Hypoxia-Reoxygenation Induced Tubular Injury

Human kidney proximal tubular cell line (HK-2) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured at 37°C in a normal atmosphere of 95% air and 5% CO₂ in keratinocyte-serum free medium supplemented with human recombinant epidermal growth factor and bovine pituitary extract (Life Technologies, Carlsbad, CA). Hypoxia-reoxygenation was induced in tubular cells to simulate ischemia-reperfusion injury as described in previous studies (30 (link), 31 (link), 36 (link)). In brief, hypoxia was induced in tubular cells by incubation for 2 h in a modified Krebs buffer (137 mM NaCl sodium chloride, 15.8 mM KCl potassium chloride, 0.49 mM MgCl₂ magnesium chloride, 0.9 mM CaCl₂ calcium chloride, 4 mM HEPES) supplemented with 10 mM 2-deoxyglucose, 20 mM sodium lactate, 12 mM KCl potassium chloride and 1 mM sodium dithionite (pH 6.4) in a hypoxia chamber (Billups-Rothenberg, Inc., Del Mar, CA) containing 95% N₂/5% CO₂ at 37°C. After 2 h hypoxia, the modified Krebs buffer was replaced with keratinocyte-serum free medium and cells were cultured for 48 h to 72 h in 95% air and 5% CO₂. Cells without induction of hypoxia were used as a control.