Renal epithelial cell growth kit components

Human primary renal proximal tubular epithelial cells were purchased from Innoprot (Derio, Spain) and American Type Culture Collection (ATCC, Manassas, USA) and were maintained in Renal Epithelial Cell Basal Medium (ATCC) supplemented with Renal Epithelial Cell Growth Kit Components (ATCC). Human primary renal fibroblasts were purchased from Innoprot and maintained in DMEM F12 (Invitrogen) containing 10% FCS and supplemented with 2 mM L-Glutamine. For experiments, epithelial cell medium was used for both cell types. Cells were grown in 100% humidity and 5% CO₂ at 37°C and were used until Passage 6. For hypoxia experiments, cells were grown in 2.5%O₂ and 5% CO₂ using a Panasonic MCO-19 M incubator. For stimulation with cytokines, cells were incubated with TGFβ1 10 ng/ml (RnD Systems, Minneapolis, USA) added at the start of the culture. Aristolochic acid and H₂O₂ were from Sigma and were used at the indicated concentrations. For TGF-β blockade, cells were incubated with anti-TGF-β MAB1835 or appropriate isotype control (RnD Systems) for the duration of the culture period.

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).