Introduction

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Organic anion transport systems of the renal proximal tubule play an important role in the elimination of a wide variety of anionic compounds, including endogenous metabolites, drugs and xenobiotics (Møller and Sheikh, 1983; Pritchard and Miller, 1993). Secretion of organic anions involves transport across the basolateral membrane and accumulation in the epithelial cells, followed by efflux from the cells across the brush-border membrane. Studies with intact kidneys, renal cortical slices, isolated renal tubules and renal membrane vesicles have provided a great deal of information about organic anion transport systems (Chatsudthipong and Dantzler, 1992; Cross and Taggart, 1950; Inui et al., 1986; Pritchard, 1990, 1995; Shimada et al., 1987). It is proposed that the transport of organic anions in the basolateral membrane is a tertiary active process. As a primary step, an inwardly directed Na⁺ gradient is created by the outward transport of Na⁺ via Na⁺-K⁺-adenosinetriphosphatase. This Na⁺ gradient drives dicarboxylate uptake into the cell by Na⁺/dicarboxylate cotransporter, thereby creating an outwardly directed dicarboxylate gradient. This dicarboxylate gradient in turn drives entry of organic anions into the cells (Pritchard and Miller, 1993). On the one hand, dicarboxylates such as -KG are also generated by intracellular metabolisms such as the citric acid cycle. Several studies with renal tubules and cortical slices have reported that uptake of PAH, a typical organic anion, is increased by preloading with -KG (Chatsudthipong and Dantzler, 1992; Pritchard, 1995). However, there is little direct evidence that -KG generated by intracellular metabolism is effluxed from the cell via PAH/dicarboxylate exchange in renal basolateral membrane.

The development of cell culture techniques has furthered the study of transcellular transport of solutes such as organic cations (Fouda et al., 1990; Inui et al., 1985; Saito et al., 1992; Takano et al., 1992) across renal epithelial monolayers (Handler, 1986). We found that the transcellular transport of PAH occurred unidirectionally from the basal to apical side across monolayers of OK cells (Hori et al., 1993), which were established from the American opossum kidney (Koyama et al., 1978). We also showed that PAH transport in the basolateral and apical membranes of OK cells is a specifically mediated, vectorial process (Takano et al., 1994). Furthermore, studies by using various dicarboxylates and -lactam antibiotics showed that the PAH transport system in the basolateral membrane of OK cells has a similar substrate specificity to that in renal proximal tubules (Fritzsch et al., 1989; Nagai et al., 1995). Based on these findings, it seems that a PAH/dicarboxylate exchange system is involved in the basolateral PAH uptake of OK cells.

In our study, we investigated the efflux of intracellular -KG via PAH/dicarboxylate exchange using OK cell monolayers. The results showed that the efflux of intracellular -KG to the basolateral side of the monolayers was increased by PAH, but the intracellular -KG concentration was not changed. These findings demonstrate that intracellular -KG is an energy source for PAH transport in the basolateral membrane of OK cells.

	Materials and Methods

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Cell culture. OK cells were cultured in medium 199 (Flow Laboratories, Rockville, MD) containing 10% fetal bovine serum (Whittaker Bioproducts Inc., Walkersville, MD) without antibiotics, in an atmosphere of 5% CO₂ - 95% air at 37°C, and subcultured every 5 to 7 days using 0.02% EDTA and 0.05% trypsin (Hori et al., 1993). OK cells were used between passages 79 and 94.

