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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Human Organic Anion Transporters and Human Organic Cation Transporters Mediate Renal Antiviral Transport

Michio Takeda, Suparat Khamdang, Shinichi Narikawa, Hiroaki Kimura, Yasuna Kobayashi, Toshinori Yamamoto, Seok Ho Cha, Takashi Sekine and Hitoshi Endou
Journal of Pharmacology and Experimental Therapeutics March 2002, 300 (3) 918-924; DOI: https://doi.org/10.1124/jpet.300.3.918

Abstract

Renal excretion is an important elimination pathway for antiviral agents, such as acyclovir (ACV), ganciclovir (GCV), and zidovudine (AZT). The purpose of this study was to elucidate the molecular mechanisms of renal ACV, GCV, and AZT transport using cells stably expressing human organic anion transporter 1 (hOAT1), hOAT2, hOAT3, and hOAT4, and human organic cation transporter 1 (hOCT1) and hOCT2. Time- and concentration-dependent uptake of ACV and GCV was observed in hOAT1- and hOCT1-expressing cells. In contrast, uptake of valacyclovir,l-valyl ester of ACV, was observed only in hOAT3-expressing cells. On the other hand, AZT uptake was observed in hOAT1-, hOAT2-, hOAT3-, and hOAT4-expressing cells. The Kmvalues of ACV uptake by hOAT1 and hOCT1 were 342.3 and 151.2 μM, respectively, whereas those of GCV uptake by hOAT1 and hOCT1 were 895.5 and 516.2 μM, respectively. On the other hand, theKm values of AZT uptake by hOAT1, hOAT2, hOAT3, and hOAT4 were 45.9, 26.8, 145.1, and 151.8 μM, respectively. In addition, probenecid weakly inhibited the hOAT1-mediated ACV uptake. In conclusion, these results suggest that hOAT1 and hOCT1 mediate renal ACV and GCV transport, whereas hOAT1, hOAT2, hOAT3, and hOAT4 mediate renal AZT transport. In addition, l-valyl ester appears to be important in differential substrate recognition between hOAT1 and hOAT3. hOAT1 may not be the molecule responsible for the drug interaction between ACV and probenecid.

Both acyclovir (ACV) and ganciclovir (GCV) are acyclic guanosine derivatives (Fig. 1, A and B) (Safrin, 2001). ACV is used in the treatment of various forms of herpes simplex infections (Safrin, 2001). Valacyclovir (VACV) is the l-valyl ester of ACV (Fig. 1C), which is active against herpes simplex virus types 1 and 2, and varicella zoster virus (Safrin, 2001). GCV is used in the treatment of cytomegalovirus infections in acquired immunodeficiency syndrome and in transplant patients (Safrin, 2001). On the other hand, 3′-azido-3′-deoxythymidine (zidovudine, AZT) is widely used for the treatment of HIV infection (de Miranda et al., 1989) (Fig. 1D). Approximately 83% of ACV, 90% of GCV, and 80% of AZT are excreted in their unchanged forms by the kidney (Laskin et al., 1982; Yarchoan et al., 1989; Morse et al., 1993). Renal excretion of ACV and AZT is reduced by probenecid, a typical inhibitor of organic anion transport (Laskin et al., 1982; Chatton et al., 1990; Mays et al., 1991). Although neither possesses a typical anionic moiety, the results suggest that the renal organic anion transport system is responsible for the tubular secretion of these drugs. On the other hand, the involvement of an organic cation transport system has also been suggested in the tubular secretion of AZT because cimetidine, an organic cation, also reduces the renal clearance of AZT (Chatton et al., 1990).

Figure 1
Figure 1

Chemical structures of ACV (A), GCV (B), VACV (C), and AZT (D).

