STING inhibition alleviates experimental peritoneal damage: potential therapeutic relevance for peritoneal dialysis

Abstract

Peritoneal dialysis (PD) is a widely used kidney replacement therapy for patients with end-stage kidney disease. Nevertheless, long-term exposure to PD fluid can damage the peritoneal membrane, leading to ultrafiltration failure and, ultimately, discontinuation of PD. Investigation of the molecular mechanisms underlying this damage is essential for identifying new therapeutic targets to mitigate peritoneal deterioration in PD patients. To this end, we employed RNA sequencing in a preclinical model of peritoneal injury, induced by prolonged chlorhexidine (CHX) exposure, which revealed cytosolic DNA-sensing signaling as a novel pathway. Next, we demonstrated that key players in this pathway, such as the stimulator of interferon genes (STING) and its downstream signaling effectors (interferon regulatory factor 3, interferon-stimulated genes, and nuclear factor-κB signaling), were upregulated in experimental peritoneal damage. Moreover, increased STING expression was observed in human peritoneal biopsies from patients with PD. Subsequent studies in STING-deficient mice showed reduced proinflammatory gene expression and immune cell infiltration, together with inhibited nuclear factor-κB pathway activation at both early (10 days) and late (30 days) stages of CHX-induced peritoneal injury. STING deficiency also reduced peritoneal membrane thickening, fibrosis, and mesothelial-to-mesenchymal transition (MMT)-related changes in advanced CHX-induced damage. Furthermore, pharmacological inhibition of STING with C-176 attenuated CHX-induced peritoneal inflammation. Macrophages were identified as one of the STING-expressing cell types in the injured peritoneum. Hence, in vitro STING blockade in activated macrophages inhibited MMT in cultured mesothelial cells, suggesting that STING activation in this population may drive peritoneal fibrosis. Additionally, STING deficiency reduced peritoneal inflammation in S. epidermidis-induced peritonitis and decreased adhesion scores in a postsurgical intra-abdominal adhesion model. These findings identify STING as a pivotal mediator of peritoneal injury and support its potential as a novel therapeutic target to prevent PD-associated ultrafiltration failure. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: STING signaling; cytosolic DNA‐sensing pathway; fibrosis; inflammation; peritoneal adhesions; peritoneal damage; peritoneal dialysis; peritonitis.

Figure 1

Transcriptomic and functional enrichment analysis in the peritoneum of mice exposed to CHX for 10 days. Sequencing analysis was performed starting from total RNA from parietal peritoneal tissue of male C57BL6/J mice that were i.p. injected with 0.1% CHX for 10 days and control mice (n = 4 per group). (A) The volcano plot shows 12,058 genes with satisfactory statistical analysis. DEGs in the CHX group versus the control group with a q‐value <0.05 are shown in green (increased) and red (decreased). The highest upregulated and downregulated DEGs are shown in cyan and purple, respectively. (B) Heatmap representing row Z‐score and the hierarchical clustering of the 1,050 DEGs with q‐value <0.05 and |Log₂(FC)| ≥ 2. The heatmap was generated using the ClustVis web tool. (C–E) Functional enrichment analysis of (C) cell type, (D) transcription factors, and (E) signaling pathways in upregulated DEGs according to Pattern Gene Database (PaGenBase), Transcriptional Regulatory Relationships Unraveled by Sentence‐based Text mining (TRRUST) database, and KEGG databases, respectively. Up: Upregulated DEGs. Down: downregulated DEGs. NS: Nonsignificant.

