Mesenchymal Stem Cells Ameliorate Interstitial Fibrosis in Adenine-induced Nephropathy Based on Renal Proteomics

Background: Tubulointerstitial brosis (TIF) is one of the main pathological features of various progressive renal damages and chronic kidney diseases. Mesenchymal stromal cells (MSCs) have been veried with signicant improvement in the therapy of brosis diseases, but the mechanism is still unclear. We attempted to explore the new mechanism and therapeutic target of MSCs against renal fibrosis based on renal proteomics. Methods: TIF model was induced by adenine gavage. Bone marrow derived MSCs was injected by tail vein after modeling. Fibrosis biomarkers or extracellular matrix proteins and histopathological change were assessed by Masson staining, Sirius red staining, immunohistochemistry, and western blot. Renal proteomics was analyzed using iTRAQ-based mass spectrometry. Results: MSCs treatment clearly decreased the expression of α-SMA, collagen type I, II, III, TGF-β1, p-Smad2/3, IL-6, IL-1β, and TNFα compared with model rats, while p38 MAPK increased. 6,213 proteins were identied, but only 40 proteins exhibited signicant differences (30 upregulated, 10 downregulated) compared MSCs group with the model group. Bioinformatics analysis revealed that these proteins play important roles in the proliferation, inammatory and immune responses, apoptosis, phagosome, etc. According to literatures and bioinformatics analysis, the most markedly downregulated protein, galectin3, was further assessed by quantitative PCR and western blot in renal tissues. Galectin3 levels were downregulated in adenine-induced renal tissues and TGF-β1 induced tubular epithelial cells and interstitial broblasts in consistent with iTRAQ after MSCs treatment. Conclusion: The founds suggest that galectin3 maybe involves in the antibrotic mechanisms of MSCs therapy for tubulointerstitial brosis as well as a possible therapeutic target. elucidated. Several recent studies show the capacity of exogenously administered MSCs or MSCs conditioned medium to dramatically reduce tubulointerstitial brosis, preserve peritubular capillary density, and prevent epithelial mesenchymal transition in multiple different models of chronic renal injury [19-21] . Our results support these evidences, and show a signicant reduction in extracellular matrix components, such as collagen type Iα, collagen type II, and collagen type III; inammation-related factors, IL-1β, IL-6, TFNα, myobroblast activation marker, α-SMA and increase in proliferation-related signal, p38 MAPK protein expression in adenine-induced nephropathy after MSCs early intervention. To date, the mechanism of MSCs contributed to the renoprotection is involved in paracrine cytokines, growth factors, and immuoregulation [4] , but no research has focused on the renal proteomic prole of MSCs therapy for adenine-induced interstitial brosis, and may make a valuable contribution towards the comprehension of the molecular mechanisms involved in MSCs which alleviated renal brosis.


Introduction
Adenine-induced nephropathy is a rat model of human tubulointerstitial brosis (TIF), which is the main pathological feature of various progressive renal damage and the nal common pathway for chronic kidney disease (CKD). TIF is characterized by in ltration of renal interstitial monocytes and lymphocytes, myo broblast activation, excessive deposition of extracellular matrix (ECM), and sclerosis [1] . Adenine is mainly used to participate in the synthesis of DNA and RNA and has been widely used for the treatment of patients with leukopenia [2] . However, it causes severe nephrotoxicity and brosis because of the high accumulation in the kidney. Adenine produces 2, 8-dihydroxyadenine through the action of xanthine oxidase, which deposits in the glomeruli and the interstitium, forming granulomatous in ammation and blocking the renal tubules leading to cystic dilatation of renal tubules and kidney failure [3] . Adenineinduced CKD rats exhibited anemia, uremia, decreased renal function, increased in ltration of in ammatory cells, tubular atrophy, and brosis [1] .
Mesenchymal stem cell-based therapy has emerged as a promising way for anti brosis in the latest 10 years [4] . MSCs secrete a wide range of soluble cytokines that are helpful for anti-brosis, anti-apoptosis, immunomodulation, and tissue repair [5] . Several studies have shown that MSCs ameliorate renal brosis in adenine, cisplatin, adriamycin-induced animal models and an ischemia-reperfusion injury model [6][7][8][9] .
However, it has not been fully elucidated that the mechanisms of MSCs for improving renal brosis and function. In this study, we examined how MSCs ameliorate renal brosis in adenine-induced rats based on isobaric tags for relative quanti cation proteomics (iTRAQ) and further veri cation in vivo and in vitro.

Isolation, culture, and identi cation 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% CO 2 incubator at 37 °C. When the cultures reached 90% con uence, MSCs were harvested using 0. 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 con rm osteogenesis.

