FIBROBLAST ACTIVATION IN URINARY OBSTRUCTION

尿路梗阻和其他刺激肾纤维化的情况会引起分化状态的改变。以几种胶原蛋白分泌显著增加为特征的实质成纤维细胞，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”。在完整肾脏中的大量研究表明，血管紧张素II、TGF- β 1和其他药物是肌成纤维细胞分化所需的关键因子。在完整肾脏中的大量研究表明血管紧张素II、tgf - β 1和其他药物是肌成纤维细胞分化所需的关键因子。相比之下，对α -平滑肌肌动蛋白在成纤维细胞和其他培养细胞中的表达的研究得出了相互矛盾的结果。本应用的初步证据表明，tgf - β 1对哺乳动物成纤维细胞的表达影响很小或没有影响。在文化。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，该启动子负责成纤维细胞中尿阻依赖性表达。Balb/c小鼠成纤维细胞将通过绿色荧光蛋白（GFP）的稳定表达进行标记，并注射到同基因小鼠的肾脏中，在那里评估它们对α -平滑肌肌动蛋白（SMA）表达的阻塞依赖性诱导。然后使用萤火虫和Renilla荧光素酶双报告系统稳定地转染标记的成纤维细胞，以鉴定阻碍依赖性表达所必需的DNA元件。然后将使用迁移位移测定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，将使用同基因成纤维细胞系统在梗阻和对侧肾脏间质中表达多组氨酸标记蛋白，以确定细胞外基质积累的决定因素。这些研究将试图确定尿路梗阻引起小管间质纤维化的必要因素。这些结果可能会导致发现新的治疗靶点，以阻止和逆转间质纤维化。

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.