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Research ArticleSpecial Section on Transporters in Drug Disposition and Pharmacokinetic Prediction
Open Access

Expression of Organic Anion Transporter 1 or 3 in Human Kidney Proximal Tubule Cells Reduces Cisplatin Sensitivity

Tom T. G. Nieskens, Janny G. P. Peters, Dina Dabaghie, Daphne Korte, Katja Jansen, Alexander H. Van Asbeck, Neslihan N. Tavraz, Thomas Friedrich, Frans G. M. Russel, Rosalinde Masereeuw and Martijn J. Wilmer
Drug Metabolism and Disposition May 2018, 46 (5) 592-599; DOI: https://doi.org/10.1124/dmd.117.079384

Abstract

Cisplatin is a cytostatic drug used for treatment of solid organ tumors. The main adverse effect is organic cation transporter 2 (OCT2)–mediated nephrotoxicity, observed in 30% of patients. The contribution of other renal drug transporters is elusive. Here, cisplatin-induced toxicity was evaluated in human-derived conditionally immortalized proximal tubule epithelial cells (ciPTEC) expressing renal drug transporters, including OCT2 and organic anion transporters 1 (OAT1) or 3 (OAT3). Parent ciPTEC demonstrated OCT2-dependent cisplatin toxicity (TC50 34 ± 1 μM after 24-hour exposure), as determined by cell viability. Overexpression of OAT1 and OAT3 resulted in reduced sensitivity to cisplatin (TC50 45 ± 6 and 64 ± 11 μM after 24-hour exposure, respectively). This effect was independent of OAT-mediated transport, as the OAT substrates probenecid and diclofenac did not influence cytotoxicity. Decreased cisplatin sensitivity in OAT-expressing cells was associated directly with a trend toward reduced intracellular cisplatin accumulation, explained by reduced OCT2 gene expression and activity. This was evaluated by Vmax of the OCT2-model substrate ASP+ (23.5 ± 0.1, 13.1 ± 0.3, and 21.6 ± 0.6 minutes−1 in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3, respectively). Although gene expression of cisplatin efflux transporter multidrug and toxin extrusion 1 (MATE1) was 16.2 ± 0.3-fold upregulated in ciPTEC-OAT1 and 6.1 ± 0.7-fold in ciPTEC-OAT3, toxicity was unaffected by the MATE substrate pyrimethamine, suggesting that MATE1 does not play a role in the current experimental set-up. In conclusion, OAT expression results in reduced cisplatin sensitivity in renal proximal tubule cells, explained by reduced OCT2-mediated uptake capacity. In vitro drug-induced toxicity studies should consider models that express both OCT and OAT drug transporters.

Introduction

The chemotherapeutic drug cisplatin is commonly used to treat solid organ tumors located in various tissues, including the head, neck, lung, testis, ovary, and bladder. Nephrotoxicity is the main adverse effect of cisplatin, causing acute kidney injury (AKI) in 30% of patients (Hartmann et al., 1999). AKI is currently the dose-limiting factor in treatment with cisplatin and is characterized by dysfunction of the proximal tubule. Cisplatin continues to be prescribed, as less toxic alternatives, including oxaliplatin, are suggested to have reduced anticancer potential (Wang and Lippard, 2005; Manohar and Leung, 2018).

Proximal tubule epithelial cells mediate active excretion of xenobiotics and metabolites, and reabsorb low-molecular-weight proteins, such as β2-microglubulin, and glucose and amino acids filtered by the glomerulus. To this end, proximal tubule cells express dedicated membrane-spanning proteins (Nigam et al., 2015). Influx of cisplatin in proximal tubule cells is critically mediated by organic cation transporter 2 (OCT2, SLC22A2) (Ciarimboli et al., 2005; Filipski et al., 2009; Ciarimboli et al., 2010) and ubiquitously expressed copper transporter 1 (CTR1, SLC31A1) (Pabla et al., 2009), both located in the proximal tubule epithelium at the basolateral membrane. Cisplatin is a substrate for efflux transporters multidrug and toxin extrusion 1 (MATE1, SLC47A1), and to a lesser extent MATE2-K (SLC47A2), both located at the apical membrane (Yonezawa et al., 2006; Nakamura et al., 2010). The combined activity of influx and efflux transporters determines the amount of cisplatin accumulation, hence correlates to the drug’s nephrotoxicity (Li et al., 2013). Recent studies indicate that drug-drug interactions and interindividual genetic differences regarding OCT2 and MATE transporters may contribute to the nephrotoxic potential of cisplatin, underlining that their concerted action is essential to maintain active clearance and avoid proximal tubule accumulation (Sauzay et al., 2016; Chang et al., 2017).

Organic anion transporters 1 (OAT1, SLC22A6) and 3 (OAT3, SLC22A8) in proximal tubule cells play a main role in clearance of organic anions, including endogenous uremic toxins and a wide range of drugs (Nigam et al., 2015). Recently, a reduction in renal adverse effects of cisplatin were demonstrated in an Oat1-knockout mouse model, even though disposition of this drug is strongly associated with cation transporters (Hu et al., 2017). The suggested mechanism involves extrarenal metabolism yielding glutathione and cysteine conjugates that act as substrates for OAT transporters. Moreover, clinical studies demonstrated that cisplatin clearance was reduced upon cotreatment with OAT-inhibitor probenecid, consistent with a complete absence of renal adverse effects (Jacobs et al., 1984, 1991). As cisplatin transport is mediated by neither OAT1 nor OAT3, their role in cisplatin-induced nephrotoxicity remains to be elucidated.

