Irisin protects against vascular calcification by activating autophagy and inhibiting NLRP3-mediated vascular smooth muscle cell pyroptosis in chronic kidney disease

Abstract

Irisin protects the cardiovascular system against vascular diseases. However, its role in chronic kidney disease (CKD) -associated vascular calcification (VC) and the underlying mechanisms remain unclear. In the present study, we investigated the potential link among Irisin, pyroptosis, and VC under CKD conditions. During mouse vascular smooth muscle cell (VSMC) calcification induced by β-glycerophosphate (β-GP), the pyroptosis level was increased, as evidenced by the upregulated expression of pyroptosis-related proteins (cleaved CASP1, GSDMD-N, and IL1B) and pyroptotic cell death (increased numbers of PI-positive cells and LDH release). Reducing the pyroptosis levels by a CASP1 inhibitor remarkably decreased calcium deposition in β-GP-treated VSMCs. Further experiments revealed that the pyroptosis pathway was activated by excessive reactive oxygen species (ROS) production and subsequent NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in calcified VSMCs. Importantly, Irisin effectively inhibited β-GP-induced calcium deposition in VSMCs in vitro and in mice aortic rings ex vivo. Overexpression of Nlrp3 attenuated the suppressive effect of Irisin on VSMC calcification. In addition, Irisin could induce autophagy and restore autophagic flux in calcified VSMCs. Adding the autophagy inhibitor 3-methyladenine or chloroquine attenuated the inhibitory effect of Irisin on β-GP-induced ROS production, NLRP3 inflammasome activation, pyroptosis, and calcification in VSMCs. Finally, our in vivo study showed that Irisin treatment promoted autophagy, downregulated ROS level and thereby suppressed pyroptosis and medial calcification in aortic tissues of adenine-induced CKD mice. Together, our findings for the first time demonstrated that Irisin protected against VC via inducing autophagy and inhibiting VSMC pyroptosis in CKD, and Irisin might serve as an effective therapeutic agent for CKD-associated VC.

Fig. 1. Pyroptosis occurs in VSMCs after exposure to β-GP.

VSMCs were treated with β-GP (10 mM) for the indicated time. A Calcium deposition in VSMCs was assessed by Alizarin red staining (positive staining: red; scale bar = 100 μm). B Quantitative analysis of calcium deposition in VSMCs normalized to the protein content. C Protein levels of pro-CASP1, cleaved CASP1, GSDMD-N, and IL1B were determined by western blotting. D Quantification of the results shown in C. E The IL1B content in VSMC culture supernatants was determined by ELISA. F The release of LDH was detected using the LDH Assay Kit. G The percentage of PI-positive cells was measured using Hoechst 33342 (blue)/PI (red) double staining (top: Representative images; bottom: Quantitative analysis of PI-positive cells). H CASP1 expression was determined by immunofluorescent staining (top: Representative images; bottom: Mean fluorescence intensity (MFI) of CASP1 was quantified). I The percentage of TUNEL-positive cells (green) was evaluated by TUNEL staining (top: Representative images; bottom: Quantitative analysis of TUNEL-positive cells). Scale bar = 50 μm for (G); Scale bar = 20 μm for (H) and (I). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01 vs. indicated group; and N.S. not significant.

Fig. 2. Inhibition of CASP1 attenuates β-GP-induced VSMC pyroptosis and calcification.

VSMCs were incubated in culture medium containing β-GP (10 mM) for 7 days with or without CASP1 inhibitor (VX-765; 10 μM). A Protein levels of pro-CASP1, cleaved CASP1, GSDMD-N, and IL1B were determined by western blotting. B Quantification of the results shown in A. C The IL1B content in VSMC culture supernatants was determined by ELISA. D The release of LDH was detected using the LDH Assay Kit. E The percentage of PI-positive cells was measured using Hoechst 33342 (blue)/PI (red) double staining (top: Representative images; bottom: Quantitative analysis of PI-positive cells). Scale bar = 50 μm. F Calcium deposition in VSMCs was assessed by Alizarin red staining (positive staining: red; scale bar = 100 μm). G Quantitative analysis of calcium deposition in VSMCs normalized to the protein content. Mouse aortic rings were incubated in the culture medium containing β-GP (10 mM) for 7 days with or without VX-765(10 μM). Calcification was assessed by Alizarin red staining (H), and calcium content of aortas was measured as described in methods (I). (positive staining: red; scale bar = 100 μm). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01 vs. indicated group; and N.S. not significant.

