A renal cell line with stable transporter expression

Abstract

The invention relates to the field of pharmacology, specifically the field of drug-drug interactions and nephrotoxicity. An engineered, stable cell line of human renal cells is provided that allows screening for drug-drug interactions and nephrotoxicity.

Claims

1.-17. (canceled)

18. A human proximal tubule epithelial cell (PTEC) that stably expresses a functional organic anion transporter (OAT) when cultured, wherein said cell is conditionally immortalized (ciPTEC).

19. The cell according to claim 18, wherein the cell is derived from ciPTEC DSM ACC 3019 or is derived from a passage or isolate thereof.

20. The cell according to claim 18, wherein said organic anion transporter is selected from the group consisting of: i) a polypeptide having at least 50% sequence identity or similarity with SEQ ID NO: 1 (organic anion transporter 1 (OAT1)), or encoded by a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 2, and ii) a polypeptide having at least 50% sequence identity or similarity with SEQ ID NO: 3 (organic anion transporter 3 (OAT3)), or encoded by a nucleotide sequence having at least 50% sequence identity with SEQ ID NO: 4.

21. The cell according to claim 18, wherein said cell further expresses at least one other relevant transporter.

22. The cell according to claim 21, wherein the relevant transported is a renal transporter.

23. The cell according to claim 22, wherein the renal transporter selected from the group consisting of SLC22A2 (OCT2), SLCO4C1 (OATP-H), ABCB1 (PgP), ABCG2 (BCRP), ABCC2 (MRP2), ABCC4 (MRP4), SLC47A1 (MATE1), SLC47A2 (MATE2-K), SLC34A1 (NaPi IIa), and SLC34A3 (NaPi IIc).

24. The cell according to claim 18, wherein said cell is obtainable by a method comprising the following steps: i) transducing a population of proximal tubule epithelial cells by a lentiviral particle comprising an expression construct that comprises a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 2 or with SEQ ID NO: 4, ii) optionally enriching the transduced population obtained in (i), and iii) subcloning the transduced population obtained in (i) of (ii) by selecting and isolating single cells and expanding these by culture.

25. The cell according to claim 24, wherein step ii) is performed by using fluorescence activated cell sorting (FACS).

26. The cell according to claim 25, wherein said expression construct has at least 50% sequence identity with an expression construct selected from the group consisting of: i) pLenti4/V5-EX-CMV-TetO2-hOAT1 (SEQ ID NO: 25), and ii) pLenti4/V5-EX-CMV-TetO2-hOAT3 (SEQ ID NO: 26).

27. The cell according claim 18, wherein said cell is ciPTEC.OAT1.4B2 DSM ACC3279 or a passage or isolate thereof.

28. The cell according to claim 18, wherein said cell is ciPTEC.OAT3.3C1 DSM ACC3280 or a passage or isolate thereof.

29. An in vitro or ex vivo method for analysis of a substance, comprising contacting said substance with at least one cell according to claim 18.

30. The method according to claim 29, wherein the substance is contacted with a mature monolayer of said cells.

31. The method according to claim 29, wherein said method is for determining the nephrotoxicity of said substance,

32. The method according to claim 31, wherein the method further comprises a subsequent step of analyzing cell viability.

33. The method according to claim 29, wherein said method is for the functional analysis of the interaction of said substance with a transporter, and wherein said contacting preferably is in the presence of a labeled anionic transporter substrate.

34. The method according to claim 33, wherein the transporter is a renal transporter.

35. The method according claim 29, wherein said method further comprises determining the drug-drug interaction of said substance.

36. The method according to claim 29, wherein said method further comprises determining whether said substance is a substrate or an inhibitor of a transporter involved in a clinically relevant drug-drug interaction.

37. A kit of parts comprising a cell as described in claim 18 and instructions for use.

Description

FIGURE LEGENDS

[0085] FIG. 1. Schematic overview of transduction procedure to obtain ciPTEC-OAT1 and ciPTEC-OAT3.

