2.1 Isolation, culture, and identification of MSCs
Rat bone marrow-derived MSCs were obtained from the femur and tibia of healthy male Sprague Dawley rats (100-120 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) in a 5% CO2 incubator at 37 °C. When the cultures reached 90% confluence, MSCs were harvested using 0.25% trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA). MSCs were subcultured at the ratio of 1:3. For all experiments, MSCs (passages 3-4) were collected and washed with sterile saline to achieve a single cell suspension for model treatment or analysis. To identify MSCs, the cells were incubated with the following antibodies against surface antigens (Abcam,UK): CD90-FITC (ab226), CD29-PE/Cy7 (ab95622), CD45-FITC (ab33916) and CD34-FITC (11-0341-82, eBioscience, USA). Finally, MSCs were washed and resuspended in 0.4 mL of PBS for analysis using a BD FACSCalibur flow cytometer and Cell Quest software (BD Biosciences, San Jose, CA, USA). Rat MSCs induction and differentiation were carried out using Oil red O staining to verify adipogenesis, using Alizarin red staining to confirm osteogenesis.
2.2 Adenine-induced nephropathy and administration of MSCs
Male Sprague Dawley rats (250-280 g, n=40) were purchased from Dashuo Biotechnology Co. LTD (Chengdu, China). Rats were housed in specific pathogen–free conditions at 24°C with 50% relative humidity under a 12 h light/dark cycle. All rats were randomly divided into three groups according to the random number table: Control group (Control), Adenine-induced nephropathy group (Adenine), and MSCs treatment group (Adenine+MSCs) (n=10 in Control group, n=15 in Adenine group and Adenine+MSCs group). Adenine group was induced by intragastric administration of 150 mg/kg adenine (Sigma Aldrich, USA) for 20 days. In the meanwhile, adenine gavage was suspended for 2 days at day 10 and continued. Adenine+MSCs group was injected with 1 mL of MSCs suspension (2.0×106 MSCs/kg) via the tail vein on day 3 after adenine gavage ends. Control group was instead injected with the same volume of saline. After MSCs treatment for 5 days, proteinuria level was measured with a kit from Thermo Fisher Scientific, and serum creatinine and urea nitrogen levels were measured with a kit from AmyJet Scientific Inc (Wuhan, Hubei, China). Serum creatinine, urea nitrogen, and proteinuria levels were measured by a Beckman Analyzer II (Beckman Instruments, Inc. USA). All animal studies were approved by the Southwest Medical University Animal Experimentation Ethics Committee and were performed in accordance with the approved guidelines.
2.3 Histological analysis and immunohistochemistry staining
The kidneys were fixed in phosphate buffer solution containing 4% paraformaldehyde and then embedded in paraffin. Tissue sections (5 µm) were stained with Hematoxylin -Eosin staining, Masson trichrome (Solarbio, Beijing, China) and Sirius Red staining (Yeasen Biotechnology Co. LTD., Shanghai, China) according to the instructions and viewed through a microscope. Tubular injury was assessed by the grading of tubular necrosis, cast formation, and tubular dilation. Scoring or positive area was quantified in 6-8 equivalent cortical HPFs (200×) from each rat by Image Pro Plus Software 6.0 (Media Cybernetics, Silver Spring, USA) and calculated as follows: 0, none; 1, ≤10%; 2, 11%-25%; 3, 26%-45%; 4, 46%-75%; and 5, >76% according to the literature.
