#3475 NOVEL OPTIMISED PROTOCOL FOR ISOLATION OF PTEC AND SYSTEMATIC CHARACTERISATION FROM HUMAN KIDNEY BIOPSY

Abstract

Abstract Background and Aims Human kidneys have a role in water homeostasis, acid-base control, reabsorption of compounds, and secretion of xenobiotics and endogenous metabolites, exposing them to substances that could cause harm. This results in an alarming number of acute kidney injuries (AKI) worldwide, estimated at 13%. Furthermore, one-quarter of hospitalised cases are due to drug-induced AKI [1]. Current methods for nephrotoxicity assays are based on animal testing and/or the use of simple human cell lines. Meta-analyses show that we can correctly predict human drug responses in only 10–50% [2]. Our work aimed to develop a novel and optimised protocol for isolating proximal tubular epithelial cells (PTEC) from human kidney biopsy to aid future research regarding AKI and nephrotoxicity studies. Method Isolation and cultivation of primary human adult PTEC obtained with biopsy during the regular diagnostic procedure was performed. We used a protocol consisting of micro-dissection of a tissue sample to get ∼1 mm3 fragments, enzymatic dissociation with 0.2% collagenase type 1, and use of selective culture media (Advanced DMEM/F12 with added insulin, transferrin, and selenite (all three together termed ITS), epidermal growth factor (EGF), and hydrocortisone). Light microscopy was used for morphologic characterisation. Some cells were cultured on Transwell inserts, and the transepithelial electric resistance (TEER) was measured in mature cells that formed a confluent culture. For phenotypic characterisation, several markers characteristic of PTEC were chosen [3], and immunocytochemical staining was performed using a fluorescent microscope to evaluate the PTEC phenotype. Results Following the described protocol resulted in isolating cells that formed first colonies after 24 h. Using light microscopy, the cells exhibited a cobblestone appearance, reached confluence after eight days, and showed dome (hemicysts) formation after 13 days. TEER reached 169 Ω/cm2 after 14 days. The isolated cells were marked positive using immunocytochemistry for sodium-glucose cotransporter 2 (SGLT2), multidrug-resistant protein 4 (MRP4), organic anionic transporter 1 and 3 (OAT1 and OAT3), organic cationic transporter 2 (OCT2), p-glycoprotein (p-gp), multidrug and toxin extrusion protein 1 (MATE1), and N-cadherin. Conclusion In this study, we developed a protocol for isolating and cultivating primary human PTEC from biopsy samples. To the best of our knowledge, we have performed the most extensive systematic characterisation following the isolation of PTEC from kidney biopsy reported to date.