2.1 MSCs primary culture and conditioned medium preparation
Bone marrow derived MSCs were obtained from the femur and tibia of healthy male Sprague-Dawley rats (130 ± 20 g). These cells were cultured in low glucose DMEM containing 10% FBS (Gibco, Invitrogen, New York, USA), 2.5 mM L-glutamine, and penicillin/streptomycin (Gibco, Invitrogen, New York, USA) at 37°C under 5% CO2. MSCs were harvested with 0.25% trypsin EDTA (Thermo Fisher Scientific, Waltham, MA, USA) when cell growth density reached 90% confluence, and then continued to be subcultured in a ratio of 1:3. In our previous studies (Tanget al. 2021), MSCs have been identified by flow cytometry. When MSCs reached 80% at passage 3, the cells were washed with phosphate buffered saline (PBS) for three time, and cultured with DMEM without serum and antibiotics for 48 h. Collect the free serum supernatant and centrifuge it at 2,000 rpm for 10 min and remove cellular debris by using a 0.22 µm disposable filter, and then store it at − 80°C till use.
2.2 Experimental grouping and processing
Normal rat renal tubular epithelial cells (NRK-52E) were purchased from Beina chuanglian (BNBIO, Beijing, China) and cultured in Dulbecco's modified Eagle's medium with low glucose (DMEM) (Gibco/Life Technologies, Grand Island, NY) with containing 10% FBS (Gibco/Life Technologies). When the cells reached 80% confluence, the cells were divided into 9 groups: normal group, DMSO group, RepSox group, RepSox + TGF-β1 group, RepSox + TGF-β1 + MSCs-cm group, TGF-β1 group, TGF-β1 + MSCs-cm group, MSCs-cm group, and RepSox + MSCs-cm group. The cells were treated with recombinant human TGF-β1 (20 ng/mL, PeproTech, Rocky Hill, NJ, USA) for 48 h to establish renal interstitial fibrosis model. RepSox (25 µM) was pretreated for 2 h until TGF-β1-induced modeling was established. MSCs-cm or SF-DMEM was added to the dishes for 48 h after modeling.
2.3 Western blot
The cells were washed twice with cold PBS buffer, and lysated by RIPA lysate containing 1.0 mmol/L of PMSF and phosphorylase inhibitor on ice. Total protein concentration was determined with a BCA protein assay kit. Protein samples were separated with 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, USA). After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with the following primary antibodies: GAPDH (1:5000, Abmart, China), α-SMA (1:1000, Affinity, China), Snail (C15D3) (1:1000, Cell Signaling, USA), Fibronectin (1:1000, Abcam, UK), E-cadherin (1:1000, Abcam, UK), MMP9 (1:1000, Abcam, UK), TIMP1 (1:1000, Abcam, UK), Kim-1 (1:1000, SAB, USA), TGF-β1 (1:1000, Affinity, China), Phospho-smad2/3 (1:1000, Affinity, China), and SMAD2/3 (1:1000, Affinity, China). HRP-conjugated goat anti-rabbit or mouse secondary antibodies were incubated at 37 ℃ for 2 hours. ChemiScope 6000 Exp Gel imaging system (CliNX, Shanghai, China) shows protein bands. The protein bands were quantified using the NIH Image J Software.
2.4 Immunofluorescence
To observe the fluorescence expression of EMT marekers, cell slides were placed into 24-well plates and the total of 10,000 or 15,000 cells were seeded in each well. When the processing was finished, the slides were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, infiltrated with 0.5% Triton X-100 for 10 min, blocked with 5% bovine serum albumin for 30 min, and incubated at 4 ℃ overnight with primary antibodies against α-SMA (1:200, Proteintech, USA) and E-Cadherin (1:100, Cell Signaling, USA). The cells were then washed with PBS three times, and then separately incubated with Alexa Fluor 594 or Alexa Fluor 488-conjugated goat anti-mouse or rabbit secondary antibodies (1:1000, Invitrogen, California, USA) for 1 h at 37 ℃. Nuclei were stained with DAPI for 5 min. Finally, the samples were rinsed with PBS and visualized using fluorescence microscopy.
2.5 Statistical analysis
Data are presented as mean ± standard deviation (s.d.) of at least three biological repetitions for data validity and authenticity. Analysis was performed using GraphPad software (San Diego, CA, USA). Statistical significance (P < 0.05) was calculated using one-way ANOVA, followed by the Bonferroni or Tukey post hoc testing to analyze differences between groups.