Gut microbiota dysbiosis promotes chronic kidney disease-associated atrial fibrillation through activation of the NLRP3 inflammasome

Abstract

Aims

Chronic kidney disease (CKD) significantly increases the risk of atrial fibrillation (AF). Although alterations in the gut microbiota have been linked to CKD progression, its exact involvement in CKD-associated AF remains unclear. We aim to investigate the role of gut microbiota in the development of CKD-associated AF and to uncover potential mechanisms that could serve as effective targets for prevention and treatment.

Methods and results

A rat model of CKD was induced by an adenine-enriched diet. 16S rRNA sequencing and faecal microbiota transplantation (FMT) were utilized to study the involvement of gut microbiota. AST-120, gut barrier protectants, and mono-colonization experiments were performed to investigate potential mechanism. CKD rats exhibited gut microbiota dysbiosis and a significantly increased susceptibility to AF. FMT from CKD rats transferred this heightened AF susceptibility to healthy recipient rats, linked to the activation of the NLRP3 inflammasome. Mechanistically, gut dysbiosis in CKD patients leads to elevated indoxyl sulphate (IS) levels, causing gut barrier dysfunction and increased circulating lipopolysaccharide (LPS). Elevated LPS activates atrial Toll-like receptor 4 (TLR4) receptors, triggering NLRP3 inflammasome activation, which contributes to AF pathogenesis. Treatment with the IS scavenger AST-120 or gut barrier protectants successfully prevented CKD-associated AF. Furthermore, supplementation with Lactobacillus gasseri reduced circulating IS levels and mitigated AF susceptibility in CKD rats.

Conclusion

This study demonstrates that gut dysbiosis-driven elevation of IS and subsequent activation of the atrial NLRP3 inflammasome are key mechanisms in CKD-associated AF. Modulating the gut microbiota could provide a new therapeutic strategy for CKD-associated AF.

Graphical Abstract

Graphical Abstract.

Gut microbiota dysbiosis promotes chronic kidney disease-associated atrial fibrillation through activation of the NLRP3 inflammasome

In chronic kidney disease (CKD), gut dysbiosis in CKD leads to elevated levels of microbiota-derived indoxyl sulphate (IS), which compromises the intestinal barrier, facilitating the translocation of lipopolysaccharides (LPS) into the bloodstream. Circulating LPS may activate the Toll-like receptor 4 (TLR4) receptor, triggering the NLRP3 inflammasome. This cascade promotes the production of pro-inflammatory cytokines IL-1β and IL-18, driving atrial structural remodelling and increasing susceptibility to atrial fibrillation (AF).

Introduction

Atrial fibrillation is the most prevalent persistent cardiac arrhythmia, with rising incidence and prevalence,^1,2 posing significant clinical and public health challenges.^3,4 Individuals with CKD have a threefold higher risk of developing AF compared with those without CKD.⁵ Once AF develops in CKD patients, the risk of complications, including stroke, heart failure, and mortality, escalates by two to five times.⁶ Despite its prevalence and clinical burden, the mechanisms underlying CKD-associated AF are not yet fully understood.

CKD is linked to significant changes in the gut microbiota profile and its associated metabolites.⁷ Disruptions in gut microbiota are pivotal in the onset and advancement of CKD, closely correlating with disease severity.^8,9 Recent studies have demonstrated that microbiome modulation following acute kidney injury (AKI) can accelerate functional recovery, reduce kidney fibrosis, and mitigate the progression from AKI to CKD.^10,11 Moreover, disturbances in gut microbiota have been linked to various adverse complications of CKD. For example, Prevotella copri has been shown to induce vascular calcification in CKD rats via the LPS–NF–κB signalling pathway.¹² Our prior work suggests that gut dysbiosis contributes to the development of age-related and cold-related AF.^13,14 However, the specific role and mechanisms of gut dysbiosis in CKD-associated AF are still not fully elucidated.

Recent studies have highlighted significant increased expression of the NLRP3 inflammasome in kidney diseases.^15,16 The NLRP3 inflammasome, which belongs to the NLR protein family of cytosolic receptors, is activated by both pathogen-associated molecular patterns and damage-associated molecular patterns, triggering inflammatory responses via activation of caspase-1, leading to the release of IL-1β and IL-18.¹⁷ Our previous work has identified NLRP3 inflammasome activation as an important mechanism in the remodelling of atrial structure and the development of AF.¹⁸ Emerging evidence suggests that the activation of the NLRP3 inflammasome is crucial in the development of CKD-associated AF. Nevertheless, the precise mechanisms underlying NLRP3 inflammasome activation in this context remain elusive and merit further investigation. We found that transplanting gut microbiota from CKD rats to healthy recipient reproduced the increased AF susceptibility.

The aim of this study was to investigate the role of gut microbiota dysbiosis in the development of CKD-associated AF. Specifically, we sought to identify key mechanisms by which microbiota-derived uraemic toxins, including IS, disrupt gut barrier integrity and activate inflammatory pathways such as the NLRP3 inflammasome, promoting atrial structural remodelling and increasing AF susceptibility. Furthermore, we assessed whether Lactobacillus gasseri supplementation could mitigate these microbiota-driven changes and reduce CKD-associated AF.

Materials and methods

Human subjects

This study was reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (Approval No. IRB-AF/SC-12/03.0) and adhered to the ethical guidelines set forth in the Declaration of Helsinki. All subjects signed an informed consent form prior to participating in the research. Faecal and plasma samples were collected cross-sectionally from patients hospitalized at the First Affiliated Hospital of Harbin Medical University. All participants had an established diagnosis of CKD. The CKD + AF group consisted of 30 patients with CKD who had subsequently developed AF, with medical records confirming that AF onset occurred after the initial diagnosis of CKD. The CKD group included 30 patients with CKD without a history of AF.

