Wnt/β-Catenin–Promoted Macrophage Alternative Activation Contributes to Kidney Fibrosis

Abstract

The Wnt/β-catenin pathway is crucial in normal development and throughout life, but aberrant activation of this pathway has been linked to kidney fibrosis, although the mechanisms involved remain incompletely determined. Here, we investigated the role of Wnt/β-catenin in regulating macrophage activation and the contribution thereof to kidney fibrosis. Treatment of macrophages with Wnt3a exacerbated IL-4– or TGFβ1-induced macrophage alternative (M2) polarization and the phosphorylation and nuclear translocation of STAT3 in vitro. Conversely, inhibition of Wnt/β-catenin signaling prevented these IL-4– or TGFβ1-induced processes. In a mouse model, induced deletion of β-catenin in macrophages attenuated the fibrosis, macrophage accumulation, and M2 polarization observed in the kidneys of wild-type littermates after unilateral ureter obstruction. This study shows that activation of Wnt/β-catenin signaling promotes kidney fibrosis by stimulating macrophage M2 polarization.

CKD, histologically characterized as excessive extracellular matrix deposition and chronic inflammation, is highly prevalent around the world.^1,2 In the diseased kidneys, persistent macrophage accumulation caused by monocytes/macrophages infiltration from blood and proliferation in situ^3–6 is closely correlated with the progression of kidney fibrosis in patients with CKD.^7–14

Both infiltrated and resident macrophages within the fibrotic kidneys display considerable plasticity and functional heterogeneity by adapting to the local microenvironment.^15,16 They may be differentiated into proinflammatory classically activated (M1) and wound healing/profibrotic alternatively activated (M2) phenotypes on stimulation.^17–23 In CKD, accumulated M2 macrophages promote kidney fibrosis through producing excessive extracellular matrix and secreting profibrotic growth factors.^23–25 However, the mechanisms for driving macrophage proliferation, migration, and polarization as well as their contribution to kidney fibrosis remain to be determined.

Wnt signaling is divided into the canonical and noncanonical signaling pathways depending on whether β-catenin is activated or not.^26–28 For the canonical Wnt signaling pathway, stabilized β-catenin is accumulated in the cytosol on activation and then translocated into the nucleus, where it binds to T cell factor (TCF)/lymphoid enhancer binding factor and stimulates the transcription of the target genes, such as Snail and PAI-1, which participate in tissue fibrogenesis.^29,30 Wnt/β-catenin activation may protect against tubular cell death and AKI in mice. However, aberrant activation of Wnt/β-catenin in podocytes or tubular cells may exacerbate proteinuria, renal dysfunction, and kidney fibrosis.^31–37 Canonical Wnt signaling may modulate the GTPase-driven plasticity that drives tumor cells to invade through varied environments.³⁸ In addition, β-catenin can regulate myeloid cell motility and adhesion, which contribute to the accumulation of mesenchymal cells after cutaneous injury.^39,40 Therefore, it is hypothesized that Wnt/β-catenin activation may promote macrophage mobility and facilitate its accumulation in the fibrotic kidneys. Recently, it was reported that Lrp5/β-catenin signaling activation in lung myeloid cells leads to differentiation of an alveolar macrophage subtype that antagonizes the resolution of lung fibrosis.⁴¹ Forced expression of a stabilized β-catenin mutant is able to promote macrophage differentiation in response to GM-CSF.⁴² In this regard, it is possible that Wnt/β-catenin signaling may modulate macrophage accumulation and activation and contribute to kidney fibrosis in CKD.

In this study, we report that Wnt/β-catenin activation can promote macrophage M2 polarization. Deletion of macrophage β-catenin in mice ameliorates kidney fibrosis accompanied by less macrophage accumulation and M2 polarization in kidneys with unilateral ureter obstructive (UUO) nephropathy.

Results

Wnt3a Exacerbates IL-4– or TGFβ1-Stimulated Macrophage M2 Polarization

Persistent M2 macrophage accumulation may promote kidney fibrosis in CKD.²³ Among all canonical Wnt molecules, Wnt3a is the one widely used for stimulating canonical Wnt signaling. To explore the role for Wnt/β-catenin signaling activation in macrophage M2 polarization, we treated Raw264.7 cells and bone marrow-derived macrophages (BMMs) with Wnt3a for 30 minutes followed by IL-4 or TGFβ1 treatment to induce macrophage M2 polarization. In Raw264.7 cells, Wnt3a alone could not induce macrophage M2 polarization, whereas Wnt3a plus IL-4 could largely increase Arg-1, MR, and chitinase 3–like 3/Ym1 expression compared with those treated with IL-4 alone. TGFβ1 alone could only induce Arg-1 mRNA expression, whereas Wnt3a plus TGFβ1 could markedly stimulate Arg-1, Fizz1, and chitinase 3–like 3/Ym1 mRNA expression (Figure 1A). Similarly, in BMMs, Wnt3a plus IL-4 or TGFβ1 could further increase the expression of Arg-1, MR, chitinase 3–like 3/Ym1, and Fizz1 mRNA compared with IL-4 or TGFβ1 treatment alone (Figure 1B). Thus, it is clear that Wnt3a may exacerbate IL-4– or TGFβ1-induced macrophage M2 polarization.

Figure 1.

