Kidney International, Vol. 56 (1999), pp. 2128–2136

Role of organic anion transporter 1 (OAT1) in cephaloridine(CER)-induced nephrotoxicity

MICHIO TAKEDA, AKIHIRO TOJO, TAKASHI SEKINE, MAKOTO HOSOYAMADA,YOSHIKATSU KANAI, and HITOSHI ENDOU

Department of Pharmacology and Toxicology, Kyorin University School of Medicine, and The Second Department ofMedicine, University of Tokyo, Tokyo, Japan

Role of organic anion transporter 1 (OAT1) in cephaloridine in the renal cortex [2]. CER is actively taken up by the(CER)-induced nephrotoxicity. proximal tubular cells from the blood via an organic

Background. Cephaloridine (CER) has been used to eluci- anion transporter (OAT) in the basolateral membrane.date the mechanisms of cephalosporin antibiotic-induced neph-However, unlike the other, less toxic cephalosporins,rotoxicity. Organic anion transporters have been thought toCER does not move readily across the luminal mem-mediate CER uptake by the proximal tubule. The purpose of

this study was to elucidate the possible involvement of organic brane, which results in the build up of high concentra-anion transporter 1 (OAT1) in CER-induced nephrotoxicity. tions of CER in and selective damage to proximal tubular

Methods. A mouse terminal proximal straight tubule (S3) cell cells [3, 4]. The accumulated CER induces toxicity vialine stably expressing rat OAT1 (S3 rOAT1) was establisheda number of molecular mechanisms including lipid per-and used in this study. The cellular uptake of [14C]-para-amino-oxidation [5], respiratory toxicity by inactivation of mito-hippuric acid (PAH), a prototype organic anion, and that of

[14C]-CER were measured. The effects of CER on the viability chondrial anion substrate carriers [6], and the activationof the cells and the amount of lipid peroxidation were estimated. of adenosine receptors [7].

Results. S3 rOAT1 expressed a functional organic anion The secretion of numerous organic anions, includingtransporter in the cytoplasmic membrane, and exhibited CERendogenous metabolites, drugs and xenobiotics, is anuptake activity. CER treatment resulted in a more significantimportant physiological function of the renal proximaldecrease in the viability and a more significant increase in the

amount of lipid peroxidation in S3 rOAT1 than in S3 cells tubules. The process of secreting organic anions throughtransfected with an expression vector lacking the rOAT1 insert. the proximal tubular cells is achieved via unidirectionalProbenecid, an inhibitor of organic anion transport, and probu- transcellular transport, involving the uptake of organiccol, an antioxidant, significantly suppressed the decrease in

anions into the cells from the blood across the basolateralviability and increase in the amount of lipid peroxidation in S3membrane, followed by extrusion across the brush borderrOAT1 treated with CER. The effects of various cephalosporin

antibiotics on the uptake of [14C]PAH were correlated signifi- membrane into the tubular fluid [8]. Recently, cDNAscantly with the effects of these drugs on cell viability. encoding renal OATs have been successively cloned, for

Conclusions. These results suggest that rOAT1 is, at least in example, OAT1, OAT2, OAT-P1, OAT-P2, and OAT-part, responsible for the cellular uptake of CER and thereforeK1 [9–13]. Among the OAT family, OAT1 is the onlyCER-induced nephrotoxicity.OAT identified to date as being responsible for the cellu-lar uptake of CER via the basolateral membrane of theproximal tubule [9].Cephaloridine (CER) is a first-generation cephalospo-

The purpose of this study was to elucidate the role ofrin antibiotic that induces acute renal failure in humansrat OAT1 (rOAT1) in CER-induced nephrotoxicity. Forand animals [1]. The nephrotoxicity induced by CER isthis purpose, we have established and utilized a mousecharacterized by acute proximal tubular necrosis and isterminal proximal straight tubule (S3) cell line stablymainly dependent on its accumulation and concentrationexpressing rat OAT1 (S3 rOAT1).

Key words: PAH, cephalosporin, proximal tubule, SV40, cell line, lipid METHODSperoxidation.Materials

Received for publication December 18, 1998[14C]-CER (13.9 mCi/mmol) and a polyclonal antibodyand in revised form May 17, 1999

Accepted for publication August 3, 1999 against rOAT1 were kind gifts from Sankyo Pharmaceu-tical Co (Tokyo, Japan). Fetal bovine serum (FBS), tryp- 1999 by the International Society of Nephrology

