Introduction

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Ionic endogenous metabolites and xenobiotics are actively secreted in the renal proximal tubule, which is physiologically and pharmacologically important for epithelial function in the kidney. The secretion of organic cations through proximal tubular cells involves two processes, i.e., uptake of organic cation into cells from blood across the basolateral membrane, and active extrusion across the brush-border membrane into the tubular fluid (Rennick, 1981). Previous studies using plasma membrane vesicles isolated from the renal cortex elucidated the transport mechanisms in the brush-border membrane and the existence of the H⁺/organic cation antiport system (Dantzler et al., 1989; Holohan and Ross, 1981; Hsyu and Giacomini, 1987; Takano et al., 1984, 1985; McKinney and Kunnemann, 1985; Wright and Wunz, 1987). Cultured epithelial cells derived from kidney have been useful in the study of transcellular transport of solutes across renal epithelial monolayers. The pig kidney epithelial cell line LLC-PK₁ has been used extensively as a model for analysis of epithelial functions in renal proximal tubules (Handler, 1986). We have shown that LLC-PK₁ cells retain the H⁺/organic cation antiport system on the apical membrane and show a unidirectional transcellular transport of tetraethylammonium, a typical substrate for this system (Inui et al., 1985; Saito et al., 1992).

Several of the quinolone antibacterial drugs developed to date have been used clinically. Levofloxacin, one such drug, is well absorbed from the intestine, distributed to tissues and mainly excreted into the urine in humans (Nakashima et al., 1992). Tubular secretion as well as glomerular filtration contribute to the renal excretion of levofloxacin (Kamiya et al., 1992). We have been interested in the mechanisms of tubular secretion of quinolones, because most of these drugs are zwitterions at physiological pH. We have previously shown that ofloxacin, a racemate of levofloxacin and its optical enantiomer, potently inhibits the H⁺-dependent tetraethylammonium uptake by renal brush-border membrane vesicles (Okano et al., 1990). We have also demonstrated that levofloxacin potently inhibits the apical H⁺/organic cation antiport system in LLC-PK₁ cells (Ohtomo et al., 1996). However, the transport mechanisms of quinolones themselves in the kidney have yet to be elucidated.

In this study, the transport of levofloxacin and grepafloxacin by LLC-PK₁ monolayers was evaluated. The findings indicate that quinolones are specifically transported from the basolateral to apical side by LLC-PK₁ monolayers and have higher affinity for the transport system in the apical membrane, a system distinct from H⁺/organic cation antiport system.

	Materials and Methods

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Cell culture. LLC-PK₁ cells (ATCC, CRL-1392, Manassas, VA) were maintained by serial passage in medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum without antibiotics. Monolayers were grown in an atmosphere of 5% CO₂-95% air at 37°C, and were subcultured every 7 days using 0.02% EDTA and 0.05% trypsin (Saito et al., 1992). In general, the plastic dishes (100 mm) were inoculated with 1 × 10⁶ cells in 10 ml of complete culture medium. For the transport studies, LLC-PK₁ cells were seeded on polycarbonate membrane filters (3-µm pores, 4.71-cm² growth area) inside Transwell cell culture chambers (Costar, Cambridge, MA) at a cell density of 5 × 10⁵ cells/cm². Transwell chambers were placed in 35-mm wells of tissue culture plates with 2.6 ml of outside (basolateral side) and 1.5 ml of inside (apical side) medium. For efflux studies, LLC-PK₁ cell monolayers grown on 60-mm plastic culture dishes (4 × 10⁶ cells/cm²) were used. The medium was renewed every 2 days, and the cells were used on the 6th day after seeding. In our study, LLC-PK₁ cells were used between passages 212 and 221.

