INTRODUCTION

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RENAL TUBULAR SECRETION OF organic cations is an important physiological transport process of the kidney that eliminates toxic compounds and drugs as well as endogenous organic cations from the body (22). The secretory process is achieved via unidirectional transcellular transport, i.e., the uptake of organic cations into the cells from blood across the basolateral membranes followed by active extrusion across the brush-border membranes into the tubular fluid (11). The mechanisms for transporting organic cations have been well characterized in the brush-border membranes, and the expression of the H⁺/organic cation antiporter, a transporter that exchanges the cellular organic cation for tubular H⁺, has been reported (11). Several organic cations such as tetraethylammonium (TEA) and cimetidine have been demonstrated to be substrates for this antiporter (11). In addition, we demonstrated that -lactam antibiotics such as cephalexin and cephradine were also transported by the H⁺/organic cation antiport system (13).

Recently, OCTN2, a new member of the organic cation transporter family, was cloned from human kidney (27) and placenta (32). OCTN2 was demonstrated to transport organic cations in a pH-dependent manner (32). It was also found to transport L-carnitine (27). OCTN2-mediated L-carnitine transport is Na⁺-dependent whereas the transport of organic cations by OCTN2 is Na⁺ independent (11). L-Carnitine is an important factor for fatty acid oxidation (3). It is usually accumulated in the body by biosynthesis and dietary intake. In the kidney, a high-affinity L-carnitine transporter expressed in the brush-border membranes of proximal tubule cells efficiently mediates reabsorption of filtrated L-carnitine. Therefore, the Na⁺-L-carnitine cotransporter contributes to maintain the L-carnitine serum level in circulation (21, 25). OCTN2 has been suggested to be responsible for this high-affinity L-carnitine transport (27). Primary L-carnitine deficiency with very low serum levels of L-carnitine appeared to be caused by genetic mutation of OCTN2 (11, 17). The clinical symptoms of this disease include cardiac myopathy and skeletal myopathy. Interestingly, OCTN2 appeared to transport several organic cations as well as L-carnitine, probably at the brush-border membranes of renal tubule cells (31, 32), but its physiological role in renal secretion of organic cations is still unknown.

The pig kidney epithelial cell line LLC-PK₁ (10) has been used extensively as a model for the analysis of epithelial functions in renal proximal tubules (8). These cells form an oriented monolayer with microvilli and tight junctions and exhibit a unidirectional transport of electrolytes and some nutrients (1, 16). We provided the first evidence that the apical membranes (corresponding to the brush-border membranes) of LLC-PK₁ cells express the H⁺/organic cation antiport system (12). In addition, LLC-PK₁ monolayers grown on collagen-coated microporous membranes show unidirectional transcellular transport of TEA (23). Expression of the Na⁺/L-carnitine transport system in LLC-PK₁ cells remains unknown.

The present study was undertaken to explore the Na⁺/L-carnitine transport system and the H⁺/organic cation antiport system in LLC-PK₁ cells on the basis of functional characteristics. The results show that an Na⁺-L-carnitine cotransporter with a function similar to that of OCTN2 is expressed in the apical membranes of LLC-PK₁ cells. Furthermore, the findings revealed that the H⁺/organic cation antiport activity in the apical membranes is independent of the Na⁺-L-carnitine cotransport.

	METHODS

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Cell culture. LLC-PK₁ cells obtained from the American Type Culture Collection (ATCC CRL-1392) were grown on plastic dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) in Dulbecco's modified Eagle's medium (GIBCO Life Technologies, Grand Island, NY), supplemented with 10% fetal calf serum (Microbiological Associates, Betheada, MD) without antibiotics in an atmosphere of 5% CO₂-95% air at 37°C. Subculturing was done every 7 days by using 0.02% EDTA and 0.05% trypsin (23). In general, 100-mm plastic dishes were inoculated with 1 × 10⁶ cells in 10 ml of complete culture medium. In this study, cells between passages 220 and 240 were used. For the transport studies, LLC-PK₁ cells were seeded on collagen-coated membrane filters (3-mm pores, 4.71-cm² growth area) inside a Transwell cell culture chamber (Costar, Cambridge, MA) at a cell density of 3.8 × 10⁵ cells/cm². Each Transwell chamber was placed in a 35-mm well of tissue culture plate with 2.6 ml of outside medium (basolateral side) and 1.5 ml of inside medium (apical side). The cell monolayers were fed fresh medium every 2 days.

