Linagliptin

NRK-52E cells, which are immortalized rat renal tubular epithelial cell lines, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Briefly, the cells were passaged every 3–4 days in 100 mm dishes (Falcon, Bedford, MA, USA) using Dulbecco’s Modified Eagle’s Medium-F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Life Technologies Inc., Gaithersburg, MD, USA), insulin–transferrin–sodium selenite media supplement (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). For experimental use, these cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C for 24 h and sub-cultured at 70%–80% confluence. After being washed extensively with phosphate-buffered saline (PBS), the cells were treated with either 0.1 µg/mL linagliptin (Santa Cruz Biotechnology Inc. Dallas, TX, USA. Cat. # sc-364721, Lot # D1921), 10 μg/mL LPS (lipopolysaccharide from Escherichia coli 0111:B4, Sigma-Aldrich, St. Louis, MO, USA, product #L2630, Lot #095M4163V), or LPS + linagliptin for 2 h. The cells were harvested at the end of treatment for molecular analysis. All cellular experiments were repeated three times for semi-quantitative real-time polymerase chain reaction (RT-PCR) and Western blot analysis.