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

Abstract

Abstract.Drug-induced kidney injury is often observed in the clinics and can lead to longterm organ failure. In this work, we evaluated a novel in vitro system that aims at detecting whether compounds can cause renal proximal tubule damage in man. For this, we implemented organotypic cultures of human conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1) in a threechannel OrganoPlate under microfluidic conditions. Cells were exposed to four known nephrotoxicants (cisplatin, tenofovir, cyclosporine A, and tobramycin). The effect on cell viability and NAG release into the medium was determined. A novel panel of four miRNAs (mir-21, mir-29a, mir-34a, and mir-192) was selected as potential biomarkers of proximal tubule damage. After nephrotoxicant treatment, miRNA levels in culture medium were earlier indicators than cell viability (WST-8 assay) and outperformed NAG for proximal tubule damage. In particular, mir-29a, mir-34a, and mir-192 were highly reproducible between experiments and across compounds, whereas mir-21 showed more variability. Moreover, similar data were obtained in two different laboratories, underlining the reproducibility and technical transferability of the results, a key requirement for the implementation of novel biomarkers. In conclusion, the selected miRNAs behaved like sensitive biomarkers of damage to tubular epithelial cells caused by several nephrotoxicity mechanisms. This biomarker panel, in combination with the 3D cultures of ciPTEC-OAT1 in the OrganoPlate, represents a novel tool for in vitro nephrotoxicity detection. These results pave the way for the application of miRNAs in longitudinal, time-course in vitro toxicity studies.

Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules

“…Both types of experiments show clear transporter functionality, whereas the latter is the most complex functional assay. We have not assessed organic anion transporter (OAT) expression by the RPTEC used in this study, but 2D evaluation by Suter-Dick et al (36) showed no response to tenofovir, indicating absence of the OAT1 transporter. This limits the use of the cell line for assessment of transport of organic ions.…”

A Multicompartment Human Kidney Proximal Tubule-on-a-Chip Replicates Cell Polarization–Dependent Cisplatin Toxicity

“…Epithelial polarization has been confirmed in these chip systems by tight junction formation and the presence of primary cilia (Jang et al, 2013;Jansen et al, 2015;Weber et al, 2016;Vedula et al, 2017;Vormann et al, 2018;Vriend et al, 2018), as well as proof-of-concept studies indicating that transepithelial transport of anionic and cationic organic compounds is feasible (Weber et al, 2016;Jansen et al, 2019;van der Made et al, 2019;Stahl et al, 2020). Kidney-on-a-chip models have been used to study drug-induced toxicity (Jang et al, 2013;Sakolish et al, 2018;Suter-Dick et al, 2018;Weber et al, 2018;Vormann et al, 2018;Maass et al, 2019), but crucially have not previously been applied to study the implications of epithelial polarization and localization of drug transporters for replication of membrane-dependent drug toxicity in vitro.…”

Preparation and Characterization of Electrospun Blend Fibrous Polyethylene Oxide:Polycaprolactone Scaffolds to Promote Cartilage Regeneration

“…After incubation, 100 μL of each sample, in three replicates each, was transferred to a 96‐well plate. The absorbance of the metabolized WST‐8 was measured using a multilabel plate reader (PHOMO, Photometer, Autobio, China) …”