The secretion of numerous organic anions and cations including endogenous metabolites, drugs, and xenobiotics is an important physiological function of the renal proximal tubules. The process of secreting organic anions and cations 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, cDNAs encoding renal organic anion transporters (OATs) have been successively cloned. The OATs cloned include OAT1 (Sekine et al., 1997; Hosoyamada et al., 1999), OAT2 (Sekine et al., 1998), OAT3 (Kusuhara et al., 1999; Cha et al., 2001), and OAT4 (Cha et al., 2000), whereas the OCTs include OCT1 (Grundemann et al., 1994;Gorboulev et al., 1997) and OCT2 (Okuda et al., 1996; Gorboulev et al., 1997). hOAT1, hOAT2, hOAT3, and hOCT2 were shown to be localized on the basolateral side of the proximal tubule (A. Enomoto, M. Takeda, S. Narikawa, Y. Kobayashi, C. H. Seok, T. Sekine, T. Niwa, and H. Endou, unpublished observation; Hosoyamada et al., 1999; Cha et al., 2001; Pietig et al., 2001), whereas hOAT4 was localized in the apical side of the proximal tubule (Babu et al., 2002). On the other hand, the localization of hOCT1 remains unclear.

The purpose of this study was to elucidate the molecular mechanisms of renal ACV, GCV, and AZT transport. For this purpose, we established and utilized the cells derived from the second segment of the proximal tubule (S2) that stably express hOAT1, hOAT2, hOAT3, hOAT4, hOCT1, and hOCT2 (S2 hOAT1, S2 hOAT2, S2 hOAT3, S2 hOAT4, S2 hOCT1, and S2 hOCT2, respectively).

Experimental Therapeutics

Materials.

[3H]ACV (1110 GBq/mmol), [3H]GCV (740 GBq/mmol), [3H]VACV (111 GBq/mmol), VACV, and [3H]AZT (558.7 GBq/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA). [14C]para-aminohippuric acid (PAH; 1.8648 GBq/mmol), [3H]estrone sulfate (1961 GBq/mmol) and [3H]prostaglandin F2α(PGF2α) (6808 GBq/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). [14C]Tetraethylamnonium (TEA) (2.035 GBq/mmol) was purchased from Muromachi Chemicals (Tokyo, Japan). ACV, GCV, AZT, and probenecid were obtained from Sigma Chemical Co. (St. Louis, MO). 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) and TfX-50 from Promega (Madison, WI).

Cell Culture and Establishment of S2 hOAT2, S2 hOAT4, S2 hOCT1, and S2hOCT2.

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 establishment and characterization of S2 hOAT1 and S2 hOAT3 have already been reported (Takeda et al., 2001). The full-length cDNA of hOAT2 was isolated by screening human kidney cDNA library using rat-OAT2 cDNA (Sekine et al., 1998) as a probe. The full-length cDNA of hOCT1 was obtained by reverse transcription and polymerase chain reaction of cDNA using primers spanning the coding region of the published sequence of hOCT1 cDNA (Gorboulev et al., 1997). The full-length cDNA of hOCT2 was isolated by screening the human kidney cDNA library using rat OCT2 cDNA (Okuda et al., 1996) as a probe. The full-length cDNAs of hOAT2, hOAT4 (Cha et al., 2000), hOCT1, and hOCT2 were subcloned into pcDNA 3.1 (Invitrogen), a mammalian expression vector. S2 hOAT2, S2 hOAT4, S2 hOCT1, and S2 hOCT2 were obtained by transfecting S2 cells with pcDNA3.1-hOAT2, pcDNA3.1-hOAT4, pcDNA3.1-hOCT1, and pcDNA3.1-hOCT2 coupled with pSV2neo, a neomycin resistance gene, using TfX-50 according to the manufacturer's instructions. S2cells transfected with pcDNA3.1 lacking an insert, and pSV2neo were designated as S2 pcDNA 3.1 and used as a control (mock). 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 mg/ml transferrin, 0.08 U/ml insulin, 10 ng/ml recombinant epidermal growth factor, and 400 mg/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 ∼10 to 35 passages. Clonal cells were isolated using a cloning cylinder and screened by determining the optimal substrate for each transporter, i.e., [14C]PAH for hOAT1 (Hosoyamada et al., 1999), [3H]PGF2α for hOAT2 (A. Enomoto, M. Takeda, S. Narikawa, Y. Kobayashi, C. H. Seok, T. Sekine, T. Niwa, and H. Endou, unpublished observation), [3H]estrone sulfate for hOAT3 (Cha et al., 2001) and hOAT4 (Cha et al., 2000), and [14C]TEA for hOCT1 and rat OCT2 (Okuda et al., 1996; Zhang et al., 1998).

Uptake Experiments.

