Introduction

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A wide variety of anionic compounds, including endogenous substances, xenobiotics, and their metabolites are excreted mainly from the liver and the kidney. Many studies have been performed regarding the organic anion transport systems, which are involved in preventing toxicity in the plasma membranes of the liver and the kidney (Tiribelli et al., 1990; Pritchard and Miller, 1993; Inui and Okuda, 1998). Recent advances in molecular techniques have identified several candidates for organic anion transporters (Inui et al., 2000; Kullak-Ublick et al., 2000).

The kidney-specific organic anion transporter OAT-K1 was isolated in our laboratory from a rat kidney cDNA library (Saito et al., 1996). In the rat kidney, OAT-K1 was localized in the brush-border membranes of the proximal straight tubules and outer medullary collecting ducts (Masuda et al., 1997). When OAT-K1 cDNA was transfected into the porcine kidney epithelial cell line LLC-PK₁ (LLC-OAT-K1), the transporter protein was localized in the basolateral membranes without post-translational processing (Masuda et al., 1997). Functional analysis by using OAT-K1-expressing Xenopus laevis oocytes and LLC-OAT-K1 cells showed that OAT-K1 specifically transported methotrexate and folic acid, but not p-aminohippuric acid or taurocholic acid (Saito et al., 1996; Masuda et al., 1999a). However, in Madin-Darby canine kidney cells stably transfected with OAT-K1 cDNA (MDCK-OAT-K1), the transporter protein was localized in the apical membranes after post-translational processing as a small molecule, which corresponded to the results obtained in the rat kidney (Masuda et al., 1997, 1999b). Therefore, functions of OAT-K1 in the renal brush-border membranes as a post-translational processed form have still to be elucidated.

The second isoform, OAT-K2, was also cloned and characterized in our laboratory (Masuda et al., 1999a). OAT-K2 was localized specifically in the brush-border membranes of the rat kidney, especially in the proximal convoluted tubules and cortical collecting ducts. By using OAT-K2-expressing oocytes and MDCK transfectant (MDCK-OAT-K2), OAT-K2 was shown to transport a wide range of substrates, including methotrexate, folic acid, taurocholic acid, and prostaglandin E2. Because OAT-K2 showed 91% amino acid identity with OAT-K1, the characteristics of these transporters may be closely related.

More recently, detailed in vivo and in vitro analyses demonstrated the roles of OAT-K1 and OAT-K2 in clinical therapy. That is, OAT-K1 and OAT-K2 represent the major excretion route of methotrexate from the proximal tubules into the urine, especially in the case of "folinic acid rescue" (Takeuchi et al., 2000, 2001). Because OAT-K1 and OAT-K2 show over 70% amino acid identity with other homologs, oatp1 (Jacquemin et al., 1994), oatp2 (Noé et al., 1997), and oatp3 (Abe et al., 1998), several substrates and transport characteristics of these transporters may overlap. A wide variety of organic compounds, including bile salts, organic anionic dyes (e.g., sulfobromophthalein), steroid conjugates (e.g., estrone 3-sulfate, dehydroepiandrosterone 3-sulfate, and estradiol 17-D-glucuronide), and thyroid hormones have been shown to be substrates for oatp1 and oatp2, mediating sinusoidal uptake of compounds into the hepatocytes (Jacquemin et al., 1994; Noé et al., 1997; Reichel et al., 1999). Because some of these compounds are excreted and/or reabsorbed from the renal proximal tubules (Honjo et al., 1976; Adlkofer et al., 1980; Longcope, 1995), it is possible that OAT-K1 and OAT-K2 are involved in the renal handling of these compounds. However, there have been few studies in this respect, and the role(s) of OAT-K1 and OAT-K2 in the kidney under physiological conditions remains to be elucidated.

In the present study, we demonstrated the polyspecific substrate recognition of OAT-K1 and OAT-K2 and the unique characteristics of these transporters in the mutual interaction between substrates with them.

