Enhanced mPGES-1 Contributes to PD-Related Peritoneal Fibrosis via Activation of the NLRP3 Inflammasome

Abstract

Background: Microsomal prostaglandin E synthase-1 (mPGES-1)-derived prostaglandin E₂ (PGE2) is a chief mediator of inflammation. However, the role and mechanism of mPGES-1 in peritoneal dialysis (PD)-associated peritoneal fibrosis have not been investigated. Material and Methods: In PD patients, mPGES-1 expression in peritoneum tissues and the levels of PGE2, IL-1β, and IL-18 in the dialysate were examined. In rat peritoneal mesothelial cells (RPMCs), the regulation and function of mPGES-1 and NLRP3 inflammasome were investigated. The expression of extracellular matrix proteins and the components of NLRP3 inflammasome were detected by Western blotting or real-time quantitative PCR. Results: In PD patients with ultrafiltration failure (UFF), mPGES-1 was enhanced in the peritoneum, which was associated with the degree of peritoneal fibrosis. Accordingly, the intraperitoneal PGE2 levels were also positively related to the PD duration, serum C-reactive protein levels, and serum creatinine levels in incident PD patients. In RPMCs, high-glucose treatment significantly induced mPGES-1 expression and PGE2 secretion without affecting the expressions of mPGES-2 and cPGES. Inhibition of mPGES-1 via short hairpin RNA significantly ameliorated the expression of extracellular matrix proteins of RPMCs induced by high glucose. Additionally, high glucose markedly activated NLRP3 inflammasome in RPMCs that was blunted by mPGES-1 inhibition. Furthermore, silencing NLRP3 with siRNA significantly abrogated the expression of extracellular matrix proteins in RPMCs treated with high glucose. Finally, we observed increased IL-1β and IL-18 levels in the dialysate of incident PD patients, showing a positive correlation with PGE2. Conclusion: These data demonstrate that mPGES-1-derived PGE2 plays a critical role in PD-associated peritoneal fibrosis through activation of the NLRP3 inflammasome. Targeting mPGES-1 may offer a novel strategy to treat peritoneal fibrosis during PD.

Keywords: NLRP3 inflammasome; PGE2; inflammation; mPGES-1; peritoneal fibrosis.

Figure 1

Enhanced expression of mPGES-1 in the peritoneal tissues of PD patients with ultrafiltration failure. (A) Representative HE and Masson staining in peritoneal tissues of inguinal hernia patients with normal renal function and PD patients with UFF. Magnification: 100×. Scale bar: 100 μm. (B) Representative image of immunohistochemical staining of mPGES-1 in the peritoneal tissues of inguinal hernia patients with normal renal function and PD patient with UFF. Analysis of mPGES-1 antibody specificity by replacing mPGES-1 antibody with isotype IgG. Magnification: 400×. Scale bar: 20μm. (C) Correlation between the expression of mPGES-1 in peritoneal tissues and the thickness of the peritoneum (Spearman's rank correlation analysis, n = 21). Symbols of black dot, red square, and blue triangle represented control, ESRD patients, and PD patients with UFF, respectively.

Figure 2

Increased PGE2 secretion during PD therapy. (A) Bivariate correlation analysis between the total secretion of PGE2 and the PD duration in incident PD patients (Spearman's rank correlation analysis, n = 116). (B) Bivariate correlation analysis between the total secretion of PGE2 and serum C-reactive protein levels in incident PD patients (Spearman's rank correlation analysis, n = 116). (C) Bivariate correlation analysis between the total secretion of PGE2 and serum creatinine levels in incident PD patients (Spearman's rank correlation analysis, n = 116). (D) Bivariate correlation analysis between the total secretion of PGE2 and daily residual urine volume levels in incident PD patients (Spearman's rank correlation analysis, n = 116).

Figure 3

Induction of mPGES-1 expression by high glucose in RPMCs. (A) Real-time quantitative PCR analysis of mPGES-1, mPGES-2, and cPGES levels in RPMCs in response to high glucose (138 mmol/L) for the indicated times. (B) RPMCs were incubated with high glucose (138 mmol/L) for the indicated times, and western blotting detected the expression of mPGES-1. (C) Quantitative analysis of mPGES-1 expression in RPMCs treated with high glucose (138 mmol/L) for the indicated times. (D) RPMCs were treated with high glucose (138 mmol/L) for the indicated times, and ELISA was performed for PGE2 in the cell culture medium. The data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

Inhibition of mPGES-1 ameliorates high-glucose-induced synthesis of extracellular matrix proteins in RPMCs. (A) RPMCs were treated with mPGES-1 shRNA plasmid or control shRNA plasmid, followed by incubation with high glucose for 48 h. ELISA of PGE2 in cell culture medium in RPMCs treated with normal glucose, high glucose, high glucose plus shRNA control or high glucose plus mPGES-1 shRNA. (B) RPMCs were transfected with the mPGES-1 shRNA plasmid or control shRNA plasmid, followed by incubation with high glucose for 48 h. Western blotting confirmed the expression of mPGES-1, FN, Collagen-I, and E-cadherin. Quantitative analyses of mPGES-1, FN, Collagen-I, and E-cadherin were displayed in the (C–F). The data are represented as the mean ± SEM. **P < 0.01, ***P < 0.001.

Figure 5

Inhibition of mPGES-1 blunts high-glucose-induced activation of the NLRP3 inflammasome. (A–C) RPMCs were transfected with the mPGES-1 shRNA plasmid or control shRNA plasmid, followed by incubation with high glucose for 24 h. Real-time quantitative PCR analysis was used to detect the mRNA levels of NLRP3 (A), caspase-1 (B), and IL-18 (C). (D–H) RPMCs were transfected with the mPGES-1 shRNA plasmid or control shRNA plasmid, followed by incubation with high glucose for 48 h. Western blotting confirmed the expression of mPGES-1, NLRP3, procaspase-1, and caspase-1. Quantitative analyses of mPGES-1 (E), NLRP3 (F), procaspase-1 (G), and caspase-1 (H) expression were displayed. The data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Inhibition of NLRP3 by siRNA abrogates high-glucose-induced synthesis of extracellular matrix proteins in RBMCs. (A) RBMCs were treated with NLRP3 siRNA or control siRNA, followed by incubation with high glucose for 48 h. Western blotting confirmed the expression of NLRP3, FN, Collagen-I, and E-cadherin. Quantitative analyses of NLRP3, FN, Collagen-I, and E-cadherin were displayed in the (B–E). The data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Correlation between the secretion of PGE2 and IL-1β or IL-18 in PD fluid. (A) Bivariate correlation analysis between the total secretion of PGE2 and IL-1β in incident PD patients' dialysate (Spearman's rank correlation analysis, n = 53). (B) Bivariate correlation analysis between the total secretion of PGE2 and IL-18 in incident PD patients' dialysate (Spearman's rank correlation analysis, n = 53).