Introduction

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Various organic anion transport inhibitors are used experimentally and clinically. Probenecid is a conventional and standard organic anion transport inhibitor experimentally, but it is used as a uricosuric drug clinically. In addition, KW-3902, developed as an adenosine A1 receptor antagonist (Shimada et al., 1991), was also shown to inhibit organic anion transport in the basolateral membrane of opossum kidney cells derived from the American opossum kidney (Nagai et al., 1999). On the other hand, betamipron and cilastatin are administered in combination with carbapenem antibiotics, panipenem, and imipenem, respectively (Birnbaum et al., 1985; Shiba et al., 1991). Betamipron inhibits the uptake of panipenem and imipenem into proximal tubule cells (Hirouchi et al., 1994). On the other hand, imipenem is degraded by human renal dehydropeptidase-I and therefore must be administered in combination with cilastatin, a dehydropeptidase-I inhibitor, to prevent loss of antimicrobial activity in urine and limit potential nephrotoxicity associated with renal metabolism (Craig, 1997).

In our previous study, we elucidated the interaction of human organic anion transporter 1 (hOAT1) and hOAT3 with organic anion transport inhibitors including probenecid, KW-3902, betamipron, and cilastatin (Takeda et al., 2001). Thus, the purpose of this study was to characterize the interaction of hOAT2 and hOAT4 with these organic anion transport inhibitors using cells derived from the second portion of the proximal tubule (S₂) from mice stably expressing hOAT2 and hOAT4 (S₂ hOAT2 and S₂ hOAT4, respectively).

	Experimental Procedures

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Materials. [³H]prostaglandin F₂ (PGF₂) (6808 GBq/mmol) and [³H]estrone sulfate (ES) (1861 GBq/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Other materials used included fetal bovine serum, trypsin, and geneticin from Invitrogen (Carlsbad, CA), recombinant epidermal growth factor from Wakunaga (Hiroshima, Japan), insulin from Shimizu (Shizuoka, Japan), RITC 80-7 culture medium from Iwaki Co. (Tokyo, Japan), probenecid from Sigma Chemicals Co. (St. Louis, MO), and TfX-50 from Promega (Madison, WI). KW-3902, betamipron, and cilastatin were kind gifts of Kyowa Hakko Kogyo Co. (Tokyo, Japan), Sankyo Pharmaceutical Co. (Tokyo, Japan), and Banyu Pharmaceutical Co. (Tokyo, Japan), respectively. The chemical structures of probenecid (A), KW-3902 (B), betamipron (C), and cilastatin (D) are listed in Fig. 1.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Chemical structures of probenecid (A), KW-3902 (B), betamipron (C), and cilastatin (D).

Cell Culture and Establishment of S₂ hOAT2 and S₂ hOAT4. S₂ cells, derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene, were established as described previously by us (Hosoyamada et al., 1996). The full-length cDNA of hOAT2 was isolated by screening the human kidney cDNA library using rat OAT2 cDNA (Sekine et al., 1998) as a probe (GenBank accession number: AF210455). The amino acids sequence homology between hOAT2 and rat OAT2 is 77%. The full-length cDNAs of hOAT2 and hOAT4 (Cha et al., 2000) were subcloned into pcDNA 3.1 (Invitrogen), a mammalian expression vector. S₂ hOAT2 and S₂ hOAT4 were obtained by transfecting S₂ cells with pcDNA3.1-hOAT2 and pcDNA3.1-hOAT4 coupled with pSV2neo, a neomycin resistance gene, using TfX-50 according to the manufacturer's instructions. S₂ cells transfected with pcDNA3.1 lacking an insert and pSV2neo were designated as S₂ pcDNA 3.1 and used as the control (mock cells). These cells were grown in a humidified incubator at 33°C and under 5% CO₂ using RITC 80-7 medium containing 5% fetal bovine serum, 10 µg/ml transferrin, 0.08 U/ml insulin, 10 ng/ml recombinant epidermal growth factor, and 400 µg/ml geneticin. The cells were subcultured in a medium containing 0.05% trypsin-EDTA solution (containing 137 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 4 mM NaHCO₃, 0.5 mM EDTA, and 5 mM HEPES, pH 7.2) and used for 25 to 35 passages. Clonal cells were isolated using a cloning cylinder and screened by determining the optimal substrate for each transporter [i.e., [¹⁴C]salicylate for OAT2 (Sekine et al., 1998) and [³H]ES for hOAT4 (Cha et al., 2000)]. S₂ hOAT4 exhibited a dose- and time-dependent increase in the uptake of ES. When S₂ hOAT2 and S₂ hOAT4 cells were cultured on permeable support (Transwell chambers; Coster, Cambridge, MA) and incubated in a solution containing D-[³H]mannitol on the apical or the basolateral side, the amounts of basal to apical and apical to basal transepithelial transport of D-[³H]mannitol were similar; thus, S₂ hOAT2 and S₂ hOAT4 cells were determined to be leaky. In addition, vertical sections of S₂ hOAT2 and S₂ hOAT4 stained with polyclonal antibodies against hOAT2 and hOAT4, respectively, showed that the subcellular localization of proteins for hOAT2 and hOAT4 was mainly on the cell membrane (unpublished observation). Both the basolateral and apical portions of the membrane showed positive staining. Therefore, the cells were cultured on a solid support for use in the following experiments.