Wnt3a exacerbates IL-4– or TGFβ1-stimulated macrophage M2 polarization. (A and B) Real-time PCR analysis showing the mRNA abundance for Arg-1, MR, Fizz1, and YM1 in (A) Raw264.7 cells and (B) BMMs stimulated with IL-4 (10 ng/ml) or TGFβ1 (2 ng/ml) with or without Wnt3a (100 ng/ml) for 24 hours. *P<0.05 versus cells treated with vehicle alone, n=3; ^#P<0.05 versus cells treated with IL-4 or TGFβ1, n=3. (C) Western blotting assay showing the induction of phosphorylated STAT3 at Y705 in BMMs stimulated with IL-4 (left panel) or TGFβ1 (right panel) with or without Wnt3a (100 ng/ml). (D) Western blotting assay showing the nuclear translocation of STAT3 in BMMs stimulated with IL-4 or TGFβ1 with or without Wnt3a (100 ng/ml). (E) Representative immunofluorescence staining images (left panel) and quantitative analysis (right panel) showing the nuclear localization of STAT3 in BMMs stimulated with IL-4 or TGFβ1 with or without Wnt3a (100 ng/ml). *P<0.05 versus BMMs treated with vehicle alone, n=4; ^#P<0.05 versus BMMs treated with IL-4 or TGFβ1, n=4. (F) Real-time PCR analysis showing that blockade of STAT3 with Stattic could inhibit macrophage M2 polarization stimulated by IL-4 or TGFβ1 with or without Wnt3a. *P<0.05 versus BMMs treated with vehicle alone, n=3; ^#P<0.05 versus BMMs treated with IL-4 or TGFβ1 alone, n=3; ^$P<0.05 versus BMMs treated with IL-4 or TGFβ1 plus Wnt3a, n=3.

Macrophage polarization is tightly controlled by the activation of various transcriptional factors, such as STATs, PPARs, KLFs, and C/EBPβ.^19,43 Overexpression of β-catenin upregulates STAT3 expression and activation in tumor cells.⁴⁴ Phosphorylation of STAT3 at Y705 stimulates its dimerization, nuclear translocation, and DNA binding.⁴⁵ To investigate whether Wnt3a exacerbates IL-4– or TGFβ1-induced macrophage M2 polarization via STAT3 signaling activation, we examined the STAT3 phosphorylation at Y705 in BMMs. STAT3 phosphorylation was induced in BMMs treated with IL-4 or TGFβ1, and Wnt3a could further increase it (Figure 1C). Western blot assay showed that Wnt3a could further enhance IL-4– or TGFβ1-induced STAT3 nuclear translocation in BMMs (Figure 1D). Also, immunostaining for STAT3 further confirmed this observation (Figure 1E). To explore the role for STAT3 activation in Wnt3a-exacerbated macrophage M2 polarization, we treated BMMs with Stattic, an STAT3 inhibitor.⁴⁶ The results showed that Stattic could largely inhibit STAT3 phosphorylation (Supplemental Figure 1) and macrophage M2 polarization induced by IL-4 or TGFβ1 plus Wnt3a, suggesting a crucial role for STAT3 activation in promoting macrophage M2 polarization (Figure 1F). Therefore, it may be concluded that Wnt3a exacerbates IL-4– or TGFβ1-stimulated macrophage M2 polarization through upregulating STAT3 expression and activation.

Blockade of β-Catenin Signaling Inhibits IL-4– or TGFβ1-Induced Macrophage M2 Polarization

We assessed β-catenin signaling activation in Raw264.7 cells and BMMs stimulated with IL-4 (10 ng/ml) or TGFβ1 (2 ng/ml). Western blot analyses and immune staining showed that β-catenin underwent nuclear translocation at 30 minutes after IL-4 or TGFβ1 treatment in both cell types (Figure 2, A–D). LRP6 phosphorylation (Figure 2, E and F) and TOP-flash luciferase activity (Figure 2G) were also markedly induced in cultured macrophages treated with IL-4 or TGFβ1.

Figure 2.

β-Catenin signaling is activated in macrophages after IL-4 or TGFβ1 treatment. (A) Western blotting analyses and (B) immunofluorescence staining showing β-catenin nuclear translocation after IL-4 or TGFβ1 treatment in Raw264.7 cells. (C) Western blotting analyses and (D) immunofluorescence staining showing β-catenin nuclear translocation after IL-4 or TGFβ1 treatment in BMMs. (E) Western blotting assay showing LRP6 phosphorylation after IL-4 or TGFβ1 treatment in Raw264.7 cells. (F) Western blotting assay showing LRP6 phosphorylation after IL-4 or TGFβ1 treatment in BMMs. (G) Graph showing the induction of TOP-flash luciferase activity in BMMs after IL-4 or TGFβ1 stimulation. *P<0.05 versus control cells treated with vehicle alone, n=4.

