Isolation and Culture of Renal Tubular Epithelial Cells

RTECs were extracted from mice using previously reported methods (Valente et al. 2011 (link)). In brief, mice were euthanized, and the kidneys were immediately excised and placed in ice-cold complete media in HBSS (Hyclone, Marlborough, MA) supplemented with 1% penicillin-streptomycin (Hyclone). Thereafter, the renal capsule was dissected out, and sagittal sections of the renal cortex were obtained and the medulla was discarded. Further, cortical tissue was minced in complete medium with collagenase I (1 mg/mL, Sigma-Aldrich, St. Louis, MO) and soybean trypsin inhibitor (100 μg/mL, Sigma-Aldrich) and incubated at 37°C for 45 min with frequent mixing. The same volume of ice-cold HBSS was added into tubules to terminate the digestion step. The digestion product was sieved successively with 150-μm and 75-μm mesh sieves, and the filtrate was collected and seeded on collagen-treated plates in a selection culture medium comprising DMEM:F12 culture media (Gibco, Waltham, MA) supplemented with 10 mL/L penicillin-streptomycin (Hyclone), 50 nmol/L hydrocortisone (Sigma-Aldrich), epidermal growth factor, insulin/transferring/selenium (10 μg/mL/5.5 μg/mL, Gibco), and tri-iodothyronine (Gibco), in an incubator (37°C, 5% CO₂) and cultured to 80% confluence for subculturing. Cells of the second and third passages were used for subsequent experiments.