2018-07, Suter-Dick, Laura, Mauch, Linda, Caj, Michaela, Vormann, Marianne K., Hutter, Simon, Lanz, Henriette, Wilmer, Martijn, Ramp, Daniela, Masereeuw, Rosalinde, Vriend, Jelle

Drug-induced kidney injury is often observed in the clinics and can lead to long-term 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 three-channel 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.

2018-07, Vriend, Jelle, Nieskens, Tom T.G., Vormann, Marianne K., van den Berge, Batholomeus T., van den Heuvel, Angelique, Russel, Frans G.M., Suter-Dick, Laura, Lanz, Henriette, Vulto, Paul, Masereeuw, Rosalinde, Wilmer, Martijn

Drug-transporter interactions could impact renal drug clearance and should ideally be detected in early stages of drug development to avoid toxicity-related withdrawals in later stages. This requires reliable and robust assays for which current high-throughput screenings have, however, poor predictability. Kidney-on-a-chip platforms have the potential to improve predictability, but often lack compatibility with high-content detection platforms. Here, we combined conditionally immortalized proximal tubule epithelial cells overexpressing organic anion transporter 1 (ciPTEC-OAT1) with the microfluidic titer plate OrganoPlate to develop a screenings assay for renal drug-transporter interactions. In this platform, apical localization of F-actin and intracellular tight-junction protein zonula occludens-1 (ZO-1) indicated appropriate cell polarization. Gene expression levels of the drug transporters organic anion transporter 1 (OAT1; SLC22A6), organic cation transporter 2 (OCT2; SLC22A2), P-glycoprotein (P-gp; ABCB1), and multidrug resistance-associated protein 2 and 4 (MRP2/4; ABCC2/4) were similar levels to 2D static cultures. Functionality of the efflux transporters P-gp and MRP2/4 was studied as proof-of-concept for 3D assays using calcein-AM and 5-chloromethylfluorescein-diacetate (CMFDA), respectively. Confocal imaging demonstrated a 4.4 ± 0.2-fold increase in calcein accumulation upon P-gp inhibition using PSC833. For MRP2/4, a 3.0 ± 0.2-fold increased accumulation of glutathione-methylfluorescein (GS-MF) was observed upon inhibition with a combination of PSC833, MK571, and KO143. Semi-quantitative image processing methods for P-gp and MRP2/4 was demonstrated with corresponding Z'-factors of 0.1 ± 0.3 and 0.4 ± 0.1, respectively. In conclusion, we demonstrate a 3D microfluidic PTEC model valuable for screening of drug-transporter interactions that further allows multiplexing of endpoint read-outs for drug-transporter interactions and toxicity