[0086] (A) ciPTEC parent was transduced with OAT1 or OAT3 lentiviral constructs and enriched by FACS using OATs' capacity to transport fluorescein. Further subcloning using radiated 3T3 fibroblasts as feeder cells resulted in a homogeneous ciPTEC-OAT1 or ciPTEC-OAT3 cell line.

[0087] (B), (C) and (D) show histograms obtained by flow cytometry of ciPTEC parent (B), and of ciPTEC-OAT1 (C) or ciPTEC-OAT3 (D) exposed to fluorescein (1 M, 10 min, dashed line), fluorescein and para-aminohippuric acid (100 dotted line), or untreated cells (continuous line). Parent cells exposed to fluorescein did not show increased fluorescence intensity, while ciPTEC-OAT1 and ciPTEC-OAT3 both showed a sub-population with increased fluorescence, which is indicative for OAT functionality. Fluorescence increase in ciPTEC-OAT1 was sensitive to inhibition induced by para-aminohippuric acid, as evidenced by the shift of the subpopulation.

[0088] (E) shows a scatter plot showing forward scatter (y-axis) and fluorescein intensity (x-axis) of transduced ciPTEC-OAT1 exposed to 1 M fluorescein for 10 min. The population with high fluorescence intensity indicated by gate P1 (8.3% of total population) was sorted to enrich successfully transduced ciPTEC-OAT1. Transduction with OAT3 was more efficient than OAT1, represented by the larger positive subpopulation in FIG. 1D compared to FIG. 1C, making the enrichment protocol redundant for ciPTEC-OAT3.

[0089] (F) shows a histogram of enriched ciPTEC-OAT1 exposed to fluorescein (1 M, 10 min) in presence (dotted line) or absence (dashed line) of the competitor para-aminohippuric acid (100 It demonstrates increased fluorescence intensity upon exposure to fluorescein as compared to non-enriched ciPTEC, but it also indicates a heterogeneous population that is sensitive to para-aminohippuric acid, pointing towards the requirement of subcloning of the enriched cells.

[0090] FIG. 2. Uptake of (4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) (1 M) by ciPTEC parent, ciPTEC-OAT1, or ciPTEC-OAT3, each when co-incubated with OCT2-substrate cimetidine for 60 min in Hank's Balanced Salt Solution (HBSS) at 37 C., relative to uptake without inhibitor. The lines represent the fit according to a one-site competition model. Values are expressed as SEM, (ciPTEC, n=3; ciPTEC-OAT1, n=4; ciPTEC-OAT3, n=2. Analysis using Two-way ANOVA indicated significant inhibition of ASP+ uptake at OCT2 with cimetidine, resulted in similar IC.sub.50 (p>0.05).

[0091] FIG. 3. OAT-mediated fluorescein uptake in ciPTEC-OAT1 and ciPTEC-OAT3.

[0092] (A) Concentration-dependent OAT1 and OAT3 mediated uptake of fluorescein after 10 min incubation in ciPTEC-OAT1 and ciPTEC-OAT3. The curve was fitted (n=4) according to a Michaelis-Menten model in combination with linear diffusion.

[0093] (B, C) Fluorescein uptake (1 M) by ciPTEC-OAT1 and (D, E) ciPTEC-OAT3 up to 60 min in absence or presence of two concentrations of the typical inhibitors para-aminohippuric acid (PAH, for ciPTEC-OAT1) or estrone sulfate (ES, for ciPTEC-OAT3). (B, D) The curves were fitted (n=4) to a standard saturation model after background subtraction. Analysis using two-way ANOVA indicated significantly decreased uptake curves in both ciPTEC-OAT1 (10 M and 100 M PAH, p<0.001)) and ciPTEC-OAT3 (3 M ES, p<0.01; 100 M ES, ***p<0.001). (C, E) Representative images of fluorescein uptake (1 M) by ciPTEC-OAT1 (C) and ciPTEC-OAT3 (E) after 10 min (magnification 20).

