The study involving both human and animals was conducted in accordance with the principles of the Helsinki Declaration and was approved by the ethical committee of Chongqing Medical University (File No. 2016-124).
Isolation, expansion, and characteristics of hucMSCs
After obtaining parental and ethics committee consent, hucMSCs were isolated as described previously . The cells were cultured in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F12) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, USA) at 37 °C with 5% CO2. The media were subsequently exchanged every 3 days. The phycoerythrin (PE)-conjugated antibodies were CD34, CD45, CD73, CD90, CD105, and human leukocyte antigen (HLA)-DR (BD Biosciences). The multilineage differentiation of hucMSCs was determined using adipogenic and osteogenic medium (Cyagen, China). Briefly, a total of 1 × 105 hucMSCs at passage 2 were seeded in a six-well plate and then incubated with induced medium. After 2 weeks, adipogenic differentiation of hucMSCs was observed by Oil Red O staining, and osteogenic differentiation of hucMSCs was examined by Alizarin Red staining (Additional file 1: Figure S1).
Collection and concentration of hucMSC-CM
Passage 3 hucMSCs were cultured to 80–90% confluence in a T75 culture flask (4 × 106 cells). After the cells were washed three times with phosphate-buffered saline (PBS), the serum-free DMEM medium was refilled (10 ml) and further incubated for 48 hours. The collected medium was centrifuged for 10 min at 3000 rpm in order to remove cell debris. CM was concentrated 25 times using 10 kDa MW cutoff filter units (Millipore) and sterilized by filtration through a 0.22-μm filter. After these steps, the mean protein concentration of the concentrated CM is about 0.5 mg/ml. All of the concentrated hucMSC-CM was stored at –80 °C until use.
In-vitro experimental treatment of NRK-52E
NRK-52E cells were purchased from China Center for Type Culture Collection (CCTCC), and cultured in DMEM medium containing 10% FBS (Gibco, Grand Island, NY, USA) and 2% penicillin/streptomycin at 37 °C with 5% CO2. NRK-52E cells with good morphology were digested and seeded in six-well plates at a cell concentration of about 1 × 105 cells per well. After the cells attached, recombinant human transforming growth factor β1 (TGF-β1) (R&D, Minneapolis, MN, USA) (5 ng/ml) was added to induce apoptosis with hucMSC-CM (40 μg) treatment. After 48 hours, the cell slide was used for immunofluorescent staining, and the proteins in cells were extracted for western blot assay.
UUO rat model and injection of hucMSC-CM
In this study, male SD rats with weight average 200 ± 10 g were provided and raised at Chongqing Medical University (SPF, License No.: SYXK (Chongqing) 2007-0001). The rats were raised in polycarbonate cages in a human-controlled environment, such as free water and food, with an illumination schedule of 12-hour light/12-hour dark.
The rats were categorized randomly into four groups (n = 8 in each group), including PBS transplanted with sham operation (Sham group), CM transplanted with sham operation (Control group), PBS transplanted with UUO (UUO group), and CM transplanted with UUO (hucMSC-CM group). The male SD rats were anesthetized with chloral hydrate. The left ureter and kidney were then exposed with the assistance of flank incision; the ligation process of the left ureter was conducted with two 4-0 silk sutures at the upper third of the ureter, then cut between the ligatures to prevent retrograde urinary tract infection. Sham-treated rats underwent an identical procedure except for ureteral ligation. The Control and hucMSC-CM groups received hucMSC-CM (100 μl) via the left renal artery after the surgery. All groups received antibiotics (0.1% amoxicillin) in their drinking water for 14 days. At days 14 post surgery, the rats were euthanized and the left kidneys were extracted. One portion of each kidney was fixed in paraformaldehyde solution (4%), and the other portions were stored in liquid nitrogen. Blood samples were collected in order to measure biochemical analysis, including serum creatinine (SCr) and blood urea nitrogen (BUN).
In order to explore the degree of renal tubular injury and renal interstitial fibrosis, the kidney was fixed with 4% paraformaldehyde, embedded in paraffin, and cut to 4 μm thick. The sections were dewaxed with xylene and then incubated sequentially for 5 min in different concentrations of alcohol and water before hematoxylin–eosin (HE), periodic acid–Schiff (PAS), and Masson’s trichrome staining. The damaged degree of tubular cells was scored and divided into four levels: 0 = normal without any damages; 1 = mild dilatation; 2 = flattened epithelial cells and loss of brush border; and 3 = denudation of basement membranes, tubular cell apoptosis and necrosis. The total score is calculated as the average of all tubular scores with 200 tubuli per section. Ten visual fields were selected randomly in digital images from each section under 40× magnification and then the percentage of fibrotic areas to overall field areas was calculated.