Measurement of L-[³H]carnitine uptake. For the uptake studies, LLC-PK₁ cell monolayers grown on 60-mm plastic culture dishes (4 × 10⁵ cells, 6 days in culture) were used. The incubation medium (pH 7.4) was Dulbecco's phosphate-buffered saline composed of (in mM) 137 NaCl (Na⁺-containing buffer) or 137 N-methyl-D-glucamine chloride (Na⁺-free buffer), 3 KCl, 8 Na₂HPO₄, 1.5 KH₂PO₄, 1 CaCl₂, 0.5 MgCl₂, and 5 D-glucose. The pH of the medium was adjusted by the addition of a solution of HCl or NaOH (Na⁺-containing buffer) and KOH (Na⁺-free buffer). In general, the monolayers were preincubated for 10 min at 37°C with 2 ml of the incubation medium (pH 7.4). After removal of the medium, the cells were incubated with 2 ml of incubation medium containing L-[³H]carnitine (5 nM, 15.5 kBq/ml) for the desired time at 37°C. After the incubation, the medium was aspirated and the dishes were rapidly washed three times with 2 ml of ice-cold incubation medium (pH 7.4). The cell monolayers were solubilized in 1.5 ml of 1 N NaOH, and radioactivity was determined in 5 ml of ACSII (Amersham International, Buckinghamshire, UK) by liquid scintillation counting.

Measurement of [¹⁴C]TEA transport and cellular accumulation. Transepithelial transport and accumulation of [¹⁴C]TEA were measured by using monolayer cultures grown in Transwell chambers (23). The incubation medium was Dulbecco's phosphate-buffered saline (pH 7.4) comprising (in mM) 137 NaCl, 3 KCl, 8 Na₂HPO₄, 1.5 KH₂PO₄, 1 CaCl₂, 0.5 MgCl₂, and 5 D-glucose. The pH of the medium was adjusted by the addition of a solution of HCl or NaOH. After removal of the culture medium from both sides of the monolayers, the cell monolayers were preincubated with 2 ml of incubation medium (pH 7.4) in each side for 10 min at 37°C. Then, 2 ml of incubation medium containing [¹⁴C]TEA (50 µM, 7.4 kBq/ml) and D-[³H]mannitol (0.05 µM, 37 kBq/ml) were added to the basolateral side, and 2 ml of nonradioactive incubation medium were added to the apical side; the monolayers were incubated for the desired time at 37°C. D-Mannitol, a compound which is not transported by the cells, was used to calculate paracellular fluxes and the extracellular trapping of TEA. For transport measurements, an aliquot (50 µl) of the incubation medium on the apical side was taken at desired times, and the radioactivity was measured. For accumulation studies, the medium was removed by aspiration at the end of the incubation period, and the monolayers were rapidly washed twice with 2 ml of ice-cold incubation medium (pH 7.4) on each side. The filters with monolayers were detached from the chambers, the cells on the filters were solubilized with 0.5 ml of 1 N NaOH, and the radioactivity in 200-µl aliquots was measured. The radioactivity of the collected media and the solubilized cell monolayers was determined as described above.

Protein assay. The protein content of the cell monolayers solubilized in 1 N NaOH was determined by the method of Braford (4) with use of a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA) with bovine -globulin as a standard. The protein contents of the monolayers ranged from 1.5 to 1.9 mg/filter (4.71cm²).

