The effect of STAT6 inhibitor AS1517499 in treatment of Renal Fibrosis
Mar 24, 2022
Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791
PART Ⅰ： Pharmacological Inhibition of STAT6 Ameliorates Myeloid Fibroblast Activation and Alternative Macrophage Polarization in Renal Fibrosis
Baihai Jiao, Changlong An, Melanie Tran, Hao Du, Penghua Wang, Dong Zhou and Yanlin Wang
INTRODUCTION
Chronic kidney disease(CKD) has become a significant public health challenge(1). Fibrosis is the ultimate common pathway for the development of CKD and is considered a major factor for the progression of all forms of CKD(2). Renal fibrosis is a pathological feature of CKD, leading to the replacement of normal kidney tissue structure with extracellular matrix(ECM)with progressive and irreversible damage to kidney function (3). However, despite increased understanding of the molecular mechanisms of renal fibrosis, there is no specific treatment to control fibrosis and restore renal function. As the origin and functional contribution of fibroblasts are not completely understood, effectively targeting fibroblasts in organ fibrosis remains a challenge.
Accumulating evidence indicates that myeloid myofibroblasts termed fibrocytes play a crucial role in the process of fibrosis(4-7). We and others have previously shown that the recruitment of bone marrow-derived fibroblasts increases the progression and development of renal fibrosis(8-12). Therefore, targeting these cells may serve as an effective therapeutic strategy to treat chronic kidney disease. Bone marrow-derived fibroblasts share the characteristics of mesenchymal cells as well as hematopoietic cells(13,14). However, various cytokines produced in the local microenvironment determine the differentiation of bone marrow-derived fibroblasts. We have previously demonstrated that Th2 cytokines(I-4 and IL-13), which are considered profibrotic cytokines, enhance the expression of type I collagen, fibronectin, and α-SMA in bone marrow-derived monocytes (15, 16).
STAT6 signaling is primarily activated by Th2 cytokines such as IL-4 and IL-13 and is associated with the pathogenesis of lung (17,18), liver(19), and renal fibrosis (15, 20). We have recently shown that JAK3/STAT6 plays a crucial role in the activation of bone marrow-derived fibroblasts and the development of renal fibrosis in obstructive nephropathy(15). Furthermore, we have shown that knockout of IL-4Ro inhibits STAT6 activation, myeloid fibroblast accumulation and transformation into myofibroblasts, M2 macrophage polarization, and development of renal fibrosis following obstructive injury or folic acid administration(16). These findings suggest that the STAT6 signaling pathway may serve as a novel therapeutic target for renal fibrosis.
In this study, we evaluated the potential therapeutic role of AS1517499, a potent and selective STAT6 inhibitor(21), in myeloid fibroblast activation and the development of renal fibrosis in two experimental murine models. Our results demonstrate that AS1517499 abolished STAT6 activation in the interstitial cells of the kidney with obstructive injury or folic acid nephropathy. Furthermore, administration of AS1517499 suppresses the activation of myeloid fibroblasts, reduces M2 macrophage polarization, and attenuates renal fibrosis. These findings suggest that inhibition of STAT6 with AS1517499 has the potential for the treatment of fibrotic kidney disease.
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RESULTS
AS1517499 Inhibits STAT6 Activation in the Kidney With Obstructive Injury
We have reported that JAK3/STAT6 signaling is activated in interstitial cells of fibrotic kidneys following obstructive injury or folic acid administration(15,16). To evaluate whether STAT6 can serve as a therapeutic target for the treatment of renal fibrosis, wild-type mice were subjected to obstructive injury and then treated with vehicle or AS1517499, a STAT6-specific inhibitor, every two days for 10 days. Immunohistochemical staining and Western blot analysis were performed to examine STAT6 activation in the kidney. Immunohistochemical staining revealed that positive phosphor-STAT6 staining was mainly detected in the interstitial cells of the kidney with obstructive nephropathy, which was reduced by AS1517499 treatment (Figures 1A, B). Consistent with these findings, Western blot analysis showed level of phospho-STAT6 was inhibited in the kidney with obstructive nephropathy following AS1517499 treatment (Figures 1C, D). These data indicate that the inhibition of STAT6 with AS1517499 prevents STAT6 activation in the kidney with obstructive nephropathy. AS1517499 Impairs Myeloid Fibroblast Accumulation in Obstructive Nephropathy Accumulating evidence indicates that the recruitment of myeloid fibroblasts contributes significantly to the pathogenesis of renal fibrosis. To investigate whether AS1517499 affects myeloid fibroblasts accumulation, kidney sections were stained for the hematopoietic marker CD45 and mesenchymal marker PDGFR-β.