Participants were excluded based on the following criteria: (i) use of antibiotics or probiotic supplements within the preceding 3 months; (ii) presence of malignant tumours, inflammatory bowel disease, or congestive heart failure; (iii) occurrence of gastrointestinal symptoms such as vomiting, diarrhoea, or atypical constipation within the past week; or (iv) significant dietary changes during the same period.

Experimental animals

All animal procedures were carried out following the institutional guidelines for laboratory animal welfare and received ethical approval from the Animal Ethics Committee of Harbin Medical University (Approval No. 2023112). The experimental protocol adhered to the Directive 2010/63/EU of the European Parliament on animal welfare in research and the current standards outlined by the US National Institutes of Health, and the study was designed and reported in accordance with the ARRIVE 2.0 guidelines for animal experiments. To ensure experimental uniformity and limit biological variability in the mechanistic studies, only male rats were used. Male Sprague–Dawley (8 weeks old) rats were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were housed under pathogen-free (SPF) conditions, with a consistent 12-hour light/dark cycle, and were provided with free access to standard food and water.

Statistical analysis

All statistical evaluations were conducted using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA). Continuous data are presented as mean ± standard error of the mean (SEM) or as median with interquartile range, depending on data distribution. Categorical data are summarized as frequencies and corresponding percentages. To assess normality, the Shapiro–Wilk test was applied. Comparisons between two independent groups were performed using an unpaired Student’s t-test for normally distributed variables or the Wilcoxon rank-sum test (also known as Mann–Whitney U test) for nonnormally distributed data. A P-value less than 0.05 was considered statistically significant.

Additional methodological details

Detailed descriptions of the experimental methodology not described here are provided in the Supplementary materials.

Results

Gut microbiota of CKD rats confers pro-AF properties

We successfully established a CKD rat model using an adenine diet (Figure 1A), as evidenced by elevated blood urea nitrogen (BUN) and creatinine levels together with characteristic renal morphological and histopathological changes (see Supplementary material online, Figures S1A–G). Compared with the control rats, CKD rats exhibited a significant increase in AF induction rate and a significantly prolonged AF duration (see Supplementary material online, Figure S2A, Figures 1B and C). Nonetheless, the atrial effective refractory period (AERP) showed no notable variation between groups (Figure 1D). Histologically, CKD rats displayed significant atrial fibrosis and apoptosis, as evidenced by increased collagen deposition and higher rates of apoptotic cell (Figure 1E and F). Consistently, fibrosis markers including TGF-β1 and α-SMA were upregulated, the antiapoptotic protein Bcl2 was downregulated, and the pro-apoptotic protein BAX was upregulated in atrial tissues of CKD rats (Figure 1G and H). The results suggest that CKD rats show a higher vulnerability to AF.

Figure 1.

Microbial transplantation of chronic kidney rats confers pro-AF properties. (A) Schematic of the experimental process. Rats were randomly divided into two groups: One group received adenine-containing diet (CKD group), and another group received standard diet (Control group) for 8 weeks. (B) AF induction of rats in Control group and CKD group (n = 10 per group). (C) AF duration of rats in Control group and CKD group (n = 10 per group). (D) Atrial effective refractory period (AERP) of rats from Control group and CKD group (n = 10 per group). (E) Representative Masson’s trichrome staining and quantified collagen deposition in Control and CKD groups (n = 5 per group). Magnification × 200. (F) Representative Tunel staining in atria of rats, and the ratio of TUNEL positive cells in the atria of rats from Control and CKD groups (n = 5 per group). Magnification × 200. (G) Representative Western blots and quantification of protein levels of TGF-β1 and α-SMA in atrial tissues of rats from Control and CKD groups (n = 6 per group). (H) Representative Western blots and quantification of protein levels of Bcl2 and BAX in atrial tissues of rats from Control and CKD groups (n = 6 per group). (I) Schematic diagram of the experimental design for microbial transplantation experiments. Before microbial transplantation, SPF rats were treated with antibiotic cocktails composed of vancomycin (100 mg/kg), ampicillin (200 mg/kg), neomycin (200 mg/kg), and metronidazole (200 mg/kg) through gavage for 1 week before FMT for exhaustion of microbiota. Then, microbiota-depleted recipient rats were randomly assigned to receive either FMT from healthy control rats (HC-FMT) or FMT from CKD rats (CKD-FMT), three times a week for 8 weeks. (J) Proportion of HC–FMT and CKD–FMT rats exhibiting reproducibly inducible AF following programmed burst pacing (n = 10 per group). (K) AF duration in HC–FMT group and CKD–FMT group (n = 10 per group). (L) Atrial effective refractory period of rats (n = 10 per group). (M) Representative Masson’s trichrome staining and quantified collagen deposition in HC–FMT and CKD–FMT groups (n = 5 per group). Magnification × 200. (N) Representative Tunel staining in atria of rats and the ratio of TUNEL positive cells in HC–FMT and CKD–FMT groups (n = 5 per group). Magnification × 200. (O) Representative Western blots and quantification graphs depicting TGF-β1 and α-SMA expression in atrial tissues (n = 6 per group). (P) Representative Western blots and quantification graphs depicting Bcl2 and BAX expression in atrial tissues (n = 6 per group). Data are expressed as mean ± SEM and compared by Student’s t test (D, E, F, G, H, O, and P) or Wilcoxon test (C and K). AF inducibility (B and J) was presented as numbers and compared by Fisher exact test. AF, atrial fibrillation; SR, sinus rhythm; HC, healthy control.