Materials. L-[Methyl-³H]L-carnitine hydrochloride (3.11 TBq/mmol) was obtained from Amersham International. [1-¹⁴C]TEA bromide (0.15 GBq/mmol) and D-[1-³H(N)]mannitol (1,110 GBq/mmol) were purchased from DuPont-New England Nuclear Reseach Products (Boston, MA). L-Carnitine hydrochloride, TEA bromide, guanidine hydrochloride, quinidine sulfonate, and cimetidine were obtained from Nacalai Tesque (Kyoto, Japan). Gentamicin, cisplatin, and p-chloromercuribenzene sulfonate (PCMBS) were purchased from Sigma (St. Louis, MO). Levofloxacin was kindly supplied by Daiichi Seiyaku (Tokyo, Japan). Cephaloridine, cephalexin, and vancomycin were kindly supplied by Shionogi (Osaka, Japan). All other chemicals used were of the highest purity available.

Statistical analysis. The statistical significance of differences among mean values was calculated by using the nonpaired t-test. Multiple comparisons were performed by using Scheffé's test.

	RESULTS

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Characteristics of L-carnitine uptake in LLC-PK₁ cells. We first investigated the time course of L-carnitine uptake in the presence or absence of NaCl in the incubation medium (Fig. 1). In the Na⁺-free medium, the NaCl of the incubation medium was replaced with N-methyl-D-glucamine chloride. The uptake of L-carnitine was several-fold greater in the presence than in the absence of NaCl. The Na⁺-dependent uptake was linear up until 60 min. Therefore, all subsequent uptake measurements were performed at an incubation time of 30 min to obtain initial uptake rates.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Na+-dependent L-[3H]carnitine uptake by LLC-PK1 cells. On day 6 after inoculation, LLC-PK1 cells were incubated at 37°C for a desired time with L-[3H]carnitine (5 nM, 15.5 kBq/ml, pH 7.4) in the presence () or absence () of NaCl. In the Na+-free medium, the NaCl of the incubation medium was replaced with N-methyl-D-glucamine. Then, the radioactivity of solubilized cells was determined. Each value represents the mean ± SE of 3 monolayers.

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View larger version (26K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of L-[3H]carnitine uptake by LLC-PK1 cells. L-[3H]carnitine uptake by LLC-PK1 cells was measured at various concentrations (0.5-100 µM) for 15 min at 37°C in the presence () or absence () of 5 mM unlabeled L-carnitine. Then, the radioactivity of solubilized cells was determined. Each value represents the mean ± SE of 3 monolayers. When the error bar is not shown, it is smaller than the symbol. Inset: Eadie-Hofstee plots of the uptake after correction for the nonsaturable component. V, uptake rate (pmol · mg protein1 · 15 min1); S, L-carnitine concentration (µM).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   pH dependence of L-[3H]carnitine uptake by LLC-PK1 cells. LLC-PK1 cells were incubated for 30 min at 37°C with incubation media of various pH containing L-[3H]carnitine (5 nM, 15.5 kBq/ml) in the presence () or absence () of 1 mM unlabeled L-carnitine. Then, the radioactivity of solubilized cells was determined. Each value represents the mean ± SE of 3 monolayers.

Effect of various compounds on L-carnitine uptake in LLC-PK₁ cells. To clarify the substrate specificity of the Na⁺-L-carnitine cotransport system, we examined the effect of various compounds on L-carnitine uptake in LLC-PK₁ cells (Table 1). L-Carnitine, TEA, quinidine, cimetidine, and cephaloridine showed potent inhibitory effects on the L-carnitine uptake. On the other hand, levofloxacin, 1-methyl-4-phenylpyridinium (MPP⁺), dopamine, vancomycin, gentamicin, and cisplatin had moderate inhibitory effects. Guanidine and cephalexin did not inhibit the uptake. Dose-dependent inhibition of L-carnitine uptake was also examined for L-carnitine, TEA, cimetidine, quinidine, and cephaloridine (Fig. 4). The apparent inhibitory constant (K_i) values of L-carnitine, quinidine, TEA, cimetidine, and cephaloridine, estimated from the transformed Michaelis-Menten equation using nonlinear least-squares regression analysis, were 5.6, 33.3, 203.3, 229.5, and 461.1 µM, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Dose-dependent inhibition of L-[3H]carnitine uptake by various compounds in LLC-PK1 cells. LLC-PK1 cells were incubated for 15 min at 37°C with incubation medium containing L-[3H]carnitine (5 nM, 15.5 kBq/ml, pH 7.4) in the presence of increasing concentrations of L-carnitine (), tetraethylammonium (TEA; ), cimetidine (), quinidine (), and cephaloridine (). Then, the radioactivity of solubilized cells was determined. Each value represents the mean ± SE of 3 monolayers. When the error bar is not shown, it is smaller than the symbol.

