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 μM. 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.

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 transcriptioncoupled polymerase chain reaction revealed that the rat OAT-K1 messenger RNA transcript is expressed predominantly in the kidney. By use of stable LLC-PK 1 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 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.
Secretion of anionic endogenous substances and xenobiotics is an important function of the liver and kidney. However, these tissues have different physiological and pharmacological roles in the secretion of organic anions. In particular, bile formation is the most important transport process of hepatocytes. Bile acids and other anionic drugs are taken up by the hepatocytes from portal blood via specific transport systems localized in the sinusoidal plasma membranes. The characteristics and mechanisms of organic anion transport have been studied by use of the perfused liver, isolated hepatocytes, and purified sinusoidal membrane vesicles (1,2). Conjugated bile acids such as taurocholate and glycocholate enter specifically hepatocytes via the Na ϩ -dependent secondary active transport process. In addition, a Na ϩ -independent transport process has been indicated as another route for hepatic extraction of a variety of organic anions.
Using the functional expression cloning strategy in Xenopus oocytes, Na ϩ -dependent taurocholate transporter (3) and Na ϩindependent organic anion transporter (oatp) 1 (4) of the rat liver have been identified. These transporter proteins are likely to constitute multispecific pathways for the hepatic extraction of anionic substances. In contrast, organic anion transport system(s) in the kidney has distinct characteristics compared with the systems in the liver. Previous studies using isolated basolateral membrane vesicles indicated that uptake of p-aminohippurate, a typical organic anion excreted by the kidney, is driven by an outwardly directed ␣-ketoglutarate gradient, an exchange system for organic anion with dicarboxylic acid (5,6). The kidney-specific organic anion transporters mediating secretion of p-aminohippurate and/or other anionic compounds have not been identified yet. The fact that a variety of structurally unrelated anionic drugs such as p-aminohippurate, furosemide, and methotrexate, which are secreted mainly by the kidney, suggests diversity of the renal organic anion transport systems. The detection of messenger RNA transcripts related to the oatp in the rat kidney also implicated localization of multispecific organic anion transporters along the nephron (4). The above findings encouraged us to investigate the expression of transporter protein(s) homologous to the rat oatp in the renal tubules.
We report here the isolation of cDNA encoding a novel rat organic anion transporter, which mediates uptake of methotrexate across the renal basolateral membranes.

EXPERIMENTAL PROCEDURES
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). One g of poly(A) ϩ RNA extracted from the kidney cortex of male Wistar rats was reverse-transcribed as reported previously (7). The synthesized cDNA was used for subsequent PCR with a set of degenerate primers (5 M) according to the following profile: 94°C for 1 min, 50°C for 1 min, 72°C for 2 min, 30 cycles. 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 (U. S. Biochemical Corp.).
Screening of cDNA Library-The oligo(dT)-primed directional rat kidney cDNA library (8) was screened by hybridization with the PCR clone labeled with [␣-32 P]dCTP (3000 Ci/mmol; 1 Ci ϭ 37 GBq; Amersham Int., Buckinghamshire, UK) as described previously (7). A positive clone (rat OAT-K1) was isolated with a 2.8-kb insert and was subcloned into SalI-and NotI-cut pSPORT1. Nested deletion clones were prepared by use of the Erase-A-Base system (Promega Corp., Madison, WI) and sequenced as described above. The complete sequence was determined on both strands with synthetic oligonucleotide primers.