Transport and cellular accumulation measurements. Transcellular transport and accumulation of [¹⁴C]levofloxacin, [¹⁴C]grepafloxacin and [¹⁴C]tetraethylammonium were measured using monolayer cultures grown in Transwell chambers. The composition of incubation medium was as follows (in mM): 145 NaCl, 3 KCl, 1 CaCl₂, 0.5 MgCl₂, 5 D-glucose, 5 MES (pH 6.0) or 5 HEPES (pH 7.4). The pH of the medium was adjusted with a solution of HCl or NaOH. After removal of the culture medium from both sides of the monolayers, the cell monolayers were preincubated with incubation medium (2 ml each side) at 37°C for 15 min. Then, 2 ml of incubation medium containing the radioactive substrate were added to either the basolateral or apical side, 2 ml of nonradioactive incubation medium to the opposite side and the monolayers were incubated for specified periods at 37°C. D-[³H]Mannitol (5 µM, 22.8 kBq/ml), a compound that is not transported by the cells, was used to calculate the paracellular fluxes and the extracellular trapping of [¹⁴C]levofloxacin (5 µM, 5.4 kBq/ml), [¹⁴C]grepafloxacin (5 or 20 µM, 5.8 kBq/ml) and [¹⁴C]tetraethylammonium (50 µM, 6.2 kBq/ml). For transport measurements, aliquots of the incubation medium on the other side were taken at specified times, and the radioactivity was counted.

Efflux measurement. The monolayers were preincubated at 37°C for 15 min with 2 ml of the incubation medium (pH 7.4). After removal of the medium, cells were incubated with 2 ml of incubation medium containing [¹⁴C]levofloxacin and D-[³H]mannitol in the presence or absence of 1 mM of unlabeled levofloxacin at 4°C for 30 min, to make the same level of intracellular [¹⁴C]levofloxacin in each condition by simple diffusion. After the incubation, the medium was aspirated and the dishes were rapidly rinsed twice with 5 ml of ice-cold incubation medium. The cells were then incubated at 37°C with the incubation medium for a specified period. After the incubation, the medium was aspirated and the dishes were rapidly rinsed twice with 5 ml of ice-cold incubation medium. The cells were solubilized in 1.5 ml of 1 N NaOH and the radioactivity was determined as described above.

Protein assay. The protein contents of the cell monolayers solubilized in 1 N NaOH were determined by the method of Bradford (1976) using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA) with bovine -globulin as a standard.

Statistical analysis. Statistical significance of differences between mean values was calculated using the nonpaired t test. Multiple comparisons were performed using the Dunnett's test after analysis of variance. P < .05 was considered significant.

Materials. D-[³H]Mannitol (828.8 GBq/mmol) and [1-¹⁴C]tetraethylammonium (124.3 MBq/mmol) were purchased from NEN-Life Sciences (Boston, MA). [¹⁴C]Levofloxacin (1.07 GBq/mmol), unlabeled levofloxacin and ofloxacin were kindly supplied by Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan), [¹⁴C]grepafloxacin (1.17 GBq/mmol) and unlabeled grepafloxacin were supplied by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan), enoxacin and sparfloxacin were by Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan), ciprofloxacin was from Bayer AG (Leverkusen, West Germany) and cyclosporin A was from Novartis Pharma Inc. (Tokyo, Japan). Cimetidine, quinidine, tetraethylammonium and p-aminohippurate were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). All other chemicals used were of the highest purity available.