We then examined macrophage M2 polarization in both cell types after blockade of β-catenin signaling. Raw264.7 cells and BMMs were treated with ICG-001, a small molecule that specifically inhibits TCF/β-catenin transcription in a cAMP response element binding protein binding protein–dependent fashion.^47,48 IL-4 or TGFβ1 could induce macrophage M2 polarization in Raw264.7 cells (Figure 3A) and BMMs (Figure 3B), respectively. ICG-001 could markedly downregulate IL-4–induced Arg-1, MR, chitinase 3–like 3/Ym1, and Fizz1 mRNA expression as well as TGFβ1-induced Arg-1 and YM1 mRNA expression in Raw264.7 cells (Figure 3A). In BMMs, IL-4– or TGFβ1-stimulated macrophage M2 polarization was markedly inhibited by ICG-001 treatment (Figure 3B). We also examined macrophage M2 polarization in BMMs with tamoxifen-inducible β-catenin deletion. BMMs isolated from Csf1r-Cre±β-catenin^fl/fl mice were treated with 4-hydroxytamoxifen (4-OHT) to induce β-catenin gene deletion (Supplemental Figure 2). Ablation of β-catenin in BMMs could prohibit IL-4– or TGFβ1-stimulated macrophage M2 polarization (Figure 3C).

Figure 3.

β-Catenin signaling is indispensable for IL-4– or TGFβ1-stimulated macrophage M2 polarization. (A) Real-time PCR analysis showing the mRNA abundance for Arg-1, MR, Fizz1, and YM1 in Raw264.7 cells. *P<0.05 versus cells treated with vehicle alone, n=4; ^#P<0.05 versus cells treated with IL-4 or TGFβ1, n=4. (B) Real-time PCR analysis showing the mRNA abundance for Arg-1, MR, Fizz1, and YM1 in BMMs cells. *P<0.05 versus cells treated with vehicle alone, n=4; ^#P<0.05 versus cells treated with IL-4 or TGFβ1, n=4. (C) Real-time PCR analysis showing the mRNA abundance for Arg-1, MR, Fizz1, and YM1 in β-catenin+/+ and β-catenin−/− BMMs cells. *P<0.05 versus β-catenin +/+ BMMs treated with vehicle, n=4; ^#P<0.05 versus β-catenin +/+ BMMs treated with IL-4 or TGFβ1, n=4. (D) Western blotting assay showing that ICG-001 could reduce IL-4– (left panel) or TGFβ1–stimulated (right panel) STAT3 phosphorylation in BMMs. (E) Western blotting assay showing that deletion of β-catenin could reduce IL-4– (left panel) or TGFβ1–stimulated (right panel) STAT3 phosphorylation in BMMs. (F) Representative immunofluorescence staining images (left panel) and quantitative analysis (right panel) showing that ICG-001 could decrease IL-4– or TGFβ1–induced STAT3 nuclear translocalization in BMMs. *P<0.05 versus BMMs treated with vehicle alone, n=4; ^#P<0.05 versus BMMs treated with IL-4 or TGFβ1, n=4. (G) Representative immunofluorescence staining images (left panel) and quantitative analysis (right panel) showing that β-catenin deletion could decrease IL-4– or TGFβ1–induced STAT3 nuclear translocalization in BMMs. *P<0.05 versus β-catenin+/+ BMMs treated with vehicle alone, n=4; ^#P<0.05 versus β-catenin+/+ BMMs treated with IL-4 or TGFβ1, n=4.

To explore the mechanisms for β-catenin in regulating macrophage M2 polarization, we examined the STAT3 phosphorylation at Y705 in both cell types. ICG-001 could largely reduce IL-4– or TGFβ1-stimulated p-STAT3 and STAT3 upregulation in BMMs (Figure 3D). The protein abundance of p-STAT3 and STAT3 in β-catenin−/− BMMs was less than that in β-catenin+/+ BMMs (Figure 3E). Immune staining showed that ICG-001 or β-catenin deletion could diminish IL-4– or TGFβ1-induced STAT3 nuclear translocation (Figure 3, F and G). Thus, it is clear that blockade of β-catenin signaling may inhibit macrophage M2 polarization through downregulating STAT3 expression and activation.

Activation of β-Catenin in Macrophages from the Fibrotic Kidneys with UUO Nephropathy

To investigate whether β-catenin signaling was activated in macrophages in the fibrotic kidneys, we sorted macrophages from bone marrow, spleens, and fibrotic kidneys with CD115 microbeads at weeks 1 and 2 after UUO in mice. Western blotting analyses showed that little β-catenin, p–β-catenin (Ser675), Snail, or Cyclin D1 were detected in macrophages from bone marrow or spleen, whereas in macrophages from the fibrotic kidneys, the abundance of β-catenin, p–β-catenin (Ser675), and β-catenin signaling target genes, including PAI-1 and Snail, was largely elevated (Figure 4A). In addition, coimmune staining results showed that, in the sham kidneys, a few F4/80-positive macrophages with little β-catenin were detected, whereas in the UUO kidneys, β-catenin underwent nuclear translocation in F4/80-positive macrophages (Figure 4B). We also extracted the nuclear protein from macrophages in bone marrow, spleen, and UUO kidneys. Western blot assay showed that β-catenin was detected in the nuclei of macrophages from the UUO kidneys but was not detected in those from bone marrow and spleens (Figure 4C). Together, these data suggest that β-catenin signaling is activated in macrophages from the UUO kidneys.

Figure 4.

