The level of the cytosolic phosphoenolpyruvate carboxykinase (PCK) mRNA is increased 4-fold within the rat renal proximal convoluted tubule in response to metabolic acidosis. This adaptation contributes to the sustained increase in renal ammoniagenesis and gluconeogenesis which are essential to partially compensate a chronic acidosis. The increased PCK activity results from increased transcription. The entire rat PCK gene has been isolated and sequenced. Many of the PCK promoter elements and the associated factors which mediate the transcriptional regulation of the PCK gene in liver have been well-characterized. Furthermore, LLC-PK1-F+ cells, a gluconeogenic line of porcine renal proximal tubule-like epithelial cells, exhibit adaptive changes in PCK mRNA levels in response to growth in acidic medium (pH = 6.9, [HCO3-]=10 mM) which model those observed in vivo. These cells were used to establish that the CRE-1 and P3(II) elements of the PCK promoter mediate both the pH-responsive and cAMP-mediated stimulations of transcription. Furthermore, the latter response was shown to utilize C/EBP and Fos/Jun transcription factors and thus differ significantly from the mechanism of cAMP stimulation of PCK induction in liver. Thus, this system is extremely well-suited to characterize the tissue specific response to cAMP and to determine how the renal proximal tubule cell senses changes in pH and transduces this information to effect expression of specific genes.
The Specific Aims of the proposed research are to characterize the mechanism of cAMP activation of the PCK gene in kidney cells; to characterize the transcription factors and the mechanism of activation which mediate the pH-responsive induction of the renal PCK gene; to determine whether the cells initially respond to changes in intracellular or extracellular pH and to define the associated signal transduction pathway; and to identify and characterize the instability elements contained in the PCK mRNA. The results of the proposed studies should provide insight into potential pharmacological approaches that may stimulate renal immunogenesis and gluconeogenesis in various clinical conditions which lead to metabolic acidosis.
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