CHARACTERIZATION OF A RENAL OXALATE TRANSPORTER

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Kidney stones afflict approximately 500,000 Americans each year, with 75% of all renal stones composed primarily of calcium oxalate. Despite its importance in the pathogenesis of nephrolithiasis, very little is known about the characteristics of the proteins responsible for oxalate excretion along the nephron. We have recently solubilized and reconstituted two oxalate transporters from the rabbit renal cortex; the basolateral sulfate/oxalate exchanger and the brush border membrane (BBM) chloride/oxalate exchanger. The objective of this proposal is to purify the reconstituted BBM chloride/oxalate exchanger and to characterize the protein in terms of its structure and function. In order to purify the reconstituted oxalate transporter to homogeneity the proposed purification protocol will expand upon the nearly complete purification that has already been achieved. We plan on following the functional activity of the oxalate transporter during each step of purification in order to confirm that the protein of interest has been purified and to assure that the purified protein remains in its native state in order to characterize its function. Additional steps proposed to achieve purification of the oxalate transporter include lectins, size exclusion chromatography and dye-ligand affinity chromatography. Once the protein has been purified to homogeneity, polyclonal and monoclonal antibodies will be generated. These will in turn be used in immunoaffinity chromatography in order to simplify the purification protocol and to increase the yield of purified protein. Antibodies will also enable us to identify immunologically related proteins in other tissues as well as in various segments of the nephron. Purification of the oxalate transporter provides us with the opportunity to examine its function in a system free of other BBM proteins. This is particularly important in the case of oxalate transport because of the multiple anion exchange pathways with overlapping substrate specificity on the apical membrane of the proximal tubule. Functional characterization will include identifying its substrate specificity, electrogenicity and pH dependence. The relationship of the oxalate transporter to chloride transport will be examined in detail because of recent evidence suggesting that oxalate is an important intermediate in the reabsorption of NaCl in different segments of the nephron. A partial amino acid sequence will be obtained for the purified protein, and in conjunction with the antibodies, will be used to clone the cDNA encoding the renal oxalate transport protein. The complete sequence will be combined with information obtained from enzymatic digestion of the protein and the role of glycosylation in order to predict its secondary structure. By purifying the oxalate transporter, characterizing its function and cloning the cDNA with expression into a cell system we will have the opportunity in the future to begin correlating the structure of the protein with its function. It is hoped that these types of studies will lead to an understanding of the role of oxalate transport in nephrolithiasis and in salt reabsorption in the kidney.