	Experimental Procedures

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Materials. [3',5',7'-³H(N)]Methotrexate, disodium salt (991.6 GBq/mmol); [3',5',7',9'-³H]folic acid, diammonium salt (1.23 GBq/mmol); [methyl-³H]zidovudine (558.7 GBq/mmol); and [³H(G)]ochratoxin A (547.6 GBq/mmol) were obtained from Moravek Biochemicals (Brea, CA). [³H(G)]Taurocholic acid (111 GBq/mmol); [1,2,6,7-³H(N)]dehydroepiandrosterone (DHEA) (2220 GBq/mmol); [1,2,6,7-³H(N)]DHEA sulfate, sodium salt (2220 GBq/mmol); [estradiol-6,7-³H(N)]estradiol 17-D-glucuronide (1628 GBq/mmol); [6,7-³H(N)]estrone sulfate, ammonium salt (1961 GBq/mmol); L-3,5,3'-[¹²⁵I]triiodothyronine (28.8 TBq/mmol); L-[¹²⁵I]thyroxine (35.9 TBq/mmol); [1-¹⁴C]tetraethylammonium bromide (88.8 MBq/mmol); and [³H(G)]digoxin (703 GBq/mmol) were from PerkinElmer Life Science Products (Boston, MA). [N-methyl-³H]Cimetidine (814 GBq/mmol) was from Amersham Pharmacia Biotech UK, Ltd. (Little Chalfont, Buckinghamshire, UK).

Cell Culture and Transfection. The MDCK cells stably expressing OAT-K1 and OAT-K2, designated MDCK-OAT-K1 and MDCK-OAT-K2, were constructed and maintained as described previously with some modifications (Masuda et al., 1999a).

Transport Studies by Cell Monolayers. The cellular uptake of radiolabeled drugs was measured using monolayer cultures grown in 12-well microplates as described previously (Takeuchi et al., 2000). The incubation medium for uptake experiments was Dulbecco's phosphate-buffered saline (137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 1 mM CaCl₂, and 0.5 mM MgCl₂; pH 7.4) containing 5 mM D-glucose (uptake buffer). Experimental procedures were performed as described previously (Takeuchi et al., 2000). Unlabeled triiodothyronine, thyroxine, DHEA, and ochratoxin A were dissolved in dimethyl sulfoxide, the final concentration of which in uptake buffer was 0.5%. After incubation, the cells were lysed in 0.5 N NaOH solution, and then the radioactivity in aliquots was determined in 5 ml of ACSII (Amersham Pharmacia Biotech UK, Ltd.). The protein content of the solubilized cells was determined by the method of Bradford (Bradford, 1976), by using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) with bovine -globulin as a standard.

Functional Expression of oatp2 in X. laevis Oocytes. Aliquots of 25 ng of capped RNA transcribed in vitro from NotI-linearized oatp2 cDNA with T7 RNA polymerase were injected into X. laevis oocytes. Injected oocytes were maintained in modified Barth's medium [88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO₃)₂, 0.4 mM CaCl₂, 0.8 mM MgSO₄, 2.4 mM NaHCO₃, and 10 mM HEPES] containing 50 µg/ml gentamicin at 18°C for 3 days. The uptake study was initiated by incubating oocytes in 500 µl of uptake buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES; pH 7.4) containing [³H]digoxin at 25°C in the presence or absence of various inhibitors for 1 h. Unlabeled triiodothyronine and digoxin was dissolved in dimethyl sulfoxide, and its final concentration in uptake buffer was 0.5%. At the end of incubation, the oocytes were washed five times with 2 ml of ice-cold uptake buffer. After washing, each oocyte was transferred to a single vial and dissolved with 500 µl of 10% sodium lauryl sulfate. The radioactivity of each solubilized oocyte was determined in 5 ml of ACSII.

Statistical Analysis. Data were analyzed statistically using one-way analysis of variance followed by Fisher's t test.

