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Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: In comparison with rOAT1
Life Sciences 70 (2002) 1861 – 1874
Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: In comparison with rOAT1 Kyu Yong Jungb, Michio Takedaa, Minoru Shimodac, Shinichi Narikawaa, Akihiro Tojod, Do Kyung Kima, Arthit Chairoungduaa, Bong Kyu Choib, Hiroyuki Kusuharae, Yuichi Sugiyamae, Takashi Sekinea, Hitoshi Endou a,* a
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka-shi, Tokyo, Japan b Department of Pharmacology, Wonkwang University School of Medicine, Iksan, Chonbuk, South Korea c Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan d The Second Department of Internal Medicine, University of Tokyo, Tokyo, Japan e Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Received 28 March 2001; accepted 19 October 2001
Abstract This study was performed to elucidate the possible involvement of organic anion transporter 3 (OAT3) in cephaloridine (CER)-induced nephrotoxicity and compare the substrate specificity between rOAT3 and rat OAT1 (rOAT1) for various cephalosporin antibiotics, using proximal tubule cells stably expressing rOAT3 (S2 rOAT3) and rOAT1 (S2 rOAT1). S2 rOAT3 exhibited a CER uptake and a higher susceptibility to CER cytotoxicity than did mock, which was recovered by probenecid. Various cephalosporin antibiotics significantly inhibited both estrone sulfate uptake in S2 rOAT3 and paraaminohippuric acid uptake in S2 rOAT1. The Ki values of CER, cefoperazone, cephalothin and cefazolin for rOAT3- and rOAT1-mediated organic anion transport ranged from 0.048 to 1.14 mM and from 0.48 to 1.32 mM, respectively. These results suggest that rOAT3, at least in part, mediates CER * Corresponding author. Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan. Tel.: +11-81-422-47-5511x3451; fax: +11-81422-79-1321. E-mail address: [email protected] (H. Endou). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 0 0 - X
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uptake and CER-induced nephrotoxicity as rOAT1. There was some difference of affinity between rOAT3 and rOAT1 for cephalosporin antibiotics. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Organic anion transporter; OAT3; Cephaloridine; Nephrotoxicity; Proximal tubule
Introduction Cephaloridine (CER) is a broad-spectrum cephalosporin antibiotic that is known to induce acute renal failure in humans and animals [1]. The nephrotoxicity induced by CER is characterized by acute proximal tubular necrosis and is mainly dependent on the accumulation and concentration of the drug in the renal cortex, more specifically, in the second segment of the proximal tubule (S2) [2–5]. CER is actively transported by the proximal tubular cells from the blood via an organic anion transporter (OAT) at the basolateral membrane. However, unlike the other less toxic cephalosporin antibiotics, CER is not transported readily across the luminal membrane, which results in the accumulation of a high concentration of CER and selective damage of these cells in the proximal tubule [6,7]. The secretion of numerous organic anions, including endogenous metabolites, drugs and xenobiotics, is an important physiological function of renal proximal tubules. The secretion of organic anions from the proximal tubular cells is achieved via unidirectional transcellular transport, involving the uptake of organic anions into the cells from the blood across the basolateral membrane, followed by their extrusion across the brush-border membrane into the tubular fluid [8]. Recently, cDNAs encoding transporters mediating renal organic anion transport have been successively cloned, namely, organic anion transporter 1 (OAT1), OAT2, OAT3, OAT4, organic anion-transporting polypeptide (oatp1), oatp2, OAT-K1, OAT-K2, multiple resistance-associated protein 2 (MRP2) and human-type I sodium-dependent inorganic phosphate transporter (NPT1) [9–19]. Among them, rat OAT3 (rOAT3) and rat OAT1 (rOAT1) have been shown to be the major OATs at the basolateral membrane of the proximal tubular cells [9,12]. We have already demonstrated that rOAT1 mediates CER transport as well as CERinduced nephrotoxicity, using cells of the mouse kidney of the third segment of the proximal tubule (S3) stably expressing rOAT1 [20]. The purpose of this study was to elucidate the possible involvement of rOAT3 in CER transport and CER-induced nephrotoxicity using mouse S2 cells stably expressing rOAT3, and compare the substrate recognition between rOAT3 and rOAT1 for various cephalosporin antibiotics.
Materials and methods Materials [14C]CER (13.9 mCi/mmol) was kindly supplied by Sankyo Co. (Tokyo, Japan). [14C]paraaminohippuric acid (PAH) (53.1 mCi/mmol) and [3H]estrone sulfate (ES) (53 Ci/mmol) were
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purchased from Perkin Elmer Life Sciences (Boston, MA, USA). All other chemicals and reagents were of analytical grade and purchased from commercial sources. Cell culture and establishment of S2 cells stably expressing rOAT3 and rOAT1 (S2 rOAT3 and S2 rOAT1) The renal tubular cells used for the study were S2 cells derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene [21]. S2 cells were cultured in RITC 80-7 medium containing 5% fetal bovine serum, 10 mg/ml transferrin, 0.08 U/ml insulin and 10 ng/ml recombinant epidermal growth factor. The cells were subcultured in the medium containing 0.05% trypsin-EDTA solution (containing in mM: 137 NaCl, 5.4 KCl, 5.5 glucose, 4 NaHCO3, 0.5 EDTA and 5 Hepes; pH 7.2) and used at passages 60100. The full-length rOAT3 [12] or rOAT1 [9] genes were subcloned into pcDNA3.1, a mammalian expression vector. The pcDNA3.1-rOAT1, pcDNA3.1-rOAT3, and pcDNA3.1, which lacked the insert coupled with pSV2neo, a neomycin resistance gene, were transfected into S2 cells using the TfX2-50 reagent according to the manufacturer’s instructions. Forty-eight hrs after the transfection, the cells were subcultured in the medium containing 400 mg/ml geneticin. The cell clones were isolated using a cloning cylinder. The organic anion transport activity in these cells was determined by measuring the uptake of the substrate preferred by rOAT3, [3H]ES [12] and that by rOAT1, [14C]PAH [9]. The cells showing the highest level of activities were selected, and designated as S2 rOAT3 and S2 rOAT1, whereas the S2 cells transfected with pcDNA 3.1 lacking the insert were designated as S2 pcDNA 3.1 and used as the control. The S2 monolayer was determined to be leaky based on the results of a study wherein we estimated paracellular secretion from cells cultured on a permeable support, using D-[3H]mannitol as an indicator. Therefore, the cells were cultured on a solid support for these experiments. Two days after inoculation, the S2 cells were employed for experiments. Immunohistochemical analysis