Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia

BACKGROUND: Kidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs. METHODS: Human conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm2 or low peak FSS of 0.5 dyne/cm2. Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined. RESULTS: A primary cilium knock-out cell model, ciPTEC-KIF3α-/-, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α-/-. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity. CONCLUSIONS: FSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane. GENERAL SIGNIFICANCE: FSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism

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

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

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

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

Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules

Proximal tubules in the kidney play a crucial role in reabsorbing and eliminating substrates from the body into the urine, leading to high local concentrations of xenobiotics. This makes the proximal tubule a major target for drug toxicity that needs to be evaluated during the drug development process. Here, we describe an advanced in vitro model consisting of fully polarized renal proximal tubular epithelial cells cultured in a microfluidic system. Up to 40 leak-tight tubules were cultured on this platform that provides access to the basolateral as well as the apical side of the epithelial cells. Exposure to the nephrotoxicant cisplatin caused a dose-dependent disruption of the epithelial barrier, a decrease in viability, an increase in effluent LDH activity, and changes in expression of tight-junction marker zona-occludence 1, actin, and DNA-damage marker H2A.X, as detected by immunostaining. Activity and inhibition of the efflux pumps P-glycoprotein (P-gp) and multidrug resistance protein (MRP) were demonstrated using fluorescence-based transporter assays. In addition, the transepithelial transport function from the basolateral to the apical side of the proximal tubule was studied. The apparent permeability of the fluorescent P-gp substrate rhodamine 123 was decreased by 35% by co-incubation with cyclosporin A. Furthermore, the activity of the glucose transporter SGLT2 was demonstrated using the fluorescent glucose analog 6-NBDG which was sensitive to inhibition by phlorizin. Our results demonstrate that we developed a functional 3D perfused proximal tubule model with advanced renal epithelial characteristics that can be used for drug screening studies