Development of an in vitro 3D Human Kidney Proximal Tubular Epithelial Tissue Model
The renal proximal tubular (PT) region is the most common site for a compound-specific kidney injury. The PT region is responsible for essential kidney functions, including reabsorption of low molecular weight proteins, solutes, and glucose, secretion of acids, and clearance of administered medications. The ultimate goal of this project is to develop a novel physiologically relevant primary human kidney-cell-based 3-dimensional (3D) organotypic tissue model for the prediction of human nephrotoxicity.
Human primary PT epithelial cells (PTEC) were isolated and expanded in a monolayer culture prior to seeding onto microporous membrane inserts to reconstruct a 3D organotypic model. 3D tissues were analyzed by histology, barrier integrity (transepithelial electrical resistance, TEER), immunostaining, and qPCR on days 5 to 30. Receptor-mediated FITC-albumin uptake, glucose uptake, and transpeptidase hydrolytic activity of glutamyl transpeptidase (GGT1) and leucine aminopeptidase (LAP) were assayed on days 10 to 28.
The PTEC organotypic tissues (EpiKidney) organize into characteristic tubular structures, develop a barrier with TEER 110.2+/-33.3 cm2 on day 9 and stain positive for tight junction proteins ZO-1, claudin-1, and occludin. The organotypic tissues differentiate into polarized epithelium expressing brush border proteins megalin, villin, and GGT1 together with water channel AQP1 on the apical side and sodium-potassium ATPase pump on the basolateral side. Real-time qPCR analysis confirmed that tissues express a panel of PTEC-specific markers that are necessary for renal clearance, secretion, and reabsorption: aminopeptidase CD13, multidrug resistance proteins MRP2/4, CYP450 enzymes, glucose transporters SGLT1/2, multidrug and toxin extrusion transporter MATE1, organic cation and anion transporters OCT1/2, OCTN1/2, and OATP4C1, urate transporter URAT1, and sodium phosphate co-transporter NP2. Specific concentration-dependent and time-dependent receptor-mediated uptake of FITC-albumin by PTEC tissues was observed by fluorescent microscopy. Significant conversion of γ-Glutamyl-p-nitroanilide (GPNA, 2.5 mM) and L-leucine-p-nitroanilide (LLNA, 3 mM), substrates for GGT1 and LAP respectively, was detected via spectrophotometric monitoring of p-nitroaniline (PNA) following 30 min incubation. Specific transpeptidase hydrolytic activity was inhibited in the presence of an irreversible inhibitor acivicin (1.2 mM) by 88.8% (GGT1) and 35.0% (LAP).
The reconstructed in vitro 3D Kidney-PT organotypic tissue morphology, barrier properties, gene expression, and tissue performance resemble the in vivo human PT region. This model is anticipated to be a valuable tool to evaluate human nephrotoxicity and its mechanisms, improve the predictivity of human responses to pharmacological substances, and help establish confidence in drug development and testing.
EpiKidney, renal proximal tubule, nephrotoxicity, TEER, albumin uptake, glucose uptake, ZO-1, claudin-1, occludin, brush border proteins, megalin, villin, GGT1, b-tubulin, water channel AQP1, sodium-potassium ATPase pump, gamma-glutamyl-p-nitroanilide, GPNA, L-leucine-p-nitroanilide, p-nitroaniline, glutamyl transpeptidase GGT1, leucine aminopeptidase LAP, acivicin, B2M, CLUSTERIN, CD13, MRP4, OATP4C1, GGT1, OCTN2, HNBC1, MDR1, AQP1, OCT3, GLUT9, OCT2, MATE1, OCTN1, MEGALIN, CYP450, SGLT2, CUBULIN, MRP2, BCRP, NP2, OAT1, OAT2, OAT3, SGLT1, renal clearance, secretion, absorption, receptor-mediated endocytosis, transpeptidase hydrolysis.
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