Test compounds used for the assays were identified from the literature. All test compounds and reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. Each compound was prepared and serially diluted in DMSO in 1536-well microplates to yield 11 concentrations. The final concentration of the 34 well-characterized compounds related to nephrotoxicity and 10 compounds showing renal toxicity in the 5 μL assay volume ranged from 31.2 nM to 184 μM and 0.7 nM to 46 μM, respectively.

Human primary kidney proximal tubule epithelial cells were obtained from Zen-Bio Inc. (Research Triangle Park, NC). Zinc finger nuclease (ZFN) pairs were delivered into the SA7K cells by nucleofection. Cells were split into two pools, one for population doubling studies and the other for FACS sorting (single-cell isolation). Cells were sorted based on negative selection for carboxyfluorescein succinimidyl ester (CFSE), a dye that distinguishes fast growing cells from slow growing ones. Cells that did not have detectable CFSE were single-cell sorted into 96-well plates. Clones with the desired mutation were identified initially by fragmentation analysis and later confirmed by sequencing of the targeted area. SA7K cells were cultured in RPTEC Complete Supplement medium while nephrotoxicity experiments were carried out using RPTEC Tox Supplement medium (Sigma-Aldrich). HK-2 cells were purchased from ATCC (Manassas, VA) and cultured in keratinocyte growth medium (Lonza Inc, Mapleton, IL).

HPTC, SA7K and HK-2 cells were seeded on 24-well plates at 0.125×10⁶ cells/well and cultured for 3 days prior to RNA extraction (RNeasy Protect Mini Kit, Qiagen, Valencia, CA). Total extracted RNA was quantified and purity verified using the Thermo Scientific NanoDrop 2000 Spectrophotometer. RNA (150 ng) were analyzed per reaction, in triplicate, using the Applied Biosystems’ TaqMan^® RNA-to-CT™ 1-step kit in MicroAmp^® optical 96-well reactions plates. GAPDH was used as the negative control. Taqman^® gene expression assay probes were acquired from Thermo Fisher Scientific (Waltham, MA).

SA7K cells were seeded on BD Falcon 24-well plates at 0.5×10⁶ cells/well for 2 days. For uptake assays (OAT1 and OCT2), cells were pre-incubated with inhibitor (probenecid or doxepin, where indicated) for 10 min at 37°C. Substrates (p-aminohippuric acid (PAH) for OAT1, amantadine for OCT2) were added and incubated for another 10 minutes at 37°C. The reactions were stopped with ice-cold buffer followed by washing 3 times with ice-cold HBSS. Samples were extracted with 250 μL methanol and analyzed by liquid chromatography–tandem mass spectrometry (LC/MS/MS). For efflux assays (P-gp and MRP2), cells were pre-incubated with inhibitor (verapamil or MK571, where indicated) for 30 minutes at 37°C, then incubated with substrate with or without the inhibitor for another 10 minutes. Substrates used were SN-38 for MRP2 and digoxin for P-gp. Samples were extracted with 250 μL methanol and dried under nitrogen. Samples were resuspended in 125 µL of 50% acetonitrile and analyzed by LC-MS/MS.

The cell viability of SA7K and HK-2 cells was measured using a CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI) after compound treatment. The change of intracellular ATP content indicates the number of metabolically competent cells. The SA7K and HK-2 cells were seeded at 2,000 cells/5 μL/well in 1536-well white, solid bottom plates (Greiner Bio-One, North America, NC) using a Multidrop Combi (Thermo Fisher Scientific, Waltham, MA). After the cells were incubated at 37°C for 5 hours, 23 nL of compounds at 11 concentrations was transferred to the assay plates by a Pintool station (Kalypsys, San Diego, CA). DMSO was used as a vehicle control and tetraoctylammonium bromide was used as a positive control. For the 15 well-known compounds the plates were incubated for 48 hours at 37°C, followed by the addition of 5 μL per well of CellTiter-Glo reagent. After 30 minutes of incubation in dark at room temperature, the luminescence intensity was measured using a ViewLux plate reader (PerkinElmer, Shelton, CT).