Disruption of gut microbiota has been linked to CKD and is strongly correlated with its progression and complications.^19–21 To better understand the role of gut microbiota in CKD-associated AF, we performed 16S rRNA sequencing of faecal samples from control and CKD rats. The analysis showed that while α-diversity did not differ significantly between the groups, principal coordinate analysis (PCoA) revealed a clear divergence in the overall microbial community structure (see Supplementary material online, Figures S3A–C). Based on these findings, we designed a faecal microbiota transplantation (FMT) experiment using two distinct donor sources. Rats in the CKD-FMT group received faecal microbiota from CKD donors, whereas rats in the HC-FMT group received faecal microbiota collected from healthy control rats (Figure  1I). Following transplantation, CKD-FMT rats exhibited aggravated renal injury, manifested by increased BUN and creatinine levels, as well as enhanced renal fibrosis, compared with HC-FMT recipients (see Supplementary material online, Figures S1H–N). Notably, recipients of CKD-derived faecal microbiota exhibited significantly increased susceptibility to AF and prolonged AF duration compared with HC-FMT recipients, without alterations in the AERP (see Supplementary material online, Figure S2B, Figures 1J–L). Histological analysis revealed that FMT with CKD microbiota markedly elevated atrial fibrosis and apoptosis, demonstrated by increased collagen deposition and a higher percentage of apoptotic cells (Figures 1M and N). Molecular assays showed a significant upregulation of TGF-β1 and α-SMA (Figure 1C). Additionally, CKD microbiota recipients exhibited increased expression of BAX and reduced expression of Bcl2 (Figure 1P). Collectively, these data demonstrate that the gut microbiota from CKD rats promotes pro-AF properties, suggesting that gut microbiota imbalance significantly contributes to the development of CKD-associated AF.

Gut microbiota dysbiosis promotes atrial fibrillation by activating atrial NLRP3 inflammasome

Recent studies have identified NLRP3 inflammasome activation as a key mechanism in CKD-associated AF.¹⁷ We found that the expression levels of pro-caspase-1 and cleaved caspase-1 (Casp1-p20) were significantly elevated in the atrial of CKD rats compared with controls (Figures 2A and B), indicating NLRP3 inflammasome activation. Notably, transplantation of CKD-derived microbiota significantly activated the NLRP3 inflammasome pathway in the atria, as indicated by increased expression of pro-caspase-1 and Casp1-p20 (Figures 2A and B). Furthermore, the level of the macrophage marker CD68 was increased in atrial tissue from CKD rats and CKD-FMT rats (see Supplementary material online, Figures S4A–D). Additionally, levels of pro-inflammatory cytokines IL-1β and IL-18 were higher in the atrial tissue of CKD microbiota recipients (Figure 2C–E).

Figure 2.

Chronic kidney disease-related gut microbiota dysbiosis promotes the occurrence of atrial fibrillation through activating the atrial NLRP3 inflammasome. (A) Representative Western blots of NLRP3, ASC, pro-cas1, and Cas1-p20 expressions in atrial tissues. (B) Quantification graphs depicting NLRP3, ASC, pro-cas1, and Cas1-p20 expressions in atrial tissues (n = 6 per group). (C) Immunohistochemical staining of IL-1β in HC–FMT group and CKD–FMT group. Magnification × 200. (D) Immunohistochemical staining of IL-18 in HC–FMT group and CKD–FMT group. Magnification × 200. (E) Quantification of IL-1β levels and IL-18 levels in Auto-FMT group and CKD-FMT group (n = 5 per group). (F) Experimental workflow for evaluating MCC950: Schematic overview of faecal microbiota transplantation from CKD donor rats followed by chronic MCC950 (NLRP3 inflammasome inhibitor) or vehicle administration at 15 mg/kg once per week for 8 weeks. (G) AF induction of rats from vehicle group and MCC950 group (n = 10 per group). (H) Duration of AF in vehicle group and MCC950 group (n = 10 per group). (I) Atrial effective refractory period of rats (n = 10 per group). (J) Representative Masson’s trichrome staining and quantified collagen deposition in vehicle and MCC950 groups (n = 5 per group). Magnification × 200. (K) Representative Tunel staining and the ratio of TUNEL positive cells in vehicle and MCC950 groups (n = 5 per group). Magnification × 200. (L) Representative Western blots and quantification graphs depicting TGF-β1 and α-SMA expressions (n = 6 per group). (M) Representative Western blots and quantification graphs depicting Bcl2 and BAX expressions (n = 6 per group). Data are expressed as mean ± SEM and compared by Student’s t test (B, E, I, J, K, L, and M) or Wilcoxon test (H). AF inducibility (G) was presented as numbers and compared by Fisher exact test.

To gain deeper insights into the involvement of NLRP3 in CKD-associated AF, we performed FMT using faecal samples from CKD rats. In this experiment, one group of recipient rats was treated with the NLRP3-specific inhibitor MCC950, while a control group received a vehicle treatment (Figure 2F). MCC950 treatment significantly reduced AF susceptibility and shortened AF duration in CKD microbiota recipients (see Supplementary material online, Figure S5A, Figures 2G–2I). Importantly, MCC950 treatment also mitigated atrial fibrosis, apoptosis, and pro-inflammatory cytokine levels (Figures 2J–M, Supplementary material online, Figures S5B and C). These results suggest that gut microbiota dysbiosis in CKD rats contributes to AF by triggering the NLRP3 inflammasome pathway.

