A Human Renal Proximal Tubule Cell Line with Stable Organic Anion Transporter 1 and 3 Expression Predictive for Antiviral-Induced Toxicity.

Tom T G Nieskens,Constanze Hilgendorf,Martijn J Wilmer,Janny G P Peters,Melanie Röring,Rob Woestenenk,Niels Smits,Marieke J Schreurs,Katja Jansen,Thom K Van Der Made,Rosalinde Masereeuw

doi:10.1208/s12248-016-9871-8

Abstract

Drug-induced nephrotoxicity still hampers drug development, because current translation from in vitro or animal studies to human lacks high predictivity. Often, renal adverse effects are recognized only during clinical stages of drug development. The current study aimed to establish a robust and a more complete human cell model suitable for screening of drug-related interactions and nephrotoxicity. In addition to endogenously expressed renal organic cation transporters and efflux transporters, conditionally immortalized proximal tubule epithelial cells (ciPTEC) were completed by transduction of cells with the organic anion transporter (OAT) 1 or OAT3. Fluorescence-activated cell sorting upon exposure to the OAT substrate fluorescein successfully enriched transduced cells. A panel of organic anions was screened for drug-interactions in ciPTEC-OAT1 and ciPTEC-OAT3. The cytotoxic response to the drug-interactions with antivirals was further examined by cell viability assays. Upon subcloning, concentration-dependent fluorescein uptake was found with a higher affinity for ciPTEC-OAT1 (Km = 0.8 ± 0.1 μM) than ciPTEC-OAT3 (Km = 3.7 ± 0.5 μM). Co-exposure to known OAT1 and/or OAT3 substrates (viz. para-aminohippurate, estrone sulfate, probenecid, furosemide, diclofenac, and cimetidine) in cultures spanning 29 passage numbers revealed relevant inhibitory potencies, confirming the robustness of our model for drug-drug interactions studies. Functional OAT1 was directly responsible for cytotoxicity of adefovir, cidofovir, and tenofovir, while a drug interaction with zidovudine was not associated with decreased cell viability. Our data demonstrate that human-derived ciPTEC-OAT1 and ciPTEC-OAT3 are promising platforms for highly predictive drug screening during early phases of drug development.Electronic supplementary materialThe online version of this article (doi:10.1208/s12248-016-9871-8) contains supplementary material, which is available to authorized users.

Highlights

Martijn J Wilmer and Rosalinde Masereeuw contributed to this work.Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.The renal proximal tubules play a major role in eliminating waste products from the body, including drugs and their metabolites
The absence of endogenous organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) expression in conditionally immortalized proximal tubule epithelial cells (ciPTEC) was demonstrated by exposure to fluorescein (1 μM) for 10 min, which did not increase the intracellular fluorescence intensity as measured by flow cytometry (Fig. 1b, red line)
We demonstrated that OAT1 or OAT3 expression is required for induction of toxicity by adefovir, cidofovir, and tenofovir in ciPTEC

Summary

Introduction

Martijn J Wilmer and Rosalinde Masereeuw contributed to this work.Electronic supplementary material The online version of this article (doi:10.1208/s12248-016-9871-8) contains supplementary material, which is available to authorized users.The renal proximal tubules play a major role in eliminating waste products from the body, including drugs and their metabolites. A robust cell-based model should include a proximal tubule epithelium stably expressing a broad range of functional transporters and metabolic enzymes that act in concert in renal drug elimination [4]. This process may be affected in concomitant drug treatment, leading to clinically relevant drug-drug interactions (DDI). As a first step in elimination of organic anions in humans, active tubular uptake is mediated by the organic anion transporter 1 (OAT1; SLC22A6) and organic anion transporter 3 (OAT3; SLC22A8) present at the, blood-facing, basolateral side [5] These transporters are characterized by their high affinity and capacity and, as a consequence, are major players in the development of druginduced nephrotoxicity [6]. Renal elimination of organic cations in the human proximal tubular epithelium is facilitated by basolateral uptake, predominantly via the organic cation transporter 2 (OCT2; SLC22A2), and apical efflux via multidrug and toxin extrusion proteins 1 and 2K (MATE1 and -2-K; SLC47A1 and -2) [8] and P-glycoprotein (P-gp; ABCB1) [9]

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