Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan
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ABSTRACT |
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We investigated expression of the
Na+-L-carnitine cotransport system and its role
in transport of tetraethylammonium in a kidney epithelial cell line,
LLC-PK1. L-Carnitine uptake in the
LLC-PK1 cells was markedly stimulated in the presence of
Na+. The uptake was saturable, with Michaelis constant and
maximal uptake velocity values of 7.8 µM and 153.7 pmol · mg
protein1 · 15 min
1, respectively.
Cationic drugs such as tetraethylammonium, cimetidine, and quinidine
inhibited L-carnitine uptake. The basolateral-to-apical transport of [14C]tetraethylammonium was enhanced
markedly in the presence of an H+ gradient on the apical
side at a pH of 5.9. Under the conditions in which
Na+/L-carnitine cotransport activity was
saturable by the addition of 100 µM L-carnitine to the
apical-side medium, the basolateral-to-apical transcellular transport
of [14C]tetraethylammonium was unaffected. These results
suggested that the Na+-L-carnitine
cotransporter is expressed in the apical membranes of
LLC-PK1 cells, and is not responsible for efflux of
tetraethylammonium from the cells. Transport of tetraethylammonium
appeared to be mediated predominantly by an H+/organic
cation antiporter in the apical membranes.
L-carnitine transporter; organic cation transporter; kidney epithelial cell line
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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.
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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.
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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.
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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 (Ki) 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.
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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).
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DISCUSSION |
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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 (Km = ~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 Km for transcellular transport of TEA in LLC-PK1 cells is ~50 µM (23). Therefore, the apparent Ki 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 Ki values of cephaloridine are similar to those of the L-carnitine transporter in LLC-PK1 cells (Ki = 461 µM and hOCTN2; IC50 = 230 µM) (7). The IC50 value in hOCTN2 is close to the Ki value for cephaloridine, because the L-carnitine concentration is much lower than the Km 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.
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ACKNOWLEDGEMENTS |
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, and by the Smoking Research Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Inui, Dept. of Pharmacy, Kyoto Univ. Hospital, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: inui{at}kuhp.kyoto-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 February 2001; accepted in final form 16 April 2001.
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