β–Catenin signaling is activated in macrophages from the fibrotic kidneys with UUO nephropathy. (A) Western blotting analyses showing the induction of β-catenin, p–β-catenin (Ser675), PAI-1, Snail, and cyclin D1 in macrophages sorted from UUO kidneys with CD115 microbeads. (B) Representative double-immune staining images showing that β-catenin underwent nuclear translocation in macrophages from the UUO kidneys. Paraffin-embedded kidney sections were costained with antibodies against F4/80 and β-catenin to identify macrophages with nuclear β-catenin translocation in the fibrotic kidneys. Arrows indicate β-catenin nuclear-positive macrophages. Scale bar, 20 μm. (C) Western blotting analysis showing that nuclear β-catenin was increased in macrophages sorted from the fibrotic kidneys after UUO. Macrophages from bone marrow (BM), spleen, and fibrotic kidneys were sorted with CD115 microbeads. Nuclear lysates were obtained by using the Nuclear Extraction Reagents (catalog no. 78833; Thermo) according to the manufacturer’s instruction. Anti-TBP (catalog no. ab818; Abcam) was probed to show the equal loading of nuclear protein among groups.

Ablation of β-Catenin in Macrophages Ameliorates UUO Nephropathy in Mice

To explore the role for macrophage β-catenin signaling activation in kidney fibrosis, we created a mouse model with inducible macrophage β-catenin deletion (Figure 5, A and B). Mice expressing tamoxifen-inducible MerCreMer fusion protein under the control of macrophage-specific mouse Csf1r promoter were used. Mice with macrophage deletion of β-catenin were generated by intraperitoneal injection of tamoxifen for 5 consecutive days in Csf1r-Cre+, β-catenin^fl/fl mice and named as Mac–β-catenin−/−. The same gender with genotyping Csf1r-Cre−, β-catenin^fl/fl littermates were also injected with tamoxifen and named as Mac–β-catenin+/+. Two days after the last injection, both control littermates and knockouts were subjected to UUO (Figure 5C). Immune staining and Western blotting analysis showed the deletion of β-catenin in macrophages from Mac–β-catenin−/− mice (Figure 5, D and E).

Figure 5.

Ablation of β-catenin in macrophages ameliorates kidney interstitial fibrosis in mice with UUO nephropathy. (A) Breeding strategy. (B) PCR analysis for genotyping the mice. Lane 1, Csf1r-Cre−/−, β-catenin^fl/fl; lane 2, Csf1r-Cre−/−, β-catenin^fl/wt; lane 3, Csf1r-Cre+/−, β-catenin^fl/fl; lane 4, Csf1r-Cre+/−, β-catenin^fl/wt. (C) Strategy for inducing β-catenin deletion in macrophages from Csf1r-Cre+/−, β-catenin^fl/fl mice. (D) Representative micrographs of immune staining showing the ablation of β-catenin in F4/80-positive macrophages in UUO kidneys from Csf1r-Cre+, β-catenin^fl/fl mice. Arrows indicate macrophages with β-catenin deletion. (E) Western blotting analyses showing the ablation of β-catenin in macrophages sorted from UUO kidneys in Csf1r-Cre+, β-catenin^fl/fl mice. BM, bone marrow. (F) Representative micrographs for periodic acid–Schiff (PAS), Masson, Sirius red, FN, and α-SMA immune staining in kidney tissues. Scale bar, 20 μm. (G) Graphic presentation showing the fibrotic area, total collagen content, FN, and α-SMA staining positive area in kidney tissues among groups as indicated. *P<0.05 versus β-catenin+/+ sham control, n=4–8; ^#P<0.05 versus β-catenin+/+ littermates after UUO, n=6–8. (H) Western blotting analyses for FN, α-SMA, and type I collagen in kidney tissues from different groups as indicated. Kidney lysates from each individual animal within the same group were pooled together to run the gel. (I) Western blotting analyses (left panel) and quantitative analysis (right panel) for FN, α-SMA, and type I collagen in UUO kidneys from the knockouts and control littermates at week 2 after surgery. Numbers (1–4) indicate each individual animal within a given group. *P<0.05 versus β-catenin+/+ littermates after UUO, n=4.

We then examined kidney histologic change in Mac–β-catenin+/+ and Mac–β-catenin−/− mice after UUO. In Mac–β-catenin+/+ mice, remarkable tubular atrophy, interstitial extracellular matrix deposition, FN, and α-SMA induction were detected after UUO, whereas in the knockout kidneys, tubular injury and interstitial fibrosis were markedly attenuated (Figure 5, F and G). FN, α-SMA, and type I collagen protein abundance were remarkably increased in kidneys from Mac–β-catenin+/+ littermates after UUO, whereas they were largely reduced in the knockouts (Figure 5, H and I). Thus, these results showed that deletion of β-catenin in macrophages diminishes kidney fibrosis in the UUO kidneys.

Ablation of β-Catenin in Macrophages Diminishes Macrophage M2 Polarization in the UUO Kidneys

A few F4/80-positive macrophages were detected, and no difference was found for macrophage number in the sham kidneys between Mac–β-catenin+/+ and Mac–β-catenin−/− mice. Macrophage accumulation was largely increased at days 7 and 14 after UUO in Mac–β-catenin+/+ kidneys, whereas it was less in the knockout kidneys (Figure 6, A and B).

Figure 6.

