Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies

Contains fulltext : 232054.pdf (Publisher’s version ) (Open Access)Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-β-d-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation)

Hydrogen concentrations in methane-forming cells probed by the ratios of reduced and oxidized coenzyme F420

Item does not contain fulltex

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

Implementation of a Human Renal Proximal Tubule on a Chip for Nephrotoxicity and Drug Interaction Studies

Proximal tubule epithelial cells (PTEC) are susceptible to drug-induced kidney injury (DIKI). Cell-based, two-dimensional (2D) in vitro PTEC models are often poor predictors of DIKI, probably due to the lack of physiological architecture and flow. Here, we assessed a high throughput, 3D microfluidic platform (Nephroscreen) for the detection of DIKI in pharmaceutical development. This system was established with four model nephrotoxic drugs (cisplatin, tenofovir, tobramycin and cyclosporin A) and tested with eight pharmaceutical compounds. Measured parameters included cell viability, release of lactate dehydrogenase (LDH) and N-acetyl-β-D-glucosaminidase (NAG), barrier integrity, release of specific miRNAs, and gene expression of toxicity markers. Drug-transporter interactions for P-gp and MRP2/4 were also determined. The most predictive read outs for DIKI were a combination of cell viability, LDH and miRNA release. In conclusion, Nephroscreen detected DIKI in a robust manner, is compatible with automated pipetting, proved to be amenable to long-term experiments, and was easily transferred between laboratories. This proof-of-concept-study demonstrated the usability and reproducibility of Nephroscreen for the detection of DIKI and drug-transporter interactions. Nephroscreen it represents a valuable tool towards replacing animal testing and supporting the 3Rs (Reduce, Refine and Replace animal experimentation)

Tubuloids derived from human adult kidney and urine for personalized disease modeling

Adult stem cell-derived organoids are three-dimensional epithelial structures that recapitulate fundamental aspects of their organ of origin. We describe conditions for the long-term growth of primary kidney tubular epithelial organoids, or 'tubuloids'. The cultures are established from human and mouse kidney tissue and can be expanded for at least 20 passages (>6 months) while retaining a normal number of chromosomes. In addition, cultures can be established from human urine. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. We apply tubuloids to model infectious, malignant and hereditary kidney diseases in a personalized fashion. BK virus infection of tubuloids recapitulates in vivo phenomena. Tubuloids are established from Wilms tumors. Kidney tubuloids derived from the urine of a subject with cystic fibrosis allow ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function

Tubuloids derived from human adult kidney and urine for personalized disease modeling

Adult stem cell-derived organoids are three-dimensional epithelial structures that recapitulate fundamental aspects of their organ of origin. We describe conditions for the long-term growth of primary kidney tubular epithelial organoids, or 'tubuloids'. The cultures are established from human and mouse kidney tissue and can be expanded for at least 20 passages (>6 months) while retaining a normal number of chromosomes. In addition, cultures can be established from human urine. Human tubuloids represent proximal as well as distal nephron segments, as evidenced by gene expression, immunofluorescence and tubular functional analyses. We apply tubuloids to model infectious, malignant and hereditary kidney diseases in a personalized fashion. BK virus infection of tubuloids recapitulates in vivo phenomena. Tubuloids are established from Wilms tumors. Kidney tubuloids derived from the urine of a subject with cystic fibrosis allow ex vivo assessment of treatment efficacy. Finally, tubuloids cultured on microfluidic organ-on-a-chip plates adopt a tubular conformation and display active (trans-)epithelial transport function