FIGURE 1|AS1517499 inhibits STAT6 activation in the kidney with obstructive injury. (A) Representative photomicrographs of kidney sections at day 10 of UUO stained for phosphorylated STAT6 (brown) and counterstained with hematoxylin (blue). Scale bar,50um. (B) Quantitative analysis of phosphorylated STAT6-positive cells in the kidney.***P<0.001 vs. Vehicle-CON;"*P<0.01 vs Vehicle-UUO;++P<0.01 vs AS-UUO.n=6 per group. (C) Representative Western blots show protein levels of p-STAT6 and STAT6 in the kidney at day 10 of UUO.(D) Quantitative analysis p-STAT6 protein levels in the kidney,***P<0.001 vs Vehicle-CON·#*P<0.05 vs Vehicle-UUO; *P< 0.05 vs AS-UUO. n =6 per group.
The number of CD45 and PDGFR-β dual positive cells was significantly enhanced in the obstructed kidneys, whereas AS1517499 treatment markedly reduced CD45 and PDGFR-βdual positive cells(Figures 2A, B). These data suggest that STAT6 inhibitor AS1517499 ameliorates myeloid fibroblasts accumulation in the kidney after obstructive injury. AS1517499 Attenuates M2 Macrophage Polarization in Obstructive Nephropathy The transformation of monocytes into bone marrow-derived fibroblasts is mediated by M2 macrophage polarization.STAT6 plays a key role in M2 macrophage polarization. Therefore, we examined whether the inhibition of STAT6 with AS1517499 mediates macrophage polarization. Kidney sections were stained for CD206(M2 macrophage marker) and PDGFR-β.CD206 and PDGFR-β dual positive cells were markedly increased in the kidney of mice with obstructive injury. In contrast, AS1517499 significantly reduced the number of CD206+ and PDGFR-β+cells in kidneys with obstructive nephropathy(Figures 2C, D). These data indicate that AS1517499 significantly attenuates the transition of monocytes into myeloid fibroblasts and reduces M2 macrophage polarization.
FIGURE 2 |AS1517499 attenuates myeloid fibroblast accumulation and macrophage polarization in the kidney with obstructive injury. (A) Representative photomicrographs of kidhey sections at day 10 of UUO stained for CD45 （red）， PDGFR-β（green）， and DAPI（blue） Scale bar，50μm. B） Quantitative analysis of CD45*and PDGFR-β* fibroblasts in the kidney.**P<0.001 vs Vehicle-CON;P<0.01 vs Vehide-UUO;+P<0.01 vs AS-UUO.n=6 per group. (C)Representative photomicroaraphs of the kidney at day 10ofUUO stained for CD206 (red).PDGFR-B (green).and DAPIblue).Scale bar.,50um.(D) Quantitative analysis of CD206t and PDGFR-B+ fibroblasts in thekidney.*P<0.01 ys Vehicle-CON:P<0.01 vs Vehicle-UUO;+P<0.01 vs AS-UUO.n=6 per group. (E)Quantitative analysis of mRNAexpression of M2 macrophage markers (MRC.FZZ1.Arg1.CCL17 in the kidney of Vehide or AS1517499 treated mice.*P<0.001,"P<0.01,*P<0.05 vs Vehide-CON;P<0.01,*P<0.05 vs Vehicle-UUO;+P<0.001,+P<0.05vs AS-UUO.n =6 per group.
To further evaluate the effect of STAT6 inhibitor AS1517499 on M2 macrophage polarization, real-time RT-PCR was performed to detect mRNA expressions of M2 macrophage markers. AS1517499 treatment suppressed mRNA expression abundance of Argl, MRCl, Fizzl, and CCL17 in obstructed kidneys(Figure 2E). These data indicate STAT6 signaling immunofluorescence staining and Western blot analysis. Immunofluorescence staining and Western blot analysis revealed the levels of fibronectin and collagen I were markedly increased in the obstructed kidney, whereas AS1517499 treatment significantly attenuated the protein levels of collagen I and fibronectin in the obstructed kidney(Figures 4C-H).
FIGURE 3 | AS1517499 reduces myofibroblast formation in the kidney with obstructive injury. (A) Representative photomicrographs of UUO treated kidney sections stained for a-SMA (green) and counterstained with DAPI (blue). Scale bar, 50mm. (B) Quantitative analysis of a-SMA positive area in the kidney. ***P < 0.001 vs Vehicle-CON, # P < 0.05 vs Vehicle-UUO, ++P < 0.01 vs AS-UUO. n = 6 per group. (C) Representative Western blots show a-SMA protein levels in the kidney. (D) Quantitative analysis of a-SMA protein expression in the kidney. ***P < 0.001 vs Vehicle-CON, ##P < 0.01 vs Vehicle-UUO, ++P < 0.01 vs AS-UUO. n = 6 per group.
