ARTICLES J Am Soc Nephrol 11: 383–393, 2000
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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 accumu- lation 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 mem- brane 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