Histone induced endothelial dysfunction in septic acute kidney injury via TLR 4 signaling

 

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Histone induced endothelial dysfunction in septic acute kidney injury via TLR 4 signaling

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Yang
Tinghang
Yang Tinghang yfxpko@qq.com West China Hospital Department of Internal Medicine-Nephrology, Department of Nephrology Chengdu China *
Li Yupei liyupei123@wchscu.cn West China Hospital Department of Internal Medicine-Nephrology, Department of Nephrology Chengdu China -
Su Baihai subaihai@scu.edu.cn West China Hospital Department of Internal Medicine-Nephrology, Department of Nephrology Chengdu China -
 
 
 
 
 
 
 
 
 
 
 
 

Building upon growing evidence of the roles of circulating histones and endothelial dysfunction in sepsis, this study investigated the mechanism by which histone-induced endothelial injury contributes to acute kidney injury (AKI) in sepsis, and explored potential therapeutic targets for sepsis-induced AKI.

Human umbilical vein endothelial cells (HUVECs) were treated with purified recombinant human histone H3. The compounds TAK-242, Forskolin, ESI-09, and selumetinib were employed to modulate the activation of TLR4, EPAC1, and MEK, respectively. Cell viability was assessed using flow cytometry and live-dead cell staining. The expression of VE-cadherin was evaluated qualitatively and quantitatively via immunofluorescence and western blot analysis. A murine sepsis model was established through intravenous histone injection. After 24 hours, serum levels of creatinine, urea nitrogen, IL-6, IL-1β, and TNF-α were measured. Western blot analysis was performed to assess the expression of renal endothelial adherens proteins and neutrophil gelatinase-associated lipocalin (NGAL). Kidney tissue pathological changes were examined by hematoxylin-eosin (H&E) staining. Endothelial permeability was quantified by measuring Evans blue dye extravasation in tissues following intravenous injection 30 minutes before sacrifice.

 We demonstrated that histones increase vascular permeability and exacerbate organ dysfunction in sepsis. Mechanistically, histones activate the TLR4-EPAC1-MEK-ERK signaling pathway, leading to VE-cadherin proteolytic cleavage and enhanced endocytosis in endothelial cells. In vivo, inhibition of TLR4 or MEK activation, or enhancement of EPAC1 expression, attenuated vascular permeability, mitigated multiple organ injury, and improved survival outcomes in sepsis.

Figure 1 Histones induce endothelial cells apoptosis and disrupt endothelial barrier disruption in vitro.(A-G,K,N) Human recombinant histone H3 induced HUVEC apoptosis in a dose-dependent manner. (E-J) Histone induced decreased expression of VE-Cadherin in a dose-dependent manner. (L) Histone disrupted the integrity of endothelial barrier in a dose-dependent manner (scale bars = 20 μm; yellow arrows indicated discontinuous VE-Cadherin staining). (M,O-Q) Histone induced decreased expression of cAMP and EPAC 1 in a dose-dependent manner. Data are represented as the mean ± SD. *P ＜ 0.05, ** P ＜0.01, *** P ＜0.001, **** P＜0.0001Figure 2 TLR-4 inhibitor alleviated histone-induced endothelial cells apoptosis and endothelial barrier disruption in vitro. (A-E, J, K) Treatment with TAK-242 successfully ameliorated histone-induced cell apoptosis. (F-H and L) Treatment with TAK-242 significantly conversed histone induced decreased expression of VE-Cadherin and stabilized the integrity of endothelial barrier (scale bars = 58 μm; yellow arrows indicated discontinuous VE-Cadherin staining). (I) Treatment with TAK-242 increased the expression of EPAC 1. Data are represented as the mean ± SD. *P ＜ 0.05, ** P ＜0.01, *** P ＜0.001, **** P＜0.0001.Figure 3 TLR-4 inhibitor significantly improved histone-induced renal dysfunction and organ hyperpermeability. (A)ALT, (B)AST, (C)Scr and (D) BUN levels in different groups of mice. (E-G) IL-6, IL-1β, and TNF-α levels in serum were determined using ELISA kits. (I-L) The protein expression level of VE-Cadherin, NGAL, p-ERK and ERK in the kidneys were analyzed by western blot analysis and quantified by densitometry. (H) Relative levels of renal Evans Blue Dye (EBD) absorbance at 610 nm in sham and septic mice with or without TAK-242 administration. (M) Representative images of HE staining (×200, scale bars = 150 μm; ×400, scale bars = 60 μm). Data are represented as the mean ± SD. *P ＜ 0.05, ** P ＜0.01, *** P ＜0.001, **** P＜0.0001.Figure 4 Upregulation of EPAC 1 alleviated histone-induced endothelial cells apoptosis and endothelial barrier disruption in vitro. (A-F, J) Treatment with Forskolin successfully ameliorated histone-induced cell apoptosis via upregulating the expression of EPAC 1, while inhibiting EPAC 1 significantly exacerbated histone-induced apoptosis. (G-I,K) Treatment with Forskolin significantly conversed histone induced decreased expression of VE-Cadherin and stabilized the integrity of endothelial barrier, whereas EPAC 1 inhibition showed opposing effects (scale bars = 58 μm; yellow arrow indicated discontinuous VE-Cadherin staining). Data are represented as the mean ± SD. *P ＜ 0.05, ** P ＜0.01, *** P ＜0.001, **** P＜0.0001.Figure 5 Upregulation of EPAC 1 significantly improved histone-induced renal dysfunction and organ hyperpermeability. (A)ALT, (B)AST, (C)Scr and (D) BUN levels in different groups of mice. (E-J) The protein expression level of EPAC 1, NGAL and VE-Cadherin in the kidneys were analyzed by western blot analysis and quantified by densitometry. (K) Relative levels of renal Evans Blue Dye (EBD) absorbance at 610 nm in sham and septic mice with or without EPAC 1 upregulation. (L-M) Representative images of HE staining (×200, scale bars = 150 μm; ×400, scale bars = 60 μm). Data are represented as the mean ± SD. *P ＜ 0.05, ** P ＜0.01, *** P ＜0.001, **** P＜0.0001.Figure 6 MEK inhibitor alleviated histone-induced endothelial barrier disruption in vitro. (A-D) Treatment with selumetinib successfully ameliorated histone-induced decreased expression of VE-Cadherin via inhibiting the activation of MEK-ERK. (E) Treatment with selumetinib significantly stabilized the integrity of endothelial barrier (scale bars = 20 μm; yellow arrow indicated discontinuous VE-Cadherin staining). Data are represented as the mean ± SD. *P ＜ 0.05, ** P ＜0.01, *** P ＜0.001, **** P＜0.0001.

Our findings reveal that the metabolic cross-talk between chromatin-derived histones and the endothelium plays a critical role in the pathophysiology of sepsis. Inhibition of the TLR4-EPAC-MEK-ERK signaling pathway confers renal protection in sepsis by attenuating histone-mediated endothelial dysfunction.

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