Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Prostglandins (PGs) play various physiological and pathophysiological roles. Among them, PGE₂ and PGF₂ are the predominant cyclooxygenase metabolites of arachidonic acid in the kidney (Breyer and Badr, 1996). PGE₂ and PGF₂ play an important role in the tubular reabsorption of salt and water as well as in the control of renal vascular resistance and the maintenance of glomerular hemodynamics. PGE₂ is known to be converted enzymatically to PGF₂ (Leslie and Levine, 1973; Terragno et al., 1976), and this conversion is enhanced in pathophysiological settings including sodium depletion (Siragy and Carey, 1997). Therapeutically, since both PGE₂ and PGF₂ have potent oxytoxic actions, the synthetic preparations of these compounds have been used to terminate pregnancy and to facilitate labor (Foegh and Ramwell, 2001). In addition, PGE₂ has also been used for the treatment of gastric and duodenal ulcers, especially those induced by nonsteroidal anti-inflammatory drugs (Altman DF, 2000). Pharmacokinetically, it has been reported that 63% of PGE₂ and 55.7% of PGF₂ administered were excreted into the urine in rats (Nishibori and Matsuoka, 1971; Nishibori et al., 1973). In addition, studies with perfused kidney tubules indicated that active vectorial PG transport across the epithelium is a two-step process consisting of active, energy-dependent basolateral uptake followed by passive apical secretion (Irish, 1979). Since PGE₂ and PGF₂ possess anionic moieties (Fig. 1, A and B), it is suggested that urinary excretion of PGE₂ and PGF₂ is mediated by the organic anion transport system. However, little is known about the molecular mechanism of the tubular secretion of PGE₂ and PGF₂.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Chemical structures of PGE2 (A) and PGF2(B).

The secretion of numerous organic anions and cations, including endogenous metabolites, drugs, and xenobiotics, is an important physiological function of the renal proximal tubule. 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 and cations 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 organic anion transporter (OAT) family have been successively cloned including OAT1 (Sekine et al., 1997; Sweet et al., 1997; Hosoyamada et al., 1999; Lu et al., 1999), OAT2 (Simonson et al., 1994; Sekine et al., 1998), OAT3 (Kusuhara et al., 1999; Cha et al., 2001) and OAT4 (Cha et al., 2000). The organic cation transporters (OCTs) isolated so far are OCT1 (Grundemann et al., 1994; Zhang et al., 1998), OCT2 (Okuda et al., 1996; Busch et al., 1998). Among these clones, human OAT1 (hOAT1), hOAT2, hOAT3, and hOCT2 were shown to be localized to the basolateral side of the proximal tubule (Gorboulev et al., 1997; Hosoyamada et al., 1999; Cha et al., 2001; Pietig et al., 2001; unpublished observation), whereas hOAT4 was localized to the apical side of the proximal tubule (Babu et al., 2002).

The purpose of this study was to elucidate the molecular mechanism of renal PGE₂ and PGF₂ transport. For this purpose, we established the proximal tubule cells stably expressing hOAT1, hOAT2, hOAT3, hOAT4, hOCT1, and hOCT2.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. [³H]PGE₂ (7159.5 GBq/mmol) and [³H]PGF₂ (7943.2 GBq/mmol) were purchased from Amersham Biosciences UK, Ltd. (Buckinghamshire, UK). ¹⁴C-Labeled para-aminohippuric acid (PAH) (1.8648 GBq/mmol), [³H]estrone sulfate (ES) (1961 GBq/mmol) and [¹⁴C]tetraethylammonium (TEA) (2.035 GBq/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). PGE₂ and PGF₂ were obtained from Sigma-Aldrich (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 the Second Portion of Proximal Tubule (S₂) Cells Stably Expressing hOAT2, hOAT4, hOCT1, and hOCT2. S₂ cells were established by culturing the microdissected S₂ segment derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene (Hosoyamada et al., 1996). During this establishment period, the functions of OATs and OCTs may have been lost. However, among the various cell lines stably expressing rat OAT3 that we have established, i.e., S₂ cells, the terminal proximal tubule (S₃) cells, Chinese hamster ovary cells and LLC-PK1 cells, we found that S₂ cells alone exhibited phorbol 12-myristate 13-acetate-induced down-regulation of organic anion uptake, which is recognized in the intact proximal tubule (Takeda et al., 2000b). Thus, we suggest that S₂ cells possess the essential machinery for the regulation of the OAT system and are the most suitable host for analyzing the OAT system. The establishment and characterization of S₂ hOAT1 and S₂ hOAT3 were reported previously (Takeda et al., 2000a). The full-length cDNA of hOAT2 was isolated by screening human kidney cDNA library using rat OAT2 cDNA as a probe (Sekine et al., 1998). 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 (Gorboulev et al., 1997). The full-length cDNA of hOCT2 was isolated by screening 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 pcDNA3.1 (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. S₂ cells transfected with pcDNA3.1 lacking an insert and pSV2neo were designated as S₂ pcDNA3.1 (mock), and used as control. 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 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]PAH for hOAT1 (Hosoyamada et al., 1999), [³H]PGF₂ for hOAT2 (unpublished observation), [³H]ES for hOAT3 and hOAT4 (Cha et al., 2000, 2001) and [³H]TEA for hOCT1 and OCT2 (Okuda et al., 1996; Zhang et al., 1998).