	Results

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Transcellular transport and cellular accumulation of levofloxacin. The transcellular transport and cellular accumulation of [¹⁴C]levofloxacin by LLC-PK₁ monolayers was evaluated. The basal-to-apical transport of [¹⁴C]levofloxacin was much larger than the apical-to-basal transport at each sampling time (fig. 1A). In the presence of 1 mM unlabeled levofloxacin, the transcellular transport of [¹⁴C]levofloxacin from the basolateral to apical side was decreased and the transport in the opposite direction was increased. The cellular accumulation of [¹⁴C]levofloxacin on both sides was significantly increased in the presence of 1 mM unlabeled levofloxacin (fig. 1B, P < .05).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Effect of unlabeled levofloxacin on the transcellular transport (A) and cellular accumulation (B) of [14C]levofloxacin by LLC-PK1 monolayers. The monolayers were incubated at 37°C for 60 min with 5 µM of [14C]levofloxacin added to either the basolateral (, ) or apical side (, ) in the absence (open symbols) or presence (solid symbols) of 1 mM of unlabeled levofloxacin. After incubation, the radioactivity on the opposite side was measured. After a 60-min transport measurement, accumulation was determined in the absence (open columns) or presence (dotted columns) of 1 mM of unlabeled levofloxacin. Each point or column represents the mean ± S.E. of three monolayers, or the mean of two monolayers at 30 and 60 min in the apical-to-basal transport in the absence of 1 mM unlabeled levofloxacin. * P < .05, significant difference from each open symbol.

Concentration-dependence of levofloxacin transcellular transport and basolateral uptake. The concentration-dependence of levofloxacin transcellular transport by LLC-PK₁ monolayers was next examined. Figure 2A shows the basal-to-apical transcellular transport of levofloxacin at 15 min as a function of an increased concentration of substrate. The relationship between concentration and the basolateral to apical flux rate approached saturation. The kinetic parameters were calculated using the following equation: V = V_max · S/(K_m + S) + K_d · S, where V is the transport rate (nmol/mg protein per 15 min), S is the substrate concentration in the medium (mM), K_m is the Michaelis-Menten constant (mM), V_max is the maximum velocity by the saturable process (nmol/mg protein per 15 min) and K_d is the coefficient of the nonsaturable transport, mainly simple diffusion (nmol/mg protein per 15 min/mM). The data were fitted to the above equation by nonlinear least squares regression analysis. The apparent K_m and V_max values for the saturable transport of levofloxacin were 0.6 mM and 3.8 nmol/mg protein per 15 min, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Kinetic analysis for concentration dependence of transcellular transport (A) and initial uptake from the basolateral side (B) of levofloxacin by LLC-PK1 monolayers. LLC-PK1 monolayers were incubated at 37°C for 15 min (A) or 1 min (B) with varying concentrations of levofloxacin added to the basolateral side. After incubation, the radioactivity on the opposite side or the uptake were measured. The solid and dotted lines represent the estimated overall and nonsaturable transport, respectively. Each point represents the mean ± S.E. of at least three monolayers from three independent experiments (A) or three monolayers (B). The insets show Eadie-Hofstee plots of levofloxacin transcellular transport (A) or initial uptake (B) after correction for nonsaturable transport. V, transport rate (nmol/mg protein per 15 min or per 1 min); S, substrate concentration (mM).

Effect of various quinolones on levofloxacin transcellular transport and cellular accumulation. To elucidate whether the transport of quinolones is mediated by specific transport systems, the effect of various quinolones on the transcellular transport and cellular accumulation of [¹⁴C]levofloxacin was examined using LLC-PK₁ monolayers. The transcellular transport of [¹⁴C]levofloxacin was inhibited by all quinolones examined, accompanied by increase of cellular accumulation (fig. 3). These results indicated that quinolones inhibited the apical efflux of levofloxacin out of the monolayers.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Effect of various quinolones on the transcellular transport (A) and cellular accumulation (B) of [14C]levofloxacin by LLC-PK1 monolayers. The monolayers were incubated at 37°C for 5 min with 5 µM of [14C]levofloxacin added to the basolateral side in the absence or presence of 1 mM of various quinolones, or 0.1 mM of sparfloxacin. After incubation, the radioactivity on the opposite side was measured. After a 5-min transport measurement, accumulation was determined in the absence or presence of 1 mM of various quinolones or 0.1 mM of sparfloxacin. Each column represents the mean ± S.E. of three monolayers. * P < .05, significant difference from control.