	Results

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Uptake of Various Compounds by MDCK-OAT-K1 and MDCK-OAT-K2 Cells. First, we examined the uptake of various compounds by MDCK-OAT-K1 and MDCK-OAT-K2 cells, in comparison with untransfected MDCK cells as a control. As shown in Table 1, uptake of structurally unrelated anionic compounds such as [³H]methotrexate, [¹²⁵I]triiodothyronine, [¹²⁵I]thyroxine, [³H]DHEA 3-sulfate, [³H]estradiol 17-D-glucuronide, [³H]estrone 3-sulfate, [³H]taurocholic acid, and [³H]ochratoxin A was significantly enhanced in MDCK-OAT-K1 and MDCK-OAT-K2 cells compared with untransfected MDCK cells. In addition, the uptake of the antiretroviral drug [³H]zidovudine, which possesses no typical anionic moiety, was also greater in MDCK-OAT-K1 and MDCK-OAT-K2 cells than that in MDCK cells. However, the uptake of unconjugated steroid [³H]DHEA and typical substrates for organic cation transporters, [¹⁴C]tetraethylammonium and [³H]cimetidine, was comparable among MDCK-OAT-K1, MDCK-OAT-K2, and untransfected MDCK cells.

View this table: [in this window] [in a new window]   TABLE 1 Substrate specificities of OAT-K1 and OAT-K2 MDCK, MDCK-OAT-K1, and MDCK-OAT-K2 cells were incubated for 30 min at 37°C with [3H]methotrexate (100 nM), [3H]DHEA (4.2 nM), [3H]DHEA 3-sulfate (4.2 nM), [3H]estrone 3-sulfate (18.9 nM), [3H]estradiol 17-D-glucuronide (11.4 nM), [125I]triiodothyronine (0.43 nM), [125I]thyroxine (0.34 nM), [3H]taurocholic acid (250 nM), [3H]ochratoxin A (33.8 nM), [14C]tetraethylammonium (50 µM), [3H]cimetidine (45 nM), and [3H]zidovudine (66.2 nM). After incubation, the radioactivity of the solubilized cells was determined. Each value represents the mean ± S.E. of three monolayers.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Chemical structures of structurally unrelated substrates for OAT-K1 and OAT-K2.

Mutual Inhibition between Substrates via OAT-K1 and OAT-K2. We then examined whether structurally unrelated substrates caused mutual inhibition on the uptake activity of OAT-K1 and OAT-K2. Figure 2 shows the effects of various compounds on [³H]methotrexate and [³H]taurocholic acid uptake via OAT-K1 and OAT-K2. [³H]Methotrexate uptake via OAT-K1 and OAT-K2 was markedly inhibited by unlabeled methotrexate but showed little or no inhibition by structurally unrelated substrates at the concentration of 100 µM. On the other hand, [³H]taurocholic acid uptake was significantly inhibited by taurocholic acid, DHEA, its conjugated anionic compounds DHEA 3-sulfate and DHEA 3-acetate, and ochratoxin A, although methotrexate and zidovudine did not have any effect at the concentration of 100 µM. Interactions of methotrexate and thyroid hormones with OAT-K1 and OAT-K2 are shown in Fig. 3. Inhibition of [³H]methotrexate uptake by unlabeled thyroid hormones at a concentration of 50 µM, which was higher than the apparent K_m value of each compound, was relatively weak or was not observed. In addition, 2.5 mM unlabeled methotrexate, which was about 1000-fold higher than the apparent K_m values for OAT-K1 and OAT-K2, inhibited only about 20% of the control uptake of ¹²⁵I-thyroid hormones. [³H]Zidovudine uptake via OAT-K1 and OAT-K2 was significantly inhibited by unlabeled zidovudine. However, unlabeled methotrexate, tetraethylammonium, and cimetidine at the concentration of 100 µM did not inhibit OAT-K1- or OAT-K2-mediated [³H]zidovudine uptake (Fig. 4).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Effects of various compounds on [3H]methotrexate (A) and [3H]taurocholic acid (B) uptake by MDCK-OAT-K1 and MDCK-OAT-K2 cells. Uptake of 100 nM [3H]methotrexate (A) and 250 nM [3H]taurocholic acid (B) by MDCK-OAT-K1 () or MDCK-OAT-K2 () cells cultured in 12-well microplates was measured for 15 min at 37°C in the absence or presence of the indicated compounds at a concentration of 100 µM. After incubation, the radioactivity of the solubilized cells was determined. Data are expressed as percentage of the control value. Each column represents the mean ± S.E. for three monolayers. **p < 0.01, significant differences from control.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of thyroid hormones on [3H]methotrexate uptake (A) and effects of methotrexate on 125I-thyroid hormones uptake (B and C) by MDCK-OAT-K1 and MDCK-OAT-K2 cells. A, uptake of 100 nM [3H]methotrexate by MDCK-OAT-K1 () or MDCK-OAT-K2 () cells cultured in 12-well microplates was measured for 15 min at 37°C in the absence or presence of the indicated compounds at a concentration of 50 µM. B and C, uptake of 0.43 nM [125I]triiodothyronine (B) and 0.34 nM [125I]thyroxine (C) by MDCK-OAT-K1 () or MDCK-OAT-K2 () cells cultured in 12-well microplates was measured for 15 min at 37°C in the absence or presence of the 2.5 mM methotrexate. After incubation, the radioactivity of the solubilized cells was determined. Data are expressed as percentage of the control value. Each column represents the mean ± S.E. for three monolayers. *p < 0.05; **p < 0.01, significant differences from control. T3, triiodothyronine; T4, thyroxine.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Effects of various compounds on [3H]zidovudine uptake by MDCK-OAT-K1 and MDCK-OAT-K2 cells. Uptake of 66.2 nM [3H]zidovudine by MDCK-OAT-K1 () or MDCK-OAT-K2 () cells cultured in 12-well microplates was measured for 15 min at 37°C in the absence or presence of the indicated compounds at a concentration of 100 µM. After incubation, the radioactivity of the solubilized cells was determined. Data are expressed as percentage of the control value. Each column represents the mean ± S.E. for three monolayers. **p < 0.01, significant differences from control. TEA, tetraethylammonium.

