ARTICLES J Am Soc Nephrol 11: 383–393, 2000

Cytotoxicity of Antiviral Nucleotides Adefovir and Cidofovir Is Induced by the Expression of Human Renal Organic Anion Transporter 1

EDMUND S. HO, DEBORAH C. LIN, DIRK B. MENDEL, and TOMAS CIHLAR Gilead Sciences, Foster City, California.

␮ ϭ 6 ⅐ Abstract. The transport of organic anions in proximal convo- 58.0 M, Vmax 103 pmol/10 cells min) such that the levels luted tubules plays an essential role in the active secretion of a of intracellular metabolites of both nucleotides were Ͼ100-fold variety of small molecules by the kidney. In addition to other higher in CHOhOAT cells than in parental CHO. Consequently, anionic substrates, the human renal organic anion transporter 1 adefovir and cidofovir were approximately 500-fold and 400- (hOAT1) is capable of transporting the nucleotide analogs fold more cytotoxic, respectively, in CHOhOAT cells compared adefovir and cidofovir. To investigate the involvement of to CHO. The cytotoxicity of both drugs in CHOhOAT cells was hOAT1 in the mechanism of nephrotoxicity associated with markedly reduced in the presence of hOAT1 inhibitors. The these two clinically important antiviral agents, Chinese ham- cyclic prodrug of cidofovir, which exhibits reduced in vivo ster ovary (CHO) cells were stably transfected with hOAT1 nephrotoxicity, was a poor substrate for hOAT1 and showed cDNA. The resulting CHOhOAT cells showed probenecid-sen- only marginally increased cytotoxicity in CHOhOAT cells. In ϭ sitive and pH-dependent uptake of p-aminohippurate (Km conclusion, these studies demonstrate that hOAT1 plays a ␮ ϭ 6 ⅐ 15.4 M, Vmax 20.6 pmol/10 cells min), a prototypical critical role in the organ-specific toxicity of adefovir and organic anion substrate. In addition, the stably expressed cidofovir, and indicates that CHOhOAT cells may represent a ϭ hOAT1 mediated efficient transport of adefovir (Km 23.8 useful in vitro model to investigate the potential nephrotoxicity ␮ ϭ 6 ⅐ ϭ M, Vmax 46.0 pmol/10 cells min) and cidofovir (Km of clinically relevant organic anion agents.

