Identification of Nephrotoxic Compounds with Embryonic Stem-Cell

Feb 4, 2014 - National University of Singapore has approved work with. HUES cells and with commercial HPTC. Drug Treatment. Cryopreserved cells were ...
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Identification of Nephrotoxic Compounds with Embryonic Stem-CellDerived Human Renal Proximal Tubular-Like Cells Yao Li, Karthikeyan Kandasamy, Jacqueline Kai Chin Chuah, Yue Ning Lam, Wei Seong Toh,† Zay Yar Oo, and Daniele Zink* Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore S Supporting Information *

ABSTRACT: The kidney is a major target for drug-induced toxicity, and the renal proximal tubule is frequently affected. Nephrotoxicity is typically detected only late during drug development, and the nephrotoxic potential of newly approved drugs is often underestimated. A central problem is the lack of preclinical models with high predictivity. Validated in vitro models for the prediction of nephrotoxicity are not available. Major problems are related to the identification of appropriate cell models and end points. As drug-induced kidney injury is associated with inflammatory reactions, we explored the expression of inflammatory markers as end point for renal in vitro models. In parallel, we developed a new cell model. Here, we combined these approaches and developed an in vitro model with embryonic stem-cell-derived human renal proximal tubularlike cells that uses the expression of interleukin (IL)-6 and IL-8 as end points. The predictivity of the model was evaluated with 41 well-characterized compounds. The results revealed that the model predicts proximal tubular toxicity in humans with high accuracy. In contrast, the predictivity was low when well-established standard in vitro assays were used. Together, the results show that high predictivity can be obtained with in vitro models employing pluripotent stem cell-derived human renal proximal tubular-like cells. KEYWORDS: stem cell, renal proximal tubular cell, embryonic stem-cell-derived human renal cell, drug-induced kidney injury, nephrotoxicity, predictive in vitro model, interleukins

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INTRODUCTION The kidney is a major target organ for drug-induced toxicity. About 5% of all hospitalized patients and ∼20%−30% of ICU patients develop acute kidney injury (AKI), and ∼20%−25% of these cases are due to nephrotoxic drugs.1−3 The nephrotoxic potential of alternative and new drugs is often underestimated.4 Typically, nephrotoxicity is detected only late during drug development and accounts for 2% of drug attrition during preclinical studies and 19% in phase 3.5 One major problem is the lack of preclinical models with high predictivity. Regulatory accepted or validated in vitro models for the prediction of nephrotoxicity are currently not available. The cells of the renal proximal tubule (PT) are often affected by drug-induced toxicity due to their roles in glomerular filtrate concentration and the transport and metabolism of organic compounds.2,3,6 Therefore, PT-derived cells are often employed in in vitro assays. However, PT-derived cell lines are often insensitive to well-known nephrotoxicants.7,8 This is due to functional changes and changes in drug transporter expression associated with immortalization.8−10 We have recently compared human primary renal proximal tubular cells (HPTC) and PT-derived human and porcine cell lines in a new model for the prediction of PT toxicity that uses the expression of interleukin (IL)-6 and IL-8 as end points.11 The mean and median values of all major performance metrics © 2014 American Chemical Society

ranged between 0.76 and 0.85 when HPTC were used. The values were significantly lower with cell lines. However, the use of primary cells like HPTC is associated with a number of problems such as limited cell source, limited proliferative capacity,9,12 functional changes during passaging,13 interdonor-variability11,14 as well as de- and trans-differentiation in vitro.15,16 Because of the problems with primary cells and cell lines, stem-cell-based approaches would be attractive. We have recently established the first protocol for the differentiation of human embryonic stem cells (hESC) into HPTC-like cells in vitro.17 The results revealed that gene and protein expression patterns of hESC-derived HPTC-like cells and HPTC were comparable. HPTC-like cells formed polarized epithelia with tight junctions and an apical brush border in vitro, and generated tubular structures in vitro and in vivo. Further, HPTC-like cells showed functional characteristics of HPTC and integrated into renal epithelia in organ cultures.17 Special Issue: Engineered Biomimetic Tissue Platforms for in Vitro Drug Evaluation Received: Revised: Accepted: Published: 1982