[0094] FIG. 4. Inhibition of OAT-mediated fluorescein uptake by a panel of OAT-perpetrators. Fluorescein uptake (1 M) by ciPTEC-OAT1 (left set of graphs) and ciPTEC-OAT3 (right set of graphs) when co-incubated with any one of para-aminohippuric acid, estrone sulfate, probenecid, furosemide, cimetidine, diclofenac, or metformin for 10 min in HBSS at 37 C., relative to uptake without inhibitor. The line represents the fit according to a one-site competition model with variable slope, except for metformin. Values are derived from experiments performed at passage x+8, x+11, x+14 and x+29 upon transduction (n=4).

[0095] FIG. 5. Inhibition of OAT-mediated fluorescein uptake by adefovir, cidofovir, tenofovir, or zidovudine. Fluorescein uptake (1 M) by ciPTEC-OAT1 (left set of graphs) and ciPTEC-OAT3 (right set of graphs) when co-incubated with any one of the antivirals for 10 min in HBSS at 37 C., relative to uptake without inhibitor. The line represents the fit according to a one-site competition model with variable slope (n=4).

[0096] FIG. 6. Antiviral-induced toxicity in ciPTEC-OAT1 and ciPTEC-OAT3.

[0097] (A) Viability of ciPTEC parent, ciPTEC-OAT1 and ciPTEC-OAT3 after exposure to antiviral agent (1 mM) for 48 h in serum free medium relative to cell viability without exposure, as measured with the MTT assay (n=3). **p<0.01, ***p<0.001.

[0098] (B) Viability of ciPTEC-OAT1 (left set of graphs) and ciPTEC-OAT3 (right set of graphs) upon tenofovir, adefovir, cidofovir, or zidovudine exposure for 24, 48 and 72 h in serum free medium, relative to cell viability without exposure. The line represents the fit according to a one-site competition model with variable slope (n>3).

[0099] FIG. 7. Expression and transport activity of OAT1 in ciPTEC-OAT1 is regulated by EGF.

[0100] OAT1 gene expression (A) and transport activity (B) was determined in isolated RNA fractions and cultured ciPTEC-OAT1, respectively. The activity of OAT1 was determined using a fluorescent OAT1 substrate (fluorescein); OAT1 expression and activity were increased upon exposure to EGF. Data are presented as mean values+/SEM, n=3. Statistical analysis was performed via unpaired Student's t-test.

EXAMPLES

[0101] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0102] Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry.

[0103] Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2.sup.nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

[0104] In the examples here below, ciPTEC (also referred to as ciPTEC cells, ciPTEC parent, parent ciPTEC or ciPTEC parent cells) is DSM ACC 3019; ciPTEC OAT1 is ciPTEC.OAT1.4B2 DSM ACC3279 and ciPTEC OAT3 is ciPTEC.OAT3.3C1 DSM ACC3280.

Example 1.Material and Methods

[0105] Cell Culture

[0106] Conditionally immortalized proximal tubule epithelial cells (ciPTEC) were developed as described by Wilmer et al. with informed consent of the donors in accordance with the approved guidelines of the Radboud Institutional Review Board. (Wilmer et al., 2010) Cells were seeded 7 days prior to the experiment at their corresponding density (55,000 cells/cm.sup.2 for ciPTEC parent cells, 63,000 cells/cm.sup.2 for ciPTEC-OAT1 and 82,000 cells/cm.sup.2 for ciPTEC-OAT3) and grown for 1 day at 33 C. and 5% v/v CO.sub.2 to allow proliferation, enabled by the temperature-sensitive mutant of SV large T antigen (SV40T). Next, cells were cultured for 6 days at 37 C. and 5% v/v CO.sub.2 to stimulate differentiation and formation of an epithelial monolayer, described as maturation. Cells were cultured using Dulbecco's modified eagle medium (DMEM HAM's F12, Life Technologies, Paisly, UK), 5 g/ml insulin, 5 g/ml transferrin, 5 g/ml selenium, 35 ng/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 40 pg/ml tri-iodothyronine (Sigma, St. Louis, USA) and 10% fetal calf serum (FCS, Greiner Bio One, Kremsmuenster, Austria). Medium was refreshed every second day, supplemented with 1% penicillin/streptomycin (pen/strep, Invitrogen, Carlsbad, USA) at 33 C. and without pen/strep at the maturation temperature of 37 C. 3T3 mouse-fibroblast (3T3) cells were cultured at 37 C. and used as feeder cells for sub-cloning procedures upon transduction, as described (Wilmer et al., 2010).

