Oxalate is excreted in the urine via a combination of both glomerular filtration and proximal tubular secretion.The intestinal absorption of oxalate may in turn be a major determinant of its excretion in the urine and thenatural history of stone disease. We and others have shown that the apical anion exchanger Slc26a6,known to be expressed in the murine proximal tubule and intestine, is capable of mediating Cl'-oxalate, SO42'-oxalate, Cl'-formate, CI'-HCCy, and CI'-OH' exchange. Direct measurement of oxalate transport in Xenopusoocytes has revealed that several other SLC26 and SLC4 exchangers are capable of oxalate transport,indicating that there is significant molecular heterogeneity of epithelial oxalate exchange. We propose inAim 1 to systematically evaluate the role of SLC26 exchangers and SLC4 exchangers in intestinal oxalatetransport, so as to define the major transporters responsible for transepithelial oxalate absorption by thistissue. The kinetics, pharmacology, and electrophysiology of oxalate transport will thus be evaluated andcharacterized by heterologous expression of individual cDNAs in Xenopus oocytes. We will alsocharacterize oxalate transport mechanisms in the Caco-2 intestinal epithelial cell line, in which we will assessthe quantitative role of specific exchangers using RNA interference. Within the kidney, the apical membraneof the proximal tubule contains mechanisms capable of Cl'-oxalate, oxalate-base, and SO42'-oxalateexchange; Slc26a6 is clearly capable of all these activities and is likely the dominant apical oxalateexchanger in these cells. Apical oxalate exchange mediated by Slc26a6 is thus thought to function inproximal tubular secretion of oxalate, in concert with basolateral oxalate exchange mediated by Slc26a1. Toclarify the role of Slc26a6 in the proximal tubule we will generate mice in Aim 2 with loxP sites flankingexons 5-7 of the Slc26a6 gene ('floxed mice'), for cell type-specific deletion of these exons. These miceand/or mice with a germline deletion of exons 5-7 will initially be utilized to examine the contribution of theSlc26a6 protein to apical oxalate exchange in the proximal tubule, using a variety of techniques. Finally, asignificant fraction of patients with calcium-oxalate stones exhibit hyperabsorption of ingested oxalate, suchthat genetic variation in the major intestinal oxalate transporters identified in Aim 1 may predispose tonephrolithiasis. Using the resources and expertise of the Genetics Core we will define the spectrum ofgenetic variability in specific SLC26 and SLC4 genes, beginning with the apical exchanger SLC26A6. Thefunctional consequence of non-conservative coding sequence variation in the SLC26A6 gene will thus bestudied using heterologous expression in Xenopus oocytes. We.will also determine the functionalconsequences of coding sequence variation in WNK4, a novel kinase with potent inhibitory effects on bothSLC26A2 and SLC26A6, apical oxalate exchangers in the small intestine. This phenotypic characterizationwill be correlated with the impact of these polymorphisms on the genetic predisposition to stone disease andhyperoxaluria, as determined in Project 3.
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