Immunohistochemical Analysis of hOAT2 Protein in Human Kidney. Human kidney tissues were collected previously from patients with urethral tract carcinoma after giving informed consent. For the generation of the antibody against hOAT2, rabbits were immunized with keyhole limpet hemocyanin-conjugated synthesized peptides, CSLQEEEMPMKQVQN, corresponding to cysteine and the 14 amino acids of the COOH terminus of hOAT2. The IgG fraction of the polyclonal antibodies for the synthesized peptide was purified from the serum of immunized rabbits using a protein A column.

Uptake Experiments. Uptake experiments were performed as previously described (Takeda et al., 1999). The cells were seeded in 24-well tissue culture plates at a cell density of 1 × 10⁵ cells/well. After a 2-day culture, they were washed three times with Dulbecco's modified phosphate-buffered saline solution (containing 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, 1 mM CaCl₂, and 0.5 mM MgCl₂, pH 7.4), and then preincubated in the same solution in a water bath at 37°C for 10 min. S₂ hOAT2 cells were incubated in a solution containing 5 nM [³H]PGF₂ for use in time course experiments and 50 nM [³H] PGF₂ for use in inhibition experiments. S₂ hOAT4 cells were incubated in a solution containing 50 nM [³H]ES for use in the inhibition experiments. The uptake was stopped by the addition of ice-cold Dulbecco's modified phosphate-buffered saline, and the cells were washed three times with the same solution. The cells in each well were lysed with 0.5 ml of 0.1 N sodium hydroxide and 2.5 ml of Aquasol-2, and radioactivity was determined using a -scintillation counter (LSC-3100; Aloka,, Tokyo, Japan).

Kinetic Analysis. After the preincubation as described above, S₂ hOAT2 and S₂ hOAT4 cells were incubated in a solution containing [³H]PGF₂ or [³H]ES at different concentrations in the absence or presence of various inhibitors at 37°C for 20 s (for hOAT2) or 2 min (for hOAT4). Probenecid and cilastatin were dissolved in H₂O, whereas KW-3902 and betamipron were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was adjusted to less than 0.1%, which did not affect the hOAT2- and hOAT4-mediated organic anion uptake in our system. Based on the [³H]PGF₂ and [³H]ES uptake under each condition, double reciprocal plot analyses were performed as previously described (Apiwattanakul et al., 1999). For accurate analysis, the transformed data points were analyzed using weighted linear regression, with a weighting factor of 1/y or 1/y². When the inhibition was competitive, the K_i values were calculated based on the following equation: K_i = the concentration of inhibitor/[(K_m of PGF₂ or ES with inhibitor/K_m of PGF₂ or ES without inhibitor)  1].

Statistical Analysis. Data are expressed as means ± S.D. or means ± S.E. Statistical differences were determined using analysis of variance with Dunnett's posthoc test. Differences were considered significant at P < 0.05.