[0107] Vector Construction

[0108] Vector construction was performed using Gateway Cloning Technology (Invitrogen), according to the manufacturer's instructions. Commercially obtained vectors containing OAT1 (pENTR201-hOAT1, Harvard Plasmids HsCD00044153) and OAT3 (pENTR201-hOAT3, HsCD00044090) were transferred into a commercially available pLenti4/V5-DEST vector by LR recombinant reaction, resulting in expression vectors pLenti4/V5-EX-hOAT1 and pLenti4/V5-EX-hOAT3. The inducible CMV-TetO2 promoter was replicated from pcDNA5-FRT-TO (Invitrogen) using two primers, one primer that introduces a ClaI restriction site (forward Cla1-CMV-TetO2: GCCGCCATCGATGCCGCCGTTGACATTGATTATTGACTSEQ ID NO: 27) and one primer that introduces an EcoRI restriction site (reverse EcoRI-CMV-TetO2: GGCGGCGAATTCGGCGGCCGGAGGCTGGATCGGTCCCGGSEQ ID NO: 28). The resulting PCR product (ClaI-CMV-TetO2-EcoRI) was purified using the High Pure PCR Product Purification kit (Roche, Basel, Switzerland). Both PCR product and expression vectors were digested by ClaI and EcoRI (New England Biolabs, Ipswich, USA) for 1 hour at 37 C. and, after purification, ligation was performed with a 1:3 (insert:vector) unit ratio using T4 ligase (Invitrogen) for 2 h at 37 C., resulting in the pLenti expression constructs (pLenti4/V5-EX-CMV-TetO2-hOAT1 (SEQ ID NO: 25) and pLenti4/V5-EX-CMV-TetO2-hOAT3 (SEQ ID NO: 26)).

[0109] OAT Transduction in ciPTEC

[0110] To obtain lentiviral particles containing the OAT constructs, lentiviral stock was produced by transfecting the pLenti expression constructs with packaging plasmid mix into the HEK293FT cell line using ViraPower Lentiviral Gateway Expression Systems (Invitrogen), according to the manufacturer's instructions. ciPTEC were cultured to 50-70% confluency and exposed to lentiviral particles for 24 h. Both ciPTEC-OAT1 and ciPTEC-OAT3 were selected and subcloned to obtain homogeneous cell populations. To this end, transduced ciPTEC-OAT3 cells were plated into 3 separate culture flasks (100, 300 and 900 cells) containing irradiated (30 Gy) 3T3-cells as described (Saleem et al., 2002). After 2-3 weeks, single cell colonies of ciPTEC-OAT3 were picked and cultured. Transduction efficiency for ciPTEC-OAT1 was too low for immediate subcloning. Therefore, the heterogeneous cell population was enriched by positive selection of fluorescein transporting cells. Only successfully transduced ciPTEC express functional OAT; hence, positive selection could be performed upon exposure to fluorescein, which is an OAT substrate, using a BD FACSAria SORP flow cytometer (BD biosciences, San Jose, USA). 20 million ciPTEC-OAT1 cells were suspended in Hank's Balanced Salt Solution (HBSS, Invitrogen) containing 1 M fluorescein and incubated for 10 min at 37 C. before fluorescence-activated cell sorting (FACS). Enriched ciPTEC-OAT1 cells were subcloned as described for ciPTEC-OAT3. Both ciPTEC-OAT1 and ciPTEC-OAT3 were cultured for up to 30 passages after transduction to study stability of OAT1 and OAT3 expression.