Caspase activity was measured in SA7K and HK-2 cells after 24 h compound treatment using a Caspase-Glo 3/7 Assay kit (Promega, Madison, WI). The SA7K and HK-2 cells were dispensed in assay medium at 2,000 cells/5 μL/well in 1536-well white plates using a Multidrop Combi. After the cells were incubated at 37°C for 5 hours, 23 nL of compounds was added via the Pintool station. Staurosporine, a caspase inducer, was used as a positive control. Treated cells were incubated for 24 hours at 37°C, followed by the addition of 5 μL per well of Caspase-Glo 3/7 reagent. After 30 minutes of incubation at room temperature, the luminescence intensity of the plates was measured using a ViewLux plate reader.

The Mitochondrial Membrane Potential Assay (Codex Biosolutions, Montgomery Village, MD) is a fluorescence-based assay that quantifies the mitochondrial membrane potential changes. The SA7K and HK-2 cells were dispensed at 2000 cells/5 µL/well in 1536-well black clear-bottom assay plates. The assay plates were incubated at 37°C overnight before 23 nL of compound was transferred from the compound plate to the assay plate via a Pintool station. The assay plates were incubated for 1 hour at 37°C, followed by the addition of 5 μL/well of dye-loading solution. After the assay plates were incubated at 37°C for 30 minutes, fluorescence intensity at 490 nm excitation and 535 nm emission wavelengths were measured using an EnVision plate reader (PerkinElmer, Shelton, CT). Data were expressed as the ratio of 590 nm/535 nm emissions.

Data were analyzed as described previously [25Xia M, Huang R, Witt KL, et al. Compound cytotoxicity profiling using quantitative high-throughput screening. Environ Health Perspect 2008; 116(3): 284-91.
[http://dx.doi.org/10.1289/ehp.10727] [PMID: 18335092] , 26Attene-Ramos MS, Huang R, Michael S, et al. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Perspect 2015; 123(1): 49-56.
[PMID: 25302578] ]. Briefly, data were normalized to DMSO controls (0%) and the maximal response of positive control compounds (100% or - 100%) and corrected by applying a NCATS in house pattern correction algorithm [27Wang Y, Huang R. (2016). Correction of microplate data from high throughput screening. In: Zhu H, Xia M, Eds. High-Throughput Screening Assays in Toxicology. Humana Press 1473.]. Concentration response data for each compound were fitted to the Hill equation, yielding concentrations of half-maximal effective or inhibitory concentration (EC₅₀/IC₅₀) and maximal response (efficacy).

In the present study, we modified human primary renal proximal tubule epithelial cells (HPTC) by knocking out a cell cycle regulatory protein using ZFN technology, thereby enabling extended cell proliferation. The resulting clones demonstrated an extended population doubling capacity compared with HPTC (passage 2) as illustrated in Fig. (1A). SA7K clone was selected based primarily on growth and morphological characterization. This clone exhibited normal epithelial morphology including dome formation, which is indicative of transepithelial solute transport (Fig. 1B). The proximal tubule origin of the cells was confirmed by expression of the transmembrane biomarker aminopeptidase N (CD13) as shown in Fig. (1C). Several additional proximal tubule specific functional properties were also evaluated in the SA7K cells. Albumin resorption from the urinary filtrate is thought to occur via cubilin/megalin receptors located on the apical membrane of proximal tubule cells [28Verroust PJ, Christensen EI. Megalin and cubilinthe story of two multipurpose receptors unfolds. Nephrol Dial Transplant 2002; 17(11): 1867-71.
[http://dx.doi.org/10.1093/ndt/17.11.1867] [PMID: 12401836] ]. SA7K cells demonstrated time- and temperature-dependent albumin uptake using FITC-labeled albumin (Fig. 1D). Proximal tubules are known to respond to the parathyroid hormone (PTH) by increasing calcium resorption and phosphate secretion through a cAMP-dependent mechanism [29Toutain H, Vauclin-Jacques N, Fillastre JP, Morin JP. Isolation of a pure suspension of proximal tubular cells from the rabbit kidney: morphological, biochemical and metabolic assessment. Cell Biol Int Rep 1989; 13(8): 701-10.
[http://dx.doi.org/10.1016/0309-1651(89)90046-5] [PMID: 2805082] ]. Exposure of SA7K cells to the PTH resulted in a concentration- and time-dependent increase in cAMP, suggesting PTH receptor expression (Fig. 1E). Gamma glutamyl transpeptidase (GGT) is an enzyme located on the brush border (apical) surface of proximal tubules [30Lebargy F, Bulle F, Siegrist S, Guellaen G, Bernaudin JF. Localization by in situ hybridization of gamma-glutamyl transpeptidase mRNA in the rat kidney using 35S-labeled RNA probes. Lab Invest 1990; 62(6): 731-5.
[PMID: 1972767] ]. GGT activity was detected by monitoring cleavage of gamma glutamyl-p-nitroanilide and was comparable to HPTC (Fig. 1F).