	Results

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Immunohistochemical Analysis of hOAT2 Protein in Human Kidney. Light-microscopic analysis of 2-µm waxed sections demonstrated that hOAT2 immunoreactivity was detected in the basolateral side of the proximal tubules (Figs. 2, A and B). Arrows indicate stained proximal tubules.

View larger version (116K): [in this window] [in a new window]   Fig. 2.   Immunohistochemical staining illustrating hOAT2 protein in human kidney stained with antibody against hOAT2. A, human kidney (100×); B, human kidney (400×). Arrows indicate stained proximal tubules.

Time- and Concentration-Dependent Uptake of PGF₂ in S₂ hOAT2. We examined the time-dependent uptake of PGF₂ in S₂ hOAT2. As shown in Fig. 3A, S₂ hOAT2 exhibited much higher PGF₂ uptake than mock cells. The kinetics of PGF₂ uptake were examined to evaluate the pharmacological characteristics of hOAT2 upon the uptake of PGF₂. We analyzed the mean data using the Michaelis-Menten equation and determined the K_m and V_max values. Using these parameters, we made a theoretical curve. As shown in Fig. 3B, S₂ hOAT2 exhibited a concentration-dependent increase in PGF₂ uptake. Eadie-Hofstee plot of the concentration dependence of PGF₂ uptake in S₂ hOAT2 after subtraction of uptake by mock cells revealed that the estimated K_m value of PGF₂ uptake by hOAT2 was 425 ± 53.0 nM (data not shown). These results suggest that hOAT2 mediates the transport of PGF₂.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time- and concentration-dependent uptake of PGF2 by hOAT2. A, time dependence. S2 hOAT2 cells and mock cells were incubated in solution containing 5 nM [3H]PGF2 at 37°C for 15 s to 4 min. B, concentration dependence. S2 hOAT2 cells were incubated in solution containing various concentrations of [3H]PGF2 for 20 s at 37°C. Each value represents the mean ± S.D. of four determinations from one typical experiment.

Effects of Various Organic Anion Transport Inhibitors on Organic Anion Uptake by hOAT2 and hOAT4. We examined the effects of probenecid, KW-3902, betamipron, and cilastatin at different concentrations on PGF₂ uptake in S₂ hOAT2 and ES uptake in S₂ hOAT4. As shown in Fig. 4, probenecid, but not KW-3902, betamipron, and cilastatin, significantly inhibited PGF₂ uptake by hOAT2 (n = 4; *P < 0.0001 versus control). In addition, probenecid exhibited a dose-dependent decrease in PGF₂ uptake by hOAT2 over the concentration range of 1 to 2000 µM (data not shown). In contrast, probenecid, KW-3902, and betamipron, but not cilastatin, significantly inhibited ES uptake by hOAT4 in a dose-dependent manner (data not shown).

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Effects of probenecid, KW-3902, betamipron, and cilastatin on PGF2 uptake by hOAT2. S2 hOAT2 cells were incubated in a medium containing 50 nM [3H]PGF2 at 37°C for 2 min in the absence or presence of 1 mM probenecid, 0.1 mM KW-3902, 1 mM betamipron, or 1 mM cilastatin. Each value represents the mean ± S.D. of four determinations from one typical experiment. , P < 0.0001 versus control.

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View larger version (18K): [in this window] [in a new window]   Fig. 5.   Kinetic analysis of the inhibitory effects of probenecid on PGF2 uptake by hOAT2. S2 hOAT2 cells were incubated in a solution containing various concentrations of [3H]PGF2 in the presence or absence of 1 mM probenecid at 37°C for 20 s, and analysis of Lineweaver-Burke plots was performed. The transformed data points were analyzed using weighted linear regression, with a weighting factor of 1/y. Each value represents the mean ± S.D. of four determinations from one typical experiment.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Kinetic analysis of the inhibitory effects of probenecid (A), KW-3902 (B), and betamipron (C) on ES uptake by hOAT4. S2 hOAT4 cells were incubated in a solution containing various concentrations of [3H]ES in the presence or absence of 200 µM probenecid, 20 µM KW-3902, or 500 µM betamipron at 37°C for 2 min, and analysis of Lineweaver-Burke plots was performed. The transformed data points were analyzed using weighted linear regression, with a weighting factor of 1/y2. Each value represents the mean ± S.D. of four determinations from one typical experiment.