of rOAT3 protein Light-microscopic analysis of the rOAT3 protein was performed as previously described [20]. S2 rOAT3 were cultured in a six-well dish. The cells were fixed with paraformaldehyde lysine periodate (PLP) solution (containing 0.01 M NaIO4, 0.075 M lysine, 0.0375 M phosphate buffer with 2% paraformaldehyde; pH 6.2) for 2 hrs at 4 C. After fixation, the cells were washed with 50 mM NH4Cl in phosphate-buffered saline (PBS) (containing in mM: 136 NaCl, 2.7 KCl, 8.2 Na2HPO412H2O and 15 KH2PO4, pH 7.4) and then with Trisbuffered saline containing 0.1% Tween 20 (TBST). The cells were incubated with polyclonal antibody against rOAT3 at a dilution of 1: 400 for 2 hrs. For generation of this antibody, rabbits were immunized with keyhole limpet hemocyanin-conjugated synthesized peptides, EAEKASQHPLKTGG, corresponding to cysteine and the 14 amino acids of the COOH terminus of rOAT3. After rinsing with TBST, the cells were incubated with biotinylated secondary antibody against rabbit immunoglobulin (LSAB kit, Dako, Carpinteria, CA) for 1 hr. The cells were washed with TBST, and incubated with horseradish peroxidase-conjugated
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streptavidine solution for 30 min. horseradish peroxidase labeling was detected using a peroxidase substrate solution, 0.8 mM diaminobenzidine. Then the cells were postfixed in 2% osmium tetroxide in PBS for 30 min and embedded in epoxy resin. Sections (0.5 mm) were cut on an ultramicrotome and observed by light microscope. Uptake experiments The uptake experiments were performed as described by us previously [20]. The cells were seeded in 24-well plates at a cell density of 1105 cells/well. After the cells grew to a confluent monolayer following cultivation for 2 days, they were washed three times with Dulbecco’s phosphate-buffered saline (D-PBS) (containing in mM: 137 NaCl, 3 KCl, 8 Na2HPO4, 1 KH2PO4, 1 CaCl2 and 0.5 MgCl2; pH 7.4) containing 5.0 mM D-glucose and preincubated with the same solution for 10 min at 37 C. The cells were then incubated in D-PBS containing 5 mM D-glucose with 50 mM [14C]CER, 50 nM [3H]ES and 5 mM [14C]PAH as indicated in each experiment. The uptake was terminated by the addition of icecold D-PBS, and the cells were washed three times with the same solution. The cells in each well were lysed with 0.5 ml of 0.1N sodium hydroxide and 2.5 ml of aquasol-2, and the radioactivity was determined using a b-scintillation counter (LSC-3100; Aloka, Tokyo, Japan). Inhibition study To investigate the inhibitory effects of cephalosporin antibiotics on the uptake of ES via rOAT3 and PAH via rOAT1, S2 rOAT3 and S2 rOAT1 were incubated for 2 min in D-PBS containing 50 nM [3H] ES or 5 mM [14C] PAH in the absence or presence of various cephalosporin antibiotics, followed by termination of the reaction and measurement of radioactivity. Cell viability assay S2 rOAT3 and S2 pcDNA 3.1 were incubated in the presence or absence of CER (0.55.0 mM) for 24 hrs at 33 C. To elucidate the protective effect of probenecid on CER-induced cytotoxicity, the cells were incubated with CER (2.5 and 5.0 mM) in the absence or presence of probenecid (0.11.0 mM) for 24 hrs at 33 C. After the incubation, 1 ml of 0.5% MTT (3-[4,5-2-yl] 2,5-diphenyltetrazolium bromide) was added to the media, and the cells were further incubated for 4 h. After the cells were lysed with isopropanol/HCl solution, the optical density was measured at 570 nm using that at 630 nm as a reference (Beckman, Du640). Kinetic analysis S2 rOAT3 and S2 rOAT1 were incubated for 2 min in D-PBS containing various concentrations of [3H]ES and [14C]PAH, respectively, in the absence or presence of various cephalosporin antibiotics. On the basis of the [3H]ES and [14C]PAH uptake under each con-
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dition, double reciprocal plot analyses were performed as previously described [22]. When the inhibition was competitive, Ki values were calculated based on the following equation, Ki = concentration of inhibitor/[(km of ES or PAH with inhibitor/km of ES or PAH without inhibitor) 1]. Statistical analysis Data were expressed as mean ± S.E. Statistical differences were determined using Student’s unpaired t-test. Differences were considered significant when P < 0.05. Results Immunohistochemical analysis of rOAT3 protein For determination of the cellular localization of rOAT3 protein, immunohistochemical analysis of S2 rOAT3 was performed using a polyclonal antibody against rOAT3. Fig. 1a and 1b present the longitudinal sections of S2 pcDNA 3.1 and S2 rOAT3 stained with a polyclonal antibody against rOAT3, respectively. As shown in Fig. 1a, no staining was observed in S2 pcDNA 3.1 ‘‘control cells’’. In contrast, Fig. 1b shows that the subcellular localization of rOAT3 protein is mainly on the cell membrane in S2 rOAT3. Both the basolateral and apical portions of the membrane show positive staining. CER uptake in S2 rOAT3 and S2 pcDNA 3.1 We examined the effect of CER on the uptake of [3H]ES, a preferred substrate of rOAT3 [12], by S2 rOAT3. As shown in Fig. 2, CER significantly inhibited the uptake of [3H] ES by S2 rOAT3 in a dose-dependent manner, suggesting that CER is taken up via rOAT3 (N = 4, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control). Moreover, we estimated CER uptake by
Fig. 1. Immunohistochemical staining illustrating rOAT3 expression in S2 rOAT3 stained with antibodies against rOAT3. a) S2 pcDNA 3.1 (350), b) S2 rOAT3 (350).
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Fig. 2. Effect of cephaloridine on the uptake of estrone sulfate by S2 rOAT3. S2 rOAT3 were incubated with 50 nM [3H]estrone sulfate in the presence or absence of cephaloridine (0 5 mM) for 2 min at 37 C. Each bar represents the means ± S.E. for four experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control.
Fig. 3. Cephaloridine uptake by S2 rOAT3 and S2 pcDNA3.1. S2 rOAT3 and S2 pcDNA3.1 were incubated with 50 mM [14C]cephaloridine for 15 min at 37 C. Each bar represents the means ± S.E. for four experiments. *p < 0.001 vs. S2 pcDNA3.1.
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Fig. 4. Effect of cephaloridine on the viability of S2 rOAT3 and S2 pcDNA3.1. The cells were incubated with various concentrations of cephaloridine for 24 hrs. Values are the means ± S.E. of the percentage changes in cell viability compared with control. N = 6, *p < 0.05 and **p < 0.01 vs. S2 pcDNA 3.1.
Fig. 5. Effect of probenecid on the viability of S2 rOAT3 incubated with cephaloridine (CER). The cells were incubated with CER (2.5 mM and 5.0 mM) and various concentrations of probenecid for 24 hrs. Each bar represents the means ± S.E. of the percentage changes of cell viability compared with the results obtained following incubation of the cells with CER alone. N = 6, *p < 0.05 and **p < 0.01 vs. CER alone.