The present study was designed to investigate the role of OATs in cisplatin-induced toxicity in conditionally immortalized proximal tubule epithelial cells (ciPTEC), a human-derived cell line manifesting proximal tubule characteristics (Wilmer et al., 2010; Nieskens et al., 2016). In this cell line, we previously demonstrated functional apical absorption of albumin and phosphate, expression of OCT2, P-glycoprotein (Pgp, ABCB1), multidrug resistance-associated protein 4 (MRP4, ABCC4), breast cancer resistance protein (BCRP, ABCG2), and after stable transductions the reintroduction of either OAT1 or OAT3 (Wilmer et al., 2010; Schophuizen et al., 2013; Jansen et al., 2014; Nieskens et al., 2016). In addition, ciPTEC express several UDP-glucuronosyltransferases and consume oxygen, suggesting intact mitochondrial energy production (Mutsaers et al., 2013; Schirris et al., 2017). Therefore, ciPTEC is a relevant proximal-tubule cell model with concerted action of different transport mechanisms and suitable for in vitro evaluation of drug-induced toxicity. The results of the present study show CTR1- and OCT2-dependent cisplatin-mediated toxicity in ciPTEC, which is alleviated in presence of active OAT1 or OAT3. Reduced sensitivity in OAT1- or OAT3-expressing cells was associated with a trend toward reduced cisplatin accumulation owing to lower OCT2 uptake capacity. Since expression of OATs affects toxicity induced by drugs that are typically imported by OCTs, simultaneous expression of basolateral solute carrier transport proteins is important for in vitro drug-induced toxicity evaluation.

Materials and Methods

Cell Culture.

Conditionally immortalized proximal tubule epithelial cells (ciPTEC-parent) were developed as described by Wilmer et al. (2010) with informed consent of the donors in accordance with the approved guidelines of the Radboud Institutional Review Board. ciPTEC-OAT1 and ciPTEC-OAT3 were derived from ciPTEC parent cells and demonstrate constitutive expression of organic anion transporters 1 (OAT1) or 3 (OAT3), respectively, as described by Nieskens et al. (2016). ciPTEC constitutively expressing enhanced yellow fluorescent protein (ciPTEC-eYFP) were developed by transduction of eYFP from the COX8-eYFP vector (Koopman et al., 2006) into the same pLenti4/V5-DEST vector as used for the OAT constructs, by LR recombinant reaction using Invitrogen Gateway cloning technology (Thermo Fisher Scientific, Carlsbad, CA) and resulting in expression vector pLenti4/V5-EX-CMV-TetO2-eYFP. Upon transduction as described for ciPTEC-OAT1 and -OAT3, ciPTEC-eYFP cells were selected for YFP fluorescence using BD FACSAria Special Order Research Program (SORP) flow cytometer (BD Biosciences, San Jose, CA). Proliferating cell culture was maintained up to 90% confluency using T75 culture flasks (Greiner Bio-One, Kremsmünster, Germany) at 33°C and 5% v/v CO2. Cells were seeded 7 or 8 days prior to the experiment in either T75 culture flasks (Greiner Bio-One), 12-well plates (Greiner Bio-One), or 96-well plates (Corning Life Sciences, Corning, New York) with a seeding density of 55,000 cells/cm2 for ciPTEC-parent, 63,000 cells/cm2 for ciPTEC-OAT1, and 82,000 cells/cm2 for ciPTEC-OAT3. Cells were subsequently incubated for 24 hours at 33°C and 5% v/v CO2 to stimulate proliferation, followed by 6 or 7 days at 37°C and 5% v/v CO2 to allow differentiation and monolayer formation, referred to as maturation. Cells were cultured using Dulbecco’s modified eagle medium/HAM’s F12 (Life Technologies/Thermo Fisher Scientific, Paisley, UK), supplemented with 5 μg/ml insulin, 5 μg/ml transferrin, 5 μg/ml selenium, 35 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor, 40 pg/ml tri-iodothyronine (MilliporeSigma, St. Louis, MO), and 10% fetal calf serum (FCS; Greiner Bio-One), and is referred to as complete medium (CM). Medium in which FCS was omitted, is referred to as serum-free medium (SFM). The medium was refreshed every 2 to 3 days, supplemented with 1% penicillin/streptomycin (Invitrogen) only at 33°C.

Viability Assay.

To evaluate cisplatin-induced toxicity, viability of ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 was evaluated by an MTT assay (Mosmann, 1983). Briefly, ciPTEC were matured in 96-well plates, washed three times with SFM, and subsequently exposed to cisplatin (8.7–222 μM; MilliporeSigma) in SFM for 24 hours. To evaluate the effect of competitive transport inhibitors on cisplatin-induced toxicity, cisplatin was coexposed with probenecid (100 μM; MilliporeSigma), diclofenac (50 μM; MilliporeSigma), cimetidine (1.25 mM; MilliporeSigma), or pyrimethamine (10 and 100 nM; MilliporeSigma). For probenecid, a pre-exposure during ciPTEC maturation was additionally performed for 6 days in CM. Following cisplatin exposure, cells were washed three times with SFM and incubated with 0.5 mg/ml MTT (MilliporeSigma) in SFM for 3 hours at 37°C. Formazan crystals formed in viable cells were dissolved in dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) on a microplate shaker (VWR, Radnor, PA) for 1 hour. Absorption was measured at 560 nm, the background subtracted at 670 nm, using the BioRad Benchmark Plus (BioRad, Hercules, CA). All values were normalized to unexposed control. Results were plotted with GraphPad Prism (version 5.03; GraphPad Software, La Jolla, CA) using nonlinear regression with four parameters (variable slope) on log-transformed x-values.

