Regulation of Renal H+ATPase by Glycolysis

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DESCRIPTION (provided by applicant): Renal acid secretion is essential for normal acid-based homeostasis, skeletal growth, and muscle metabolism. Abnormal urinary acidification is an important contributing factor in the morbidity of acute and chronic kidney disease, nephrolithiasis, and electrolyte disorders. Vacuolar H+ATPases (V-ATPases) are electrogenic ATP-driven proton pumps responsible for 1/3 or more of proximal tubule proton secretion, and for the majority of collecting duct acid secretion. Acid secretion is coupled to cellular metabolism, but the mechanisms for metabolic control of the H+ATPase are poorly understood. We recently discovered that the E subunit of the of the vacuolar H+ATPase binds directly to the glycolytic enzyme aldolase. We demonstrated that the V-ATPase colocalizes with aldolase at he apical membrane in the proximal tubule, and that disruption of the interaction of the H+ATPase with adolase and other glycolytic enzymes has important physiologic effects on the H+ATPase. In preliminary data included in this application, we demonstrate that glucose is a potent activator of V-ATPase proton secretion in cultured renal epithelial cells, and that several subunits of the H+ATPase bind to aldolase. The long-term objective of this proposal is to examine the structural basis and regulation of the interaction of the H+ATPase with aldolase, and its role in control of renal proton secretion. The Specific Aims are 1) to identify and characterize the aldolase binding site on the H+ATPase E, B1 and a4 subunits using binding assays with H+ATPase subunit-fusion protein constructs; 2) to examine the functional significance of the aldolase-V-ATPase interaction in vitro by studying the determinants and enzymatic effects of aldolase binding to the V-ATPase; and 3) to study the functional significance and regulation of the V-ATPase-aldolase interaction in intact renal epithelial cells by expressing normal and mutated forms of aldolase to study the physiologic effects on H+ATPase function. These studies should lead to important new insights on the control of renal acid secretion, on the coupling of ion transport to metabolism, and on mechanisms that contribute to abnormal glucose metabolism and complications of diabetes mellitus.