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

Hideyuki Salto, Satohiro Masuda, Ken Ichi Inui

Research output: Contribution to journalArticle

187 Citations (Scopus)

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 K(m) value of 1.0 μM. Folate, sulfobromophthalein, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the methotrexate accumulation markedly. These findings suggest that the rat OAT-KI is localized in the basolateral membranes of renal tubules, where it mediates renal clearance of methotrexate from the blood.

Original languageEnglish
Pages (from-to)20719-20725
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number34
DOIs
Publication statusPublished - Sep 5 1996

Fingerprint

Organic Anion Transporters
Cloning
Methotrexate
Rats
Organism Cloning
Kidney
Complementary DNA
LLC-PK1 Cells
Sulfobromophthalein
p-Aminohippuric Acid
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid
Leukotriene C4
Taurocholic Acid
Polymerase chain reaction
Molecular mass
Transcription
Folic Acid
Dinoprostone
Base Pairing
Liver

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney. / Salto, Hideyuki; Masuda, Satohiro; Inui, Ken Ichi.

In: Journal of Biological Chemistry, Vol. 271, No. 34, 05.09.1996, p. 20719-20725.

Research output: Contribution to journalArticle

@article{f9f0cefd4b9a4580b12de101ae4b2bfc,
title = "Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney",
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 K(m) value of 1.0 μM. Folate, sulfobromophthalein, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the methotrexate accumulation markedly. These findings suggest that the rat OAT-KI is localized in the basolateral membranes of renal tubules, where it mediates renal clearance of methotrexate from the blood.",
author = "Hideyuki Salto and Satohiro Masuda and Inui, {Ken Ichi}",
year = "1996",
month = "9",
day = "5",
doi = "10.1074/jbc.271.34.20719",
language = "English",
volume = "271",
pages = "20719--20725",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "34",

}

TY - JOUR

T1 - Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney

AU - Salto, Hideyuki

AU - Masuda, Satohiro

AU - Inui, Ken Ichi

PY - 1996/9/5

Y1 - 1996/9/5

N2 - 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 K(m) value of 1.0 μM. Folate, sulfobromophthalein, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the methotrexate accumulation markedly. These findings suggest that the rat OAT-KI is localized in the basolateral membranes of renal tubules, where it mediates renal clearance of methotrexate from the blood.

AB - 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 K(m) value of 1.0 μM. Folate, sulfobromophthalein, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited the methotrexate accumulation markedly. These findings suggest that the rat OAT-KI is localized in the basolateral membranes of renal tubules, where it mediates renal clearance of methotrexate from the blood.

UR - http://www.scopus.com/inward/record.url?scp=0029786681&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029786681&partnerID=8YFLogxK

U2 - 10.1074/jbc.271.34.20719

DO - 10.1074/jbc.271.34.20719

M3 - Article

C2 - 8702823

AN - SCOPUS:0029786681

VL - 271

SP - 20719

EP - 20725

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 34

ER -