Cisplatin Accumulation.

To evaluate the effect of OCT2- and MATE-mediated transport on intracellular cisplatin accumulation, ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 were matured in T75 culture flasks, washed once in CM, and exposed to cisplatin (250 μM; MilliporeSigma), with and without OCT2 and MATE substrates cimetidine (1.25 mM; MilliporeSigma) or pyrimethamine (100 nM; MilliporeSigma) in CM for 90 minutes. Cells were washed three times in Hanks’ buffered salt solution (HBSS) (37°C), harvested using Accutase (Invitrogen), and placed on ice. Cell suspensions were centrifuged at 1500 relative centrifugal force (rcf) for 5 minutes (4°C), and cell pellets were resuspended in 200 μl nitric acid (100 mM; Merck). Lysates were obtained by applying three freeze-thaw cycles of liquid nitrogen and a 37°C water bath, followed by centrifugation at 10,000 rcf for 10 minutes (4°C). Cisplatin content of cell samples was evaluated using an atomic absorption spectrophotometer, AAnalyst800 (PerkinElmer, Waltham, MA), equipped with a transversally heated graphite tube atomizer and a platinum hollow cathode lamp (Photron, Melbourne, Australia), loaded with 20 μl of each sample, as described in detail before (Durr et al., 2013; Eljack et al., 2014). All values were corrected for blank (unexposed), normalized to ciPTEC-parent (for uninhibited control) or uninhibited control of each respective cell line (for competitive inhibitors), and results were plotted with GraphPad Prism (version 5.03; GraphPad Software).

Gene Expression.

To evaluate expression of drug transporter genes involved in cisplatin influx and efflux, ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 were matured in 12-well plates, washed three times, and exposed to SFM for 24 hours, before total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. When evaluating Nrf2 pathway induction, SFM was supplemented with 50 nM bardoxolone methyl (CDDO-Me; MilliporeSigma), and cDNA was synthesized using M-MLV Reverse Transcriptase (Promega, Madison, WI), according to the manufacturer’s instructions. mRNA expression levels of transporters were determined using gene-specific primer probe sets obtained from Life Technologies: OCT2 (SLC22A2, hs01010723_m1), MATE1 (SLC47A1, hs00217320_m1), MATE2-K (SLC47A2, hs00945650_m1), MRP2 (ABCC2, hs00166123_m1), NQO1 (hs01045993_g1), GCLC (hs00155249_m1), GAPDH (hs99999905_m1), and TaqMan Universal PCR Master Mix (Applied Biosystems Inc., Foster City, CA). Real-time quantitative reverse transcription–polymerase chain reactions were performed using CFX96 Touch Real-Time PCR system (BioRad) according to the manufacturer’s instructions, and analyzed by CFX Manager software (version 1.6; BioRad). mRNA levels were calculated using GAPDH as reference gene and expressed as either −ΔΔCt or fold change (2−ΔΔCt) compared with ciPTEC-parent. Results were plotted with GraphPad Prism (version 5.03; GraphPad Software).

ASP Accumulation.

To evaluate the accumulation of hOCT2 model substrate ASP+ (Pietruck and Ullrich, 1995; Cetinkaya et al., 2003), ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 were matured in 96-well plates (Schophuizen et al., 2013). Prior to the experiment, cells were washed three times in HEPES-Tris buffer, consisting of 132 mM NaCl, 4.2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.5 mM glucose, 10 mM Hepes, buffered to pH 7.4 using Tris (pH 8.8). Cells were next exposed to ASP+ (4-di-1-ASP+, 0.8–50 μM; Thermo Fisher Scientific, Waltham, MA) with and without cimetidine (1.25 mM; MilliporeSigma), pyrimethamine (10 and 100 nM; MilliporeSigma), or probenecid (100 μM; MilliporeSigma) for 1 hour at 37°C. For probenecid, a pre-exposure during ciPTEC maturation was additionally performed for 6 days in CM to study the effect of long-term OAT activity inhibition on ASP+-transporting proteins. After accumulation for 1 hour, the fluorescent signal was measured (excitation 485 nm; emission 590 nm) using a Victor ×3 plate reader (PerkinElmer). Values were corrected for extracellular signal (empty wells with corresponding ASP+ concentration) and either presented as arbitrary units (for kinetic curves) or normalized to control (for competitive inhibitors). Results were plotted with GraphPad Prism (version 5.03; GraphPad Software) using Michaelis-Menten regression analysis for concentration-dependent transport rate curves.

Glutathione Level.

To evaluate total cellular glutathione level, ciPTEC were matured in six-well plates and glutathione level was evaluated using the Glutathione Assay Kit (MilliporeSigma), according to the manufacturer’s instructions. Briefly, cells were harvested using Accutase (Invitrogen), washed with HBSS, and deproteinized with 5% sulfosalicylic acid (MilliporeSigma). Lysates were obtained by applying two freeze-thaw cycles of liquid nitrogen and a 37°C water bath, followed by a 5-minute incubation on ice and centrifugation at 10,000 rcf for 10 minutes (4°C). Samples were measured undiluted and absorption was evaluated at 412 nm every minute for 5 minutes, using Benchmark Plus (BioRad). All values were corrected for blank, normalized to ciPTEC-parent, and results plotted with GraphPad Prism (version 5.03; GraphPad Software).