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Takeda et al: OAT1 in cephaloridine nephrotoxicity 2129

sin and geneticin were obtained from Gibco/BRL (Gai- [14C]-PAH and [14C]-CER uptake and effluxthersburg, MD, USA), transferrin, para-aminohippuric Cellular uptake of [14C]-PAH, a prototype organicacid (PAH), probenecid, probucol, thiobarbituric acid anion, and that of [14C]-CER were measured as pre-(TBA) and various cephalosporin antibiotics from Sigma viously described [15]. S3 rOAT1 and S3 pcDNA 3.1(St. Louis, MO, USA), recombinant epidermal growth (1 3 105 cells) were plated in 24-well dishes and culturedfactor (EGF) from Wakunaga (Hiroshima, Japan), insu- for two days. After removal of the medium, the celllin from Shimizu (Shizuoka, Japan), Celldisk from Sumi- monolayers were washed twice with Dulbecco’s modifiedtomo (Osaka, Japan), RITC 80-7 from Kyokuto Pharma- phosphate-buffered saline (D-PBS) (containing in mm:ceutical Industries (Tokyo, Japan), pcDNA3.1 from 137 NaCl, 3 KCl, 8 NaHPO4, 1 KH2PO4, 1 CaCl2 and 0.5Invitrogen (San Diego, CA, USA), TfXe-50 reagent MgCl2; pH 7.4) supplemented with 5 mm d-glucose, andfrom Promega (Madison, WI, USA), [14C]-PAH (53.1 preincubated for 10 minutes. After the incubation, themCi/mmol) from New England Nuclear (Boston, MA, medium was aspirated immediately, and the cell mono-USA), diaminobenzidine from Dojindo Laboratories layers were washed three times with the medium and(Kumamoto, Japan) and horseradish peroxidase (HRP)- solubilized in 0.5 ml of 0.1 n sodium hydroxide. Theconjugated secondary antibody against rabbit immuno- amount of substrate accumulated within the cells was

then measured by counting the radioactivity.globulin from Dako (Glostrup, Denmark). All otherThe CER efflux study was performed as follows. S3chemicals were of analytical grade and available from

rOAT1 (1.0 3 105 cells) were plated in 24-well dishescommercial sources.and cultured for two days. After removal of the medium,

Establishment of S3 rat organic anion transporter 1 the cell monolayers were washed twice with D-PBS sup-plemented with 5 mm d-glucose, and pre-incubated forThe renal tubular cells used for the study were mouse10 minutes. Thereafter, the monolayers were incubatedS3 cells derived from transgenic mice harboring the tem-with 500 ml of medium containing 50 mm [14C]-CER forperature-sensitive simian virus (SV)40 large T-antigen30 minutes at 378C, washed immediately three times withgene [14]. The S3 cells retained the hydroxylase activitiesmedium, and incubated at 378C with 500 ml of mediumassociated with intact proximal tubules, including alka-in the absence or presence of 1 mm CER. After theline phosphatase, g-glutamyl transferase and leucine-incubation for the indicated periods of time, 50 ml of theaminopeptidase activities, and the levels of these activi-supernatant was collected. The amount of [14C]-CERties were higher than those in LLC-PK1 cells but lowerwithin the supernatant was measured by counting thethan those in freshly isolated S3 segments (unpublishedradioactivity.observation). In addition, we also observed morphologi-

cal features specific to polarized epithelia, such as micro- Immunohistochemical analysis of rOAT1 proteinvilli and tight junctions. S3 cells were cultured in RITC

Light microscopic analysis of the rOAT1 protein was80-7 medium containing 5% FBS, 10 mg/ml transferrin,performed as previously described [9]. The antibody0.08 U/ml insulin and 10 ng/ml recombinant EGF. Theused in this study has previously been shown to detectcells were subcultured in a medium containing 0.05%a specific band that corresponds to the rOAT1 proteintrypsin-EDTA solution (containing in mm: 137 NaCl, 5.4with a molecular weight of approximately 57 kDa. TheKCl, 5.5 glucose, 4 NaHCO3, 0.5 EDTA and 5 Hepes;cells were cultured on the cell disk. After the cells were

pH 7.2) and used at passages 60,100. The full-lengthwashed three times with 50 mm NH4Cl in phosphate-

