Fig 2: Mitochondrial ROS (mitoROS) generation is increased by cytosolic LPS, mitoROS promotes the caspase-4/GSDMD pathway, and casp4/GSDMD also promotes mitoROS generation.(A) In total, 165 ROS regulator genes from GSEA were screened in WT aorta RNA-Seq data and casp11–/– CKD+HFD aorta RNA-Seq data. (B) The mitoROS level was detected using mitoSOX (5 μM) in LPS-transfected HAECs in the presence or absence of a GSDMD cleavage inhibitor (8 μM). (C–E) Casp4 activity (C). The expression levels of N-GSDMD (D) and adhesion molecule VCAM-1 (E) were detected in LPS-transfected HAECs in the presence or absence of mitoROS inhibitor mitoTempo (1 μM) using flow cytometry. The Kruskal-Wallis test with Benjamini and Hochberg multiple-comparison method was used to control the overall FDR of 5% (B–E).

Fig 3: Our working model.HFD+CKD (UTs) promote extracellular LPS enter aortic cell cytosol, increase intracellular gram-negative bacterial infections in CKD, increase intracellular crystallization of CKD-elevated palmitic acid, activate casp4/11 and N-GSDMD membrane expression, increase secretion of IL-1β and other casp11-GSDMD secretome, and upregulate TI genes in aortic cells; after sensing intracellular LPS, palmitic acid stimulation, and UT indoxyl sulfate stimulation, casp11 gets activated and cleaves N-GSDMD and promotes N-GSDMD membrane expression in aortic endothelial cells.

Fig 4: Caspase-11 deficiency decreases the cleavage of N-GSDMD in the aortas of HFD+CKD mice.(A) Five endothelial cell activation genes were identified in Venn diagram of 584 significantly downregulated genes in casp11–/– aortas and 1,311 endothelial cell activation genes identified in the literature (P < 0.05, log2FC < –1). (B and C) Intravital microscopy was used to examine peripheral blood cell rolling and adhesion in the cremaster muscle vein in male mice (n = 4–6). (D) Western blot analysis showed that HF+CKD increased the expression of endothelial cell adhesion molecule VCAM-1 in aortas compared with CKD and HFD-sham controls, suggesting that HFD+CKD activates aortic endothelial cells. (E) Flow cytometry gating analysis was used on mouse aorta cells to examine membrane GSDMD expression (n = 3–4). (F) Quantification of N-GSDMD expression was performed in endothelial cells (CD45–CD31+) in WT and casp11–/– HFD+CKD and HFD-Sham mouse aortas. (G) IL-1β secretion in the aorta of WT and casp11–/– HFD+CKD and HFD-Sham mice were quantified by ELISA. The Mann Whitney U test was used in B and C. The Kruskal-Wallis test with Benjamini and Hochberg multiple-comparison method was used to control the overall FDR of 5% (F and G).

Fig 5: The expression and activity of caspase-4 are positively associated with the progression of CKD-accelerated vascular inflammation.(A) Casppase-1 (casp1) expression in the kidneys of patients with CKD compared with that of healthy donors. (B) Caspase-4 (CASP4) expression in the kidneys of patients with CKD compared with that of healthy donors. (C and D) CASP1 and CASP4 were negatively correlated with GFR (Pearson analysis). (E) The top 20 CASP4/Gasdermine D–related (GSDMD-related) secretion genes were significantly increased in patients with CKD (GSE66494). (F) The LPS level in the aorta of HFD+CKD, HFD-Sham, ND-CKD, and ND-Sham mice was detected by an ELISA kit (n = 4–6). (G) Casp11 activation scales in the HFD+CKD aorta were higher than those of CASP1. Western blot analysis of WT mouse aortic tissue for CASP1 and casp11. (H) Casp11 activities in the aorta of HFD+CKD, HFD-sham, ND-CKD, and ND-sham were detected by the casp4 activity assay. In total, 50 μg protein from each sample was used to detect casp11 activities (n = 4–6). (I) The proinflammatory cytokines in the plasma of HFD+CKD, HFD-sham, ND-CKD, and ND-sham were analyzed by cytokine array. Each sample was pooled from 3 mice in each group (n = 3). ImageJ was used to quantify the bands, and the significantly changed proteins were indicated. Two-tailed Student’s t test was used in A and B. Pearson correlations were used in C and D. The Kruskal-Wallis test with Benjamini and Hochberg multiple-comparison method was used to control the overall FDR of 5% (F, H, and I). *P < 0.05.