Figure 2

Expression and localization of STING and its downstream signaling mediators in injured peritoneal tissue from mice and patients. (A–E) Male C57BL/6J mice were daily i.p. injected with 0.1% CHX for 30 days. (A and B) Protein levels of STING, total and phosphorylated TBK1 (p‐TBK1), and total and phosphorylated IRF3 (p‐IRF3) in the peritoneum of 30‐day‐CHX‐exposed mice. Protein levels were assessed by western blotting from total proteins of parietal peritoneal tissue, using α‐tubulin or GAPDH as loading control. (C) Relative expression levels of ISGs were analyzed by RT‐qPCR from total RNA of parietal peritoneal tissue, using Gapdh as a housekeeping gene. Protein and gene results are expressed as n‐fold compared to control and represented as the mean ± SEM of five to eight animals per group. *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Immunohistochemistry microscopy images showing STING⁺ cells within infiltrated and thickened areas of peritoneal membrane. (E) Immunofluorescence staining of STING (red) and F4/80 (green) shows presence of double‐labeled F4/80⁺STING⁺ (merged) macrophages in peritoneum of 30‐day‐CHX‐exposed mice. (F and G) Immunohistochemical staining of STING sections of parietal peritoneal tissue from 30‐day‐PDF‐exposed mice (F) and 60‐day‐PDF‐exposed 5/6 nephrectomized mice (G). Microscopy images in panels (D–G) correspond to a representative animal from each group. Higher‐magnification images of squared areas are presented alongside original micrographs. Nx: nephrectomy. (H) Immunohistochemical staining of STING in peritoneal biopsies from a control ESKD and an ESKD‐PD patient (Patient C1 and Patient PD1, respectively; see supplementary material, Table S1). Red arrows indicate STING⁺ stain in endothelium. Yellow arrows indicate STING⁺ stain in submesothelial zone. Scale bars, 50 μm for all micrographs.

Figure 3

Peritoneal damage in WT and STING‐KO mice exposed to CHX for 10 days. Male C57BL/6J WT and STING‐deficient (STING‐KO) mice were i.p. injected with 0.1% CHX for 10 days. (A) Peritoneal macrophage infiltrates and peritoneal membrane thickness in CHX‐exposed mice. Upper panel: F4/80 immunohistochemistry images from a representative animal from each group (left) and the corresponding quantification of F4/80⁺ cells (right). Bottom panel: representative Masson's trichrome stained images (left) and corresponding quantification of submesothelial zone thickness (right). Yellow lines indicate width measured. Scale bar, 50 μm. Results are represented as mean ± SEM of four to eight animals per group. (B) Heatmap representing row Z‐score of relative expression levels of inflammatory and MMT/fibrosis markers. Relative gene expression was analyzed by RT‐qPCR from total RNA of parietal peritoneal tissue, using Gapdh as a housekeeping gene. *p < 0.05, **p < 0.01, and ****p < 0.0001. A.U.: arbitrary unit

Figure 4

Peritoneal membrane thickening, fibrosis, and inflammation in WT and STING‐KO mice exposed to CHX for 30 days. Male C57BL/6J WT and STING‐deficient (STING‐KO) mice were i.p. injected with 0.1% CHX for 30 days. (A) Masson's trichrome staining in parietal peritoneal tissue. Yellow lines in Masson's trichrome images indicate width measured. (B) Heatmap representing row Z‐score of relative expression levels of mesothelial marker calretinin (Calb2) and several MMT/fibrosis and angiogenesis markers. (C) Fibronectin protein levels in parietal peritoneum of mice (D) α‐SMA immunostaining of parietal peritoneal tissue from mice. (E) Flow cytometry analysis in peritoneal lavages from mice to identify cells present in peritoneal cavity. Peritoneal cells were labeled with specific antibodies tagged with different fluorophores to identify CD3⁺, CD4⁺, CD8⁺, CD11b⁺, Ly6G⁺, and F4/80⁺ cells. Cytometry results are represented in scattered box plots with min‐to‐max whiskers, and the median and quartiles are shown. (F) Heatmap representing row Z‐score of relative expression levels of inflammatory markers. (G) Protein levels of phosphorylated p65 subunit of NF‐κB (p‐p65 NF‐κB) and phosphorylated IκBα (p‐IκBα) in parietal peritoneum of mice. (H) Immunohistochemical detection of phosphorylated (p)‐p65 NF‐κB protein in parietal peritoneal tissue sections. Yellow arrows indicate p‐p65⁺ nuclei. Microscopy images in panels (A), (D), and (H) correspond to a representative animal from each group. Higher‐magnification images of the squared areas in panels (D) and (H) are presented alongside the original micrographs. Scale bars, 50 μm for all micrographs. Relative gene expression was analyzed by RT‐qPCR from total RNA of parietal peritoneal tissue, using Gapdh as a housekeeping gene. Protein levels were assessed by western blotting from total proteins of parietal peritoneal tissue, using GAPDH as loading control. Western blotting results are expressed as fold change (n‐fold) relative to control group. Results are represented as mean ± SEM of five to nine animals per group. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5

