Renal epithelial cell basal media

Primary hPTECs were maintained in Renal Epithelial Cell Basal Media from ATCC, supplemented with Renal Epithelial Cell Growth Kit components (PCS-400-040) in a humidified atmosphere at 37 °C with 5% CO₂. Cells were treated with TGF-β1 or ATPγS ± a 30-minute pre-incubation with Cx43 hemichannel blocker Peptide5 (25µM) or P2X7R antagonist A438079 (50µM − 100µM) for 48 h. For real time ATP biosensing studies, human kidney (HK2) cells (passage 18–30) were maintained in DMEM/Hams F12 medium, supplemented with 10% foetal calf serum, glutamine (2mmol/L) and epidermal growth factor (5ng/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. Cells were seeded in low-glucose DMEM/F12 (5mmol/L) for 48 h and serum-starved overnight prior to treatment with TGF-β1 (2–10ng/mL) for 48 h, ± a 30-minute pre-incubation with Peptide 5 (25 μm), NLRP3 inhibitor CY-09 (1–20µM) or caspase 1 inhibitor AC-YVAD-CMK (0.1–10 µg/mL).

American type culture collection (ATCC)® Normal Human Primary Renal Proximal Tubule Epithelial Cells (hPTC) were grown in Renal Epithelial Cell Basal Media (PCS-400-030,ATCC) supplemented with Renal Epithelial Cell Growth Kit components (PCS-400-040). The final medium included fetal bovine serum (0.5%), triiodothyronine (10 nM), recombinant human epidermal growth factor (10 ng/mL), hydrocortisone hemisuccinate (100 ng/mL), recombinant human insulin (5 mg/mL), epinephrine (1.0 mM), transferrin (5 mg/mL), and L-alanyl-L-glutamine (2.4 mM). The experimental group was treated with 80 ng/mL rGDF11 (Perprotec) in 0.1% BSA, and the control group was administered an equal volume of 0.1% BSA. To measure migration, hPTCs were grown to confluence, and monolayers were wounded with a rubber policeman to produce a linear 4-mm swipe. After one wash with PBS, cells were cultured in medium in the presence of 80 ng/mL rGDF11 or 0.1% BSA. After 36 h, cell migration was determined using a microscope and camera, and the wound area was calculated using NIH Image J software. To measure hPTC proliferation, 2,500 cells/well were seeded into 96-well plates, and the relative cell viability at each experimental time was determined by using a cell counting kit (DojinDo).