Sirtuin 1 deficiency mediates chronic kidney disease-induced inflammaging cardiovascular calcification

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  Fig. 1. Chronic kidney disease promoted senescence phenotype and calcification in aortic valves. a Strategy for establishing the UKB population cohort, including exclusion or access criteria and population composition. b Chronic kidney disease (CKD) population (n = 13,118) exhibited significantly elder PhenoAge than non-CKD population (n = 262,481) in UKB. c CKD population (n = 13,118) exhibited significantly younger adjusted age of AS occurrence than non-CKD population in UKB. d Restricted cubic spline regression of immuneage and AS OR. e CKD population exhibited significantly elder immuneage than non-CKD population in UKB. f CKD population exhibited significantly elder proteinage than non-CKD population in UKB. g Restricted cubic spline regression of proteinage and AS OR. h Proteomic analyses showed significant upregulation of aging-related indicators in the CKD population, more prominent in the group suffering from AS. i Odds ratios (ORs) of CKD by stage on the onset of AS. j Restricted cubic spline regression of plasma creatinine level and AS OR. k Schematic diagram of establishing and monitoring chronic kidney disease (CKD) model. l Echocardiography showed that Apoe−/− CKD mice had…

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  Fig. 2. Single-cell RNA sequence revealed chronic kidney disease decreased the expression of SIRT1 in aortic valves. a Schematic diagram of single cell RNA (scRNA) sequencing in normal valves from control and calcified valves from CKD patients. n = 3 per group. b ScRNA atlas of aortic valves from control and CKD patients. c Major markers for cell types in aortic valves. d Differential gene expression volcano plots of the four major aortic valve cell subclusters. e Trajectory analysis of various subpopulations of VICs from normal versus calcified aortic valves, with myofibroblasts-VICs emerging as a critical and late state in the calcification process. The number of cells in each VIC subpopulation is listed aside. f Changes in aging and calcification-related genes with trajectory progression. g GSVA pathway enrichment analysis revealed that senescence-related pathways are upregulated in myofibroblast-VICs. h GSEA pathway enrichment analysis revealed that the cellular senescence pathway significantly upregulated in myofibroblast-VICs. i Violin plot of SIRT1 levels in myofibroblast-VICs. j Representative western blot band of SIRT1 expression in calcified and normal human aortic valves.…

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  Fig. 3. Intervention of the SIRT1 influenced aortic valve calcification and senescence in vivo and in vitro. a Echocardiography showed that Sirt1+/− Apoe−/− and Sirt1mfKO Apoe−/− mice had significantly higher transvalvular peak jet velocity, and Sirt1mfOV Apoe−/− mice and those supplied with resveratrol had significantly lower transvalvular peak jet velocity than Apoe−/− CKD mice. n = 10 per group. b Representative images of von Kossa staining of calcium in aortic valves of CKD mice. n = 10 per group. Calcium deposition was significantly increased in aortic valves of Sirt1+/− Apoe−/− and Sirt1mfKO Apoe−/− mice and decreased in Sirt1mfOV Apoe−/− mice and those supplied with resveratrol, compared with Apoe−/− CKD mice. n = 10 per group. Scale bar = 100 μm. c Representative images of immunofluorescence staining and quantification analysis showed that Sirt1+/− Apoe−/− and Sirt1mfKO Apoe−/− mice had significantly higher expression of Runx2 (red), and Sirt1mfOV Apoe−/− mice and those supplied with resveratrol had significantly lower expression of Runx2 (red) than Apoe−/− CKD mice, DAPI (blue) was used for nuclear counterstaining. n = 10 per group. Scale bar = 100 μm. d Representative images of…

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  Fig. 4. NLRP3 inflammasome mediated the effects of SIRT1 deficiency on CKD-induced aortic valve calcification. a Differential gene volcano plots of high SIRT1-expressing subpopulations versus low SIRT1-expressing subpopulations in myofibroblasts from the aforementioned single-cell dataset. b GSVA pathway enrichment analysis revealed that NOD-like pathways were significantly upregulated in the SIRT1 low-expression subpopulation. c Scatter plots showed a significant negative correlation between SIRT1 expression and NLRP3 pathway score. d Representative images of immunofluorescence staining and quantification analysis showed that Sirt1+/− Apoe−/− CKD mice had significantly higher expression of Nlrp3 (red) than Apoe−/− CKD mice. DAPI (blue) was used for nuclear counterstaining. n = 10 per group. Scale bar = 100 μm. e Representative western blot bands showed ALP, RUNX2, and NLRP3 expression in VICs treated with hyperphosphate culture medium for 72 h after SIRT1 siRNA transfection; densitometric quantification is shown for NLRP3. n = 5 per group. f Representative images of immunofluorescence staining and quantification analysis showed that silencing SIRT1 VICs had significantly higher…

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  Fig. 5. SIRT1 suppressed high phosphorous-associated NF-κB-NLRP3 pathway activation by inhibiting glycolysis. a Reduction plot of glycolysis pathway score in myofibroblast-VICs. b Violin plot of glycolysis pathway in myofibroblast-VICs. c Volcano plot comparing gene expression between siNC/siSIRT1-treated VICs (n = 3). d Principal component analysis (PCA) revealing inter-sample (siNC and siSIRT1) agreement. e Heatmap comparing the enrichment score of key metabolic pathways; analysis of RNA-seq expression (n = 3). f GSEA reveals that glycolysis/glyconeogenesis pathway was enriched in siSIRT1 group. g Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) plot of the metabolomic profiles. h Volcano plot of untargeted metabolomic profiling of siNC/siSIRT1-treated VICs (n = 5). i Relative expression of L-Lactic acid (n = 5). j Relative expression of 3-Phosphoglyceric acid (n = 5). k The enrichment analysis of the metabolomic data. l-m Real-time changes in the OCR (l) and ECAR (m) of VICs, pretreated with siSIRT1 or siNC, measured using Seahorse. Oligo, oligomycin; FCCP, carbonyl cyanide4-(trifluoromethoxy) phenylhydrazone; R/A, rotenone plus antimycin A; 2-DG, 2-deoxy-d-glucose.…

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  Fig. 6. Anti-diabetic compound screening revealed semaglutide attenuated CKD-induced aortic valve calcification. a Schematic diagram of high content anti-diabetic compound screening on VICs. b Representative images of immunofluorescence staining screening after different compound treatment. NLRP3 (red), SIRT1 (green), and DAPI (blue) was used for nuclear counterstaining. n = 3 per group. c Representative heatmap of SIRT1 expression after different compound treatment. d Representative heatmap of NLRP3 expression after different compound treatment. e Scatterplot showed the statistical level change of SIRT1 and NLRP3 after treatment. f Venn diagram indicated 6 compounds were candidate for CAVD treatment. g Statistical level change of SIRT1/NLRP3 ratio of 6 compounds after treatment. n = 5 per group. h Representative images of immunofluorescence staining and quantification analysis showed that semaglutide had significantly lowered NLRP3 (red) and elevated SIRT1 (green) level in VICs. DAPI (blue) was used for nuclear counterstaining. n = 5 per group. Scale bar = 25 μm. i SIRT1/NLRP3 ratio and separate levels after semaglutide treatment on VICs under a sequenced concentration gradient. j LOESS…