Transport measurements. The efflux of -KG and the uptake of PAH were measured in OK cell monolayers cultured in Transwell chambers (Costar, Cambridge, MA). To prepare cell monolayers, cells were seeded at a density of 4 × 10⁵ cells/cm² on polycarbonate membranes (3-µm pore size) in Transwell cell chambers (4.71 cm² surface area), which were placed in six-well, cluster plates. The volume of medium inside and outside the Transwell chambers was 1.5 and 2.6 ml, respectively. The medium was renewed every 2 or 3 days, and the cells were used on between the 5th and 7th day after seeding. The transport study was performed at 37°C in PBS (buffer containing in mM, 137 NaCl, 3 KCl, 8 Na₂HPO₄, 1.5 KH₂PO₄, 1 CaCl₂, and 0.5 MgCl₂) supplemented with 5 mM D-glucose. After removal of the culture medium, the cell monolayers were washed twice with PBS buffer containing 5 mM D-glucose. The measurements of -KG efflux from OK cells were initiated by adding 0.5 ml of the medium either with or without PAH to the basolateral and the apical side of OK cell monolayers. Then, the cell monolayers were incubated for the specified period of time, and the medium -KG concentration in the basolateral and the apical side was assayed by the fluorimetric method as stated below. At the end of the incubation, the filter was washed rapidly three times with PBS buffer containing 5 mM D-glucose. The filters with monolayers were detached from the chambers and immersed in 0.5 ml of 3% (v/v) perchloric acid for 30 min on ice. The extracts were neutralized with 3 M sodium hydroxide and -KG concentration was determined with the fluorimetric method. To measure PAH transport across the basolateral side of OK cell monolayers, the reaction was initiated by adding PBS buffer containing 5 mM D-glucose, [¹⁴C]PAH and [³H]mannitol to the basolateral side of the monolayers. D-[³H]Mannitol was used to estimate extracellular trapping and nonspecific uptake. After incubation for 1 min, the medium was aspirated immediately and the filter was washed rapidly three times with ice-cold PBS buffer containing 5 mM D-glucose. Then, the cell monolayers on the filter were solubilized in 0.5 ml of 0.1 M sodium hydroxide, and the amount of substrate taken up by the cells was measured by counting the radioactivity.

Analytical methods. Intracellular and medium -KG concentrations were determined by the fluorimetric method of Williamson and Corkey (1979). The conversions of -KG and aspartate to glutamate and oxaloacetate, respectively, were catalyzed by aspartate aminotransferase. The oxaloacetate was then converted to malate by malate dehydrogenase. The associated conversion of NADH to NAD⁺ was determined fluorimetrically (excitation, 360 nm; emission, 460 nm) at 37°C with a Shimadzu spectrofluorophotometer RF-5000 (Kyoto, Japan). Intracellular -KG concentration was calculated by using 9.5 µl/mg of protein as the intracellular volume of OK cells (Yuan et al., 1991).

Materials. p-[glycyl-1-¹⁴C]Aminohippurate (PAH 1.6 to 2.0 GBq/mmol) and D-[³H]mannitol (832.5 GBq/mmol) were obtained from Du Pont-New England Nuclear (Boston, MA). Aspartate aminotransferase, probenecid, antimycin A and NADH were purchased from Sigma Chemical Co. (St. Louis, MO). PAH and aspartate were purchased from Nacalai Tesque (Kyoto, Japan). Malate dehydrogenase was purchased from Toyobo Co. (Osaka, Japan). All other chemicals used were of the highest purity available.

	Results

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Effect of PAH application on the -KG efflux from OK cells and the intracellular level of -KG. We examined the efflux of -KG from the OK cell to the basolateral side in the absence or presence of PAH in the basolateral medium. As shown in figure 1A, the -KG efflux to the basolateral side was markedly increased by PAH. However, the incubation with PAH did not significantly affect the intracellular -KG concentration (fig. 1B), despite the increase in the -KG efflux from the OK cells (fig. 1A).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of p-aminohippurate (PAH) application on -ketoglutarate (-KG) efflux to the basolateral side (A) and the intracellular level of -KG (B) in OK cell monolayers. A, After incubation without () or with () PAH (100 µM) in basolateral medium for 5, 15, 30 and 60 min at 37°C, -KG efflux to the basolateral side was measured. B, After incubation for 5, 15, 30 and 60 min, the intracellular level of -KG in OK cells was determined. Each point represents the mean ± S.E. of four monolayers from two experiments. *P < .05, significant difference from each control.