Effect of unlabeled levofloxacin on levofloxacin efflux out of LLC-PK₁ monolayers. To clarify further the transport across apical membrane, we measured the efflux of [¹⁴C]levofloxacin out of the monolayers in the presence of unlabeled levofloxacin. As shown in figure 4, the intracellular remaining of [¹⁴C]levofloxacin decreased rapidly with time and was significantly greater in the presence of unlabeled levofloxacin (P < .05).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of unlabeled levofloxacin on [14C]levofloxacin efflux out of LLC-PK1 monolayers grown on dishes. After 30 min incubation at 4°C with 5 µM of [14C]levofloxacin in the absence () or presence () of 1 mM of unlabeled levofloxacin, the monolayers were incubated at 37°C. The amount of [14C]levofloxacin remaining in the monolayers after the specified incubation period was determined. The cellular amount of levofloxacin at time zero for the absence and presence of unlabeled levofloxacin was 6.6 ± 0.5 and 7.8 ± 0.4 pmol/mg protein, respectively. Each value represents the mean ± S.E. of three monolayers. * P < .05, significant difference from each open symbol.

Effects of ionic drugs and cyclosporin A on levofloxacin transcellular transport and cellular accumulation. To determine the interaction of organic cations with the unidirectional transport of quinolones, we examined the effect of various organic cations and an organic anion, p-aminohippurate, on the transport of levofloxacin. As shown in figure 5, cimetidine and quinidine inhibited the basal-to-apical transcellular transport of [¹⁴C]levofloxacin without affecting cellular accumulation of [¹⁴C]levofloxacin. However, tetraethylammonium and p-aminohippurate affected neither the transcellular transport nor accumulation of [¹⁴C]levofloxacin.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of organic cations on the transcellular transport (A) and cellular accumulation (B) of [14C]levofloxacin by LLC-PK1 monolayers. The monolayers were incubated at 37°C for 5 min with 5 µM of [14C]levofloxacin added to the basolateral side in the absence or presence of 1 mM of various drugs. The experiment was done as described in figure 3. Each column represents the mean ± S.E. of three monolayers. * P < .05, significant difference from control.

Effect of apical pH and PCMBS on transcellular transport and cellular accumulation of levofloxacin. It has previously been reported that the transcellular transport of tetraethylammonium is highly dependent on the pH of the apical side (Saito et al., 1992). Therefore, to reveal whether levofloxacin is transported by H⁺/organic cation antiporter responsible for tetraethylammonium flux, the effect of apical pH on the transcellular transport and cellular accumulation of [¹⁴C]levofloxacin was examined. Lowering the pH of the apical side did not affect the transcellular transport of [¹⁴C]levofloxacin, although it decreased the cellular accumulation significantly (transport at pH 7.4, 102.9 ± 7.1; at pH 6.0, 108.2 ± 14.5 pmol/cm² per 60 min: accumulation at pH 7.4, 15.5 ± 0.8; at pH 6.0, 11.2 ± 0.5 pmol/mg protein per 60 min, mean ± S.E. of at least three monolayers).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Effect of PCMBS on the transcellular transport (A) and cellular accumulation (B) of [14C]levofloxacin and [14C]tetraethylammonium by LLC-PK1 monolayers. LLC-PK1 monolayers were preincubated at 4°C for 10 min with (dotted columns) or without (open columns) 0.1 mM PCMBS added to the apical side; incubation buffer without PCMBS was added to the basolateral side. After preincubation, LLC-PK1 monolayers were incubated at 37°C for 60 min with 5 µM of [14C]levofloxacin or 50 µM of [14C]tetraethylammonium added to the basolateral side. The experiment was done as described in figure 3. The transcellular transport of levofloxacin and tetraethylammonium was 67.6 ± 0.8 and 442 ± 52 pmol/cm2/60 min, respectively. The accumulation of levofloxacin and tetraethylammonium was 9.6 ± 0.4 and 665 ± 93 pmol/mg protein/60 min, respectively. Each value represents the mean ± S.E. of three monolayers. * P < .05, significant difference from control.