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S2 rOAT3 directly using [14C]CER. For this purpose, S2 rOAT3 and S2 pcDNA3.1 were incubated with 50 mM [14C]CER for 15 min. As shown in Fig. 3, S2 rOAT3 exhibited a CER uptake activity to a greater extent than S2 pcDNA3.1 (N = 4, *p < 0.001 vs. S2 pcDNA 3.1). Although S2 pcDNA3.1 exhibited a high basal uptake of [14C]CER, S2 pcDNA 3.1 did not show a significant uptake of PAH nor ES (data not shown). Thus, we think that the high basal uptake of [14C]CER by S2 pcDNA3.1 may not be a specific uptake via OAT, but due to the characteristics of [14C]CER, including a nonspecific binding of [14C]CER to the cell surface. These results suggest that rOAT3 mediates CER uptake. CER-induced cytototoxicity and its prevention in S2 rOAT3 Fig. 4 shows the changes in cell viability of S2 rOAT3 and S2 pcDNA3.1 incubated with various concentrations of CER for 24 hrs. When the cells were incubated with CER at concentrations higher than 1.0 mM, the viability of S2 rOAT3 was significantly decreased compared with that of S2 pcDNA3.1 (N = 6, *p < 0.05 and **p < 0.01 vs. control). In addition, as shown in Fig. 5, the decrease in the viability of S2 rOAT3 incubated with CER (2.5 mM and 5.0 mM) was recovered by simultaneous incubation with probenecid in a dose-dependent manner (N = 6, *p < 0.05 and **p < 0.01 vs. CER). Inhibitory effects of various cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 To determine the inhibitory effects of cephalosporin antibiotics on the uptake of organic anions via rOAT3 and rOAT1 [9,12], S2 rOAT3 and S2 rOAT1 were incubated with 50 nM
Table 1 Effect of various cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 rOAT3 Drug
% of control
cephaloridine cefadroxil cefotaxime cefamandole cefoperazone ceftriaxone cephalexin cephalothin cefazolin
25.3 46.5 22.5 16.7 37.5 56.7 33.7 23.7 36.0
± ± ± ± ± ± ± ± ±
3.25* 4.50* 2.40* 1.04* 1.23* 2.10* 2.30* 2.47* 1.50*
rOAT1 IC50 (mM) 1.26 1.78 0.80 0.09 1.05 2> 0.63 0.08 0.74
% of control 45.0 68.0 52.2 14.7 31.7 33.3 70.3 25.2 33.0
± ± ± ± ± ± ± ± ±
0.87* 1.62** 2.32* 0.30* 0.76* 2.80* 2.65** 2.45* 1.36*
IC50 (mM) 1.58 2> 2> 0.45 0.78 0.84 2> 0.57 0.96
S2 rOAT3 and S2 rOAT1 were incubated for 2 min in solution containing 50 nM [3H]ES or 5 mM [14C]PAH in the absence or presence of various concentrations of cephalosporin antibiotics. The inhibitory effects of various cephalosoprin antibiotics (2 mM) on the organic anion uptake were expressed as % of control and IC50 values were also counted out. Values are the means ± S.E. of the percentage changes in uptake compared with control. N = 4, *p < 0.001, and **p < 0.01 vs. control.
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[3H]ES and 5 mM [14C]PAH, respectively, in the presence or absence of various cephalosporin antibiotics. As shown in Table 1, [3H] ES uptake via rOAT3 was significantly inhibited by all cephalosporin antibiotics tested; CER, cefadroxil, cefotaxime, cefamandole, cefoperazone, ceftriaxone, cephalexin, cephalothin and cefazolin, at 2 mM, respectively (N = 4, *p < 0.001 vs. control). Similarly, these cephalosporin antibiotics at 2 mM also exhibited significant inhibitory effects on [14C] PAH uptake by S2 rOAT1 (Table 1; N = 4,
Fig. 6. Kinetic analysis of the effects of various cephalosporin antibiotics on estrone sulfate (ES) uptake in S2 rOAT3. The cells were incubated with various concentrations of [3H] ES in the absence or presence of various cephalosporin antibiotics for 2 min. Double reciprocal plots of [3H] ES uptake uptake in the presence or absence of various cephalosporin antibiotics. (A) cephaloridine, (B) cefoperazone, (C) cephalothin and (D) cefazolin. Each bar represents the means ± S.E. for three experiments.
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*p < 0.001 and **p < 0.01 vs. control). The IC50 values of various cephalosporin antibiotics on rOAT1- and rOAT3-mediated organic anion uptake are also listed in Table 1. Kinetic analysis of inhibitory effects of various cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 Among nine cephalosporin antibiotics tested in the above experiments, the inhibitory kinetics of five cephalosporin antibiotics were analyzed with respect to the [3H]ES uptake in
Fig. 7. Kinetic analysis of the effects of various cephalosporin antibiotics on para-aminohippuric acid (PAH) uptake in S2 rOAT1. The cells were incubated with various concentrations [14C]PAH in the absence or presence of various cephalosporin antibiotics for 2 min. Double reciprocal plots of [14C]PAH uptake in the presence or absence of various cephalosporin antibiotics. (A) cephaloridine, (B) cefoperazone, (C) cephalothin and (D) cefazolin. Each bar represents the means ± S.E. for three experiments.
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Table 2 Ki values of various cephalosporin antibiotics which competitively inhibit the uptake of ES in S2 rOAT3 and PAH in S2 rOAT1 rOAT3 Drug cephaloridine cefoperazone cephalothin cefazolin
rOAT1
Ki value (mM)
Concentrations of drugs (mM)
Ki value (mM)
Concentrations of drugs (mM)
1.14 0.67 0.048 0.78
2 1 0.1 1
1.32 0.48 0.53 0.56
2 1.5 0.5 1
S2 rOAT3 and S2 rOAT1 were incubated with solution containing various concentrations of [3H]ES and [14C]PAH, respectively, in the absence or presence of various cephalosporin antibiotics. The Ki values were estimated by Lineweaver-Burk.
S2 rOAT3 and the [14C]PAH uptake in S2 rOAT1. Four cephalosporin antibiotics tested were CER, cefoperazone, cephalothin and cephazolin. The concentrations of these cephalosporin antibiotics tested were similar to IC50 values listed in Table 1. As shown in Figs. 6 and 7, analysis of the Lineweaver-Burk plot of the effects of these cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 demonstrated that these drugs inhibited rOAT3- and rOAT1-mediated organic anion uptake in a competitive manner. The Ki values of these drugs for rOAT3 and rOAT1-mediated organic anion uptake are listed in Table 2.
