RENAL EPITHELIAL UREA SENSOR

描述(改编自《调查者摘要》)：肾脏细胞 延髓暴露在极高浓度的强效物质中 变性剂，尿素，作为肾脏浓缩机制的结果。 肾髓质细胞适应和调节的分子机制 忍受这种恶劣的环境对 对水和尿素生理学的理解，以及对 代谢应激对细胞保护作用的潜在增强 不同的病理生理背景。申请者已经获得了丰富的 证据表明，与其他主要的髓质溶质形成鲜明对比的是， NaCI，尿素激活肾脏一种新的受体酪氨酸激酶途径 上皮细胞。两个候选尿素敏感分子表现出提示， 溶质和组织特异性，尿素诱导的自动磷酸化或酪氨酸 申请人通过以下途径部分纯化了磷酸化 与尿素反应信号分子的免疫共沉淀，并通过 凝集素亲和沉淀。《公约》的总体目标 项目是了解分子机制和生理 尿素诱导的蛋白激酶信号通路激活的后果 在体外和体内的肾髓质细胞中，并鉴定这些细胞和 其他潜在的新型尿素传感和效应器分子。的能力 已知的SH2结构域受体酪氨酸激酶效应器分子 肾上皮细胞对尿素适应性反应的中介分子 应激(目标1)将通过抑制效应器通路来确定 并通过瞬时和稳定的转染法 具有可诱导的显性-负性表达结构。两位候选人 尿素传感器将进行部分生化提纯(和其他潜在的 如有必要，确定候选人)使用从32PI制备的裂解物 代谢标记的对照和尿素处理的细胞，通过 结合2-D凝胶的层析和批次亲和策略 电泳法(AIM II)。最后，候选尿素感应蛋白将 通过串联质谱学鉴定，或者，如果是新的，通过 肽显微测序及文库筛选(AIM III)。 此后，他们的生理调节在转录， 翻译水平和翻译后水平将在肾脏中确定 骨髓细胞在培养和啮齿动物模型中与异常相关 水分和尿素代谢。

DESCRIPTION (Adapted from Investigator's Abstract): Cells of the renal medulla are exposed to extraordinarily high concentrations of the potent denaturant, urea, as a consequence of the renal concentrating mechanism. The molecular mechanisms through which renal medullary cells adapt to and tolerate this harsh environment have important implications for the understanding of water and urea physiology, and for the understanding of and potential enhancement of cellular protection from metabolic stress in diverse pathophysiological contexts. The applicant has obtained abundant evidence that, in marked contrast to the other principal medullary solute, NaC1, urea activates a novel receptor tyrosine kinase pathway in renal epithelial cells. Two candidate urea sensing molecules exhibiting prompt, solute- and tissue-specific, urea-inducible autophosphorylation or tyrosine phosphorylation have been partially purified by the applicant through co-immunoprecipitation with urea-responsive signaling molecules, and through lectin affinity precipitation, respectively. The overall objective of the project is to understand the molecular mechanism and physiological consequences of the activation of urea-inducible kinase signaling pathways in renal medullary cells in vitro and in vivo, and to identify these and other potentially novel urea sensing and effector molecules. The ability of known SH2 domain-containing receptor tyrosine kinase effector molecules to mediate elements of the renal epithelial cell adaptive response to urea stress (Aim 1) will be determined through inhibition of effector pathways with pharmacological agents and through transient and stable transfection with inducible dominant-negative expression constructs. The two candidate urea sensors will be partially biochemically purified (and other potential candidates identified, if necessary) using lysates prepared from 32Pi metabolically labeled control- and urea-treated cells, through chromatographic and batch affinity strategies in conjunction with 2-D gel electrophoresis (Aim II). Lastly, the candidate urea sensing proteins will be identified through tandem mass spectrometry, or, if novel, cloned through peptide microsequencing followed by cDNA library screening (Aim III). Thereafter, their physiological regulation at the transcriptional, translational, and post-translational levels will be determined in renal medullary cells in culture and in rodent models associated with abnormal water and urea metabolism.