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 speci c 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 (2.0×10 6 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 Scienti c, and serum creatinine and urea nitrogen levels were measured with a kit from AmyJet Scienti c 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.
For immunohistochemistry staining, kidney sections were blocked with 3% H 2 O 2 in PBS for 15 min and nonspeci c 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 quanti ed in 6 equivalent cortical HPFs (200×) by Image Pro Plus 6.0 software. Protein semiquantitative analysis was presented as integrated optical density (IOD sum).

The Cytokine Antibody Array
The serum specimens were processed according to the guidelines speci ed 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 rst calculated as the signal value of each factor, then the signal value was normalized to a positive control to allow comparison between subarrays, and nally the concentration was quanti ed 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 sacri ced at day 5 after MSCs intervention to carry out renal proteomic analysis based on isobaric tags for relative and absolute quanti cation (iTRAQ). The work ow of the study is presented in Figure 1. Forty-ve 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 lter-aided sample preparation procedure described by Wisniewski [11] , and the resulting peptide mixture was labeled using the 8-plex iTRAQ reagent according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Brie y, 30 μL of protein digestion for each sample were added into DTT to the nal 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 ultra ltration. 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 lters 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 ltrate. 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.

Mass spectrometry analysis
All the labeled samples were mixed with equal amount. Next, the labeled samples were fractionated using a Agilent 1260 in nity 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 Scienti c, Waltham, Massachusetts, USA).

Bioinformatic analysis
The mass spectrometry data was analyzed using Proteome Discoverer 2.1 (Thermo Scienti c) and Mascot 2.5 sortware, peptide identi cations 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 quanti cation, and proteins with an average fold change larger than 1.2 times were considered signi cantly differentially expressed for further analysis. To determine the biological and functional properties of the identi ed proteins, the identi ed protein sequences were mapped with Gene Ontology Terms (http://geneontology.org/). In brief, a homology search was rstly performed for all identi ed sequences with a localized NCBI BLAST+ (ncbiblast-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 signi cant pathways.

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 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 quanti ed using the NIH ImageJ Software.

Fluorescence quantitative polymerase chain reaction
For further veri cation, we selected the most clearly downregulated protein (galectin3) compared adenine group with adenine+MSCs group according to bioinformatics analysis and references [12] . 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 ampli cations 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. Speci city of the PCR products was con rmed by analysis of the dissociation curve. In addition, the amplicons' expected size was con rmed 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. To test the effect of MSCs on galectin3 protein expression induced by TGF-β1 in two cells. We rst prepared MSCs conditioned medium (MSCs-CM). MSCs (Passages 3, 80% con uence) 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 lter. NRK-49F and NRK-52E were, respectively, seeded into 6 cm sterile dishes and randomly divided into ve groups: (1) Normal group; (2) TGF-β1-induced group; (3) TGF-β1+TD139 (a speci c 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.

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 signi cance, 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 signi cant.

MSCs features
Bone marrow-derived MSCs showed a typical spindle-shaped appearance ( Fig. 2A), and the ability to differentiate into osteocytes and adipocytes (Fig. 2B, 2C). Flow cytometric analysis revealed MSCs showed high expression levels of CD29 and CD90 and low expression levels of CD34 and CD45 (Fig. 2D). These ndings con rmed the presence of a MSC phenotype.

MSCs ameliorated renal function in Adenine-induced nephropathy
We examined the therapeutic effects of MSCs on renal injury in adenine-treated rats. Adenine was carried out intragastric administration continuously for 20 days and MSCs were transplanted on day 3, and creatinine, urea nitrogen, and 24h urinary protein were analyzed on days 0, 20, and 30. Kidney index (Kidney weight/Body weight ×100%) of adenine-treated rats increased, exhibited typically large white kidneys (Fig. 3E), and MSCs prevented these increases (Fig. 3A). Adenine increased the levels of serum urea nitrogen (Fig. 3B), creatinine (Fig. 3C), and 24 h urinary proteins (Fig. 3D). These effects were prevented by MSCs.

MSC treatment ameliorated renal brosis in Adenine-induced rats.
Adenine leaded to cystic dilatation of renal tubules, in ammatory cell in ltration, and renal damage scores increase, in contrast, the pathologic changes in MSC-treated rats were milder (Fig. 4A, D). The Masson's trichrome staining (Fig. 4B, E) and Sirus red staining (Fig. 4C, F) of renal tissues showed that renal interstitial extracellular matrix increased in the Adenine-induced group, while the renal brosis was improved by MSC treatment in Adenine+MSC group. The immunohistochemical staining revealed that Collagen I, II, III was majorly expressed in the tubulointerstitium in the Adenine group, while the expression of Collagen I, II, III was greatly reduced in Adenine+MSC group (Fig. 5A-C, E-G).
To further explore the role of MSCs against renal brosis, we examined the typical molecules involved in TGF-β/Smad signaling pathway. The immunohistochemistry staining showed that the expressions of α-SMA in the kidneys of adenine-induced rats were clearly increased and then decreased after MSCs administration (Fig. 5D, H). Western blot also showed that adenine signi cantly increased the expression of α-SMA, TGF-β1, and phosphorylated Smad2/3, and MSCs markedly inhibited this enhancement (Fig.  6). These data suggest that adenine impaired renal tubules and induced renal interstitial brosis, which can be ameliorated by MSCs.