Data Analysis and Statistic.

All data analysis and statistics were performed using GraphPad Prism (version 5.03; GraphPad Software) and presented as mean ± S.E.M. of three independent experiments (n = 3) performed in experimental triplicate, unless stated otherwise. For calculation of EC50 values, log cisplatin concentration-versus-viability was plotted after background subtraction. Statistics were performed by Student’s t test (two-tailed, α = 0.05) or by two-way analysis of variance (two-tailed, α = 0.05) using the Bonferroni post test (P < 0.05), as indicated in the figures.

Results

The toxic potential of cisplatin was evaluated in parent ciPTEC by exposure for up to 72 hours, which resulted in a concentration- and time-dependent loss in cell viability as measured via the MTT assay, presented in Fig. 1A. To elucidate the role of influx transporter activity on cisplatin-induced toxicity, ciPTEC-parent was coexposed to cisplatin in presence of the OCT2 substrate cimetidine or CTR1 substrate CuSO4 for 24 hours. Both substrates significantly attenuated toxicity of cisplatin, demonstrating their involvement in uptake and related toxicity of the drug (Fig. 1B). Next cisplatin exposure to ciPTEC expressing either OAT1 or OAT3 demonstrated reduced sensitivity compared with ciPTEC-parent, reflected by increased TC50 values, for which the largest shift was observed for ciPTEC-OAT3 (Fig. 1C; Table 1). The involvement of OAT-mediated transport in cisplatin-induced toxicity was investigated further by cisplatin exposure in the presence of OAT substrates probenecid and diclofenac. These did not affect the cytotoxicity after 24 hours nor after 7 days of pre-exposure, suggesting that OAT-mediated influx is not directly involved in reducing cisplatin sensitivity, nor that OAT transport results in the induction of protective cellular changes in the long term (Table 1). It should be noted that 7-day exposure to competitive inhibitor solvent (DMSO, 0.1% v/v) slightly reduced cisplatin-induced toxicity, which may be explained by inactivation of the drug by prolonged exposure to DMSO (Table 1) (Hall et al., 2014). To avoid possible adaptive responses that may have affected drug sensitivity, cisplatin preconditioning at shorter incubation times was not performed. Moreover, longer term exposures usually take place in vivo when patients are treated with cisplatin, making 24- or 48-hour exposures clinically more relevant. To investigate the intracellular capacity to handle oxidative stress induced by cisplatin, total glutathione content was analyzed in the three cell lines. No significant changes were observed in glutathione content between ciPTEC-parent, ciPTEC-OAT1 (P = 0.44), and ciPTEC-OAT3 (P = 0.56) (Supplemental Fig. 1). As decreased sensitivity to cisplatin might have resulted from the transduction process used to overexpress OAT1 or OAT3, we evaluated cisplatin sensitivity in ciPTEC-eYFP generated by the same transduction methodology. No difference in toxicity (P = 0.53) was observed between ciPTEC-eYFP (TC50 39 ± 7 μM) and ciPTEC-parent (TC50 34 ± 1 μM, Table 1), supporting our hypothesis that OAT protein expression is responsible for diminished cisplatin-sensitivity in OAT1- or OAT3-expressing cells and excluding nonspecific aberrations induced by the transduction methods.

Fig. 1.
Fig. 1.

Cisplatin sensitivity in ciPTEC is reduced by OAT1 and OAT3 expression, and is dependent on OCT2- and CTR1-mediated transport. (A) Time- and dose-dependent decrease in viability of ciPTEC-parent following exposure to cisplatin (8.7–222 μM) for 24, 48, or 72 hours as determined by MTT assay (mean ± S.E.M., n = 3). (B) Decreased viability of ciPTEC-parent upon cisplatin exposure could be partially restored by competitive inhibition of OCT2 (1.25 mM cimetidine) or CTR1 (1.25 mM CuSO4) for 24 hours, determined by MTT assay (mean ± S.E.M., n = 3, *P < 0.05; **P < 0.01; compared with control by Student’s t test). (C) Reduced sensitivity of ciPTEC-OAT1 and ciPTEC-OAT3 following exposure to cisplatin (8.7–222 μM) for 24 hours compared with ciPTEC-parent, as determined by MTT assay (mean ± S.E.M., n = 3). Results are normalized to unexposed ciPTEC.

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TABLE 1

Cisplatin-induced toxicity is independent of OAT-mediated transport in ciPTEC-OAT1 and ciPTEC-OAT3

TC50 values (represented in micromolar) calculated from MTT viability assays following cisplatin (8.7–222 μM) exposure for 24 h are not affected by 24 h (n = 3) and 7-day (n = 6) competitive inhibition of OAT (probenecid, 100 μM; diclofenac, 50 μM) in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 (mean TC50 ± S.E.M., *P < 0.05; ***P < 0.001; compared with ciPTEC-parent, no significant differences were found for treatments by two-way analysis of variance, N.D. is not determined).