The effect of L-carnitine on transcellular transport and accumulation of tetraethylammonium. We previously reported that the transport of TEA across the apical membrane of LLC-PK₁ cells was stimulated by acidification of the medium on the apical side, i.e., an inwardly directed H⁺ gradient acts as a driving force for the extrusion (23). To ascertain whether the Na⁺-L-carnitine cotransporter is involved in the pH-dependent organic cation secretion, we examined the effect of L-carnitine on the apical side medium on the pH-dependent transcellular transport and accumulation of TEA in LLC-PK₁ cell monolayers. As shown in Fig. 5A, when the pH of the apical incubation buffer was decreased to 5.9, the basolateral-to-apical transport of [¹⁴C]TEA was increased compared with the transport at pH 7.4. Conversely, the accumulation of [¹⁴C]TEA was lower at pH 5.9 than at pH 7.4 (Fig. 5B). The transport and accumulation of TEA were not affected in the presence of 100 µM L-carnitine on the apical side (Fig. 5, A and B).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of L-carnitine on the apical side on pH-dependent transcellular transport (A) and accumulation (B) of [14C]TEA by LLC-PK1 cell monolayers. A: on day 6 after inoculation, LLC-PK1 cell monolayers were incubated at 37°C with 50 µM [14C]TEA (2 ml, pH 7.4) added to the basolateral side in the absence (, pH 5.9; , pH 7.4) or presence (, pH 5.9; , pH 7.4) of 100 µM L-carnitine on the apical side of the monolayers. The radioactivity on the apical side (2 ml) was measured periodically. B: after 60-min incubation, monolayers were rapidly washed twice with 2 ml of ice-cold incubation medium (pH 7.4) on both sides, and the radioactivity of solubilized monolayers was determined (open bars, without L-carnitine; solid bars, with L-carnitine). Each point or bar represents the mean ± SE of 3 monolayers.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effect of L-carnitine on both apical and basolateral sides on transcellular transport (A) and accumulation (B) of [14C]TEA by LLC-PK1 cell monolayers. On day 6 after inoculation, LLC-PK1 cell monolayers were incubated at 37°C with 50 µM [14C]TEA (2 ml, pH 7.4) added to the basolateral side in the absence () or presence of 20 (), 100 (), and 500 µM () L-carnitine on both the apical and the basolateral sides of monolayers. The radioactivity on the apical side (2 ml, pH 7.4) was measured periodically. B: after 60-min incubation, monolayers were rapidly washed twice with 2 ml of ice-cold incubation medium on both sides, and the radioactivity of solubilized cells was determined (open bar, without L-carnitine; solid bars, with L-carnitine). Each point or bar represents the mean ± SE of 3 monolayers.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

It was previously demonstrated that organic cation secretion in the brush-border membranes of the renal tubule cells is mediated by the H⁺/organic cation antiport system (11). However, the recently cloned OCTN2 (27, 32), an organic cation/L-carnitine transporter, mediates Na⁺-dependent L-carnitine and Na⁺-independent organic cation transport (11). Therefore, to distinguish OCTN2 from the H⁺/organic cation antiporter based on functional analysis, we examined the transport characteristics of L-carnitine and TEA in LLC-PK₁ cell monolayers, which exhibit unidirectional transcellular transport of TEA, a transport process corresponding to renal tubular secretion (23).