Gut dysbiosis and a potential atrial LPS–TLR4–NLRP3 inflammasome axis

Lipopolysaccharide, a well-established gut-derived metabolite, can interact with TLR4 and has been reported to trigger NLRP3 inflammasome activation.^12,22 In our study, rats that received faecal transplantation from CKD donors exhibited elevated circulating LPS levels, whereas no significant changes in LPS levels were observed in faecal samples (Figures 3A and 3B). Furthermore, CKD-FMT rats had higher atrial TLR4 expression (Figure 3C).

Figure 3.

Gut dysbiosis activates atrial NLRP3 inflammasome via LPS–TLR4 signalling. (A) The levels of LPS in plasma of rats from HC–FMT and CKD–FMT groups (n = 10 per group). (B) The levels of LPS in faecal sample of rats from HC-FMT and CKD-FMT groups (n = 10 per group). (C) Representative Western blots and quantification graphs depicting TLR4 expression in HC–FMT and CKD–FMT groups (n = 6 per group). (D) The examples of HE staining of proximal colon and quantification of the colonic villi length rats from HC–FMT and CKD–FMT groups (n = 5 per group). Magnification × 200. (E) Representative Masson’s trichrome staining and quantified collagen deposition in HC–FMT and CKD–FMT groups (n = 5 per group). Magnification × 200. (F) Representative Western blots and quantification of protein levels of ZO-1, OCC, and Clud4 in colon tissues of rats from HC–FMT and CKD–FMT groups (n = 6 per group). (G) The levels of LPS in plasma of rats from Control and CKD groups (n = 10 per group). (H) The levels of LPS in faecal sample of rats from Control and CKD groups (n = 10 per group). (I) The examples of HE staining and quantification of the colonic villi length of proximal colon in rats from Control and CKD groups. Magnification × 200. (J) The examples of Masson staining and quantified collagen volume fraction in rats from Control and CKD groups. Magnification × 200. (K) Representative Western blots and quantification of ZO-1, OCC, and Clud4 expression in colon tissues of rats from Control and CKD groups (n = 6 per group). (L) Representative Western blots and quantification graphs depicting TLR4 expression in Control and CKD groups (n = 6 per group). Data are expressed as mean ± SEM and compared by Student’s t test (A-L).

Intestinal barrier dysfunction is a key mechanism driving the increase in circulating levels of LPS. Notably, FMT from CKD rats exacerbated gut barrier damage in healthy recipients, as evidenced by shortened intestinal villi and increased intestinal collagen deposition (Figures 3D and E), accompanied by significant reductions in the colonic expression of tight junction proteins such as Zonula Occludens-1 (ZO-1), Occludin (OCC), and Claudin-4 (Clud4) (Figure 3F). Similarly, CKD rats exhibited elevated LPS and TLR4 expression, accompanied by evidence of impaired intestinal barrier function (Figures 3G–L). Taken together, although we did not directly assess the requirement of TLR4 for inflammasome activation, these observations support a plausible model in which microbiota dysbiosis and impaired intestinal barrier integrity may contribute to atrial NLRP3 inflammasome activation, possibly through LPS–TLR4 signalling.

Gut barrier dysfunction is responsible for elevated lipopolysaccharide and increased AF susceptibility

To determine whether microbiota dysbiosis-induced intestinal barrier damage is responsible for AF susceptibility, we administered the intestinal barrier protectant glutamine to recipient rats subjected to FMT from CKD rats (Figure 4A). Glutamine supplementation significantly increased the expression of ZO-1, OCC, and Clud4 in intestinal tissues (Figure 4B). Circulating LPS levels were significantly reduced in glutamine-treated rats (Figure 4C). Consistently, glutamine supplementation enhanced villus length and reduced intestinal fibrosis compared with control rats (Figures 4D and E). Notably, glutamine treatment markedly reduced AF susceptibility, as supported by a decrease in both AF induction and AF duration (see Supplementary material online, Figures S6A and B, Figures 4F and G). Glutamine treatment suppressed the atrial expression of both pro-caspase-1 and its cleaved product, Casp1-p20 (Figure  4H), accompanied by a decrease in IL-1β and IL-18 levels (Figure 4I and J). Furthermore, glutamine supplementation significantly alleviated atrial structural remodelling, as demonstrated by reduced collagen deposition and a lower percentage of apoptotic cells in the atrial tissue (Figure 4K and 4L). A concurrent reduction in TGF-β1 and α-SMA was observed, along with enhanced antiapoptotic signalling, as indicated by increased Bcl-2 expression and decreased BAX levels (Figure 4M and 4N). These results highlight that gut barrier dysfunction is responsible for elevated LPS and increased AF susceptibility.

Figure 4.

Glutamine alleviates intestinal barrier dysfunction and prevents atrial fibrillation in CKD rats. (A) Experimental workflow for glutamine intervention: rats were treated with antibiotics for 1 week, followed by faecal microbiota transplantation from CKD rats. One group received a control vehicle, while the other group was administered glutamine for 8 weeks. Glutamine was dissolved in water, administered continuously throughout the intervention period. (B) Representative Western blots and quantification of protein levels of ZO-1, OCC, and Clud4 in colon tissues of rats from Vehicle and Glutamine groups (n = 6 per group). (C) The levels of LPS in plasma of rats (n = 10 per group). (D) The examples of HE staining and quantification of the colonic villi length of proximal colon in rats from Vehicle and Glutamine groups (n = 5 per group). (E) The examples of Masson staining and collagen volume fraction of proximal colon in rats from Vehicle and Glutamine groups (n = 5 per group). (F) Atrial fibrillation induction in Vehicle and Glutamine groups (n = 10 per group). (G) Atrial fibrillation duration of rats in Vehicle and Glutamine groups (n = 10 per group). (H) Representative Western blots and quantification graphs depicting NLRP3, ASC, pro-cas1, and Cas1-p20 expressions (n = 6 per group). (I–J) Immunohistochemical staining and quantitation of IL-1β (I) and IL-18 (J) in atrial tissues of rats from Vehicle and Glutamine groups. (K) Representative Masson’s trichrome staining and quantified collagen deposition in Vehicle and Glutamine groups (n = 5 per group). Magnification × 200. (L) Representative Tunel staining and frequency of Tunel-positive cells in Vehicle and Glutamine Groups (n = 5 per group). (M) Representative bands and quantification showing TGF-β1 and α-SMA expressions (n = 6 per group). (N) Representative bands and quantification showing Bcl2 and BAX expressions (n = 6 per group). Data are expressed as mean ± SEM and compared by Student’s t test (B, C, D, E, H, I, J, K, L, M, and N) or Wilcoxon test (G). Atrial fibrillation inducibility (F) was presented as numbers and compared by Fisher exact test.

