Recent studies from the principal investigator's laboratory have shown that mitochondrial glutaminase (GA) activity is increased 7- to 20-fold within the rat renal proximal tubule during chronic metabolic acidosis. This adaptation contributes to the sustained increase in renal ammoniagenesis and gluconeogenesis which are essential to partially compensate the acidosis. The increased GA activity results from stabilization of the GA mRNA. This response is modeled by the expression of a chimeric construct containing the 3'-nontranslated region of the GA mRNA in LLC-PK1-F+ cells, a pH-responsive line of porcine renal proximal tubule-like cells. Deletion analysis has mapped the functional element to a 340-base sequence within the 2446-base 3'-nontranslated region of the GA mRNA. This sequence confers an enhanced liability and a pH-responsive stabilization when incorporated into a chimeric mRNA. RNA gel-shift analysis established that rat renal cortical cytosolic extracts contains a protein which exhibits high affinity and specific binding to this sequence. The level of binding is decreased significantly in extracts prepared from rats made acutely acidotic. The LLC-PK1-F+ cells express four GA mRNAs, only one of which is induced by transfer of the cells to acidic medium (pH 6.9, 10 mM HCO3-). RNase H mapping indicates that the 4GA mRNAs differ in the length and sequence of their 3'-nontranslated regions. This system will be used to pursue the functional analysis of the multiple isoforms of GA mRNA and the mechanism of the pH-responsive stabilization.
The specific aims of the proposed research are: 1) to clone and characterize the multiple isoforms of GA mRNA; 2) to define the organization and mechanism of expression of the GA genome; 3) to further define the pH-responsive instability elements and clone and characterize the associated binding proteins; and 4) to determine the mechanism of GA mRNA degradation and stabilization. The proposed analysis is designed to provide insight into potential pharmacological approaches, which may stimulate renal ammoniagenesis in various clinical conditions leading to metabolic acidosis.
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