Active renal secretion of a wide variety of small molecules including rat (rROAT1, OAT1) (3,4), mouse (mOAT) (5), and occurs through the cooperative function of specific transport winter flounder (fROAT) (6). In all cases, these transporters proteins located in the basolateral and apical membranes of the mediated uptake of p-aminohippuric acid (PAH), a prototypi- tubular epithelium (1). The renal organic anion transporter, cal organic anion substrate, and they functioned as organic which has been identified in many diverse animal species, is a anion/dicarboxylate exchangers. Initial studies with OAT1 component of a specific secretory pathway of organic anions. have demonstrated its broad substrate specificity with the ca- This basolateral transport system mediates the active uptake of pability of transporting urate, methotrexate, prostaglandins, a wide variety of endogenous metabolites, toxins, xenobiotics, and cyclic nucleotides (3). More recently, OAT1 has been therapeutics, and other negatively charged molecules from the reported to mediate uptake of nonsteroidal anti-inflammatory systemic circulation into the proximal tubular epithelium (2). drugs such as salicylate, acetylsalicylate, and salicylurate (7). Once accumulated within the convoluted tubule, the anionic Consistent with the proposed function of OAT1, immunohis- substances are subsequently secreted into the tubular lumen via tochemical analysis has demonstrated that it is specifically less well characterized membrane carrier(s) or channel(s) (2). localized to the basolateral membrane of S2 segments of the Although the enormous physiologic importance of renal secre- proximal convoluted tubules (8). tion of organic anions was recognized years ago, significant progress toward the molecular characterization of membrane Recently, we (9) and others (10,11) have independently proteins involved in this process has only recently occurred reported the cloning and expression of the human renal organic with the cloning, expression, and functional characterization of anion transporter 1 (hOAT1 or hPAHT). Similar to the organic renal organic anion transporters from several animal species anion transporters from other species, hOAT1 functions as a PAH/dicarboxylate exchanger when transiently expressed in Xenopus laevis oocytes or HeLa cells. Despite one study sug- Received July 12, 1999. Accepted August 18, 1999. gesting a much narrower substrate specificity of hOAT1 com- Correspondence to Dr. Tomas Cihlar, Gilead Sciences, 333 Lakeside Drive, pared to rROAT1 (11), we have shown that hOAT1 efficiently Foster City, CA 94404. Phone: 650-522-5637; Fax: 650-522-5890; E-mail: transports various acyclic nucleotide analogs including adefo- [email protected] vir [9-(2-phosphonylmethoxyethyl)adenine] and cidofovir 1046-6673/1103-0383 Journal of the American Society of Nephrology [(S)-1-(3-hydroxy-2-phophonylmethoxypropyl)cytosine], two Copyright © 2000 by the American Society of Nephrology clinically important antiviral therapeutics (9). Interestingly, 384 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 383–393, 2000 hOAT1 showed markedly higher affinity toward this type of sites (in bold), respectively. Using the sense primer, a truncated 5Ј- molecule than did rROAT1 (9). untranslated sequence (5Ј-UTR) of alfalfa mosaic virus (19) (underlined) and Both adefovir and cidofovir are nucleoside phosphonate a favorable Kozak consensus (20) (in italic) were introduced upstream from Ј analogs, a class of novel antivirals structurally related to nat- the 5 end of the hOAT1 open reading frame to optimize initiation of ural nucleotides (Figure 1). Cidofovir has been approved for translation. Plasmid pIRES-hOAT was generated by the cloning of the EcoRI/BamHI-digested PCR product into a pIRESneo expression vector the treatment of cytomegalovirus retinitis in AIDS patients (Clontech, Palo Alto, CA). Upon completion of the cloning, the correct (12), and adefovir dipivoxil, an orally available prodrug of nucleotide sequence of the entire fragment generated by PCR was verified. adefovir, is currently undergoing clinical evaluation as an For the transfection, CHO cells were seeded into a 100-mm Petri dish (6 ϫ anti-HIV and anti-hepatitis B virus agent (13). Both drugs are 106 cells). After 24 h, medium was aspirated and 6 ml of fresh growth actively secreted by the kidney (14,15), presumably via medium containing 12 ␮g of pIRES-hOAT and 60 ␮g of Cytofectin GSV hOAT1, and for both of them, the main dose-limiting toxicity (Glen Research, Sterling, VA) was added to the cells followed by an addi- is nephrotoxicity (16,17). To reduce this adverse event, cido- tional 6 ml of medium 4 h later. After an overnight incubation, stably fovir is used in conjunction with probenecid, an inhibitor of transfected cells were selected in phenol red-free growth medium supple- organic anion transport (12). The organic anion transport sys- mented with 1 mg/ml G418 (Clontech). Growing colonies were isolated and tem has also been implicated in the nephrotoxicity of other tested for PAH uptake in the presence and absence of 1 mM probenecid as agents, for example, cephaloridine and cephaloglycin, two described below. A clone showing the highest probenecid-sensitive accumulation of PAH was designated CHOhOAT. Under the same conditions, CHO cephalosporin antibiotics (18), suggesting that hOAT1 may cells were also transfected with the empty pIRESneo vector. The pool of cells also be directly involved in the induction of nephrotoxicity harvested after G418 selection (CHOpIRES) was used as the control for initial associated with adefovir and cidofovir. experiments. In the cytotoxicity experiments, V-79 cells (ATCC CCL-93) In an attempt to understand the involvement of hOAT1 in stably transfected with hOAT1 cDNA were used in addition to CHOhOAT drug-induced nephrotoxicity, we studied the effect of hOAT1 cells. The transfection and cultivation of V-79 cells were carried out under the expression on the transport, intracellular metabolism, and cy- same conditions as defined for the CHO cells. totoxicity of the two antiviral nucleotide analogs. Using a cell line stably expressing hOAT1, we have been able to demon- Northern Blot Analysis strate a direct role of hOAT1 in the etiology of organ-specific Total RNA was extracted from CHO and CHOhOAT cells using toxicity associated with adefovir and cidofovir. Trizol Reagent (Life Technologies, Rockville, MD). After separation on 1.2% agarose gel, RNA was transferred onto a Hybond-N mem- Materials and Methods brane (Amersham Pharmacia Biotech, Piscataway, NJ) and hybridized Stable Expression of hOAT1 in Chinese Hamster Ovary with hOAT1-specific 32P-labeled probe (9) for1hat68°C in Ex- Cells pressHyb hybridization buffer (Clontech). Subsequently, the membrane was washed twice in 2ϫ SSC with 0.05% sodium dodecyl Chinese hamster ovary (CHO) cells (American Type Culture Collection sulfate for 30 min at room temperature followed by a single wash in [ATCC] CCL61, Manassas, VA) were grown in F-12 medium supplemented 0.1ϫ SSC with 0.1% sodium dodecyl sulfate at 50°C. After autora- with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ␮g/ml strepto- diography, the membrane was stripped and reprobed with a ␤ mycin. Plasmid pIRES-hOAT used for stable transfection of hOAT1 cDNA -actin into CHO cells was constructed as follows. The hOAT1 coding sequence control probe (Clontech) under the same conditions. was amplified by PCR from plasmid pOAT-8 (9) under standard conditions using Expand High Fidelity PCR system (Boehringer Mannheim, Transport Assays Indianapolis, IN). Oligonucleotides 5Ј-ACCGTCTAGAATTC- The assays were carried out in 12-well plates with nearly confluent TTTTTATTTTTAATTTTCTTTCAAATACGTCCACCATGGCCTTTA- cells seeded 48 h before each experiment. On the day of the experi- ATGACCTCCTGCAGCAGG-3Ј and 5Ј-TACTCACGTGGATCCT- ment, growth medium was aspirated and the cells were washed twice GATCAGACGTCTGTAGGACCTTCCCTCCCTTTAGG-3Ј were used with phosphate-buffered saline (PBS). The uptake of radiolabeled as a sense and antisense primer to introduce EcoRI and BamHI restriction substrates was determined at 37°C in Waymouth buffer (135 mM

