The proto-oncogene tyrosine protein kinase Src is essential for macrophage-myofibroblast transition during renal scarring

Src activation has been associated with fibrogenesis after kidney injury. Macrophage-myofibroblast transition is a newly identified process to generate collagen-producing myofibroblasts locally in the kidney undergoing fibrosis in a TGF-β/Smad3-dependent manner. The potential role of the macrophage-myofibroblast transition in Src-mediated renal fibrosis is unknown. In studying this by RNA sequencing at single-cell resolution, we uncovered a unique Src-centric regulatory gene network as a key underlying mechanism of macrophage-myofibroblast transition. A total of 501 differentially expressed genes associated with macrophage-myofibroblast transition were identified. However, Smad3-knockout largely reduced the transcriptome diversity. More importantly, inhibition of Src largely suppresses ureteral obstruction-induced macrophage-myofibroblast transition in the injured kidney in vivo along with transforming growth factor-β1-induced elongated fibroblast-like morphology, α-smooth muscle actin expression and collagen production in bone marrow derived macrophages in vitro. Unexpectedly, we further uncovered that Src serves as a direct Smad3 target gene and also specifically up-regulated in macrophages during macrophage-myofibroblast transition. Thus, macrophage-myofibroblast transition contributes to Src-mediated tissue fibrosis. Hence, targeting Src may represent as a precision therapeutic strategy for macrophage-myofibroblast transition-driven fibrotic diseases. Src activation has been associated with fibrogenesis after kidney injury. Macrophage-myofibroblast transition is a newly identified process to generate collagen-producing myofibroblasts locally in the kidney undergoing fibrosis in a TGF-β/Smad3-dependent manner. The potential role of the macrophage-myofibroblast transition in Src-mediated renal fibrosis is unknown. In studying this by RNA sequencing at single-cell resolution, we uncovered a unique Src-centric regulatory gene network as a key underlying mechanism of macrophage-myofibroblast transition. A total of 501 differentially expressed genes associated with macrophage-myofibroblast transition were identified. However, Smad3-knockout largely reduced the transcriptome diversity. More importantly, inhibition of Src largely suppresses ureteral obstruction-induced macrophage-myofibroblast transition in the injured kidney in vivo along with transforming growth factor-β1-induced elongated fibroblast-like morphology, α-smooth muscle actin expression and collagen production in bone marrow derived macrophages in vitro. Unexpectedly, we further uncovered that Src serves as a direct Smad3 target gene and also specifically up-regulated in macrophages during macrophage-myofibroblast transition. Thus, macrophage-myofibroblast transition contributes to Src-mediated tissue fibrosis. Hence, targeting Src may represent as a precision therapeutic strategy for macrophage-myofibroblast transition-driven fibrotic diseases. Src is a nonreceptor tyrosine kinase that can be activated by a number of cytokines, including transforming growth factor-β1 (TGF-β1).1Tanaka Y. Kobayashi H. Suzuki M. et al.Transforming growth factor-beta1-dependent urokinase up-regulation and promotion of invasion are involved in Src-MAPK-dependent signaling in human ovarian cancer cells.J Biol Chem. 2004; 279: 8567-8576Crossref PubMed Scopus (75) Google Scholar Src activation is critically involved in tissue fibrosis. For example, Src is activated in fibroblasts from patients with systemic sclerosis by profibrotic cytokines, and inhibition of Src effectively reduced the production of extracellular matrix in vitro and in experimental dermal fibrosis in vivo.2Skhirtladze C. Distler O. Dees C. et al.Src kinases in systemic sclerosis: central roles in fibroblast activation and in skin fibrosis.Arthritis Rheum. 