Ablation of β-catenin in macrophages attenuates macrophage accumulation, proliferation, and M2 polarization in the UUO kidneys. (A) Representative images and (B) quantitative analysis for F4/80-positive cells in kidney tissues. Scale bar, 20 μm. *P<0.05 versus β-catenin+/+ sham control, n=5; ^#P<0.05 versus β-catenin+/+ control littermates after UUO, n=5. (C) Representative images for costaining of F4/80 and cleaved caspase 3 (CCP3) as well as F4/80 and MR and (D) quantitative analysis for cleaved caspase 3–positive macrophages and MR-positive macrophages in kidney tissues. White arrows indicate costaining positive macrophages. Scale bar, 20 μm. *P<0.05 versus β-catenin+/+ sham control, n=5; ^#P<0.05 versus β-catenin+/+ control littermates after UUO, n=5. (E) Western blotting analyses (left panel) and quantitative analysis (right panel) for MR in macrophages from the UUO kidneys at week 2 after surgery. Numbers (1–3) indicate individual samples that were pooled from two individual animals within the same group. *P<0.05 versus macrophages from β-catenin+/+ control littermates after UUO, n=3. (F) Real-time PCR analysis showing the mRNA abundance for Arg-1, MR, Fizz1, and YM1 in macrophages from sham and UUO kidneys at week 2 after surgery. Each sample was pooled from two individual animals within the same group. *P<0.05 versus macrophages from β-catenin +/+ sham control, n=3; ^#P<0.05 versus macrophages from β-catenin+/+ control littermates at week 2 after UUO, n=3. (G) FACS analysis showing macrophage M2 polarization for macrophages from UUO kidneys. *P<0.05 versus macrophages from Mac–β-catenin+/+ UUO kidneys, n=3.

We then costained kidney tissues with antibodies against F4/80 and cleaved caspase 3 to identify macrophage apoptosis. Very few F4/80 staining–positive cells with cleaved caspase 3 positive was detected in the sham kidneys. At day 14 after UUO, about 1.5% of the macrophages were cleaved caspase 3 positive in Mac–β-catenin+/+ kidneys, which was comparable with those in Mac–β-catenin−/− kidneys (Figure 6, C and D). Together, these data suggests that ablation of β-catenin in macrophages may not affect macrophage apoptosis in the UUO kidneys.

In the UUO kidneys, F4/80 and MR costaining positive cell number was increased in Mac–β-catenin+/+ mice, which was much less in the knockouts (Figure 6, C and D). Flow cytometry analysis for CD11b and CD206 confirmed the results of immunostaining (Figure 6G). Western blot analysis showed that the MR abundance in macrophages enriched from Mac–β-catenin−/− kidneys after UUO was much less compared with that from Mac–β-catenin+/+ mice. Similarly, Arg-1, MR, chitinase 3–like 3/Ym1, and Fizz1 mRNA expressions were markedly increased in macrophages from Mac–β-catenin+/+ kidneys, which were much less in macrophages from Mac–β-catenin+/+ kidneys after UUO (Figure 6, E and F). In addition, the mRNA abundance for the profibrotic cytokines, including PDGFA, PDGFB, PDGFC, PDGFD, VEGFC, TGFβ1, TGFβ2, TGFβ3, and CTGF, was largely upregulated in macrophages from Mac–β-catenin+/+ kidneys after UUO, and the abundance was much less in those from Mac–β-catenin−/− kidneys after UUO (Supplemental Figure 3). Interestingly, mRNA abundance for M1 macrophage markers, including iNOS, IL-6, IL-1β, and TNFα, was significantly increased in macrophages from Mac–β-catenin+/+ kidneys after UUO, and the abundance was much higher in macrophages from Mac–β-catenin−/− kidneys after UUO (Supplemental Figure 4). Collectively, these data showed that ablation of β-catenin in macrophages reduces macrophage M2 polarization in the fibrotic kidneys with UUO nephropathy.

Discussion

We report here that Wnt3a could exacerbate IL-4– or TGFβ1-induced macrophage M2 polarization involving STAT3 activation. Ablation of β-catenin in macrophages attenuated macrophage M2 polarization and kidney fibrosis in UUO kidneys. This study identifies a new mechanism for Wnt/β-catenin signaling in promoting kidney fibrosis in CKD.

The Wnt/β-catenin pathway is crucial in both normal development and throughout life.^27,29,30 Wnt/β-catenin signaling is relatively silenced in adult kidneys and reactivated in various forms of experimental animal models and CKD in patients. Accumulated evidence showed that Wnt/β-catenin signaling is involved in kidney injury and repair after a variety of insults. Appropriate activation of Wnt/β-catenin in tubule protects against tubular cell death and AKI, whereas persistent activation of Wnt/β-catenin in tubular cells may result in progressive kidney fibrosis.³⁵ Macrophages are accumulated at sites of renal fibrosis in both patients and animal models with CKD.⁶ β-Catenin activation is critical for regulating the macrophage accumulation in the healing wound due to modulating cell motility and adhesion.⁴⁰ After recruitment to the injured kidneys, monocytes/macrophages may be differentiated into different subtypes in response to various stimuli in the local microenvironment.^16,22,23 M1 macrophages produce a large amount of proinflammatory mediators; in contrast, M2 macrophages exhibit anti-inflammatory features and are involved in renal repair and fibrosis.²¹ In this study, the ablation of β-catenin in macrophages facilitated macrophage M1 polarization in the fibrotic kidneys. In our previous study, we found that activated β-catenin may bind with p65 NF-κB, which prevented p65 binding to the κB site, sequestered its transactivating activity, and repressed p65-mediated gene transcription.⁴⁹ However, the underlying mechanisms and the contribution for β-catenin deletion-enhanced macrophage M1 polarization in kidney fibrosis remain to be determined. On the contrary, ablation of β-catenin diminished macrophage M2 polarization and profibrotic cytokine expression. In cultured macrophages, Wnt3a treatment could exacerbate IL-4– or TGFβ1-induced macrophage M2 polarization. Blockade of β-catenin signaling could inhibit macrophage M2 polarization. Regarding the profibrotic role for accumulated M2 macrophages in the profession of CKD,⁵⁰ it may be concluded that β-catenin–mediated macrophage M2 polarization contributes to kidney fibrosis.