Trans-Stimulation Effects of Intracellular Methotrexate on Uptake of Various Labeled Compounds via OAT-K1 and OAT-K2. To obtain more information about the characteristics of substrate recognition of OAT-K1 and OAT-K2, we examined the trans-stimulation effects of intracellular unlabeled methotrexate on the uptake of various labeled substrates via OAT-K1 and OAT-K2. Although the uptake of [³H]folic acid was stimulated in the presence of the counterdirected transmembrane gradient of unlabeled methotrexate, the uptake of [³H]DHEA, [³H]DHEA 3-sulfate, [¹²⁵I]triiodothyronine, and [¹²⁵I]thyroxine by OAT-K1 and OAT-K2 was not stimulated (Fig. 5).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Trans-stimulation effects of intracellular unlabeled methotrexate on the uptake of various labeled compounds by MDCK-OAT-K1 and MDCK-OAT-K2 cells. MDCK-OAT-K1 () and MDCK-OAT-K2 () cells were preloaded with methotrexate at a concentration of 1 µM for 30 min at 37°C and then incubated with uptake buffer in the absence (control) or presence of each labeled compounds for 5 min. After incubation, the radioactivity of the solubilized cells was determined. Data are expressed as percentage of the control value. Each column represents the mean ± S.E. for three monolayers. **p < 0.01, significant differences from control. T3, triiodothyronine; T4, thyroxine.

Inhibition Study by Using oatp2-Expressing Oocytes. To examine whether these phenomena were unique features of OAT-K1 and OAT-K2, or were observed for other transporters belonging to the oatp-gene family, we performed inhibition experiments by using oatp2-expressing oocytes. To compare the inhibition potencies of unlabeled digoxin, triiodothyronine, and taurocholic on the oatp2-mediated uptake of [³H]digoxin, the dose dependence of the inhibition was examined (Fig. 6). The [³H]digoxin uptake via oatp2 was inhibited by unlabeled digoxin, triiodothyronine, and taurocholic acid in this order of inhibitory potency. The estimated inhibition constant (IC₅₀) values for [³H]digoxin uptake were 0.81 µM for unlabeled digoxin, 50.4 µM for triiodothyronine, and 1.48 mM for taurocholic acid.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Dose-dependent inhibition of various compounds on [3H]digoxin uptake by oatp2-expressing oocytes. The oatp2-mediated 58.8 nM [3H]digoxin uptake was determined in the absence or presence of unlabeled digoxin (), triiodothyronine (), or taurocholic acid () for 1 h. After incubation, the radioactivity of the solubilized oocytes was determined. Each point represents the mean ± S.E. for 5 to 10 oocytes. Data are expressed as percentage of the control value.