Uptake experiments were performed as previously described (Takeda et al., 2001). The S2 cells were seeded in 24-well tissue culture plates at a cell density of 1 × 105cells/well. After the cells were cultured for 2 days, they were washed three times with Dulbecco's modified phosphate-buffered saline (D-PBS) solution containing 137 mM NaCl, 3 mM KCl, 8 mM NaHPO4, 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. The cells were then incubated in D-PBS with either [3H]ACV, [3H]GCV, [3H]VACV, or [3H]AZT at various concentrations as indicated in each experiment at 37°C. The uptake was stopped by the addition of ice-cold D-pbs, 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 β-scintillation counter (LSC-3100, Aloka, Tokyo, Japan).

Inhibition Study.

To evaluate the inhibitory effects of antiviral agents on the organic anion transport by hOATs and the organic cation transport by hOCTs, the cells were incubated in D-pbs containing either 5 μM[14C]PAH, 50 nM [3H]PGF2α, 50 nM [3H]estrone sulfate, or 5 μM [14C]TEA in the absence or presence of either ACV, GCV, or AZT at 37°C for 2 min, as described above. In addition, to examine the effects of probenecid on the hOAT1-mediated ACV uptake, S2 hOAT1 was incubated in a solution containing 50 nM [3H]ACV in the absence or presence of various concentrations of probenecid at 37°C for 2 min. Probenecid was dissolved in distilled water, whereas ACV, GCV, and AZT were dissolved in dimethyl sulfoxide, and diluted with the incubation medium. The final concentration of dimethyl sulfoxide in the incubation medium was adjusted to less than 1%.

Statistical Analysis.

Data are expressed as means ± S.E. Statistical differences were determined using Student's unpairedt test. Differences were considered significant atP < 0.05.

Results

Inhibitory Effects of ACV, GCV, and AZT on the Uptakes of Organic Anion and Organic Cation.

We examined the inhibitory effects of ACV, GCV, and AZT on the organic anion uptake mediated by hOATs, and the organic cation uptake mediated by hOCTs. Table1 shows the results. ACV significantly inhibited the organic anion uptake by hOAT1 and hOAT3, but not hOAT2 and hOAT4 and the organic cation uptake by hOCT1 but not hOCT2 (n = 4; *P < 0.001 and ***P < 0.05 versus control). In addition, GCV significantly inhibited the organic anion uptake by hOAT1, hOAT2, and hOAT3, but not hOAT4 and the organic cation uptake by hOCT1 but not hOCT2 (n = 4; *P < 0.001, **P < 0.01, and ***P < 0.05 versus control). Furthermore, AZT significantly inhibited the organic anion uptake by hOAT1, hOAT2, hOAT3, and hOAT4 (n = 4; *P < 0.001 and ***P < 0.05 versus control), but did not significantly inhibited the organic cation uptake by hOCT1 and hOCT2 (n = 4; N.S.).

View this table:
Table 1

The percentage of remaining uptakes of organic anion by hOCTs and organic cation by hOCTs in the presence of ACV, GCV, and AZT

ACV, GCV, and AZT Uptake Mediated by hOATs and hOCTs.

We examined ACV, GCV, and AZT uptake by hOATs and hOCTs using [3H]ACV, [3H]GCV, and [3H]AZT. As shown in Fig.2, A and B, S2hOAT1 and S2 hOCT1, but not S2 hOAT2, S2 hOAT3, S2 hOAT4, and S2 hOCT2, exhibited significantly higher uptake activities of ACV (A) and GCV (B) than mock (n = 4; *P < 0.001, **P < 0.01, and ***P < 0.05 versus mock). In contrast, the amount of AZT uptake by S2 hOAT1, S2 hOAT2, S2 hOAT3, and S2 hOAT4, but not S2 hOCT1 and S2 hOCT2, was significantly larger than that by mock. (Fig. 2D; n= 4; *P < 0.001 and **P < 0.01 versus mock). Since ACV, GCV, and AZT uptake by mock were relatively high, we examined the effects of 1 mM probenecid, an organic anion transport inhibitor, or 1 mM quinine, an organic cation transport inhibitor, on ACV, GCV, and AZT uptake by mock. The inhibitory effects of probenecid on ACV, GCV, and AZT uptake by mock were 101 ± 4.33% of control (n = 4; N.S.), 112 ± 3.25% of control (n = 4; N.S.), and 86.3 ± 3.79% of control (n = 4; *P < 0.05 versus control), respectively, and those of quinine on ACV, GCV, and AZT uptake by mock were 88.7 ± 3.22% of control (n = 4; N.S.), 84.3 ± 5.98% of control (n = 4; *P < 0.05 versus control), and 89.0 ± 7.65% of control (n = 4; N.S.), respectively. Thus, AZT uptake by mock may be mediated in part by endogenously expressed OATs, whereas GCV uptake by mock may be mediated in part by endogenously expressed OCTs.

