The levels of the mitochondrial glutaminase (GA) and glutamate dehydrogenase and of the cytoplasmic phosphoenolpyruvate carboxykinase (PCK) are increased within the rat renal proximal convoluted tubule in response to metabolic acidosis. This adaptation is necessary to sustain increased renal ammoniagenesis and gluconeogenesis during a compensated chronic acidosis. The increase in GA and PCK activities result from increased rates of synthesis that correlate with increased levels of their respective mRNAs. However, only the prompt increase in PCK mRNA that occurs following acute onset of acidosis is due to an increased rate of transcription. Increased stability accounts for the 6- to 8-fold increases in GA mRNA that are initiated 4-7 h after onset of acute acidosis and sustained during chronic acidosis. This process also contributes to the maintenance of increased PCK mRNA during chronic acidosis. The 3' non- translated regions of both mRNAs contain palindromic sequences, AU-rich segments and AUUUA motifs. Such domains may bind specific proteins that regulate mRNA stability. Furthermore, LLC-PK-F+ cells, an established gluconeogenic line of renal proximal tubular epithelial cells, exhibit an increased level of GA mRNA in response to growth in acidic medium. This adaptation occurs with kinetics that are identical to those observed in vivo. Experiments using actinomycin D indicate that the observed changes are due to altered stability of the GA mRNA. In addition, the level of GA mRNA in LLC-PK-F+ cells is superinduced by cycloheximide (16-fold) and is decreased 30-fold following treatment with a phorbol ester (PMA). Thus, this system is well suited to characterize the mechanism by which specific cells within the kidney sense changes in extracellular pH and/or HCO3 and transduce this information to alter the stability of specific mRNAs.
The specific aims of the proposed research are to establish the mechanism of altered GA mRNA levels in LLC-PK-F+ cells; to map the cis-elements that are responsible for the increased stability of GA mRNA, to identify and clone the factors that affect GA mRNA stability; to identify and characterize the promoters of the GA gene; and to characterize the mechanisms of signal recognition and transduction that initiate and mediate the renal response to acidosis. The results of the proposed study are likely to uncover novel mechanisms by which the kidney, and possibly other tissues that participate in the interorgan metabolism of glutamine, regulate gene expression in response to altered acid-base balance. This information should provide insight into potential pharmacologic approaches that may stimulate ammoniagenesis in various clinical conditions which cause metabolic acidosis.
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