Uptake Experiments. Uptake experiments were performed as previously described (Takeda et al., 1999, 2000a,b, 2002). The S₂ cells were seeded in 24-well tissue culture plates at a cell density of 1 × 10⁵ cells/well. After the cells were cultured for 2 days, the cells 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 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 a solution containing either [³H]PGE₂ or [³H]PGF₂ at various concentrations as indicated in each experiment at 37°C for 1 min. 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 -scintillation counter (LSC-3100; Aloka, Tokyo, Japan).

Inhibition Study. To evaluate the inhibitory effects of PGE₂ and PGF₂ on organic anion uptake in S₂ hOAT1, S₂ hOAT2, S₂ hOAT3, and S₂ hOAT4, and organic cation uptake in S₂ hOCT1 and S₂ hOCT2, the cells were incubated in a solution containing either 5 µM [¹⁴C]PAH for 2 min (for hOAT1), 50 nM [³H]PGF₂ for 1 min (for hOAT2), 50 nM [³H]ES for 2 min (for hOAT3 and hOAT4), or 5 µM [³H]TEA for 5 min (for hOCT1 and hOCT2) in the absence or presence of PGE₂ and PGF₂ at 37°C as described above. PGE₂ and PGF₂ 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 the Student's unpaired t test. Differences were considered significant at P < 0.05.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

PGE₂ and PGF₂ Uptake Mediated by hOATs and hOCTs. We have already observed that S₂ hOAT2 exhibited a time- and dose-dependent uptake of PGF₂ with a K_m value of 425 nM (unpublished observation). We further elucidated the time-dependent uptake of PGE₂ and PGF₂ in S₂ cells stably expressing hOATs and hOCTs. As shown in Fig. 2, S₂ hOAT1 (A), S₂ hOAT2 (B), S₂ hOAT3 (C), S₂ hOAT4 (D), S₂ hOCT1 (E), and S₂ hOCT2 (F) exhibited higher PGE₂ uptake than mock. Similarly, as shown in Fig. 3, S₂ hOAT1 (A), S₂ hOAT3 (B), S₂ hOAT4 (C), S₂ hOCT1 (D), and S₂ hOCT2 (E) exhibited significantly higher PGF₂ uptake than mock. The kinetics of PGE₂ and PGF₂ uptake were examined to evaluate the pharmacological characteristics of hOATs and hOCTs on the uptake of PGE₂ and PGF₂. Figure 4 shows the Eadie-Hofstee plots of the concentration dependence of PGE₂ uptake by S₂ hOAT1 (A), S₂ hOAT2 (B), S₂ hOAT3 (C), S₂ hOAT4 (D), S₂ hOCT1 (E), and S₂ hOCT2 (F) after subtraction of uptake by mock. The estimated K_m values of PGE₂ uptake by hOAT1, hOAT2, hOAT3, hOAT4, hOCT1, and hOCT2 are listed in Table 1. On the other hand, Fig. 5 shows the Eadie-Hofstee plots of concentration dependence of PGF₂ uptake by S₂ hOAT1 (A), S₂ hOAT3 (B), S₂ hOAT4 (C), S₂ hOCT1 (D), and S₂ hOCT2 (E) after subtraction of uptake by mock. The estimated K_m values of PGF₂ uptake by hOAT1, hOAT3, hOAT4, hOCT1, and hOCT2 are also listed in Table 1. These results suggest that PGE₂ and PGF₂ transport is mediated by hOAT1, hOAT2, hOAT3, hOAT4, hOCT1, and hOCT2.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Time course of PGE2 uptake by hOATs and hOCTs. S2 hOAT1 (A), S2 hOAT2 (B), S2 hOAT3 (C), S2 hOAT4 (D), S2 hOCT1 (E), S2 hOCT2 (F), and mock were incubated in solution containing 5 nM [3H]PGE2 at 37°C up to 4 min. Each value represents the mean ± S.E. of four determinations from one typical experiment.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Time course of PGF2 uptake by hOATs and hOCTs. S2 hOAT1 (A), S2 hOAT3 (B), S2 hOAT4 (C), S2 hOCT1 (D), S2 hOCT2 (E), and mock were incubated in solution containing 5 nM [3H]PGF2 at 37°C up to 4 min. Each value represents the mean ± S.E. of four determinations from one typical experiment.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Kinetic analysis of PGE2 uptake by hOATs and hOCTs. S2 hOAT1 (A), S2 hOAT2 (B), S2 hOAT3 (C) S2 hOAT4 (D), S2 hOCT1 (E), S2 hOCT2 (F), and mock were incubated in solution containing various concentrations of [3H]PGE2 at 37°C for 1 min. The values for the uptake by mock were subtracted. Analysis of Eadie-Hofstee plots was performed. Each value represents mean ± S.E. of three determinations from one typical experiment.

