Onset of metabolic acidosis activates transcription of the genes that encode the enzymes and transport proteins that sustain the adaptive increases in renal ammonia excretion, HCO3- synthesis and reabsorption, and gluconeogenesis. A well-characterized example of this response is the 6-fold increase in the level of the phosphoenolpyruvate carboxykinase (PEPCK) mRNA that occurs within the rat renal proximal convoluted tubule. The full-length PEPCK cDNA and genome have been isolated and sequenced. Many of the promoter elements and associated transcription factors that mediate its transcriptional regulation in liver, kidney, and adipose tissues have been characterized. In addition, various segments and specific mutations of the PEPCK promoter and 3'-untranslated region have been expressed as reporter constructs or transgenes. Furthermore, LLC-PK1-F+ cells, a porcine line of renal proximal tubule-like cells, exhibit a 3-4-fold increase in expression of the endogenous PEPCK gene or the CRC362 PEPCK transgene when transferred to acidic medium [pH 6.9, 10 mM HCO3-]. The PEPCK mRNA also contains a unique instability element that accounts for its rapid turnover and cAMP-dependent stabilization. Thus, the PEPCK gene and this cell line serve as an effective paradigm to investigate the mechanism by which changes in pH activate transcription of specific genes within the proximal tubule.
The specific aims of the proposed research are: to use the CRC362 transgene to map and characterize the pH-response element that regulates transcription of the PEPCK gene; to characterize the role of the p38a SAPK/ATF-2 signaling pathway in the pH-responsive induction of the PEPCK gene; and to identify the binding proteins and the mechanism that mediate the turnover of PEPCK mRNA. The results of the proposed experiments should significantly increase understanding of the molecular mechanism that regulates this essential adaptive response and provide insight that may lead to improved clinical treatment of chronic acidosis.
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