Figure 2
Figure 2

ACV (A), GCV (B), VACV (C), and AZT (D) uptake by hOATs and hOCTs. S2 hOAT1, S2 hOAT2, S2 hOAT3, S2 hOAT4, S2 hOCT1, S2 hOCT2, and mock were incubated in solution containing 50 nM [3H]ACV, 100 nM [3H]GCV, 1.5 μM [3H]VACV, or 200 nM [3H]AZT at 37°C for 5 min. Each value represents the mean ± S.E. of four determinations. ★, P < 0.001, ★★,P < 0.01, and ★★★, P < 0.05 versus mock.

For further analysis of ACV, GCV, and AZT uptake, we examined the time-dependent uptake of ACV, GCV, and AZT. Figure3 shows the time course of ACV and GCV uptake by hOAT1 and hOCT1. S2 hOAT1 and S2 hOCT1 exhibited higher amounts of ACV (A, B) and GCV (C, D) uptake than mock. On the other hand, as shown in Fig.4, S2 hOAT1 (A), S2 hOAT2 (B), S2 hOAT3 (C), and S2 hOAT4 (D) exhibited higher amounts of AZT uptake than mock.

Figure 3
Figure 3

Time courses of ACV and GCV uptake by hOAT1 and hOCT1. S2 hOAT1, S2 hOCT1, and mock were incubated in solution containing 50 nM[3H]ACV or 100 nM[3H]GCV at 37°C for 1 to 45 min. (A) ACV uptake by hOAT1, (B) ACV uptake by hOCT1, (C) GCV uptake by hOAT1, and (D) GCV uptake by hOCT1. Each value represents the mean ± S.E. of four determinations.

Figure 4
Figure 4

Time courses of AZT uptake by hOATs. S2hOAT1 (A), S2 hOAT2 (B), S2 hOAT3 (C), S2 hOAT4 (D), and mock were incubated in solution containing 200 nM[3H]AZT at 37°C for 1 to 45 min. Each value represents the mean ± S.E. of four determinations.

To further elucidate the mechanism of ACV, GCV, and AZT uptake, the kinetic analysis of concentration-dependent uptake of ACV, GCV, and AZT was performed. Figure 5 shows the Eadie-Hofstee plot of the concentration dependence of ACV and GCV uptake in S2 hOAT1 and S2hOCT1 after subtraction of uptake by mock. The estimatedKm values of ACV and GCV uptake by hOAT1 and hOCT1 are listed in Table 2. Figure 6 shows the Eadie-Hofstee plot of the concentration dependence of AZT uptake in S2hOAT1 (A), S2 hOAT2 (B), S2hOAT3 (C), and S2 hOAT4 (D) after subtraction of uptake by mock. The estimated Kmvalues of AZT uptake by hOAT1, hOAT2, hOAT3, and hOAT4 are also listed in Table 2. These results suggest that hOAT1 and hOCT1 mediate the transport of ACV and GCV, whereas hOAT1, hOAT2, hOAT3, and hOAT4 mediate the transport of AZT.

Figure 5
Figure 5

Kinetic analyses of ACV and GCV uptake by hOAT1 and hOCT1. S2 hOAT1, S2 hOCT1, and mock were incubated in solution containing various concentrations of either [3H]ACV or [3H]GCV at 37°C for 1 min. The values for mock were subtracted. (A) ACV uptake by hOAT1, (B) ACV uptake by hOCT1, (C) GCV uptake by hOAT1, and (D) GCV uptake by hOCT1. Each value represents the mean ± S.E. of three determinations from one typical experiment. Eadie-Hofstee plot of the uptake of ACV and GCV was performed.

