Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway

Abstract

Background: Pyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI.

Methods and results: The expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1β production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis.

Conclusion: Our results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.

Keywords: Acetyl-CoA synthetase 2; Acute kidney injury; Pyroptosis; Sepsis.

Fig. 1

The NLRP3 inflammasome and pyroptosis are activated in an LPS-induced mouse sepsis model. A Serum creatinine levels in vehicle-treated mice and LPS-induced sepsis mice were measured (n = 5-6). B H&E staining of kidney tissues from the AKI model mice and control mice (Scale bars, 50 μm). C F4/80 staining of kidney tissues of the noted mice (Scale bars, 200 μm). D Expression of pro-inflammatory factors at the mRNA level in the kidneys of vehicle- or LPS-treated mice were determined using RT-qPCR (n = 5). E The protein expression of NLRP3, cleaved GSDMD, and caspase-1 in the kidney were detected by immunofluorescent staining (green, caspase-1; red, NLRP3 or cleaved GSDMD; blue, DAPI) and Western blotting (n = 3). Data were presented as mean ± SD. ^*P < 0.05, ^**P < 0.01 vs. Ctrl

Fig. 2

ACSS2 contributes to the inflammatory response and pyroptotic cell death in LPS-treated HK-2 cells. A Representative immunohistochemical staining and Western blotting analysis of ACSS2 in the kidneys of control and LPS-induced AKI mice. (Scale bars, 50 μm; n = 4). B Immunofluorescent staining of ACSS2 in HK-2 cells with or without LPS treatment (green, ACSS2; blue, DAPI; scale bars, 200 μm). C Western blotting analysis of ACSS2 protein in HK-2 cells treated with LPS for 24 or 48 hours (n = 3). D Relative mRNA expression levels of the pro-inflammatory cytokines (MCP-1, IL-6, and IL-1β) in HK-2 cells with or without pre-treatment with an ACSS2 inhibitor (ACSS2i) prior to LPS treatments for 24 or 48 hours (n = 3). E Relative mRNA expression level of kidney injury molecule-1 (KIM-1) in HK-2 cells with or without pre-treatment with ACSS2i prior to LPS treatments for 24 or 48 hours (n = 3). F The mRNA and protein levels of ACSS2 in HK-2 cells transfected with an ACSS2-specific siRNA or scrambled siRNA prior to LPS treatments for 24 hours (n = 3). G Relative mRNA expression levels of the pro-inflammatory cytokines (MCP-1, IL-6, and IL-1β) in HK-2 cells transfected with an ACSS2-specific siRNA or scrambled siRNA prior to LPS treatments for 24 hours (n = 3). H: TEM observation of the ultrastructure of HK-2 cells with or without pre-treatment with ACSS2i prior to LPS treatments for 24 hours (Scale bars, 5 μm). I: Immunofluorescence detection of the expression of cleaved GSDMD in HK-2 cells with or without LPS (1 μg/mL) and with or without co-treatment with ACSS2i (10 μmol/L) for 24 hours (Scale bars, 100 μm). J: Western blotting analysis of GSDMD in HK-2 cells which were stimulated with LPS (1 μg/mL) or vehicle and co-incubated with ACSS2i (10 μmol/L) for 24 or 48 hours (n = 3). Data were presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.005 vs. Ctrl; ^#P < 0.05, ^##P < 0.01, ^###P < 0.005 vs. LPS or LPS + siControl; ^$P < 0.05 vs. LPS for 48 hours

