Endothelial cell dysfunction (ECD), premature senescence and apoptosis are important contributors to capillary drop-out, glomeruosclerosis, tubulointerstitial scarring and progression of chronic renal disease. Previous studies showed decreased availability of nitric oxide (NO), decreased levels of tetrahydrobiopterin (BH4) and glutathione (GSH), oxidative stress, and accumulation of peroxides as leading causes of ECD. Elevated levels of asymmetric dimethylarginine (ADMA) further compound ECD by inhibiting NO synthease. Preliminary Studies showed induction of a profibrotic phenotype of endothelial cells subjected to elevated levels of ADMA. NOS inhibition resulted in the in vitro and in vivo manifestations of the loss of differentiated endothelial cell functions and acquisition of myofibroblast phenotype. The concept to be tested and further developed in this proposal ascribes to NO deficiency and ECD-induced endothelial-mesenchymal transdifferentiation the role of a trigger of 1) initial functional insufficiency of microcirculation followed by 2) structural phase of microvascular regression - prerequisites of tissue hypoxia and progressive nephrosclerosis. These goals will be accomplished through 1) elucidating profibrotic sequelae of NOS inhibition in vivo; 2) assessing the contribution of vascular non-perfusion and structural regression to glomerulosclerosis and tubulointerstitial scarring; 3) investigations into transdifferentiation of endothelial cells as a potentially important contributor to vascular drop-out; its stimulators and mediators, like TGF-beta, angiostatin and endostatin; 4) detection and identification of intracellular proteins deficient in nitrosothiol modification, as a cause of the downstream endothelial-mesenchymal transdifferentiation; and 5) attempts to ameliorate endothelial-mesenchymal transdifferentiation, vascular regression and nephrosclerosis using an intermediate in NO synthesis, hydroxy-L-arginine, which combines the properties of superoxide scavenger and NO donor. These studies will be performed using established approaches for studies into cell physiology, renal morphology, proteomics, and renal physiology, all available to the investigative team. It is anticipated that proposed set of studies should provide a broad view on the potentially important mechanism of vascular regression in chronic kidney disease - endothelial-mesencymal transdifferentiation, its contribution to nephrosclerosis and therapeutic actions of hydroxy-L-arginine, which can ameliorate NO deficiency, accompanied by endothelial-mesenchymal transdifferentiation and vascular drop-out, thus reducing progression of chronic renal diseases. ? ?
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