Fig. 3. ROS-mediated NLRP3 inflammasome activation is involved in β-GP-induced VSMC pyroptosis.

VSMCs were treated with β-GP (10 mM) for the indicated time. A The protein levels of NLRP3 and PYCARD were determined by western blotting. B Quantification of the results shown in A. The data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. control group; ^#P < 0.05, ^##P < 0.01 vs^. indicated group. VSMCs were incubated in the presence of β‐GP (10 mM) medium with the ROS inhibitor NAC (20 μM) or NLRP3 inhibitor MCC950 (100 μM) as indicated. C The protein levels of pyroptosis-related markers were determined by western blotting. D Quantification of the results shown in C. E The IL1B content in VSMC culture supernatants was determined by ELISA. F The release of LDH was detected using the LDH Assay Kit. G The percentage of PI-positive cells was measured using Hoechst 33342 (blue)/PI (red) double staining (top: Representative images; bottom: Quantitative analysis of PI-positive cells). Scale bar = 50 μm. H The intracellular ROS level was detected using the fluorescent probe DCFH-DA and measured by flow cytometry. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01 vs. β-GP group; and N.S. not significant.

Fig. 4. Overexpression of Nlrp3 attenuates the inhibitory effect of Irisin on β-GP-induced VSMC pyroptosis and calcification.

VSMCs were transfected with the pcDNA empty vector (pcDNA) or Nlrp3-expressed plasmid (NLRP3) for 48 h, and were subsequently treated with β-GP (10 mM) in the presence or absence of 100 ng/ml of Irisin for 7 days. A The protein levels of pyroptosis-related markers were determined by western blotting. B Quantification of the results shown in A. C The IL1B content in VSMC culture supernatants was determined by ELISA. D The release of LDH was detected using the LDH Assay Kit. E The percentage of PI-positive cells was measured using Hoechst 33342 (blue)/PI (red) double staining (top: Representative images; bottom: Quantitative analysis of PI-positive cells). F The percentage of TUNEL-positive cells (green) was evaluated by TUNEL staining (top: Representative images; bottom: Quantitative analysis of TUNEL-positive cells). Scale bar = 50 μm for (E); Scale bar = 20 μm for (F). G Calcium deposition in VSMCs was assessed by Alizarin red staining (positive staining: red; scale bar=100 μm). H Quantitative analysis of calcium deposition in VSMCs normalized to the protein content. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01 vs. β-GP group;^&P < 0.05, ^&&P < 0.01 vs. β-GP + Irisin+pcDNA group; and N.S. not significant.

Fig. 5. Irisin treatment induces autophagy and restores autophagic flux in calcified VSMCs.

A Pathway analysis of the 277 target genes based on KEGG database. The top 10 positively enriched pathways are shown in the bubble chart. The x-axis represents the enrichment score, y-axis represents the enriched pathways. The color of the bubble represents the P value of the pathway enrichment. The size of each bubble represents the number of different genes contained in the pathway. B VSMCs were treated with Irisin (100 ng/ml) for 72 h and the mRNA level of Map1lc3b, Becn1, Atg7, Atg5, Lamp1, and Tfeb was assessed by qRT-PCR. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. control group. C–F VSMCs were treated with Irisin (100 ng/ml) or β-GP (10 mM) for the indicated time. Protein levels of MAP1LC3B and SQSTM1were determined by western blotting. G, H VSMCs were incubated with or without Irisin (100 ng/ml) in the presence of β-GP (10 mM) for 72 h. Protein levels of MAP1LC3B and SQSTM1 were evaluated by western blotting. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs corresponding control group; ^#P < 0.05, ^##P < 0.01 vs corresponding β-GP group. I Confocal microscopy observation of mRFP-GFP-LC3 adenovirus transfected VSMCs treated as indicated (Magnification: ×63, Scale bars = 10 μm). J Autophagosome (yellow dots) and autolysosome (red dots) numbers in each group were calculated. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. indicated group; ^#P < 0.05, ^##P < 0.01 vs. indicated group.