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compounds (Table 1 and Supporting Information, Table S1) dissolved in the same vehicles. Vehicle controls were always included, and all data were normalized to the respective vehicle controls. qPCR and Immunostaining. qPCR and immunostaining were performed as described11,17 with the same primers and antibodies. In addition, antibodies against GLUT1, SGLT2 (Abcam, Cambridge, MA, USA), and PEPT1 (Santa Cruz Biotechnology, Dallas, TX, USA) were used, as well as a primer set for SLC22A2/organic cation transporter (OCT)2 with the following sequence: forward, 5′-GCTGTACCCCACATTCATTAGGA-3′; reverse, 5′-GGGAGCTCAAGCCAGATGTTA-3′. The primer set was purchased from Sigma-Aldrich (Singapore), and the amplicon length was 120 base pairs. Standard in Vitro Assays. Cells were treated in the same way as that for the IL-6/IL-8-based assay. Cellular ATP depletion was measured with the Molecular Probes ATP determination kit (Life Technologies, Carlsbad, CA, USA). Compound 11 was also tested with the CellTiter-Glo Assay (Promega, Madison, WI, USA). Assay kits for determining GSH depletion (GSH-Glo glutathione assay) and LDH leakage (CytoTox-ONE homogeneous membrane integrity assay) were purchased from Promega. All assays were performed according to the manufacturers’ instructions. Definitions and Data Analysis. Analysis of results obtained with the IL-6/IL-8-based assay was performed as described.11 For standard definitions for performance metrics, see Figure S1 (Supporting Information). Statistics. Microsoft Excel 2010 was used for all calculations. The unpaired t test was used for statistics. The normal distribution of the data was confirmed using SigmaStat (3.5) (Systat Software Inc., Chicago, IL, USA). Z′ values were calculated as described.27

Here, we tested the predictive performance of an in vitro model employing hESC-derived HPTC-like cells and using IL6 and IL-8 expression as end points. These end points were further explored because their use in combination with HPTC resulted in high predictivity.11 In contrast, the prediction of PT toxicity has been difficult with end points that are associated with general cytotoxicity, such as cell death or ATP depletion.11,18 Also, the use of potential novel biomarkers for AKI, such as neutrophil-gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), has not been convincing in in vitro models (refs 11 and 19 and Predict-IV Project, third, fourth, and fifth periodic reports http://www. predict-iv.toxi.uni-wuerzburg.de/periodic_reports/). This is probably due to changes in the cell state and up-regulation of such markers in vitro even in the absence of drug treatment.11,20 In vivo, kidney injury and disease are associated with upregulation of IL-6 and IL-8.21−23 Increasing evidence suggests that pro-inflammatory cytokines play a central role in the pathophysiology of AKI, including nephrotoxin-induced AKI.24 For instance, cisplatin triggers increases in pro-inflammatory cytokine expression in the kidney.25 In concordance with these in vivo results, significant up-regulation of IL-6 and other inflammatory markers after exposure to nephrotoxicants has been demonstrated in a kidney culture model employing murine purified PTs.26 These findings are in agreement with the observation that IL-6 and IL-8 are up-regulated in vitro in human PT-derived cells after exposure to compounds that are specifically toxic for this cell type.11 Here, we characterized the predictive performance of an in vitro model that used IL-6 and IL-8 expression as an end point and employed hESC-derived HPTC-like cells.

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EXPERIMENTAL SECTION Cell Culture. HUES 7 cells were obtained from the Harvard Stem Cell Institute (Harvard University, Cambridge, MA, USA) at passage 11. They were differentiated at passage 16 into HPTC-like cells as described.17 Briefly, for differentiation the HUES 7 cells were seeded onto Matrigel and cultivated for 20 days in renal epithelial growth medium (REGM) containing various supplements and growth factors (REGM BulletKit, Lonza BioScience, Singapore). In addition, the medium was supplemented with 0.5% fetal bovine serum, 10 ng/mL of bone morphogenetic protein (BMP)2 and 2.5 ng/mL of BMP7 (R&D Systems, Minneapolis, MN, USA). HUES 7-derived HPTC-like cells were harvested and cryopreserved on day 20. HPTC (Lot.-Nr. 58488852; American Type Culture Collection (ATCC), Manassas, VA, USA; this lot is called HPTC 1 in our studies) were cultivated and used at passages 4 and 5 as before.11 The Institutional Review Board of the National University of Singapore has approved work with HUES cells and with commercial HPTC. Drug Treatment. Cryopreserved cells were thawed and seeded into multiwell plates at a density of 5 × 104 cells/cm2 (HPTC) or 1 × 105 cells/cm2 (HPTC-like). HPTC-like cells proliferated more slowly than HPTC after thawing. Therefore, HPTC-like cells were seeded at a higher density to ensure the formation of confluent epithelia after seeding. Before drug treatment, cells were cultivated for 3 days in commercial renal cell medium purchased from ATCC (HPTC) or Lonza BioScience (Singapore; HPTC-like cells). Both media contained 0.5% fetal bovine serum. Drug treatment was performed for 16 h as described before11 with the same set of 41