rOAT1 gene [9] was subcloned into pcDNA3.1. The buffered saline (PBS) (containing in mm: 136 NaCl, 2.7pcDNA3.1-rOAT1 and pcDNA3.1 lacking the insert KCl, 8.2 NaHPO4 · 12H2O and 15 KHPO4; pH 7.4), theywere transfected into S3 cells using the TfXe-50 reagent were fixed with a paraformaldehyde lysine periodateaccording to the manufacturer’s instructions. Forty-eight (PLP) solution (containing 0.01 m NalO4, 0.075 m lysine,hours after the transfection, the cells were subcultured in 0.0375 m phosphate buffer with 2% paraformaldehyde;medium containing 400 mg/ml geneticin. The cell clones pH 6.2) for two hours at 48C. After fixation, the cellswere isolated using a cloning cylinder. Among the cell were washed with PBS, and incubated with 3% H2O2 forclones obtained, the cell line showing the highest level 15 minutes to eliminate endogenous peroxidase activity,of PAH uptake was selected and designated S3 rOAT1. and thereafter with blocking serum for 15 minutes. Sub-Electron microscopic observation revealed only occa- sequently, the cells were incubated with a polyclonalsional tight junctions in S3 rOAT1 (data not shown). antibody against rOAT1 at a dilution of 1:200 or 1:400Consistent with this, the cell monolayer was determined for two hours, rinsed with Tris-buffered saline containingto be leaky based on the results of the study wherein 0.1% Tween 20 (TBST) and again incubated with HRP-we estimated the paracellular secretion from S3 rOAT1 conjugated streptavidine solution for 30 minutes. HRPcultured on permeable support, using D-[3H]-mannitol labeling was detected using a peroxidase substrate solu-

tion, 0.8 mm diaminobenzidine.as the indicator (data not shown).

Takeda et al: OAT1 in cephaloridine nephrotoxicity2130

Electron microscopic analysis of rOAT1 was per- vortexed, followed by centrifugation at 2,000 3 g for 10minutes. The organic layer was removed for malondial-formed as follows. The cells were grown on the celldisk

and fixed with periodate-lysine-paraformaldehyde (PLP) dehyde (MDA) determination. The absorbance of thesamples and standards (1,1,3,3-tetramethoxypropane atsolution (containing 0.01 m NalO4, 0.075 m lysine, 0.0375

m phosphate buffer with 2% paraformaldehyde; pH 6.2) concentrations ranging from 0.01 to 10 nmol) was mea-sured using a spectrophotometer at a wavelength of 535for four hours at 48C. After rinsing with TBST, the cells

were incubated with HRP-conjugated secondary anti- nm, and the amount of MDA extrapolated from thestandard curve.body against rabbit immunoglobulin for one hour, rinsed

and fixed with 1% glutaraldehyde for 30 minutes. AfterStatistical analysisrinsing, the HRP labeling was detected by incubation

with a peroxidase substrate solution containing 0.8 mm The results are presented as means 6 se. Statisticalanalyses were performed using the unpaired Student’sdiaminobenzidine. The cells were postfixed in 2% os-

mium tetroxide in 0.1 m PBS buffer for 30 minutes at t-test. Regression analysis was used to test the correlationbetween variables. Differences were considered signifi-48C and embedded in epoxy resin. Ultrathin sections

were cut on an ultramicrotome and observed under a cant when P , 0.05.transmission EM (Hitachi H-7000, Tokyo, Japan).

RESULTSExperimental designEstablishment of S3 rat organic anion transporter 1S3 rOAT1 and S3 pcDNA 3.1 were cultured for two

days. Thereafter, the cells were exposed to various con- S3 rOAT1 exhibited a dose- and time-dependent in-crease in PAH uptake. Eadie-Hofstee analysis showedcentrations of CER in the absence or presence of probu-

col or probenecid for the indicated periods of time. Pro- that the Michaelis constant (Km) was 43.2 6 3.1 mm(N 5 4), which is similar to that reported in an experi-benecid and probucol were added at the same time as

CER and at 24 hours after CER addition. ment using Xenopus oocytes, 14.3 6 2.9 mm [9]. In addi-tion, we examined the effects of PAH and probenecid,

Cell viability an organic anion transport inhibitor, on [14C]-PAH up-take in S3 rOAT1. Both PAH (data not shown) andCell viability was assessed by the trypan blue exclusion

test as previously described [16]. Briefly, S3 rOAT1 and probenecid (Fig. 1A) inhibited [14C]-PAH uptake in S3rOAT1 in a dose-dependent manner at concentrationsS3 pcDNA 3.1 (1 3 105 cells) were plated in P-60 dishes

and exposed to various agents for the indicated periods between 0.01 mm and 1 mm, suggesting that PAH uptakeis mediated by OAT. For determination of the cellularof time. Then, the cells were detached by trypsinization.