Transcellular transport and cellular accumulation of grepafloxacin. Using LLC-PK₁ monolayers, the transcellular transport and cellular accumulation of another quinolone antibacterial drug, [¹⁴C]grepafloxacin (5 µM), which is mainly metabolized in the liver then eliminated in bile (Akiyama et al., 1995) were studied. The basal-to-apical transcellular transport of [¹⁴C]grepafloxacin was much larger than the apical-to-basal transport similar to levofloxacin (fig. 7A). The accumulation of [¹⁴C]grepafloxacin from the apical side was 1.7-fold that from the basolateral side (fig. 7B).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Transcellular transport (A) and cellular accumulation (B) of [14C]grepafloxacin by LLC-PK1 monolayers. The monolayers were incubated at 37°C for 60 min with 5 µM of [14C]grepafloxacin added to either the basolateral () or apical side (). The experiment was done as described in figure 1. Each point or column represents the mean ± S.E. of three monolayers.

Effect of unlabeled grepafloxacin concentration on transcellular transport and cellular accumulation of grepafloxacin. To clarify the interaction of grepafloxacin with both apical and basolateral membranes, we examined the effect of unlabeled grepafloxacin concentration on transport of [¹⁴C]grepafloxacin. As shown in figure 8, the transcellular transport of [¹⁴C]grepafloxacin was suppressed by unlabeled grepafloxacin in a concentration-dependent manner. However, the cellular accumulation was significantly increased at 80 and 180 µM unlabeled grepafloxacin, but decreased with increasing concentration thereafter. The apparent K_m value for grepafloxacin transcellular transport could not be determined, because the relationship between the substrate concentration and the basolateral to apical flux rate of grepafloxacin showed a convex curve.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Effect of unlabeled grepafloxacin on transcellular transport (A) and cellular accumulation (B) of [14C]grepafloxacin by LLC-PK1 monolayers. The monolayers were incubated at 37°C for 15 min with 20 µM [14C]grepafloxacin added to the basolateral side in the absence or presence of varying concentrations of unlabeled grepafloxacin. The experiment was done as described in figure 3. Each point represents the mean ± S.E. of three monolayers. * P < .05, significant difference from each open symbol.

	Discussion

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Most quinolone antibacterial drugs are excreted via glomerular filtration and tubular secretion (Fish and Chow, 1997). In our study, to investigate the mechanisms of the renal tubular secretion of quinolones, the transport of levofloxacin and grepafloxacin by LLC-PK₁ monolayers was examined.

Levofloxacin was transported unidirectionally from the basolateral to apical side by LLC-PK₁ monolayers, which corresponds to renal secretion. The unidirectional transport of [¹⁴C]levofloxacin was inhibited by unlabeled levofloxacin, and both the transcellular transport and initial uptake from the basolateral side of levofloxacin showed concentration-dependent saturation. It is likely that the unidirectional transcellular transport of levofloxacin is a consequence of two specific transport processes, namely basolateral uptake and apical efflux. The apparent K_m values for the basal-to-apical transport and the uptake from the basolateral side were 0.6 and 13 mM, respectively. Various quinolones (1 mM) inhibited the transcellular transport of [¹⁴C]levofloxacin, accompanied by a marked increase of cellular accumulation. In addition, unlabeled levofloxacin inhibited the efflux of [¹⁴C]levofloxacin out of monolayers across the apical membrane. These results indicated that levofloxacin had higher affinity for the apical membrane compared with the basolateral membrane of LLC-PK₁ monolayers.

