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The renal proximal tubule is responsible for re-absorption of the majority of the glomerular filtrate and its proper function is necessary for whole-body homeostasis. Aging, certain diseases and chemical-induced toxicity are factors that contribute to proximal tubule injury and chronic kidney disease progression. To better understand these processes, it would be advantageous to generate renal tissues from human induced pluripotent stem cells (iPSC). Here, we report the differentiation and characterization of iPSC lines into proximal tubular-like cells (PTL). The protocol is a step wise exposure of small molecules and growth factors, including the GSK3 inhibitor (CHIR99021), the retinoic acid receptor activator (TTNPB), FGF9 and EGF, to drive iPSC to PTL via cell stages representing characteristics of early stages of renal development. Genome-wide RNA sequencing showed that PTL clustered within a kidney phenotype. PTL expressed proximal tubular-specific markers, including megalin (LRP2), showed a polarized phenotype, and were responsive to parathyroid hormone. PTL could take up albumin and exhibited ABCB1 transport activity. The phenotype was stable for up to 7 days and was maintained after passaging. This protocol will form the basis of an optimized strategy for molecular investigations using iPSC derived PTL.

Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.

Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62–1000 nM) or diethyl maleate (5.62–1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities.

The polarised expression of specific transporters in proximal tubular epithelial cells is important for the renal clearance of many endogenous and exogenous compounds. Thus, ideally, the in vitro tools utilised for predictions would have a similar expression of apical and basolateral xenobiotic transporters as in vivo. Here, we assessed the functionality of organic cation and anion transporters in proximal tubular-like cells (PTL) differentiated from human induced pluripotent stem cells (iPSC), primary human proximal tubular epithelial cells (PTEC), and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1). Organic cation and anion transport were studied using the fluorescent substrates 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP) and 6-carboxyfluorescein (6-CF), respectively. The level and rate of intracellular ASP accumulation in PTL following basolateral application were slightly lower but within a 3-fold range compared to primary PTEC and RPTEC/TERT1 cells. The basolateral uptake of ASP and its subsequent apical efflux could be inhibited by basolateral exposure to quinidine in all models. Of the three models, only PTL showed a modest preferential basolateral-to-apical 6-CF transfer. These results show that organic cation transport could be demonstrated in all three models, but more research is needed to improve and optimise organic anion transporter expression and functionality.

The utilisation of genome-wide transcriptomics has played a pivotal role in advancing the field of toxicology, allowing the mapping of transcriptional signatures to chemical exposures. These activities have uncovered several transcriptionally regulated pathways that can be utilised for assessing the perturbation impact of a chemical and also the identification of toxic mode of action. However, current transcriptomic platforms are not very amenable to high-throughput workflows due to, high cost, complexities in sample preparation and relatively complex bioinformatic analysis. Thus, transcriptomic investigations are usually limited in dose and time dimensions and are, therefore, not optimal for implementation in risk assessment workflows. In this study, we investigated a new cost-effective, transcriptomic assay, TempO-Seq, which alleviates the aforementioned limitations. This technique was evaluated in a 6-compound screen, utilising differentiated kidney (RPTEC/TERT1) and liver (HepaRG) cells and compared to non-transcriptomic label-free sensitive endpoints of chemical-induced disturbances, namely phase contrast morphology, xCELLigence and glycolysis. Non-proliferating cell monolayers were exposed to six sub-lethal concentrations of each compound for 24 h. The results show that utilising a 2839 gene panel, it is possible to discriminate basal tissue-specific signatures, generate dose–response relationships and to discriminate compound-specific and cell type-specific responses. This study also reiterates previous findings that chemical-induced transcriptomic alterations occur prior to cytotoxicity and that transcriptomics provides in depth mechanistic information of the effects of chemicals on cellular transcriptional responses. TempO-Seq is a robust transcriptomic platform that is well suited for in vitro toxicity experiments.

Transcriptomic analysis is a powerful method in the utilization of New Approach Methods (NAMs) for identifying mechanisms of toxicity and application to hazard characterization. With this regard, mapping toxicological events to time of exposure would be helpful to characterize early events. Here, we investigated time-dependent changes in gene expression levels in iPSC-derived renal proximal tubular-like cells (PTL) treated with five diverse compounds using TempO-Seq transcriptomics with the aims to evaluate the application of PTL for toxicity prediction and to report on temporal effects for the activation of cellular stress response pathways. PTL were treated with either 50 μM amiodarone, 10 μM sodium arsenate, 5 nM rotenone, or 300 nM tunicamycin over a temporal time course between 1 and 24 h. The TGFβ-type I receptor kinase inhibitor GW788388 (1 μM) was used as a negative control. Pathway analysis revealed the induction of key stress-response pathways, including Nrf2 oxidative stress response, unfolding protein response, and metal stress response. Early response genes per pathway were identified much earlier than 24 h and included HMOX1 , ATF3 , DDIT3 , and several MT1 isotypes. GW788388 did not induce any genes within the stress response pathways above, but showed deregulation of genes involved in TGFβ inhibition, including downregulation of CYP24A1 and SERPINE1 and upregulation of WT1 . This study highlights the application of iPSC-derived renal cells for prediction of cellular toxicity and sheds new light on the temporal and early effects of key genes that are involved in cellular stress response pathways.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

Accurate detection and prediction of renal injury are central not only to improving renal disease management but also for the development of new strategies to assess drug safety in pre-clinical and clinical testing. In this study, we utilised the well-characterised and differentiated human renal proximal tubule cell line, RPTEC/TERT1 in an attempt to identify markers of renal injury, independent of the mechanism of toxicity. We chose zoledronate as a representative nephrotoxic agent to examine global transcriptomic alterations using a daily repeat bolus protocol over 14 days, reflective of sub-acute or chronic injury. We identified alterations in targets of the cholesterol and mevalonate biosynthetic pathways reflective of zoledronate specific effects. We also identified interleukin-19 (IL-19) among other inflammatory signals such as SERPINA3 and DEFB4 utilising microarray analysis. Release of IL-19 protein was highly induced by an additional four nephrotoxic agents, at magnitudes greater than the characterised marker of renal injury, lipocalin-2. We also demonstrate a large increase in levels of IL-19 in urine of patients with chronic kidney disease, which significantly correlated with estimated glomerular filtration rate levels. We suggest IL-19 as a potential new translational marker of renal injury.

Potassium bromate (KBrO 3 ) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO 3 in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO 3 for 6, 24 and 72 h. Pathway analysis identified “glutathione metabolism”, “Nrf2-mediated oxidative stress” and “tight junction (TJ) signalling” as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO 3 exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N -Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.