The cell suspensions were washed once with ice-cold PBS localization of rOAT1, immunohistochemical analysis ofS3 rOAT1 was performed using a polyclonal antibodyand resuspended with 10 ml of PBS. Ninety microliters of

4% trypan blue was added to the cell suspension, which against rOAT1, which revealed expression of rOAT1 inthe cytoplasmic membrane as well as in the cytoplasmwas then examined using a hemacytometer by light mi-

croscopy. Cells failing to exclude the dye were consid- of S3 rOAT1 (Fig. 2a). However, no staining was ob-served in S3 pcDNA 3.1 (Fig. 2b). Figure 2c presents aered to be nonviable, and the data are expressed as a

percentage of viable cells. longitudinal section of an S3 rOAT1 cell stained withan antibody against rOAT1. Electron microscopic obser-

Lipid peroxidation vation revealed rOAT1 expression predominantly in thecytoplasmic membrane, and only weakly in the cyto-The amount of lipid peroxidation was measured as pre-

viously described [17, 18]. S3 rOAT1 and S3 pcDNA3.1 plasm. There was no positive staining of the nucleus (Fig.2d). These results suggest that S3 rOAT1 expressed a(2 3 105 cells) were plated in six well dishes and exposed

to various agents. The cells were then scraped off the functional OAT in the cytoplasmic membrane of S3rOAT1, with similar characteristics to that originally de-culture dishes, suspended, and an antioxidant (butylated

hydroxytoluene, BHT) was added to each sample at a scribed [9].final concentration of 0.05 g/100 ml. The supernatants

Cephaloridine uptake and efflux in S3 rOAT1and the cells were transferred into glass tubes and centri-fuged at 1,300 3 g for 20 minutes. After aspirating and The effect of CER on PAH uptake in S3 rOAT1 was

examined. As shown in Figure 1B, CER significantlydiscarding the supernatant, the cell pellets were sus-pended in 1.5 ml of a solution containing 0.18% TBA, inhibited PAH uptake in S3 rOAT1 in a dose-dependent

manner at concentrations between 0.5 mm and 5.0 mm,0.24% SDS and 4.4% acetic acid. Each sample was indi-vidually titrated to pH 3.5, for 50 minutes at 958C and suggesting that CER is also taken up via OAT1. More-

over, CER uptake was directly estimated by using [14C]-then sonicated. After the addition of 1.5 ml of n-butanol/pyridine (15:1), the contents of each tube were vigorously CER. As shown in Figure 3A, S3 rOAT1 exhibited a

Takeda et al: OAT1 in cephaloridine nephrotoxicity 2131

Fig. 1. Effect of probenecid (A) and cepha-loridine (CER) (B) on para-aminohippuricacid (PAH) uptake in S3 rat organic aniontransporter 1 (rOAT1). Each bar representsthe mean 6 se for four experiments. *P ,0.05, **P , 0.01 and ***P , 0.001 vs. control.

time-dependent increase in CER uptake to a greater S3 pcDNA 3.1. We also examined the effects of probene-extent from S3 pcDNA 3.1 (N 5 4, P , 0.01 vs. control). cid and probucol on the amount of lipid peroxidation inIn addition, we measured the CER efflux from S3 rOAT1 S3 rOAT1 treated with 1 mm CER. Neither probenecidpreloaded with [14C]-CER was. As shown in Figure 3B, nor probucol exerted any significant effect on the amount[14C]-CER efflux increased rapidly with time, peaked at of lipid peroxidation in S3 rOAT1 (data not shown). As15 minutes, and the increase was significantly enhanced shown in Figure 6, incubation with either probenecid atin the presence of extracellular unlabeled CER (N 5 4, 0.05 and 0.25 mm or probucol at 0.1 and 1 mm suppressedP , 0.05 vs. control). the increase in the amount of lipid peroxidation in S3

rOAT1 treated with 1 mm CER (N 5 4, P , 0.05 vs.Effect of CER on the viability of S3 rOAT1 and S3 CER), whereas probenecid at 0.01 mm and probucol atpcDNA3.1 in the absence or presence of probenecid 0.01 mm did not (data not shown).or probucol

Effects of various cephalosporin antibiotics on theThe effect of CER on the viability of S3 rOAT1 andPAH uptake and viability of S3 rOAT1S3 pcDNA3.1 was studied. As shown in Figure 4, CER

at concentrations of both 0.5 mm and 1.0 mm induced a The effects of various cephalosporin antibiotics includ-time-dependent decrease in the viability of S3 rOAT1 ing CER, cefadroxil, cefotaxime, cefoperazone, ceftriax-to a greater extent than in that of S3 pcDNA3.1. In one, cephalexin, cephalothin, cefazolin and cefamandoleaddition, we also examined the effects of probenecid and on the PAH uptake and viability of S3 rOAT1 wereprobucol, an antioxidant, on the viability of S3 rOAT1 studied. As shown in Figure 7, the effects of these cepha-exposed to 1 mm CER. Neither probenecid nor probucol losporin antibiotics on PAH uptake were correlated sig-exerted any effect on either PAH uptake or the viability nificantly with the effects of these drugs on cell viabilityof S3 rOAT1 (data not shown). As shown in Figure 5, (N 5 4, r 5 0.626, P , 0.05).both probenecid at 0.05 and 0.25 mm and probucol at0.1 and 1 mm significantly suppressed the decrease in the