View this table:
Table 2

Kinetic parameters of ACV, GCV, and AZT uptake by hOATs and by hOCTs

Figure 6
Figure 6

Kinetic analyses of AZT uptake by hOATs. S2 hOAT1 (A), S2 hOAT2 (B), S2hOAT3 (C), S2 hOAT4 (D), and mock were incubated in solution containing various concentrations of [3H]AZT at 37°C for 1 min. The values were subtracted from that in mock. Each value represents the mean ± S.E. of three determinations from one typical experiment. Eadie-Hofstee plot of the uptake of AZT was performed.

VACV Uptake Mediated by hOAT3.

We examined VACV uptake by hOATs and hOCTs using [3H]VACV. As shown in Fig. 2D, S2 hOAT3, but not S2 hOAT1, S2 hOAT2, S2 hOAT4, S2 hOCT1, or S2 hOCT2, exhibited significantly higher uptake activity of VACV than mock. (n = 4; *P< 0.01 versus mock). For further analysis of hOAT3-mediated VACV uptake, we examined the time-dependent uptake of VACV. As shown in Fig.7A, S2 hOAT3 exhibited higher amounts of VACV uptake than mock. In addition, Fig. 7B shows the Eadie-Hofstee plot of the concentration dependence of VACV uptake in S2 hOAT3 after subtraction of uptake by mock. The estimated Km value of VACV uptake by hOAT3 was 57.9 μM. These results suggest that hOAT3, but not hOAT1, hOAT2, and hOAT4, mediates the transport of VACV.

Figure 7
Figure 7

Time- and concentration-dependent uptake of VACV by hOAT1. A, time dependence. S2 hOAT3 and mock were incubated in solution containing 1.5 μM [3H]VACV at 37°C for 1 to 45 min. Each value represents the mean ± S.E. of four determinations. B, kinetic analysis of concentration-dependent uptake of VACV by hOAT3. S2 hOAT3 and mock were incubated in solution containing various concentrations of [3H]VACV at 37°C for 1 min. The values for mock were subtracted. Each value represents the mean ± S.E. of three determinations from one typical experiment. Eadie-Hofstee plot of the uptake of VACV was performed.

Inhibitory Effect of Probenecid on hOAT1-Mediated ACV Uptake.

To elucidate the molecular mechanism for the interaction between ACV and probenecid (Laskin et al., 1982), we examined the effects of probenecid on the hOAT1-mediated ACV uptake. As shown in Fig. 8, probenecid weakly but significantly inhibited the hOAT1-mediated ACV uptake (n = 4; *P < 0.01 versus control). In addition, we also examined the effects of 1 mM probenecid on hOAT1-mediated ACV uptake in 15-min incubation. The result was similar to that in a 2-min incubation, i.e., 76.4 ±3.46% of control (n = 4; *P < 0.01 versus control).

Figure 8
Figure 8

Inhibitory effects of probenecid on hOAT1-mediated ACV uptake. S2 hOAT1 was incubated in solution containing 50 nM [3H]ACV in the absence or presence of various concentrations of probenecid at 37°C for 2 min. Each value represents the mean ± S.E. of four determinations. ★,P < 0.01 versus control.

Discussion

hOAT1 and hOAT3 mediate the transport of a wide variety of drugs and xenobiotics, including nonsteroidal anti-inflammatory drugs, antitumor drugs, histamine H2-receptor antagonist, prostaglandins, diuretics, angiotensin-converting enzyme inhibitors, and β-lactam antibiotics (Hosoyamada et al., 1999; Cha et al., 2001). Some differences in characteristics exist between hOAT1 and hOAT3, such as substrate specificity and localization; hOAT1 is localized in the basolateral side of the S2segment of the proximal tubule (Hosoyamada et al., 1999), whereas hOAT3 is localized in the first, second, and third segments (S1, S2 and S3) of the proximal tubule (Cha et al., 2001). In addition, hOAT1, but not hOAT3, exhibits transport properties as an exchanger (Hosoyamada et al., 1999; Cha et al., 2001). hOAT2, which has also shown to be localized in the basolateral side of the proximal tubule, mediates the transport of organic anions including salicylate and prostaglandin F2α (A. Enomoto, M. Takeda, S. Narikawa, Y. Kobayashi, C. H. Seok, T. Sekine, T. Niwa, and H. Endou, unpublished observation). hOAT4 mediates the apical transport of various anionic drugs; however, this transporter exhibits a relatively narrow substrate recognition compared with hOAT1 and hOAT3 (Cha et al., 2000).

hOCT1 has been shown to be mainly localized in the liver. hOCT1 also has been shown to mediate polyspecific pH independent transport of organic cations, whereas that by hOCT2 is pH independent, electrogenic, and polyspecific (Gorboulev et al., 1997).