[0111] OAT-Mediated Fluorescein Uptake

[0112] To evaluate OAT transporter function and to evaluate the inhibition properties of several known OAT substrates, fluorescein uptake was measured by flow cytometry and multiplate reader assays. Mature monolayers of sub-cloned ciPTEC were co-incubated with fluorescein (1 M, unless stated otherwise) and a test compound in HBSS for 10 min at 37 C. Compounds known for their inhibitory effect on OAT-mediated transport were tested. The following is a list of tested compounds: para-aminohippuric acid (PAH), estrone sulfate, probenecid, furosemide, cimetidine, diclofenac, adefovir, cidofovir, tenofovir, and zidovudine. The organic cation metformin was included as a negative control. All chemicals were obtained from Sigma, unless stated otherwise. Uptake was stopped by washing 3 times with cold HBSS (4 C.). For flow cytometry, samples were harvested following fluorescein exposure using trypsin-EDTA, then washed, fixed using 0.5% paraformaldehyde, and measured using FACS calibur (Becton Dickinson, Franklin Lakes, USA). For 96 well plate assay, cells were lysed by 200 l 0.1 M NaOH for 10 min at 37 C. and fluorescence was measured (excitation 485 nm, emission 535 nm) using the multiplate reader Victor X3 (Perkin Elmer, Waltham, USA).

[0113] Viability Assays

[0114] To evaluate toxicity induced by antivirals, viability of ciPTEC was evaluated by an MTT assay (Moghadasali et al., 2013). Briefly, monolayers of ciPTEC (96-wells) were exposed to antivirals in serum-free medium (SFM) on day 6 of maturation. Cell toxicity was analyzed further in presence of multidrug resistance protein (MRP) efflux inhibitor MK571 (5 M) and breast cancer resistance protein (BCRP) efflux inhibitor KO143 (10 M). After incubation for 24, 48 and 72 h at 37 C., ciPTEC were washed and incubated with 0.5 mg/ml thiazolyl blue tetrazolium bromide (MTT, Sigma) for 3 h at 37 C. in absence of antivirals. Formazan crystals formed in viable cells were dissolved in dimethyl sulfoxide (DMSO, Merck, Whitehouse Station, USA) and optical density was measured (560 nm, background at 670 nm was subtracted) using Benchmark Plus (Bio-Rad, Hercules, USA).

[0115] Gene Expressions in ciPTEC

[0116] Total RNA was isolated from matured ciPTEC (6-well plates) using TRIzol (Life Technologies Europe BV) and chloroform extraction. cDNA was synthesised using M-MLV Reverse Transcriptase (Promega, Madison, USA), according to the manufacturer's instructions. The mRNA expression levels were evaluated using gene-specific primer-probe sets obtained from Life Technologies: OAT1 (SLC22A6, hs00537914), OAT3 (SLC22A8, hs00188599), GAPDH (hs99999905) and TaqMan Universal PCR Master Mix (Applied Biosystems). The quantitative PCR reactions were performed using CFX96-Touch Real Time PCR System (BioRad) and analyzed using BioRad CFX Manager (version 1.6). Fold differences in mRNA-levels for ciPTEC-OAT1 and ciPTEC-OAT3 were calculated using GAPDH as a reference gene and normalized to parent ciPTEC.

[0117] Data Analysis

[0118] A Michaelis-Menten equation was combined with linear diffusion to fit fluorescein uptake data after background subtraction with GraphPad Prism (version 5.03). For calculation of IC.sub.50 values, log (concentration inhibitor) versus fluorescein uptake was plotted after background subtraction using GraphPad Prism.

[0119] For MTT and fluorescein inhibition assays, data were normalized to the viability or activity of untreated control cells. Non-linear regression with variable slope constraining the top to 100% was used to fit the data after background subtraction with GraphPad Prism. Statistics was performed by two-way ANOVA (two-tailed, =0.05) using GraphPad Prism as well. All data is presented as meanSEM of at least three separate experiments (n=3) performed in triplicate, unless stated otherwise.