Fig. (1) Growth, morphological and functional characterization of SA7K cells. (A) Normal epithelial morphology of SA7K cells, including dome formation; (B) Expression of the proximal tubule marker aminopeptidase N (CD13) in SA7K cells; (C) Extended population doublings in ZFN-modified SA7K versus HPTC cells; (D) Uptake of Albumin-FITC; (E) Response to Parathyroid Hormone (PTH); (F) GGT Activity in HPTC versus SA7K cells. Scale bar: 50 µm.

Transporters play an important role in the uptake/secretion mechanisms of proximal tubule cells, and may contribute to elevated, potentially toxic intracellular concentration of drugs or metabolites. As noted above, immortalized renal cell lines have been reported to have low or non-detectable levels of one or more drug transporters [21Jenkinson SE, Chung GW, van Loon E, Bakar NS, Dalzell AM, Brown CD. The limitations of renal epithelial cell line HK-2 as a model of drug transporter expression and function in the proximal tubule. Pflugers Arch 2012; 464(6): 601-11.
[http://dx.doi.org/10.1007/s00424-012-1163-2] [PMID: 23014881] ]. Therefore, the mRNA expression levels of the major uptake and efflux drug transporters were measured in SA7K and HK-2 cells (Fig. 2). Nine transporters were detected at various levels in the SA7K cells, with the notable exception of OAT1 and OAT3. The highest levels of expression were observed for P-gp, MRP4, OATP4C1 OCTN1 and OCTN2 (threshold cycle, CT, values ranging from 21-26), while lower levels were observed for MATE1, MATE2-K, MRP2 and OCT2 (CT values from 27-32). Seven out of the nine transporters measured were higher in SA7K cells than HK-2 (Fig. 2B) . In addition, the expression profile of uptake and efflux transporters in SA7K cells was similar to that of the early passage human primary donor cells used as source material.

Fig. (2) Expression profile of transporters. (A) HPTC versus SA7K cells; (B) SA7K versus HK-2 cells. The mRNA levels of nine known proximal tubule transporters were measured by RT-PCR in HPTC, SA7K and HK-2 cells.

It is well-known that mRNA levels do not always correlate with transporter protein levels or activity [31Ohtsuki S, Schaefer O, Kawakami H, et al. Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities. Drug Metab Dispos 2012; 40(1): 83-92.
[http://dx.doi.org/10.1124/dmd.111.042259] [PMID: 21994437] ]. To functionally assess the transporters, we measured the activities of two uptake and two efflux transporters. Uptake of the OCT2 substrate amantadine and the OAT1 substrate PAH were readily observed in SA7K cells (Fig. 3A, B). While doxepin (10 µM) inhibited amantadine uptake by 46%, probenecid inhibited PAH uptake by 64% and 78% at concentrations of 10 µM and 100 µM, respectively. Interestingly, OAT-dependent uptake was observed in spite of undetectable mRNA levels. The activity of the OATs was highly dependent on the culture conditions used. For the efflux transporters, P-gp-mediated efflux of digoxin was inhibited with 50 and 100 µM verapamil, leading to a 1.8-fold and 2.5-fold increase in intracellular digoxin, respectively (Fig. 3C). Similarly, MRP2-mediated efflux of SN-38 was inhibited using 10 µM MK571, leading to a 50% increase in intracellular SN-38 (Fig. 3D). Thus, SA7K cells maintained functional activity for both uptake and efflux transporters, suggesting their potential use in studying drug-transporter interactions and evaluating renal toxicants.