So far, limited information is available concerning the interaction between various OAT or OCT molecules and antiviral agents. Adefovir and cidofovir are transported via hOAT1 and rat OAT1 (Cihlar et al., 1999;Ho et al., 2000). AZT, ACV, zalcitabine, didanosine, lamivudine, stavudine, trifluridine, and foscarnet are transported via rat OAT1 (Wada et al., 2000). Zhang et al. (2000) showed that HIV protease inhibitors including indinavir, nelfinavir, ritonavir, and saquinavir are potent inhibitors of hOCT1; however, they are not the substrates for hOCT1-mediated transport.

In the current study, it was demonstrated that hOAT1 and hOCT1 mediate ACV and GCV uptake, whereas hOAT1, hOAT2, hOAT3, and hOAT4 mediate AZT uptake. These results may serve as a molecular background for previous results that suggest that ACV transport is mediated by OAT in vivo (Laskin et al., 1982; Chatton et al., 1990; Mays et al., 1991) and that AZT transport is mediated by OAT in vivo (Chatton et al., 1990) and in vitro (Griffiths et al., 1991). However, we cannot exclude the possibility that transporters other than those analyzed in this study are also involved in renal ACV, GCV, and AZT transport. As shown in Table 1, the results of the inhibition studies are consistent with those of uptake experiments shown in Fig. 2 except that GCV inhibited hOAT2-mediated organic anion uptake, and ACV and GCV inhibited hOAT3-mediated organic anion uptake. These results suggest that GCV and/or ACV interact with hOAT2 and hOAT3; however, they were not transported by these transporters.

In contrast, since ACV and GCV possess no cationic moiety, the hOCT1-mediated uptake of ACV and GCV is unexpected. Various substrates are shown to be transported via OAT as well as OCT system, and called “bisubstrates” (Ullrich et al., 1993). In this regard, AZT could also be regarded as bisubstrates. However, since the localization of hOCT1 remains unclear, it could not be determined whether AZT is transported via OAT and OCT at the same time. Further studies should be performed to elucidate the underlying mechanisms for ACV and GCV transport by hOCTs.

As shown in Fig. 1, A and B, both ACV and GCV are guanine derivatives and structurally similar except the side chain of ACV is 9-[(2-hydroxyethoxymethyl)methyl]guanine and that of GCV is 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine. Comparing the Km values of hOAT1 and hOCT1 for ACV or GCV uptake, the Km values of hOAT1 and hOCT1 for ACV uptake was about 2.5 and 3 times larger than those for GCV, respectively. Based on these data, since the structure of side chain appears to be important for substrate recognition, further structure-function analysis of ACV and GCV transport by hOAT1 and hOCT1 should be performed.

Several investigators regarded AZT as a cationic compound (Henry et al., 1988; Kornhauser et al., 1989), and renal clearance of AZT was shown to be reduced by cimetidine (Chatton et al., 1990). This is consistent with the results that cimetidine is an efficient inhibitor of hOAT3 (Cha et al., 2001), and AZT is a substrate for hOAT3. In addition, AZT was shown to be a low-affinity substrate for OCT in rat brush-border membrane vesicles from renal cortex (Griffiths et al., 1991), but not rat basolateral membrane vesicles from renal cortex (Griffiths et al., 1992). Furthermore, Aiba et al. (1995) demonstrated that AZT is transported via OAT system in the basolateral membrane, whereas it is transported via the OCT system in the brush-border membrane. Since hOCT2 is localized in the basolateral side of the proximal tubule (Pietig et al., 2001), these pieces of evidence are consistent with the current results that hOCT2 does not mediate AZT uptake. Further studies should be performed to elucidate the interaction of AZT with OCTN2, which mediates the transport of organic cations in the apical side of the proximal tubule (Tamai et al., 1998).