Figure 1. Structure of adefovir, cidofovir, and cyclic prodrug of cidofovir. J Am Soc Nephrol 11: 383–393, 2000 Induction of Drug Cytotoxicity by hOAT1 385

14 NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 0.8 mM MgSO4, thoxypropyl)cytosine (cidofovir; 56 mCi/mmol), and [ C] [((S)-1 to 28 mM glucose, and 13 mM Hepes, pH 7.2). At the end of incubation, 2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methyl]cytosine (cy- the cells were washed 3 times with ice-cold PBS (2 ml/well) and lysed clic prodrug of cidofovir; 55 mCi/mmol) were purchased from directly on the plate by adding 0.3% Triton X-100 (0.5 ml/well) for 15 Moravek Biochemicals (Brea, CA). Nonradioactive adefovir, cidofo- min. Subsequently, the wells were washed with an additional 0.5 ml vir, and cyclic prodrug of cidofovir were synthesized at Gilead Sci- of the detergent, the lysate and wash were combined, and the radio- ences according to previously published procedures (24). Nonradio- activity in each sample was determined after addition of scintillation active PAH, probenecid, N-benzoyl-␤-alanine (betamipron), and fluid (Beckman Instruments, Fullerton, CA). In parallel plates, the glutarate were obtained from Sigma. number of cells was determined and the substrate uptake was expressed as pmol/106 cells. For determination of transport kinetics, the assays were performed at various substrate concentrations, and the Results kinetic constants were estimated by the linear regression from double Stable Expression of hOAT1 in CHO Cells reciprocal plots using Enzyme Kinetics software (ChemSW, Fairfield, Initially, appropriate cells for the expression and study of CA). Inhibition experiments were carried out in the presence of hOAT1 were selected. From a number of established cell lines various inhibitor concentrations at the substrate concentration equal to suitable for transfection, CHO cells showed the lowest uptake

its Km. Fifty percent inhibitory concentration (IC50) values were of both PAH and adefovir. The uptake of both compounds was estimated from semilogarithmic plots of inhibitor concentration ver- not saturable and was insensitive to probenecid, indicating the sus percentage of uptake relative to uninhibited control. absence of a carrier-mediated transport system that could po- tentially interfere with the determination of hOAT1-specific Intracellular Metabolism transport. Confluent CHO and CHOhOAT cells in 6-well plates were incu- After transfection with the pIRES-hOAT plasmid, a number bated in phenol red-free growth medium containing 5 ␮M[3H]ade- of G418-resistant CHO clones were isolated and assayed for 14 fovir or 10 ␮M[ C]cidofovir. After 12 h at 37°C, the cells were PAH uptake in the presence and absence of probenecid. A washed 3 times with 3 ml of ice-cold PBS and extracted with 0.5 ml transfectant of CHO cells capable of accumulating PAH to a 5% TCA as described previously (21). The number of cells was level more than 30-fold higher than that determined in CHO determined in parallel samples incubated with unlabeled drugs under cells was identified and designated CHOhOAT. Northern anal- identical conditions. The extracts were analyzed using a Separon SGX C18 HPLC column (Melcor Technologies, Sunnyvale, CA) with a ysis with a hOAT1-specific DNA probe detected a 5-kb bicys- linear gradient of acetonitrile (0 to 20% over 40 min at 1 ml/min) in tronic transcript (containing both the hOAT1 and G418-resis- hOAT the presence of 50 mM potassium phosphate, pH 7.0, and 3 mM tance marker sequences) in the RNA extract from CHO , t-butylammonium hydrogen sulfate. One-milliliter fractions were col- but not CHO cells, indicating the specific expression of lected, and the amount of 3H- or 14C-radioactivity was determined hOAT1 in the stably transfected cells (Figure 2A). The uptake after addition of 6 ml of scintillation fluid (Beckman Instruments). of PAH into CHO cells stably transfected with the empty The identity of each radioactive metabolite was confirmed by com- pIRESneo vector (CHOpIRES) was similar to that determined parison of its retention time with that of a corresponding unlabeled for parental CHO cells (Figure 2B). Preloading CHOhOAT cells standard, and by HPLC analysis of extracts treated with alkaline with 5 mM glutarate, a counterion accepted by the renal phosphatase or phosphodiesterase I as described previously (22). organic anion transporter (1), trans-stimulated PAH uptake; thus, the stably expressed hOAT1 functions as a PAH/dicar- Drug Cytotoxicity Assays boxylate exchanger (Figure 2B). hOAT CHO and CHO cells were seeded in parallel into 96-well Inhibition experiments (Figure 2C) showed that probenecid ϫ 3 plates at a density of 3 10 cells/well. After 24 h, various concen- efficiently blocks the uptake of PAH by CHOhOAT cells with trations of the tested drugs were added in triplicate, and the cells were Ϯ ␮ ϭ an IC50 value of 6.5 1.0 M(n 3). Betamipron (N- incubated for an additional 120 h. At the end of the incubation, cell ␤ viability was determined by a modified colorimetric assay using benzoyl- -alanine) also inhibited hOAT1-mediated PAH up- 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; take, but was less potent than probenecid with an IC50 of Ϯ ␮ ϭ Sigma, St. Louis, MO) (23). Briefly, medium with the drug was 16.2 2.9 M(n 3). removed and replaced by fresh medium (200 ␮l/well) containing 0.1 mg/ml MTT. After a 90-min incubation at 37°C, the medium was High Efficiency of Adefovir and Cidofovir Transport ␮ aspirated and the cells were extracted with 150 l/well of DMSO. The Mediated by hOAT1 concentration of the extracted formazan metabolite was determined by In addition to PAH, CHOhOAT cells exhibited efficient in- measurement of the absorbance at 560 nm (A ) in a 96-well plate 560 tracellular accumulation of adefovir and cidofovir. As shown reader. Average A560 values were calculated from triplicates after subtraction of the blank values, and the 50% cytotoxic concentration in Figure 3A, hOAT1-mediated uptake of adefovir was linear of each drug (CC ) was determined from a semilogarithmic plot of during the initial 20 min. After 60 min, adefovir accumulated 50 hOAT drug concentration versus percentage of A560 relative to untreated in CHO cells to a level almost 80-fold higher than that control. detected in parental CHO cells under the same conditions. Uptake of cidofovir into CHOhOAT cells showed similar char- Materials acteristics (data not shown). Efficiency of hOAT1-mediated [3H]PAH (3.7 Ci/mmol) was obtained from Dupont New England uptake of nucleotide analogs and PAH was compared in trans- Nuclear (Boston, MA). [3H]9-(2-phosphonylmethoxyethyl)adenine port kinetic experiments. To approximate the initial rate of (adefovir; 30 Ci/mmol), [14C](S)-1-(3-hydroxy-2-phosphonylme- uptake, the experiments were carried out within 3 min. Under 386 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 383–393, 2000

