PROGRAMMED RENAL CELL RESPONSE TO HYPEROSMOLALITY

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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.