Indoxyl sulphate induces gut barrier dysfunction

CKD-related microbiota dysbiosis leads to an increase in microbiota-derived uraemic toxins, which are key contributors to intestinal barrier damage. Plasma samples from CKD individuals, with or without AF, were analysed for several microbiota-derived toxins, including IS, p-Cresyl sulphate (PCS), and trimethylamine-N-oxide (TMAO) through ELISA. Strikingly, circulating IS levels were significantly increased in CKD patients with AF compared with those without AF, while no significant variation in PCS or TMAO levels was observed (Figure  5A). Consistent with this, circulating levels of IS were significantly elevated in both CKD rats and those receiving CKD-FMT (see Supplementary material online, Figures S7A and B). To investigate whether IS directly induces intestinal barrier dysfunction, human colonic epithelial cells (Caco-2) were treated with IS, resulting in a marked decrease in the expression of tight junction proteins (Figures  5B and C). Next, to determine whether IS is a key mediator of intestinal barrier damage and CKD-associated AF, rats receiving CKD-derived FMT were treated with AST-120, an oral adsorbent of IS (Figure  5D). Rats that received AST-120 exhibited significant decreased level of IS (Figure  5E) and increased expression of ZO-1, OCC, and CL4 in colonic tissues (Figure 5F). Notably, AST-120 treatment significantly decreased AF susceptibility, as evidenced by lower AF induction rate and shortened AF duration (Figure  5G and H, Supplementary material online, Figures S8A and B). Moreover, AST-120 treatment attenuated atrial pro-caspase-1 and Cas1-p20 expression, along with lower levels of IL-1β and IL-18 (Figure 5I–K). Histological analysis revealed a decrease in atrial collagen deposition and a lower percentage of apoptotic cells (Figure 5L and M). Collectively, the data indicate that elevated IS levels are intimately involved in intestinal barrier impairment and CKD-associated AF, likely through the activation of the LPS–TLR4–NLRP3 axis.

Figure 5.

Indoxyl sulphate induces gut barrier dysfunction, and elimination of indoxyl sulphate prevents the development of AF in CKD rats. (A) The level of indoxyl sulphate, PCS, and TMAO in CKD patients and CKD patients with atrial fibrillation. (B) Schematic illustration of the experiment: Caco2 cells were treated with control solvent or indoxyl sulphate for 24 h. (C) Representative Western blots and quantification of ZO-1, OCC, and Clud4 expression in Caco2 cells (n = 6 per group). (D) Schematic illustration of the experimental design for AST-120 intervention: rats were treated with antibiotics for one week, followed by faecal microbiota transplantation from CKD rats. Rats were gavaged with AST-120 (40 mg/day) or its vehicle (PBS) for 8 weeks. (E) The level of indoxyl sulphate in rats from Vehicle group and AST-120 group (n = 10 per group). (F) Representative western blots and quantification of ZO-1, OCC, and Clud4 in Vehicle and AST-120 groups (n = 6 per group). (G) Atrial fibrillation induction of rats from Vehicle and Glutamine groups (n = 10 per group). (H) Atrial fibrillation duration of rats in Vehicle and Glutamine groups (n = 10 per group). (I) Representative bands and quantification of NLRP3, ASC, pro-cas1, and Cas1-p20 in atrial tissue of rats from Vehicle and AST-120 groups (n = 6 per group). (J and K) Immunohistochemical staining and quantitation of IL-1β (J) and IL-18 (K) in atrial tissues of rats from Vehicle and AST-120 groups (n = 6 per group). (L) Representative Masson’s trichrome staining and collagen volume fraction in Vehicle and AST-120 groups (n = 5 per group). Magnification × 200. (M) Representative Tunel staining and the ratio of Tunel-positive cells in Vehicle and AST-120 groups (n = 5 per group). Magnification × 200. Data are expressed as mean ± SEM and compared by Student’s t test (A, B, E, F, I, J, K, L, and M) or Wilcoxon test (H). Atrial fibrillation inducibility (G) was presented as numbers and compared by Fisher exact test.

