RENAL CELL RESPONSE TO HYPEROSMOLAR UREA

肾髓质积聚了高浓度的尿素和氯化钠。 根据水合状态和膳食蛋白质摄入量。然而，在那里 对细胞对高渗透压的反应知之甚少 尽管尿素看起来与 Nac1。与氯化钠不同，高渗尿素(25-250 mM)不会抑制 细胞生物合成，增强应激蛋白的表达，也不增加 有机渗透膜转运蛋白的表达。最重要的是，我们的工作 已经证明尿素能特异性地激活转录和翻译 转录因子Egr-1。这种影响只发生在肾脏。 上皮细胞，而不是由尿素类似物模仿导致我们 提出了存在低亲和力、高特异性的尿素“传感器”。 此外，尿素诱导的Egr-1转录似乎是由一种 PKC依赖的途径涉及启动子元件的激活 Egr-1基因5‘侧翼区最近的1.2kb部分 吉恩。这项研究的目标将是定义细胞 肾内髓内集合管(IMCD)细胞的突起 对高渗尿素的适应。我们将使用一只永生的小鼠IMCD 本实验室建立的mIMCD-3细胞系。具体目标是：1. 确定尿素诱导Egr-1基因的启动子元件(S) 抄写。我们将使用瞬时转基因荧光素酶报告 5“侧翼区缺失和截短突变的基因分析 定义“尿素反应元件(S)”。2.定义信号 Egr-1激活尿素的信号转导途径 抄写。我们将检测DNA结合蛋白并确定 PKC和PLC在尿素信号转导过程中的作用3.识别 表达上调或下调的下游效应基因 尿素。Northern分析、差异显示RT-PCR和Western分析 分析将用于识别和表征下游基因。 被尿素处理激活或抑制。4.尿素诱导的特征 体内基因表达的变化。在建立了尿素之后- 细胞培养中的反应因子和基因，我们将定义它们的 两种已知促进肾髓质尿素的动物模型的意义 积聚、高蛋白饮食和脱水。这项研究将是 对了解肾细胞特异性基因激活的价值 抄写。此外，我们还将确定新的下游效应器 基因以及尿素反应的信号转导过程，我们 假设涉及尿素受体。

Renal medulla accumulates high concentrations of urea and NaC1 that vary according to hydration state and dietary protein intake. However, there is relatively little known about the cellular response to hyperosmolar urea though it appears to be distinctly different from that seen with NaC1. Unlike NaC1, hyperosmolar urea (25-250 mM) does not inhibit cellular biosynthesis, enhance expression of stress proteins, nor increase expression of organic osmolyte transporters. Most importantly, our work has shown that urea specifically activates transcription and translation of the transcription factor Egr-1. This effect occurs only in renal epithelial cells and is not mimicked by urea analogues leading us to propose the existence of a low affinity, high specificity urea "sensor." Furthermore, urea-induced Egr-1 transcription appears to be mediated by a PKC-dependent pathway involving activation of a promoter element within the most proximal 1.2 kb portion of the 5' flanking region of the Egr-1 gene. The objective of this study will be to define the cellular processes involved in renal inner medullary collecting duct (IMCD) cell adaptation to hyperosmolar urea. We will use an immortalized mouse IMCD cell line (mIMCD-3) created in our laboratory. Specific Aims are: 1. Define the promoter element(s) responsible for urea-induced Egr-1 transcription. We will use a transient transfection luciferase reporter gene assay with deletion and truncation mutants of the 5" flanking region of Egr-1 to define the "urea response element(s)." 2. Define the signal transduction pathway responsible for urea activation of Egr-1 transcription. We will examine DNA binding proteins and determine the roles of PKC and PLC in the urea signal transduction process. 3. Identify downstream effector genes whose expression is up- or down-regulated by urea. Northern analyses, differential display RT-PCR, and western analyses will be used to identify and characterize downstream genes activated or repressed by urea treatment. 4. Characterize urea-induced changes in gene expression in vivo. Having established the urea- responsive factors and genes in cell culture, we will define their significance in two animal models known to promote renal medullary urea accumulation, high protein diet and dehydration. This study will be valuable in understanding renal cell-specific activation of gene transcription. In addition, we will identify novel downstream effector genes as well as a urea-responsive signal transduction process that we hypothesize involves a urea receptor.