2008; 58: 1475-1484Crossref PubMed Scopus (102) Google Scholar A genomic analysis identified an Src family tyrosine kinase is upregulated in allografts of chronic renal allograft injury.3Wei C. Li L. Menon M.C. et al.Genomic analysis of kidney allograft injury identifies hematopoietic cell kinase as a key driver of renal fibrosis.J Am Soc Nephrol. 2017; 28: 1385-1393Crossref PubMed Scopus (24) Google Scholar Src phosphorylation of β-catenin triggers p-β-catenin-Y654/p-Smad2 complex mediated integrin-linked kinase transcription in mice, thereby promoting kidney fibrosis.4Zheng G. Zhang J. Zhao H. et al.α3 Integrin of cell-cell contact mediates kidney fibrosis by integrin-linked kinase in proximal tubular E-cadherin deficient mice.Am J Pathol. 2016; 186: 1847-1860Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Inhibition of Src effectively attenuated the severity of bleomycin-induced lung fibrosis in mice.5Hu M. Che P. Han X. et al.Therapeutic targeting of SRC kinase in myofibroblast differentiation and pulmonary fibrosis.J Pharmacol Exp Ther. 2014; 351: 87-95Crossref PubMed Scopus (61) Google Scholar Src also participated in renal fibroblast activation and proliferation in vitro, as well as fibrosis in the injured kidney of mice with unilateral ureteral obstruction (UUO).6Yan Y. Ma L. Zhou X. et al.Src inhibition blocks renal interstitial fibroblast activation and ameliorates renal fibrosis.Kidney Int. 2016; 89: 68-81Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar However, the pathogenic role of Src in renal fibrosis is still largely unexplored. Fibrosis is a hallmark and common pathway that contributes to the development of end-stage organ diseases, including chronic cardiovascular disease, chronic liver disease, chronic pulmonary disease, and chronic kidney disease.7Rockey D.C. Bell P.D. Hill J.A. Fibrosis-a common pathway to organ injury and failure.N Engl J Med. 2015; 372: 1138-1149Crossref PubMed Scopus (675) Google Scholar Myofibroblast, a subset of activated fibroblasts characterized by expression of alpha-smooth muscle actin (α-SMA), is the principal cell type responsible for organ deformation through triggering fibrillar collagen deposition during tissue fibrosis.8Klingberg F. Hinz B. White E.S. The myofibroblast matrix: implications for tissue repair and fibrosis.J Pathol. 2013; 229: 298-309Crossref PubMed Scopus (443) Google Scholar Myofibroblasts are heterogeneous and can originate from a number of sources, including epithelial-mesenchymal transition (EMT),9Jinde K. Nikolic-Paterson D.J. Huang X.R. et al.Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis.Am J Kidney Dis. 2001; 38: 761-769Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 10Ng Y.Y. Huang T.P. Yang W.C. et al.Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.Kidney Int. 1998; 54: 864-876Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar endothelial-mesenchymal transition,11Zeisberg E.M. Tarnavski O. Zeisberg M. et al.Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.Nat Med. 2007; 13: 952-961Crossref PubMed Scopus (1627) Google Scholar proliferation of local resident fibroblasts or pericytes,12Humphreys B.D. Lin S.L. Kobayashi A. et al.Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (1093) Google Scholar as well as a newly identified phenomenon macrophage-myofibroblast transition (MMT).13Nikolic-Paterson D.J. Wang S. Lan H.Y. Macrophages promote renal fibrosis through direct and indirect mechanisms.Kidney Int Suppl (2011). 2014; 4: 34-38Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Wang S. Meng X.M. Ng Y.Y. et al.TGF-β/Smad3 signalling regulates the transition of bone marrow derived macrophages into myofibroblasts during tissue fibrosis.Oncotarget. 2016; 7: 8809-8822Crossref PubMed Scopus (136) Google Scholar, 15Meng X.M. Wang S. Huang X.R. et al.Inflammatory macrophages can transdifferentiate into myofibroblasts during renal fibrosis.Cell Death Dis. 2016; 7: e2495Crossref PubMed Scopus (152) Google Scholar, 16Wang Y.Y. Jiang H. Pan J. et al.Macrophage-to-myofibroblast transition contributes to interstitial fibrosis in chronic renal allograft injury.J Am Soc Nephrol. 