FIGURE 4 | AS1517499 attenuates renal fifibrosis and ECM protein production in the kidney with obstructive injury. (A) Representative photomicrographs of kidney sections stained with Sirius red for evaluation of total collagen deposition in the kidney. Scale bar, 50mm. (B) Quantitative analysis of interstitial collagen deposition in the kidney. ***P <0.001 vs Vehicle-CON. ##P < 0.01 vs Vehicle-UUO, ++P < 0.01 vs AS-UUO. n = 6 per group. (C) Representative photomicrographs of the kidney sections stained for fibronectin (green) and counterstained with DAPI (blue). Scale bar, 50mm. (D) Quantitative analysis of the fibronectin-positive area in the kidney. ***P < 0.001 vs Vehicle-CON. ##P < 0.01 vs Vehicle-UUO, + P < 0.05 vs AS-UUO. n = 6 per group. (E) Representative photomicrographs of the kidney sections stained for collagen I (green) and counterstained with DAPI (blue). Scale bar, 50mm. (F) Quantitative analysis of collagen I positive area in the kidney. ***P < 0.001 vs Vehicle-CON. # P < 0.05 vs Vehicle-UUO, ++P < 0.01 vs AS-UUO. n = 6 per group. (G) Representative Western blots show protein expression of fibronectin and collagen I in the kidney. (H) Quantitative analysis of protein levels of fibronectin and collagen I in the kidney. ***P < 0.001 vs Veh-CON; ##P < 0.01, # P < 0.05 vs Veh-UUO; ++P < 0.01 vs AS-UUO. n = 6 per group.
AS1517499 Inhibits STAT6 Activation in Folic Acid Nephropathy
Folic acid nephropathy is another commonly used murine model of renal fibrosis. Therefore, we examined whether AS1517499 can inhibit STAT6 activation and pathogenesis of renal fibrosis in folic acid nephropathy. Kidney sections were stained for phospho-STAT6.STAT6 phosphorylation was significantly induced in interstitial cells of the kidney with FA nephropathy, whereas AS1517499 treatment markedly inhibited the expression of phospho-STAT6(Figures 5A, B). These findings were further confirmed by Western blot analysis(Figures 5C, D). These data indicate that STAT6 is activated in the interstitial cells of the damaged kidney with folic acid nephropathy, which is significantly inhibited following AS1517499 treatment.
FIGURE 5 | AS1517499 inhibits STAT6 activation in folic acid nephropathy. (A) Representative images of immunohistochemical staining of phosphorylated STAT6 in the kidney 2 weeks after vehicle or AS1517499 treatment. Scale bar, 50mm. (B) Quantitative analysis of phosphorylated STAT6-positive cells in the kidney 2 weeks after vehicle or AS1517499 treatment. ***P < 0.001 vs. CON; ##P < 0.01 vs FA+Veh. n = 6 per group. (C) Representative Western blot of p-STAT6 and STAT6 in the kidney. (D) Quantitative analysis p-STAT6 protein levels in the kidney. ***P < 0.001 vs. CON; ##P < 0.01 vs FA+Veh. n = 6 per group.
AS1517499 Suppresses Myeloid Fibroblast Accumulation in Folic Acid Nephropathy To examine the role of AS1517499 in the myeloid fibroblast accumulation in the kidney with folic acid nephropathy, we performed double immunofluorescence staining for CD45 and PDGFR-β. The number of CD45 and PDGFR-β dual positive cells increased in the kidney with folic acid nephropathy. AS1517499 considerably suppressed the number of CD45 and PDGFR-β dual positive cells in the kidney with folic acid nephropathy (Figures 6A, B).
AS1517499 Attenuates M2 Macrophage Polarization in Folic Acid Nephropathy We then examined whether the pharmacological inhibition of STAT6 with AS1517499 regulates M2 macrophage polarization in FA nephropathy. As confirmed by immunofluorescence staining analysis, treatment of AS1517499 significantly inhibited the number of CD206 and PDGFR-β dual positive cells compared with vehicle-treated mice with FA administration (Figures 6C, D).In addition, M2 macrophage markers, Argl, MRC1,Fizzl,and CCL17 were induced in folic acid nephropathy. These M2 macrophage markers were reduced following AS1517499 treatment(Figure 6E). These data indicate that the pharmacological inhibition of STAT6 with AS1517499 attenuates M2 macrophage polarization in the kidney with FA nephropathy.