Macrophage polarization is tightly controlled by the activation of various transcriptional factors, such as STATs, PPARs, KLFs, and C/EBPβ.^19,43 Overexpression of β-catenin may increase STAT3 expression and activation in tumor cells.⁴⁴ In this study, Wnt3a alone could up regulate STAT3 expression but not its phosphorylation at Y705 in macrophages. However, it could synergistically stimulate IL-4– or TGFβ1-induced STAT3 phosphorylation. Blocking β-catenin signaling inhibited IL-4– or TGFβ1-induced STAT3 phosphorylation. In addition, blockade of STAT3 with Stattic could largely inhibit macrophage M2 polarization. Putting all data together, it may be concluded that Wnt/β-catenin signaling exacerbates IL-4– or TGFβ1-induced macrophage M2 polarization through STAT3 induction.

Except for Wnt proteins, proximal tubule may produce colony-stimulating factor (CSF-1), which promotes macrophage proliferation and M2 polarization. Genetic or pharmacologic inhibition of macrophage CSF-1 signaling could inhibit the recovery after AKI.¹⁴ In this study, a progressive kidney fibrosis model induced by UUO was used. Persistent accumulation of M2 macrophages plays a crucial role for promoting extracellular matrix deposition and fibrosis in this model. In this regard, it is possible that CSF-1–regulated macrophage activation may contribute to kidney fibrosis in such model.

Together, this study showed that Wnt/β-catenin signaling activation promotes macrophage M2 polarization, which contributes to kidney fibrosis in mice with UUO nephropathy. Targeting the Wnt/β-catenin pathway in macrophages may provide a new strategy for retarding kidney fibrosis in patients with CKD.

Concise Methods

Mice and Animal Models

Male C57BL/6 mice weighing approximately 18–20 g were acquired from the specific pathogen-free laboratory animal center of Nanjing Medical University and maintained according to the guidelines of the Institutional Animal Care and Use Committee at Nanjing Medical University. UUO was performed as previously reported.⁵¹ Mice were euthanized. Kidneys, bone marrow, and spleen were harvested at days 0, 7, and 14 after UUO.

Homozygous β-catenin floxed mice [022775; B6(Cg)-Ctnnb1^tm1Knw/J] and mice expressing tamoxifen-inducible MerCreMer fusion protein under the control of macrophage-specific mouse Csf1r promoter [019098; FVB-Tg(Csf1r-cre/Esr1*)] were ordered from Jackson Laboratories (Bar Harbor, ME). FVB-Tg(Csf1r-cre/Esr1*) mice were crossed with C57BL/6J mice for eight generations to get Csf1r-Cre transgenic mice on C57BL/6J background. All animals were housed in the specific pathogen-free laboratory animal center of Nanjing Medical University according to the guidelines of the Institutional Animal Care and Use Committee at Nanjing Medical University. By mating β-catenin floxed mice with Csf1r-Cre/Esr1* transgenic mice, mice that were heterozygous for the β-catenin floxed allele were generated (genotype: Csf1r-Cre+/−, β-catenin^fl/wt). These mice were crossbred with homozygous β-catenin floxed mice (genotype: β-catenin^fl/fl) to generate offspring with different littermates (Csf1r-Cre+/−, β-catenin^fl/fl; Csf1r-Cre+/−, β-catenin^fl/wt; Csf1r-Cre−/−, β-catenin^fl/wt; Csf1r-Cre−/−, β-catenin^fl/fl). Mice with genotyping Csf1r-Cre+/−, β-catenin^fl/fl and the same sex littermates with genotyping Csf1r-Cre−/−, β-catenin^fl/fl were used in the study. Genotyping was performed by PCR assay using DNA extracted from the mouse tail. The primers used for genotyping were as follows: Cre transgene, sense: 5′-AGATGCCAGGACATCAGGAACCTG-3′; antisense: 5′-ATCAGCCACACCAGACACAGAGATC-3′; β-catenin floxed, sense: 5′-AAGGTAGAGTGATGAAAGTTGTT-3′; antisense: 5′-CACCATGTCCTCTGTCTATTC-3′. Csf1r-Cre+/−, β-catenin^fl/fl mice and control littermates were intraperitoneally injected with tamoxifen (T5648; Sigma-Aldrich, St. Louis, MO) at 25 mg/kg for 5 consecutive days, and 2 days after the last injection, the mice were subjected to UUO operation.