For immunohistochemistry staining, kidney sections were blocked with 3% H2O2 in PBS for 15 min and nonspecific sites were blocked with 5% goat serum albumin for 30 min at room temperature. Then, slices were incubated overnight at 4 °C with 1:100 diluted rabbit anti-α-SMA antibody (ab5694, Abcam, UK), 1: 50 diluted rabbit collagen I (ab90395, Abcam, UK), 1:200 diluted rabbit Collagen II antibody (ab34712, Abcam, UK), 1:100 diluted rabbit Collagen III antibody (ab34710, Abcam, UK), and and 1:400 diluted rabbit p38 MAPK antibody (#8690, Cell Signaling, USA), respectively. After that, the HRP conjugated goat anti-rabbit IgG secondary antibody (ab6721, Abcam, UK) was added and incubated for 1.5 hours at room temperature. After washing with PBS and counterstaining with 3, 3’-diamnobenzidine (DAB), then the slices were placed in xylene to penetrate, concentration gradient alcohol to dehydrate, and images were examined by a light microscope. Positive staining was quantified in 6 equivalent cortical HPFs (200×) by Image Pro Plus 6.0 software. Protein semiquantitative analysis was presented as integrated optical density (IOD sum).
2.4 The Cytokine Antibody Array
The serum specimens were processed according to the guidelines specified by the Raybiotech Antibody Microarray protocol (GSR-CYT-3-1; Raybiotech, Atlanta, Georgia State, USA). All chemical reagents and solvents were obtained from the Raybiotech Antibody Microarray kit and Wayne Biotechnologies Inc.,（Shanghai, China）. The protein concentration of each specimen was determined using a BCA protein assay kit (Beyotime, Shanghai, China). A rat cytokine array kit was used to compare cytokine expression in serum between control group, adenine group and adenine+MSCs group, n=5 in each group. Standardized quantities of each protein specimen were loaded into identical cytokine antibody subarrays. Following incubation at room temperature for 2 hours, the subarrays were washed with the included wash buffer according to the operating instructions. The detection process was then performed using a biotin antibody cocktail as well as Cy3-Streptavidin. The subarrays were scanned on a GenePix 4000B microarray scanner and the raw data were readed by GenePix Pro 6.0 software (Axon Instruments, USA). A total of 27 cytokines were detected on the GSR-CYT-3-1 chip, and each antibody on the chip was set up four technical repetitions. During the data analysis, the mean value of four replicates was first calculated as the signal value of each factor, then the signal value was normalized to a positive control to allow comparison between subarrays, and finally the concentration was quantified by using the normalized data. The intergroup ratio of 27 factors was calculated, and the P values between groups were analyzed by t-TEST (double-tailed).
2.5 Renal tissue proteomics
2.5.1 Protein isolation, digestion, labeling with iTRAQ reagents
Rats were sacrificed at day 5 after MSCs intervention to carry out renal proteomic analysis based on isobaric tags for relative and absolute quantification (iTRAQ). The workflow of the study is presented in Figure 1. Forty-five kidney tissues were washed twice with ice-cold PBS and then homogenized with MP homogenizer (MP Fastprep-24, 5G) in SDT pyrolysis solution (4% SDS, 1 mM DTT, 150 mM Tris-HCl pH 8.0, and protease inhibitor). After ultrasonication, the homogenate was incubated for 15 min in boiling water. The crude extract was then centrifugated at 14,000 × g at 25 ℃ for 15 min, the protein concentration was measured by a BCA protein assay kit. The samples were stored at -80 ℃.
Protein digestion was performed according to the filter-aided sample preparation procedure described by Wisniewski, and the resulting peptide mixture was labeled using the 8-plex iTRAQ reagent according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Briefly, 30 μL of protein digestion for each sample were added into DTT to the final concentration of 100 mM, boiling water for 5 min, cooling to room temperature. The detergent, DTT, and other low-molecular-weight components were removed using 200 μL UA Buffer (8 M Urea and 150 mM Tris-HCl, pH 8.0) by repeated ultrafiltration. Next, 100 μL IAA buffer (0.1 M iodoacetamide in UA Buffer) was added to block to reduce cysteine residues and the samples were incubated for 30 min in darkness. The filters were washed with 100 μL UA buffer two times, washed twice with 100 μL dissolution buffer. Finally, the protein suspensions were digested with 4 μg trypsin (Promega, Madison, WI) in 40 μL dissolution overnight at 37℃ and the resulting peptides were collected as a filtrate. The peptide content was estimated by UV light spectral density at 280 nm with Nano Drop 2000. 100 μg peptides from each sample were labeled according to the iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex, UK). The peptides were, respectively, mixed as a pool. The peptides from all groups were, respectively, mixed as a pool and then equally divided into three fractions (Control group: A1 and A2; Adenine group: B1, B2 and B3; Adenine+MSCs group: C1, C2 and C3). A standard pool comprising a mixture of equal amounts of protein derived from all samples served as an internal control. The samples were labeled as A1-113, A2-114, B1-115, B2-116, B3-117, C1-118, C2-119, and C3-121, and were multiplexed and vacuum dried. For labeling, each iTRAQ reagent was dissolved in 70 μL of ethanol and added to the respective peptide mixture. This experiment was done with three biological replicates.
2.5.2 Mass spectrometry analysis
All the labeled samples were mixed with equal amount. Next, the labeled samples were fractionated using a Agilent 1260 infinity II high-performance liquid chromatography (HPLC) system (Thermo Dinoex Ultimate 3000 BioRS) equipped with a XBridge Peptide BEH C18 (130Å, 5 µm, 4.6 mm × 100 mm) column. LC-MS/MS analysis was performed with an Q Exactive Plus LC-MS/MS system (Thermo Scientific, Waltham, Massachusetts, USA).
2.5.3 Bioinformatic analysis
The mass spectrometry data was analyzed using Proteome Discoverer 2.1 (Thermo Scientific) and Mascot 2.5 sortware, peptide identifications were made using the Paragon algorithm searching against the uniprot Rattus Norvegicus protein database (http://www.uniprot.org/). Only the peptide FDR value which set to less than 0.01 was contained in iTRAQ labeling quantification, and proteins with an average fold change larger than 1.2 times were considered significantly differentially expressed for further analysis. To determine the biological and functional properties of the identified proteins, the identified protein sequences were mapped with Gene Ontology Terms (http://geneontology.org/). In brief, a homology search was firstly performed for all identified sequences with a localized NCBI BLAST+ (ncbi-blast-2.2.28+-win32.exe) program against NCBI database. The e-value was set to less than 1e-3, and the best hit for each query sequence was accounted for GO term matching. The informations on molecular function, cellular components, and biological process were acquired by searching with terms and gene products. The GO term matching was performed with Blast2go Command Line. Pathway analysis specifying the relationships between the interacting molecules was made by keyword search in the KEGG GENES database (http://www.kegg.jp/) and KAAS (KEGG Automatic Annotation Server Ver. 2.1) online tool (http://www.genome.jp/tools/kaas/). The pathway enrichment statistics were performed by Fisher’s exact test, and those with a corrected p value< 0.05 were regarded as the most significant pathways.
2.6 Western Blot
About 100 mg of kidney tissue was lysated in ice-cold RIPA lysis buffer (Beyotime, China) containing 1 mM PMSF. The concentration of protein was determined by a BCA assay kit. About 50 μg protein samples were loaded in 10% SDS-PAGE gels and transferred onto the PVDF membrane (Millipore, USA). After blocking with 5% skim milk, the membrane was incubated overnight at 4 °C with the following antibodies: TGFβ1 (1:2000, Proteintech, USA), GAPDH (1:50000, Proteintech, USA), α-SMA (1:5000, Proteintech, USA), Galectin3 (1:1000, Cell Signaling, USA), p38 MAPK (1:1000, Abcam, UK), phospho-Samd2/3 (1:1000, Cell Signaling, USA), Smad2/3 (1:1000, Cell Signaling, USA), IL-6 (1:1000, Proteintech, USA), IL-1β (1:1000, Bioworld, China), and TNFα (1:1000, Proteintech, USA) antibodies. The bolts were incubated with horseradish peroxidase (HRP)-conjugated goat-anti rabbit or mouse secondary antibodies for 1 h, and the membranes reacted with chemiluminescence HRP substrate (Solarbio, China) and exposed to the ChemiScope 6000 Exp image system (CliNX, Shanghai, China) for visualization of protein bands. The protein bands were quantified using the NIH ImageJ Software.
