Abstract
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.
Highlights
Secretion of anionic endogenous substances and xenobiotics is an important function of the liver and kidney
The deduced amino acid sequence of OAT-K1 has 72% identity with the rat oatp [4], another Naϩ-independent organic anion transporter identified in the liver
Previous studies on clearance experiments, renal slice uptake, and isolated renal tubules indicated that methotrexate secretion was affected by other anionic drugs such as p-aminohippurate [21], probenecid [22], and penicillin [23], suggesting the contribution of the organic anion transport system
Summary
Reverse Transcription-coupled PCR and cDNA Sequencing—Degenerated PCR primers based on the amino acid sequence of rat oatp [4] are as follows: sense strand, 5Ј-CCGAATTCTG(T/C)GC(A/C/T)TG(T/C)(T/ C)T(A/G/T)AC(A/C/T)AA-3Ј (corresponding to amino acid sequence 28 – 33); antisense strand, 5Ј-CCGGATCCCCCAT(A/G)AA(A/G)AA(A/G) TG(A/G/T)GG-3Ј (corresponding to amino acid sequence 106 –111). The PCR products (approximately 270 bp) were cut with EcoRI and BamHI on both ends and ligated into the EcoRI- and BamHI-cut pSPORT1 (Life Technologies, Inc.). Both strands of the subcloned cDNA inserts were sequenced by the chaintermination method with a Sequenase version 2.0 DNA sequencing kit
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