Investigating Metabolic Regulation in HK-2 Cells

HK-2 cells (ATCC, American Type Culture Collection, Manassas, VA) were cultured in MEM medium (#31985, Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS, #10099-141, Gibco, Grand Island, USA) and 1% Penicillin-Streptomycin solution in 95% air and 5% CO₂ atmosphere. For siRNA transfections of HK-2 cells (VDUP-1 siRNA or scrambled siRNA as a nonspecific control), cells were seeded (2 × 10⁵ per well) in 6-well plates and transfected using with Lipofectamine 2000 reagent (#11668-019, Invitrogen, USA) according to the manufacturer's instructions. The cells were used for further experiments 48 hours after transfection. After this time period, the cells were randomly divided into eight groups: NG group (stimulated with NG (5.6 mM)); HG group (30 mM); NG + mannitol (24.4 mM) group (M) as an osmotic control; NH/R (hypoxia 4 hours and reoxygenation 2 hours) group; HH/R group; HH/R treated with scrambled siRNA group; HH/R treated with TXNIP siRNA group; and HH/R treated with RES (50 µM) during the 72 hours of high glucose (HG) incubation [34 (link)].