RESULTS Predictive Performance Determined with 41 Compounds. hESC were differentiated into HPTC-like cells as described.17 HPTC-like cells were harvested and cryopreserved after 20 days of differentiation. After thawing, cells were cultivated for 3 days. They were then either exposed to the test compounds for 16 h or were assessed in the untreated state for marker expression by quantitative real-time reverse transcription polymerase chain reaction (qPCR). Expression of 18 epithelial and HPTC-specific markers was assessed in untreated HPTC-like cells and HPTC (HPTC 1, this batch had been extensively characterized11,17). Eleven markers were expressed at similar or significantly higher levels in HPTClike cells (compared to HPTC, Figure 1). The genes that showed the highest expression levels in HPTC-like cells were the PT-specific markers aquaporin (AQP) 1, glucose transporter (GLUT) 5, and N-cadherin. Seven markers were expressed at lower levels in HPTC-like cells (compared to HPTC, Figure 1). In agreement with our previous results,17 these included megalin and the organic anion transporter (OAT) 3. For OAT1 and OAT3 > 100 widely overlapping substrates have been identified.28 OAT1 was expressed at similar levels in HPTC and HPTC-like cells (Figure 1). The expression levels of various transporters of 3-fold and in addition significant (P < 0.05) were marked with asterisks.

41 well-characterized drugs and environmental toxins was tested. Therefore, the results obtained with the different cell types were directly comparable. Most of the 41 compounds were drugs that are routinely and widely applied in clinical practice. Some compounds, like CdCl2 or lindane, were wellcharacterized environmental toxins. For all of the compounds or their derivatives, a wealth of human and animal in vivo and in vitro data were available (see Table S1 (Supporting Information) and ref 11). The 41 compounds were classified based on their clinical effects in humans (Table S1 (Supporting Information)). Compounds 1−22 were nephrotoxicants that are directly toxic for PT cells (group 1, Table 1; some of these compounds have also different negative effects on the kidney in addition to PT-specific toxicity). Compounds 23−33 were nephrotoxicants that are not directly toxic for PT cells and injure the kidney by other mechanisms (group 2, Table 1). In addition, 8 nonnephrotoxic drugs were included (group 3, Table 1, compounds 34−41). After drug exposure, IL-6 and IL-8 expression levels were determined by qPCR. All data were

a

The table lists the 41 compounds used, which were divided into three groups. Group 1 (compounds 1−22) represents nephrotoxicants that are directly toxic for PT cells. Group 2 (compounds 23−33) comprises nephrotoxicants that are not directly toxic for PT cells and injure the kidney by different mechanisms. Group 3 (compounds 34−41) represents non-nephrotoxic compounds. HPTC-like cells were exposed to these compounds at concentrations ranging from 1 μg/mL to 1000 μg/mL. Tables S2 and S3 (Supporting Information) display in detail for each compound and marker the expression levels obtained at all drug concentrations tested. The highest expression levels of IL-6 and IL-8 that were observed at any given concentration of a compound within the concentration range tested are highlighted (bold) in Tables S2 and S3 (Supporting Information). These highest expression levels of IL-6 and IL-8 are listed here in this table. The numbers show the mean fold expression ± s.d. (n = 3) relative to the vehicle control.

performed here with HPTC-like cells in the same way as before with HPTC, HK-2, and LLC-PK1 cells,11 and the same set of 1984

