Vitamin C transport and SVCT1 transporter expression in chick renal proximal tubule cells in culture

Abstract

The characteristics of vitamin C (ascorbic acid, ASC) transport were studied in polarized cultured monolayers of the chick (Gallus gallus) renal proximal tubule in Ussing chambers. Under voltage clamp conditions, monolayers responded to apical addition of ASC in a dose-dependent manner, with positive short circuit currents (I(SC)), ranging from 3 microA/cm(2) at 5 microM ASC to a maximal response of 27 microA/cm(2) at 200 microM, and a half-maximal response at 40 microM. There was no effect of basolateral addition of ASC, indicating a polarized transport process. The oxidized form of ASC, dehydroascorbic acid had negligible effects. The I(SC) response to ASC was completely eliminated with Na(+) ion replacement, and was also eliminated by bilateral reduction of bath Cl(-), from 137 to 2.6 mM. There was significant inhibition of the I(SC) responses to 30 microM ASC by the flavanoid quercetin (50 microM) and by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-ethylisopropylamiloride (EIPA), blockers of anion exchangers and sodium-proton exchangers, respectively. There was no inhibition, however, by the chloride channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB). Phorbol 12-myristate 13 acetate (PMA), the phorbol ester activator of protein kinase C, caused a 37% decrease in the I(SC) response to ASC. Chicken-specific primers to an EST homolog of the human vitamin C transporter SVCT1 (SLC23A1) were designed and used to probe transporter expression in these cells. RT-PCR analysis demonstrated the presence of chicken SVCT1 in both cultured cells and in freshly isolated proximal tubule fragments. These data indicate the presence of an electrogenic, sodium-dependent vitamin C transporter (SVCT1) in the chick renal proximal tubule. Vitamin C transport and conservation by the kidney is likely to be especially critical in birds, due to high plasma glucose levels and resulting high levels of reactive oxygen species.