Immortalized mouse podocyte culture

Conditionally immortalized mouse podocytes were kindly provided by Professor John Cijiang He (Department of Nephrology, Icahn School of Medicine at Mount Sinai, New York City, NY, USA) and cultured as previously described (14). Briefly, cells were cultured on Corning Collagen I plates (Corning Incorporated, Corning, NY, USA) at 33°C in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with the presence of 20 U/ml mouse recombinant interferon (IFN)-γ (R&D Systems, Inc., Minneapolis, MN, USA) and 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) to enhance the expression of thermosensitive T antigen. Immortalized mouse podocyte cell lines are routinely characterized in the laboratory on the basis of morphology and gene expression patterns, prior to the experimentation. Immortalized cells for all experiments in the current study were between passages 4 and 12. To induce differentiation, podocytes were maintained in RPMI medium and FBS at 37°C without IFN-γ for 10–12 days. Podocytes prepared for experiments were serum deprived for 24 h prior to intervention, followed by culture with D-glucose (Sigma-Aldrich; Merck KGaA) or D-mannitol (Sigma-Aldrich; Merck KGaA) added to the cultures at 30 mM glucose as high-glucose stimulation or at 5.5 mM glucose and 24.5 mM mannitol as isotonic control for 24 or 48 h.