Urinary tract obstruction and other conditions that stimulate renal fibrogenesis induce a change in the state of differentiation. of parenchymal fibroblasts characterized by a marked increase in the secretion of several classes of collagen, and the acquisition of a contractile phenotype associated with expression of alpha-smooth muscle actin: the """"""""myofibroblast."""""""" A number of studies in the intact kidney have implicated angiontensin II, TGF- beta1, and other agents as key factors required for myofibroblast differentiation. A number of studies in the intact kidney have implicated angiotensin II, TGF-beta1, and other gents as key factors required for myofibroblast differentiation. In contrast, studies on alpha-smooth muscle actin expression in fibroblasts and other cells in culture have yielded conflicting results. Preliminary evidence presented in this application indicate that TGF-beta1 has little or not effect on the expression of mammalian fibroblast. in cultures. The focus of this project will be to identify the DNA regulatory elements and DNA-binding proteins in the alpha-smooth focus of this project will be to identify the DNA regulatory elements and DNAS-binding proteins in the alpha-smooth muscle actin promoter responsible for urinary obstruction-dependent expression in fibroblasts. Balb/c mouse fibroblasts will be marked by stable expression of green fluorescent protein (GFP), and injected into the kidneys of syngeneic mice, where they be assessed for obstruction-dependent induction of alpha- smooth muscle actin (SMA) expression. The marked fibroblasts will then be stably transfected with SMA promoter-reporter constructs using the firefly and Renilla luciferase dual reporter systems to allow identification of DNA elements essential for obstruction-dependent expression. Mobility shift assays and other techniques will then be used to identify the DNA-binding proteins interacting with these sites. Finally, the syngeneic fibroblast system will be used to express polyhistidine-tagged proteins in the interstitium of obstructed and contralateral kidneys, to identify the determinants of extracellular matrix accumulation. These studies will attempt to identify factors essential for the initiation of tubulointerstitial fibrosis in urinary tract obstruction. The results may lead to the identification of new therapeutic targets for arresting and reversing interstitial fibrosis.