␮ these conditions, Km values of 15.4, 23.8, and 58.0 M were determined for PAH, adefovir, and cidofovir, respectively, indicating a slightly lower affinity of hOAT1 for nucleotide

analogs compared to PAH (Table 1). In contrast, the Vmax values for the uptake of the nucleotide analogs were higher than that for PAH, resulting in comparable efficiencies (i.e.,

Vmax/Km ratio) of hOAT1-mediated uptake of all substrates. Interestingly, the cyclic prodrug of cidofovir (Figure 1) was also a substrate for hOAT1, even though one of the negative charges in the molecule is blocked by an intramolecular ester bond. However, compared with cidofovir, hOAT1-mediated

uptake of the cyclic prodrug showed a significantly higher Km and lower Vmax, resulting in an almost 15-fold decrease in the transport efficiency for this molecule (Table 1). Similar to what was observed with PAH, the hOAT1-mediated uptake of adefovir was trans-stimulated by preloading CHOhOAT cells with glutarate. Optimization of the preloading conditions showed maximal stimulation of adefovir uptake after a 2-h preincubation of CHOhOAT cells in Waymouth buffer containing 5 mM glutarate. Under these conditions, preloading resulted in an approximately threefold increase in the efficiency of hOAT1-mediated uptake of adefovir (Figure 3B). Based on the observation that hOAT1 is able to transport molecules that differ in terms of their net charge, we studied the pH dependence of hOAT1-mediated uptake of adefovir and PAH by performing the transport assay at pH values ranging from 5 to 9. As shown in Figure 4, PAH uptake was highest at pH 5 to 6. In contrast, adefovir showed highest accumulation in CHOhOAT cells at pH 6.5 to 7.5. At pH Ͼ 8, there was only very limited uptake (Ͻ15% of that at pH optimum) of both PAH and adefovir by CHOhOAT cells.

Metabolism of Adefovir and Cidofovir in CHOhOAT Cells As analogs of nucleoside monophosphates, both adefovir and cidofovir are intracellularly metabolized to the corresponding mono- and diphosphoryl derivatives (22,25,26). In addition, cidofovir forms an adduct with choline, cidofovir-phos-

and reprobed with a 32P-labeled ␤-actin-specific probe (bottom panel). (B) Uptake of [3H] p-aminohippuric acid ([3H]PAH) into CHO, CHOpIRES, and CHOhOAT cells. The cells in 12-well plates were incubated with 5 ␮M substrate for 60 min at 37°C. Where indicated, the cells were preincubated with 5 mM glutarate for 2 h before determination of PAH uptake. The uptake was terminated by washing the cells with ice-cold phosphate-buffered saline (PBS), and the samples were further processed as described in Materials and Meth- ods. The data are means Ϯ SEM from two independent experiments. (C) hOAT1-mediated uptake of PAH is sensitive to inhibitors of Figure 2. Stable expression of human renal organic anion transporter organic anion transport. CHOhOAT cells were incubated with 15 ␮M 1 (hOAT1) in Chinese hamster ovary (CHO) cells. (A) Northern blot [3H]PAH in the presence of various concentrations of probenecid (F) analysis of RNA extracted from CHO cells (lane 1) and CHOhOAT or betamipron (E), and the substrate uptake was determined. The data cells (lane 2). RNA was separated on 1.2% agarose gel, transferred represent a mean of duplicates in a representative experiment. In all onto a membrane, and hybridized with a 32P-labeled hOAT1 DNA cases, standard error was Ͻ5% of the uptake in the absence of fragment (top panel). After film exposure, the membrane was stripped inhibitor. J Am Soc Nephrol 11: 383–393, 2000 Induction of Drug Cytotoxicity by hOAT1 387

