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 E2, and leukotriene C4. 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 Km value of 1.0 microM. 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.
Saito
H
H
Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan.
Masuda
S
S
Inui
K i
Ki
D000595:Amino Acid Sequence; D000818:Animals; D027321:Anion Transport Proteins; D000838:Anions / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D016764:Cell Polarity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D007672:Kidney Cortex / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism*; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014018:Tissue Distribution
D027321:Anion Transport Proteins; D000838:Anions; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D008565:Membrane Proteins; D027361:Organic Anion Transporters; D012333:RNA, Messenger; C101140:Slc21a4 protein, rat; D008727:Methotrexate