Effect of the side of PAH application on the -KG efflux from OK cells. Figure 2 shows the -KG efflux to the basolateral and apical side, after applying PAH to the basolateral and apical side, respectively. To estimate PAH-dependent -KG efflux, the efflux in the absence of PAH was subtracted from that in the presence of PAH. PAH-independent -KG efflux to the basolateral and apical side was 92.9 ± 6.0 and 144.4 ± 31.6 pmol·cm²·15 min¹, respectively. The basolateral application of PAH induced a larger increase in PAH-dependent -KG efflux than did the apical application.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Effect of side of PAH application on -KG efflux from OK cell monolayers. PAH (100 µM) was added to either the basolateral or apical side. After incubation for 15 min at 37°C, -KG efflux to the PAH-applied side was measured. To estimate PAH-dependent -KG efflux, the efflux in the absence of PAH was subtracted from that in the presence of PAH. The -KG efflux to the basolateral and apical side in the absence of PAH was 92.9 ± 6.0 and 144.4 ± 31.6 pmol·cm2·15 min1, respectively. Each column represents the mean ± S.E. of four monolayers from two experiments.

Effect of probenecid on PAH-dependent -KG efflux to the basolateral side. We next examined the effect of probenecid on the -KG efflux from the OK cell to the basolateral side (fig. 3). The basolateral application of 100 µM probenecid slightly increased the -KG efflux, but the increase induced by probenecid was lower than that by 100 µM PAH. Moreover, coincubation with 100 µM PAH and 100 µM probenecid did not increase the -KG efflux to the basolateral side.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of probenecid on PAH-dependent -KG efflux to the basolateral side of OK cell monolayers. After incubation in the absence or presence of PAH (100 µM) and/or probenecid (100 µM) in the basolateral medium, -KG efflux to the basolateral side was measured. -KG efflux to the basolateral side in the absence of both PAH and probenecid (control) was 143.3 ± 30.1 pmol·cm2·15 min1. Each column represents the mean ± S.E. of four monolayers from two experiments. *P < .05, significant difference from control.

-KG efflux to the basolateral side with varying concentrations of PAH. The efflux of -KG from OK cells to the basolateral side was saturable with increasing PAH concentration in the basolateral medium (fig. 4). However, the simultaneously measured concentrations of intracellular -KG were nearly constant level (data not shown). Eadie-Hofstee analysis of the PAH-dependent -KG efflux to the basolateral side showed that the Michaelis constant (K_m) was 33.6 µM, and the maximum uptake rate (Vmax) was 355.4 pmol ·cm²·15 min¹. A Hill plot of the PAH-dependent -KG efflux to the basolateral side depicted in figure 4 yielded a Hill coefficient of 0.96, suggesting that one PAH molecule is exchanged for one -KG molecule (fig. 4, inset). Furthermore, the correlation was examined between the basolateral PAH uptake and the PAH-dependent -KG efflux to the basolateral side when the PAH concentrations were varied. As shown in figure 5, the basolateral PAH uptake was linearly correlated with the PAH-dependent -KG efflux. However, the slope obtained from figure 5 was 2.4, suggesting that the stoichiometry is 2 PAH: 1 -KG by this analysis.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   -KG efflux to the basolateral side of OK cell monolayers with varying concentrations of PAH. After incubation with various concentrations of PAH in the basolateral medium for 15 min, -KG efflux to the basolateral side was measured. To estimate PAH-dependent -KG efflux, the efflux in the absence of PAH was subtracted from that in the presence of PAH. The -KG efflux to the basolateral side in the absence of PAH was 125.3 ± 11.2 pmol·cm2·15 min1. Inset, Hill plot of the data. Each point represents the mean ± S.E. of four monolayers from two experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Correlation between the basolateral PAH uptake and the PAH-dependent -KG efflux to the basolateral side of OK cell monolayers with varying PAH concentration. For PAH uptake, [14C]PAH and [3H]mannitol were added to the basolateral side of the monolayers. PAH uptake was measured for 1 min at 37°C. For PAH-dependent -KG efflux to the basolateral side, data in figure 4 were expressed as pmol/mg of protein per min. PAH concentrations in the basolateral medium of OK cells are (in µM) 15 (), 50 (), 100 (), 200 () and 400 (). The linear regression was as follows: y = 2.4x - 15.6 (r = 0.94).