DISCUSSIONviability of S3 rOAT1 induced by 1 mm CER (N 5 4,Cephaloridine-induced nephrotoxicity has been stud-P , 0.05 vs. CER), whereas probenecid at 0.01 mm and

ied in whole animals [7], renal slices [19], isolated proxi-probucol at 0.01 mm did not.mal tubules [20], primary cultures of proximal tubular

Effect of CER on the amount of lipid peroxidation in cells [21], and cell lines such as LLC-PK1 [22–24]. How-S3 rOAT1 and S3 pcDNA3.1 in the absence or ever, the specific OATs expressed in these tissues havepresence of probenecid or probucol not been identified, and absence of their expression has

been confirmed in the LLC-PK1 cell line (data notThe effects of CER on the amount of lipid peroxida-shown), where CER may accumulate intracellularly bytion in S3 rOAT1 and S3 pcDNA3.1 were examined. Aspassive diffusion. In this regard, the cell line used in theshown in Figure 6, exposure to CER for 24 hours resultedcurrent study expresses a distinct OAT, thus providingin a dose-dependent increase in the amount of lipid per-

oxidation in S3 rOAT1 to a greater extent than that in a reliable method for analyzing CER-induced nephro-

Takeda et al: OAT1 in cephaloridine nephrotoxicity2132

Fig. 2. Immunocytochemical staining illustratingrOAT1 expression in S3 rOAT1 and S3 pcDNA3.1stained with antibody against rOAT1. (a) S3rOAT1 (3450), (b) S3 pcDNA 3.1 (3450), (c)high magnification of longitudinal section of S3rOAT1 (31000), (d) immunoelectron micrographof S3 rOAT1 (36000).

toxicity. Using this cell line, we found evidence sug- S3 rOAT1 exhibited a time-dependent increase in CERuptake activity. In addition, CER was effluxed from S3gesting that OAT1 mediates CER uptake, and observed

CER-induced lipid peroxidation and cytotoxicity in S3 rOAT1 and the presence of extracellular unlabeled CERenhanced the efflux of CER from S3 rOAT1 (trans-rOAT1. The CER concentrations used for the current

study were similar to the plasma concentrations of CER stimulation). These results indicate that rOAT1 couldact as an exchanger and CER could be transported bidi-in rats injected with CER [25, 26].

Rat OAT1, isolated in our laboratory in 1997 [9], was rectionally via rOAT1. rOAT1 has already been shownto act as a heteroexchanger (dicarboxylate/PAH ex-found to be predominantly expressed in the basolateral

membrane of the S2 segment of the proximal tubule, change) [27] and homoexchanger PAH/PAH; (unpub-lished observation).whereas faint expression was also observed in the brain.

Since OAT1 mediates the transport of various endoge- Although OAT1 was shown to mediate CER uptakeand CER-induced nephrotoxity in S3 rOAT1, we do notnous and exogenous organic anions including PAH, it is

considered to be the classical organic anion transporter rule out the possibility that OATs other than OAT1 maybe involved in the uptake of CER and CER-inducedresponsible for the basolateral uptake of organic anions

in renal epithelial cells. nephrotoxicity observed in the S2 segment of the proxi-

Takeda et al: OAT1 in cephaloridine nephrotoxicity 2133

the proximal tubule [28, 29]. Since the current studyfocused on the contribution of basolateral OAT to CERuptake and CER-induced nephrotoxicity, the determina-tion of whether OAT-P1 and OAT-K1 mediate CERtransport and CER-induced nephrotoxicity by establish-ing and using cell lines expressing these OATs is beyondthe scope of the study. Moreover, there are several linesof evidence indicating that the cellular uptake of CERresulting from secretory transport plays a primary patho-genic role in CER-induced nephrotoxicity [30]. In addi-tion, we have also confirmed that CER exhibited noinhibitory effect on organic cation transport in an S3 cellline stably expressing rat organic cation transporter 1[31], which is now thought to be a major basolateralorganic cation transporter (data not shown).

Since probenecid treatment has been shown to resultin a decrease in intracellular accumulation of CER andan attenuation of its nephrotoxicity both in vivo andin vitro [32, 33], renal accumulation of CER has beenidentified as a critical factor for inducing CER-inducednephrotoxicity. In the current study, probenecid alsosuppressed accumulation and cytotoxicity of CER inCER-treated S3 rOAT1, confirming the importance ofaccumulation of CER in CER-induced nephrotoxicity.Probenecid significantly, but not completely, inhibitedCER uptake after 45 minutes incubation (data notshown). Thus, this incomplete inhibition of CER uptakeby probenecid may lead to cellular accumulation of CERand partial inhibition of cytotoxicity (Fig. 5).