Cell Culture and Transfection-The parental LLC-PK 1 cells were cultured in the complete medium consisted of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum (Whittaker Bioproducts Inc., Walkersville, MD) in an atmosphere of 5% CO 2 , 95% air at 37°C (9). OAT-K1 cDNA was subcloned into the SalI-and NotI-cut mammalian expression vector pBK-CMV (Stratagene) (10). LLC-PK 1 cells were transfected with pBK-CMV/OAT-K1 or pBK-CMV using the calcium phosphate coprecipitation technique (11). Fifteen hours after the transfection, cells were rinsed with Ca 2ϩ -and Mg 2ϩ -free Dulbecco's phosphate-buffered saline (pH 7.4) (PBS(Ϫ) buffer comprising 137 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 , and 1.5 mM KH 2 PO 4 ) containing 15% glycerol for 3 min and then cultured under the normal condition. Twenty-four hours later, the cells split 1:75 were cultured in the complete medium containing G418 (1 mg/ml) (Life Technologies, Inc.). Ten to fifteen days after the transfection, single colonies appeared and were picked up for subsequent screening. G418-resistant clonal cells were analyzed by both reverse transcription-PCR and Northern blotting for the expression of rat OAT-K1 mRNA. For the transport experiments, cells were seeded on microporous membrane filters (3-m pores, 4.71 cm 2 -growth area) inside a Transwell cell culture chamber (Costar, Cambridge, MA) at a cell density of 5 ϫ 10 5 cells/cm 2 with the complete medium (9). In this study, LLC-PK 1 cells between the 225th and 235th passages were used.
Uptake Study-Cellular uptake of radioactive drugs was measured using monolayer cultures grown in the Transwell chamber. The incubation medium for uptake experiments was Dulbecco's phosphate-buffered saline (pH 7.4) (PBS buffer, 137 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , 1 mM CaCl 2 , and 0.5 mM MgCl 2 ), containing 5 mM D-glucose. In Na ϩ -free medium, NaCl and NaH 2 PO 4 of PBS buffer were replaced with N-methyl-D-glucamine and KH 2 PO 4 , respectively. In Cl Ϫ -free medium, NaCl, KCl, CaCl 2 , and MgCl 2 were replaced with sodium gluconate, potassium gluconate, calcium gluconate, and MgSO 4 , respectively. In general, uptake measurements were performed as described previously (9). Under the ATP-depleted condition, cells were preincubated for 20 min and incubated for 15 min at 37°C in the PBS buffer containing 20 mM 2-deoxy-D-glucose and 10 mM NaN 3 without D-glucose (12). The protein content of the solubilized cell monolayers was determined by the method of Bradford (13), using a Bio-Rad Protein Assay kit (Bio-Rad) with the bovine ␥-globulin as a standard. The protein content of the intact monolayers was 0.9 -1.1 mg/filter (4.71 cm 2 ).
Tissue Distribution of Methotrexate in Rats-Male Wistar rats weighing 200 -220 g were anesthetized with sodium pentobarbital (40 mg/kg intraperitoneal). A tracer amount of [ 3 H]methotrexate (5 mol/kg, 7.4 MBq/ml) was administered as a bolus via catheterized left femoral vein. Five and 30 min after the injection, blood and several tissue specimens were collected immediately after sacrificing of rats. The excised tissues were gently washed with 0.9% NaCl, and the wet weight was determined. After homogenizing the tissues in 3 volumes of 0.9% NaCl and separating plasma from blood, an aliquot (100 l) of each sample was solubilized in 0.5 ml of NCS II (Amersham Corp.) and then the radioactivity was determined in 5 ml of ACS II (Amersham Corp.) by liquid scintillation counting.
Statistical Analysis-Data were analyzed statistically using one-way analysis of variance followed by Fisher's t test.  Japan). Sulfobromophthalein, taurocholate, p-aminohippurate, furosemide, NaN 3 , and 2-deoxy-D-glucose were purchased from Nacalai Tesque (Kyoto, Japan). All other chemicals used for the experiment were of the highest purity available.