Effect of antimycin A on the basolateral PAH uptake, -KG efflux to the basolateral side and intracellular -KG concentration. Figure 6 shows the effect of antimycin A, a metabolic inhibitor, on the basolateral [¹⁴C]PAH uptake in OK cells. Pretreatment of antimycin A for 30 min inhibited PAH uptake across the basolateral membrane of OK cells in a dose-dependent manner with an apparent half-maximal inhibitory concentration (IC₅₀) of approximately 28 nM. To confirm whether the inhibition of the basolateral PAH uptake correlates with the decrease in the efflux of -KG to the basolateral side, the -KG efflux in the presence of PAH in the basolateral medium was examined after pretreatment with antimycin A for 30 min. As shown in figure 7A, a dose-dependent inhibitory effect on the efflux by antimycin A was observed, and the apparent IC₅₀ was approximately 51 nM. The simultaneously measured intracellular concentration of -KG was also decreased by antimycin A in a dose-dependent manner (fig. 7B), and the apparent IC₅₀ was approximately 45 nM.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Dose-dependent effect of antimycin A on PAH uptake from the basolateral side of OK cell monolayers. Confluent monolayers were incubated for 30 min with various concentrations of antimycin A (1010 -106 M). After washing the cells, [14C]PAH (15 µM) and D-[3H]mannitol (15 µM) were added to the basolateral side of the monolayers, and [14C]PAH uptake for 1 min at 37°C was measured. Each point represents the mean ± S.E. of four monolayers from two experiments.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effect of antimycin A on -KG efflux to the basolateral side (A) and the intracellular -KG concentration (B) in OK cell monolayers. A, Confluent monolayers were incubated for 30 min with various concentrations of antimycin A (1010-106 M). After washing the cells, PAH (100 µM) was added to the basal side, and the -KG efflux to the applied side for 15 min was measured. B, After incubation for 15 min, -KG in OK cells was determined. Each point represents the mean ± S.E. of four monolayers from two experiments.

	Discussion

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We reported that OK cells are a useful in vitro model system to study renal organic anion transport across intact epithelial cells (Hori et al., 1993), and both apical and basolateral transport systems for PAH in OK cells are vectrial and specifically mediated processes (Takano et al., 1994). Studies with basolateral membrane vesicles, renal cortex slices and isolated renal tubules have shown that PAH is transported via PAH/dicarboxylate exchange in renal basolateral membrane, and that -KG could be a physiological substrate for PAH/dicarboxylate exchange (Chatsudthipong and Dantzler, 1992; Pritchard, 1990; Shimada et al., 1987). Subsequently, we showed that the basolateral PAH transport system in OK cells has a similar substrate specificity to that in renal proximal tubules (Nagai et al., 1995). Based on these findings, we suggested that the PAH/dicarboxylate exchange system is involved in the basolateral uptake of OK cells. However, it is not clear whether -KG, which is generated by intracellular metabolism, is effluxed via PAH/dicarboxylate exchange in renal basolateral membrane. Therefore, PAH-dependent efflux of intracellular -KG was examined using OK cell monolayers on this study.

The presence of PAH in the medium of the basolateral side of OK cell monolayers significantly increased the -KG efflux to the basolateral side. The -KG was effluxed linearly with time during a 60-min incubation with PAH (fig. 1A), whereas the intracellular -KG concentration was nearly constant whether PAH was present or not in the medium (fig. 1B). These results indicate the involvement of the PAH/dicarboxylate exchange for PAH transport across the basolateral membrane in OK cells, and that the intracellular -KG concentration was maintained by metabolic -KG production. The maintenance of the -KG concentration level by intracellular metabolism supports the previous observation that the basal-to-apical transport of [¹⁴C]PAH in OK cells was linear up to 60 min, even in the absence of an exogenous -KG supply (Hori et al., 1993; Nagai et al., 1995).