may result in the saturation of intracellular metabolism with subsequent changes in the proportions of metabolites. To explore the effect of hOAT1 expression on the intracellular metabolism of the two nucleotide analogs, CHO and CHOhOAT cells were incubated for 12 h with 5 ␮M[3H]adefovir or 10 ␮M[14C]cidofovir followed by analysis of the metabolites. The total intracellular accumulation of adefovir and cidofovir was almost 140-fold and 190-fold higher, respectively, in CHOhOAT cells compared with CHO cells (Table 2). The levels of all adefovir metabolites in CHOhOAT increased proportionally to the total drug accumulation, with adefovir- diphosphate being the most abundant metabolite both in CHOhOAT and CHO cells. In contrast, the most abundant metabolite of cidofovir was cidofovir-phosphocholine. A more than 100-fold increase in the level of each cidofovir and adefovir metabolite upon hOAT1 expression indicated that nucleotide analogs, and presumably also the other substrates taken up via hOAT1, are subjected to the intracellular metabolism similar to that observed in hOAT1-negative cells.

Selective Cytotoxicity of Adefovir and Cidofovir in Cells Expressing hOAT1 Consistent with the limited intracellular accumulation of adefovir and cidofovir, CHO cells exhibited a relatively low susceptibility to the cytotoxic effects of the two drugs. Thus, we investigated whether the enhanced intracellular uptake and metabolism of both nucleotide analogs due to hOAT1 expression would translate into increased susceptibility of CHOhOAT cells toward the two drugs. Both CHO and CHOhOAT cells were incubated for 5 d with various concentrations of adefovir or cidofovir, and the cytotoxic effect was determined by MTT colorimetric assay. As shown in Figure 5A, both nucleotide analogs were markedly more cytotoxic in CHOhOAT cells compared with CHO cells. In the case of adefovir, expression of hOAT1 enhanced its cytotoxicity approximately 500-fold. Similarly, cidofovir showed 400-fold higher cytotoxicity in Figure 3. hOAT1-mediated uptake of adefovir. (A) Time course of CHOhOAT cells compared with CHO cells. In contrast, adefovir intracellular accumulation. Confluent CHO () and CHOhOAT cells were only fourfold more susceptible to the hOAT F ␮ 3 CHO ( ) cells were incubated with 25 M[H]adefovir for 5 to cyclic prodrug of cidofovir, which corresponds with the low 60 min at 37°C. The uptake was terminated by washing the cells with efficiency of hOAT1-mediated uptake of this compound. ice-cold PBS, and the samples were processed as described in Mate- To address whether hOAT1 expression can also induce the rials and Methods. The inset shows uptake of adefovir into CHOhOAT cells during the interval of 0 to 5 min under the same conditions. The cytotoxicity of adefovir and cidofovir in other cell types, data represent a mean of duplicates in a representative experiment. (B) Chinese hamster lung fibroblasts (V-79) were also stably trans- Stimulation of hOAT-mediated uptake of adefovir by glutarate pre- fected with hOAT1 cDNA. A transfectant of V-79 cells exhib- loading. CHOhOAT cells were incubated in Waymouth buffer in the iting Ͼ50-fold enhancement in the uptake of adefovir was absence or presence of 5 mM glutarate for 2 h. After extensive isolated and its susceptibility to nucleotide analogs was com- washing, 3-min uptake of 25 ␮M[3H]adefovir has been determined as pared with parental V-79 cells. Figure 5B shows that hOAT1 described in Materials and Methods. The data are means Ϯ SEM from expression in V-79 cells also caused a marked shift in the two independent experiments. susceptibility to adefovir and cidofovir, but not to the cyclic prodrug of cidofovir. phocholine (22). Although the metabolism of both nucleotide hOAT1-Mediated Cytotoxicity Is Reduced by analogs has been extensively studied in a variety of cell types, Probenecid and Betamipron the mechanism of cellular uptake in the vast majority of these Similar to PAH, the uptake of nucleoside phosphonate ana- models was fluid-phase endocytosis (27–29), and none of the logs by CHOhOAT cells was inhibited in the presence of the investigated cells apparently expressed hOAT1. In addition, hOAT1 inhibitors probenecid and betamipron (Figure 6). Both highly efficient uptake of adefovir and cidofovir via hOAT1 compounds showed similar inhibitory potency with IC50 values 388 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 383–393, 2000

Table 1. Kinetics of hOAT1-mediated transport of PAH and antiviral nucleotide analogsa

␮ Vmax Transport Efficiency Substrate Km ( M) 6 ⅐ (pmol/10 cells min) (Vmax/Km) PAH 15.4 Ϯ 2.5 20.6 Ϯ 3.8 1.34 Adefovir 23.8 Ϯ 4.2 46.0 Ϯ 4.4 1.93 Cidofovir 58.0 Ϯ 5.7 103 Ϯ 10 1.77 Cyclic prodrug of cidofovir 309 Ϯ 106 42.2 Ϯ 10.3 0.137

a Confluent CHOhOAT cells were incubated with labeled substrates at various concentrations. After 3 min at 37°C, the uptake was stopped by washing cells with ice-cold PBS. Cells were lysed, radioactivity in cell lysates was counted, and kinetic constants for each substrate were estimated from a double-reciprocal plot. The data represent mean values Ϯ SD from at least two independent experiments. hOAT1, human renal organic ion transporter 1; PAH, p-aminohippuric acid; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.

