Immortalized Mouse Podocyte Culture

The conditionally immortalized mouse podocyte cell line was provided by Professor Maria Pia Rastaldi (S. Carlo Hospital, University of Milan, Italy) and cultured as described previously.[12 ] Well-differentiated immortalized mouse podocytes were used for experiments. The podocytes were incubated with the RPMI-1640 medium with 5% fetal bovine serum and grouped as follows: medium alone; medium containing leptin (Abcam, UK); medium containing A438079 (Tocris Bioscience, UK) or KN-62 (Sigma-Aldrich, USA); and medium containing both leptin and A438079 or leptin and KN-62 (cells were preincubated with A438079 or KN-62 for 30 min, and then leptin was added in the culture medium). The concentrations of leptin, KN-62, and A438079 in the medium were 20 ng/ml, 2.5 μmol/L, and 10 μmol/L, respectively, of which all had no cytotoxic effects on podocytes confirmed by lactate dehydrogenase (LDH) release assay [Supplementary Material 1].[21 (link)]