In the present study, we first demonstrated that the Na⁺-L-carnitine cotransporter is expressed in the apical membranes of the LLC-PK₁ cells (Fig. 1). This transporter mediated high-affinity L-carnitine transport (K_m = ~10 µM, Fig. 2). Because the plasma concentration of L-carnitine is ~40 µM (14), the high-affinity L-carnitine transporter plays an important role in the reabsorption of glomerular filtrated L-carnitine. L-Carnitine uptake in the LLC-PK₁ cells was sensitive to pH. When the medium pH was acidic, L-carnitine uptake was depressed. Ohashi et al. (18) has indicated that L-carnitine transport via hOCTN2 has low activity at acidic pH. Therefore, the pH dependence of L-carnitine uptake in the LLC-PK₁ cells is comparable to that in hOCTN2. Because intracellular acidification decreased L-carnitine currents in oocytes expressing hOCTN2, an allosteric regulation of L-carnitine transport by protons might be involved in the decreased uptake (30). The activity of the L-carnitine transporter was not stimulated by acidification of the medium on the apical side, i.e., an inwardly directed H⁺ gradient, suggesting that the transporter should be discriminated from the apical membrane H⁺/organic cation antiporter.

L-Carnitine uptake in LLC-PK₁ cells was inhibited significantly by various organic cations (Table 1). Among the compounds tested, TEA, cimetidine and quinidine had potent inhibitory effects on the uptake. Thus the Na⁺-L-carnitine cotransporter in LLC-PK₁ cells has sensitivity not only for L-carnitine but also for a variety of organic cations. In addition, the intensity of the inhibition by various compounds of the Na⁺-L-carnitine cotransport activity was comparable to that for hOCTN2, indicating that the Na⁺-L-carnitine cotransporter in LLC-PK₁ cells has similar substrate specificities to hOCTN2. We reported that the K_m for transcellular transport of TEA in LLC-PK₁ cells is ~50 µM (23). Therefore, the apparent K_i of TEA (203.3 µM) indicates that the affinity of the L-carnitine transporter for TEA is much lower than that of the organic cation transport system in LLC-PK₁ cells. MPP⁺ moderately inhibited this L-carnitine transporter (~30% inhibition, Table 1). Sokol et al. (24) showed that MPP⁺ (0.5 mM) inhibited H⁺-driven N¹-methylnicotinamide transport by ~80% in canine renal brush-border membrane vesicles. In addition, using rabbit renal brush-border membrane vesicles, Lazaruk et al. (15) indicated that MPP⁺ shares a common H⁺/organic cation antiporter with TEA and N¹-methylnicotinamide. Therefore, our data indicate that MPP⁺ inhibits the H⁺/organic cation antiporter more strongly than does the Na⁺-L-carnitine cotransporter in LLC-PK₁ cells. Furthermore, levofloxacin, a pyridonecarboxylic acid antibacterial drug, inhibited L-carnitine uptake slightly in LLC-PK₁ cells (~10% inhibition) (Table 1). Okano et al. (20) demonstrated that ofloxacin, an enantiomer of levofloxacin, inhibits ~70% of TEA uptake in rat renal brush-border membrane vesicles. Ohtomo et al. (19) also showed that levofloxacin drastically interacts with the apical H⁺/organic cation antiporter rather than with the basolateral organic cation transport system. The inhibitory effect of levofloxacin on the L-carnitine transporter is lower than that on the H⁺/organic cation antiporter. Accordingly, these results suggest that the substrate affinity of the L-carnitine transporter would be different from that of the brush-border H⁺/organic cation antiporter, although the L-carnitine transporter in LLC-PK₁ cells recognizes, at least in part, L-carnitine as well as organic cations.