to interfere with cellular efflux of various organic anions (30,31). Consequently, in the presence of probenecid, adefovir and cidofovir were only 2.5-fold and 4-fold more cytotoxic, respectively, in CHOhOAT cells than in CHO cells. Betamipron, in accordance with its inhibitory effect on hOAT1-specific transport, also markedly reduced hOAT1-mediated cytotoxicity of both nucleotide analogs (Table 3).

Discussion A recent study utilizing protein expression in Xenopus laevis oocytes has demonstrated that both adefovir and cidofovir are high-affinity substrates for hOAT1 (9). Based on this finding, an active role of hOAT1-mediated uptake in the mechanism of nephrotoxicity associated with these two clinically important antivirals has been suggested (9). However, although the Xe- nopus laevis system is widely used for the study of membrane transport proteins because it offers fast and efficient expression as well as low background in the transport assays, one of its Figure 4. pH dependence of hOAT1-mediated uptake of adefovir and PAH. CHOhOAT cells were incubated with 15 ␮M[3H]PAH (F,Œ)or limitations is the inability to study the effects of the expressed 25 ␮M[3H]adefovir (E,‚) in Waymouth buffer at various pH, and the transport protein on other cellular functions. Therefore, we intracellular substrate accumulation was determined after 3 min. Phos- stably expressed hOAT1 in CHO cells, which have been cho- phate buffer (F,E) and TAPS buffer (Œ,‚) was used at pH 5 to 7 and sen because of their low background accumulation of hOAT1 pH 6.5 to 9, respectively. substrates. To maximize the efficiency of expression, hOAT1 cDNA was cloned downstream from the truncated version of AMV 5Ј-UTR, which has been shown previously to stimulate of 7.0 Ϯ 2.7 ␮M(n ϭ 2) and 6.0 Ϯ 1.3 ␮M(n ϭ 3), in vitro protein expression (19). This approach resulted in 30- respectively, against adefovir, and 5.6 Ϯ 0.3 ␮M(n ϭ 2) and to 100-fold enhancement in the uptake of hOAT1 substrates in 4.5 Ϯ 0.8 ␮M(n ϭ 2), respectively, against cidofovir, sug- stably transfected CHO cells, i.e., similar to that achieved by gesting that they may have a protective effect against hOAT1- the expression of hOAT1 in Xenopus oocytes (9). mediated cytotoxicity of the nucleotide analogs. Therefore, the Kinetic experiments carried out using CHOhOAT cells dem- cytotoxicity of adefovir and cidofovir was determined simul- onstrated that the efficiency (Vmax/Km ratio) of hOAT1-specific taneously in the presence or absence of 1 mM probenecid. At uptake of adefovir and cidofovir is comparable to that of PAH. this concentration, probenecid itself showed no cytotoxicity Consistent with the proposed function of hOAT1 as an organic hOAT either in CHO or CHO cells. Notably, the CC50 values of anion/dicarboxylate exchanger, hOAT1-specific uptake of adefovir in CHOhOAT cells in the presence and absence of PAH and adefovir in CHOhOAT cells was trans-stimulated by probenecid were 16 and 0.33 ␮M, respectively, accounting for glutarate. Further functional characterization of hOAT1 dem- an approximately 50-fold reduction in the drug cytotoxicity onstrated a marked dependence of its transport activity on (Table 3). Similarly, probenecid also decreased the cytotoxic extracellular pH. The uptake of PAH and adefovir showed effect of cidofovir in CHOhOAT cells. In parental CHO cells, a different pH optima, most likely reflecting the differences in slight increase (two- to fourfold) in the cytotoxicity of adefovir the ionization of the two substrates. Comparison of pKa esti- ϭ and cidofovir was observed in the presence of probenecid. This mates for the carboxyl moiety of PAH (pKa 3.2) and the ϭ may be due to the effect of probenecid on efflux of both drugs phosphonate moiety of adefovir (pKa2 7.8) (32) is consistent from CHO cells, since probenecid has been shown previously with the lower pH optimum of PAH uptake and supports J Am Soc Nephrol 11: 383–393, 2000 Induction of Drug Cytotoxicity by hOAT1 389

Table 2. Comparison of intracellular metabolism of adefovir and cidofovir in CHO and CHOhOAT cellsa

Intracellular Level (pmol/106 cells) Metabolite Fold Changeb CHO CHOhOAT

Adefovir treatment total 4.5 620 138 ADV 1.4 (31%)c 155 (25%) 111 ADV-phosphate 0.7 (16%) 130 (21%) 185 ADV-diphosphate 2.4 (53%) 335 (54%) 140

