Immortalized Human Renal Proximal Tubular Cells

Immortalized hRPTCs were generated from the normal portions of renal surgical specimens from normotensive Caucasian male patients who had renal cell carcinoma (8 (link),15 (link),20 (link)–22 (link)). Human embryonic kidney (HEK) 293 cells (ATCC, Manassas, VA, USA) and hRPTCs were maintained at 37°C in 95% air/5% CO₂ in DMEM/F-12 medium (Gibco, Grand Island, NY, USA) supplemented with 5% penicillin-streptomycin-amphotericin (Corning Life Sciences, Manassas, VA) and 10% FBS (Gibco, Grand Island, NY, USA). When 90% confluent, the cells were subcultured for use in experimental protocols using trypsin-EDTA (ThermoFisher; Rockford, IL, USA). The cells tested negative for Mycoplasma infection. Utilization of cells was limited to <25 passages to avoid the effects of cellular senescence. The cells were transfected with the plasmids using FuGENE® 6 (Cat# E2691; Promega, Madison, WI, USA) with a 3:1 reagent to plasmid DNA ratio. Stable transfection was done through treatment with 500 μg/μl of G418 (Invivogen, San Diego, CA, USA).