	Discussion

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Over the past several years, considerable progress has been made in the molecular identification and characterization of organic anion transporters in the kidney, providing useful information for pharmaceutical research (Inui et al., 2000). At present, organic anion transporters expressed in the kidney are classified as belonging to two distinct gene families, OAT and oatp/OAT-K (Inui et al., 2000; Dresser et al., 2001). OAT-K1 and OAT-K2 are members of the oatp-gene family, and their roles in the renal excretion of methotrexate have been clarified (Saito et al., 1996; Masuda et al., 1999a,b; Takeuchi et al., 2000, 2001).

In the present study, we expanded the substrate spectrum of OAT-K1 and OAT-K2, by using MDCK cells stably transfected with these transporter cDNAs. This broad substrate spectrum of OAT-K1 and OAT-K2 includes methotrexate, ochratoxin A, and various endogenous organic anions (thyroid hormones, taurocholic acid, and conjugated steroids) (Tables 1 and 2). We previously reported that OAT-K1 specifically transports methotrexate and folic acid, but not taurocholic acid, by using LLC-OAT-K1 cells and OAT-K1-expressing oocytes (Masuda et al., 1996, 1999a). These discrepancies are probably related to the possibility that membrane-sorting mechanism and the proteolytic processing and/or excision of OAT-K1 found in renal tubular cells are retained in MDCK-OAT-K1 cells, but not in LLC-OAT-K1 cells (Inui et al., 2000). By proteolytic processing and/or excision, OAT-K1 protein in the MDCK-OAT-K1 cells and in the renal tubular cells may lose some structural features, which determine the strict substrate recognition in LLC-OAT-K1 cells and in OAT-K1-expressing oocytes. The present study indicated that both OAT-K1 and OAT-K2 possess the properties of multispecific substrate recognition in the brush-border membranes of the renal tubular cells.

Among the newly identified substrates for OAT-K1 and OAT-K2, ochratoxin A, thyroid hormones, taurocholic acid, and conjugated steroids are overlapping substrates for other members of the oatp-gene family, oatp1, oatp2, and oatp3 (Jacquemin et al., 1994; Kontaxi et al., 1996; Noé et al., 1997; Abe et al., 1998; Reichel et al., 1999). However, OAT-K1 and OAT-K2 are unique in mediating the transport of methotrexate. In addition, OAT-K1 and OAT-K2 transported the antiretroviral drug zidovudine, which possesses no typical anionic moiety (Fig. 1). Montfoort et al. (1999) reported that oatp1 and oatp2 mediate the uptake of cationic compounds N-(4,4-azo-n-pentyl)-21-deoxyajmalinium and rocuronium. These results indicated that although the majority of substrates for members of the oatp-gene family are amphipathic organic anions, these transporters can also transport cationic compounds as characteristics common among members of the oatp-gene family. Some of the substrates of OAT-K1 and OAT-K2, methotrexate, ochratoxin A, and zidovudine, also overlap with those of another organic anion transporter, rat OAT1 (rOAT1), which belongs to a distinct gene family from OAT-K1 and OAT-K2 (Inui et al., 2000; Dresser et al., 2001). rOAT1 is expressed in the basolateral membranes of the proximal tubules and exhibits the transport characteristics of a classic organic anion, p-aminohippuric acid, at the basolateral membranes of the proximal tubules (Sekine et al., 1997; Sweet et al., 1997). By considering the membrane localization of these transporters, rOAT1 at the basolateral membranes and OAT-K1 and OAT-K2 at the brush-border membranes may be responsible for the vectorial transport of several organic compounds via the proximal tubular cells. We previously demonstrated that both OAT-K1 and OAT-K2 mediate bidirectional transport of substrates, methotrexate and folic acid for OAT-K1, and taurocholic acid for OAT-K2, respectively (Masuda et al., 1999a,b; Takeuchi et al., 2000). Most recently, the role of OAT-K1 and OAT-K2 in the excretion of methotrexate from the proximal tubules into urine has been clarified using the rat model of renal failure (Takeuchi et al., 2001). Therefore, a wide variety of newly identified substrates of OAT-K1 and OAT-K2 may interact with these transporters from both inside and outside of the brush-border membranes. However, further characterization of these transporters, such as evaluation of substrate recognition from inside as well as outside of the brush-border membranes, is needed to elucidate their distinct roles in the renal handling of organic compounds.