Discussion CER-induced nephrotoxicity has been studied in whole animals [23], renal slices [24], isolated proximal tubules [25], primary cultures of proximal tubular cells [26] and established cell lines such as LLC-PK1, a pig kidney cell line [27,28]. However, the specific activity of OATs expressed in proximal tubular cells of the kidney has not been completely clarified, and in which the absence of their expression has been confirmed in the LLC-PK1 cell line, where CER may accumulate intracellularly by passive diffusion. In this regard, the cell line used for the current study expresses a distinct rOAT3, thus providing a reliable method for analyzing CER-induced nephrotoxicity. Using these cells, we obtained results suggesting that rOAT3 mediates CER transport as well as CER-induced nephrotoxicity. rOAT1 and rOAT3 were isolated in our laboratory in 1997 [9] and 1999 [12], respectively. Since both transporters mediate basolateral uptake of various drugs and endogenous substances such as nonsteroidal anti-inflammatory drugs, anti-tumor drugs, H2-blockers, prostaglandins, diuretics, angiotensin-converting enzyme inhibitors and beta-lactam antibiotics [9,12], rOAT1 and rOAT3 appeared to be the major OATs at the basolateral membrane of the proximal tubular cells. However, some differences in characteristics have been noted between rOAT1 and rOAT3, such as substrate specificity and localization: rOAT3 is localized in cells of the first (S1), S2 and S3 segments of the proximal tubule (unpublished observation), whereas rOAT1 is localized at the basolateral membrane of cells of the S2 of the proximal
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tubule [9,29]. In addition, rOAT1, but not rOAT3, exhibits transport properties as an exchanger [9,12]. Although rOAT3 appears to mediate CER uptake and CER-induced nephrotoxicity as also dose rOAT1 [20], we do not rule out the possibility that basolateral OATs other than rOAT3 and rOAT1, which remain unidentified, may be involved in the uptake of CER and the CER-induced nephrotoxicity. On the other hand, it is also possible that OAT4, oatp1, OAT-K1, OAT-K2, MRP2 and NPT1 which have been shown to be localized at the apical membrane of the proximal tubule [13,14,16–19,30], participate in the secretion of CER from the proximal tubular cells into the tubular lumen. Since the minimal efflux of CER into the apical fluid has been shown to be associated with the highest nephrotoxicity of CER among cephalosporin antibiotics [31], the role of these apical OATs in CER-induced nephrotoxicity should be examined. Since probenecid treatment has been shown to cause a decrease in the intracellular accumulation of CER and an attenuation of its nephrotoxicity both in vivo and in vitro [20,32,33], renal accumulation of CER has been identified as a critical factor for the development of CER-induced nephrotoxicity. In the current study, probenecid suppressed CER-induced cytotoxicity in S2 rOAT3, confirming the importance of CER accumulation in CER-induced nephrotoxicity. All of the cephalosporin antibiotics tested exhibited inhibitory effects on both ES uptake via S2 rOAT3 and PAH uptake via S2 rOAT1 (Table 1). The present results are in good agreement with those obtained in membrane vesicles using CER, cephalexin and cefazolin [34] and those using cefazolin and CER [35]. In addition, these inhibitory effects were also consistent with those observed for cefoperazone in Xenopus oocytes injected with rOAT3 cRNA [12] and those for the same series of drugs in Xenopus oocytes injected with rOAT1 cRNA [36]. These results suggest that rOAT3 interacts with various cephalosporin antibiotics as also dose rOAT1. There was difference in affinity between rOAT3 and rOAT1 only for cephalothin (more than 3-fold) [37]. The further studies should be perfomed to discriminate the transport property between rOAT3 and rOAT1 using other cephalosporin antibiotics. In addition, the Ki values for rOAT1 in the current studies were similar to those in the previous studies using Xenopus oocytes injected with rOAT1 cRNA, where the Ki values for CER, cefazolin and cephalothin were 2.33 mM, 0.45 mM and 0.29 mM respectively [36]. In addition to S2 rOAT3, we also established cells stably expressing hOAT3 (S2 hOAT3). S2 hOAT3 could be used not only as a tool for elucidating the mechanisms of CER-induced nephrotoxicity in a similar way to S2 rOAT3, but also as an effective screening system for evaluating the renal transport and nephrotoxic potential of newly developed cephalosporin antibiotics, as well as investigating the interactions between cephalosporin antibiotics and other drugs. In conclusion, the current results suggest that rOAT3 is, at least in part, responsible for the cellular uptake of CER at the basolateral membrane of proximal tubular cells, and this mechanism may play an important role in the development of CER-induced nephrotoxicity. In the basolateral membrane of the proximal tubule, both rOAT3 and rOAT1 may participate in CER transport and CER-induced nephrotoxicity. There was some difference of affinity for various cephalosporin antibiotics between rOAT3 and rOAT1.
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Acknowledgments This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture (No. 11671048, No. 11694310, No. 13671128) and a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation.
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25. 26. 27. 28. 29.
30.
31. 32.
33. 34. 35.
36.
37.
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nohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1. Biochemical and Biophysical Research Communications 2000;270:254 – 9. Leier I, Hummel-Eisenbeiss J, Cui Y, Keppler D. ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2. Kidney International 2000;57:1636 – 42. Takeda M, Tojo A, Sekine T, Hosoyamada M, Kanai Y, Endou H. Role of organic anion transporter 1 (OAT1) in cephaloridine induced nephrotoxicity. Kidney International 1999;56:2128 – 36. Hosoyamada M, Obinata M, Suzuki M, Endou H. Cisplatin-induced toxicity in immortalized renal cell lines established from transgenic mice harboring temperature sensitive SV40 large T-antigen gene. Archives of Toxicology 1996;70:284 – 92. Apiwattanakul N, Sekine T, Chairoungdua A, Kanai Y, Nakajima N, Sophasan S, Endou H. Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in xenopus laevis oocytes. Molecular Pharmacology 1999;55:847 – 54. Nagashima K, Kusaka H, Sato K, Karasawa A. Effects of KW-3902, a novel adenosine A1-receptor antagonist, on cephaloridine-induced acute renal failure in rats. Japanese Journal of Pharmacology 1994;64:9 – 17. Goldstein RS, Pasino DA, Hewitt WR, Hook JB. Biochemical mechanisms of cephaloridine nephrotoxicity: Time and concentration dependence of peroxidative injury. Toxicology and Applied Pharmacology 1986;83: 261 – 70. Rush GF, Ponsler GD. Cephaloridine-induced biochemical changes and cytotoxicity in suspensions of rabbit isolated proximal tubules. Toxicology and Applied Pharmacology 1991;109:314 – 26. Bolon C, Gauthier C, Simonnet H, Baverel G. Advantages of a two-chamber culture system to test drug nephrotoxicity: The example of cephaloridine. Kidney Blood Pressure Research 1997;20:264 – 70. Steinmassl D, Pfaller W, Gstraunthaler G, Hoffmann W. LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity. In Vitro Cellular Developemnet and Biology 1995;31:94 – 106. Williams PD, Laska DA, Tay LK, Hottendorf GH. Comparative toxicities of cephalosporin antibiotics in a rabbit kidney cell line (LLC-RK1). Antimicrobial Agents Chemotherapy 1988;32:314 – 8. Tojo A, Sekine T, Nakajima N, Hosoyamada M, Kanai Y, Endou H. Immunohitochemical localization of multispecific renal organic anion transporter 1 in rat kidney. Journal of American Society of Nephrology 1999;10:464 – 71. Bergwerk AJ, Shi X, Ford AC, Kanai N, Jacquemin E, Burk RD, Bai S, Novikoff PM, Stieger B, Meier PJ, Schuster VL, Wolkoff AW. Immunologic distribution of an organic anion transport protein in rat liver and kidney. American Journal of Physiology 1996;271:G231 – 8. Tune BM. Nephrotoxicity of beta-lactam antibiotics mechanisms and strategies for prevention. Pediatric Nephrology 1997;11:768 – 72. Tune BM, Wu KY, Kempson RL. Inhibition of transport and prevention of toxicity of cephaloridine in the kidney. Dose-responsiveness of the rabbit and guinea pig to probenecid. Journal of Pharmacology and Experimental Therapeutics 1997;202:466 – 71. Cojocel C, Laeschke KH, Inselmann G, Baumann K. Inhibition of cephaloridine-induced lipid peroxidation. Toxicology 1985;35:295 – 305. Takano M, Okano T, Inui K, Hori R. Transport of cephalosporin antibiotics in rat renal basolateral membrane. Journal of Pharmacy and Pharmacology 1989;41:795 – 6. Tsuji A, Terasaki T, Tamai I, Takeda K. In vivo evidence for carrier-mediated uptake of b-lactam antibiotics through organic anion transport systems in rat kidney and liver. Journal of Pharmacology and Experimental Therapeutics 1990;253:315 – 20. Jariyawat S, Sekine T, Takeda M, Kanai Y, Apiwattanakul N, Tsuda M, Nakajima N, Sosphasan S, Endou H. The interaction and transport of b-lactam antibiotics with organic anion transporter 1(OAT1). Journal of Pharmacology and Experimental Therapeutics 1999;290:672 – 7. Zhang L, Schaner ME, Giacomoni KM. Functional characterization of an organic cation transporter (hOCT1) in a transiently transfected human cell line (HeLa). Journal of Pharmacology and Experimental Therapeutics 1998;286:354 – 61.
Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: In comparison with rOAT1 Kyu Yong Jungb, Michio Takedaa, Minoru Shimodac, Shinichi Narikawaa, Akihiro Tojod, Do Kyung Kima, Arthit Chairoungduaa, Bong Kyu Choib, Hiroyuki Kusuharae, Yuichi Sugiyamae, Takashi Sekinea, Hitoshi Endou a,* a
Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka-shi, Tokyo, Japan b Department of Pharmacology, Wonkwang University School of Medicine, Iksan, Chonbuk, South Korea c Department of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan d The Second Department of Internal Medicine, University of Tokyo, Tokyo, Japan e Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Received 28 March 2001; accepted 19 October 2001
Abstract This study was performed to elucidate the possible involvement of organic anion transporter 3 (OAT3) in cephaloridine (CER)-induced nephrotoxicity and compare the substrate specificity between rOAT3 and rat OAT1 (rOAT1) for various cephalosporin antibiotics, using proximal tubule cells stably expressing rOAT3 (S2 rOAT3) and rOAT1 (S2 rOAT1). S2 rOAT3 exhibited a CER uptake and a higher susceptibility to CER cytotoxicity than did mock, which was recovered by probenecid. Various cephalosporin antibiotics significantly inhibited both estrone sulfate uptake in S2 rOAT3 and paraaminohippuric acid uptake in S2 rOAT1. The Ki values of CER, cefoperazone, cephalothin and cefazolin for rOAT3- and rOAT1-mediated organic anion transport ranged from 0.048 to 1.14 mM and from 0.48 to 1.32 mM, respectively. These results suggest that rOAT3, at least in part, mediates CER * Corresponding author. Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan. Tel.: +11-81-422-47-5511x3451; fax: +11-81422-79-1321. E-mail address: [email protected] (H. Endou). 0024-3205/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 5 0 0 - X
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uptake and CER-induced nephrotoxicity as rOAT1. There was some difference of affinity between rOAT3 and rOAT1 for cephalosporin antibiotics. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Organic anion transporter; OAT3; Cephaloridine; Nephrotoxicity; Proximal tubule
Introduction Cephaloridine (CER) is a broad-spectrum cephalosporin antibiotic that is known to induce acute renal failure in humans and animals [1]. The nephrotoxicity induced by CER is characterized by acute proximal tubular necrosis and is mainly dependent on the accumulation and concentration of the drug in the renal cortex, more specifically, in the second segment of the proximal tubule (S2) [2–5]. CER is actively transported by the proximal tubular cells from the blood via an organic anion transporter (OAT) at the basolateral membrane. However, unlike the other less toxic cephalosporin antibiotics, CER is not transported readily across the luminal membrane, which results in the accumulation of a high concentration of CER and selective damage of these cells in the proximal tubule [6,7]. The secretion of numerous organic anions, including endogenous metabolites, drugs and xenobiotics, is an important physiological function of renal proximal tubules. The secretion of organic anions from the proximal tubular cells is achieved via unidirectional transcellular transport, involving the uptake of organic anions into the cells from the blood across the basolateral membrane, followed by their extrusion across the brush-border membrane into the tubular fluid [8]. Recently, cDNAs encoding transporters mediating renal organic anion transport have been successively cloned, namely, organic anion transporter 1 (OAT1), OAT2, OAT3, OAT4, organic anion-transporting polypeptide (oatp1), oatp2, OAT-K1, OAT-K2, multiple resistance-associated protein 2 (MRP2) and human-type I sodium-dependent inorganic phosphate transporter (NPT1) [9–19]. Among them, rat OAT3 (rOAT3) and rat OAT1 (rOAT1) have been shown to be the major OATs at the basolateral membrane of the proximal tubular cells [9,12]. We have already demonstrated that rOAT1 mediates CER transport as well as CERinduced nephrotoxicity, using cells of the mouse kidney of the third segment of the proximal tubule (S3) stably expressing rOAT1 [20]. The purpose of this study was to elucidate the possible involvement of rOAT3 in CER transport and CER-induced nephrotoxicity using mouse S2 cells stably expressing rOAT3, and compare the substrate recognition between rOAT3 and rOAT1 for various cephalosporin antibiotics.