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normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression levels to account for changes in cell numbers. All compounds were tested at concentrations of 1 μg/mL, 10 μg/mL, 100 μg/mL, and 1000 μg/mL. As outlined before,11 this covered the widest useful range of concentrations, with a lack of drug-induced changes at concentrations below the lower limit and compromised solubility of many compounds at concentrations exceeding the upper limit. All results were normalized to the respective vehicle controls and expressed as fold change of IL-6 and IL-8 expression relative to the vehicle control. Detailed results on IL-6 and IL-8 expression for each drug at every concentration tested are listed in Tables S2 and S3 (Supporting Information). The highest levels of IL-6 and IL-8 expression determined for each compound within the concentration range tested are highlighted in Tables S2 and S3 (Supporting Information). These highest expression levels were summarized in Table 1. A drug was classified as positive and predicted as a PT-specific nephrotoxin if the highest increase in gene expression (Table 1) of at least one of the markers (IL-6 and IL-8) was equal to or higher than a cutoff value. The predictive performance was analyzed with a range of cutoff values from 0.1 to 5.0. Examples that illustrate the processing of the data and the way true positives (TP), true negatives (TN), and sensitivity and specificity were determined with different cutoff values are shown in Tables S4 and S5 (Supporting Information). The results obtained with the full range of cutoff values are summarized in Table 2 and are graphically displayed in Figure 2a. In addition, Figure 2a shows the overall concordance with clinical results on the PT toxicity of the compounds. The results showed that a cutoff value of 4.0 was most appropriate. Table 2. Determination of True Positives (TP), True Negatives (TN), Sensitivity, and Specificitya cutoff

TP

sensitivity

TN

specificity

0.1 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

22 22 20 17 16 16 15 15 14 14

100% 100% 91% 77% 73% 73% 68% 68% 64% 64%

0 2 11 12 13 13 14 16 16 16

0% 11% 58% 63% 68% 68% 74% 84% 84% 84%

Figure 2. Sensitivity, specificity, overall concordance, and ROC curves. (a) The figure displays graphically the values for sensitivity and specificity shown in Table 2. A value of 80% (y-axis) is indicated by a dashed line to facilitate perception. The figure shows also the overall concordance of the in vitro results with the PT toxicity of the compounds in humans (see classification in Table S1 (Supporting Information)). Cutoff values (x-axis) ranged from 0.1−5.0. (b) On the basis of the results obtained with HPTC-like cells, the ROC curves were calculated with respect to each single marker (IL-6 and IL-8, gray graphs) or the combination of both markers (black). The respective AUC values ranging from 71% to 82% are indicated. An AUC value of 50% would delineate a model with no predictive value. (c) The ROC curves were calculated based on the results obtained with HPTC-like cells and HPTC, when ATP depletion (dashed and dotted gray graphs) or GSH depletion (solid gray graph) were assayed. For comparison, the ROC curve obtained with respect to HPTC-like cells and the combination of IL-6 and IL-8 is displayed again (black; identical with the black graph in panel b). The AUC values (%) obtained with the different cell types and end point combinations are indicated.

a

TP were defined as true PT-specific nephrotoxicants (compounds 1− 22, group 1) that were correctly detected as positives by the in vitro assay. TN were defined as non-PT-specific nephrotoxicants and nonnephrotoxic compounds (compounds 23−41, groups 2 and 3) that remained negative in the in vitro assay. How positive and negative test results were obtained at different cutoff levels is shown in detail in Tables S3 and S4 (Supporting Information). TP and TN were determined at the indicated cutoff range from 0.1 to 5.0, and the numbers are displayed. From these numbers, the percentages of sensitivity (number of TP/total number of 22 group 1 compounds × 100%) and specificity (number of TN/total number of 19 group 2 + 3 compounds × 100%) were calculated. The numbers of TP and TN and respective percentages of sensitivity and specificity were determined with the whole set of 41 compounds. The percentages of sensitivity and specificity shown here are graphically displayed in Figure 2a. 1985

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Table 3. Comparison of Different Assays Performed with HPTC-Like Cells and HPTCa HPTC-like

HPTC

compound

IL-6/IL-8

ATP depletion

ATP depletion

GSH depletion

LDH leakage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 sensitivity specificity

− − + + + + + − − + + + + + − + + − + + + − − − − − + − − − − − + − − − − − − − + 68% 84%

>1000 >1000 981 ± 78 >1000 1±0 1±0 >1000 679 ± 16 >1000 551 ± 129 >1000 >1000 82 ± 5 555 ± 53 >1000 229 ± 122 >1000 4±1 654 ± 44 >1000 ND >1000 >1000 >1000 >1000 618 ± 6 >1000 >1000 >1000 >1000 >1000 >1000 >1000 667 ± 80 >1000 >1000 496 ± 59 216 ± 111 >1000 >1000 >1000 48% 79%