RESULTS
Sequencing of several PCR products originating from the rat kidney cortex revealed that the existence of a PCR clone that was homologous (ϳ80% nucleotide identity) to the oatp cDNA. A single cDNA clone coding OAT-K1 was isolated from the rat kidney cDNA library, subcloned into a plasmid (pSPORT1), and sequenced. The OAT-K1 cDNA consists of 2788 bp with an open reading frame encoding a 669-amino acid protein (calculated molecular mass of 74 kDa) and with a poly(A) ϩ tail (Fig. 1). Based on the Kozak consensus sequence (14), we assigned the initiation site to the first ATG codon at position 159. Fig. 2A shows the deduced amino acid sequence of rat OAT-K1 and its alignment with the rat oatp. Rat OAT-K1 showed amino acid identity of 72% with the rat oatp and of 35% with the rat matrin F/G (recently defined as a prostaglandin transporter, PGT) (15,16), but a search of available data bases (GenBank, EMBL and SWISS-PROT, February 1996) revealed no significant homology with any other cloned membrane transporter proteins. Kyte-Doolittle (17) hydropathy analysis suggested that rat OAT-K1 has 12 putative membrane-spanning ␣-helices (Fig. 2B), thereby indicating four potential Nlinked glycosylation sites in the extracellular loop. There are three potential cAMP-dependent kinase phosphorylation sites (18) at positions 290, 383, and 644. Three potential protein kinase C phosphorylation sites (18) are present at positions 383, 644, and 648.
We examined the tissue distribution of OAT-K1 mRNA transcripts by Northern blot analysis (Fig. 3A). Under both low and high stringent conditions, the whole OAT-K1 cDNA probe hybridized with mRNA transcripts from the rat liver (ϳ3 and ϳ4 kb), kidney cortex (ϳ3 kb), and kidney medulla (ϳ3 kb). For PCR analysis of rat oatp and OAT-K1 mRNA expression, a set of specific primers for the cDNA of oatp and OAT-K1 was used, respectively. As shown in Fig. 3B, the PCR product with the expected size for rat oatp was found in the liver, kidney cortex, and kidney medulla but not in a template of the rat OAT-K1 cRNA (upper). On the other hand, a PCR product of the expected size for rat OAT-K1 was found in both the kidney cortex and kidney medulla as well as in the OAT-K1 cRNA but not in other tissues examined (lower). Therefore, the signal detected in the liver by Northern blotting was suggested to be the oatp-related mRNA (4).
To characterize the transport function of OAT-K1, the accumulation of various anionic drugs was measured by use of the stable transfectant LLC-OAT-K1 monolayers grown on membrane filters. Six transfectants, which appeared to express the rat OAT-K1 mRNA, were isolated, and a transfectant showing the highest mRNA expression was used for the subsequent experiments. As shown in Fig. 4, methotrexate accumulation in the LLC-OAT-K1 monolayers from the ba-solateral side was enhanced markedly compared with that in the cell monolayers (LLC-pBK) transfected with the expression vector lacking an insert of the rat OAT-K1 cDNA. Neither taurocholate, p-aminohippurate, prostaglandin E 2 , nor leukotriene C 4 was accumulated into the LLC-OAT-K1 monolayers. The other five transfectants obtained also showed transport activity of methotrexate (data not shown). In a separate experiment, cellular accumulation of  Fig. 5 illustrates the time course of methotrexate accumulation from the basolateral side and the apical side in the monolayers of LLC-OAT-K1 and LLC-pBK cells. The accumulation from the basolateral side was much higher in the LLC-OAT-K1 monolayers than in the LLC-pBK monolayers. In contrast, the accumulation from the apical side in LLC-OAT-K1 monolayers was comparable with that in the LLC-pBK monolayers. Therefore, the rat OAT-K1 was suggested to be expressed functionally in basolateral membranes of the monolayers. In addition, transepithelial flux of [ 3 H]methotrexate was less than that of D-[ 14 C]mannitol in both directional experiments (Ͻ1.5% of the applied amount for 60 min). Fig. 6 shows the accumulation of methotrexate from the basolateral side in the LLC-OAT-K1 monolayers as a function of the substrate concentration. The nonspecific component was estimated from the uptake in the presence of excess unlabeled methotrexate (1 mM). The curve for the accumulation of methotrexate showed a saturability, whereas that of nonspecific uptake was almost linear over the concentration range examined. The specific component of the uptake was evaluated kinetically using nonlinear least-squares regression analysis (19) following the Michaelis-Menten equation. The values of the apparent K m and V max for the methotrexate transport were 1.0 M and 31.7 pmol/mg protein/15 min, respectively.