Materials and methods Materials [14C]CER (13.9 mCi/mmol) was kindly supplied by Sankyo Co. (Tokyo, Japan). [14C]paraaminohippuric acid (PAH) (53.1 mCi/mmol) and [3H]estrone sulfate (ES) (53 Ci/mmol) were
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purchased from Perkin Elmer Life Sciences (Boston, MA, USA). All other chemicals and reagents were of analytical grade and purchased from commercial sources. Cell culture and establishment of S2 cells stably expressing rOAT3 and rOAT1 (S2 rOAT3 and S2 rOAT1) The renal tubular cells used for the study were S2 cells derived from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene [21]. S2 cells were cultured in RITC 80-7 medium containing 5% fetal bovine serum, 10 mg/ml transferrin, 0.08 U/ml insulin and 10 ng/ml recombinant epidermal growth factor. The cells were subcultured in the medium containing 0.05% trypsin-EDTA solution (containing in mM: 137 NaCl, 5.4 KCl, 5.5 glucose, 4 NaHCO3, 0.5 EDTA and 5 Hepes; pH 7.2) and used at passages 60100. The full-length rOAT3 [12] or rOAT1 [9] genes were subcloned into pcDNA3.1, a mammalian expression vector. The pcDNA3.1-rOAT1, pcDNA3.1-rOAT3, and pcDNA3.1, which lacked the insert coupled with pSV2neo, a neomycin resistance gene, were transfected into S2 cells using the TfX2-50 reagent according to the manufacturer’s instructions. Forty-eight hrs after the transfection, the cells were subcultured in the medium containing 400 mg/ml geneticin. The cell clones were isolated using a cloning cylinder. The organic anion transport activity in these cells was determined by measuring the uptake of the substrate preferred by rOAT3, [3H]ES [12] and that by rOAT1, [14C]PAH [9]. The cells showing the highest level of activities were selected, and designated as S2 rOAT3 and S2 rOAT1, whereas the S2 cells transfected with pcDNA 3.1 lacking the insert were designated as S2 pcDNA 3.1 and used as the control. The S2 monolayer was determined to be leaky based on the results of a study wherein we estimated paracellular secretion from cells cultured on a permeable support, using D-[3H]mannitol as an indicator. Therefore, the cells were cultured on a solid support for these experiments. Two days after inoculation, the S2 cells were employed for experiments. Immunohistochemical analysis of rOAT3 protein Light-microscopic analysis of the rOAT3 protein was performed as previously described [20]. S2 rOAT3 were cultured in a six-well dish. The cells were fixed with paraformaldehyde lysine periodate (PLP) solution (containing 0.01 M NaIO4, 0.075 M lysine, 0.0375 M phosphate buffer with 2% paraformaldehyde; pH 6.2) for 2 hrs at 4 C. After fixation, the cells were washed with 50 mM NH4Cl in phosphate-buffered saline (PBS) (containing in mM: 136 NaCl, 2.7 KCl, 8.2 Na2HPO412H2O and 15 KH2PO4, pH 7.4) and then with Trisbuffered saline containing 0.1% Tween 20 (TBST). The cells were incubated with polyclonal antibody against rOAT3 at a dilution of 1: 400 for 2 hrs. For generation of this antibody, rabbits were immunized with keyhole limpet hemocyanin-conjugated synthesized peptides, EAEKASQHPLKTGG, corresponding to cysteine and the 14 amino acids of the COOH terminus of rOAT3. After rinsing with TBST, the cells were incubated with biotinylated secondary antibody against rabbit immunoglobulin (LSAB kit, Dako, Carpinteria, CA) for 1 hr. The cells were washed with TBST, and incubated with horseradish peroxidase-conjugated
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streptavidine solution for 30 min. horseradish peroxidase labeling was detected using a peroxidase substrate solution, 0.8 mM diaminobenzidine. Then the cells were postfixed in 2% osmium tetroxide in PBS for 30 min and embedded in epoxy resin. Sections (0.5 mm) were cut on an ultramicrotome and observed by light microscope. Uptake experiments The uptake experiments were performed as described by us previously [20]. The cells were seeded in 24-well plates at a cell density of 1105 cells/well. After the cells grew to a confluent monolayer following cultivation for 2 days, they were washed three times with Dulbecco’s phosphate-buffered saline (D-PBS) (containing in mM: 137 NaCl, 3 KCl, 8 Na2HPO4, 1 KH2PO4, 1 CaCl2 and 0.5 MgCl2; pH 7.4) containing 5.0 mM D-glucose and preincubated with the same solution for 10 min at 37 C. The cells were then incubated in D-PBS containing 5 mM D-glucose with 50 mM [14C]CER, 50 nM [3H]ES and 5 mM [14C]PAH as indicated in each experiment. The uptake was terminated by the addition of icecold D-PBS, and the cells were washed three times with the same solution. The cells in each well were lysed with 0.5 ml of 0.1N sodium hydroxide and 2.5 ml of aquasol-2, and the radioactivity was determined using a b-scintillation counter (LSC-3100; Aloka, Tokyo, Japan). Inhibition study To investigate the inhibitory effects of cephalosporin antibiotics on the uptake of ES via rOAT3 and PAH via rOAT1, S2 rOAT3 and S2 rOAT1 were incubated for 2 min in D-PBS containing 50 nM [3H] ES or 5 mM [14C] PAH in the absence or presence of various cephalosporin antibiotics, followed by termination of the reaction and measurement of radioactivity. Cell viability assay S2 rOAT3 and S2 pcDNA 3.1 were incubated in the presence or absence of CER (0.55.0 mM) for 24 hrs at 33 C. To elucidate the protective effect of probenecid on CER-induced cytotoxicity, the cells were incubated with CER (2.5 and 5.0 mM) in the absence or presence of probenecid (0.11.0 mM) for 24 hrs at 33 C. After the incubation, 1 ml of 0.5% MTT (3-[4,5-2-yl] 2,5-diphenyltetrazolium bromide) was added to the media, and the cells were further incubated for 4 h. After the cells were lysed with isopropanol/HCl solution, the optical density was measured at 570 nm using that at 630 nm as a reference (Beckman, Du640). Kinetic analysis S2 rOAT3 and S2 rOAT1 were incubated for 2 min in D-PBS containing various concentrations of [3H]ES and [14C]PAH, respectively, in the absence or presence of various cephalosporin antibiotics. On the basis of the [3H]ES and [14C]PAH uptake under each con-
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dition, double reciprocal plot analyses were performed as previously described [22]. When the inhibition was competitive, Ki values were calculated based on the following equation, Ki = concentration of inhibitor/[(km of ES or PAH with inhibitor/km of ES or PAH without inhibitor) 1]. Statistical analysis Data were expressed as mean ± S.E. Statistical differences were determined using Student’s unpaired t-test. Differences were considered significant when P < 0.05. Results Immunohistochemical analysis of rOAT3 protein For determination of the cellular localization of rOAT3 protein, immunohistochemical analysis of S2 rOAT3 was performed using a polyclonal antibody against rOAT3. Fig. 1a and 1b present the longitudinal sections of S2 pcDNA 3.1 and S2 rOAT3 stained with a polyclonal antibody against rOAT3, respectively. As shown in Fig. 1a, no staining was observed in S2 pcDNA 3.1 ‘‘control cells’’. In contrast, Fig. 1b shows that the subcellular localization of rOAT3 protein is mainly on the cell membrane in S2 rOAT3. Both the basolateral and apical portions of the membrane show positive staining. CER uptake in S2 rOAT3 and S2 pcDNA 3.1 We examined the effect of CER on the uptake of [3H]ES, a preferred substrate of rOAT3 [12], by S2 rOAT3. As shown in Fig. 2, CER significantly inhibited the uptake of [3H] ES by S2 rOAT3 in a dose-dependent manner, suggesting that CER is taken up via rOAT3 (N = 4, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control). Moreover, we estimated CER uptake by
Fig. 1. Immunohistochemical staining illustrating rOAT3 expression in S2 rOAT3 stained with antibodies against rOAT3. a) S2 pcDNA 3.1 (350), b) S2 rOAT3 (350).