To evaluate the nephrotoxicity, SA7K and HK-2 cells were both exposed to a group of 34 well-characterized compounds, 15 of which are known to be toxic to proximal tubule cells, 10 of which are not directly toxic for PT cells and nine of which are non-nephrotoxic compounds (20); all 34 compounds were used to treat cells at various concentrations ranging from 31.2 nM to 184 µM. After exposure, cell viability, cell apoptosis and mitochondrial membrane potential (MMP) were determined. To determine cell viability, the endpoint for evaluating cytotoxicity was the measurement of cellular ATP content. Caspase 3/7 induction in cells is an indicator of cell apoptosis, which can be employed to evaluate the toxicity of compounds. Mitochondrial damage and dysfunction are known to be closely related to kidney diseases [32Che R, Yuan Y, Huang S, Zhang A. Mitochondrial dysfunction in the pathophysiology of renal diseases. Am J Physiol Renal Physiol 2014; 306(4): F367-78.
[http://dx.doi.org/10.1152/ajprenal.00571.2013] [PMID: 24305473] ]. Therefore, mitochondrial function represents an initial step in predicting exposure-related toxicity of compounds that potentially reduce the mitochondrial membrane potential (MMP) [26Attene-Ramos MS, Huang R, Michael S, et al. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Perspect 2015; 123(1): 49-56.
[PMID: 25302578] ]. Out of the 15 nephrotoxicants tested, 10 showed nephrotoxic effects in the three endpoints tested (Table 1). The other five compounds including gentamicin, 5-fluorouracil, tobramycin, ifosfamide and tenofovir were negative in our assays (data not shown); however, the reported IC₅₀s of these 5 compounds were much higher than the highest concentration used in our study [20Li Y, Kandasamy K, Chuah JK, et al. Identification of nephrotoxic compounds with embryonic stem-cell-derived human renal proximal tubular-like cells. Mol Pharm 2014; 11(7): 1982-90.
[http://dx.doi.org/10.1021/mp400637s] [PMID: 24495215] ]. Among the 10 positive compounds confirmed in our study, eight and seven compounds showed cytotoxic effects in HK-2 and SA7K cells respectively. In the caspase 3/7 and MMP assays, SA7K cells showed higher sensitivity than HK-2 cells, and the three endpoints were complementary in predicting nephrotoxicity. The other 19 compounds were inactive (supplemental Table 1) in viability and caspase 3/7 assays except atorvastatin and triiodothyronine; atorvastatin showed cytotoxicity and apoptosis induction in both SA7K cells and HK-2 cells and triiodothyronine showed cytotoxicity only in HK-2 cells. Moreover, four compounds out of 19, including triiodothyronine, atorvastatin, ibuprofen and lindane decreased MMP in SA7K and HK-2 cells, indicating good specificity of SA7K cells. Ibuprofen and lindane are nephrotoxic compounds that are not directly toxic to PT cells, implicating their induction of renal toxicity may occur through the mitochondria related pathway. Therefore, MMP assays might be a good endpoint through which to evaluate nephrotoxicity. Thus, multiple assays employing cell viability, cell apoptosis and MMP may be a promising avenue for early detection of nephrotoxicity for compounds with a high potency.

