Interaction of human organic anion transporters with various cephalosporin antibiotics

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Abstract

Cephalosporin antibiotics are thought to be excreted into the urine via organic anion transporters (OATs). The purpose of this study was to elucidate the interaction of human-OATs with various cephalosporin antibiotics, using proximal tubule cells stably expressing human-OAT1, human-OAT3 and human-OAT4. Human-OAT1 and human-OAT3 are localized to the basolateral side of the proximal tubule, whereas human-OAT4 is localized to the apical side. The cephalosporin antibiotics tested were cephalothin, cefoperazone, cefazolin, ceftriaxone, cephaloridine, cefotaxime, cefadroxil and cefamandole. All of these cephalosporin antibiotics significantly inhibited organic anion uptake mediated by human-OAT1, human-OAT3 and human-OAT4. Kinetic analysis revealed that these inhibitions were competitive. The inhibition constant (Ki) values of cefoperazone, cefazolin, ceftriaxone and cephaloridine for human-OAT1 were much lower than those for human-OAT3 and human-OAT4, whereas the Ki values of cephalothin and cefotaxime for human-OAT3 were much lower than those for human-OAT1 and human-OAT4. Human-OAT4 mediated the bidirectional transport of estrone sulfate, an optimal substrate for human-OAT4. These results suggest that human-OAT1, human-OAT3 and human-OAT4 interact with various cephalosporin antibiotics, and that human-OAT1 and human-OAT3 play a distinct role in the basolateral uptake of cephalosporin antibiotics. Since the Ki value of cephaloridine for human-OAT4-mediated organic uptake was much higher than that for human-OAT1, the results indicate the possibility that human-OAT4 limits the efflux of cephaloridine, leading to the accumulation of cephaloridine and the induction of nephrotoxicity.

Introduction

The secretion of numerous organic anions, including endogenous metabolites, drugs and xenobiotics, is an important physiological function of the renal proximal tubules. The secretion of organic anions through 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 extrusion across the brush-border membrane into the tubular fluid (Pritchard and Miller, 1993). Recently, cDNAs encoding members of the organic anion transporter (OAT) family have been successively cloned, including OAT1 Sekine et al., 1997, Hosoyamada et al., 1999, OAT2 (Sekine et al., 1998), OAT3 Kusuhara et al., 1999, Cha et al., 2001 and OAT4 (Cha et al., 2000). Among these clones, human-OAT1 and human-OAT3 were shown to be localized to the basolateral side of the proximal tubule (Hosoyamada et al., 1999; Cha et al., 2001), whereas human-OAT4 is localized to the apical side of the proximal tubule (Babu et al., in press).

Cephalosporin antibiotics are suggested to be not only filtered through the glomeruli but also actively secreted by the proximal tubules. Cephalosporins inhibited para-aminohippuric acid uptake in rat renal slices (Hori et al., 1982) and renal plasma membrane vesicles (Takano et al., 1989). Cephaloridine, a cephalosporin that possesses both anionic and cationic moieties, inhibited para-aminohippuric acid transport but not N-methylnicotinamide transport in basolateral membrane vesicles (Kasher et al., 1983). Thus, cephalosporin antibiotics are considered to be secreted by the proximal tubule via the para-aminohippuric acid transporter system Moller and Sheikh, 1983, Ulrich et al., 1989. Consistent with these results, we have recently observed that rat-OAT1 as well as rat-OAT3 interacts with various cephalosporin antibiotics Jariyawat et al., 1999, Takeda et al., 1999, Jung et al., in press.

The purpose of this study was to elucidate the interactions of human-OATs with various cephalosporin antibiotics, using cells derived from the second segment of the proximal tubule (S2) that stably express human-OAT1, human-OAT3 and human-OAT4 (S2 human-OAT1, S2 human-OAT3 and S2 human-OAT4, respectively).

Section snippets

Materials

[14C]Para-aminohippuric acid (53.1 mCi /mmol) and [3H]estrone sulfate (53 Ci/mmol) were purchased from Perkin Elmer Life Sciences (Boston, MA, USA). Cephalosporin antibiotics were obtained from Sigma (St. Louis, MO, USA). Other materials used included fetal bovine serum, trypsin and geneticin from Invitrogen (Carlsbad, CA, USA) recombinant epidermal growth factor from Wakunaga (Hiroshima, Japan), insulin from Shimizu (Shizuoka, Japan), RITC 80-7 culture medium from Iwaki (Tokyo, Japan) and

Effects of various cephalosporin antibiotics on organic anion uptake by S2 human-OAT1, S2 human-OAT3 and S2 human-OAT4

We elucidated the effects of various cephalosporin antibiotics on organic anion uptake mediated by human-OAT1, human-OAT3 and human-OAT4. The cephalosporin antibiotics tested were cephalothin, cefoperazone, cefazolin, ceftriaxone, cephaloridine, cefotaxime, cefadroxil and cefamandole. As shown in Fig. 1, all of these cephalosporin antibiotics at 2 mM significantly inhibited the organic anion uptake mediated by human-OAT1 (A), human-OAT3 (B) and human-OAT4 (C) (N=4, *P<0.001, **P<0.01 and ***P

Discussion

Human-OAT1 and human-OAT3 were recently cloned and characterized as multispecific OATs and were found to mediate the active transport of organic anions from the interstitium to the cells in the basolateral membrane of the proximal tubule Hosoyamada et al., 1999, Cha et al., 2001. Both transporters mediate the basolateral uptake of various drugs and endogenous substances such as nonsteroidal anti-inflammatory drugs, antitumor drugs, H2-receptor antagonists, prostaglandins, diuretics,

Acknowledgements

This study was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (No. 11671048, No. 11694310 and No. 13671128), the Science Research Promotion Fund of the Japan Private School Promotion Foundation and Research on Health Sciences Focusing on Drug Innovation from Japan Health Sciences Foundation.

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      OAT4 (SLC22A11) and Oat5 (SLC22A10/19) proteins have been detected on the apical membrane of human and rat renal proximal tubules, respectively [10,11]. OATs are responsible for the uptake of organic anions, such as beta-lactam antibiotics and nonsteroidal anti-inflammatory drugs [12,13]. Endogenous dicarboxylate anions (such as alpha-ketoglutarate) are considered to be the driving force for organic anion exchange [14–18].

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