MOLECULAR BASIS OF RENAL URATE TRANSPORT

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The kidneys play an essential role in the maintenance of urate homeostasis, providing the major route for elimination of urate from the body. Although two mechanisms of urate transport, electroneutral anion exchange and electrogenic uniport have been well described, neither transporter has been identified and characterized at the molecular level. The long term goal of this proposal is to understand the molecular mechanisms by which urate is transported across renal tubular cell membranes. We have recently cloned a full-length cDNA (designated UAT), prepared recombinant protein from the UAT cDNA, and functionally reconstituted the recombinant protein in planar lipid bilayers as a urate transporter/channel. The studies detailed in this proposal will test the hypothesis that the unique cDNA that we have cloned serves as a transporter/channel in intact cell membranes, functioning within the kidney as the electrogenic urate transporter that contributes importantly to urate excretion. Four specific aims have been developed to test this hypothesis. The first aim is to more extensively characterize the urate transporter/channel encoded by the UAT cDNA subsequent to fusion of the recombinant protein in planar lipid bilayers: the selectivity of the channel, and the influence of pH, sulfhydryl groups, calcium, and phosphorylation on channel activity will be examined. The second aim is to evaluate and characterize the biologic activity of the urate transporter/channel encoded by the UAT cDNA subsequent to its translation and expression in intact cells including Xenopus laevis oocytes and immortalized human renal cells: two microelectrode voltage clamp and patch clamp studies will be employed. The third aim is to modify the coding sequence of the UAT cDNA and correlate alterations in structure with the functional activity of the encoded protein. Functional effects of mutations in specific amino acids in UAT will be evaluated with electrophysiologic techniques in planar lipid bilayers and/or oocytes. The final aim is to characterize the cellular and subcellular sites of expression of the UAT cDNA along the length of the nephron. These studies will utilize primary cultures and immortalized cells from defined segments of the nephron of the human kidney.