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Fig. 2. Effect of cephaloridine on the uptake of estrone sulfate by S2 rOAT3. S2 rOAT3 were incubated with 50 nM [3H]estrone sulfate in the presence or absence of cephaloridine (0 5 mM) for 2 min at 37 C. Each bar represents the means ± S.E. for four experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control.
Fig. 3. Cephaloridine uptake by S2 rOAT3 and S2 pcDNA3.1. S2 rOAT3 and S2 pcDNA3.1 were incubated with 50 mM [14C]cephaloridine for 15 min at 37 C. Each bar represents the means ± S.E. for four experiments. *p < 0.001 vs. S2 pcDNA3.1.
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Fig. 4. Effect of cephaloridine on the viability of S2 rOAT3 and S2 pcDNA3.1. The cells were incubated with various concentrations of cephaloridine for 24 hrs. Values are the means ± S.E. of the percentage changes in cell viability compared with control. N = 6, *p < 0.05 and **p < 0.01 vs. S2 pcDNA 3.1.
Fig. 5. Effect of probenecid on the viability of S2 rOAT3 incubated with cephaloridine (CER). The cells were incubated with CER (2.5 mM and 5.0 mM) and various concentrations of probenecid for 24 hrs. Each bar represents the means ± S.E. of the percentage changes of cell viability compared with the results obtained following incubation of the cells with CER alone. N = 6, *p < 0.05 and **p < 0.01 vs. CER alone.
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S2 rOAT3 directly using [14C]CER. For this purpose, S2 rOAT3 and S2 pcDNA3.1 were incubated with 50 mM [14C]CER for 15 min. As shown in Fig. 3, S2 rOAT3 exhibited a CER uptake activity to a greater extent than S2 pcDNA3.1 (N = 4, *p < 0.001 vs. S2 pcDNA 3.1). Although S2 pcDNA3.1 exhibited a high basal uptake of [14C]CER, S2 pcDNA 3.1 did not show a significant uptake of PAH nor ES (data not shown). Thus, we think that the high basal uptake of [14C]CER by S2 pcDNA3.1 may not be a specific uptake via OAT, but due to the characteristics of [14C]CER, including a nonspecific binding of [14C]CER to the cell surface. These results suggest that rOAT3 mediates CER uptake. CER-induced cytototoxicity and its prevention in S2 rOAT3 Fig. 4 shows the changes in cell viability of S2 rOAT3 and S2 pcDNA3.1 incubated with various concentrations of CER for 24 hrs. When the cells were incubated with CER at concentrations higher than 1.0 mM, the viability of S2 rOAT3 was significantly decreased compared with that of S2 pcDNA3.1 (N = 6, *p < 0.05 and **p < 0.01 vs. control). In addition, as shown in Fig. 5, the decrease in the viability of S2 rOAT3 incubated with CER (2.5 mM and 5.0 mM) was recovered by simultaneous incubation with probenecid in a dose-dependent manner (N = 6, *p < 0.05 and **p < 0.01 vs. CER). Inhibitory effects of various cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 To determine the inhibitory effects of cephalosporin antibiotics on the uptake of organic anions via rOAT3 and rOAT1 [9,12], S2 rOAT3 and S2 rOAT1 were incubated with 50 nM
Table 1 Effect of various cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 rOAT3 Drug
% of control
cephaloridine cefadroxil cefotaxime cefamandole cefoperazone ceftriaxone cephalexin cephalothin cefazolin
25.3 46.5 22.5 16.7 37.5 56.7 33.7 23.7 36.0
± ± ± ± ± ± ± ± ±
3.25* 4.50* 2.40* 1.04* 1.23* 2.10* 2.30* 2.47* 1.50*
rOAT1 IC50 (mM) 1.26 1.78 0.80 0.09 1.05 2> 0.63 0.08 0.74
% of control 45.0 68.0 52.2 14.7 31.7 33.3 70.3 25.2 33.0
± ± ± ± ± ± ± ± ±
0.87* 1.62** 2.32* 0.30* 0.76* 2.80* 2.65** 2.45* 1.36*
IC50 (mM) 1.58 2> 2> 0.45 0.78 0.84 2> 0.57 0.96
S2 rOAT3 and S2 rOAT1 were incubated for 2 min in solution containing 50 nM [3H]ES or 5 mM [14C]PAH in the absence or presence of various concentrations of cephalosporin antibiotics. The inhibitory effects of various cephalosoprin antibiotics (2 mM) on the organic anion uptake were expressed as % of control and IC50 values were also counted out. Values are the means ± S.E. of the percentage changes in uptake compared with control. N = 4, *p < 0.001, and **p < 0.01 vs. control.
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[3H]ES and 5 mM [14C]PAH, respectively, in the presence or absence of various cephalosporin antibiotics. As shown in Table 1, [3H] ES uptake via rOAT3 was significantly inhibited by all cephalosporin antibiotics tested; CER, cefadroxil, cefotaxime, cefamandole, cefoperazone, ceftriaxone, cephalexin, cephalothin and cefazolin, at 2 mM, respectively (N = 4, *p < 0.001 vs. control). Similarly, these cephalosporin antibiotics at 2 mM also exhibited significant inhibitory effects on [14C] PAH uptake by S2 rOAT1 (Table 1; N = 4,
Fig. 6. Kinetic analysis of the effects of various cephalosporin antibiotics on estrone sulfate (ES) uptake in S2 rOAT3. The cells were incubated with various concentrations of [3H] ES in the absence or presence of various cephalosporin antibiotics for 2 min. Double reciprocal plots of [3H] ES uptake uptake in the presence or absence of various cephalosporin antibiotics. (A) cephaloridine, (B) cefoperazone, (C) cephalothin and (D) cefazolin. Each bar represents the means ± S.E. for three experiments.