FIGURE 6 | AS1517499 attenuates myeloid fifibroblast accumulation and macrophage polarization in folic acid nephropathy. (A) Representative photomicrographs of kidney sections 2 weeks after vehicle or AS1517499 treatment stained for CD45 (red), PDGFR-b (green), and DAPI (blue). Scale bar, 50mm. (B) Quantitative analysis of CD45+ and PDGFR-b+ fifibroblasts in the kidney. **P < 0.01 vs CON. # P < 0.05 vs FA+Veh. n = 6 per group. (C) Representative photomicrographs of kidney sections 2 weeks after vehicle or AS1517499 treatment stained for CD206 (red), PDGFR-b (green), and DAPI (blue). Scale bar, 50mm. (D) Quantitative analysis of CD206+ and PDGFR-b+ fifibroblasts in the kidney. ***P < 0.001 vs CON. ##P < 0.01 vs FA+Veh. n = 6 per group. (E) Quantitative analysis of mRNA expression of M2 macrophage makers (MRC, FIZZ1, Arg1, CCL17) in the kidney 2 weeks after vehicle or AS1517499 treatment. ***P < 0.001 vs CON; ###P < 0.001 vs FA+Veh; ##P < 0.01 vs FA+Veh. n = 6 per group.
AS1517499 Attenuates Myofibroblast Formation in Folic Acid Nephropathy To determine whether AS1517499 affects myofibroblast transformation, α-SMA expression was examined in kidney sections and by western blot analysis. Consistent with our findings in the UUO model, α-SMA positive myofibroblasts and the expression of α-SMA protein was enhanced in the kidney after treatment with FA compared with vehicle-treated mice. Moreover, co-administration of FA with AS1517499 significantly reduced α-SMA positive cells and α-SMA protein level in the kidney compared with FA co-administrated vehicle group (Figures 7A-D). These results demonstrate that AS1517499 inhibits myofibroblast formation in the kidney with folic acid nephropathy.
FIGURE 7 | AS1517499 reduces myofibroblast formation in folic acid nephropathy. (A) Representative photomicrographs of kidney sections 2 weeks after vehicle or AS1517499 treatment stained for a-SMA (green) and counterstained with DAPI (blue). Scale bar, 50mm. (B) Quantitative analysis of a-SMA positive area in the kidney. ***P < 0.001 vs CON; ###P < 0.001 vs FA+Veh. n = 6 per group. (C) Representative Western blots showing a-SMA protein levels in the kidney 2 weeks after vehicle or AS1517499 treatment. (D) Quantitative analysis of a-SMA protein expression in the kidney. ***P < 0.001 vs CON, ##P < 0.01 vs FA+Veh. n = 6 per group.
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AS1517499 Ameliorates Renal Fibrosis and Preserves Renal Function in Folic Acid Nephropathy
To evaluate the effect of AS1517499on renal fibrosis,picrosiriusred staining was performed on kidney sections. FA-treated mice had markedly elevated levels of collagen deposition, whereas AS1517499 treatment significantly reduced the amounts of collagen deposition in the kidney(Figures 8A, B). Additionally, immunofluorescence staining and Western blot analysis further demonstrated that AS1517499 significantly inhibits the expression of ECM proteins(fibronectin and collagen I) in the kidney with folic acid nephropathy(Figures 8C-H). These data suggest that AS1517499 inhibits ECM production in the kidney and the development of renal fibrosis in folic acid nephropathy.
To assess the effect of STAT6 deficiency on kidney function in folic acid nephropathy, serum creatinine was measured. Folic acid-treated mice displayed a significant elevation of serum creatinine (Figure 8I). In contrast, mice treated with AS1517499 exhibited much lower serum creatinine in folic acid nephropathy. These results indicate that inhibition of STAT6 with AS1517499 protects the kidney from folic acid nephropathy.
FIGURE 8 | AS1517499 reduces kidney fifibrosis and preserves kidney function in folic acid nephropathy. (A) Representative photomicrographs of kidney sections 2 weeks after vehicle or AS1517499 treatment stained with Sirius red for evaluation of total collagen deposition in the kidney. Scale bar, 50mm. (B) Quantitative analysis of interstitial collagen deposition in the kidney. ***P < 0.001 vs CON, ##P < 0.01 vs FA+Veh. n = 6 per group. (C) Representative photomicrographs of kidney sections stained for fibronectin (green) and counterstained with DAPI (blue). Scale bar, 50mm. (D) Quantitative analysis of the fifibronectin-positive area in FA treated kidneys. ***P < 0.001 vs CON, ##P < 0.01 vs FA+Veh. n = 6 per group. (E) Representative photomicrographs of kidney sections stained for collagen I (green) and counterstained with DAPI (blue). Scale bar, 50mm. (F) Quantitative analysis of collagen I positive area in the kidney. ***P < 0.001 vs CON, # P < 0.05 vs FA+Veh. n = 6 per group. (G) Representative Western blots showing protein expression of fibronectin and collagen I in the kidney. (H) Quantitative analysis of protein levels of fibronectin and collagen I in the kidney. ***P < 0.001 vs CON; ##P < 0.01 vs FA+Veh. n = 6 per group. (I) Effect of AS1517499 on serum creatinine. **P < 0.001 vs CON; # P < 0.05 vs FA+Veh. n = 6 per group.
The traditional Chinese medicinal herb: cistanche
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