2.7 Fluorescence quantitative polymerase chain reaction
For further verification, we selected the most clearly downregulated protein (galectin3) compared adenine group with adenine+MSCs group according to bioinformatics analysis and references. Total RNA were extracted using trizol (Invitrogen, USA) from renal tissues in all groups. First-strand cDNA syntheses were performed from total RNA by reverse transcription using the Eastep RT Master Mix Kit (Promega, Shanghai, China). Fluorescence quantitative PCR amplifications were performed at 95℃ for 10 sec, 60℃ for 15 sec using rat Galectin3 using the Eastep qPCR Master Mix kit (Promega, Shanghai, China). GAPDH was used as an internal control. The design of the oligonucleotide primer sequences based on: Galectin3, sense 5’-aacgacatcgccttccac-3’, and antisense 5’-cccagttattgtcctgcttc-3’; GAPDH, sense 5’-gcaagttcaacggcacag-3’ and antisense 5’-gccagtagactccacgacat-3’. Fluorescence quantitative PCR was performed on LightCycle480 (Roche, Basel, Switzerland) in triplicate. Specificity of the PCR products was confirmed by analysis of the dissociation curve. In addition, the amplicons' expected size was confirmed by analysis of the PCR products on 1% agarose gels, subsequently visualized under UV light. The relative mRNA levels were calculated using the 2-ΔΔCt method after normalization with GAPDH as a housekeeping gene. All data for RNA expression analysis were calculated using the 2-ΔΔCt method.
2.8 NRK-49F and NRK-52E Cells culture and treatment
Rat renal fibroblast cells (NRK-49F) and rat renal tubular epithelial cells (NRK-52) were purchased from the Beina Chuanglian Biotechnology Research Institute (BNBIO, Beijing, China) and separately maintained in MEM and DMEM medium (Gibco/Life Technologies, Grand Island, NY) supplemented with 10% FBS (Gibco/Life Technologies). Cells that reached approximately 50% confluence were used for in vitro experiments.
To test the effect of MSCs on galectin3 protein expression induced by TGF-β1 in two cells. We first prepared MSCs conditioned medium (MSCs-CM). MSCs (Passages 3, 80% confluence) were incubated with serum-free DMEM low-glucose for 24 h at 37˚C before MSCs-CM supernatant collection. Supernatants were then centrifuged at 2, 000 rpm at 4˚C for 5 min and the cell debris were removed with a 0.22 μm disposable filter. NRK-49F and NRK-52E were, respectively, seeded into 6 cm sterile dishes and randomly divided into five groups: (1) Normal group; (2) TGF-β1-induced group; (3) TGF-β1+TD139 (a specific galectin3 inhibitor, MCE, NJ, USA) group; (4) TGF-β1+50% MSC-CM group; (5) TGF-β1+TD139+50% MSC-CM group. TGF-β1-induced group was serum starved for 12 h, followed by incubation with recombinant human TGF-β1 (20 ng/mL，PeproTech, Rocky Hill, NJ, USA) for 48 h. TD139 was pretreated for 2h before TGF-β1 treatment. 50% MSCs-CM was added to the dishes for 48 h. The cell lysates were used for western blot analysis.
2.9 Statistical analysis
Data are presented as mean±SD of at least three biological repeation. Normal distribution of data was checked by means of the Shapiro-Wilk test and a Levene statistic test was performed to check the homogeneity of variances. To determine statistical significance, statistical analysis was performed using one-way ANOVA (GraphPad Software, San Diego, CA, USA), followed by the Bonferroni post hoc testing to analyze differences between groups. P< 0.05 was considered significant.