Nrk 52e

A rat renal proximal tubular cell line (NRK-52E cells) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). NRK-52E cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 0.15% sodium bicarbonate (Sigma-Aldrich), 4 mM l-glutamine (Sigma-Aldrich), and 1% streptomycin/penicillin (Invitrogen, CA, Carlsbad, USA) in a humidified atmosphere chamber containing 5% CO₂ at 37 °C. For hypoxia experiments, cells were incubated in serum-free medium for 24 h in a hypoxic chamber (AnaeroPack; Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan) containing 1% O₂, followed by reoxygenation for a further 24 h to generate hypoxia/reoxygenation (H/R) cell models. The control group was exposed to normoxic conditions for 48 h.
miR-214 mimic, mimic control (miR-con), miR-214 inhibitor (anti-miR-214), inhibitor control (anti-miR-con), siRNA against Dkk3 (si-Dkk3), and siRNA control (si-con) were obtained from GenePharma Co. These oligonucleotides were transfected into NRK-52E cells using Lipofectamine 2000 (Invitrogen).

Kidney culture line (NRK-52E) was obtained through ATCC (USA). NRK-52E cultures were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and streptomycin (100 μg/ml; Invitrogen™) and penicillin (100 units/ml) at 37°C and CO₂ (5%). Human recombinant TGF-β1 was acquired from PeproTech™. Cultures (40% confluence) were exposed to recombinant human TGF-β1 (10 ng/ml, R&D Systems™) or negative control in DMEM for as long as required.

The normal rat kidney epithelial-derived cell line NRK52E (CRL-1571) was obtained from the Bioresource Collection and Research Center, Food Industry Research Development Institute, Hsinchu, Taiwan. NRK52E cells were cultured in 5% bovine calf serum-supplemented Dulbecco’s modified eagle medium (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere with 5% CO₂. Upon reaching 80% confluence, the cells were trypsinized with 0.25% trypsin–0.02% ethylenediaminetetraacetic acid (EDTA) for 5 min at 37 °C and then repassaged.

Rat renal proximal tubular epithelial cell lines (NRK52e) were purchased from ATCC (Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Hyclone, UT, USA) supplemented with 5% FBS (Gibco, MA, USA) at 37℃ in the humidified incubator with 5% CO₂.
NRK52e cells were divided into the following groups: the control group (Control), the high NaCl-induced model group (High-NaCl) (treated with 200 mmol/L NaCl for 6 h), the HCTZ group (High-NaCl + HCTZ) (treated with 200 mmol/L NaCl and 10 μmol/L HCTZ for 6 h), the different dose PA group (treated with 200 mmol/L NaCl and 1, 5, 10 μmol/L PA seperately for 6 h). At the same time, each group was set with corresponding SB203580 control group, and 10 µM SB203580 was added 1 h before modeling.
NRK52e cells were seeded in 96-well plates at the density of 2 × 10⁴ cells/mL. After the cells became adherent, we removed the original medium and washed the cells. The medium of the normal control group was replaced with DMEM containing 10% FBS. The medium of the model group was replaced with DMEM containing 10% FBS with 200 mmol/L NaCl. Other group replaced the medium with corresponding component. After the cells were cultured for 6 h, we detected the cell survival rate using MTT in each group [18 (link)].

The rat renal tubular cells line NRK-52E was purchased from National Collection of Authenticated Cell Cultures. NRK-52E cells were maintained in DMEM (GIBCO, USA) containing 10% fetal bovine serum, 100 U mL⁻¹ penicillin, and 100 mg mL⁻¹ streptomycin in 5% CO₂ at 37 °C.
Mesenchymal stem cells were isolated from human umbilical cord according to a previously described method. In brief, take the umbilical cord from a full-term newborn, wash with PBS and cut off the arteries and veins. Cut it into 2 mm sized tissue blocks and stick them on a 6-well plate with an interval of 5 mm. Add basic DMEM (GIBCO, USA) medium containing 10% fetal bovine serum and 100 U mL⁻¹ penicillin, and 100 mg mL⁻¹ streptomycin in 5% CO₂ at 37 °C. Thereafter, the medium was refreshed every 3 days and continuous culture until the purity of mesenchymal stem cells reached 85%.