LLC-PK₁ monolayers retain the H⁺/organic cation antiport system on the apical membrane and are useful for the study of organic cation secretion and interaction of drugs with this transporter in renal proximal tubules (Inui et al., 1985; Saito et al., 1992). It has previously been reported (Ohtomo et al., 1996) that levofloxacin potently inhibited the transcellular transport of tetraethylammonium, whereas tetraethylammonium did not show inhibitory effects on levofloxacin transport and accumulation by LLC-PK₁ monolayers. In our study, the transcellular transport and cellular accumulation of levofloxacin, in the therapeutic plasma concentration range, were not inhibited by tetraethylammonium, consistent with the previous results. In addition, the transcellular transport and accumulation of levofloxacin were not influenced by either the pH of the apical side or pretreatment of the apical side with PCMBS. It is likely that the transport systems distinct from the H⁺/organic cation antiport system for tetraethylammonium on the apical membranes are involved in the unidirectional transport of levofloxacin. Because cimetidine and quinidine decreased the transcellular transport of levofloxacin from the basolateral to apical side without influencing its accumulation, it is considered that these drugs inhibited the transport of levofloxacin at least across the apical membrane, and probably across both membranes. Previously, using an in vivo clearance method, cimetidine was shown to decrease the renal clearance of levofloxacin and did not affect the tissue/plasma concentration ratio in the kidney (Yano et al., 1997). It has been reported that cimetidine reduced the renal clearance of levofloxacin and enoxacin in healthy subjects (Fish and Chow, 1997, Misiak et al., 1993). These findings are consistent with our results.

P-glycoprotein, which functions as an adenosine triphosphate-dependent drug-efflux pump, has been found on the brush-border membranes of proximal tubules of the kidney (Thiebaut et al., 1987). Quinolones are transported by P-glycoprotein in a study using LLC-GA5-COL150 monolayers that overexpress human P-glycoprotein on the apical membrane (Ito et al., 1997). Because cyclosporin A, a P-glycoprotein modulator, did not influence the transcellular transport or cellular accumulation of levofloxacin, P-glycoprotein did not contribute to the transport of levofloxacin across the apical membrane of LLC-PK₁ cells, and the expression level of P-glycoprotein in these cells might be negligible.

Ullrich et al. (1993) reported that quinolones inhibited the uptake of cationic compounds, N-methylnicotinamide and tetraethylammonium, an anionic compound p-aminohippurate through the basolateral membrane of renal tubular cells. They also reported that the uptake of ofloxacin across the basolateral membrane was not inhibited by tetraethylammonium or probenecid. In the liver, the hepatic uptake of quinolones is via carrier-mediated active transport, which is distinct from the systems involved in the transport of bile acid, organic anions, organic cations or natural steroids, although grepafloxacin could potently inhibit the uptake of substrates for these transport systems (Sasabe et al., 1997). These observations would suggest that quinolones interact with various transport systems, but it is not clear which transport systems contribute to the transport of quinolones themselves. In the human intestinal Caco-2 cells, it has been reported that transepithelial secretion of quinolones involved a common accumulative transport at the basolateral membrane followed by facilitated exit across the apical membrane (Griffiths et al., 1994).

In our study, using a kidney epithelial cell line LLC-PK₁, all examined quinolones were found to interact with the apical transport systems of levofloxacin. Furthermore, grepafloxacin, which is mainly metabolized in the liver then eliminated in bile (Akiyama et al., 1995), showed unidirectional transport similar to levofloxacin. The transcellular transport of [¹⁴C]grepafloxacin was inhibited by unlabeled grepafloxacin and the cellular accumulation was increased by the lower concentration and decreased with increasing concentration thereafter. These results suggested that grepafloxacin also interacted with both membranes and had higher affinity for the apical membrane. Therefore, it is likely that quinolones are transported by common transport systems and have higher affinity for the apical than the basolateral membrane of LLC-PK₁ monolayers. However, these findings could not explain the difference of elimination routes of quinolones and further studies are needed to elucidate the mechanisms of selective tissue distribution of these drugs.

In conclusion, quinolones could be transported in LLC-PK₁ cells by a specific mechanism and have higher affinity for the transport system on the apical membrane, a system distinct from H⁺/organic cation antiport system. Our results should be useful for studies on the renal tubular secretion of quinolones.