Cell Culture and Treatment

Raw264.7 cells were cultured in DMEM containing 10% (vol/vol) FBS (Invitrogen, Grand Island, NY) and 1% (vol/vol) antibiotics (100 U/ml penicillin) at 37°C in 5% CO₂. BMMs were isolated as previously described.⁵² BMMs were cultured in DMEM containing 10% (vol/vol) FBS, 10 ng/ml mouse M-CSF (catalog no. 416-ML-050; R&D Systems, Minneapolis, MN), and 1% (vol/vol) antibiotics for 9 days. The medium was changed every other day. To generate BMMs with β-catenin gene deletion, BMMs from Csf1r-Cre+/−, β-catenin^fl/fl mice were treated with 1 μM 4-OHT (H6278; Sigma-Aldrich) at the beginning of the culture. BMMs from Csf1r-Cre−/−, β-catenin^fl/fl mice were treated with 4-OHT as control. On day 9 of culture, BMMs were cultured with serum-free medium and treated with IL-4 (10 ng/ml; catalog no. 93–4138–10; Biovision) or TGFβ1 (2 ng/ml; catalog no. 100-B-010-CF; R&D Systems). To block STAT3 signaling, BMMs were treated with Stattic (10 μM; catalog no. ab120952; Abcam, Cambridge, United Kingdom) for 30 minutes, and then, they were treated with Wnt3a (100 ng/ml) followed by IL-4 (10 ng/ml) or TGFβ1 (2 ng/ml) treatment for various durations as indicated.

Nuclear and Cytoplasmic Fractionation

Raw264.7 cells or BMMs were cultured with serum-free medium and treated with IL-4 (10 ng/ml) or TGFβ1 (2 ng/ml) with or without Wnt3a (100 ng/ml) for various durations as indicated. Macrophages from bone marrow, spleen, and fibrotic kidneys were sorted with CD115 microbeads. Nuclear and cytoplasmic lysates were obtained by using the Nuclear and Cytoplasmic Extraction Reagents (catalog no. 78833; Thermo) according to the manufacturer’s instruction for Western blotting assay. The primary antibodies used were anti–β-catenin (catalog no. 610154; BD Transduction Laboratories), anti–β-catenin (Ser675; catalog no. 9567s; Cell Signaling Technology), anti-STAT3 (catalog no. 4904; Cell Signaling Technology), anti-TBP (catalog no. ab818; Abcam), and anti–β-actin (catalog no. sc1616; Santa Cruz Biotechnology).

Semiquantitative Analyses of Fibrotic Area in the Kidney Tissue

Kidney sections at 3-μm thickness was stained with the Masson Trichrome kit (catalog no. HT15–1KT; Sigma-Aldrich) according to the manufacturer’s protocol. Accumulated collagen in the interstitial area was stained with aniline blue. Ten fields under ×400 microscopes were randomly selected in the cortical area for each kidney section. The percentage of interstitial fibrotic area to the selected field was analyzed with Image Pro plus 6.0, and an average percentage of kidney fibrotic area for each section was calculated.

Quantitative Analyses for Total Collagen in Kidney Tissue

Total collagen within kidney tissue was quantitated as previously reported.⁵³ Briefly, 3-μm sections of paraffin-embedded tissue were stained with Sirius red F3BA and Fast Green FCF (Sigma-Aldrich) overnight. After washing three times with 1× PBS buffer, the dye was eluted from tissue sections with 0.1 N sodium hydroxide methanol. Absorbance values at 540 and 605 nm were determined for Sirius red F3BA and Fast Green FCF binding protein, respectively. Measurement of the ratio of collagen to total protein was expressed as micrograms per milligram of total protein.

Western Blot Analyses

Raw264.7 cells and BMMs were lysed in 1× SDS sample buffer. The kidneys were lysed with RIPA buffer containing 1% NP-40, 0.1% SDS, 100 mg/ml PMSF, 1% protease inhibitor mixture, and 1% phosphatase I and II inhibitor mixture on ice. The supernatants were collected after centrifugation at 13,000×g at 4°C for 30 minutes. The primary antibodies used were anti–β-catenin (catalog no. 610154; BD Transduction Laboratories), anti–β-catenin (Ser-675; catalog no. 9567s; Cell Signaling Technology), anti–PAI-1 (catalog no. sc-5297; Santa Cruz Biotechnology), anti-Snail (catalog no. 3879s; Cell Signaling Technology), anti-FN (catalog no. F3648; Sigma-Aldrich), anti–α-SMA (catalog no. A5228; Sigma-Aldrich), anti-type I collagen (catalog no. AB765P; Millipore, Billerica, MA), anti–p-LRP6 (catalog no. 2568P; Cell Signaling Technology), anti–β-actin (catalog no. sc1616; Santa Cruz Biotechnology), anti-GAPDH (catalog no. sc25778; Santa Cruz Biotechnology), anti-MR (catalog no. ab64693; Abcam), anti-STAT3 (catalog no. 4904; Cell Signaling Technology), and anti–p-STAT3 (Y705; catalog no. 9145; Cell Signaling Technology). Quantification was performed by measuring the intensity of the signals with the aid of the National Institutes of Health ImageJ software package.

Real-Time RT-PCR Assay

Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s instruction. cDNAs were synthesized using 1 μg total RNA, ReverTra Ace (Toyobo, Osaka, Japan), and oligo(dT)12–18 primers according to the protocol specified by the manufacturer. Gene expression was measured by real-time PCR using cDNA, real-time PCR Master Mix Reagents (catalog no. Q141–02; Vazyme, Nanjing, China), and 7300 real-time PCR system (Applied Biosystems). GAPDH was detected as an internal control. The relative amount of mRNA or gene to internal control was calculated using the equation 2 ∆CT, in which ∆CT = CTgene − CTcontrol.