2017; 28: 2053-2067Crossref PubMed Scopus (170) Google Scholar Indeed, Src activation is important for promoting EMT,17Tanjore H. Cheng D.S. Degryse A.L. et al.Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress.J Biol Chem. 2011; 286: 30972-30980Crossref PubMed Scopus (180) Google Scholar but its potential role in MMT is still unknown. Recently, bone marrow–derived fibroblasts have been found in the injured kidney during fibrogenesis.18Li J. Deane J.A. Campanale N.V. et al.The contribution of bone marrow-derived cells to the development of renal interstitial fibrosis.Stem Cells. 2007; 25: 697-706Crossref PubMed Scopus (112) Google Scholar, 19Xia Y. Yan J. Jin X. et al.The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis.Kidney Int. 2014; 86: 327-337Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 20Cao Q. Wang Y. Zheng D. et al.Failed renoprotection by alternatively activated bone marrow macrophages is due to a proliferation-dependent phenotype switch in vivo.Kidney Int. 2014; 85: 794-806Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Furthermore, we revealed that these myofibroblasts can be differentiated from bone marrow–derived macrophages (BMDMs) locally in the injured kidney via MMT in a TGF-β1–dependent manner.14Wang S. Meng X.M. Ng Y.Y. et al.TGF-β/Smad3 signalling regulates the transition of bone marrow derived macrophages into myofibroblasts during tissue fibrosis.Oncotarget. 2016; 7: 8809-8822Crossref PubMed Scopus (136) Google Scholar TGF-β1, a profibrogenic cytokine, is an important initiator for organ inflammation and fibrosis via activating the downstream Smad signaling cascade, especially on Smad3.21Huang X.R. Chung A.C. Yang F. et al.Smad3 mediates cardiac inflammation and fibrosis in angiotensin II-induced hypertensive cardiac remodeling.Hypertension. 2010; 55: 1165-1171Crossref PubMed Scopus (122) Google Scholar, 22Lan H.Y. Chung A.C. TGF-beta/Smad signaling in kidney disease.Semin Nephrol. 2012; 32: 236-243Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 23Meng X.M. Huang X.R. Chung A.C. et al.Smad2 protects against TGF-beta/Smad3-mediated renal fibrosis.J Am Soc Nephrol. 2010; 21: 1477-1487Crossref PubMed Scopus (272) Google Scholar Smad3 is a critical transcription factor for the canonical TGF-β1 signaling; however, targeting Smad3 systemically may induce autoimmune disease by impairing host immune system.24Yang X. Letterio J.J. Lechleider R.J. et al.Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar Thus, alternative approach specifically blocks TGF-β1–induced MMT should be achieved by further elucidating the underlying regulatory mechanism of MMT. Single-cell high-throughput RNA sequencing is a newly emerging approach to resolve cell-to-cell transcriptome profile on a genomic scale. It helps to dissect the reconstitute temporal transcription networks during developmental processes or external stimuli that can be masked on a population level.25Shalek A.K. Satija R. Adiconis X. et al.Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.Nature. 2013; 498: 236-240Crossref PubMed Scopus (825) Google Scholar, 26Deng Q. Ramskold D. Reinius B. et al.Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.Science. 2014; 343: 193-196Crossref PubMed Scopus (770) Google Scholar, 27Saliba A.E. Westermann A.J. Gorski S.A. et al.Single-cell RNA-seq: advances and future challenges.Nucleic Acids Res. 2014; 42: 8845-8860Crossref PubMed Scopus (476) Google Scholar In a previous study, we successfully obtained the unique profile of Smad3-dependent transcriptomes and long noncoding RNAs in chronic injury kidney by using RNA sequencing, in which gene expression pattern of the diseased kidney was largely altered by Smad3-knockout.28Zhou Q. Chung A.C. Huang X.R. et al.Identification of novel long noncoding RNAs associated with TGF-β/Smad3-mediated renal inflammation and fibrosis by RNA sequencing.Am J Pathol. 2014; 184: 409-417Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 29Zhou Q. Xiong Y. Huang X.R. et al.Identification of genes associated with Smad3-dependent renal injury by RNA-seq-based transcriptome analysis.Sci Rep. 2015; 5: 17901Crossref PubMed Scopus (17) Google Scholar In addition, Smad3-knockout BMDMs are substantially resistant to TGF-β1–induced MMT, reflecting by the reduction of α-SMA and collagen-I expressions in vitro,14Wang S. Meng X.M. Ng Y.Y. et al.TGF-β/Smad3 signalling regulates the transition of bone marrow derived macrophages into myofibroblasts during tissue fibrosis.Oncotarget. 2016; 7: 8809-8822Crossref PubMed Scopus (136) Google Scholar can serve as negative control in the mechanistic study of MMT. In this work, we uncovered the underlying mechanism of MMT by RNA sequencing at single-cell resolution. Unexpectedly, Src is identified as the centric gene of regulatory gene network reconstructed with the differentially expressed genes (DEGs) of MMT. More importantly, we demonstrated that inhibition of Src effectively blocks MMT in UUO kidney as well as macrophages under TGF-β1 stimulation. Surprisingly, we revealed Src is a direct Smad3 target gene and acts as the key regulator of TGF-β1–mediated MMT. Our findings revealed the crucial relationship between Src activation and MMT, whereby targeting Src may also represent a precise therapeutic target for MMT-associated fibrotic diseases. MMT is a newly identified mechanism that occurs in injured tissues undergoing fibrogenesis (characterized by CD68+α-SMA+ cells), which are found in day 7 UUO-injured kidneys in vivo, TGF-β1–stimulated BMDM in vitro, as well as fibrotic lesions in human fibrotic kidneys.13Nikolic-Paterson D.J. Wang S. Lan H.Y. Macrophages promote renal fibrosis through direct and indirect mechanisms.Kidney Int Suppl (2011). 2014; 4: 34-38Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Wang S. Meng X.M. Ng Y.Y. et al.TGF-β/Smad3 signalling regulates the transition of bone marrow derived macrophages into myofibroblasts during tissue fibrosis.Oncotarget. 2016; 7: 8809-8822Crossref PubMed Scopus (136) Google Scholar, 15Meng X.M. Wang S. Huang X.R. et al.Inflammatory macrophages can transdifferentiate into myofibroblasts during renal fibrosis.Cell Death Dis. 2016; 7: e2495Crossref PubMed Scopus (152) Google Scholar, 16Wang Y.Y. Jiang H. Pan J. et al.Macrophage-to-myofibroblast transition contributes to interstitial fibrosis in chronic renal allograft injury.J Am Soc Nephrol. 2017; 28: 2053-2067Crossref PubMed Scopus (170) Google Scholar Spindle-like morphology and α-SMA expression are signature phenotypes of MMT cells, which were strongly induced in BMDMs by TGF-β1 (Figure 1a). MMT is largely dependent on Smad3, as population of F4/80+α-SMA+ MMT cells were dramatically diminished in the UUO-kidneys of Smad3-knockout mice in vivo (Supplementary Figure S1A) and BMDM lacking Smad3 or Samd3 inhibitor SIS3-pretreated (S0447; Sigma, St. Louis, MO30Jinnin M. Ihn H. Tamaki K. Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta1-induced extracellular matrix expression.Mol Pharmacol. 2006; 69: 597-607Crossref PubMed Scopus (350) Google Scholar) in vitro (Figure 1a; Supplementary Figure S1B). To uncover the underlying mechanism of MMT, F4/80+α-SMA+ (MMT) and F4/80+α-SMA− (BMDM) cells derived from TGF-β1–stimulated Smad3+/+ and Smad3−/− BMDMs on day 5 were collected by flow sorting and subjected to single-cell RNA-seq (Figure 1b; Supplementary Figure S2). According to the results, the independent replicates are clearly clustered and surprisingly homologous in each group according to their principal component analysis plot, gene expression profiles, and overall expression similarity (Figure 1c–e). It is interesting that the number of DEGs were largely reduced in cells lacking Smad3, where 279 upregulated and 222 downregulated DEGs were detected in the Smad3+/+ cells but only 121 and 133 were observed in the Smad3−/− cells (Figure 1f). Surprisingly, only 14 genes were commonly regulated in both of the Smad3+/+ and Smad3−/− groups during MMT (Figure 1g). In this study, the gene expression profile of MMT was further elucidated by bioinformatics analyzing the DEGs detected from single-cell high-throughput RNA sequencing. Here, the Smad3−/− group was served as MMT-negative control representing the BMDMs resistant to TGF-β1–induced MMT (unique and common DEGs of Smad3+/+ and Smad3−/− groups are summarized in Supplementary Table S1). Under the catalog of biology process, up- and downregulated MMT-dependent DEGs (unique DEGs of Smad3+/+ group) were highly enriched with Gene Ontology (GO) terms “regulation of transcription” and “transcriptional” including transcription factors (e.g., Tcfcp2, Pbx4, STAT4, MYBL2), whereas transcriptional regulation-related GO terms was suggested by using the downregulated DEGs (e.g., ELL2, SMAD6, ercc2) and immune-related GO terms (e.g., “immune response” and “acute inflammatory response,” including TNFSF9, ADORA3, C1QB, Cx3cr1, H2-Q10, C1QC) were obtained from the upregulated DEGs in BMDM lacking Smad3 (unique DEGs of Smad3−/− group) (Figure 2a and b; Supplementary Tables S2 and S3). Surprisingly, the GO terms enriched for molecular function from upregulated MMT-dependent DEGs were mainly related to “ion binding,” whereas similar terms “transition ion binding” and “zinc ion binding” were associated with the downregulated DEGs of BMDM lacking Smad3 (Supplementary Tables S2 and S3). Nevertheless, annotation analysis further indicated that the total gene set, including both the up- and downregulated MMT-dependent DEGs was highly enriched with GO terms “regulation of cell proliferation” and “regulation of cellular process” (Figure 2c; Supplementary Table S4). In contrast, GO terms related to T-cell activation and immunity were the result of the data of the Smad3−/− group, in which only 6 genes (i.e., HLA-A3, HLA-A11, HLA-A68, HLA-A2, MHC class I, HLA-A) were used by MetaCore analytical suite (Thomson Reuters, Canada) with statistical significance (false discovery rate < 0.01, fold change >2, Figure 2d; Supplementary Table S5). These data revealed that transcriptional regulation and ion homeostasis are predominantly regulated during the MMT process. As targeting Smad3 may induce autoimmune disease by impairing the host immune system,24Yang X. Letterio J.J. Lechleider R.J. et al.Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar an alternative approach that specifically blocks MMT should be identified. A number of gene regulatory actions are working at the protein level, which cannot be detected by RNA sequencing, so we conducted network analysis to reveals significantly connected DEGs together with other gene targets, sub-networks and important regulators (transcription factors and receptors) based on the published experimental evidence.31Subramanian A. Tamayo P. Mootha V.K. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression analysis.Proc Natl Acad Sci U S A. 2005; 102: 15545-15550Crossref PubMed Scopus (26834) Google Scholar Thus, we reconstructed the early regulatory events with the MMT-dependent DEGs by using unbiased gene network analysis to identify the key regulator of MMT. In the analysis, unbiased gene regulatory networks of MMT were constructed based on the shortest path algorithm interaction networks found between the MMT-dependent DEGs by MetaCore analytical suite.31Subramanian A. Tamayo P. Mootha V.K. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression analysis.Proc Natl Acad Sci U S A. 2005; 102: 15545-15550Crossref PubMed Scopus (26834) Google Scholar Three gene networks were suggested, as shown in Table 1, whereby the Src-centric network “Ephrin-B, PAK1, NCK2 (Grb4), EGFR, FISH” contained the highest numbers of DEGs and most outstanding significance (g score 172.03, P value 8.55e-10). This Src-centric regulatory network is associated with biological processes, including axonogenesis, cell morphogenesis involved in neuron differentiation, axon development, neuron projection morphogenesis, and axon guidance, supporting by the published experimental evidence in the MetaCore database (Figure 3; Supplementary Table S6).Table 1Unbiased gene regulatory networks of macrophage-myofibroblast transitionNetwork nameProcessesPathwayg scoreP valueEphrin-B, PAK1, NCK2 (Grb4), EGFR, FISHAxonogenesis (76.0%), cell morphogenesis involved