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*p < 0.001 and **p < 0.01 vs. control). The IC50 values of various cephalosporin antibiotics on rOAT1- and rOAT3-mediated organic anion uptake are also listed in Table 1. Kinetic analysis of inhibitory effects of various cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 Among nine cephalosporin antibiotics tested in the above experiments, the inhibitory kinetics of five cephalosporin antibiotics were analyzed with respect to the [3H]ES uptake in
Fig. 7. Kinetic analysis of the effects of various cephalosporin antibiotics on para-aminohippuric acid (PAH) uptake in S2 rOAT1. The cells were incubated with various concentrations [14C]PAH in the absence or presence of various cephalosporin antibiotics for 2 min. Double reciprocal plots of [14C]PAH uptake in the presence or absence of various cephalosporin antibiotics. (A) cephaloridine, (B) cefoperazone, (C) cephalothin and (D) cefazolin. Each bar represents the means ± S.E. for three experiments.
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Table 2 Ki values of various cephalosporin antibiotics which competitively inhibit the uptake of ES in S2 rOAT3 and PAH in S2 rOAT1 rOAT3 Drug cephaloridine cefoperazone cephalothin cefazolin
rOAT1
Ki value (mM)
Concentrations of drugs (mM)
Ki value (mM)
Concentrations of drugs (mM)
1.14 0.67 0.048 0.78
2 1 0.1 1
1.32 0.48 0.53 0.56
2 1.5 0.5 1
S2 rOAT3 and S2 rOAT1 were incubated with solution containing various concentrations of [3H]ES and [14C]PAH, respectively, in the absence or presence of various cephalosporin antibiotics. The Ki values were estimated by Lineweaver-Burk.
S2 rOAT3 and the [14C]PAH uptake in S2 rOAT1. Four cephalosporin antibiotics tested were CER, cefoperazone, cephalothin and cephazolin. The concentrations of these cephalosporin antibiotics tested were similar to IC50 values listed in Table 1. As shown in Figs. 6 and 7, analysis of the Lineweaver-Burk plot of the effects of these cephalosporin antibiotics on organic anion uptake in S2 rOAT3 and S2 rOAT1 demonstrated that these drugs inhibited rOAT3- and rOAT1-mediated organic anion uptake in a competitive manner. The Ki values of these drugs for rOAT3 and rOAT1-mediated organic anion uptake are listed in Table 2.
Discussion CER-induced nephrotoxicity has been studied in whole animals [23], renal slices [24], isolated proximal tubules [25], primary cultures of proximal tubular cells [26] and established cell lines such as LLC-PK1, a pig kidney cell line [27,28]. However, the specific activity of OATs expressed in proximal tubular cells of the kidney has not been completely clarified, and in which the absence of their expression has been confirmed in the LLC-PK1 cell line, where CER may accumulate intracellularly by passive diffusion. In this regard, the cell line used for the current study expresses a distinct rOAT3, thus providing a reliable method for analyzing CER-induced nephrotoxicity. Using these cells, we obtained results suggesting that rOAT3 mediates CER transport as well as CER-induced nephrotoxicity. rOAT1 and rOAT3 were isolated in our laboratory in 1997 [9] and 1999 [12], respectively. Since both transporters mediate basolateral uptake of various drugs and endogenous substances such as nonsteroidal anti-inflammatory drugs, anti-tumor drugs, H2-blockers, prostaglandins, diuretics, angiotensin-converting enzyme inhibitors and beta-lactam antibiotics [9,12], rOAT1 and rOAT3 appeared to be the major OATs at the basolateral membrane of the proximal tubular cells. However, some differences in characteristics have been noted between rOAT1 and rOAT3, such as substrate specificity and localization: rOAT3 is localized in cells of the first (S1), S2 and S3 segments of the proximal tubule (unpublished observation), whereas rOAT1 is localized at the basolateral membrane of cells of the S2 of the proximal
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tubule [9,29]. In addition, rOAT1, but not rOAT3, exhibits transport properties as an exchanger [9,12]. Although rOAT3 appears to mediate CER uptake and CER-induced nephrotoxicity as also dose rOAT1 [20], we do not rule out the possibility that basolateral OATs other than rOAT3 and rOAT1, which remain unidentified, may be involved in the uptake of CER and the CER-induced nephrotoxicity. On the other hand, it is also possible that OAT4, oatp1, OAT-K1, OAT-K2, MRP2 and NPT1 which have been shown to be localized at the apical membrane of the proximal tubule [13,14,16–19,30], participate in the secretion of CER from the proximal tubular cells into the tubular lumen. Since the minimal efflux of CER into the apical fluid has been shown to be associated with the highest nephrotoxicity of CER among cephalosporin antibiotics [31], the role of these apical OATs in CER-induced nephrotoxicity should be examined. Since probenecid treatment has been shown to cause a decrease in the intracellular accumulation of CER and an attenuation of its nephrotoxicity both in vivo and in vitro [20,32,33], renal accumulation of CER has been identified as a critical factor for the development of CER-induced nephrotoxicity. In the current study, probenecid suppressed CER-induced cytotoxicity in S2 rOAT3, confirming the importance of CER accumulation in CER-induced nephrotoxicity. All of the cephalosporin antibiotics tested exhibited inhibitory effects on both ES uptake via S2 rOAT3 and PAH uptake via S2 rOAT1 (Table 1). The present results are in good agreement with those obtained in membrane vesicles using CER, cephalexin and cefazolin [34] and those using cefazolin and CER [35]. In addition, these inhibitory effects were also consistent with those observed for cefoperazone in Xenopus oocytes injected with rOAT3 cRNA [12] and those for the same series of drugs in Xenopus oocytes injected with rOAT1 cRNA [36]. These results suggest that rOAT3 interacts with various cephalosporin antibiotics as also dose rOAT1. There was difference in affinity between rOAT3 and rOAT1 only for cephalothin (more than 3-fold) [37]. The further studies should be perfomed to discriminate the transport property between rOAT3 and rOAT1 using other cephalosporin antibiotics. In addition, the Ki values for rOAT1 in the current studies were similar to those in the previous studies using Xenopus oocytes injected with rOAT1 cRNA, where the Ki values for CER, cefazolin and cephalothin were 2.33 mM, 0.45 mM and 0.29 mM respectively [36]. In addition to S2 rOAT3, we also established cells stably expressing hOAT3 (S2 hOAT3). S2 hOAT3 could be used not only as a tool for elucidating the mechanisms of CER-induced nephrotoxicity in a similar way to S2 rOAT3, but also as an effective screening system for evaluating the renal transport and nephrotoxic potential of newly developed cephalosporin antibiotics, as well as investigating the interactions between cephalosporin antibiotics and other drugs. In conclusion, the current results suggest that rOAT3 is, at least in part, responsible for the cellular uptake of CER at the basolateral membrane of proximal tubular cells, and this mechanism may play an important role in the development of CER-induced nephrotoxicity. In the basolateral membrane of the proximal tubule, both rOAT3 and rOAT1 may participate in CER transport and CER-induced nephrotoxicity. There was some difference of affinity for various cephalosporin antibiotics between rOAT3 and rOAT1.
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Acknowledgments This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture (No. 11671048, No. 11694310, No. 13671128) and a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation.
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