Screening of Drug-Transporter Interactions in a 3D Microfluidic Renal Proximal Tubule on a Chip

Abstract

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.

Perspective on the Application of Microphysiological Systems to Drug Transporter Studies

“…Coculturing of hRPTECs and endothelial cells was also shown to improve cell viability, mitochondrial activity, and sodium-dependent glucose reabsorption under flow conditions (Vedula et al, 2017). Immortalized human proximal tubule cells showed a high level of membrane polarization in a microplateformat MPS model, while retaining similar levels of P-gp and MRP activity and gene expression (Vriend et al, 2018). In one of the first proof-of-concept studies of renal MPS models, the activity of the efflux transporter P-gp was increased compared with static culture, shown by using the fluorescent probe substrate Calcein-AM, in the presence of the P-gp inhibitor verapamil.…”

Bioengineering strategies for nephrologists: kidney was not built in a day

“…The most diffusely and appealing application of tubule-on -a-chip is drug-induced nephrotoxicity screening [134][135][136]. This technology was helpful to reproduce the in vivo cisplatin and gentamicin toxicity, demonstrating that the once-a-day bolus dosing method was less nephrotoxic than the continuous infusion method [137].…”

“…This technology was helpful to reproduce the in vivo cisplatin and gentamicin toxicity, demonstrating that the once-a-day bolus dosing method was less nephrotoxic than the continuous infusion method [137]. Moreover, the combination of tubule-on -a-chip systems with innovative detection methods to analyze novel biomarkers specific for nephrotoxicity identifies this technology as a fully compatible platform with automation and high content screening equipment [134][135][136][137][138][139][140][141]. However, the goal standard in understanding nephrotoxicity is the integration of multiple cell types from different organs.…”

Kidney-based in vitro models for drug-induced toxicity testing

“…Advances in microelectromechanical systems (MEMs) allowed researchers to apply microfluidics in cell and tissue cultures that resulted in the so-called Ryan et al (1994), Zhao et al (2017), Jenkinson et al (2012b) and Murphy et al (2017) Nki-2 organ-on-a-chip (Table 3). Vriend et al demonstrated that culturing ciPTEC-OAT1 in an OrganoPlate, a 3D platform consisting of 96 chips that provides fluid shear stress by placing the plate on a rocker platform, it was possible to create a high-throughput screening method compatible with advanced imaging techniques (Vriend et al 2018). Following a similar approach, Schutgens et al developed a new microfluidic in vitro system using kidney tubular epithelial organoids, or "tubuloids", derived from adult stem cells.…”