The next step was to determine cisplatin accumulation in the three cell lines, which showed a reduced trend in ciPTEC-OAT1 (P = 0.29) and ciPTEC-OAT3 (P = 0.12) compared with ciPTEC-parent after exposure to excess cisplatin concentrations for 90 minutes (Fig. 2A). Hence, cisplatin accumulation in the cell lines was directly associated with their sensitivity to cisplatin as assessed by cell viability. This could have been the result of decreased OCT2 expression levels or increased MATE1 and MATE2-K expression, which facilitate cisplatin influx and efflux, respectively. ciPTEC expressing OAT3 showed slightly but significantly decreased OCT2 (SLC22A2) gene expression of 0.4 ± 0.1-fold, whereas the 0.5 ± 0.1-fold reduction in ciPTEC-OAT1was not significant. On the other hand, MATE1 (SCL47A1) was 16.2 ± 0.3-fold increased in ciPTEC-OAT1 and 6.1 ± 0.7-fold in ciPTEC-OAT3 (Fig. 2B; Ct values are shown in Supplemental Table 1). In addition, MATE2-K gene expression was increased in ciPTEC-OAT1 but decreased in ciPTEC-OAT3. Although MRP2 (ABCC2) expression has also been associated with reduced cisplatin sensitivity (Kawabe et al., 1999), we did not find differential regulation of this drug transporter between the three cell lines studied (Supplemental Table 2). To evaluate the functional contribution of influx and efflux transporters further, cisplatin accumulation was analyzed in the presence of the competitive OCT2 inhibitor cimetidine and resulted in a reduction of intracellular cisplatin levels that was significant for ciPTEC-OAT1 but not for ciPTEC-parent (P = 0.32) or -OAT3 (P = 0.33). Pyrimethamine did not affect cisplatin accumulation at concentrations for which it is a selective MATE1 substrate (Ito et al., 2010), suggesting a limited role of MATE-mediated efflux in the current experimental conditions (Fig. 2C). Competitive MATE inhibition by pyrimethamine did not affect cisplatin-induced toxicity following coexposure for 24 hours, again supporting the limited role of MATE in the current conditions (Table 2).

Fig. 2.
Fig. 2.

Decreased intracellular cisplatin accumulation and OCT2 gene expression in ciPTEC-OAT1 and ciPTEC-OAT3. (A) Whole-cell cisplatin content is decreased in ciPTEC-OAT1 and ciPTEC-OAT3 compared with ciPTEC-parent, following exposure to cisplatin (250 μM) for 90 minutes. Results are normalized to ciPTEC-parent [mean ± S.E.M., n = 3, performed with two experimental replicates, no significant differences were found by one-way analysis of variance (ANOVA)]. (B) Gene expression of OCT2 (SLC22A2) was decreased, but gene expression of MATE1 (SLC47A1) and MATE2-K (SLC47A2) was increased in ciPTEC-OAT1 and ciPTEC-OAT3 compared with ciPTEC-parent, using GAPDH as reference gene (mean ± S.E.M., n = 3, performed with one experimental replicate, *P < 0.05; ***P < 0.001; compared with ciPTEC-parent by one-way ANOVA). (C) Whole-cell cisplatin content is decreased by coexposure to OCT2-substrate cimetidine (1.25 mM) but is not affected by coexposure to MATE-substrate pyrimethamine (100 nM) during exposure to cisplatin for 90 minutes in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3. Results are normalized to control without substrate (mean ± S.E.M., n = 3, performed with two experimental replicates, **P < 0.01; compared with control by Student’s t test).

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TABLE 2

Cisplatin-induced toxicity is independent of MATE-mediated transport in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3

TC50 values (represented in micromolar) calculated from MTT viability assays following cisplatin (8.7–222 μM) exposure for 24 h are not affected by competitive MATE inhibition (pyrimethamine, 10 and 100 nM) in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 (mean TC50, n = 2).

To study the influence of OAT expression on OCT2 protein activity in more detail, we investigated uptake of ASP+ as specific substrate for the transporter in all ciPTEC cell lines in presence and absence of OCT2, MATE, and OAT substrates. Vmax of ASP+ uptake was significantly reduced in ciPTEC-OAT1 (13.1 ± 0.3 minutes−1) and ciPTEC-OAT3 (21.6 ± 0.6 minutes−1) compared with ciPTEC-parent (23.5 ± 0.1 minutes−1), suggesting that OCT2-mediated uptake capacity of ASP+ is reduced in ciPTEC expressing OAT1 or OAT3 (Fig. 3A; Table 3). As expected, competitive inhibition of OCT2 by cimetidine reduced ASP+ accumulation (Fig. 3B), whereas the competitive MATE inhibitor pyrimethamine did not affect ASP+ accumulation, suggesting that ASP+ is transported by OCT2 and not by MATE, which is in line with cisplatin accumulation assays (Fig. 3C). Competitive OAT inhibition by probenecid did not affect ASP+ accumulation after 24 hours (Fig. 3D), nor after 7 days (Fig. 3E) of treatment, suggesting that OAT-mediated uptake did not regulate OCT2 transport capacity.

Fig. 3.
Fig. 3.

Reduced ASP+ accumulation reflects attenuated OCT2 transport capacity in ciPTEC-OAT1 and ciPTEC-OAT3. (A) ASP+ (0.8–50 μM) accumulation for 60 minutes is decreased in ciPTEC-OAT1 and ciPTEC-OAT3 compared with ciPTEC-parent, evaluated by intracellular fluorescence intensity after 60 minutes (mean ± S.E.M., n = 3). (B–D) ASP+ (3.1 μM) accumulation for 60 minutes in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 is reduced by (B) competitive inhibition of OCT (cimetidine, 1.25 mM) but not affected by competitive inhibition of (C) MATE (pyrimethamine, 10 and 100 nM) or (D) OAT (probenecid, 100 μM), evaluated by intracellular fluorescence intensity. Results are normalized to unexposed ciPTEC-parent (mean ± S.E.M., n = 3, *P < 0.05; **P < 0.01; ***P < 0.001; compared with control by Student’s t test). (E) ASP+ (3.1 μM) accumulation for 60 minutes in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 is not affected by 7-day competitive OAT inhibition (probenecid, 100 μM), evaluated by intracellular fluorescence intensity. Results are normalized to unexposed ciPTEC-parent (mean ± S.E.M., n = 2, no significant differences were found for treatment by Student’s t test).

