4.4. Cell Culture Experiments

In vitro experiments were performed using a rat renal fibroblast cell line (NRK-49F, American Type Culture Collection, Manassas, VA). We cultured NRK-49F cells in Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L of sodium bicarbonate and 4.5 g/L of glucose supplemented with 5% (vol/vol) heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin G and 100 μg/mL streptomycin) at 37 °C with 5% CO₂ in 95% air. To investigate the effect of VP on myofibroblast activation, ECM expression, and the activation of the TGF-β1/Smad signaling pathway, we incubated sub-confluent NRK-49F cells with VP (50, 100, and 250 nM) for 30 min and then stimulated them with TGF-β1 (2 ng/mL, Sigma Chemical Co.) for the indicated periods.