Pcs 400 040

Normal renal tissues were collected from patients who provided informed consent as stipulated by the Yonsei University Health System, Severance Hospital, Institutional Review Board, and the study protocol was approved by the same institutional review board (approval number 4-2015-0104). Primary normal human RPTECs were purchased from the American Type Culture Collection (ATCC, PCS-400-010) and were maintained (37°C, 5% CO2) in renal epithelial cell growth media (ATCC, PCS-400-040) containing 0.5% fetal bovine serum (FBS), 10 nM triiodothyronine, 10 ng/ml recombinant human EGF, 100 ng/ml hydrocortisone, 5 μg/ml recombinant human insulin, 1 μM epinephrine, 5 μg/ml transferrin, and 2.4 mM L-alanyl-L-glutamine.

Human kidney (HK2) cells (passage 18–30) were maintained in DMEM/Hams F12 medium, supplemented with 10% FCS wt/vol, glutamine (2 mmol/l) and EGF (5 ng/ml), in a humidified atmosphere at 37 °C with 5% CO₂. Cells are proximal tubular epithelial cells, immortalized by the transduction of human papilloma virus 16 (HPV-16) E6/E7 genes and are mycoplasma-free. For all treatments, cells were seeded in low-glucose DMEM/F12 (5 mmol/L) for 48 h, then serum-starved overnight prior to treatment with Transforming Growth Factor Beta-1 (TGF-β1) (2-10 ng/mL) or ATPγS (1-100 μM) for 48 h. For ATP experiments, cells were incubated with either TGF-β1 (10 ng/mL) or ATPγS (100uM) ± ATP-diphosphohydrolase apyrase (5Units/ml); Suramin (100 μM), A438079 (50 μM) or A804598 (50 nM) for 48 h.
Primary human proximal tubule epithelial cells (hPTECs) were maintained in the basal medium obtained from the American Type Culture Collection (ATCC), supplemented with the renal epithelial cell growth kit (PCS-400-040) in a humidified atmosphere at 37 °C with 5% CO₂. Cells were treated with TGF-β1 (10 ng/mL) +/− Peptide 5 (25 μM). A scrambled version was used as a control.