Measurement of TNF-α, IL-6, IL-1β, and MCP-1
In order to determine the levels of TNF-α, IL-6, and IL-1β in blood serum and kidney tissues of each group, ELISA kits from R&D Systems were used and operated according to the manufacturer’s instructions. The levels of TNF-α and MCP-1 (BIOSOURCE, USA) in the cell supernatant were measured by the same method.
Immunofluorescent and immunohistochemistry
NRK-52E cells were treated as already described, fixed with 4% paraformaldehyde, and permeabilized with Triton X100 (Solarbio, China). The cell slides were then blocked with 5% BSA (BD, USA) and incubated with cytokeratin 8 (1:100; Abcam, USA), E-cadherin (1:200; Abcam, USA), α-SMA (1:200; Abcam, USA), TLR4 (1:200; Santa Cruz, USA), and p-NF-κB (1:200; CST, USA) at 4 °C overnight. After washing three times with PBS, the corresponding fluorescent secondary antibody (ZSGB-BIO, China) was added to incubate for 1 hour. After nuclear counterstaining with DAPI (Beyotime, China), images were examined under a fluorescence microscope (K10587; Nikon, Japan).
The kidney tissue slices were underwent dewaxing and hydration, and antigen was retrieved by citric acid buffer (PH 6.0) microwave antigen retrieval. After natural cooling, the endogenous peroxidase was blocked with 3% H2O2 for 10 min, and then the nonspecific protein binding site was blocked with 0.5% BSA for 1 hour. Slices were incubated at 4 °C with 1:200 diluted mouse anti-E-cadherin antibody (Santa Cruz, USA), rabbit anti-Collagen-1 antibody (Abcam, USA), mouse anti-α-smooth muscle actin antibody (Abcam, USA), rabbit anti-CD3 antibody (Abcam, USA), rabbit anti-CD68 antibody (Abcam, USA), rabbit anti-MCP-1 antibody (Abcam, USA), rabbit anti-TNF-α (Genetex, USA), mouse anti-TLR-4 antibody (Santa Cruz, USA), and rabbit anti-phosphorylated-NF-κB antibody (CST, USA), respectively. Slices were washed three times with PBS, then with the goat anti-rabbit or anti-mouse antibody (Zhongshan, Beijing, China) at 37 °C for 1 hour. After sections were counterstained with hematoxylin, positive staining was developed by incubation with 3,3′-diamnobenzidine (DAB). The intensity of E-cadherin, Collagen-I, α-SMA, MCP-1, TNF-α, TLR-4, and phosphorylated-NF-κB was assessed under five randomly selected high-power fields (×400). Image Pro Plus 6.0 was used as a tool to quantify the integrated optical density (IOD) of positive areas.
Western blot analysis
Kidney tissues and cells were lysed in RIPA Lysis Buffer (Beyotime, China) containing PMSF, and centrifuged at 4 °C at 12,000 rpm for 20 min. The protein concentration was measured using the BCA assay kit (Beyotime, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was mixed with protein samples and boiled last for 10 min. The proteins were electrophoresed through SDS-polyacrylamide gels. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). In order to block the nonspecific protein background, membranes were blocked in 5% skim milk for 1 hour with a shaker. After that, the protein bands were incubated overnight at 4 °C with the following primary antibodies: mouse anti-E-cadherin (1:1000; Santa Cruz, USA), rabbit anti-Collagen-I (1:1000; Abcam, USA), mouse anti-α-SMA (1:1000; Abcam, USA), mouse anti-cytokeratin 8 (1:1000; Abcam, USA), rabbit anti-CD68 (1:500; Abcam, USA), rabbit anti-MCP-1 (1:2000; Abcam, USA), rabbit anti-TNF-α (1:1000; Genetex, USA), mouse anti-TLR-4 (1:1000; Santa Cruz, USA), rabbit anti-NF-κB (1:1000; Proteintech, USA), rabbit anti-phosphorylated-NF-κB (1:1000; CST, USA), rabbit anti-IκBα (1:400; Bioss, China), rabbit anti-phosphorylated-IκBα (1:400; Bioss, China) and β-actin (1:500; Zhongshan, China). The membrane was then washed three times with Tris-buffered saline/Tween (TBST) and incubated in goat anti-rabbit or mouse antibodies (Zhongshan, China) for 2 hours at 37 °C. Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) were used to detect positive immune reactions.
The data obtained from the experiment were expressed as mean ± standard deviation (SD). The mean value of each group was compared with one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. When P < 0.05, the difference was considered statistically significant. All data were analyzed by SPSS 16.0 software.