The production of indoxyl sulphate in gut microbiota is enhanced in chronic kidney disease patients with AF

Indoxyl sulphate is a metabolic byproduct of tryptophan, produced by intestinal microbiota via the enzyme tryptophanase (TnaA). To identify the key gut bacteria responsible for TnaA expression, we comprehensively assessed TnaA distribution in human gut microbes by profiling TnaA-encoding genes in 1520 gut-bacterial reference genomes.²³ Notably, we observed that TnaA was predominantly expressed in specific bacteria, including Bacteroides, Butyricimonas, and Escherichia (Figure 6A). We analysed faecal samples from CKD individuals, both with and without AF, to examine the expression of TnaA (Figure 6B). Strikingly, TnaA levels were significantly higher in CKD patients with AF than those without AF. Furthermore, TnaA levels were also increased in CKD rat and CKD-FMT rats compared with their controls (see Supplementary material online, Figures S9A and B). This was accompanied by an increased abundance of Bacteroides and Escherichia, both key producers of IS. In contrast, the abundance of Butyricimonas remained unchanged (Figure 6B). These findings indicate that the overgrowth of Bacteroides and Escherichia may participate in elevated circulating IS levels in CKD patients with AF. Further analysis of 16S rRNA sequencing revealed that CKD rats exhibited a marked decrease in Lactobacillus and an increase in Bacteroides (Figure 6C). Lactobacillus is a well-known probiotic that is essential for gut homeostasis and fundamentally involved in the treatment of a wide range of diseases.^24,25 Notably, we found that Lactobacillus gasseri was significantly depleted in both CKD rats and CKD patients with AF (Figure 6D and E). Given the role of bacterial interactions in maintaining microbial stability, we further examined the interplay between Lactobacillus gasseri and IS-producing bacteria, such as Escherichia coli and Bacteroides uniformis. In in vitro inhibition assays, Lactobacillus gasseri demonstrated strong suppression of Escherichia coli and Bacteroides uniformis growth (Figure 6F). These results suggest that the depletion of Lactobacillus gasseri abundance in CKD disrupts its inhibitory effects on Escherichia coli and Bacteroides uniformis, promoting their overgrowth and contributing to elevated IS levels.

Figure 6.

The production of indoxyl sulphate in gut microbiota is enhanced in CKD patients with AF. (A) Distribution of TnaA gene in 1520 bacterial reference genomes. The bars in the inner ring represent strains with a higher copy number of the TnaA gene, while the bars in the outer ring represent the distribution of TnaA copy numbers across different strains. Certain bacteria, such as Bacteroides, Butyricimonas, and Escherichia a higher copy number of the TnaA gene. (B) The relative abundance of TnaA, Bacteroides, Butyricimonas, and Escherichia in CKD patients and CKD patients with AF (n = 13 per group). (C) Heat map showing the relative abundance of key gut microbiota in CKD rats (blue) and controls (red). (D) Relative expression levels of Lactobacillus species, including L. gasseri, L. salivarius, L. acidophilus, L. johnsonii, and L. reuteri in rats from Control and CKD groups (n = 10 per group). (E) The relative abundance of L. gasseri in CKD patients with AF (n = 10 per group). (F) Antimicrobial activity of L. gasseri against Escherichia coli and Bacteroides uniformis demonstrated by inhibition zone assays (red arrows indicate inhibition zones). Data are expressed as mean ± SEM and compared by Wilcoxon test (B, D, and E).

Supplementation with Lactobacillus gasseri reduces circulating IS levels and prevents chronic kidney disease-associated atrial fibrillation

To explore the possible therapeutic impact of Lactobacillus gasseri in CKD-associated AF, we administered Lactobacillus gasseri supplementation or a control solvent to rats that had received faecal microbiota from CKD rats (Figure 7A). Notably, Lactobacillus gasseri supplementation effectively increased the abundance of Lactobacillus gasseri (Figure 7B), reduced the abundance of Bacteroides and Escherichia (see Supplementary material online, Figures S10A and B), and significantly reduced both intestinal TnaA levels and circulating IS levels (Figure 7C and D). Furthermore, Lactobacillus gasseri treatment notably improved gut barrier integrity, manifested by prolonged intestinal villi and decreased fibrosis (Figure 7E and F). Importantly, Lactobacillus gasseri supplementation led to a shortened duration and lower AF incidence in rats that received CKD-derived microbiota (Figure 7G-7H, Supplementary material online, Figures S11A and B). Furthermore, Lactobacillus gasseri supplementation inhibited the NLRP3 inflammasome pathway, supported by reduced expression of pro-caspase-1 and Cas1-p20 in atrial tissue (Figure 7I), along with decreased levels of IL-1β and IL-18 (Figure 7J-7K). Moreover, Lactobacillus gasseri supplementation attenuated atrial fibrosis and apoptosis, as indicated by reduced collagen deposition and a lower percentage of apoptotic cells in atrial tissue (Figure 7L-7M), along with reduced expression of TGF-β1, α-SMA, and BAX, and increased expression of Bcl2 (Figure 7N-7O). Furthermore, supplementation with Lactobacillus gasseri markedly improved renal function in rats, as evidenced by reduced BUN and creatinine levels, as well as decreased renal fibrosis (see Supplementary material online, Figures S12A–C). Our results indicate that Lactobacillus gasseri supplementation holds therapeutic potential in mitigating and managing CKD-associated AF. Collectively, this work provides evidence that gut dysbiosis in CKD leads to elevated levels of microbiota-derived IS, which damages the intestinal barrier and increases circulating LPS levels. Lipopolysaccharide may activate the TLR4–NLRP3 inflammasome pathway, promoting atrial structural remodelling and heightened susceptibility to AF (Figure 8).

Figure 7.