>1000 >1000 856 ± 22 >1000 13 ± 1 699 ± 243 >1000 889 ± 21 >1000 875 ± 111 >1000 >1000 14 ± 5 315 ± 36 >1000 232 ± 4 >1000 9±1 49 ± 5 371 ± 114 >1000 >1000 >1000 >1000 >1000 858 ± 13 >1000 >1000 >1000 >1000 >1000 >1000 >1000 619 ± 330 >1000 >1000 >1000 94 ± 64 940 ± 3 >1000 907 ± 49 50% 74%

>1000 >1000 769 ± 133 >1000 1±0 837 ± 48 >1000 742 ± 17 >1000 >1000 >1000 944 ± 133 >1000 708 ± 4 >1000 869 ± 35 >1000 81 ± 2 54 ± 22 >1000 634 ± 100 >1000 >1000 >1000 755 ± 8 239 ± 125 >1000 >1000 >1000 >1000 >1000 >1000 733 ± 316 >1000 >1000 >1000 >1000 814 ± 12 541 ± 112 >1000 >1000 45% 74%

105% ± 13% 110% ± 4%* 128% ± 4%* 107% ± 2%* 106% ± 1%* 117% ± 5%* 146% ± 7%* 104% ± 4% 111% ± 5%* 111% ± 3%* 115% ± 6%* 108% ± 4%* 146% ± 6%* 100% ± 3% 95% ± 2% 96% ± 1% 109% ± 3%* 106% ± 22% 136% ± 29% 105% ± 9% 109% ± 2%* 118% ± 7%* 102% ± 3% 97% ± 4% 90% ± 3% 102% ± 29% 98% ± 4% 95% ± 5% 110% ± 5%* 107% ± 4%* 109% ± 3%* 108% ± 5% 114% ± 13% 110% ± 3%* 115% ± 2%* 106% ± 3%* 101% ± 10% 113% ± 17% 101% ± 25% 110% ± 2%* 122% ± 2%* 64% 58%

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All assays were performed with the set of 41 compounds (left column). For comparison, positive (+) and negative (−) results that were obtained with HPTC-like cells and the IL-6/IL-8-based assay at a cutoff value of 4.0 are listed. The ATP-depletion assay was performed with HPTC-like cells, and HPTC and IC50 values (μg/mL) are listed. If ATP levels remained >50% of the vehicle control up to the highest concentration tested (1000 μg/ mL), an IC50 value of >1000 μg/mL was indicated. GSH depletion and LDH leakage were determined with HPTC, and with respect to GSH depletion, IC50 values were listed. In the case of LDH leakage, the percentages of the vehicle control were listed. Significant increases in comparison to the vehicle control are marked with an asterisk. All values represent the mean ± s.d. (n = 3). Sensitivity and specificity are displayed at the bottom below each respective column. For these calculations, all results were classified as positives where IC50 values of 80% (HPTC, Table S6 (Supporting Information)) were obtained with the IL6/IL-8-based assay, AUC values of 65% were obtained with the ATP depletion assay with both cell types (Figure 2c; Table S6 (Supporting Information)). Next, we addressed glutathione (GSH) depletion. In proximal tubular cells, GSH is required for drug metabolism and protection from reactive oxygen species.6 Batch (HPTC 1) and passage numbers of HPTC were the same as those used for determining ATP depletion. The results obtained with the ATP depletion assay and the GSH depletion assay were overall similar, and the predictivity of the GSH depletion assay was also low (Table 3; Figure 2c; Table S6 (Supporting Information)). Another widely used assay measures membrane damage by determining leakage of lactate dehydrogenase (LDH). When performed with HPTC, specificity and other performance

Figure 3. Immunostaining of HPTC-like cells (batch 2). The indicated markers were detected by immunostaining (green; cell nuclei, blue). The epifluorescence images display the x,y plane. The lateral enrichment of the tight junctional protein ZO-1 and the baso-lateral markers GLUT1 and URO10 is discernible. In contrast, proteins that are predominantly localized at the brush border (AQP1, PEPT1, CD13, and SGLT2) display a more uniform distribution over the cell body. Scale bars: 100 μm.

