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

Abstract

Freel RW, Morozumi M, Hatch M. Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1. Am J Physiol Gastrointest Liver Physiol 297: G918 -G929, 2009. First published August 27, 2009 doi:10.1152/ajpgi.00251.2009.-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 2Ϫ , Cl Ϫ , and SO 4 2Ϫ whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO 3 Ϫ exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (Ͼ60%) and mRNA (Ͼ75%) expression in A6KD. No net flux of Ox 2Ϫ , Cl Ϫ , or SO 4 2Ϫ was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl Ϫ -dependent HCO 3 Ϫ 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 (EC 50 ϳ5 M) and that mucosal Cl Ϫ inhibits oxalate uptake with an EC50 Ͻ 20 mM. Transepithelial Cl Ϫ gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl Ϫ 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 CALCIUM OXALATE NEPHROLITHIASIS is a relatively common disorder that may result from an imbalance in oxalate input (absorption from dietary sources and endogenous hepatic oxalogenesis) and oxalate output (renal excretion, intestinal secretion) (12). Intestinal epithelia play an important role in oxalate homeostasis by mediating bidirectional and net oxalate transport in a segment-specific manner (11, 12). Recent interest in identifying possible mediators of oxalate transport has been successfully focused on anion exchanger proteins encoded by the SLC26A family of genes (1,3,23). For example, the protein encoded by SLC26A6 (PAT-1, putative anion transporter-1; also referred to as CFEX, chloride-formate exchanger) has been shown to be an important mediator of oxalate secretion in the mouse ileum (10) and duodenum (13), since SLC26A6 knockout mice excrete less oxalate enterically and develop hyperoxaluria, which can promot...

Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation

“…In addition, DIDS (100 M) also significantly inhibited [ 14 C]oxalate uptake by C2 cells grown on plastic supports by Ͼ93% (10.26 Ϯ 2.07 and 0.69 Ϯ 0.20 pmol·cm Ϫ2 ·min Ϫ1 for control and DIDS, respectively). These results strongly indicate that the oxalate uptake by C2 cells is an active transport process mediated by one or more of the involved anion exchanger(s), with SLC26A6 expected to be the main player, as described above (33).…”

“…C2 cells closely resemble the native epithelium, and they express several purinergic receptors (23,60). C2 cells express SLC26A6 on the apical surface, where Ն50% of Cl Ϫ /oxalate exchange has been shown using siRNA knockdown studies, resulting in Ͼ60% reduction of its protein expression (33), indicating that SLC26A6 mediates most of the Cl Ϫ /oxalate exchange activity in C2 cells. We assessed oxalate uptake by C2 cells by imposing an outward Cl Ϫ gradient by removing extracellular Cl Ϫ (intracellular Cl Ϫ Ͼ extracellular Cl Ϫ ) and measuring DIDS (anion-exchange inhibitor)-sensitive influx of [ 14 C]oxalate in exchange for intracellular Cl Ϫ (i.e., Cl Ϫ /oxalate exchange activity).…”

Regulated transport of sulfate and oxalate by SLC26A2/DTDST

“…In oocytes, SLC26A2-mediated oxalate influx and efflux were both at considerably lower rates than SLC26A6-mediated oxalate influx and efflux (6,8). Moreover, small interfering RNA (siRNA) knockdown of hSLC26A6 by ϳ60% in Caco2 cells decreased unidirectional oxalate fluxes in both directions by 50% (16), suggesting that most oxalate is carried by SLC26A6 in this cell model. The direction of colonic oxalate/Cl Ϫ exchange will depend on translumenal membrane gradients of oxalate and Cl Ϫ .…”

Oxalate: From the Environment to Kidney Stones

“…Evidence suggests that this transporter should be expressed in the epithelial cell apical membrane of the human small and large intestine (114,115,126), but thus far its presence at the level of mRNA has been proven only in the rat intestine (114). The affi nity of DTDST for oxalate and its role in handling them still need confi rmation, but recent fi ndings suggest that this transporter could be responsible for the residual intestinal secretion of oxalate in CFEX KO mice (115,127,128). The localization of various members of the Slc26 family along the mammalian intestine is depicted in Fig.…”