Fig. 6. Irisin protects VSMCs from β-GP-induced pyroptotic cell death and calcification by promoting autophagic flux.

VSMCs were cultured in the presence of β-GP (10 mM) with or without of Irisin, and were subsequently treated with rapamycin (Rapa, 200 nM), 3-methyladenine (3-MA, 5 mM) or chloroquine (CQ,25 µM) for 72 h (autophagy) or 7 days (calcification). A The protein levels of autophagy- and pyroptosis-related markers were determined by western blotting. B Quantification of the results shown in A. C Confocal microscopy observation of mRFP-GFP-LC3 adenovirus transfected VSMCs treated as indicated (Magnification: ×63, Scale bars=10 μm). D Autophagosome (yellow dots) and autolysosome (red dots) numbers in each group were calculated. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. indicated group; ^#P < 0.05, ^##P < 0.01 vs. indicated group. E The intracellular ROS level was detected using the fluorescent probe DCFH-DA and measured by flow cytometry. F The IL1B content in VSMC culture supernatants was determined by ELISA. G The release of LDH was detected using the LDH Assay Kit. H The percentage of PI-positive cells was measured using Hoechst 33342 (blue)/PI (red) double staining (top: Representative images; bottom: Quantitative analysis of PI-positive cells). Scale bar = 50 μm. I Calcium deposition in VSMCs was assessed by Alizarin red staining. Scale bar = 100 μm. J Quantitative analysis of calcium deposition in VSMCs normalized to the protein content. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. corresponding β-GP group; ^&P < 0.05, ^&&P < 0.01 vs. corresponding β-GP + Irisin group; and N.S. not significant.

Fig. 7. Irisin prevents medical VC ex vivo and in vivo.

A Scheme of the construction of the CKD-associated VC animal model. B-C Mouse aortic rings were treated with Irisin in medium containing β-GP (10 mM) for 7 days. Calcification was assessed by Alizarin red staining (B), and the calcium content in the aortas was quantified (C). (positive staining: red; scale bar=100 μm). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01 vs. β-GP group. D Mice serum levels of blood urea nitrogen (BUN), creatinine (Cr), calcium (Ca), and phosphate (P) were measured. Values are expressed as mean ± SEM (n = 6 per group). *P < 0.05, **P < 0.01 vs. the age-marched controls. ^#P < 0.05, ^##P < 0.01 vs. the age-marched CKD group. CKD mice were injected subcutaneously with vitamin D3(Vit-D3), fed a high Ca and P diet, and treated with Irisin or normal saline (NS) for 12 weeks. Normal mice with the same age were used as controls. E Calcium deposition in the abdominal aortas was detected by Von Kossa staining (D). (positive staining: brown to black; scale bar = 100 μm). F The calcium content in the abdominal aortas was quantified. G The ROS level in aortic tissues was detected using the fluorescent probe DCFH-DA and visualized using confocal microscopy. (shown in red, Magnification: ×400, scale bar = 100 μm). Cell nuclei were visualized using DAPI (blue). H Serum Irisin level was evaluated by ELISA. I Serum IL1B level was evaluated by ELISA. J The protein levels of autophagy- and pyroptosis-related markers were determined by western blotting. K Quantification of the results shown in J. Representative immunofluorescent staining images for NLRP3 (green, L), CASP1 (green, M), and SQSTM1 (green, N). The sections were co-stained with ACTA2/α-SMA to outline the VSMC area. (Magnification: ×400, scale bar=100 μm). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; ^#P < 0.05, ^##P < 0.01 vs. CKD group. Calcium: Ca, phosphate: P.

Fig. 8. A graphic illustration of the protective role of Irisin in CKD-associated VC.

High phosphate (Pi) induces the production of ROS, which activates NLRP3 inflammasome, leads to the activation of CASP1-dependent pyroptotic cell death, and release of inflammatory factors in VSMCs, and ultimately promotes the formation of VC in CKD (Red arrows). Irisin induces autophagy and reduces ROS production. These effects lead to inhibition of NLRP3-mediated pyroptotic cell death, proinflammatory response, and calcium deposition in VSMCs, and ameliorate the progression of VC in CKD (Blue arrows).