Example 2.Functional OAT Expression in ciPTEC

[0120] The absence of endogenous OAT1 and OAT3 expression in ciPTEC was demonstrated by exposure to fluorescein (1 M) for 10 min, which did not increase the intracellular fluorescence intensity as measured by flow cytometry (FIG. 1B, dashed line). Therefore, OAT transporters were introduced separately by lentiviral transduction. A schematic overview of the experimental approach is provided in FIG. 1A. The transporter genes SLC22A6 and SLC22A8 were cloned under regulation of a CMV promoter and a TetO2 site to conditionally induce their expression. Remarkably, basal expression and function upon transduction of both OAT transporters was positive without tetracycline induction, and was not influenced by this inducer (data not shown). Fluorescein uptake capacity (without induction by tetracycline) was used to discriminate between successfully transduced cells and non-transduced cells, reflected by two sub-populations in the flow cytometer histograms (ciPTEC-OAT1, FIG. 1C, ciPTEC-OAT3, FIG. 1D). When exposed to 1 M fluorescein for 10 min, a small cell population accumulated the fluorescent substrate, which was immediately selected using FACS. The fraction of OAT1 positive cells selected (FIG. 1E) accounted for only 8.3% of the total population, suggesting that a down-regulation in OAT expression upon culturing can be part of a survival mechanism. The enriched population accumulated fluorescein efficiently, and was sensitive to inhibition by para-aminohippuric acid, a known OAT1 substrate and/or inhibitor (FIG. 1F). The ciPTEC-OAT1 population enriched by FACS and the non-enriched ciPTEC-OAT3 population were subcloned to obtain homogeneous cell populations with high functional OAT transporter expression, demonstrated by qPCR. Expression of OAT1 and OAT3 in the respective cell lines was semi-quantified in relation to GAPDH expression and appeared to be 0.80.1 for ciPTEC-OAT1 and 0.090.01 for ciPTEC-OAT3, which was comparable to the relative levels in human kidney tissue homogenates (1.00.1 and 0.20.01 for OAT1 and OAT3, respectively; experiments performed in duplicate). Intact tubular phenotype was further demonstrated by functionally active OCT2, for which a drug-interaction with cimetidine was shown to be similar to the parent cell line (FIG. 2B).

Example 3Drug-Interaction at the Site of OAT1 and OAT3

[0121] Pharmacokinetics of OAT-mediated fluorescein transport was investigated by studying the time- and concentration-dependent uptake of the substrate. Fluorescein uptake demonstrated partial saturation in OAT1 and OAT3 expressing cells (FIGS. 3A, B and D) for which a K.sub.m and a V.sub.max value were determined, taking a passive diffusion component k.sub.d into account (Table 1). Fluorescein affinity was approximately 5-fold higher for OAT1 than for OAT3. Upon fluorescein exposure (10 min, 1 M), confocal fluorescent imaging confirmed uptake in ciPTEC-OAT1 and ciPTEC-OAT3 (FIGS. 3C and E). To demonstrate that the uptake was indeed transporter mediated, specific inhibition of fluorescein uptake in the presence of two concentrations of para-aminohippuric acid (10 M or 100 M) or estrone sulfate (3 M or 100 M) in ciPTEC-OAT1 and ciPTEC-OAT3 respectively, was studied (FIGS. 3B and 3D). CiPTEC-OAT1 and ciPTEC-OAT3 were validated further by determination of IC.sub.50 values using concentration-dependent inhibition of fluorescein uptake in presence of any one of para-aminohippuric acid, estrone sulfate, probenecid, furosemide, cimetidine, or diclofenac (FIG. 4, Table 2). Overall, IC.sub.50 values calculated in these models are in close agreement with previously reported values (Table 2). Further confirmation of specificity was obtained by using metformin, which did not affect OAT-mediated fluorescein uptake in both ciPTEC-OAT1 and ciPTEC-OAT3, as metformin is an OCT substrate and not an OAT substrate (Kimura et al., 2005). The experiments depicted in FIG. 4 were performed in cells spanning 29 passages after transduction. The small variations in this data and the maintained fluorescein uptake both indicate stable transduction and high robustness of transporter function in ciPTEC-OAT1 and in ciPTEC-OAT3.