View this table: [in this window] [in a new window]   TABLE 1 PGE2 and PGF2 uptake by hOATs and hOCTs S2 hOAT1, S2, hOAT2, S2 hOAT3, S2 hOAT4, S2 hOCT1, S2 hOCT2, and mock were incubated in a solution containing either [3H]PGE2 or [3H]PGF2 at various concentrations at 37°C for 1 min. Each value represents the mean ± S.E. of three determinations from one typical experiment.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Kinetic analysis of PGF2 uptake by hOATs and hOCTs. S2 hOAT1 (A), S2 hOAT3 (B), S2 hOAT4 (C), S2 hOCT1 (D), S2 hOCT2 (E), and mock were incubated in solution containing various concentrations of [3H]PGF2 at 37°C for 1 min. The values for the uptake by mock were subtracted. Analysis of Eadie-Hofstee plots was performed. Each value represents mean ± S.E. of three determinations from one typical experiment.

Inhibitory Effects of PGE₂ and PGF₂ on Organic Anion and Organic Cation Uptake. We examined the inhibitory effects of PGE₂ and PGF₂ on organic anion uptake mediated by hOAT1, hOAT2, hOAT3, and hOAT4, and organic cation uptake mediated by hOCT1 and hOCT2. Table 2 shows that PGE₂ significantly inhibited organic anion uptake mediated by hOAT1, hOAT2, hOAT3, and hOAT4 (n = 4, *P < 0.001 versus control), and organic cation uptake mediated by hOCT1 and hOCT2 (n = 4, *P < 0.001 and **P < 0.01 versus control). Similarly, PGF₂ exerted a significant inhibitory effect on hOAT1-, hOAT2-, hOAT3-, and hOAT4-mediated organic anion uptake (n = 4, *P < 0.001 versus control), and hOCT1- and hOCT2-mediated organic cation uptake (hOCT1: n = 4, *P < 0.001 versus control; hOCT2: n = 4, **P < 0.01 versus control).

View this table: [in this window] [in a new window]   TABLE 2 Effects of PGE2 and PGF2 on organic anion uptake by hOATs and the organic cation uptake by hOCTs S2 hOAT1, S2 hOAT2, S2 hOAT3, S2 hOAT4, S2 hOCT1, and S2 hOCT2 were incubated in a solution containing either 5 µM [14C]PAH for 2 min (for hOAT1), 50 nM [3H]PGF2 for 1 min (for hOAT2), 50 nM [3H]ES for 2 min (for hOAT3 and hOAT4), or 5 µM [3H]TEA for 5 min (for hOCT1 and hOCT2) in the absence or presence of PGE2 and PGF2 at 37°C. Each value represents the mean ± S.E. of four determinations from one typical experiment.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

hOAT1 and hOAT3 were shown to mediate the transport of nonsteroidal anti-inflammatory drugs, antitumor drugs, histamine H₂ receptor antagonist, PGs, diuretics, angiotensin-converting enzyme inhibitors, and -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 at the basolateral side of the S₂ segment of the proximal tubule (Hosoyamada et al., 1999) versus hOAT3 at 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, also shown to be localized to the basolateral side of the proximal tubule, mediates the transport of organic anions including salicylate and PGF₂ (unpublished observation). HOAT4 also mediates the apical transport of various anionic drugs; however, this transporter exhibits relatively narrow substrate recognition compared with hOAT1 and hOAT3 (Cha et al., 2000; Babu et al., 2002).