View this table: [in this window] [in a new window]   TABLE 1 Ki values of inhibitory effects of various organic anion transport inhibitors on the uptake of PGF2 in S2 hOAT2 and ES in S2 hOAT4. S2 hOAT2 and S2 hOAT4 cells were incubated with solution containing various concentrations of [3H]PGF2 or [3H]ES in the absence or presence of various organic anion transport inhibitors at 37°C for 20 s (hOAT2) or 2 min (hOAT4). The Ki values were estimated from a Lineweaver-Burke plot. The transformed data points were analyzed using weighted linear regression, with a weighting factor of 1/y (hOAT2) or 1/y2 (hOAT4). Each value represents the mean ± S.E. of eight determinations from two separate experiments.

	Discussion

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The secretion and reabsorption of numerous organic anions, including endogenous metabolites, drugs, and xenobiotics, are important physiological functions of the renal proximal tubule. The process of secreting organic anions through the proximal tubule cells is achieved via unidirectional transcellular transport involving the uptake of organic anions into the cells from the blood across the basolateral membrane, followed by extrusion across the brush-border membrane into the proximal tubule fluid (Pritchard and Miller, 1993). Recently, cDNA-encoding transporters mediating renal organic anion transport have been successively cloned, including OAT1 (Sekine et al., 1997; Reid et al., 1998; Hosoyamada et al., 1999), OAT2 (Sekine et al., 1998), OAT3 (Kusuhara et al., 1999; Cha et al., 2001), OAT4 (Cha et al., 2000), OAT-K1 (Saito et al., 1996), OAT-K2 (Masuda et al., 1999), organic anion-transporting polypeptide (oatp1) (Jacquemin et al., 1994), oatp2 (Noe et al., 1997), oatp3 (Abe et al., 1998), multidrug resistance-associated protein 2 (MRP2) (Leier et al., 2000) and human-type I sodium-dependent inorganic phosphate transporter (NPT1) (Uchino et al., 2000). Among them, hOAT1 and hOAT3 are thought to be the major OATs responsible for the basolateral uptake of various organic anions (Hosoyamada et al., 1999; Cha et al., 2001). HOAT4, localized to the apical side of the proximal tubule (Babu et al., 2002), was reported to mediate the transport of various anionic drugs, including para-aminohippuric acid (PAH), ES, methotrexate, and ochratoxin A (Cha et al., 2000).

At first, we characterized the localization and functional properties of hOAT2. Immunohistochemically, hOAT2 was shown to be localized to the basolateral side of the proximal tubule. Although salicylate was found to be the best substrate for rat OAT2 (Sekine et al., 1998), the background uptake by mock cells was high (unpublished observation). In the current study, we found that PGF₂ was the better substrate for hOAT2, whereas the background uptake by mock cells was much lower. HOAT2 mediated a time- and dose-dependent increase in PGF₂ uptake. Based on these observations, we used PGF₂ as a substrate to elucidate the interaction of hOAT2 with various organic anion transport inhibitors. In addition, since hOAT1, hOAT2, and hOAT3 (Hosoyamada et al., 1999; Cha et al., 2001) are localized to the basolateral side of the proximal tubule, the functional difference among these transporters should therefore be elucidated.

Probenecid, KW-3902, and betamipron, but not cilastatin, significantly inhibited ES uptake by hOAT4, whereas probenecid alone inhibited the hOAT2-mediated PGF₂ uptake. These results are in contrast to the previous results that probenecid, KW-3902, betamipron, and cilastatin significantly inhibited PAH uptake by hOAT1 and ES uptake by hOAT3 (Takeda et al., 2001). However, the rank order of the inhibitory effects on hOAT4-mediated ES uptake (i.e., KW-3902 > probenecid > betamipron) was the same as that for hOAT1-mediated PAH uptake and hOAT3-mediated ES uptake (Takeda et al., 2001).