Cidofovir treatment total 10.0 1860 186 CDV 3.7 (37%) 428 (23%) 116 CDV-phosphate 0.5 (5%) 108 (6%) 216 CDV-diphosphate 0.9 (9%) 353 (19%) 392 CDV-phosphocholine 4.9 (49%) 969 (52%) 198

a CHO and CHOhOAT cells were treated for 12 h with 5 ␮M[3H]adefovir (ADV) or 10 ␮M[14C]cidofovir (CDV). Cell extracts were separated by ion-pair HPLC, radioactivity in collected fractions was counted, and the amount of each metabolite was quantified. Cell numbers were determined in separate samples treated with unlabeled drugs under the same conditions. The data represent the average of two independent experiments. b Fold increase in the intracellular level of each metabolite in CHOhOAT cells relative to that in CHO cells incubated under the identical conditions. c Numbers in parentheses represent percent contribution of each metabolite to the total amount of the intracellular drug. previous findings demonstrating a direct relationship between their toxicity may be unique for each compound despite their the pKa values of various organic anions and their interference close structural similarity. In the case of cidofovir, the major with PAH uptake in isolated kidney proximal tubules (33). metabolite in CHOhOAT cells is cidofovir-phosphocholine, an Notably, both the uptake of adefovir and PAH were almost analog of the phospholipid synthesis intermediate cytidine completely suppressed at pH Ͼ8. This effect is opposite to that 5Ј-diphosphocholine. At high intracellular concentrations gen- observed with some of the organic cation transporters. For erated upon hOAT1 expression, this metabolite may possibly example, transport activity of placental organic cation trans- interfere with synthesis or degradation of membrane phospho- porter type 3 is highest at pH Ͼ8, but is almost completely lipids, similar to what has been shown with arabinofuranosyl- abolished at pH Ͻ6 due to the electrogenic character of the cytosine 5Ј-diphosphocholine (35). In contrast, adefovir, which transporter, which makes it strongly sensitive to changes in the is an adenine nucleotide analog, undergoes only mono- and membrane potential induced by a shift to acidic pH (34). diphosphorylation in all cells tested thus far (13). Hence, Because the sharp decline in hOAT1 transport activity at following the accumulation via hOAT1, the drug itself or some higher pH could not be explained only by the suppression of of its metabolites may interfere with ATP synthesis, specific charge-charge interactions between the substrate and hOAT1, a ATP-dependent processes, and/or transport of adenine nucle- further electrophysiologic characterization of hOAT1 is neces- otides in various cellular compartments, e.g., the Golgi (36) or sary to explore its sensitivity to membrane potential and fully mitochondria (37). explain the observed pH dependence. It is important to recognize that in vivo, the hOAT1-medi- The finding that the efficiency of hOAT1-mediated uptake ated cytotoxic effect of adefovir and cidofovir in proximal of adefovir and cidofovir is as high as that of PAH supports the tubules may not be as dramatic as that seen in our cell culture proposed role of hOAT1 in the mechanism of nephrotoxicity model due to the secretion of these two drugs from the tubular associated with the two antiviral nucleotides. However, in this epithelium into the lumen as demonstrated by pharmacokinetic study, we have also obtained direct evidence of the involve- studies (14,15). Recently, the presence of multidrug resistance ment of hOAT1 in the organ-specific toxicity induced by protein 2 (MPR2) in the apical membrane of human proximal adefovir and cidofovir. After comparing the cytotoxic effect of tubules has been demonstrated (38). MRP2 is an ATP-depen- both drugs in parental cells and cells stably expressing hOAT1, dent efflux pump for organic anion substances, and thus may we concluded that hOAT1 expression markedly increases the mediate the apical efflux of adefovir and cidofovir into the sensitivity of cells to the cytotoxic effect of the two antivirals. lumen. For most substrates of the classical organic anion This sensitivity increase appears to be proportional to the transport system, basolateral uptake appears to be the rate- elevation of intracellular metabolites of adefovir and cidofovir limiting step in the entire tubular secretion pathway. However, in cells expressing hOAT1. an accumulation of cidofovir in the superficial kidney cortex, Based on the observed differences in the metabolism of where proximal tubules are located, has been detected previ- adefovir and cidofovir, the intracellular target associated with ously (39), indicating that the luminal efflux is most likely the 390 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 383–393, 2000

Figure 5. Effect of hOAT1 expression on cytotoxicity of adefovir and cidofovir. Parental and hOAT-1-transfected cells in 96-well plates were incubated in parallel for 5 d with various concentrations of tested drugs. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed, and drug concentration inhibiting cell growth by 50% (CC50) was determined. (A) Cytotoxicity in parental CHO cells (E) and in CHOhOAT cells (F). (B) Cytotoxicity in parental V-79 cells (‚) and in V-79hOAT cells (Œ). Data are from representative experiments carried out simultaneously for the parental and hOAT1-transfected cells.

