Bicarbonate Transport by the Maturing Renal Tubule

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DESCRIPTION (provided by applicant): Metabolic acidosis is a common but potentially life-threatening disorder that occurs when too much acid accumulates in the blood. The kidney is the organ that is primarily concerned with controlling the level of acid in the blood. The cortical collecting duct (CCD) is a key segment of kidney tubule that adapts to the level of acid and pumps it out of the body. The long term goal is to determine how intercalated cells of the CCD adapt to acid-base disturbances by changing their transport of acid and bicarbonate. Rabbit CCDs, which normally secrete bicarbonate, exposed to 3h incubation at pH 6.8 secrete acid. The novel protein hensin is expressed in the extracellular matrix (ECM) surrounding adapting B-intercalated cells (ICs). Aim 1 examines how Cyclosporin A (CsA) causes a distal renal tubular acidosis by preventing adaptation of B-ICs. CsA inhibits hensin polymerization via an inhibition of the proline cis/trans isomerase activity of cyclophilin. Studies using specific inhibitors of these functions are performed in cultured ICs and CCDs incubated at pH 6.8 x 3 h to simulate acidosis. A "knockdown" of cyclophilin will determine which cyclophilin is important in polymerizing hensin in cultured ICs and whether a defect in this cyclophilin causes acidosis. Aim 2 examines the role of galectin-3 in hensin polymerization. Galectin-3 can help polymerize hensin in vitro. This aim will determine which cells express galectin-3 and whether there is stimulation during acidosis. The aim will determine if galectin-3 associates with hensin in the ECM in the adapting CCD and whether a dominant negative galectin-3 prevents hensin polymerization and adaptation of B-ICs in CCD. This aim will establish whether expressed cyclophilin and galectin-3 can cooperate in the in vitro polymerization of hensin. Aim 3 determines which integrins are expressed in ICs and which interact with hensin. Aim 4 examines the role of hensin polymerization in the adaptation of CCD ICs to metabolic alkalosis, with the hypothesis that alkalosis reduces hensin polymerization by degrading hensin and allowing B-ICs to secrete bicarbonate to correct the abnormality. These studies should show how hensin and associated proteins mediate the kidney's adaptation to acid-base disturbances.