Supplementation with Lactobacillus gasseri prevents the development of atrial fibrillation in CKD rats. (A) Schematic diagram for L. gasseri intervention: Rats received faecal microbiota transplantation from CKD rats, one of which was given control vehicle, and the other was given Lactobacillus gasseri supplementation by gavage three times a week at a dose of 1*10⁹/CFU for 8 weeks. (B) The relative abundance of Lactobacillus gasseri in rats from Vehicle group and L. gasseri group (n = 10 per group). (C) The relative abundance of TnaA in faecal samples of rats from Vehicle group and L. gasseri group (n = 10 per group). (D) The levels of indoxyl sulphate in rats from Vehicle group and L. gasseri group (n = 10 per group). (E) The examples of HE staining and quantification of the colonic villi length of proximal colon in rats from Vehicle group and L. gasseri group (n = 5 per group). Magnification × 200. (F) The examples of Masson staining and collagen volume fraction of proximal colon in rats from Vehicle group and L. gasseri group (n = 5 per group). Magnification × 200. (G) Atrial fibrillation induction of rats from Vehicle and L. gasseri groups (n = 10 per group). (H) Atrial fibrillation duration of rats in Vehicle and L. gasseri groups (n = 10 per group). (I) Representative bands and quantification of expressions of NLRP3, ASC, pro-cas1, and Cas1-p20 in atrial tissue of rats from Vehicle and L. gasseri groups (n = 6 per group). (J–K) Immunohistochemical staining and quantitation of IL-1β (J) and IL-18 (K) in atrial tissues of rats from Vehicle and L. gasseri groups (n = 6 per group). (L) Representative Masson’s trichrome staining and collagen volume fraction in Vehicle and L. gasseri groups (n = 5 per group). Magnification × 200. (M) Representative Tunel staining and the ratio of Tunel-positive cells in Vehicle and L. gasseri groups (n = 5 per group). Magnification × 200. (N) Representative Western blots and quantification graphs depicting TGF-β1 and α-SMA expressions (n = 6 per group). (O) Representative Western blots and quantification graphs depicting Bcl2 and BAX expressions (n = 6 per group). Data are expressed as mean ± SEM and compared by Student’s t test (B, C, D, E, F, I, J, K, L, M, N, and O) or Wilcoxon test (H). AF inducibility (G) was presented as numbers and compared by Fisher exact test.

Figure 8.

Gut microbiota dysbiosis promotes CKD-associated atrial fibrillation through activation of the NLRP3 inflammasome. In CKD, gut dysbiosis in CKD leads to elevated levels of microbiota-derived IS, which compromises the intestinal barrier, facilitating the translocation of lipopolysaccharides into the bloodstream. Circulating LPS activates the TLR4 receptor, triggering the NLRP3 inflammasome. This cascade promotes the production of pro-inflammatory cytokines IL-1β and IL-18, driving atrial structural remodelling and increasing susceptibility to atrial fibrillation.

Discussion

The pathogenesis of CKD-associated AF is shaped by several factors, including increased cardiac volume load, activation of RAAS, heightened inflammatory responses, ion imbalances, and the accumulation of uraemic toxins.^17,26 Although the gut microbiota has been linked to the progression of CKD, its specific role in CKD-associated AF remains incompletely understood. Our present study highlights the crucial role of microbiota-induced IS elevation in the pathogenesis of CKD-associated AF. Transplanting gut microbiota from CKD rats induced an increased predisposition to AF in healthy recipients. Mechanistically, CKD-related gut dysbiosis led to elevated circulating levels of IS, which impaired gut barrier integrity, triggering an increase in circulating LPS and initiating NLRP3 inflammasome activation. The activation of this pathway contributed to atrial structural remodelling and an enhanced vulnerability to AF. Importantly, supplementation with L. gasseri significantly inhibited the growth of E. coli and Bacteroides, two key IS producers, thereby lowering circulating IS levels and preventing AF in CKD rats. Our findings demonstrate that gut dysbiosis promoted CKD-associated AF by activating NLRP3 through IS. These findings suggest that gut microbiota could represent a novel therapeutic target for the prevention and treatment of CKD-associated AF.

Emerging evidence suggests that gut microbiota dysbiosis facilitates the progression of CKD and associated complications, with several studies pointing to the therapeutic potential of targeting the microbiome.^11,27 Cao et al.²⁴ demonstrated Lactobacillus johnsonii reverses CKD by modulating the aryl hydrocarbon receptor signalling pathway and increasing serum IAld levels. Additionally, administration of Lactobacillus casei Zhang has been shown to correct gut microbial imbalance and alleviate kidney injury in ischemia–reperfusion-induced CKD models.²⁸ Our study reveals notable shifts in the gut microbiome composition of CKD rats, characterized by a marked diminution of Lactobacillus gasseri abundance and enrichment of Bacteroides. Importantly, FMT from CKD rats to healthy recipients significantly heightened AF susceptibility. Supplementation with Lactobacillus gasseri effectively suppressed the growth of Bacteroides and other dysbiotic bacteria, leading to a decrease in circulating IS levels and preventing CKD-associated AF. Notably, our findings align with the contemporary holistic approach to AF management, as underscored in the latest 2024 ESC/EACTS guidelines and the ABC (Atrial fibrillation Better Care) pathway.^29,30 By addressing the complex interplay of multiple comorbidities, our data suggest that gut dysbiosis represents a novel and critical component within this integrated care framework, potentially offering a new frontier for personalized patient management.

Gut microbiota-derived uraemic toxins have been linked to CKD progression and poor prognosis.^31,32 Indoxyl sulphate, a key uraemic toxin, is produced by gut microbiota via TnaA-mediated conversion of tryptophan to indole, which is then processed into IS by the liver.³³ Our study highlights the role of IS in CKD-associated AF. We found that CKD patients with AF exhibited significantly higher intestinal TnaA levels and elevated circulating IS compared with CKD patients without AF. In animal models, oral supplementation with AST-120, a therapeutic agent that adsorbs IS, effectively reduced circulating IS levels and prevented AF, further emphasizing the involvement of IS in the development of CKD-associated AF. IS, a protein-bound uraemic toxin, resists efficient clearance by haemodialysis, complicating its management in CKD.³⁴ Intriguingly, we found that Lactobacillus gasseri supplementation effectively inhibited the growth of IS-producing bacteria, reducing IS production and its systemic levels, suggesting that modulation of the gut microbiome could be an effective strategy to address IS accumulation and its cardiovascular consequences.