In addition to basolateral transporters, the characterization of the interaction between ACV, GCV, or AZT and apical OATs is also important. These apical OATs other than hOAT4 may include OAT-K1 (Saito et al., 1996), OAT-K2 (Masuda et al., 1999), organic anion-transporting peptide-1 (oatp1) (Jacquemin et al., 1994), multiple drug resistance protein (MRP2) (Leier et al., 2000), and human inorganic phosphate transporter (NPT1) (Uchino et al., 2000). However, this issue is beyond the scope of this study, and further research should be performed to elucidate this.

After oral administration, VACV is rapidly absorbed from the gastrointestinal tract via intestinal dipeptide transporter and almost completely converted to ACV and l-valine by first pass intestinal and/or hepatic metabolism (Perry and Faulds, 1996; Wang et al., 1996). Although hOAT3 is not localized to the gastrointestinal tract, hOAT3 but not hOAT1 was shown to mediate the transport of VACV. Clinical implication of this phenomenon remains unknown. However, since hOAT1 but not hOAT3 mediated the uptake of ACV, it was suggested thatl-valyl may be important for differential substrate recognition between hOAT1 and hOAT3. Interestingly, among various substrates examined, VACV was the only substrate that is transported only via hOAT3.

We previously found that probenecid inhibited the organic anion uptake mediated by hOAT1 and hOAT3 up to ∼10 to 20% of control (Takeda et al., 2001). In contrast to this, in the current study, probenecid exerted weak inhibitory effects on the hOAT1-mediated ACV uptake, i.e., up to approximately 80% of control (Fig. 8). At present, the precise reason for this discrepancy remains unknown; however, this may be associated with the difference of affinity in hOAT1 for PAH and ACV, i.e., the Km values of hOAT1 for PAH and ACV were 20.1 and 342.3 μM, respectively (Takeda et al., 2001; Table 1). The steady-state maximum plasma concentration was reported to be 170 μM (Nierenberg, 1983). Since the IC50 value of probenecid for hOAT1-mediated ACV uptake was over 1 mM, which is higher than the therapeutically relevant concentration of probenecid in the plasma (within 5-fold of the maximum steady-state concentration of probenecid in the plasma) (Zhang et al., 2000). The results suggest that hOAT1 is not responsible for the drug interaction between ACV and probenecid.

In conclusion, the current results suggest that hOAT1 and hOCT1 are responsible for renal ACV and GCV transport, whereas hOATs, but not hOCTs, are responsible for renal AZT transport. The current results provide important information that will lead to safer and more efficient clinical use of ACV, GCV, and AZT. Particular attention must be taken when these antivirals are concomitantly used with other drugs that share common transporters as these antivirals for urinary excretion. Otherwise, concomitant administration of such drugs potentially induces the increase in plasma concentrations of these antivirals, resulting in adverse drug reactions.

Footnotes

  • This study was supported in part by Grants-in-Aid 11671048, 11694310, and 13671128 from the Ministry of Education, Culture, Sports, Sciences and Technology, the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and Research on Health Sciences focusing on Drug Innovation from Japan Health Sciences Foundation.

  • Abbreviations:
    ACV
    acyclovir
    hOAT
    human organic anion transporter
    hOCT
    human organic cation transporter
    GCV
    ganciclovir
    VACV
    valacyclovir
    AZT
    zidovudine
    S1
    S2, and S3, the first, second, and third segment of proximal tubule
    TEA
    tetraethylammonium
    D-PBS
    Dulbecco's modified phosphate-buffered saline
    PGF2α
    prostaglandin F2α
    PAH
    para-aminohippuric acid
    HIV
    human immunodeficiency virus
    • Received August 28, 2001.
    • Accepted November 11, 2001.

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Human Organic Anion Transporters and Human Organic Cation Transporters Mediate Renal Antiviral Transport

Michio Takeda, Suparat Khamdang, Shinichi Narikawa, Hiroaki Kimura, Yasuna Kobayashi, Toshinori Yamamoto, Seok Ho Cha, Takashi Sekine and Hitoshi Endou
Journal of Pharmacology and Experimental Therapeutics March 1, 2002, 300 (3) 918-924; DOI: https://doi.org/10.1124/jpet.300.3.918
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