It is reported that Na⁺/-KG cotransporter would play an important role in the maintenance of intracellular -KG level or the outward -KG gradient as a driving force for the basolateral PAH transport in renal proximal tubules (Pritchard, 1995), and we are also interested in the contribution of Na⁺/-KG cotransporter in the basolateral PAH uptake. However, the contribution seems to be small, if any, in OK cells, judging from the results stated below. First, we previously showed that lithium, which inhibits the Na⁺/dicarboxylate cotransport, had no effect on the uptake of -[¹⁴C]KG across the basolateral membrane in OK cells. In addition, the basolateral uptake of -[¹⁴C]KG was only about 2% of the applied dose in OK cells even after incubation for 30 min (Takano et al., 1994). Furthermore, we examined whether the intracellular -KG concentration was changed by applying -KG in the basolateral or apical side. OK cells were incubated for 30 min in the presence of 400 µM -KG in the apical or the basolateral medium, but intracellular -KG concentration did not change in either case (data not shown). Based on these results, it is likely that OK cells have little activity of Na⁺/-KG cotransport in the apical and the basolateral membrane. Therefore, reuptake of -KG would be small and may not affect the present results. In addition, inhibition of the PAH-dependent -KG efflux by probenecid in figure 3 supports that the increase in -KG efflux by PAH application is due to the PAH/-KG exchange, and not to the inhibition of -KG reuptake. Further studies are needed to clarify the relative importance of Na⁺/-KG cotransport and metabolic production of -KG for the maintenance of intracellular -KG level as a driving force for PAH transport in renal proximal tubules.

Schmitt and Burckhardt (1993) reported that similar or identical PAH transporters were located in brush-border and basolateral membranes of bovine kidney proximal tubule cells. We examined the difference of PAH/-KG exchange activity at the basolateral and apical membrane of OK cells. As shown in figure 2, PAH-dependent -KG efflux to the basolateral side was clearly larger than that to the apical side. This difference of exchange activity between the basolateral and the apical membrane may explain the previous observation that PAH uptake by OK cells occurred preferentially across the basolateral membrane (Takano et al., 1994).

The PAH-dependent -KG efflux to the basolateral side was saturable with increasing PAH concentration in the basolateral medium (fig. 4). The kinetic analysis revealed an apparent K_m of 33.6 µM, which was not so different from the K_m value (64.0 µM) for the PAH uptake across the basolateral membrane of OK cells (Takano et al., 1994). Furthermore, the Hill plot showed a 1: 1 stoichiometry for PAH/-KG exchange in the basolateral membrane of OK cells, which corresponded with that in basolateral membrane vesicles reported by Schmitt and Burckhardt (1993). However, the stoichiometry obtained from figure 5 was 2 PAH: 1 -KG. The reason for the difference is unknown at present. The difference would not be due to the error in the transformation from cm² to mg protein because the amount of protein per well was nearly constant in all the experiments in our study. One possibility is that the Hill analysis overestimated the number of -KG molecules being exchanged for PAH. Another possibility is that one PAH molecule is exchanged with one -KG molecule via the PAH/-KG exchange and another PAH molecule is transported by an independent, yet unknown, mechanism. Further studies are needed to clarify the true stoichiometry of the PAH/-KG exchange.

In early in vitro studies of organic anion transport, it was shown that metabolic inhibitors and anaerobic conditions inhibited the PAH transport across the basolateral membrane (Cross and Taggart, 1950; Dominguez and Shideman, 1955; Ross and Weiner, 1972). In OK cells, antimycin A, a metabolic inhibitor, inhibited in a dose-dependent manner both the PAH uptake across the basolateral membrane and the -KG efflux to the basolateral side. The apparent IC₅₀ values of the PAH uptake and the -KG efflux induced by PAH application were 28 and 51 nM, respectively (figs. 6 and 7A). Furthermore, the intracellular -KG concentration was decreased by antimycin A pretreatment in a dose-dependent manner with an apparent IC₅₀ value of 45 nM (fig. 7B). These findings suggest that the inhibitory effect on the PAH uptake by antimycin A is, at least in part, due to the decrease in the intracellular concentration or the outwardly oriented gradient of -KG. Recently, Pritchard (1995) reported that PAH transport in rat renal cortical slices was modulated by changes in intracellular -KG concentration or the outward -KG gradient. Thus, alteration of -KG production and/or metabolism by endogenous or exogenous factors may influence organic anion secretion in renal proximal tubules.

In conclusion, PAH/dicarboxylate exchange is involved in PAH transport across the basolateral membrane of OK cells, and -KG is an intracellular substrate that exchanges for PAH across the basolateral membrane of OK cells. In addition, it seems likely that the inhibitory effect on the basolateral PAH transport in OK cells by the metabolic inhibitor antimycin A is, at least in part, due to the decrease in intracellular -KG concentration or the outward -KG gradient. These findings indicate that the -KG generated by intracellular metabolism is an energy source for PAH transport in the basolateral membrane of OK cells.