Ly294

Human renal proximal tubular epithelial cells (HK-2, ATCC, Manassas, VA, USA) were cultured and passaged every 3 to 4 days in 100-mm dishes containing Dulbecco’s modified Eagle’s medium-F-12 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich). Cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37°C for 24 hours and sub-cultured at 70% to 80% confluence. For experiments, HK-2 cells were plated onto 60-mm dishes in medium containing 10% fetal bovine serum for 24 hours, and cells were then switched to Dulbecco’s modified Eagle’s medium F12 serum-free media for 24 hours. Cells were harvested at the end of treatment for further analysis. IS was obtained from Sigma-Aldrich (Steinheim, Germany), and paricalcitol was obtained from Abbott Laboratories (North Chicago, IL, USA). PD98059 (a mitogen-activated protein kinase [MAPK]/extracellular signal-related kinase [Erk] inhibitor), SP600125 (a specific Jnk inhibitor), and SB203580 (a p38 MAPK inhibitor) were obtained from Calbiochem (San Diego, CA, USA). Ly294 (a phosphoinositide 3-kinase/protein kinase B [Akt] inhibitor) and N-acetyl-L-cysteine were obtained from Sigma-Aldrich (Steinheim). Bay 11-7082 was obtained from Biomol (Plymouth Meeting, PA, USA).