Probenecid significantly inhibited an increase in lipidperoxidation in S3 rOAT1, suggesting that the CER ac-cumulated within the cells may induce an increase inlipid peroxidation. In addition, since probucol blockedcytotoxicity and the increase in lipid peroxidation in S3rOAT1 treated with 1 mm CER, the CER-induced in-crease in lipid peroxidation may play a role in CER-induced cytotoxicity observed in S3 rOAT1. These re-sults are consistent with previous reports that lipid perox-idation is involved in CER-induced nephrotoxicity both

Fig. 3. Cephaloridine (CER) uptake and efflux. (A) Uptake studies in in vivo and in vitro [5, 20, 22, 25, 34, 35].S3 rOAT1 (h) and S3 pcDNA 3.1 (j). (B) CER efflux from S3 rOAT1

The significant correlation observed between the ef-preincubated with [14C]-CER. After 30 minutes incubation at 378C with50 mM [14C]-CER, the monolayers were incubated at 378C in the absence fects of various cephalosporin antibiotics on PAH uptake(h) or presence (j) of 1 mm CER. The amount of [14C]-CER within and cell viability suggests that renal accumulation ofthe supernatant was determined. Each bar represents the mean 6 se

these drugs via OAT1 plays an important role not onlyfor four experiments. *P , 0.05 vs. control and **P , 0.01 vs. control.in CER-induced nephrotoxicity, but also in the nephro-toxicity induced by other, less nephrotoxic cephalosporinantibiotics. However, in the current study, the rank order

mal tubule. In addition, CER-induced proximal tubular of the effects of these drugs on the viability of S3 rOAT1damage has been shown to be not only restricted to S2, was different from that reported previously in vivo [23,but also involves S3 of the proximal tubule [1], whereas 36], and CER was not the most cytotoxic of the drugsthe OATs responsible for CER uptake and CER-in- investigated. In vivo, renal accumulation of cephalospo-duced damage in the S3 segment of the proximal tubule rin antibiotics is determined by basolateral uptake ofremain unknown. In this regard, the other OATs identi- these drugs by an organic anion transporter in additionfied thus far, such as OAT-P1 and OAT-K1, have been to apical efflux of these drugs by an organic cation trans-

porter [4, 37]. Furthermore, unlike other less toxic cepha-shown to be localized at the brush border membrane of

Takeda et al: OAT1 in cephaloridine nephrotoxicity2134

Fig. 4. Effect of cephaloridine (CER) on theviability of S3 rOAT1 (j) and S3 pcDNA3.1(h). The cells were exposed to 0.5 mm (A) or1.0 mm (B) CER for 6,72 hours. Each barrepresents the mean 6 se for four experi-ments. *P , 0.05 and **P , 0.01 vs. S3 pcDNA3.1.

Fig. 5. Effect of probenecid and probucol on the viability of S3 rOAT1exposed to 1 mM cephaloridine (CER) for 48 hours. Each bar represents Fig. 6. Effect of cephaloridine (CER) on the amount of lipid peroxida-the mean 6 se for four experiments. *P , 0.01 vs. control and **P , tion in S3 rOAT1 and S3 pcDNA3.1 in the absence or presence of0.05 vs. CER. probenecid or probucol. The cells were exposed to various concentra-

tions of CER for 24 hours in the absence or presence of probenecid orprobucol. Each bar represents the mean 6 se for five experiments.*P , 0.05 vs. control, **P , 0.01 vs. control and ***P , 0.05 vs. CER.

losporin antibiotics, CER has been shown not to movereadily across the luminal membrane, which results inthe accumulation of high concentrations of CER in and clinical use, and the investigation of interactions betweenselective damage to proximal tubular cells [3, 4]. Thus, cephalosporin antibiotics and other drugs.we suggest that the discrepancy between the results of In conclusion, the current results suggest that OAT1the current study and those reported previously in vivo is, at least in part, responsible for the cellular uptake of[23, 36] may be due to the absence of an intact apical CER in the basolateral membrane and CER-inducedefflux system for these drugs in S3 rOAT1. nephrotoxicity.