Blinded compounds were selected from the set of 41 compounds, in order to enable comparison with the results obtained before. The nature of the compounds was not revealed to the persons who did the sample preparation, qPCR, and data analysis. The selected test compounds were cephalosporin C, tacrolimus, and acarbose (compounds 6, 19, and 40). These compounds were tested at concentrations of 1 μg/mL, 10 μg/mL, 100 μg/mL, and 1000 μg/mL as before. In addition, dexamethasone (100 μg/mL) and puromycin (100 μg/mL) were selected as negative and positive controls. Also, the nature and plate positions of the positive and negative controls were not revealed to the persons who did the sample preparation and qPCR. On the basis of the controls, the Z′ values were calculated to ensure overall proper assay performance (Table 4). Table 4 also displays the highest IL-6 and IL-8 expression levels (the detailed results obtained at all compound concentrations are displayed in Table S7 (Supporting Information)). For the prediction of PT toxicity, a cutoff value of 4.0 was applied (Table 4), which had been determined independently with a different cell batch (Figure 2a). With this cutoff value compound 40 (acarbose) was predicted to be nontoxic for the cells of the PT in humans, whereas compounds 6 and 19 (cephalosporin C and tacrolimus) were predicted to be toxic for this cell type (Table 4). These results were in agreement with the results obtained with the first batch of HPTC-like cells (Tables 3 and 4). 1987

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the differentiation protocol and the use of alternative stem cell lines. Together, the results of this and the previous study11 showed that neither the use of established PT cell lines with IL-6 and IL-8 expression as the end point nor the use of HPTC and HPTC-like cells with well-established standard in vitro assays resulted in high predictivity. The low sensitivity of the ATP depletion assay observed here (∼50%) was in agreement with the high false-negative rate (∼50%) observed in a recent Pfizer study on organ-specific toxicity, including nephrotoxicity.18 A problem of our current model is the lack of dose-related information and the fact that some true PT-specific toxicants triggered positive responses only at high concentrations. This is a common problem of in vitro models. It would be surprising if such altered dose−response relationships would not exist, given the altered levels of transporters and other cellular changes in PT-derived cell lines8−10 and primary and hESC-derived cells13,15,17,20 in vitro. Probably, the further development of biomimetic cell models will result in improvements in the future, although it will be difficult to fully recapitulate in vivo conditions and cell states. Notably, the specificity of our model was high, and true negatives remained negative up to the highest concentrations. This would provide the opportunity to select compounds with a low probability of PT-specific toxicity during early preclinical testing, even in the absence of further dose-related information. Furthermore, our model allows for the identification of potential PT-specific nephrotoxicants. This would allow one to flag potential PT toxicants early and monitor specifically for effects on the PT if it should be decided to further develop such a compound. Our current and previous11 results, as well as recent data from another group,26 showed that major inflammatory pathways are triggered in PT cells upon treatment with PTspecific nephrotoxicants in vitro. This is in agreement with the inflammatory reactions induced by nephrotoxicants in vivo.24,25,35,36 As pro-inflammatory cytokines and inflammatory processes play a major role in drug-induced kidney injury in vivo,24,35,36 it would be important to further explore cytokine and chemokine expression and other pro-inflammatory reactions by renal cells in response to drug exposure. As different cell types express respective markers, in vitro models using only one or few cell types would be helpful to further study inflammatory mechanisms and to elucidate cell typespecific responses. Respective studies would also help to improve predictive in vitro models employing inflammatory markers as end points. Although pro-inflammatory cytokines play an important role in drug-induced kidney injury in vivo, using such end points in vitro provides limited insights into the mechanism of how a drug injures the cell type of interest. However, end points for in vitro assays that should have high predictivity when tested with libraries of different compounds must be relatively unspecific with respect to the mechanisms of toxicity. Otherwise, the rates of false negatives would be high if compounds were tested that would injure the cell type of interest by various different mechanisms. This view is supported by the results of our current study. For instance, tacrolimus decreases oxidative phosphorylation,37 and in agreement with this, we observed ATP depletion in HPTC-like cells and HPTC treated with this drug. However, in the current study and a previous study18 a high false-negative rate was obtained when ATP levels were measured after exposure to various nephrotoxicants that injure

Table 4. Results Obtained with Three Blinded Compounds and Prediction of PT Toxicitya prediction compound

IL-6

Z′

IL-8

Z′

(cutoff = 4.0)

6 19 40

3.9 ± 1.0 70.5 ± 10.5 2.4 ± 0.8

0.7 0.9 0.8

67.8 ± 23.1 206.1 ± 51.5 3.7 ± 1.2

1.0 0.9 0.8

+ + −

a A second batch of HPTC-like cells was tested with compounds 6, 19, and 40. The nature of the three test and the control compounds was not revealed to the persons who performed the qPCR and data analysis. The table displays the highest expression levels of IL-6 and IL-8 in HPTC-like cells (batch 2) at any given concentration of a compound within the concentration range tested (detailed results obtained at all compound concentrations tested are displayed in Table S7 (Supporting Information)). The numbers show the mean fold expression ± s.d. (n = 3) relative to the vehicle control. Z′ values were calculated for each plate and marker gene with the results from the negative (100 μg/mL dexamethasone) and positive (100 μg/mL puromycin) controls. A cutoff value of 4.0 was applied in order to predict whether a compound would be toxic (+) or not toxic (−) for proximal tubular cells in humans.

