NOS and calpain or by infusing antisense-oligos intravenously. Also certain experiments will be performed in iNOS and eNOS knockout mice. Besides the parameters used to asses the renal functions, such as, determination of BUN, creatinine and immuno-electron-microscopy, translational blockade of NOS, its isoforms and of calpain will be assessed. The second specific aim is to determine the induction of enzyme protein acitivity of iNOS and of calpain following ischemia. Various immunoblotting experiments are proposed to assess induction of the enzyme activities. The studies will be extended to study the translocation of iNOS in the subcellular fractions and status of phosphorylation of NOS during renal ischemia. Immuno-electron microscopy and cell fractionation studies will be performed to asses the translocation of iNOS. To study the phosphorylation of NOS, activity of calcineurin in FK506-treated animal undergoing ischemia will be determined, and various phosphotyrosine antibodies will be utilized. The third specific aim includes a series of experiments to identify the potential intracellular and cell-matrix interface targets of NO and calpain which are adversely affected by the ischemic injury to the tubules. Experiments to determine the effect of NO on actin polymerization, on actin-associated cytoskeletal proteins and cell-matrix interactions and the role of ADP ribosylation in actin polymerization will be determined. Finally, the experiments to determine the interaction between Tamm-Horsfall protein (THP), a secretory protein of cells lining the thick ascending limb of loop of Henle, and the sloughed off epithelia from ischemic tubules. The last set of experiments will be initially carried out in vitro with one of the renal epithelial cell line, i.e., LLCPK1 cells, and the effect of cyclic RGD peptide on their interactions with the THP would be assessed.
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