limiting step in the tubular secretion of nucleoside phospho- transporter. Indeed, probenecid-sensitive active transport of PAH nates. Thus, in addition to highly efficient basolateral uptake, and a variety of other organic anions has been detected previously the slower rate of apical secretion may be another factor in the choroid plexus of several mammalian species (40,41). involved in the mechanism of nephrotoxicity associated with However, both cell culture (42) and in vivo experiments (43) cidofovir and adefovir. suggest that the organic anion transport activity in the choroid We demonstrated that the cytotoxicity enhancement upon plexus is localized apically rather than basolaterally and partici- hOAT1 expression is cell type-independent, because a similar pates in the active secretion of anionic drugs from cerebrospinal effect was observed both in the cells of epithelial origin (CHO) fluid into the systemic circulation. The apical membrane localiza- and in fibroblasts (V-79). In vivo, high-level expression of hOAT1 tion of hOAT1 would preclude its active role in organ-specific has been detected specifically in kidney (9,10) corresponding with drug toxicity correlating with the lack of neurotoxicity of adefovir the kidney-specific toxicity of adefovir and cidofovir. However, and cidofovir. Nevertheless, the precise localization and function based on sensitive detection using reverse transcription-PCR, the of hOAT1 in brain remains to be confirmed by molecular and presence of a hOAT1-specific transcript has also been detected in immunohistochemical analysis. skeletal muscles and brain (9). In skeletal muscles, only a trun- Characterization of the effect of hOAT1 expression on up- cated form of hOAT1-specific mRNA has been found, suggesting take and cytotoxicity of the cyclic prodrug of cidofovir pro- the expression of a modified form of hOAT1 that may lack or may vided additional support for the involvement of hOAT1 in the have altered transport activity. This hypothesis correlates with the mechanism of drug-associated nephrotoxicity. The efficiency observed lack of accumulation of cidofovir in skeletal muscles as of hOAT1-mediated uptake of the prodrug was 15-fold lower well as with the lack of the muscle-specific toxicity of the two than that of cidofovir, presumably due to differences in the antiviral nucleotides (39). In contrast, detection of the full-length polarity of the two molecules. Accordingly, the cytotoxicity of hOAT1-specific mRNA in brain suggests expression of the active the prodrug was enhanced only three- to fourfold by hOAT1 J Am Soc Nephrol 11: 383–393, 2000 Induction of Drug Cytotoxicity by hOAT1 391

Table 3. Effect of nephroprotective agents on hOAT1- mediated cytotoxicity of adefovir and cidofovira ␮ CC50 ( M) Treatment Fold Changeb CHO CHOhOAT

Adefovir 155 0.33 470 Adefovir ϩ 1mM 39 16 2.43 probenecid Adefovir ϩ 0.5 mM ND 7.5 ND betamipron Cidofovir 278 0.72 386 Cidofovir ϩ 1mM 193 59 3.27 probenecid Cidofovir ϩ 0.5 mM ND 19 ND betamipron

a CHO and CHOhOAT cells were treated with various concentrations of adefovir or cidofovir in the presence or absence of nephroprotective agents. After 5 d, MTT cytotoxicity assay was performed and drug concentration inhibiting cell growth by 50%

(CC50) was determined. The data represent the average of two independent experiments. MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; ND, not determined. Other abbreviations as in Table 1. b Fold increase in the sensitivity of CHOhOAT cells to adefovir and cidofovir compared with parental CHO cells incubated under identical conditions.

Finally, the uptake of adefovir and cidofovir in CHOhOAT cells was sensitive to probenecid and betamipron, two potent hOAT1 inhibitors, which have exhibited in vivo nephroprotective effects against various nephrotoxic organic anions (46– 48). Both compounds protected CHOhOAT cells from hOAT1- induced cytotoxicity of both nucleotide analogs. In fact, probenecid showed a corresponding nephroprotective effect in cynomolgus monkeys treated with cidofovir (49) and is currently used in conjunction with cidofovir therapy in AIDS patients to reduce the potential for nephrotoxicity. In conclusion, using a cell line stably expressing hOAT1, we have extended the functional characterization of this physio- logically important renal transport protein and have provided compelling evidence that hOAT1 plays a key role in the mechanism of organ-specific toxicity associated with adefovir Figure 6. hOAT1-mediated uptake of adefovir and cidofovir is sensitive and cidofovir antiviral therapy. In addition, the presented data to inhibitors of organic anion transport. CHOhOAT cells were incubated support the utility of this in vitro model to explore the involve- with [3H]adefovir (A) or [14C]cidofovir (B) at a concentration equal to ment of hOAT1 in the nephrotoxicity of other agents, to study ␮ ␮ their Km value (24 M and 60 M, respectively) in the presence of potential drug-drug pharmacokinetic interactions and mecha- various concentrations of probenecid (F) or betamipron (E), and the nisms of renal drug secretion, and to screen for inhibitors that intracellular accumulation of substrates was determined. The data repre- could serve as efficient nephroprotectants. sent a mean of duplicates in a representative experiment. In all cases, SEM was Ͻ5% of the uptake in the absence of inhibitor. Acknowledgments We thank Norbert Bischofberger, Mick Hitchcock, and Jay Toole of Gilead Sciences for helpful discussions and critical reading of the expression compared to 400-fold enhancement determined for manuscript. cidofovir under identical conditions. This observation strongly correlates with significantly lower renal accumulation of the References cyclic prodrug as well as with its much lower nephrotoxic 1. Pritchard JB, Miller DS: Renal secretion of organic anions and potential in various species compared to cidofovir (44,45). cations. Kidney Int 49: 1649–1654, 1996 392 Journal of the American Society of Nephrology J Am Soc Nephrol 11: 383–393, 2000

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