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TABLE 3

ASP+ accumulation is decreased in ciPTEC-OAT1 and ciPTEC-OAT3

Km (represented in micromolar concentration) and Vmax values (represented in min−1) calculated from Michaelis-Menten fitted ASP+ accumulation assays in ciPTEC-parent, ciPTEC-OAT1, and ciPTEC-OAT3 (mean ± S.E.M., n = 3, *P < 0.05; **P < 0.01; ***P < 0.001; compared with ciPTEC-parent by one-way analysis of variance).

The Nrf2 pathway is known to attenuate cisplatin-mediated toxicity by regulation of cisplatin transporters (Atilano-Roque et al., 2016). Therefore, the involvement of this pathway in the regulation of OCT2 and MATE transporters was evaluated in ciPTEC. Bardoxolone stimulated the Nrf2 pathway in ciPTEC-parent upon 24-hour exposure by factors of 6.1 ± 0.1 and 4.1 ± 0.2, respectively, as assessed by increasing expression of both target genes, NQO1 and GCLC (Fig. 4A). This was accompanied by a 1.7 ± 0.2-fold increase in MATE1, an 8.5 ± 0.6-fold increase in MATE2-K, and a 0.3 ± 0.1-fold decrease in OCT2 expression, supporting the nephroprotective effect of Nrf2. A similar decrease in OCT2 expression was observed for ciPTEC-OAT1 and ciPTEC-OAT3, as described above. The involvement of Nrf2 in differential regulation of cisplatin transporters in ciPTEC-OAT1 and ciPTEC-OAT3 compared with ciPTEC-parent could not be confirmed, however, as only the Nrf2 target NQO1 was increased 2.7 ± 0.2-fold in ciPTEC-OAT1, whereas GCLC was neither upregulated in ciPTEC-OAT1 nor ciPTEC-OAT3 (Fig. 4B). This illustrates that stimulation of Nrf2 via bardoxolone is distinct from the gene expression pattern observed in ciPTEC-OAT1 and ciPTEC-OAT3, indicating that the differences in OCT2 and MATE expression observed are not solely caused by Nrf2 pathway induction.

Fig. 4.
Fig. 4.

Nrf2 pathway activation by bardoxolone in ciPTEC-parent is associated with cisplatin transporter regulation but is not increased in ciPTEC-OAT1 and -OAT3, compared with parent ciPTEC. (A) Gene expression of Nrf2 target genes NQO1 and GCLC is increased after 24-hour pre-exposure to bardoxolone (50 nM), which is associated with decreased OCT2 (SLC22A2) gene expression, whereas gene expression of MATE1 (SLC47A1) and MATE2-K (SLC47A2) is increased in parent ciPTEC, using GAPDH as reference gene (mean ± S.E.M., n = 3, performed with one experimental replicate, *P < 0.05; **P < 0.01; ***P < 0.001; compared with control by Student’s t test). (B) Gene expression of Nrf2 target genes NQO1 and GCLC is not consistently increased in ciPTEC-OAT1 and ciPTEC-OAT3, compared with ciPTEC-parent, using GAPDH as reference gene (mean ± S.E.M., n = 3, performed with one experimental replicate, ***P < 0.001; compared with ciPTEC-parent by one-way analysis of variance).

Discussion

Cisplatin is a well-described nephrotoxicant, affecting the proximal tubule in particular. Here, we demonstrate that the human-derived proximal tubule epithelial cell model, ciPTEC, is sensitive to OCT2- and CTR1-mediated cisplatin-induced toxicity. Expression of OAT1 and OAT3 reduced ciPTEC sensitivity to cisplatin, which is independent of OAT transport activity, and is associated with a trend toward reduced intracellular cisplatin accumulation, explained by reduced OCT2-mediated uptake capacity.

