FIBROBLAST ACTIVATION IN URINARY OBSTRUCTION

尿路梗阻和其他刺激肾纤维化的条件会导致分化状态的改变。实质成纤维细胞的特征是几种胶原蛋白的分泌显著增加，并获得与α-平滑肌肌动蛋白表达相关的收缩表型：“肌成纤维细胞”。许多关于完整肾脏的研究表明，血管紧张素II、转化生长因子-β1和其他因子是肌成纤维细胞分化所必需的关键因素。许多关于完整肾脏的研究表明，血管紧张素II、转化生长因子-β1和其他激素是肌成纤维细胞分化所必需的关键因素。相比之下，对成纤维细胞和培养的其他细胞中α-平滑肌肌动蛋白表达的研究却产生了相互矛盾的结果。本应用的初步证据表明，转化生长因子-β1对哺乳动物成纤维细胞的表达几乎没有或几乎没有影响。在文化上。本项目的重点将是确定α-平滑肌中的DNA调控元件和DNA结合蛋白。本项目的重点将是鉴定α-平滑肌肌动蛋白启动子中的DNA调控元件和DNA结合蛋白，这些调控元件和DNA结合蛋白在成纤维细胞中负责尿路梗阻依赖性表达。BALB/c小鼠成纤维细胞将被标记为稳定表达绿色荧光蛋白(GFP)，并被注射到同基因小鼠的肾脏中，在那里它们被评估为梗阻依赖的诱导α-平滑肌肌动蛋白(SMA)的表达。然后，使用Frefly和Renilla荧光素酶双重报告系统，将SMA启动子-报告构建稳定地导入标记的成纤维细胞，以鉴定阻碍依赖表达所必需的DNA元件。随后将使用迁移率改变分析和其他技术来鉴定与这些位点相互作用的DNA结合蛋白。最后，同基因成纤维细胞系统将用于在梗阻和对侧肾脏的间质中表达多组氨酸标记的蛋白，以确定细胞外基质积累的决定因素。这些研究将试图确定在尿路梗阻中启动肾小管间质纤维化的基本因素。这一结果可能导致发现阻止和逆转间质纤维化的新的治疗靶点。

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.