Further, these predictions were in agreement with results on PT toxicity. Tacrolimus (FK506) has various negative effects on the human kidney, including direct toxic effects on renal PT cells.30,31 Cephalosporin antibiotics are semisynthetic derivatives of cephalosporin C, and these compounds are substrates of the organic anion transport system of the PT. Several cephalosporins were found to be associated with acute tubular necrosis, and there is a consistent risk of PT toxicity associated with cephalosporin-derived compounds.32,33 Acarbose is an αglucosidase inhibitor used for the improvement of glycemic control in adults with type 2 diabetes mellitus. Adverse effects on various human organ systems including liver, lung, and skin have been reported (see, for instance, http://chem.sis.nlm.nih. gov/chemidplus/cas/56180-94-0), but to our knowledge, no adverse effects on the proximal tubule have been observed.

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DISCUSSION Here, we developed an in vitro model for the prediction of PT toxicity in humans. The model employed hESC-derived HPTC-like cells and used IL-6 and IL-8 expression as the end point. The predictivity of this model was high with an AUC value of 0.8. Although use of HPTC-like cells resulted in high predictivity, even better results in terms of sensitivity and overall predictivity had been obtained with HPTC11 (Table S6 (Supporting Information)). This was in agreement with the observation that some compounds, for instance, gentamicin, gave positive results with HPTC11 but yielded false-negative results with HPTC-like cells. Such differences in drug responses can be explained by the observed biological differences between HPTC and HPTC-like cells. For instance, megalin, which is essential for gentamicin uptake,34 is expressed at low levels in HPTC-like cells (current study and ref 17). Although the sensitivity of HPTC-like cells was reduced compared to that of HPTC, the predictivity obtained with HPTC or HPTC-like cells was superior in comparison to that of PT-derived cell lines (ref 11 and Table S6 (Supporting Information)). In contrast to HPTC, stem cell-derived cells are available in unlimited amounts, and functional changes during passaging can be avoided. Also, it would be expected that the sensitivity of HPTC-like cells can be further improved by modifications of 1988

dx.doi.org/10.1021/mp400637s | Mol. Pharmaceutics 2014, 11, 1982−1990

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PT cells by different mechanisms. In contrast, the predictivity was high when IL-6 and IL-8 expression was used as the end point. It would be useful to employ end points that are relatively unspecific in terms of toxicity mechanisms but provide high predictivity and combine them with end points that provide more mechanistic insights. This strategy was applied in the current study and provides both high predictivity and mechanistic insights. This would be more efficient than focusing only on end points that provide mechanistic insights but have necessarily limited predictivity when tested with compounds that injure cells by various different mechanisms. This view is supported by the fact that, so far, the development of predictive in vitro models for nephrotoxicity has been difficult by using such end points.

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ASSOCIATED CONTENT

S Supporting Information *

Terms and definitions; detailed information on the nephrotoxic effects of the 41 compounds used; detailed results of IL-6 and IL-8 expression levels; determination of positive and negative results at cutoff values of 3.0 and 4.0 and calculation of sensitivity and specificity; summary of results obtained with different end points and cell types; and detailed results of IL-6 and IL-8 expression levels in batch 2 of HPTC-like cells. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +65 6824 7107. Fax: +65 6478 9080. E-mail: dzink@ ibn.a-star.edu.sg. Present Address †

(W.S.T.) Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore 119083, Singapore.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Karthikeyan Narayanan (Institute of Bioengineering and Nanotechnology (IBN), A*STAR, Singapore) for advice and Yun Ting Soong (IBN) and Chun Siang Chia (IBN) for help with the experimental work. This work was supported by a grant from the Joint Council Office (Agency for Science, Technology and Research (A*STAR)) Development Program and the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, A*STAR, Singapore).

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