A Quantitative Approach to Screen for Nephrotoxic Compounds In Vitro

Abstract

Nephrotoxicity due to drugs and environmental chemicals accounts for significant patient mortality and morbidity, but there is no high throughput in vitro method for predictive nephrotoxicity assessment. We show that primary human proximal tubular epithelial cells (HPTECs) possess characteristics of differentiated epithelial cells rendering them desirable to use in such in vitro systems. To identify a reliable biomarker of nephrotoxicity, we conducted multiplexed gene expression profiling of HPTECs after exposure to six different concentrations of nine human nephrotoxicants. Only overexpression of the gene encoding heme oxygenase-1 (HO-1) significantly correlated with increasing dose for six of the compounds, and significant HO-1 protein deregulation was confirmed with each of the nine nephrotoxicants. Translatability of HO-1 increase across species and platforms was demonstrated by computationally mining two large rat toxicogenomic databases for kidney tubular toxicity and by observing a significant increase in HO-1 after toxicity using an ex vivo three-dimensional microphysiologic system (kidneyon-a-chip). The predictive potential of HO-1 was tested using an additional panel of 39 mechanistically distinct nephrotoxic compounds. Although HO-1 performed better (area under the curve receiver-operator characteristic curve [AUC-ROC]=0.89) than traditional endpoints of cell viability (AUC-ROC for ATP=0.78; AUC-ROC for cell count=0.88), the combination of HO-1 and cell count further improved the predictive ability (AUC-ROC=0.92). We also developed and optimized a homogenous time-resolved fluorescence assay to allow high throughput quantitative screening of nephrotoxic compounds using HO-1 as a sensitive biomarker. This cell-based approach may facilitate rapid assessment of potential nephrotoxic therapeutics and environmental chemicals. Drugs and environmental chemicals, such as aminoglycoside antibiotics, analgesics, chemotherapeutic agents, and heavy metals, as well as endemic toxins like aristolochic acid are a common cause of AKI or CKD. 1,2 Current approaches to conduct safety and risk assessment of compounds rely predominantly on animal studies, and these results are extrapolated to dose effects in humans despite knowledge that the typical responses of animal models and humans can differ greatly. 3 The lack of adequate models to accurately predict human toxicity contributes to an underestimation of the kidney toxic potential of new therapeutic candidates, which also explains why nephrotoxic effects in patients are often only detected during late phase

RNA-binding Protein Musashi Homologue 1 Regulates Kidney Fibrosis by Translational Inhibition of p21 and Numb mRNA

“…Cell Culture, siRNA, and Plasmid Transfections-Primary human proximal tubular epithelial cells (HPTECs) were purchased from Biopredic (Paris, France) and cultured as described previously (15 …”

“…Because Msi1 is expressed in tubular epithelial cells (Fig. 1G), we performed siRNA-mediated knockdown of MSI1 in previously characterized HPTECs (15). As shown in Fig.…”

Combining Extracellular miRNA Determination with Microfluidic 3D Cell Cultures for the Assessment of Nephrotoxicity: a Proof of Concept Study

“…Currently used systems apply either simple twodimensional (2D) cell cultures or more complex, 3D cell cultures combined with microfluidics devices (3,9). Some published results on human proximal tubule epithelial cells (PTEC) grown on filter inserts or on microphysiologic systems (kidney-on-a-chip) used gene expression analysis for the detection of drug-induced cell injury (10,11). The results indicate that gene expression changes are associated with PTEC damage in vitro.…”

An Array of Gut-on-a-Chips for Drug Development

“…In new US regulations, e.g. Toxicology in the 21st Century (Tox21) program calls to move away from animal studies 5 .…”