MSCs activated p38 MAPK signaling and reduced in ammation in Adenine-induced rats
The immunohistochemical staining and western blot showed adenine decreased p38 MAPK expression compared with the control group, and MSCs treatment surely enhanced p38 MAPK expression compared with the Adenine group (Fig. 7A). The expressions of IL-6, IL-1β, and TNF-α in kidney tissues were all signi cantly elevated in the adenine group, whereas these cytokine levels were indeed reduced by MSC treatment (Fig. 7B). Microarray analysis of cytokine antibodies revealed that a total of 17 cytokines increased in the serum, such as CINC-1, CINC-2, CINC-3, GM-CSF, ICAM-1, IFNγ, IL-1α, IL-2, IL-6, IL-10, L-Selectin, MCP-1, PDGF-AA, Prolactin R, RAGE, TCK-1, TIMP-1, and VEGF, and a total of 8 cytokines decreased, including β-NGF, CNTF, Fractalkine, IL-1β, IL-4, IL-13, LIX, and TNFα in adenine group. Among them, there were signi cantly statistical differences both increases in ICAM-1, IL-10, and L-Selectin and reduction in IL-1β. While MSCs treatment reduced the serum levels of 18 upregulated cytokines and increased the serum levels of 8 downregulated cytokines, but there was no statistical difference. In addition, adenine increased the serum level of B7-2 and decreased the serum level of TCK-1, but MSCs treatment further increased B7-2 level and decreased TCK-1 level without statistical differences (Fig. 7C).

Effect of MSCs on the Adenine-induced rat kidney proteome
Using untargeted proteomic analysis, a total of 58,016 peptides matching 6,213 proteins (≥ one or more unique peptides with an FDR less than 1% ) were identi ed. Using a threshold of 1.2-fold change and p<0.05 between groups was considered as differentially expressed protein. A total of 5,873 proteins were identi ed in the quantitative proteomic study. MSCs treatment nitely affected the pro le of the renal proteome, only 40 proteins were found differentially expressed compared with adenine group (Table 1). Using Cluster 3.0 software classi ed the two dimensions of sample and protein expression simultaneously (distance algorithm: Euclid; connection mode: average linkage). Finally, Java Trewview software was used to generate the volcano plots and hierarchical clustering heatmaps between adenine group and adenine+MSCs group ( Figure 8A, B). Compared with the Adenine group, 30 proteins were upregulated and 10 downregulated in the MSCs group ( Table 2). The majority of biological process that upregulated proteins involved are biological process, cellular process, metabolic process, cellular metabolic process, organic substance metabolic process, single-organism process, primary metabolic process, single-organism cellular process, macromolecule metabolic process and cellular component organization or biogenesis. Meanwhile, the downregulated proteins involved in response to stimulus, biological process, cellular process, single-organism process, biological regulation, regulation of biological process, single-organism cellular process, cellular response to stimulus, regulation of cellular process, and multicellular organismal process. The GO term of these differently expressed proteins was classi ed between adenine group and adenine+MSCs group ( Figure 8C). The pathway analysis of all upregulated or down-regulated proteins was also shown in Figures 9A and 9B. The top seven pathways identi ed were the N-Glycan biosynthesis (Mgat3), MAPK signaling pathway (Nme6), bisphenol degradation (Pon3), aminobenzoate degradation (Pon3), thiamine metabolism (Thtpa), steroid hormone biosynthesis (Cyp21a1), and signaling pathways regulating pluripotency of stem cells (Pcgf2) ( Table 3).
Among them, enrichment analysis showed that there were signi cantly statistical differences of bisphenol degradation and aminobenzoate degradation in adenine group and adenine+MSCs group, the p value was 0.02 and 0.04, respectively (Figures 9C, 10).  To verify whether MSCs which protected NRK-52E and NRK-49F against TGF-β1 induced brosis have a relationship with galectin3, quantitative PCR and western blot showed that adenine increased mRNA and protein expression of galectin3 compared with adenine group and MSCs treatment markedly decreased these in renal tissues, which were consistent with iTRAQ-based and western blot results (p<0.05, Figure  11). In vitro, western blot showed MSCs-CM also downregulated galectin3 expression in TGF-β1 induced NRK-52E and NRK-49F. TD139 pretreatment further reduced galectin3 expression.

