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.

Introduction Endogenous biomarkers are promising tools to assess transporter-mediated drug-drug interactions (DDI) early in humans.Methods We evaluated on a common and validated in vitro system the selectivity of 4-pyridoxic acid (PDA), homovanillic acid (HVA), glycochenodeoxycholate-3-sulfate (GCDCA-S) and taurine towards different renal transporters, including multidrug resistance-associated protein (MRP), and assessed the in vivo biomarker sensitivity towards the strong organic anion transporters (OAT) inhibitor probenecid at 500mg every 6h to reach close to complete OAT inhibition.Results PDA and HVA were substrates of the OAT1/2/3, OAT4 (PDA only) and MRP4; GCDCA-S was more selective, having affinity only towards OAT3 and MRP2. Taurine was not a substrate of any of the investigated transporters under the in vitro conditions tested. Plasma exposure of PDA and HVA significantly increased and the renal clearance of GCDCA-S, PDA and HVA decreased; the magnitude of these changes was comparable to the ones of known clinical OAT probe substrates. PDA and GCDCA-S were the most promising endogenous biomarkers of the OAT pathway activity: PDA plasma exposure was the most sensitive to probenecid inhibition, and, in contrast, GCDCA-S was the most sensitive OAT biomarker based on renal clearance, with higher selectivity towards the OAT3 transporter. ConclusionThe current findings illustrate a clear benefit of measuring PDA plasma exposure during Phase 1 studies when a clinical drug candidate is suspected to be an OAT inhibitor based on in vitro data.Subsequently, combined monitoring of PDA and GCDCA-S in both urine and plasma is recommended in order to tease out the involvement of OAT1/3 in the inhibition interaction.

Quantitative translation of microfluidic transporter in vitro data to in vivo reveals impaired albumin-facilitated indoxyl sulfate secretion in chronic kidney disease.

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Active tubular secretion of drugs from the blood to urine is mediated by multiple drug transporters expressed on both basolateral and apical membrane of the proximal tubule. Human proximal tubule microphysiological system (MPS) has been proposed as a more physiologically relevant in vitro system to assess nephrotoxicity and tubular reabsorption.[1,2] However, drug‐transporter studies in MPSs are currently sparse and quantitative translation of MPS data to in vivo is not yet established. This study aimed to assess the feasibility of NORTIS MPS to study quantitatively OAT1‐mediated transport of pyridoxic acid (PDA) in the absence and presence of 1mM probenecid (prototypical OAT inhibitor). Conditionally immortalized proximal tubule epithelial cells overexpressing OAT1 (ciPTEC‐OAT1) were seeded in the NORTIS MPS and were cultured for at least 5 days to allow cell maturation and tight tubule formation prior to transport experiments. PDA secretion rate in the apical channel effluent was linear up to 8h and plateaued between 8‐24h. Inhibition of PDA secretion rate by probenecid ranged between 14 – 46% over 2‐8h time period. PDA CLu,int,invitro obtained from the initial linear uptake phase was 0.07 and 0.09 µL/min/mm2 in the absence and presence of probenecid, respectively. PDA CLu,int,invitro was quantitatively scaled via total human proximal tubule surface area to CLu,int,invivo which was subsequently used to predict PDA CLR. Translation of MPS transporter data resulted in 5‐fold under‐prediction of PDA CLR obtained from clinical data (48.2 vs. 215 mL/min). The findings of this study demonstrate a first attempt of quantitative translation of MPS transporter data. The study also highlighted that the extent of inhibition by probenecid was not fully captured in the MPS‐ciPTEC‐OAT1. Further investigations of the feasibility to derive additional scaling factors for in vitro‐in vivo extrapolation of transporter data from MPS are ongoing. [1] Weber, E. J., Chapron, A., Chapron, B. D., Voellinger, J. L., Lidberg, K. A., Yeung, C. K., … & Shen, D. D. (2016). Development of a microphysiological model of human kidney proximal tubule function. Kidney international, 90(3), 627‐637. [2] Sakolish, C., Chen, Z., Dalaijamts, C., Mitra, K., Liu, Y., Fulton, T., … & Chiu, W. A. (2020). Predicting tubular reabsorption with a human kidney proximal tubule tissue‐on‐a‐chip and physiologically‐based modeling. Toxicology in Vitro, 63, 104752.