Three-dimensional Culture of Human Proximal Tubular Epithelial Cells for an in vitro Evaluation of Drug-induced Kidney Injury.

Drug-induced kidney injury (DIKI) is the major cause of acute kidney injury (AKI). Renal proximal tubular epithelial cells (RPTECs) are the primary target sites of DIKI and express transporters involved in renal drug disposition. In the present study, we focused on three-dimensionally cultured human RPTECs (3D-RPTECs) with elevated expression of drug transporters to investigate their utility in DIKI evaluation. Intracellular ATP levels in 3D-RPTECs are reduced by tenofovir and cisplatin that are substrates of an organic anion transporter 1 and an organic cation transporter 2, respectively. In addition, 3D-RPTECs were exposed to 17 and 15 drugs that are positive and negative to RPTEC toxicity, respectively, for up to 28 d. The 20 % decreasing concentration of drugs for ATP amount (EC20) was obtained, and the ratio of EC20 values and clinical maximum concentration (Cmax) ≤100 were used as cut-off value to evaluate potential of DIKI. The sensitivities of 3D-RPTECs were 82.4 % and 88.2 % after 7 d and 28 d of drug exposure, respectively, and the specificities were 100 % and 93.3 %, respectively. The predictive performance of 3D-RPTECs was higher than that of two-dimensional cultured RPTECs and the kidney cell line HK-2. In conclusion, 3D-RPTECs are useful for in vitro evaluation of RPTEC injury by measuring intracellular ATP levels.

Cytochrome P450 and UDP-Glucuronosyltransferase Expressions, Activities, and Induction Abilities in 3D-Cultured Human Renal Proximal Tubule Epithelial Cells.

The role of the kidney as an excretory organ for exogenous and endogenous compounds is well recognized, but there is a wealth of data demonstrating that the kidney has significant metabolizing capacity for a variety of exogenous and endogenous compounds that in some cases surpass the liver. The induction of drug-metabolizing enzymes by some chemicals can cause drug-drug interactions and intraindividual variability in drug clearance. In this study, we evaluated the expression and induction of cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) isoforms in 3D-cultured primary human renal proximal tubule epithelial cells (RPTEC) to elucidate their utility as models of renal drug metabolism. CYP2B6, CYP2E1, CYP3A4, CYP3A5, and all detected UGTs (UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) mRNA levels in 3D-RPTEC were significantly higher than those in 2D-RPTEC and HK-2 cells and were close to the levels in the human kidney cortex. CYP1B1 and CYP2J2 mRNA levels in 3D-RPTEC were comparable to those in 2D-RPTEC, HK-2 cells, and the human kidney cortex. Midazolam 1'-hydroxylation, trifluoperazine N-glucuronidation, serotonin O-glucuronidation, propofol O-glucuronidation, and morphine 3-glucuronidation in the 3D-RPTEC were significantly higher than the 2D-RPTEC and comparable to those in the HepaRG cells, although bupropion, ebastine, and calcitriol hydroxylations were not different between the 2D- and 3D-RPTEC. Treatment with ligands of the aryl hydrocarbon receptor and farnesoid X receptor induced CYP1A1 and UGT2B4 expression, respectively, in 3D-RPTEC compared with 2D-RPTEC. We provided information on the expression, activity, and induction abilities of P450s and UGTs in 3D-RPTEC as an in vitro human renal metabolism model. SIGNIFICANCE STATEMENT: This study demonstrated that the expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in 3D-cultured primary human renal proximal tubule epithelial cells (3D-RPTEC) was higher than those in 2D-cultured primary human renal proximal tubule epithelial cells and HK-2 cells. The results were comparable to that in the human kidney cortex. 3D-RPTEC are useful for evaluating the induction of kidney P450s, UDP-glucuronosyltransferases, and human renal drug metabolism in cellulo.

Improvement of Protein Expression Profile in Three-Dimensional Renal Proximal Tubular Epithelial Cell Spheroids Selected Based on OAT1 Gene Expression: A Potential In Vitro Tool for Evaluating Human Renal Proximal Tubular Toxicity and Drug Disposition.

The proximal tubule plays an important role in the kidney and is a major site of drug interaction and toxicity. Analysis of kidney toxicity via in vitro assays is challenging, because only a few assays that reflect functions of drug transporters in renal proximal tubular epithelial cells (RPTECs) are available. In this study, we aimed to develop a simple and reproducible method for culturing RPTECs by monitoring organic anion transporter 1 (OAT1) as a selection marker. Culturing RPTECs in spherical cellular aggregates increased OAT1 protein expression, which was low in the conventional two-dimensional (2D) culture, to a level similar to that in human renal cortices. By proteome analysis, it was revealed that the expression of representative two proximal tubule markers was maintained and 3D spheroid culture improved the protein expression of approximately 7% of the 139 transporter proteins detected, and the expression of 2.3% of the 4,800 proteins detected increased by approximately fivefold that in human renal cortices. Furthermore, the expression levels of approximately 4,800 proteins in three-dimensional (3D) RPTEC spheroids (for 12 days) were maintained for over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroids developed by monitoring OAT1 gene expression are a simple and reproducible in vitro experimental system with improved gene and protein expressions compared with 2D RPTECs and were more similar to that in human kidney cortices. Therefore, it can potentially be used for evaluating human renal proximal tubular toxicity and drug disposition. SIGNIFICANCE STATEMENT: This study developed a simple and reproducible spheroidal culture method with acceptable throughput using commercially available RPTECs by monitoring OAT1 gene expression. RPTECs cultured using this new method showed improved mRNA/protein expression profiles to those in 2D RPTECs and were more similar to those of human kidney cortices. This study provides a potential in vitro proximal tubule system for pharmacokinetic and toxicological evaluations during drug development.

Proarrhythmia risk prediction using human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to become a useful tool for proarrhythmia risk prediction in the non-clinical drug development phase. Several features including electrophysiological properties, ion channel expression profile and drug responses were investigated using commercially available hiPSC-CMs, such as iCell-CMs and Cor.4U-CMs. Although drug-induced arrhythmia has been extensively examined by microelectrode array (MEA) assays in iCell-CMs, it has not been fully understood an availability of Cor.4U-CMs for proarrhythmia risk. Here, we evaluated the predictivity of proarrhythmia risk using Cor.4U-CMs. MEA assay revealed linear regression between inter-spike interval and field potential duration (FPD). The hERG inhibitor E-4031 induced reverse-use dependent FPD prolongation. We next evaluated the proarrhythmia risk prediction by a two-dimensional map, which we have previously proposed. We determined the relative torsade de pointes risk score, based on the extent of FPD with Fridericia's correction (FPDcF) change and early afterdepolarization occurrence, and calculated the margins normalized to free effective therapeutic plasma concentrations. The drugs were classified into three risk groups using the two-dimensional map. This risk-categorization system showed high concordance with the torsadogenic information obtained by a public database CredibleMeds. Taken together, these results indicate that Cor.4U-CMs can be used for drug-induced proarrhythmia risk prediction.

CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information.

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs.

CSAHi study: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes -assessment of inter-facility and cells lot-to-lot-variability.

In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of drug-induced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9- to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia.