Abstract

   Hyperuricemia is an essential risk factor in chronic kidney disease
   (CKD), while urate‐lowering therapy to prevent or delay CKD is
   controversial. Alternatively activated macrophages in response to local
   microenvironment play diverse roles in kidney diseases. Here, we aim to
   investigate whether and how macrophage integrin αM (ITGAM) contributes
   to hyperuricemia‐related CKD. In vivo, we explored dynamic
   characteristics of renal tissue in hyperuricemia‐related CKD mice. By
   incorporating transcriptomics and phosphoproteomics data, we analyzed
   gene expression profile, hub genes and potential pathways. In vitro, we
   validated bioinformatic findings under different conditions with
   interventions corresponding to core nodes. We found that
   hyperuricemia‐related CKD was characterized by elevated serum uric acid
   levels, impaired renal function, activation of macrophage alternative
   (M2) polarization, and kidney fibrosis. Integrated bioinformatic
   analyses revealed Itgam as the potential core gene, which was
   associated with focal adhesion signaling. Notably, we confirmed the
   upregulated expression of macrophage ITGAM, activated pathway, and
   macrophage M2 polarization in injured kidneys. In vitro, through
   silencing Itgam, inhibiting p‐FAK or p‐AKT1 phosphorylation, and
   concurrent inhibiting of p‐FAK while activating p‐AKT1 all contributed
   to the modulation of macrophage M2 polarization. Our results indicated
   targeting macrophage ITGAM might be a promising therapeutic approach
   for preventing CKD.

   Keywords: chronic kidney disease, hyperuricemia, integrin αM,
   macrophage M2 polarization
     __________________________________________________________________

   Schematic diagram of macrophage ITGAM and related mechanisms in
   hyperuricemia‐related chronic kidney disease in mice. Through the
   integration and analysis of transcriptomic and phosphoproteomic data
   from renal tissue stimulated by hyperuricemia, we identified ITGAM as a
   central node. ITGAM on macrophages activates FAK/AKT/GSK‐3β, resulting
   in the stabilization of β‐catenin, and promotion of M2 polarization and
   kidney fibrosis in hyperuricemic CKD mice. The findings were confirmed
   through both in vivo and in vitro experiments by manipulating Itgam,
   p‐FAK, and p‐AKT1.

   graphic file with name MCO2-5-e580-g006.jpg

1. INTRODUCTION

   Hyperuricemia prevalence has been rising in recent years, attributable
   in part to shifts toward unhealthy dietary patterns and lifestyles.[32]
   ^1 , [33]^2 The kidneys in healthy individuals are responsible for the
   excretion of two‐third uric acid (UA), thus playing an essential role
   in the pathogenesis and progression of chronic kidney disease (CKD).
   Kidney damage related to hyperuricemia typically manifests as chronic
   interstitial nephritis, the formation of urate crystals or stones, and
   subsequent kidney fibrosis.[34] ^3 , [35]^4 As a modifiable metabolite,
   UA represents a viable therapeutic target for mitigating kidney damage
   induced by hyperuricemia. But in the setting of CKD, it is
   controversial whether UA‐lowering is an effective strategy to prevent
   or delay CKD progression.[36] ^5 Investigations into therapeutic
   targets that participate in hyperuricemia‐related CKD are of great
   clinical significance.

   Integrin constitutes the largest family of cell adhesion molecules and
   is involved in kidney development and diseases.[37] ^6 , [38]^7 Renal
   fibrosis could be induced by integrins through cell–matrix or cell–cell
   interactions.[39] ^8 Integrins are αβ heterodimeric transmembrane
   glycoproteins and mainly divided into integrin β1, β2, and β3 families
   according to β subunits.[40] ^9 As transmembrane receptors, integrins
   participate in cell proliferation, survival and migration,
   differentiation, and matrix homeostasis.[41] ^8 Due to lack of
   enzymatic activity, integrins need to bind adaptor proteins for
   intracellular signal propagation, such as focal adhesion kinase (FAK),
   a key tyrosine kinase of intracellular signaling binding to a number of
   downstream molecules.[42] ^10

   αMβ2, also known as macrophage antigen 1 (Mac‐1), is the predominant
   leukocyte‐specific β2 integrin abundantly expressed in
   monocytes/macrophages and dendritic cells.[43] ^11 The αMI‐domain
   within αMβ2 mediates ligand binding and is responsible for substrate
   specificity, thus integrin αM (ITGAM) mainly determined diverse
   functions of Mac‐1.[44] ^12 Previous studies reported that ITGAM
   overexpression was associated with macrophage infiltration and renal
   fibrosis.[45] ^13 , [46]^14 Macrophages accumulate in injured kidneys
   and present as polarized M1 or M2 phenotype for proinflammatory or
   profibrotic functions, respectively.[47] ^15 , [48]^16 Macrophage
   alternative (M2) polarization is considered as an essential feature of
   fibrosis.[49] ^17 However, it is not well elucidated whether ITGAM
   regulated macrophage M2 polarization in renal fibrosis and signaling
   pathways involved.

   Here, integrin ITGAM is reported as the hub gene promoting macrophage
   M2 polarization in hyperuricemia‐related CKD. Through comprehensive
   bioinformatic analysis, we have confirmed that ITGAM contributes to the
   development of kidney disease by modulating the FAK/AKT1/GSK‐3β
   signaling pathway. Our findings shed light on the molecular mechanism
   underlying kidney fibrosis in hyperuricemia‐related CKD, highlighting
   the significance of ITGAM expression, and signaling as potential
   therapeutic targets for the prevention and delay of CKD progression.

2. RESULTS

2.1. Progressive renal function decline and kidney fibrosis in mice wth
hyperuricemia‐related CKD

   To investigate the phenotypic dynamic changes of hyperuricemia‐related
   CKD, we conducted animal model in mice and performed biochemical and
   renal tissue analyses. From day 0 to day 21 under gavage feeding of
   adenine and potassium oxonate, we set several time points and described
   dynamic characteristics of blood, urine, and kidney samples
   (Figure [50]1A). Serum UA kept rising and reached peak at day 21
   (Figure [51]1B). Similar trend to UA was observed in renal function
   measurements. Urine albumin‐to‐creatinine ratio (UACR) rose rapidly and
   became five fold level at day 7 compared with day 0, and then slowly
   declined but still at high level at days 14 and 21 (Figure [52]1C).
   Serum urea and serum creatinine (SCr) went up quickly from day 0 to day
   7, kept stable at high level at day 14, and then sharply increased to
   three and four times of baseline level respectively
   (Figures [53]1D and E). Histologic and quantitative changes observed in
   periodic acid‐Schiff (PAS)‐staining and Trichrome‐staining sections
   indicated progressive tubular and glomerular injuries, as well as
   increased collagen deposition in the tubular interstitial and
   glomerular mesangial compartments, respectively (Figures [54]1F and G).
   We further observed progressive inflammatory cell recruitment indicated
   by chemokine MCP‐1, proinflammatory cytokine TNF‐α, IL‐6, IL‐1β
   (Figure [55]1H), as well as profibrotic marker fibronectin, collagen‐I,
   and α‐SMA (Figure [56]1I). The dynamic evolution of
   hyperuricemia‐related CKD is characterized by a progressive increase in
   blood UA levels and a decline in renal function. The renal tissue
   phenotype predominantly exhibits persistent inflammation and
   progressive fibrosis.

FIGURE 1.

   FIGURE 1
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   Progressive renal dysfunction and kidney fibrosis in
   hyperuricemia‐related CKD mice. (A) Brief instruction on model
   establishment and sample collection. (B) Quantification of serum uric
   acid from day 0 to day 21. (C) Quantification of urine
   albumin‐to‐creatinine ratio (UACR) from day 0 to day 21. (D)
   Quantification of serum urea from day 0 to day 21. (E) Quantification
   of serum creatinine (SCr) from day 0 to day 21 (F) Representative
   images of periodic acid‐Schiff (PAS) and Masson trichrome staining,
   indicating progressive tubular atrophy, glomerulosclerosis, and
   interstitial collagen deposition. (G) Quantification and comparison of
   tubular injuries and interstitial fibrosis across time points. (H)
   Protein expression changes in inflammatory indicators (MCP‐1, TNF‐α,
   IL‐1β, and IL‐6) from day 0 to day 21 in renal tissue. (I) Protein
   expression changes of profibrotic indicators (fibronectin, collagen‐1,
   and α‐SMA) from day 0 to day 21 in renal tissue. Each experiment had a
   sample size of n = 6; ****p < 0.0001, ***p < 0.001, **p < 0.01,
   *p < 0.05.

2.2. Itgam is the hub gene potentially activating focal adhesion pathway in
mice with hyperuricemia‐related CKD

   To extract essential molecules from a data‐driven perspective, we
   conducted mRNA and protein sequencing on mouse kidneys and performed
   comprehensive integrated analyses. Differentially expressed genes
   (DEGs) and differentially expressed proteins (DEPs) in mouse kidneys
   were filtered followed by standardized analysis workflow
   (Figures [58]2A and [59]S1A). Details of quality control were provided
   in Figures [60]S1B and [61]C. The details about digging out hub genes
   and pathways related are presented in Figure [62]S1D. In brief, we
   performed the enrichment analysis using upregulated and downregulated
   DEGs/DEPs separately and presented functional characteristics
   accordingly (Figure [63]S1E). After molecular complex detection (MCODE)
   analysis, we picked out the top five clusters consisting of 334 DEGs
   followed by CentiScaPe analysis to obtain 49 hub genes (Figure
   [64]S2A). By matching the top ingenuity pathway analysis (IPA)
   canonical pathways with hub genes, we found two most significantly
   enriched genes—Itgam and Itgb2, that encoded two subunits constituting
   the heterodimer Mac‐1 (Figure [65]2B). The key functional role of Itgam
   was also highlighted by IPA canonical pathway and biomarker analysis,
   where the integrin signaling pathway and Itgam ranked at the top,
   respectively (Figures [66]2B and [67]S2B, C). In day‐21 group, Itgam
   and ITGAM were upregulated to 19‐fold and 3.3‐fold of their respective
   levels in the day‐0 group, both at the mRNA and protein level
   (Figures [68]2C and D). To investigate the potential regulatory
   pathways of ITGAM, we identified significant DEGs and DEPs that were
   positively correlated with Itgam/ITGAM. Subsequently, Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis
   was conducted (Figure [69]2E). Gene Set Enrichment Analysis (GSEA)
   analyses were also performed using these DEGs and DEPs, which revealed
   that the Focal Adhesion pathway ranked highest (Figure [70]2F).
   Considering the extensive literature on tyrosine kinases as key signal
   transducers for integrins,[71] ^18 we hypothesized that ITGAM
   significantly activated focal adhesion signaling. Collectively,
   multiple biostatistical analyses identified Itgam as a hub gene, with
   significant upregulation in the day‐21 group compared with the day‐0
   group, suggesting its potential role in regulating focal adhesion
   signaling pathway.

FIGURE 2.

   FIGURE 2
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   Bioinformatic analyses revealed Itgam as the hub gene and downstream
   focal adhesion pathway. (A) Sketch map of how we incorporated mRNA and
   protein sequencing data to select core genes and pathways related. In
   brief, we found out 1337 DEGs both at mRNA and protein levels and
   further adopted hub gene analyses (MCODE followed by CentiScape in
   Cytoscape) to acquire simplified 49 genes. (B) There are 6 of 49 hub
   genes corresponding to top canonical pathways conducted by IPA. Itgam
   and Itgb2, as two heterodimers of Mac‐1, were top‐ranked. (C and D)
   Itgam on day 21, at mRNA and protein level, was upregulated to 19 and
   3.3 times of day 0. (E) KEGG results based on genes significantly
   positively correlated with Itgam at both mRNA and protein levels; (F)
   Top1 GSEA analysis of DEGs and DEPs is focal adhesion pathway.

2.3. Macrophage Itgam is highly expressed with alternative M2 polarization in
mice with hyperuricemia‐related CKD

   Based on findings from integrated bioinformatic analysis, we further
   verified the expression and location of Itgam in hyperuricemia‐related
   CKD. Compared with day 0, Itgam expression significantly increased on
   day 7, 14, and 21 with a trend continuous growth at both of mRNA and
   protein levels (Figure [73]3A). Considering Itgam mainly but not only
   expressed in macrophage, we investigated its location in kidney tissue
   and found it highly colocated with macrophage marker F4/80 in
   tubulointerstitial and glomerular mesangial space (Figure [74]3B).
   Furthermore, upregulation of ITGAM was confirmed by human renal biopsy
   section in CKD patients with primary hyperuricemia (Figure [75]3C).
   Both of transcriptomics and proteomics data revealed activated
   macrophage M2 polarization in kidney tissue on day 21, clarified by
   greatly increased mRNA expression of Arg1 and Mr (Figure [76]3D). We
   further performed qPCR and compare M1‐trait and M2‐triat membrane
   markers, and found activation of M1 polarization on day 7, followed by
   increasingly M2 polarization till day 21 (Figures [77]3E and F).
   Similar trends were also observed in M1 and M2 specific cytokines
   (Figures [78]3G and H). The evidence presented above, from both animal
   and human studies, as well as from multiple time points, together
   suggests that the high expression of Itgam is associated with
   macrophage M2 polarization and is also correlated with
   hyperuricemia‐mediated renal fibrosis.

FIGURE 3.

   FIGURE 3
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   ITGAM expression and macrophage M2 polarization in vivo. (A) ITGAM
   exhibited increased upregulation in vivo from day 0 to day 21 at both
   mRNA and protein levels. (B) Expression of ITGAM showed a strong
   correlation with the macrophage marker F4/80, supported by
   colocalization of FITC‐labeled ITGAM and TRITC‐labeled F4/80 on the
   macrophage surface. (C) ITGAM was significantly upregulated in renal
   biopsy tissue from patients diagnosed with hyperuricemia‐induced CKD
   compared with human peritumoral kidney tissue. (D) Almost all the
   M2‐trait genes or proteins (Arg‐1/ARG‐1, Fizz‐1/FIZZ1, Mr/MR, and
   Ym1/YM1) in mice renal tissue were significantly upregulated from the
   day‐21 group compared with the day‐0 group, as verified by qPCR. (E)
   Dynamic changes of representative M1‐trait markers (Cxcl10 and Cd80)
   were examined from day 0 to day 21. (F) Dynamic changes of
   representative M2‐trait markers (Arg‐1, Fizz‐1, Mr, and Ym1) were
   examined from day 0 to day 21. (G) Classic M1‐trait (Il6 and Tnfa) and
   M2‐trait (Il4 and Il10) genes were examined from day 0 to day 21 using
   renal tissue supernate. (H) Classic M1‐trait (IL‐6 and TNF‐α) and
   M2‐trait (IL‐4 and IL‐10) cytokines were examined from day 0 to day 21
   using renal tissue supernate. The sample size for Figure 3A was n = 5,
   and the rest had a sample size of n = 3. ****p < 0.0001, ***p < 0.001,
   **p < 0.01, *p < 0.05.

2.4. UA activated ITGAM and focal adhesion signaling pathway

   To reveal disease mechanism from various dimensions, we integrated
   phosphoproteomics sequencing to further elucidate the main pathway at
   the protein modification level. Based on results from KEGG enrichment,
   pathway analysis and kinase perturbation analysis (Figure [80]4A), we
   observed focal adhesion pathway (Wikipathway: WP85) was greatly
   activated by multiple integrins and extracellular matrix components,
   with phosphorylation of FAK and AKT1 significantly upregulated and
   GSK‐3β downregulated (Figures [81]4B–D). Phosphorylation data
   demonstrated Ser722, Ser124, and Tyr279 were mostly fluctuated in FAK,
   AKT1, and GSK‐3β, respectively (Figure [82]4E). These findings were
   supported by phosphorylation quantification using renal tissue from day
   21 compared with day 0 (Figure [83]4F). Highly colocated p‐FAK and
   ITGAM indicated the hypothesis that activation of FAK signaling might
   be related to ITGAM and they coworked together mediating macrophage
   polarization in hyperuricemia‐related CKD (Figure [84]4G).

FIGURE 4.

   FIGURE 4
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   Phosphoproteomics data reveals focal adhesion pathway as essential
   downstream of ITGAM. (A) The workflow for phosphoproteomics sequencing
   and statistical analysis was implemented, integrating proteomics and
   phosphorylation modification data from mice renal tissue in the day‐21
   group compared with the day‐0 group. (B and C) KEGG enrichment and
   pathway analyses both indicated focal adhesion pathway predominantly
   activated as downstream pathway of ITGAM. (D) Kinase perturbation
   analysis revealed activation of GSK‐3β and AKT1 were greatly
   downregulated and upregulated, respectively. (E) Phosphorylation site
   of enriched molecules in focal adhesion pathway. (F) FAK/AKT1/GSK‐3β
   pathway was activated in vivo on day 21 versus day 0. (G) ITGAM and
   p‐FAK expression were highly colocated in the tubulointerstitial
   compartment.

2.5. UA directly activated macrophage M2 polarization

   We performed in vitro experiments to explore how UA functions on
   macrophage. Since UA at high concentration causes tubular damages,[86]
   ^19 , [87]^20 we designed two in vitro models: model 1, using UA to
   stimulate Raw 264.7 macrophage; and model 2, using UA to stimulate the
   coculture environment of Raw 264.7 + proximal tubular cell TCMK1.
   Macrophages were collected after 24 and 48 h of stimulation
   (Figure [88]5A). Itgam mRNA expression in model 1 quickly increased to
   peak after 24 h, and Itgam in model 1 was higher or equal to that in
   model 2 at time points of 24 and 48 h, respectively (Figure [89]5B).
   Although not highly consistence in M2 polarization markers, two models
   both presented obvious M2 polarization especially after 48 h
   (Figures [90]5C–F). Cytokine changes also indicated M1 polarization at
   early stage and progressively M2 polarization from 24 to 48 h
   (Figures [91]5G and H). ITGAM expression and hypothesized downstream
   FAK signaling were activated since 24 h of UA stimulation. As indicated
   by KEGG enrichment, we confirmed the higher phosphorylation of FAK and
   AKT1, lower phosphorylation of GSK‐3β, and less degraded β‐catenin
   (Figure [92]5I). Evidence above indicated that UA strongly activated
   ITGAM/FAK signaling in macrophage and M2 polarization, regardless of
   existence of tubular epithelial cells.

FIGURE 5.

   FIGURE 5
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   Two experimental models in vitro validated overexpression of ITGAM, M2
   polarization of macrophages, and pathway activation. (A) study design
   of uric acid at 800 µM stimulating Raw 264.7 vs. Raw 264.7 +TCMK1; (B)
   Raw 264.7 alone, under stimulation of uric acid, greatly overexpressed
   ITGAM and the expression levels in two models at 48 hours were not
   significantly different; (C–F) Either Raw 264.7 or co‐culture system
   presented obvious activation of macrophage M2 activation after uric
   acid exposure; (G) M1‐trait cytokine IL‐6 in cell culture supernatant
   was upregulated after 24 hours of uric acid stimulation and remained
   unchanged thereafter; (H) M2‐trait cytokine IL‐10 continued to rise
   after uric acid treatment and showed a significant increase from 24
   hours onwards; (I) Two models at different time points showed
   activation of ITGAM/FAK/Akt/β‐catenin pathway. ****P < 0.0001, ***P <
   0.001, **P < 0.01, *P < 0.05.

2.6. ITGAM induced macrophage M2 polarization through activating
FAK/AKT1/GSK‐3β signaling

   Based on omics data, we adopted several interventions targeting key
   molecules in focal adhesion pathway to verify our hypothesis. The
   mechanism was verified by silencing Itgam mRNA, inhibiting p‐FAK, and
   p‐AKT1 phosphorylation, respectively, followed by combination of
   inhibiting p‐FAK and activating p‐AKT1 (Figure [94]6A). As shown by
   pre‐experiment, we picked the Itgam siRNA with the highest knockdown
   efficiency (mean difference = 77.6%; Figure [95]S3). After knocking
   down Itgam, we observed the decreased phosphorylation of FAK and AKT1,
   and reversed the upregulation of phosphorylated GSK‐3β, together with
   downregulation of β‐catenin protein (Figure [96]6B). Meanwhile, M2
   polarization was greatly attenuated after Itgam knockdown, indicated by
   significant decrease in Arg1, Mr, Fizz1, and Ym1 mRNA expression
   (Figure [97]6C). Similar effects were found after p‐FAK and p‐AKT1
   intervention. After inhibiting phosphorylation of these two proteins,
   the downstream signaling was significantly suppressed together with
   reduced M2 polarization (Figures [98]6D–G). The suppression of M2
   polarization achieved by inhibiting p‐FAK, was reversed upon activation
   p‐AKT1, which revealed the downstream involvement of AKT1 regulated by
   FAK (Figures [99]6H and I). The consistent result from the intervention
   experiments above indicates that ITGAM functions through the
   FAK/AKT1/GSK‐3β signaling within the focal adhesion pathway, leading to
   the accumulation of β‐catenin and promoting M2 polarization.

FIGURE 6.

   FIGURE 6
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   Interventions of ITGAM, FAK, and AKT1 verified the participation of
   focal adhesion pathway in macrophage M2 polarization in
   hyperuricemia‐related CKD. (A) Overall design of ITGAM and pathway
   intervention. (B and C) Silencing of Itgam significantly inhibited
   phosphorylation of FAK and AKT1, while activating phosphorylation of
   GSK‐3β and attenuating M2 markers including Arg1, Mr, Fizz‐1, and Ym1.
   (D and E) Inhibition of p‐FAK greatly inhibited downstream pathway and
   macrophage M2 polarization. (F and G) Inhibition of p‐Akt downregulated
   β‐catenin and macrophage M2 polarization. (H and I) Concurrent
   inhibition of p‐FAK and activation of p‐AKT1 revealed p‐AKT1 as the
   downstream of p‐FAK. ****p < 0.0001, ***p < 0.001, **p < 0.01,
   *p < 0.05.

3. DISCUSSION

   Macrophages promotes renal fibrosis and M2 macrophages is strongly
   associated with kidney fibrosis in both human and experimental
   diseases. In this study, we report that macrophage ITGAM contributes to
   renal fibrosis in hyperuricemia‐related CKD. Mechanistically, ITGAM
   promotes macrophage M2 polarization through activating FAK/AKT1/GSK‐β
   pathway.

   ITGAM, also known as CD11b, was commonly used as a monocyte/macrophage
   surface marker.[101] ^21 , [102]^22 Gradually, ITGAM diverse functions
   were reflected by its rapid confirmational change that alters affinity
   for its more than 40 ligands, including ICAM‐1,[103] ^23
   fibrinogen,[104] ^24 fibronectin,[105] ^25 GPIbα,[106] ^26 RAGE,[107]
   ^27 JAM‐c,[108] ^13 and others.[109] ^28 We examined the expression of
   frequently reported main ligands and found a significant upregulation
   of ICAM‐1 and fibronectin (Figures [110]1F and [111]S4A, B), which
   indicated potential role of ITGAM in cell–ECM and cell–cell
   interactions in kidneys of hyperuricemia‐related CKD. Further studies
   are needed to clarify how ITGAM mediates crosstalk between macrophage
   and other cells or compartments in kidney tissue. ITGAM was reported to
   be involved in various immune responses but with bidirectional
   effects.[112] ^29 Negative regulation of ITGAM could be observed in
   systemic lupus erythematous and acute infectious diseases.[113] ^30 ,
   [114]^31 On the contrary, ITGAM positively regulated chronic
   inflammatory diseases.[115] ^32 Lange‐Sperandio et al.[116] ^13
   reported upregulated Mac‐1 and its ligands ICAM‐1 and JAM‐3 in murine
   unilateral ureteric obstruction model, and found knockout of Mac‐1
   greatly attenuated renal fibrosis. Taking our results together, the
   functional role of ITGAM in contributing to renal fibrosis through
   promoting M2 polarization could be inferred.

   Each of the α and β subunits of integrins comprises a single
   transmembrane domain and a short cytoplasmic tail, necessitating
   interaction with downstream tyrosine kinases to facilitate “outside‐in”
   signal transduction.[117] ^9 , [118]^33 Binding to ligands (e.g.,
   extracellular matrix) induces integrins clustering at focal adhesions
   and connecting to intracellular molecules. FAK as a pivotal mediator in
   the focal adhesion signaling pathway, is one of the initially
   identified key elements involving central integrin signaling
   mechanisms. Once activated, FAK undergoes autophosphorylation, and
   triggers the activation of multiple downstream effectors associated
   with diverse signaling pathways. These include SRC, AKT1, Grb7, and
   others.[119] ^34 Numerous studies reported FAK/AKT1 pathway
   participated in chronic inflammation and fibrosis, such as
   atherosclerosis,[120] ^35 and lung and liver fibrosis.[121] ^36 ,
   [122]^37 Of notes, we additionally observed the change in SRC
   phosphorylation and found p‐SRC were both attenuated after silencing
   Itgam and inhibiting p‐FAK. Our finding indicated that SRC participated
   in ITGAM/FAK signaling and might act as the downstream molecular. It is
   to be further explored whether SRC together with FAK stimulates AKT1,
   or acts through other pathways, such as STAT3 and ERK.[123] ^38 ,
   [124]^39 , [125]^40 β‐Catenin activation, as the key component of Wnt
   signaling, is associated with macrophage M2 polarization in tumor
   malignant and renal fibrosis.[126] ^17 , [127]^41 Multiple studies have
   demonstrated that the activation of AKT1 and subsequently decreased
   phosphorylation of GSK‐3β result in the stabilization of β‐catenin.
   Accumulated β‐catenin in cytosol then translocates into nucleus and
   stimulates transcription of target genes related to tissue
   fibrosis.[128] ^42 , [129]^43

   To our best acknowledgement, this is the first paper investigating the