In addition to the proximal tubule cell line stably ex-pressing OAT1, we also established an S3 cell line stably ACKNOWLEDGMENTSexpressing human OAT1 (S3 hOAT1) [38]. S3 hOAT1 This study was supported in part by Grants-in-Aid from the Ministrycould be used not only as a tool for elucidation of the of Education, Science, Sports and Culture (07557317 and 08671297),

a Program Project Grant from the Ministry of Health and Welfaremechanisms of CER-induced nephrotoxicity in a similarof Japan, and grants from the Suzuken Memorial Foundation, the

way to S3 rOAT1, but also as an effective screening Foundation of Life Science Research, and the Science Research Pro-motion Fund of the Japan Private School Promotion Foundation. Partsystem of newly developed cephalosporin antibiotics for

Takeda et al: OAT1 in cephaloridine nephrotoxicity 2135

transporter 2 expressed predominantly in the liver. FEBS Lett429:179–182, 1998

11. Jacquemin E, Hagenbuch B, Stieger B, Wolkoff AW, Meier PJ:Expression cloning of a rat liver Na1-independent organic aniontransporter. Proc Natl Acad Sci USA 91:133–137, 1994

12. Noe B, Hagenbuch B, Stieger B, Meier PJ: Isolation of a multispe-cific organic anion and cardiac glycoside transporter from rat brain.Proc Natl Acad Sci USA 94:10346–10350, 1997

13. Saito H, Masuda S, Inui K: Cloning and functional characteriza-tion of a novel rat organic anion transporter mediating basolateraluptake of methotrexate in the kidney. J Biol Chem 271:20719–20275, 1996

14. Hosoyamada M, Obinata M, Suzuki M, Endou H: Cisplatin-induced toxicity in immortalized renal cell lines established fromtransgenic mice harboring temperature sensitive SV40 large T-antigen gene. Arch Toxicol 70:284–292, 1996

15. Takano M, Kato M, Takayama A, Yasuhara M, Inui K, HoriR: Transport of procainamide in a kidney epithelial cell line LLC-PK1 Biochem Biophys Acta 1108:133–139, 1992

16. Walker PD, Shah SV: Hydrogen peroxide cytotoxicity in LLC-PK1 cells: A role for iron. Kidney Int 40:891–898, 1991

17. Ohkawa H, Ohoshi N, Yagi K: Assay for lipid peroxides in animaltissues by thiobarbituric acid reaction. J Biol Chem 262:1098–1104,1987

18. Sheridan AM, Fitzpatrick S, Wang C, Wheeler DC, LieberthalFig. 7. Relationship between the effects of various cephalosporin anti-W: Lipid peroxidation contributes to hydrogen peroxide inducedbiotics on PAH uptake by and viability of S3 rOAT1. Each bar repre-cytotoxicity in renal epithelial cells. Kidney Int 49:88–93, 1996sents the mean 6 se for four experiments. Symbols are: (1) CER,

19. Goldstein BS, Pasino DA, Hewitt WR, Hook JB: Biochemical(2) cefadroxil, (3) cefotaxime, (4) cefamandole, (5) cefoperazone, (6)mechanisms of cephaloridine nephrotoxicity: Time and concentra-ceftriaxone, (7) cephalexin, (8) cephalothin, and (9) cefazolin (N 5 4;tion dependence of peroxidative injury. Toxicol Appl Pharmacolr 5 0.626; P , 0.05).83:261–270, 1986

20. Rush GF, Ponsler GD: Cephaloridine-induced biochemicalchanges and cytotoxicity in suspensions of rabbit isolated proximaltubules. Toxicol Appl Pharmacol 109:314–326, 1991

of this work was presented at the annual meeting of the American 21. Bolon C, Gauthier C, Simonnet H, Baverel G: Advantages ofSociety of Nephrology in 1998. a two-chamber culture system to test drug nephrotoxicity: The

example of cephaloridine. Kidney Blood Press Res 20:264–270,1997.Reprint requests to Hitoshi Endou, M.D., PhD., Department of Phar-

22. Kawai Y, Gemba M: Amelioration by cAMP of cephaloridine-macology and Toxicology, Kyorin University School of Medicine,induced injury in the porcine kidney cell line LLC-PK1. Jpn J6-20-2, Shinkawa, Mitaka-shi, Tokyo 181, Japan.Pharmacol 72:67–70, 1996E-mail: endouh@Kyorin-u.ac.jp.

23. Williams PD, Laska DA, Tay LK, Hottendorf GH: Comparativetoxicities of cephalosporin antibiotics in a rabbit kidney cell line

REFERENCES (LLC-RK1). Antimicrob Agents Chemother 32:314–318, 198824. Steinmassl D, Pfaller W, Gstraunthaler G, Hoffmann W:

1. Atkinson RM, Currie JP, Davis B, Pratt DAH, Sharpe HM, LLC-PK1 epithelia as a model for in vitro assessment of proximalTomich EG: Acute toxicity of cephaloridine, an antibiotic derived tubular nephrotoxicity. In Vitro Cell Dev Biol 31:94–106, 1995from cephalosporin C. Toxicol Appl Pharmacol 8:398–406, 1966 25. Rush GF, Heim A, Ponsler D, Engelhardt J: Cephaloridine-