Histology and Immunohistochemical Staining

Paraffin-embedded mouse kidney sections (3-μm thickness) were stained with periodic acid–Schiff, Masson, and Sirius red. The antibodies used for immunohistochemical staining were as the following: anti–β-catenin (catalog no. 610154; BD Transduction Laboratories) and anti-type I collagen (catalog no. AB765P; Millipore). After incubation with primary antibodies at 4°C overnight, the slides were stained with secondary antibody for 1 hour at room temperature. The sections were then incubated with ABC reagents for 1 hour at room temperature before being subjected to DAB staining (Vector Laboratories, Burlingame, CA). Slides were viewed under a Nikon Eclipse 80i microscope equipped with a digital camera (DS-Ri1; Nikon, Shanghai, China).

Immunofluorescence Staining

Kidney cryosections at 3-μm thickness were fixed for 15 minutes with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100 in 1× PBS for 5 minutes at room temperature. After blocking with 2% donkey serum for 60 minutes, slides were immune stained with the following antibodies: anti-FN (catalog no. F3648; Sigma-Aldrich), anti–α-SMA (catalog no. A5228; Sigma-Aldrich), anti-F4/80 (catalog no. 14–4801; eBioscience, San Diego, CA), anticleaved caspase 3 (catalog no. 9664; Cell Signaling Technology), and anti-CD3 (catalog no. 555273; BD Pharmingen). Raw 264.7 cells or BMMs cultured on coverslips were washed with cold 1× PBS and fixed with cold methanol:acetone (1:1) for 10 minutes at −20°C. After three extensive washings with 1× PBS, cells were treated with 0.1% Triton X-100 for 5 minutes, blocked with 2% normal donkey serum in 1× PBS buffer for 40 minutes at room temperature, and incubated with the following antibodies: anti–β-catenin (catalog no. 610154; BD Transduction Laboratories) and anti-STAT3 (catalog no. 4904; Cell Signaling Technology) followed by staining with FITC or tetramethylrhodamine-conjugated secondary antibodies. Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i Epi-fluorescence microscope equipped with a digital camera. The F4/80-positive macrophage number was counted from ten randomly selected fields in the cortical area for each sample under ×400 microscope, and an average number of positive cells for each section was calculated. For the quantitative analysis of FN and α-SMA expression in kidney tissues, ten fields under ×400 microscope were randomly selected in the cortical area for each kidney section. The percentage of FN or α-SMA staining positive area to the selected field was analyzed with Image Pro plus 6.0, and an average percentage of staining positive area for each section was calculated.

Kidney Monocytes/Macrophages Enrichment

After perfusion with cold 1× PBS, kidneys were removed, minced into fragments, and digested in DMEM containing 1 mg/ml collagenase (catalog no. 17018–029; Gibco) and 0.1 mg/ml DNAase (catalog no. 10104159001; Roche) for 1 hour at 37°C with intermittent agitation. The fragments were filtered through 40-μm mesh (Falcon; BD Biosciences) to get single-cell suspension. Macrophages were enriched from the single-cell suspension with CD115 Microbeads and MACS column (Miltenyi Biotech, Bergisch-Gladbach, Germany) according to the manufacturer’s instruction.

Flow Cytometry

FACS analysis was performed according to the previous reports.¹⁴ Briefly, after perfusion with cold 1× PBS, kidneys was removed, minced into fragments, and digested with 1 mg/ml collagenase (catalog no. 17018–029; Gibco) and 0.1 mg/ml DNAase for 1 hour at 37°C with intermittent agitation. Kidney fragments were passed through a 40-μm mesh (Falcon; BD Biosciences), and approximately 1×10⁷ cells were incubated in 2.5 μg/ml Fc blocking solution, centrifuged (800×g, 10 minutes, 8°C), and resuspended with FACS buffer. Approximately 1×10⁶ cells were stained for 20 minutes at room temperature with antibodies including anti–CD45-APC (561018; BD Biosciences), anti–CD11b-FITC (553310; BD Biosciences), and anti–CD206-PE (141705; Biolegend), washed, and resuspended in FACS buffer. The suspensions were washed twice with FACS buffer, resuspended in FACS buffer, and analyzed on BD Canto II Flow Cytometer with FlowJo software.

Luciferase Activity Assay

The effect of IL-4 or TGFβ1 on β-catenin–mediated gene transcription was detected by using the TOP-flash TCF reporter plasmid containing two sets of three copies of the TCF binding site upstream of the thymidine kinase minimal promoter and luciferase open reading frame (Millipore). BMMs were seeded on 12-well culture plates to 90%–95% confluence in complete medium containing 10% FBS plus M-CSF (10 ng/ml) and changed to serum-free medium after washing twice with serum-free medium. BMMs were transfected with TOP-flash or FOP-flash plasmid (1 μg) and an internal control reporter plasmid (0.1 μg) Renilla reniformis luciferase driven under thymidine kinase promoter (pRL-TK; Promega) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Twenty-four hours later, cells were treated with TGFβ1 (2 ng/ml) or IL-4 (10 ng/ml) for 24 hours. Luciferase assay was performed using a dual luciferase assay system kit according to the manufacturer’s protocol (Promega). Relative luciferase activity (arbitrary units) was reported as fold induction over the controls.

Statistical Analyses

All data examined are presented as mean±SEM. Statistical analyses of the data were performed using SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparison between groups was made using one-way ANOVA followed by the Student–Newman–Keuls test. Paired or unpaired t test was used to compare two groups. A P value of 0.05 or lower was considered statistically significant.

Disclosures

None.