The principal focus of this project continues to be the mechanisms of anion transport in the proximal tubule. Studies during the past decade have supported a model by which transcellular CI-absorption in the proximal tubule involves uphill CI-uptake across the luminal membrane by exchange with formate and oxalate. Recycling of formate occurs by H/+- couple formate transport in parallel with Na/+-H/+ exchange, whereas next project period we propose to investigate two aspects of proximal tubule anion transport. First, we plan to complete the cDNA cloning and carry out the physiological characterization of a novel transporter that is likely to play a role in mediating apical membrane anion transport. Specifically, we will isolate, clone and sequence cDNAs encoding the transporter, determine anion specificity and transport modes by functional expression in Xenopus oocytes; general specific antibodies, and determine cell and membrane sites of expression; determine whether different isoforms of the transporter exist; examine structure-function relationships by use of chimeric constructs; and estimate the contribution of the transporter to integrated tubule function in microperfusion studies in mice with targeted disruption of the transporter gene. Second, in collaboration with Gerhard Giebisch (project#1), we propose to continue studies of the mechanisms of regulation of transcellular NaHCO/3 and NaCI reabsorption in the proximal tubule. Specifically, we will measure the activities of both anion exchanges (CI-formate and CI-oxalate) and recycling pathways (H+-coupled formate transport, Na/+- sulfate co-transport, oxalate-sulfate exchange) in renal brush border vesicles isolated from rats subjects to conditions that regulate proximal NaHCO/3 and NaCI reabsorption (eg. metabolic acidosis, hypokalemic alkalosis, furosemide-induced volume contraction). Activities of these pathways in membrane vesicles will be correlated with rates of transtubular HCO/3- and CI- reabsorption in the intact tubule under similar conditions. We will thereby test the hypothesis that activities of luminal membrane anion transporters are appropriate altered tot permit independent regulation of proximal tubule NaHCO/3 and NaCI reabsorption. The proposed project will provide new information on the molecular mechanisms and regulation of renal CI- transport, and is therefore of relevance for understanding clinical disorders of NaCI balance.
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