In vivo and in vitro effects of STING pharmacological inhibition. (A–C) Male C57BL/6J WT mice were i.p. injected with 0.1% CHX together with pharmacological STING inhibitor C‐176 or its vehicle (Veh, corn oil) for 10 days. Untreated and vehicle‐treated mice were used as controls. (A) Peritoneal membrane thickness (upper panel) and immunohistochemical staining of F4/80 (bottom panel). Microscopy images correspond to a representative animal from each group. Yellow lines indicate thickness measured in Masson's trichrome images. Scale bar, 50 μm. (B) Graphs showing corresponding quantification of submesothelial zone thickness (left) and F4/80⁺ cells (right). Results are represented as mean ± SEM of four to seven animals per group. *p < 0.05, **p < 0.01, ****p < 0.0001 compared to control group. ++++p < 0.0001 compared to CHX group. ###p < 0.001, ####p < 0.0001 compared to CHX + Veh group. (C) Heatmap representing row Z‐scores of relative expression levels of inflammatory and MMT/fibrosis markers. Relative gene expression was analyzed by RT‐qPCR from total RNA of parietal peritoneal tissue using Gapdh as housekeeping gene. (D) Schedule of in vitro study design for assessment of macrophage‐mesothelial cell interaction. Created using elements from Servier Medical Art (Servier; https://smart.servier.com/). (E) Relative gene expression of MMT and fibrosis markers in MeT‐5A cells incubated for 48 h with pooled conditioned medium from LPS‐activated macrophages (AMCM) with or without C‐176 treatment. Relative gene expression levels were analyzed by RT‐qPCR from total RNA using GAPDH as housekeeping gene, expressed as fold‐change (n‐fold) relative to control condition (first column) and represented as mean ± SEM of three to five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. A.U.: arbitrary unit.

Figure 6

Effect of STING deficiency on peritoneal adhesion formation and peritonitis development in mice. (A–C) For peritoneal adhesion model, three IBs were surgically made in peritoneum of male C57BL/6J WT and STING‐deficient (STING‐KO) mice. Adhesion formation on IBs was assessed 5 days after surgery. (A) Macroscopic and microscopic analyses of adhesions formed on IBs surgically made in mice. Yellow arrows in top panel indicate IB to which correspond lower Masson's trichrome micrographs (scale bar, 200 μm). Microscopy images correspond to a representative animal from each group. (B) Peritoneal adhesion grade, tenacity, and total score values are represented in scattered box plots with min‐to‐max whiskers, and the median and quartiles are shown for each experimental group (n = 5–6 mice per group, 3 IBs/mouse). (C) Heatmap representing row Z‐scores of relative expression levels of inflammatory markers. (D–F) For peritonitis model, female C57BL/6J WT and STING‐deficient (STING‐KO) mice were injected with a single dose of live S. epidermidis bacteria (S. epi, 5 × 10⁸ cfu/mouse) and evaluated 72 h after injection. (D) Peritoneal membrane thickness assessment. The figure shows Masson's trichrome‐stained parietal peritoneal tissue sections of representative mice from each group (left; scale bar, 50 μm) and the corresponding quantification of submesothelial zone thickness (right). Yellow lines indicate width measured. (E) Heatmap representing row Z‐scores of relative expression levels of inflammatory markers. (F) Protein levels of phosphorylated IκBα (p‐IκBα) were assessed by western blotting from total proteins of parietal peritoneum using GAPDH as loading control. Protein‐level results are expressed as fold‐change (n‐fold) relative to control group. Relative gene expression was analyzed by RT‐qPCR from total RNA of parietal peritoneal tissue, using Ppia as a housekeeping gene. Results are represented as mean ± SEM of three to six animals per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. A.U.: Arbitrary unit.