The mechanism responsible for reduced cisplatin sensitivity in ciPTEC-OAT1 and ciPTEC-OAT3 involves reduced OCT2-dependent influx of the drug. ASP+ is a model substrate for OCT2 and was used to evaluate OCT2 transport capacity, which was decreased in ciPTEC expressing OAT. We can only speculate on how this is regulated, but this could involve the Nrf2 pathway, as Nrf2-null mice developed more extensive nephrotoxicity after cisplatin treatment compared with wild types (Aleksunes et al., 2010). Nrf2-mediated signaling reduces expression of OCT2 in rat cortical tissue and Madin-Darby canine kidney cells, and increases expression of MATE1 in primary human proximal tubule cells, reducing renal cisplatin accumulation and toxicity (Shu et al., 2001; Atilano-Roque et al., 2016; Huang et al., 2017). In ciPTEC-parent, the involvement of Nrf2 in downregulation of OCT2 and upregulation of MATE1 could be confirmed, supporting previous in vivo and in vitro studies (Aleksunes et al., 2010; Atilano-Roque et al., 2016). Remarkably, we found a profound increase of MATE2-K gene expression, whereas the effect for MATE1 was less pronounced. Regulation of MATE2-K via Nrf2 when proximal tubule cells were exposed to flow was recently published (Fukuda et al., 2017). With respect to cisplatin-induced toxicity, OAT expression affected OCT2 and MATE1/2-K, for which the direct involvement of Nrf2 remains elusive, as established target genes of this pathway were not consistently regulated. Therefore, the protective effect on cisplatin toxicity via bardoxolone-mediated Nrf-2 stimulation was not tested in the current study focusing on the differential effect of OAT expression. Alternatively, tyrosine kinase inhibitors reduced OCT2 activity and oxaliplatin-induced acute neuropathy in vivo, suggesting a role for tyrosine phosphorylation in regulation of OCT2. (Sprowl et al., 2016). It is important to note that reduction in cisplatin sensitivity and accumulation was most pronounced for ciPTEC-OAT3, whereas ASP+ accumulation was reduced mostly in ciPTEC-OAT1. This suggests that additional, nonelucidated cell-protecting mechanisms in parallel to the Nrf2 pathway or tyrosine kinase activity might play a role in the protection against cisplatin toxicity.

In addition to drug transport function, OAT1 and OAT3 are associated with several metabolite pathways, including fatty acid, amino acid, nucleic acid, glucose, pyruvate, and glutathione metabolism (Ahn et al., 2011; Wu et al., 2013). As a result, a hypothesis was postulated on the role of drug transporters in detecting and maintaining levels of endogenous metabolites, antioxidants, signaling molecules, hormones, and nutrients (Nigam, 2015; Bush et al., 2017). OAT-mediated transport is bidirectional and therefore its activity influences the intracellular concentration of counter-transported compounds, including metabolites of the citric acid cycle, predominantly α-ketoglutarate (Burckhardt and Burckhardt, 2003; Sweet et al., 2003; Kaufhold et al., 2011). Reduced or competitively inhibited OAT function might therefore reduce energy metabolism, illustrated by reduced active transport of para-aminohippuric acid upon inhibition of mitochondrial ATP production (Nagai et al., 1998). This, in turn, may affect drug-induced toxicity response in proximal tubules by predisposing to a redox state and supplementing or depleting the energy pool. Since reactive oxygen species (ROS) are key players in cisplatin-induced nephrotoxicity, the proximal tubule redox state and cellular content of the ROS scavenger glutathione are highly important for sensitivity to drug-induced toxicity (Wang and Lippard, 2005; Manohar and Leung, 2018). No significant differences in total glutathione content were observed among the ciPTEC cell lines, suggesting that redox metabolism is not directly affected by (lack of) OAT function in ciPTEC. The expression of OAT could have influenced the intracellular proton gradient, thereby decreasing the membrane potential, which drives transport by OCTs (Okuda et al., 1999; Budiman et al., 2000). This might explain the increased Km values for ASP+ accumulation in ciPTEC-OAT1 and ciPTEC-OAT3 as observed in the present study. Metabolomic analysis of compounds involved in energy production, especially of the citric acid cycle, may generate a more profound understanding of the role of OAT transporters in cellular homeostasis and drug sensitivity.

The present findings contradict previously reported in vivo studies. Jacobs et al. (1984, 1991) suggested that cisplatin cotreatment with probenecid competitively inhibited OAT-mediated transport, thereby reducing the renal clearance of cisplatin and protecting patients from nephrotoxicity, even at dose-escalation. In line with this, reduced cisplatin excretion and attenuated nephrotoxicity were observed in cisplatin-treated Oat1 knockout mice and probenecid cotreated wild-type mice (Hu et al., 2017). The authors of the latter study suggested that cisplatin is converted into a highly reactive mercapturic acid metabolite, which is a substrate for OAT1 and OAT3, in a way similar to that described for cadmium and mercury (Zalups, 2000a,b; Cannon et al., 2001). However, the first conversion step involves extrarenal conjugation of cisplatin to glutathione, generating a water-soluble metabolite filtered by the glomerulus. Proximal tubule cells are therefore mainly apically exposed to the conjugated metabolite, suggesting limited involvement of basolaterally located OAT1 and OAT3. This indicates that current in vitro models underestimate the complexity of drug-induced renal injury. To account for the contribution of metabolism, our in vitro model could be extended to include pre-exposure of cisplatin to cultured hepatocytes or precision-cut liver slices (Starokozhko et al., 2017). In contrast to studies in which OAT function contributes to nephrotoxicity, the beneficial effects of OAT-mediated transport on renal function have also been described. Ischemia reperfusion resulted in severe kidney injury in rats, which was accompanied by reduced expression of Oat1 and Oat3. Indomethacin prevented ischemia-induced downregulation of Oat1 and Oat3, whereas probenecid abolished indomethacin-mediated attenuation (Schneider et al., 2015). As cisplatin-induced proximal tubule toxicity can induce a similar decrease of Oat1 and Oat3 expression (Liu et al., 2012), investigating indomethacin-mediated regulation of OAT1 and OAT3 might provide an interesting mechanism that can explain reduced renal adverse effects.