PCMBS inhibited L-carnitine uptake strongly in LLC-PK₁ cells (Table 1). Because of its hydrophilicity, PCMBS cannot permeate across the cell membrane (29). Therefore, PCMBS does not react with sulfhydryl groups on the inside of LLC-PK₁ cells. In consideration of these findings, it was concluded that the sulfhydryl groups of the Na⁺-L-carnitine cotransport system are essential in the apical membranes of the LLC-PK₁ cells, and that these functional sulfhydryl groups should be localized to the outside of the cells. In fact, the amino acid sequence of hOCTN2 appears to include four sulfhydryl groups in the external region between the first and second transmembrane domains (32).

Among aminocephalosporins, cephaloridine has a potent inhibitory effect, whereas cephalexin has no inhibitory effect (Table 1). Similar sensitivity of hOCTN2 against cephalosporins has been reported (7, 18). In addition, the apparent K_i values of cephaloridine are similar to those of the L-carnitine transporter in LLC-PK₁ cells (K_i = 461 µM and hOCTN2; IC₅₀ = 230 µM) (7). The IC₅₀ value in hOCTN2 is close to the K_i value for cephaloridine, because the L-carnitine concentration is much lower than the K_m of L-carnitine for hOCTN2. Therefore, these findings suggest that the L-carnitine transport activity in LLC-PK₁ cells is similar to the activity of hOCTN2. Cephalexin appeared to be transported, in part, by the H⁺/organic cation antiporter in rat renal brush-border membranes (13). However, the L-carnitine transporter does not recognize cephalexin, demonstrating that the H⁺/organic cation antiport system and L-carnitine transport system have a different substrate specificity. Inhibition of L-carnitine transport by cephaloridine seems to be very interesting because it might be responsible for the cephaloridine-induced nephrotoxicity (2). It was reported that the nephrotoxicity of cephaloridine may be caused by impairment of the fatty acid oxidation associated with L-carnitine (28). The increase in urinary L-carnitine excretion evoked by cephaloridine could be related to the nephrotoxicity. Takeda et al. (26) also reported that rOAT1 is responsible, in part, for the cellular accumulation of cephaloridine from basolateral sides, and therefore, for cephaloridine-induced nephrotoxicity. Because cephaloridine is transported, in part, by OCTN2 (7), cephaloridine-induced nephrotoxicity might be due to accumulation from not only basolateral membranes but also apical membranes. Cisplatin, vancomycin, and gentamicin (2, 5, 6) had moderate inhibitory effects on the L-carnitine uptake (Table 1). We speculate that the interaction of these drugs with the L-carnitine transport system in the kidney is responsible for the renal failure associated with their use.

Previously, we showed that the transcellular transport and cellular accumulation of TEA corresponding to the renal tubular secretion by LLC-PK₁ cell monolayers were dependent on the pH of the medium on the apical side, with greater transport at lower pH and greater accumulation at higher pH (23). In the present study, pH-dependent transcellular transport and accumulation of TEA were observed in LLC-PK₁ cell monolayers (Fig. 5). This pH-dependent TEA transport across LLC-PK₁ cell monolayers was not linked to L-carnitine uptake from apical sides (Fig. 5). We also found that L-carnitine on both the apical and basolateral sides had no effect on TEA transport or accumulation (Fig. 6). These results suggest that the Na⁺-L-carnitine cotransporter in LLC-PK₁ cells does not contribute significantly to organic cation secretion from renal epithelial cells under physiological conditions. The role of the L-carnitine transport system in the secretion of organic cations remains unclear. Because many of the organic cations recognized as substrates by the L-carnitine transporter are pharmacologically active, this transporter might play a significant role in the disposition and pharmacokinetics of these drugs in the body (31).

In conclusion, the apical membranes of LLC-PK₁ cells express a Na⁺-L-carnitine cotransporter that is similar to OCTN2 but is not involved in the H⁺/organic cation antiport activity. Our findings suggest that the L-carnitine transport system is physiologically important for renal reabsorption of L-carnitine, whereas the secretion of organic cations is mediated mainly by the H⁺/organic cation antiporter in LLC-PK₁ cells.