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MUCOSAL BIOLOGY

Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1

¹Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, Florida; and ²Division of Urology, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan

Submitted June 26, 2009 ; accepted in final form August 24, 2009

The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6–8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox^2–, Cl^–, and SO₄^2– whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO₃^– exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (>60%) and mRNA (>75%) expression in A6KD. No net flux of Ox^2–, Cl^–, or SO₄^2– was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl^–-dependent HCO₃^– efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC₅₀ 5 µM) and that mucosal Cl^– inhibits oxalate uptake with an EC₅₀ < 20 mM. Transepithelial Cl^– gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl^– gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.

chloride; bicarbonate; sulfate; intestine; transport; anion flux; nephrolithiasis; putative anion transporter-1; small interfering RNA