Nrk 52e

The normal rat renal tubular epithelial cell line NRK-52E was purchased from American Type Culture Collection (ATCC, Manassas, United States), and routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM, Wisent, Nanjing, China) containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, United States), 100 U/ml penicillin (Gibco, Carlsbad, CA, United States), and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, United States) at 37°C with 5% CO₂. In the first experiment, NRK-52E cells were pretreated with serum-free medium for 24 h, then exposed to 40 μM or 80 μM emodin (Shanghai Yuanye Bio-Technology Co., Ltd., China) for 24 h. In the second experiment, NRK-52E cells were pretreated with serum-free medium for 24 h, then exposed to 10 ng/mL TGF-β1 (R&D Systems, MN, United States) with or without 40 μM or 80 μM emodin for 24 h. In the third experiment, HEK293 cells were transfected with miR-490-3p mimics, miR-490-3p inhibitors, and negative controls (NCs), then treated with or without 10 ng/mL TGF-β1 for 24 h. In the fouth experiment, NRK-52E cells transfected with miR-490-3p mimics, miR-490-3p inhibitors, and NCs were treated with or without 80 μM emodin for 24 h, then exposed to 10 ng/mL TGF-β1 for 24 h. All control groups were treated with the same volume of vehicle.

Rat renal proximal tubular epithelial cells (NRK-52E) and rat cardiomyocyte cells (H9C2) were purchased from ATCC (Manassas, VA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM/F12), cultured with 5% CO2 and 95% humidity, supplemented with 10% fetal bovine serum. After 24 h, NRK-52E cells were cultured in FBS-free medium for another 16 h, then incubated with or without recombined human transforming growth factor β1 (rhTGF-β1) (R&D Systems, MN) at a dose of 5 ng/mL [10 (link)]. Donor NRK-52E cells were incubated with rhTGF-β1 for 48 h and maintained in FBS-free DMEM/F12 for another 48 h in experimental group. To generate conditioned medium, rhTGF-β1-free medium was collected and sequentially centrifuged at 300, 1,200 and 10,000 g for 5, 20 and 30 min, respectively. The control medium was generated in the same way but without exposing to rhTGF-β1 in the first 48 h. Then the recipient cardiomyocytes were incubated with control or conditioned medium and collected for further characterization.