To characterize the substrate specificity of the rat OAT-K1, we examined the methotrexate transport by LLC-OAT-K1 monolayers by the cis inhibition method. All drugs were used at a concentration of 100 M except for methotrexate which was used at both 10 and 100 M. As illustrated in Fig. 7, [ 3 H]methotrexate accumulation was inhibited markedly in the presence of either folate, unlabeled methotrexate, or DIDS. Probenecid, p-aminohippurate, furosemide, and valproate, which are substrates for the renal organic anion transport system, had relatively weak but significant inhibitory effects on the [ 3 H]methotrexate accumulation. Sulfobromophthalein and taurocholate, which are substrates for the rat oatp (4), also showed significant inhibitory potencies on the [ 3 H]methotrexate accumulation.
As summarized in Table I, replacement of Na ϩ by N-methyl-D-glucamine had no significant effect on the methotrexate accumulation into LLC-OAT-K1 monolayers. Furthermore, replacement of Cl Ϫ with gluconate caused no significant effect on the accumulation. To examine whether the rat OAT-K1-mediated transport of methotrexate is an energy-dependent process, the effects of temperature dependence and cellular ATP depletion on its accumulation were also examined. As shown in Table I, when the monolayers were incubated at 4°C, methotrexate accumulation was decreased to 45% of the control at 37°C. Under intracellular ATP-depleted condition, the methotrexate accumulation was decreased to 81% of the control.
The tissue distribution of methotrexate in vivo was examined. At 5 min after the bolus injection, [ 3 H]methotrexate concentrations in both the kidney and liver were much higher than in any of the other tissues (Table II). The tissue to plasma concentration ratio at 5 min after the administration indicated that [ 3 H]methotrexate was accumulated in the following order: kidney medulla, kidney cortex Ͼ liver Ͼ Ͼ brain, heart, lung, small intestine, and spleen. At 30 min following the administration, the ratio in the kidney cortex, kidney medulla, and liver was increased extensively to 923, 584, and 224, respectively. DISCUSSION We have isolated and characterized cDNA encoding a novel organic anion transporter OAT-K1 expressed specifically in the kidney of rats. The deduced amino acid sequence of OAT-K1 has 72% identity with the rat oatp (4), another Na ϩ -independent organic anion transporter identified in the liver.
The rat OAT-K1 mediates basolateral uptake of methotrexate with a relatively high affinity (apparent K m value of 1.0 M) in the stable transfectant cell monolayers, suggesting the transporter contributes to renal elimination of the drug. Urinary excretion is the predominant pathway of methotrexate elimination from the body, inevitably reaching high concentrations in the nephron to cause nephrotoxicity, a major limitation of methotrexate therapy (20). 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. Recent findings with isolated basolateral membrane vesicles from the kidney suggest that p-aminohippurate is transported via an organic anion/dicarboxylate exchange system (6). However, methotrexate has not been demonstrated to be recognized and transported by the "organic anion/dicarboxylate exchange system" in the renal basolateral membranes. In the present studies, both Northern hybridization and PCR analyses revealed that the rat OAT-K1 mRNA appeared to be expressed specifically in the kidney but not in the liver (Fig. 3). The uptake studies of methotrexate by use of monolayers of the rat OAT-K1 transfectant cells indicated clearly that the transporter is localized functionally at the basolateral membranes but not at the apical membranes (Fig.  5). Each anionic drug examined in the uptake studies, such as taurocholate, p-aminohippurate, prostaglandin E 2 , and gluta-thione S-conjugate leukotriene C 4 , a substrate with the highest affinity for multidrug resistance protein (MRP) (24), was not taken up by the rat OAT-K1-expressing cells (Fig. 4). Although taurocholate and p-aminohippurate inhibited weakly the methotrexate transport (Fig. 7), both anions were not substrates for the rat OAT-K1. Therefore, the rat OAT-K1 has substrate specificity that is distinct from that of the oatp, PGT, and MRP and of the typical renal p-aminohippurate transporter. These findings suggest that the OAT-K1 mediates methotrexate uptake across the basolateral membranes in the renal tubules and is functionally independent of the systems known to transport other anionic drugs.