Fig. 3

Promotion of NLRP3 inflammasome activation and GSDMD cleavage by ACSS2 depends on NF-κB signaling activation. A The immunofluorescence staining of NF-κB p65 was detected in HK-2 cells which were treated with LPS (1 μg/mL) or vehicle along with ACSS2i (10 μmol/L) for 24 hours. (green, p65; blue, DAPI; scale bars, 100 μm). B The effects of ACSS2 siRNA (siACSS2) on the protein expression and phosphorylation of p65 in HK-2 cells stimulated with LPS (1 μg/mL) were analyzed by Western blotting (n = 3). C and D Western blotting and immunofluorescence analyses of NLRP3 in HK-2 cells which were stimulated with LPS (1 μg/mL) or vehicle and co-incubated with ACSS2i (10 μmol/L) for 24 or 48 hours (red, NLRP3; blue, DAPI, scale bars, 100 μm; n = 3). E and F Western blotting analyses of the protein expression of (E) NLRP3 or (F) pro-caspase-1, caspase-1, pro-IL-1β, and IL-1β in HK-2 cells which were treated with LPS (1 μg/mL) or vehicle and ACSS2 siRNA (siACSS2) for 24 hours (n = 3). G The expression of IL-1β in mouse kidney was determined by immunofluorescence (green, IL-1β; blue, DAPI; scale bars, 50 μm). Data were presented as mean ± SD. ^*P < 0.05, ^***P < 0.005 vs. control; ^#P < 0.05, ^##P < 0.01 vs. LPS or LPS + siControl

Fig. 4

ACSS2 ablation protects kidney function and ameliorates renal tubular injury in LPS-induced mice. A Schematic diagram of the strategy leading to the LPS-induced sepsis mouse model. B Western blotting analyses of the protein expression levels of ACSS2 in renal cortex of mice (n = 3). C Representative immunofluorescent images of ACSS2 and Lotus Tetragonolobus Lectin (LTL) in the kidney tissues from mice (Scale bars, 200 μm). D and E Representative images of (D) H&E staining and (E) immunofluorescent staining of KIM-1 in the kidneys of mice (Scale bars, 100 μm). F The serum levels of BUN and serum creatinine were measured (n = 5-6). G The expressions of F4/80 (a marker for macrophages) in the kidneys were analyzed (Scale bars, 100 μm). Data were presented as mean ± SD. ^*P < 0.05, ^****P < 0.0001 vs. Ctrl; ^#P < 0.05, ^##P < 0.01, ^###P < 0.005 vs. LPS

Fig. 5

ACSS2 deletion decreases inflammasome activation and GSDMD-mediated cell pyroptosis in renal tubular cells of LPS-induced mice. A Representative transmission electron microscopy (TEM) images of kidney tissues (Scale bars, 1 μm or 0.5 μm). B Representative immunofluorescent staining of cleaved GSDMD and IL-1β in the kidneys (green, LTL; red, IL-1β or cleaved GSDMD; blue, DAPI; Scale bars, 200 μm). C Western blotting analyses of the protein expression levels of NLRP3, IL-1β, and the cleavage of GSDMD in the kidney tissues of the noted mice (n = 3). Data were presented as mean ± SD. ^*P < 0.05, ^***P < 0.005 vs. Ctrl; ^#P < 0.05, ^##P < 0.01 vs. LPS

Fig. 6

ACSS2 induces KLF5-mediated p65 activation and downstream cell pyroptosis in LPS-induced kidney tubular injury. A RNA-seq analysis of kidney tissues from ACSS2 gene knockout (KO) mice and wild-type mice was performed. The colored dots indicated differentially expressed genes (DEGs). B Relative protein expression levels of KLF5 in the mouse kidney (n = 3). C Representative immunofluorescent images of KLF5 and LTL in the renal cortex of mice (green, LTL; red, KLF5; blue, DAPI; Scale bars, 100 μm). D Western blotting was used to compare expression of KLF5 in HK-2 cells treated with LPS (1 μg/mL) or vehicle and with ACSS2 siRNA or scrambled siRNA for 24 hours (n = 3). E Immunofluorescence staining was used to detect KLF5 (green, KLF5; blue, DAPI; scale bars, 50 μm). F and G HK-2 cells were pre-treated with ML264 (10 μmol/L) or vehicle for 1 hour, then with LPS (1 μg/ml) or vehicle for 24 hours. Western blotting was used to analyze (F) the expression of KLF5 or (G) the phosphorylation of p65, the expression of NLRP3, and the cleavage of GSDMD (n = 3). H The levels of cleaved GSDMD were determined by immunofluorescence staining (green, cleaved GSDMD; blue, DAPI; scale bars, 50 μm). Data were presented as mean ± SD. ^*P < 0.05, ^***P < 0.005 vs. Ctrl, siControl or LPS + vehicle; ^#P < 0.05, ^##P < 0.01 vs. LPS + siControl or LPS + vehicle