Abstract
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.
Highlights
IntroductionDrug Research, Einsteinweg 55, 2333CC, Leiden, the Netherlands
Guest Editors: Rosalinde Masereeuw and Raymond LaiElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.box 9101, 6500 HB, Nijmegen, the Netherlands. 3 Division of Analytical Biosciences, Leiden Academic Center forDrug Research, Einsteinweg 55, 2333CC, Leiden, the Netherlands. 4 Division Pharmacology, Department of Pharmaceutical Sciences, Renal proximal tubules play a crucial role in reabsorbing salt, water, and organic solutes such as glucose from the glomerular filtrate as well as eliminating endogenous and exogenous waste products from the body [1]
Renal proximal tubule epithelial cells (RPTEC; Kidney PTEC Control Cells, SA7K Clone, Sigma, Germany, MTOX1030) were cultured on PureCol-coated (Advanced BioMetrix, 5005-B, diluted with 1:30 in HBSS (Sigma H6648), 20 min incubation at 37°C) T75 flasks in MEME alpha Modification (Sigma, M4526) supplemented with renal proximal tubule epithelial cells (RPTECs) Complete Supplement (Sigma, MTOXRCSUP), L-glutamine (1.87 mM, Sigma, G7513), gentamicin (28 μg/mL, Sigma, G1397), and amphotericin B (14 ng/mL, Sigma, A2942)
Summary
Drug Research, Einsteinweg 55, 2333CC, Leiden, the Netherlands. 4 Division Pharmacology, Department of Pharmaceutical Sciences, Renal proximal tubules play a crucial role in reabsorbing salt, water, and organic solutes such as glucose from the glomerular filtrate as well as eliminating endogenous and exogenous waste products from the body [1]. The transepithelial transport of substrates and the concentration of xenobiotics in the tubular lumen make the proximal tubule a target for drug-induced toxicity [2]. Due to ethical concerns and the limited translatability of these models to the human situation, in vitro modeling is rapidly becoming important for studying solute transport, drug-induced toxicity, and disease-related kidney failure [3]. Current-day in vitro models typically comprise human renal proximal tubule epithelial cells (RPTECs) grown on a permeable membrane. The permeable support membrane is a crucial barrier influencing both transport parameters and physiology of cells
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