RENAL EPITHELIAL UREA SENSOR

描述（改编自研究者摘要）：肾细胞 髓质暴露在极高浓度的 变性剂，尿素，作为肾浓缩机制的结果。 肾髓质细胞适应和分化的分子机制 容忍这种恶劣的环境对人类的 了解水和尿素生理学，并了解和 潜在的增强代谢应激的细胞保护作用， 不同的病理生理学背景。 申请人已取得丰富的 证据表明，与其他主要髓溶质形成鲜明对比， NaC 1，尿素激活肾组织中一种新的受体酪氨酸激酶途径 上皮细胞 两个候选的尿素敏感分子表现出迅速， 溶质和组织特异性、尿素诱导的自磷酸化或酪氨酸 磷酸化已被申请人通过以下方式部分纯化： 与尿素反应性信号分子的免疫共沉淀， 凝集素亲和沉淀。 的总体目标 项目是了解分子机制和生理 尿素诱导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， 其他潜在的新型尿素传感和效应分子。 的能力 已知的含有SH 2结构域的受体酪氨酸激酶效应分子 肾上皮细胞对尿素适应性反应的介导因素 将通过抑制效应物途径来确定应激（目标1） 通过瞬时和稳定转染， 用可诱导的显性负性表达构建体。 两个候选 尿素传感器将被部分地生物化学纯化（以及其它可能的纯化）。 如有必要，使用由32 Pi制备的裂解物鉴定候选物 代谢标记的对照和尿素处理的细胞，通过 结合2-D凝胶的色谱和批量亲和策略 电泳（Aim II）。 最后，候选的尿素敏感蛋白将 通过串联质谱鉴定，或者，如果是新的，通过 肽微测序，然后进行cDNA文库筛选（Aim III）。 此后，它们在转录水平的生理调节， 翻译和翻译后水平将在肾组织中测定。 在培养物中和与异常相关的啮齿动物模型中的髓细胞 水和尿素代谢。

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.