Discussion
Mesenchymal stem cell-based therapies have been shown to confer renal protection in several models of acute kidney injury (AKI) and chronic kidney disease (CKD) [13,14] . Most preclinical and clinical trials have demonstrated the safety and e cacy of MSCs in protecting against renal dysfunction [15][16][17] . MSC therapy is becoming an attractive strategy for renal repair, and the potential of MSCs based therapy or ameliorating CKD or AKI is only beginning to be elucidated. Several recent studies show the capacity of exogenously administered MSCs or MSCs conditioned medium to dramatically reduce tubulointerstitial brosis, preserve peritubular capillary density, and prevent epithelial mesenchymal transition in multiple different models of chronic renal injury [19][20][21] . Our results support these evidences, and show a signi cant reduction in extracellular matrix components, such as collagen type Iα, collagen type II, and collagen type III; in ammation-related factors, IL-1β, IL-6, TFNα, myo broblast activation marker, α-SMA and increase in proliferation-related signal, p38 MAPK protein expression in adenine-induced nephropathy after MSCs early intervention. To date, the mechanism of MSCs contributed to the renoprotection is involved in paracrine cytokines, growth factors, and immuoregulation [4] , but no research has focused on the renal proteomic pro le of MSCs therapy for adenine-induced interstitial brosis, and may make a valuable contribution towards the comprehension of the molecular mechanisms involved in MSCs which alleviated renal brosis.
In this study, iTRAQ combined with Q Exactive Plus LC-MS/MS was used to identify the differentially regulated proteins based on a threshold of 1.2-fold change and p<0.05 between groups. We found that MSCs had a very limited in uence on the renal proteome comparing adenine group with adenine+MSCs group, only a total of 40 proteins were detected, of which 30 proteins were signi cantly upregulated and 10 proteins downregulated. According to the reference [22][23][24][25] and bioinformatics analysis, galectin3 was one of the signi cantly downregulated proteins after MSCs treatment. Thus, glaectin3 was further veri ed by uorescence quantitative PCR analysis and western blot in renal tissues, and further determined in renal tubular epithelial cells (NRK-52E) and renal interstitial broblasts (NEK-49F) induced by human recombinant TGF-β1. Our results were in consistent with the iTRAQ results. Besides that, these differentially expressed proteins were involved in a multitude of biological processes including acute in ammatory responses, proliferation, in ammatory response, apoptosis, phagosome, immune response, regulation of the biosynthesis and biological function of glycoprotein oligosaccharides, and so on. Next, we focus on galectin3, MSCs and renal interstitial brosis. Therefore, the mechanism of anti-brosis of MSCs has not been concerned about galectin3.
Galectin-3 proteins (also known as Lgals3) were localized to the nucleus and mitochondrion, which involved in acute in ammatory responses including neutrophil activation and adhesion, chemoattractant of monocytes and macrophages, opsonization of apoptotic neutrophils, and activation of mast cells (https://www.genecards.org/). Galectin3 protein has recently been deemed as a possible biomarker of in ammation and renal brosis and plays a pivotal role in interstitial brosis and progression of chronic kidney disease, and it promotes nephrogenesis during development [26] . Elevated serum levels of galectin-3 have been associated with a higher risk of incident CKD and renal dysfunction [27] . Galectin3 can be secreted extracellularly but can also shuttle into the nucleus. Extracellular galectin3 modulates important interactions between epithelial cells and the extracellular matrix and plays a role in the embryonic development of collecting ducts. In contrast, intracellular galectin3 is important for cell survival due to its ability to block the intrinsic apoptotic pathway, while intranuclear galectin3 promotes cell proliferation [28][29][30] . Our experiments proved that MSCs reduced the mRNA and protein expression of galectin3 in adenineinduced interstitial brosis kidney. We also determined that galectin3 was downregulated in renal tubular epithelial cells and renal interstitial broblasts induced by TGF-β1 after MSCs-CM treatment. A speci c galectin3 inhibitor, TD139 pretreatment and MSCs further reduced galectin3 expression. However, little is known about the regulatory mechanisms of galectin3 during anti brosis of mesenchymal stem cells and need to be further explored. Our data provided a new insight and possible therapeutic targets in the anti brosis of MSCs, which may have a certain valuable in the understanding of the pathogenesis of adenine-induced interstitial brosis.

Conclusions
In summary, we depicted the differentially expressed proteins in the early period of MSCs treatment for adenine-induced nephropathy by iTRAQ-based proteomic analysis. We found MSCs had very limited in uence on the renal proteome, only 40 proteins were differentially expressed compared adenine group with MSCs treatment. The anti brosis effect of MSCs may attribute to the proliferation, immune response, in ammatory response, apoptosis, phagosome, and the like. MSCs, which protect against adenine-induced interstitial brosis, have a certain relationship with galectin3 downregulation. Our data provided a new insight into MSCs treatment for interstitial brosis and may be valuable in furthering our understanding of the pathogenesis of adenine-induced kidney injury and need to be further explored.