Intestinal barrier dysfunction is a well-documented feature of CKD, contributing to the systemic circulation of uraemic toxins, including IS, which may facilitate CKD progression.³⁵ In our research, we confirmed that both CKD rats and those receiving CKD faecal microbiota exhibited intestinal barrier dysfunction. Treatment with AST-120 reduced circulating IS levels and mitigated intestinal barrier damage, thereby preventing AF. In vitro, IS treatment significantly downregulated tight junction protein expression in Caco-2 cells, highlighting its role in disrupting intestinal integrity. Consistently, it has been reported that IS disrupt intestinal barrier integrity by impairing mitophagy.³⁶ Moreover, supplementation with an intestinal barrier-protective agent significantly ameliorated intestinal damage, reduced circulating IS levels, and prevented the onset of CKD-associated AF, suggesting that strategies aimed at preserving gut barrier function could have therapeutic potential in CKD-associated AF.

The NLRP3 inflammasome, a critical mediator of inflammation, has been implicated in a variety of cardiovascular diseases, such as atherosclerosis, myocardial ischemia–reperfusion injury, and AF.^37–41 Li et al.¹⁷ established the critical role of the NLRP3 inflammasome in CKD-associated AF. Our study further corroborates these findings, showing that NLRP3 inflammasome activation in the atria of CKD rats and CKD faecal microbiota-transplanted rats is linked to increased AF susceptibility. Notably, our data support gut dysbiosis as a potential contributor to atrial NLRP3 inflammasome activation. In our model, microbiota dysbiosis is associated with elevated IS, disruption of the intestinal barrier, increased circulating LPS, and upregulation of atrial TLR4 expression. However, these findings support, but do not establish, LPS–TLR4 signalling as a plausible upstream pathway for atrial NLRP3 inflammasome activation.

While our study provides important insights into the association between gut dysbiosis, LPS–TLR4 signalling, and NLRP3 inflammasome activation, further mechanistic investigations are needed to establish causality. Future research could involve the use of cardiomyocyte-specific TLR4 knockout models to more directly assess the role of TLR4 in atrial inflammation and the activation of the NLRP3 inflammasome. Additionally, pharmacologic inhibition of TLR4 in the CKD–FMT rat model could help determine whether TLR4 blockade prevents the observed inflammasome activation and AF development. Moreover, combining LPS inhibitors with CKD–FMT in rats could provide a more definitive evaluation of the LPS–TLR4–NLRP3 pathway in the pathogenesis of AF. These studies would be essential for providing a clearer mechanistic understanding of how gut-derived LPS and TLR4 signalling contribute to AF in CKD and could ultimately identify novel therapeutic targets to prevent AF in CKD patients.

Conclusion

In summary, our findings highlight the critical involvement of gut dysbiosis in the development of CKD-associated AF. We show that dysbiosis leads to elevated IS levels, which disrupts the intestinal barrier and increases circulating LPS. This increase in LPS triggers NLRP3 inflammasome activation, contributing to atrial remodelling and increased AF susceptibility. Notably, L. gasseri supplementation effectively reduces LPS and prevents CKD-associated AF. These findings highlight the gut microbiota as a novel therapeutic target for the prevention and treatment of CKD-associated AF, paving the way for new clinical strategies.

Limitations

While our study provides compelling evidence that gut dysbiosis and elevated IS contribute to CKD-associated AF, several limitations should be acknowledged. First, the efficacy of L. gasseri in humans remains unproven, and the therapeutic potential of probiotics in CKD patients with AF warrants validation in larger randomized clinical trials. Second, the sample size for uraemic toxin measurements was not determined by an a priori power calculation, which may limit the precision of the observed associations. Therefore, future studies with larger sample sizes will be required to further validate these findings. Third, the absence of a non-CKD control group limits our ability to distinguish AF-related changes from those attributable to CKD alone, highlighting the need for non-CKD controls in future investigations. Finally, although we assessed inflammasome activation markers, the lack of comprehensive immune cell profiling represents a limitation; future studies should include detailed immune phenotyping to better elucidate cellular contributors to NLRP3-driven inflammation.

Supplementary material

Supplementary material is available at Europace online.

Authors’ contributions

Xuejie Han: Writing-original draft, Methodology, Data curation. Hui Yu and Qianhui Gao: Methodology and Data curation. Xiaoyu Wang, Linwei Zhang, and Qian Zhao: Methodology. Luyang Yu: Revision. Manshu sui: Resources, Methodology, Writing—review & editing. Yue Li and Yun Zhang: Conceptualization, Resources, Writing—review & editing.

Funding

This work was financially supported by the National Key Research and Development Program of China (2024YFA1307001) to Yue Li; the National Natural Science Foundation of China (82400375), and the Heilongjiang Provincial Natural Science Foundation (PL2024H031) to Xuejie Han; the State Key Program of National Natural Science Foundation of China (82330014) to Yue Li; and the National Natural Science Foundation of China (82200413) and the China Postdoctoral Science Foundation (2022M721525) to Yun Zhang.

Data availability

The datasets, analytical procedures, and materials used in this study can be obtained from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All animal experiments were performed in accordance with the ARRIVE Guidelines and were approved by the Animal Care and Utilization Committee of Harbin Medical University (Ethical approval number: 2023112). All participants provided written informed consent prior to their inclusion in the study. In parallel, the Research Ethics Committees of the First Affiliated Hospital of Harbin Medical University authorized the human study (Ethical approval number: IRB-AF/SC-12/03.0). All experiments were conducted under the national animal welfare guidelines.