If several substrates interact with a transporter at the same site in its molecule, the expected inhibition constant values should be comparable with the K_m values. Interestingly, the apparent inhibition constant value of taurocholic acid for [³H]methotrexate uptake via OAT-K1 is 183 µM (Takeuchi et al., 2000), and that of methotrexate for [³H]taurocholic acid uptake via OAT-K2 is 252 µM (A. Takeuchi, S. Masuda, H. Saito, and K. Inui, unpublished data), which are about 100-fold higher than the apparent K_m value of each compound, respectively (Table 2). The present study confirmed this phenomena. That is, uptake of structurally unrelated substrates via OAT-K1 and OAT-K2 showed little or no mutual inhibition even when concentrations of inhibitors were much higher than their apparent K_m values (Figs. 2-4). Moreover, not only OAT-K1 and OAT-K2 but also oatp2 had unique characteristics in substrate recognition. Although apparent K_m values of triiodothyronine and taurocholic acid for oatp2 were reported to be 5.87 and 35 µM, respectively (Reichel et al., 1999), the IC₅₀ value of triiodothyronine for [³H]digoxin uptake via oatp2 was 50.4 µM, and that of taurocholic acid was 1.48 mM, which were about 9- and 40-fold higher than the apparent K_m value of each compound, respectively (Fig. 6). There is a discrepancy between K_m value and IC₅₀ value if these substrates interact at the same substrate recognition site in the transporter molecules. One possible explanation that accounts for this discrepancy is that there may be several substrate recognition sites in the OAT-K1, OAT-K2, and oatp2 molecules. The existence of several substrate recognition sites was further confirmed by the trans-stimulation experiment. Intracellular unlabeled methotrexate trans-stimulated the uptake of [³H]folic acid, but not of structurally unrelated substrates ¹²⁵I-thyroid hormones and [³H]DHEA 3-sulfate (Fig. 5). This was comparable with our previous report describing the trans-stimulation effects of extracellular folic acid derivatives but not taurocholic acid on [³H]methotrexate efflux from MDCK-OAT-K1 cells (Takeuchi et al., 2000). Therefore, OAT-K1, OAT-K2 and oatp2 were suggested to transport a wide variety of structurally diverse organic compounds, not via a single interaction site in their molecules, but via several different interaction sites, preserving the transport activity even when one substrate occupies one interaction site. Such multispecificity of substrate recognition of OAT-K1, OAT-K2, and oatp2 may contribute to renal and hepatic transport systems in the elimination of a wide range of compounds. If these various compounds interact with a transporter at the same single interaction site, competition between substrates with the transporter should occur frequently. Therefore, the multiple substrate recognition sites in the OAT-K1, OAT-K2, and oatp2 molecules may minimize the severe drug-drug or drug-endogenous compound interactions via the transporter, reducing adverse effects on the body such as elevated plasma concentration or delayed excretion of drugs, toxins, and metabolites. Although the precise characteristics determining which kinds of substrates inhibit the transport activities of these transporters and which do not are not yet fully understood, the present results provide useful information for the comprehension of the pharmacokinetics of drugs in the kidney and the liver.

In conclusion, OAT-K1 and OAT-K2 were suggested to serve as multispecific organic anion transporters, mediating transport of a wide variety of xenobiotics and their metabolites, presumably via several substrate interaction sites.