Methotrexate also undergoes biliary excretion by the liver. Horne and Reed (25) reported that methotrexate uptake into rat liver basolateral membrane vesicles is a carrier-mediated process, is independent of imposed Na ϩ or H ϩ gradients, is electrogenic, and is inhibited by various structurally unrelated anions and the folate analog. Although the rat oatp would be a functional member of the liver organic anion transporters, whether the rat oatp recognizes and transports methotrexate remains unknown. The present findings that methotrexate is accumulated extensively in both the kidney and liver in vivo demonstrate the involvement of high affinity transport system(s) for methotrexate clearance at the basolateral membranes of these organs. The functional characterization of the rat OAT-K1 suggests that this transporter contributes to efficient basolateral uptake of methotrexate in the kidney but not in the liver. Further studies are needed to define a role of the rat oatp and OAT-K1 in the hepatic and/or renal elimination of anionic drugs.
In the present study, we observed that the structure analog folate has a potent inhibitory effect on the methotrexate uptake and is transported by the rat OAT-K1-expressing cells. A specific transporter for folate in rat intestinal basolateral mem-branes which is electroneutral and Na ϩ -independent has been reported to exist (26). The rat OAT-K1 is unlikely an intrinsic folate transporter, because the OAT-K1 mRNA expression is not detected in the small intestine. The findings that the OAT-K1-mediated methotrexate uptake was inhibited by structurally unrelated organic anions, such as sulfobromophthalein, taurocholate, and DIDS suggest that the OAT-K1 is a multispecific anion transporter involved in renal detoxification process. Furthermore, the rat OAT-K1 shows no significant amino acid identity and no structural homology with the intrinsic folate transporter proteins (27,28). It should be further studied whether the rat OAT-K1 participates physiologically in the renal handling of folate.
Methotrexate accumulation in the LLC-OAT-K1 cells was unaffected in both the Na ϩ -and Cl Ϫ -free incubation medium, suggesting that methotrexate transport is not mediated by either a methotrexate/Cl Ϫ exchanger or a Na ϩ -coupling process, Na ϩ -independent transport system as for the case of rat oatp and PGT. Furthermore, the incubation temperature at

FIG. 3. Northern blot analysis (A) and detection by PCR amplification (B) of OAT-K1 mRNA in rat tissues.
A, poly(A) ϩ RNA (2.5 g) from the indicated tissues was electrophoresed, blotted, and hybridized with the whole OAT-K1 cDNA as a probe at low stringency (upper) and high stringency (lower). B, poly(A) ϩ RNA (1 g) from the indicated tissues and of OAT-K1 cRNA (50 ng) was reverse-transcribed, and the cDNA synthesized was amplified using a set of primers specific for the rat oatp (upper) or for the rat OAT-K1 (lower) as described in the text. The PCR products were separated by electrophoresis through 1% agarose gels and stained with ethidium bromide. added to the basolateral side (2 ml, pH 7.4) of the monolayers. Unlabeled incubation medium was added to the apical side (2 ml, pH 7.4). After incubation, monolayers were rapidly washed twice with 2 ml of ice-cold incubation medium in both sides, and the radioactivity of solubilized cells was determined. Accumulation of each drug is expressed as uptake clearance. Each column represents the mean Ϯ S.E. of three monolayers.
4°C led a marked decrease in the methotrexate transport, whereas the condition in which the cellular ATP was depleted caused only a small depression of the methotrexate transport (Table I). These findings suggest that the process of OAT-K1mediated methotrexate uptake might be a facilitated transport process but not a secondary active transport process. Precise transport mechanisms including its coupling with other ions of the OAT-K1 should be further studied.
In conclusion, cDNA encoding a novel member of organic anion transporter proteins, OAT-K1, was isolated from the kidney of rats. Predominant expression of the OAT-K1 in the kidney among rat tissues, its functional properties, and localization at the basolateral membranes of the transfected cell line suggest that the OAT-K1 contributes to renal clearance of methotrexate from the blood.