Probenecid has been widely used to analyze organic anion transport systems. In the current study, probenecid was shown to inhibit organic anion uptake mediated by hOAT2-mediated PGF₂ uptake and hOAT4-mediated ES uptake in vitro. The maximum steady-state plasma concentration and unbound fraction of probenecid were reported to be 170 µM (Nierenberg, 1983) and 11.0% (Dayton et al., 1963), respectively. Thus, the maximum steady-state concentration of unbound probenecid in the plasma is estimated to be approximately 18.7 µM. Since the therapeutically relevant plasma concentrations of a drug is thought to be within 5-fold of the maximum steady-state plasma concentration of a drug (Zhang et al., 2000), the therapeutically relevant concentration of unbound probenecid in the plasma is thought to be 93.5 µM. In addition, since the unbound drug could be filtered through the glomerulus, the concentration of unbound probenecid with the tubular fluid in the apical side of the proximal tubule would be within 93.5 µM. Based on these observations, since the K_i value of probenecid for hOAT2-mediated PGF₂ uptake and hOAT4-mediated ES uptake were 766 and 54.9 µM, respectively (Table 1), it was predicted that probenecid could inhibit the reabsorption of organic anions by hOAT4 on the apical side of the proximal tubule in vivo.

KW-3902 is selective and is the most potent adenosine A1 receptor antagonist known to date (Suzuki et al., 1992). In animal studies, this compound was shown to have diuretic activity and renal-protective effect against cephaloridine-induced nephrotoxicity (Mizumoto et al., 1993; Nagashima et al., 1994). However, this compound was excluded from clinical development based on the results of a phase II trial study, which showed that this compound did not exert sufficient diuretic action (K. Hakko and K. Co, unpublished observation). In this study, KW-3902 was shown to inhibit hOAT4-mediated ES uptake but not hOAT2-mediated PGF₂ uptake in vitro. Based on the rank order of the K_i values of various inhibitors shown in Table 1, KW-3902 was the most potent inhibitor of hOAT4-mediated ES uptake. Thus, it was suggested that KW-3902 could be a powerful pharmacological agent for analyzing hOAT4-mediated organic anion transport in vitro. However, the K_i values of KW-3902 for hOAT4-mediated PGF₂ uptake (20.7 µM) were much higher than the maximum plasma concentration of KW-3902 in a healthy volunteer [i.e., 0.196 µM (KW-3902 product brochure; Kyowa Hakko Kogyo Co.) (more than 3-fold; Zhang et al., 1998)]. Thus, it was predicted that this compound would exhibit no significant inhibitory effects on hOAT4-mediated organic anion uptake in vivo.

Betamipron significantly inhibited ES uptake by hOAT4 but not PGF₂ uptake by hOAT2 in vitro. However, the K_i value of betamipron for hOAT4-mediated ES uptake was much higher than the therapeutically relevant concentration of unbound betamipron in the plasma (25.5 µM) (Shiba et al., 1991; Zhang et al., 2000). These results suggest that betamipron exerts no significant inhibitory effects on hOAT4-mediated ES transport in vivo.

In the current study, cilastatin was found to have no significant inhibitory effects on hOAT2-mediated PGF₂ uptake and hOAT4-mediated ES uptake. In contrast, cilastatin significantly inhibited hOAT1-mediated PAH uptake and hOAT3-mediated ES uptake in vitro, whereas it was predicted that cilastatin exhibits significant inhibitory effects on hOAT1- but not hOAT3-mediated organic anion uptake in vivo (Takeda et al., 2001).

In addition to the hOATs used in the current study, it is important to understand the interaction of apical transporters mediating organic anion transport (i.e., OAT-K1, OAT-K2, oatp1, MRP2, and NPT1) with various organic anion inhibitors (Inui et al., 2000). However, these topics are beyond the scope of this study, and further study should be performed to elucidate them.

In conclusion, the results suggest that in vitro hOAT2 interacts with only probenecid, whereas hOAT4 interacts with probenecid, KW-3902, and betamipron. In addition, by comparing the K_i values of the inhibitors with their therapeutically relevant concentrations of unbound inhibitors in the plasma, it was predicted that hOAT4-mediated organic anion uptake would be inhibited by probenecid in vivo, whereas none would inhibit hOAT2-mediated organic anion uptake in vivo. The cells used in this study would serve as a good tool to characterize newly developed organic anion transport inhibitors.