Isolation and Culture of Primary Renal Tubular Cells

Primary RTECs from WT and KO mice were isolated in sterile conditions according to previously stated methods with slight modifications (8 (link), 47 (link), 48 (link)). Renal cortices were dissected in ice-cold HBSS and sliced into small fragments and then digested in PBS buffer with 1 mg/ml type I collagenase (Worthington, Lakewood, NJ) and 125 μg/ml defined trypsin inhibitor (Gibco) at 37°C for 30 min. The resulting supernatant was sieved through two nylon sieves (pore size: 200 um and 70 μm) and tubular fragments caught by the sieve were flushed in the reverse direction with PBS and centrifuged at 200×g for 5 min. The resulting pellet was resuspended and kept in DMEM/F12 medium containing 5% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin, 1×insulin-transferrin-selenium, and 1× MEM nonessential amino acids. Incubation of the plate was done in a humidified incubator under 5% CO₂ at 37°C and the medium was changed every other day until 90% of cell cultures had been organized as a confluent monolayer.