Example 4OATs Mediate Antiviral-Induced Toxicity

[0122] Toxicity of antivirals was reported to be associated with renal tubular uptake mediated by OAT1 and OAT3 (Kohler et al., 2011; Takeda et al., 2002; Ciglar et al., 1999). Therefore, the effects of antivirals on OAT function and on cell viability was investigated upon drug exposures. Concentration-dependent inhibition of fluorescein uptake via OAT1 was observed by adefovir, cidofovir, tenofovir and zidovudine, while OAT3 was only associated with zidovudine-fluorescein interactions (FIG. 5, Table 3). Next, the DDI indices were determined. The United States Food and Drug Administration (FDA) draft DDI guideline (Huang & Zhang, 2012) states that a ratio between unbound plasma concentration and IC.sub.50 (C.sub.max,u/IC.sub.50) higher than 0.1 corresponds to a high chance of clinical drug interaction and a low potential for false negative results. For adefovir, cidofovir, and zidovudine, the IC.sub.50 value was less than 10 times the maximal free plasma concentration (C.sub.max,u/IC.sub.50>0.1), and, therefore, at clinically relevant plasma concentrations, inhibition of OAT1 is likely and DDI with OAT1 transporter substrates were defined as clinically relevant in this study.

[0123] Next, cytotoxicity caused by all four antivirals was evaluated after exposure of ciPTEC to the drugs for 24-72 h. As a measure of cytotoxicity, cell viability was analyzed by cellular dehydrogenase capacity, metabolizing MTT into purple formazan. In the parent ciPTEC, viability was not affected by any of the antivirals (48 hr, 1 mM), while adefovir, cidofovir and tenofovir significantly affected cell viability in ciPTEC-OAT1 and only tenofovir slightly decreased ciPTEC-OAT3 viability (FIG. 6A). Antiviral-induced toxicity was evaluated in more detail, demonstrating a concentration- and time-dependent decrease in viability by adefovir, cidofovir and tenofovir in ciPTEC-OAT1, while the effect was less pronounced in ciPTEC-OAT3 (FIG. 6B and Table 4). These findings indicate the direct involvement of the OAT transporters in antiviral-mediated nephrotoxicity, although IC.sub.50 values found in the current study are higher compared to those obtained in previous studies (Table 4). In this regard, it should be noted that the present system involves a highly relevant set of functional transporters, whereas previous studies often only overexpressed OATs or at least did not express all transporters that are active in ciPTEC-OAT cells, which makes ciPTEC-OAT a more relevant model system. The cytotoxic effect of the antivirals correlated nicely with the inhibitory effect on fluorescein uptake shown in FIG. 5, except for zidovudine. Despite a clear inhibition of fluorescein uptake by zidovudine, which suggests OAT-mediated uptake, this compound did not affect cell viability as determined by the MTT assay. To investigate a potential protective effect via intact efflux transporters in ciPTEC, cells were exposed to zidovudine at 10C.sub.max (50 M) in the presence of the MRP4 inhibitor MK571, and the BCRP inhibitor KO143. This did not affect cell viability in ciPTEC, ciPTEC-OAT1, or ciPTEC-OAT3, indicating that efflux transporters did not counteract intracellular exposure to zidovudine, thus that efflux transporters did not reduce the cytotoxic potential of zidovudine.