Most in vitro models applied in drug-induced toxicity prediction lack human- and tissue-relevant expression of transporters and metabolizing enzymes (Shaw et al., 2002; Chu et al., 2013; Van der Hauwaert et al., 2014; Wilmer et al., 2016). The proximal tubule model used here circumvents these issues as it demonstrates a tissue-specific expression profile in a human-derived stable cell line. To enable drug-induced toxicity evaluation of compounds that require influx by OAT transporters, OAT1 or OAT3 were reintroduced by lentiviral transfection of parent ciPTEC, generating ciPTEC-OAT1 and ciPTEC-OAT3 (Nieskens et al., 2016). As this method may have induced genetic aberrations, a similar transduction process was performed with eYFP to show that the transfection procedure did not significantly affect cisplatin sensitivity.

Functional MATE-mediated transport was lacking in our experimental conditions, illustrated by the inability of pyrimethamine to modulate ASP+ and cisplatin accumulation, and subsequent cisplatin-induced toxicity. This may be the result of limited polarization of ciPTEC under the current culture conditions, although activity of OCT2, OAT1, and OAT3, which are exclusively expressed at the basolateral membrane in vivo, was demonstrated. Although pyrimethamine is offered extracellularly, which may compromise inhibition of MATE-mediated cisplatin extrusion, MATE protein levels were below the limit of detection with Western blotting in ciPTEC (data not shown). Therefore, MATE transport function was not studied further in ciPTEC. Additional experiments using a polarized culture system could be used to enhance the expression levels and possibly transport activity of MATE1 and MATE2-K.

ASP+ and cisplatin have been described as MATE1-substrates in vitro, and MATE1 has been shown to mediate efflux of cisplatin in rodents in vivo (Yonezawa et al., 2006; Ito et al., 2010; Nakamura et al., 2010; Wittwer et al., 2013). The driving force for MATE-mediated transport is a counter-directed gradient of H+ (Tsuda et al., 2007). Transport of cisplatin was demonstrated upon intracellular acidification, resulting in a nonphysiologic outward-directed proton gradient and MATE-mediated influx (Nakamura et al., 2010). All experiments in ciPTEC were performed at a buffered pH of 7.4, and thus lacked the driving force for MATE. It is important to consider the limited cationic charge of cisplatin in buffers that contain a physiologic chloride concentration, reflecting blood plasma, whereas it is 2-fold positively charged when exposed to low chloride, reflecting the cytoplasm (Andersson and Ehrsson, 1995; Jennerwein and Andrews, 1995). Apparent transport affinities might be different for the neutral or charged form, possibly affecting accumulation of the drug and further increasing the mechanistic complexity of cisplatin-induced toxicity. In short, the in vivo contribution of MATE-mediated efflux to cisplatin-induced nephrotoxicity in humans remains to be elucidated. Evaluating cisplatin-induced toxicity in bicompartmental Transwell or three-dimensional culture models that allow for mimicking of the pH gradient—as is experienced in the physiologic proximal tubule—may help to elucidate the involvement of MATE in this process.

In conclusion, we demonstrated that expression of OAT1 or OAT3 in a human-derived proximal tubule epithelial cell model resulted in reduced sensitivity to cisplatin. This was independent of OAT transport function and is most probably explained by a reduced OCT2-mediated influx capacity. As organic anion transporters influence sensitivity to drugs that are typically handled by organic cation transporters, reliable in vitro drug-induced nephrotoxicity evaluation requires simultaneous expression of basolateral solute carrier transport proteins.

Authorship Contributions

Participated in research design: Nieskens, Peters, Van Asbeck, Russel, Masereeuw, Wilmer.

Conducted experiments: Nieskens, Peters, Dabaghie, Korte, Jansen, Van Asbeck, Tavraz.

Contributed new reagents or analytic tools: Peters, Friedrich, Tavraz.

Performed data analysis: Nieskens, Dabaghie, Korte, Jansen, Van Asbeck.

Wrote or contributed to the writing of the manuscript: Nieskens, Jansen, Dabaghie, Van Asbeck, Friedrich, Russel, Masereeuw, Wilmer.

Footnotes

    • Received November 8, 2017.
    • Accepted February 28, 2018.

  • This work was financially supported by a grant from the Dutch Kidney Foundation [Grant no. KJPB 11.023], by the CRACK-IT NephroTube Challenge funded by NC3Rs [Project no. 37497-25920], and by the German Research Foundation [Cluster of Excellence “Unifying Concepts in Catalysis,” EXC 314, Project Area E3]. The authors declare no potential conflict of interest.

  • https://doi.org/10.1124/dmd.117.079384.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

ASP+
4-di-1-ASP (4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide)
ciPTEC
conditionally immortalized proximal tubule epithelial cells
CM
complete medium, supplemented with 10% fetal calf serum
CTR
copper transporter
DMSO
dimethyl sulfoxide
eYFP
enhanced yellow fluorescent protein
HBSS
Hanks’ buffered salt solution
MATE
multidrug and toxin extrusion
MTT
thiazolyl blue tetrazolium bromide (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)
OAT
organic anion transporter
OCT
organic cation transporter
rcf
relative centrifugal force
SFM
serum-free medium

This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

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Research ArticleSpecial Section on Transporters in Drug Disposition and Pharmacokinetic Prediction

OAT Expression Reduces Proximal Tubule Cisplatin Sensitivity

Tom T. G. Nieskens, Janny G. P. Peters, Dina Dabaghie, Daphne Korte, Katja Jansen, Alexander H. Van Asbeck, Neslihan N. Tavraz, Thomas Friedrich, Frans G. M. Russel, Rosalinde Masereeuw and Martijn J. Wilmer
Drug Metabolism and Disposition May 1, 2018, 46 (5) 592-599; DOI: https://doi.org/10.1124/dmd.117.079384
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