in neuron differentiation (76.0%), axon development (76.0%), neuron projection morphogenesis (76.0%), axon guidance (70%)125172.038.55E-10SMAD3, FasR(CD95), p15, TGF-β3, PADI2Phosphatidylethanolamine acyl-chain remodeling (18.8%), phosphatidylcholine acyl-chain remodeling (18.8%), phosphatidylglycerol metabolic process (16.7%), positive regulation of programmed cell death (37.5%), aging (31.2%)66116.131.25E-24SSX2IP, SAMD10, TRM61, TUBGCP2, D330028D13RikProtein-chromophore linkage (15.2%), phototransduction (19.6%), visual perception (21.7%), sensory perception of light stimulus (21.7%), detection of light stimulus (19.6%)046.374.97E-36EGFR, epiderman growth factor receptor; FITC, fluorescein isothiocyanate; TGF, transforming growth factor. Open table in a new tab EGFR, epiderman growth factor receptor; FITC, fluorescein isothiocyanate; TGF, transforming growth factor. Inhibition of Src effectively attenuated tissue fibrosis in mice with bleomycin-injured lung or UUO-damaged kidney,5Hu M. Che P. Han X. et al.Therapeutic targeting of SRC kinase in myofibroblast differentiation and pulmonary fibrosis.J Pharmacol Exp Ther. 2014; 351: 87-95Crossref PubMed Scopus (61) Google Scholar, 6Yan Y. Ma L. Zhou X. et al.Src inhibition blocks renal interstitial fibroblast activation and ameliorates renal fibrosis.Kidney Int. 2016; 89: 68-81Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar but the underlying mechanism is still largely unknown. Blockade of MMT may contribute to the antifibrotic effects of Src-targeted therapy. Thus, we further investigated the pathogenic role of Src in MMT. We found that inhibition of Src with a specific inhibitor SKI-1 (#567805; Calbiochem, San Diego, CA) successfully blocked the elongated fibroblast-like morphology and α-SMA induction in BMDM after 5-day TGF-β1 stimulation (Figure 4a). Nevertheless, signatures of myofibroblasts including α-SMA and collagen-I expression were significantly inhibited in TGF-β1–stimulated BMDMs by Src inhibitor in a dose-dependent manner, shown by real-time polymerase chain reaction (PCR) and Western blot analysis (Figure 4b–d). More importantly, inhibition of Src effectively blocked UUO-induced MMT in the fibrosing kidney. As shown in Figure 5a, UUO dramatically triggered Src activation in injured kidney on day 7, which was significantly inhibited by daily treatment with 2 mg/kg/i.p. of Src inhibitor PP1 (#S7060; Selleckchem, Houston, TX) following the protocol of Yan et al. [6]. Pharmacologic inhibition of Src produced a significant inhibition of MMT, as shown in Figure 5b and c, UUO largely increased α-SMA–expressing macrophages in the injured kidney on day 7 but significantly reduced by the PP1 treatment. Moreover, the MMT-driven renal fibrosis was effectively ameliorated by targeting Src with PP1, immunohistochemistry, Western blot analysis, and real-time PCR, also evidence that treatment with Src inhibitor significantly suppressed UUO-induced renal fibrosis, including α-SMA expression and collagen-I accumulation (Figure 6). Our findings demonstrated the pathogenic role of Src in MMT and the potential of Src as a therapeutic target in MMT-associated tissue scarring.Figure 6Src inhibition effectively reduces macrophage-myofibroblast transition–driven renal fibrosis in unilateral ureteral obstruction (UUO) kidney on day 7. (a) Immunohistochemistry, (b) Western blot analysis, and (c,d) real-time polymerase chain analysis shows that UUO dramatically increases the protein and mRNA expression levels of α-smooth muscle actin (SMA) and collagen-I (Col-I) in the injured kidney on day 7, which are significantly suppressed by treatment with Src inhibitor PP1. Each bar represents the mean ± SEM for groups of at least 3 mice. *P < 0.05, ***P<0.001 compared with sham-operated mice; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the solvent control-treated mice with UUO. Bar = 50 μm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The regulation of Src in renal fibrosis is mainly by posttranslational modifications, it is shown by the increment of p-Src protein