To further evaluate SA7K cells, we tested 10 more compounds, which have been reported to be associated with renal dysfunction, including camptothecin [33Li W, Lam M, Choy D, Birkeland A, Sullivan ME, Post JM. Human primary renal cells as a model for toxicity assessment of chemo-therapeutic drugs. Toxicol In Vitro 2006; 20(5): 669-76.
[http://dx.doi.org/10.1016/j.tiv.2005.09.016] [PMID: 16289493] ], sunitinib malate [34Jhaveri KD, Flombaum CD, Kroog G, Glezerman IG. Nephrotoxicities associated with the use of tyrosine kinase inhibitors: a single-center experience and review of the literature. Nephron Clin Pract 2011; 117(4): c312-9.
[http://dx.doi.org/10.1159/000319885] [PMID: 21051905] ], sulfinpyrazone [35Docci D, Mambelli M, Manzoni G, Turci F, Salvi G. Acute renal failure secondary to sulfinpyrazone treatment after myocardial infarction. Nephron 1984; 37(3): 213-4.
[http://dx.doi.org/10.1159/000183248] [PMID: 6738772] ], mitomycin C [36Wen MC, Chen CH, Ho WL. Mitomycin C-induced renal insufficiency: a case report. Kaohsiung J Med Sci 2003; 19(6): 317-21.
[http://dx.doi.org/10.1016/S1607-551X(09)70479-6] [PMID: 12873041] ], vinblastine sulfate [37Hejazi S. Study of nephrotoxic effects of vincristine treatment in mice. Eur J Cancer 2013; 49: S99.], doxorubicin [38Lahoti TS, Patel D, Thekkemadom V, Beckett R, Ray SD. Doxorubicin-induced in vivo nephrotoxicity involves oxidative stress-mediated multiple pro- and anti-apoptotic signaling pathways. Curr Neurovasc Res 2012; 9(4): 282-95.
[http://dx.doi.org/10.2174/156720212803530636] [PMID: 22873725] ], taxol [39Rabah SO. Acute Taxol nephrotoxicity: Histological and ultrastructural studies of mice kidney parenchyma. Saudi J Biol Sci 2010; 17(2): 105-14.
[http://dx.doi.org/10.1016/j.sjbs.2010.02.003] [PMID: 23961065] ], ochratoxin A [40Hope JH, Hope BE. A review of the diagnosis and treatment of Ochratoxin A inhalational exposure associated with human illness and kidney disease including focal segmental glomerulosclerosis. J Environ Public Health 2012; 2012: 835059.], ergotamine [41Damstrup L, Jensen TT. Retroperitoneal fibrosis after long-term daily use of ergotamine. Int Urol Nephrol 1986; 18(3): 299-301.
[http://dx.doi.org/10.1007/BF02082717] [PMID: 3771129] ], and digoxin [42Pincus M. Management of digoxin toxicity. Aust Prescr 2016; 39(1): 18-20.
[http://dx.doi.org/10.18773/austprescr.2016.006] [PMID: 27041802] ]. The potencies of these compounds are listed in Table 2. Among these 10 compounds tested in the viability assay, seven compounds displayed toxicity utilizing the HK-2 cells, while six compounds showed toxicity in the SA7K cells. In the caspase 3/7 assay, five compounds exhibited an increase in caspase 3/7 activity when treating the HK-2 cells, whereas six compounds showed an increase in this endpoint in the SA7K cells. In the MMP assay, five compounds decreased MMP in both HK-2 and SA7K cells. With respect to the IC₅₀ values, there were several compounds that showed differences in sensitivity between HK-2 and SA7K. For example, Ochratoxin A, camptothecin, and doxorubicin showed different dose-response curves in HK-2 and SA7K cells (Fig. 4A-C). These different responses may be related to differences in expression of the renal transporters or other related components of nephrotoxicity pathways.

Table 2
Evaluation of nephrotoxic compounds in HK-2 and SA7K Cells.

Fig. (4) Concentration response curves of selected compounds in HK-2 and SA7K cells. (A) Effect of ochratoxin A on cell viability; (B) Effect of campothecin on caspase 3/7; (C) Effect of doxorubicin on MMP.