HOCT1 was shown to be mainly localized to the liver and to mediate polyspecific pH independent transport of organic cations, whereas organic cation transport by hOCT2 was pH independent, electrogenic, and polyspecific. (Gorboulev et al.,1997; Busch et al., 1998; Zhang et al., 1998).

At present, limited information is available concerning renal tubular excretion of PGE₂ and PGF₂; PG transporter-mediated transport of PGE₂ and the inhibitory effect of PGE₂ on rat-OAT1-mediated organic anion transport were reported (Kanai et al., 1995; Sekine et al., 1997). In this regard, the current results revealed that multiple pathways exist concerning the transport of PGs in the basolateral side of the proximal tubule, i.e., hOAT1, hOAT2, hOAT3, and hOCT2, whereas we could not exclude the possibility that transporters other than those analyzed in this study are also involved in PGE₂ and PGF₂ transport.

In addition to basolateral transporters, the characterization of the interaction between PGE₂ or PGF₂ and apical OATs is also important. In this regard, we found that hOAT4, an apical transporter of renal tubules, mediates the uptake of PGE₂ as well as and PGF₂. In addition, we already observed that hOAT4 mediates the efflux of ES (Babu et al., 2002). Thus, it is possible that hOAT4 mediates the bidirectional transport of PGs on the apical side of the proximal tubule. On the other hand, we could not find the functional characteristics of hOAT4 as an exchanger (Cha et al., 2000). Instead, when the amount of PG reabsorbed from the tubular lumen together with that taken up from the basolateral side reaches a certain threshold that activates the hOAT4-mediated efflux, PG reabsorption from the tubular lumen may contribute to the secretion of PG into the tubular lumen. At present, the role of other apical transporters mediating organic anion transport remains unknown, including OAT-K1 (Saito et al., 1996), OAT-K2 (Masuda et al., 1999), organic anion-transporting peptide 1 (Jacquemin et al., 1994), multiple drug resistance protein 2 (Leier et al., 2000), and human type I sodium-dependent inorganic phosphate transporter (Uchino et al., 2000), and further study should be performed to elucidate this.

As shown in Fig. 1, PGE₂ and PGF₂ possess anionic moieties. This is consistent with the current results that hOAT1, hOAT2, hOAT3, and hOAT4 mediate the transport of PGE₂ and PGF₂. In contrast, since both PGE₂ and PGF₂ possess no cationic moiety, hOCT1- and hOCT2-mediated PGE₂ and PGF₂ uptakes were unexpected. Various substrates were shown to be transported via the OAT as well as the OCT system, and are called bisubstrates (Ullrich et al., 1993a,b). In this regard, PGE₂ and PGF₂ could also be regarded as bisubstrates. Recently, we also found that acyclovir and ganciclovir are transported by hOAT1 as well as hOCT1 (Takeda et al., 2002). In addition, hOATs including hOAT1, hOAT2, and hOAT3 and hOCT2 are colocalized in the basolateral side of the proximal tubule (Gorboulev et al., 1997; Hosoyamada et al., 1999; Cha et al., 2001; Pietig et al., 2001; unpublished observation), and it is possible to design a model in which PGE₂ and PGF₂ are transported via OAT and OCT at the same time. Further studies should be performed to identify the mechanism underlying the involvement of OCTs in the transport of PGE₂ and PGF₂.

In conclusion, the current results suggest that hOATs as well hOCTs are responsible for the transport of PGE₂ and PGF₂ in the basolateral side as well as the apical side of the renal tubule. The current results provide important information for safer and more efficient clinical use of PGE₂ and PGF₂. Particular attention must be taken when these PGE₂ and PGF₂ are concomitantly used with other drugs that share common transporters with these PGs for tubular excretion. Otherwise, concomitant administration of PGE₂ or PGF₂ with other drugs may induce the increase in plasma concentrations of these PGs or other drugs, resulting in adverse drug reactions.