[0124] Antiviral-induced nephrotoxicity was shown to be associated with OAT-mediated uptake and further evaluated in the present disclosure (Izzedine et al., 2009; Kohler et al., 2011; Cihlar et al., 2009; Zhang et al., 2015). It is demonstrated here that OAT1 or OAT3 expression is required for induction of toxicity by adefovir, cidofovir and tenofovir in ciPTEC. The relation between OAT1 transporter affinity and toxicity was described earlier using HeLa cells that transiently expressed hOAT1, in which cidofovir showed a higher affinity as well as a higher toxicity compared to tenofovir (Mandikova et al., 2013). In agreement, when the cytotoxic potential of NtRTIs in ciPTEC-OAT1 at 72 h of exposure was ranked, it was found that cidofovir has a higher potency compared to tenofovir and adefovir (Cihlar et al., 2009; Zhang et al., 2015). On the other hand, the low potency of adefovir shown in the present disclosure contrasts to the cytotoxicity reported for other cell models (Khamdang et al., 2004; Wang & Sweet, 2012). In general, the toxic potency of the antivirals in ciPTEC is lower as compared to hOAT1-CHO and HEK-OAT1, possibly due to the presence of functional metabolic enzymes and an intact efflux machinery in ciPTEC (Cihlar et al., 2009; Zhang et al., 2015; Imaoka et al., 2007). Activity of phase I and phase II metabolizing enzymes was demonstrated in ciPTEC of which the UGT2B7 subfamily is a possible cause of the tolerance for zidovudine observed in the present study (Mutsaers et al., 2013). While adefovir, cidofovir and tenofovir are largely excreted unchanged by the kidneys, only 23% of zidovudine is eliminated via the urine without metabolic alterations (Varma et al., 2009). Zidovudine undergoes either phase II metabolism into the non-toxic 5-zidovudine-O-glucuronide or the antiviral is phosphorylated resulting in mitochondrial toxicity (Blum et al., 1988; Lewis et al., 2003). As both glucuronidation and phosphorylation take place at the same functional group of zidovudine (5-OH), the low toxicity of zidovudine suggests a favour for glucuronidation in ciPTEC. Although glucuronidation predominantly takes place in the liver, UGT2B7 expression in ciPTEC likely contributes to zidovudine detoxification. The efflux pumps MRP4 and BCRP in ciPTEC, which can minimize intracellular exposure, appeared to be of minor importance, as efflux inhibition did not reduce viability of ciPTEC upon antiviral exposure.

Example 5Regulated OAT Expression in ciPTEC

[0125] Results and Conclusion

[0126] Epidermal growth factor (EGF) dependent regulation of OAT1 drug transport expression and activity was determined in a representative ciPTEC-OAT1 (ciPTEC.OAT1.4B2 DSM ACC3279). The expression of OAT1 was significantly increased in the presence of EGF (FIG. 7A). As a result, the uptake of fluorescein (an OAT1 substrate) was significantly increased upon EGF exposure (calculated Vmax of 14.6 vs 9.7 A.U.; FIG. 7B). These data depict that OAT1 expression and transport activity is regulated in ciPTEC-OAT1 by EGF, likely via the EGF receptor. These data demonstrates that the human renal background of ciPTEC-OAT1 provides a physiological relevant model with intact regulation of OAT transport.

[0127] Methods

[0128] Cell Culture and EGF Exposure

[0129] ciPTEC-OAT1. 4B2 DSM ACC3279 was cultured in phenol red-free DMEM/F12 (Invitrogen, Breda, The Netherlands), as described in example 1 here above. Cells were seeded at a density of 63,000 cell/cm.sup.2, cultured for 24 hours at 33 C. and subsequently at 37 C. for 7 days. To study the effects of EGF, matured ciPTEC-OAT1 were treated for 48 hours in the presence or absence of EGF (10 ng/mL).

[0130] Fluorescein Uptake

[0131] The uptake of fluorescein was used to determine the changes in activity of OAT1 mediated transport. Cells were washed twice before incubation at 37 C. for 10 minutes with fluorescein. After incubation, plates were washed twice and cells were lysed with 0.1 M NaOH. Subsequently, intracellular fluorescence was measured via an Ascent Fluoroskan FL microplate reader (excitation: 494 nm, emission: 512 nm). To calculate Vmax, a Michaelis-Menten equation was combined with linear diffusion to fit fluorescein uptake data after background subtraction with GraphPad Prism (version 5.03).

[0132] Gene Expression

[0133] OAT1 gene expression profiling was performed by isolating total RNA from cells grown in six-well plates, using an RNeasy Mini kit (Qiagen, Venlo, The Netherlands), according to the manufacturers specifications. Subsequently, cDNA was synthesized using the Omniscript RT-kit (Qiagen). Subsequently, quantitative PCR was performed in a CFX96 Real-Time PCR detection system (Bio-rad, Veenendaal, The Netherlands) according to the manufacturers conditions. GAPDH was used as reference gene for normalization and relative expression levels were calculated as fold change using the 2.sup.CT method. The primer-probe sets for the quantitative PCR were obtained from Applied Biosystems: GAPDHhs99999905 ml and OAT1hs00537914.

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