Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney

We have cloned a cDNA coding for a novel member of organic anion transporter, designated OAT-K1, expressed specifically in the kidney of rats, The rat OAT-K1 cDNA (2788 base pairs) had an open reading frame encoding for a 669-amino acid protein (calculated molecular mass of 74 kDa) which shows 72% identity with the cloned rat liver organic anion transporter, oatp, Northern hybridization and reverse transcription coupled polymerase chain reaction revealed that the rat OAT-K1 messenger RNA transcript is expressed predominantly in the kidney. By use of stable LLC-PK1 cell monolayers transfected with the rat OAT-K1 cDNA, the transporter was suggested to mediate basolateral uptake of methotrexate, an anionic anticancer drug, but not taurocholate, p-aminohippurate, prostaglandin E(2), and leukotriene C-4, The methotrexate transport by rat OAT-K1 was unaffected by the presence of Na+ or Cl(-)gradient. The methotrexate accumulation by the OAT-K1-expressing cells showed saturability with the apparent K-m value of 1.0 mu m. Folate, sulfobromophthalein, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the methotrexate accumulation markedly, These findings suggest that the rat OAT-K1 is localized in the basolateral membranes of renal tubules, where it mediates renal clearance of methotrexate from the blood.