2. Foord RD: Cephaloridine, cephalothin and the kidney. J Antimi- induced renal pathological and biochemical changes in female rab-crob Chemother (Suppl 1): 119–133, 1975 bits and isolated proximal tubules in suspension. Toxicol Pathol

3. Tune BM, Fernholt M: Relationship between cephaloridine and 20:155–168, 1992p-aminohippurate transport in the kidney. Am J Physiol 225:1114– 26. Wold JS: Cephalosporin nephrotoxicity, in Toxicology of the Kid-1117, 1973 ney, edited by Hook JB, New York, Raven Press, 1988, pp 251–266

4. Wold JS, Turnispeed SA, Miller BL: The effect of renal cation 27. Sekine T, Cha SH, Hosoyamada M, Kanai Y, Watanabe N, Fu-transport on cephaloridine nephrotoxicity. Toxicol Appl Pharma- ruta Y, Fukuda K, Igarashi T, Endou H: Cloning, functionalcol 45:115–122, 1979 characterization, and localization of a rat renal Na1-dicarboxylate

5. Kuo CH, Hook JB: Depletion of renal GSH content and nephro- transporter. Am J Physiol 275:F298–F305, 1998toxicity of cephaloridine in rabbits, rats and mice. Toxicol Appl 28. Bergwerk AJ, Shi X, Ford AC, Kanai N, Jacquemin E, BurkPharmacol 63:292–302, 1982 RD, Bai S, Novikoff PM, Stieger B, Meier PJ, Schuster VL,

6. Tune M, Hsu CY: Toxicity of cephaloridine to carnitine transport Wolkoff AW: Immunologic distribution of an organic anion trans-and fatty acid metabolism in rabbit renal cortical mitochondria: port protein in rat liver and kidney. Am J Physiol 271:G231–G238,Structure-activity relationship. J Pharmacol Exp Ther 270:873–880, 19961994 29. Masuda S, Saito H, Nonoguchi H, Tomita K, Inui K: mRNA

7. Nagashima K, Kusaka H, Sato K, Karasawa A: Effects of KW- distribution and membrane localization of the OAT-K1 organic3902, a novel adenosine A1-receptor antagonist, on cephaloridine- anion transporter ion rat renal tubules. FEBS Lett 407:127–131,induced acute renal failure in rats. Jpn J Pharmacol 64:9–17, 1994 1997

8. Pritchard JB, Miller DS: Mechanisms mediating renal secretion 30. Tune BM: Nephrotoxicity of beta-lactam antibiotics: mechanismsof organic anions and cations. Physiol Rev 73:765–796, 1993 and strategies for prevention. Pediatr Nephrol 11:768–772, 1997

9. Sekine T, Watanabe N, Hosoyamada M, Kanai Y, Endou H: 31. Grundemann D, Gorboulev V, Gambaryan S, Veyhl M, Koep-Expression cloning and characterization of a novel multispecific sell H: Drug excretion mediated by a new prototype of polyspecificorganic anion transporter. J Biol Chem 272:18526–18529, 1997 transporter. Nature 372:549–552, 1994

10. Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, 32. Tune BM, Wu KY, Kempson RL: Inhibition of transport andprevention of toxicity of cephaloridine in the kidney. Dose-respon-Kanai Y, Endou H: Identification of multispecific organic anion

Takeda et al: OAT1 in cephaloridine nephrotoxicity2136

siveness of the rabbit and guinea pig to probenecid. J Pharmacol 36. Tune BM: Renal tubular transport and nephrotoxicity of betaExp Ther 202:466–471, 1977 lactam antibiotics: Structure-activity relationships. Miner Electro-

33. Cojocel C, Laeschke KH, Inselmann G, Baumann K: Inhibition lyte Metab 20:221–231, 1994of cephaloridine-induced lipid peroxidation. Toxicology 35:295– 37. Inui K, Takano M, Okano T, Hori R: H1 gradient-dependent305, 1985 transport of aminocephalosporins in rat renal brush border mem-

34. Suzuki Y, Sudo J: Changes in lipid peroxidation and activities of brane vesicles: Role of H1/organic cation antiport system. J Phar-xanthine oxidase, superoxide dismutase and catalyse in kidneys ofmacol Exp Ther 233:181–185, 1985cephaloridine-administered rats. Jpn J Pharmacol 49:43–51, 1989

38. Hosoyamada M, Sekine T, Kanai Y, Endou H: Molecular cloning35. Suzuki Y, Sudo J: Lipid peroxidation and generations of oxygenand functional characterization of a novel multispecific organicradicals induced by cephaloridine in renal cortical microsomes of

rats. Jpn J Pharmacol 52:233–243, 1990 transporter from human kidney. Am J Physiol 276:F122–F128, 1999