in kidney-infiltrating macrophages (Figure 7a) as well as in UUO-kidney compared with the sham control (Figure 7b). Unexpectedly, we found that the expression level of Src protein was obviously decreased in UUO kidney of Smad3−/− mice (Figure 7b) as well as TGF-β1–stimulated BMDM with Smad3 inhibition (Figure 4d). Interestingly, we detected that transcriptional level of Src mRNA was Smad3-dependently increased in UUO kidney (Figure 7c), and Src-expressing macrophages were found in the day 7 UUO kidney shown by immunofluorescence microscopy (Figure 7d). It triggered us to hypothesize that Src may also be cell-type specifically increased in macrophages undergoing MMT. To confirm this interesting phenomenon, we stimulated BMDM with TGF-β1 and detected that expression levels of Src mRNA and protein were significantly increased in the TGF-β1–treated BMDM, but largely reduced by Smad3-knockout (Figure 8a and b), detected by real-time PCR and Western blotting. Mechanistically, a Smad3 binding site is predicted on the 3′ untranslated region (UTR) of the Src gene by ECR browser (National Center for Biotechnology Information, USA)32Ovcharenko I. Nobrega M.A. Loots G.G. et al.ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes.Nucleic Acids Res. 2004; 32: W280-W286Crossref PubMed Scopus (404) Google Scholar (Figure 8c). Thus, we further hypothesized that Src may serve as a direct Smad3 target gene during the process of MMT. By chromatin immunosuppression (ChIP) assay, we demonstrated that TGF-β1 significantly enhances the physical binding of Smad3 protein on the 3′ UTR region of the Src gene in BMDM undergoing MMT (Figure 8d). Furthermore, Src protein level was significantly triggered in BMDM by 3 hours of TGF-β1 stimulation (Figure 8e), as well as the overexpression of Smad3 protein (Figure 8f–h), identifying an important role of TGF-β1/Smad3 signaling in Src expression during MMT. Our previous study reported the importance of MMT in tissue fibrosis [13–16] in a TGF-β1/Smad3-dependent manner, but the underlying mechanism is largely unknown. Targeting Smad3 may induce autoimmune disease by impairing the host immune system.24Yang X. Letterio J.J. Lechleider R.J. et al.Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar Thus, we applied RNA sequencing to further elucidate MMT process at single-cell resolution to identify a key regulator for blocking MMT. Eventually, we uncovered a unique Src-centric gene network representing the key regulatory mechanism at the early stage of MMT. Most importantly, inhibition of Src significantly blocked MMT in TGF-β1 stimulated BMDM in vitro as well UUO kidney in vivo, which largely contributed to the amelioration of renal fibrosis in mice. Unexpectedly, we identified that Src is a direct Smad3 target gene during MMT, and demonstrated the importance of TGF-β1/Smad3 signaling in Src induction during MMT. Thus, our work uncovered the crucial role of Src in MMT that may contribute to the Src-mediated renal fibrosis and represent as a precise therapeutic target for MMT-driven tissue scaring. In this work, gene expression profile at the late phase of MMT was resolved from day 5 TGF-β1–stimulated BMDM. Indeed, we planned to look at the heterogeneity in the MMT cells, but their gene expression patterns are surprisingly homologous in each group according to the principal component analysis plot. Eventually, we changed to identify the key regulator of MMT by performing gene network analysis. As a number of gene regulatory actions are at the protein level, which cannot be detected by RNA sequencing, so we conducted network analysis to reveal significantly connected DEGs together with other gene targets, sub-networks and important regulators (transcription factors and receptors) based on the published experimental evidence. The earlier regulatory gene network that represents the key mechanism of MMT formation were predicted and reconstructed by using the MetaCore analytical suite.31Subramanian A. Tamayo P. Mootha V.K. et al.Gene set enrichment analysis: a knowledge-based approach for interpreting