Chronic kidney disease promotes atrial fibrillation via inflammasome pathway activation

Abstract

Chronic kidney disease (CKD) is associated with a higher risk of atrial fibrillation (AF). The mechanistic link between CKD and AF remains elusive. IL-1β, a main effector of NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, is a key modulator of conditions associated with inflammation, such as AF and CKD. Circulating IL-1β levels were elevated in patients with CKD who had AF (versus patients with CKD in sinus rhythm). Moreover, NLRP3 activity was enhanced in atria of patients with CKD. To elucidate the role of NLRP3/IL-1β signaling in the pathogenesis of CKD-induced AF, Nlrp3-/- and WT mice were subjected to a 2-stage subtotal nephrectomy protocol to induce CKD. Four weeks after surgery, IL-1β levels in serum and atrial tissue were increased in WT CKD (WT-CKD) mice versus sham-operated WT (WT-sham) mice. The increased susceptibility to pacing-induced AF and the longer AF duration in WT-CKD mice were associated with an abbreviated atrial effective refractory period, enlarged atria, and atrial fibrosis. Genetic inhibition of NLRP3 in Nlrp3-/- mice or neutralizing anti-IL-1β antibodies effectively reduced IL-1β levels, normalized left atrial dimensions, and reduced fibrosis and the incidence of AF. These data suggest that CKD creates a substrate for AF development by activating the NLRP3 inflammasome in atria, which is associated with structural and electrical remodeling. Neutralizing IL-1β antibodies may be beneficial in preventing CKD-induced AF.

Keywords: Arrhythmias; Cardiology; Chronic kidney disease.

Figure 1. Enhanced NLRP3 inflammasomes activity in CKD patients with AF.

Levels of the cytokines IL-1β (A), IL-18 (B), TNF-α (C), and IL-10 (D) in serum samples from dialysis-dependent patients with CKD-SR or CKD-AF. Representative Western blots and quantification of protein levels of NLRP3 (E) and ASC (F). Representative Western blots (G) and quantification of pro–caspase-1 (Pro-Casp1) (H) and mature caspase-1 (p20) (I). Representative Western blots (J) and quantification of pro–IL-1β (K) and mature IL-1β (L). Representative Western blots (M) and quantification of pro–IL-18 (N) and mature IL-18 (O). Data are expressed as the mean ± SEM. *P < 0.05, by unpaired 2-tailed Student’s t test (A, F, H, I, and O).

Figure 2. Murine model of CKD exhibits increased NLRP3 inflammasome activity.

(A) Timeline of the 2-stage subtotal nephrectomy to generate the CKD mouse model. The schematic was created with BioRender.com. (B) BUN levels and (C) BWs in WT and Nlrp3^–/– mice subjected to sham or CKD procedures. (D and E) Serum levels of IL-1β and IL-18. (F) Representative Western blots and (G) quantification of IL-1β in kidney tissue. (H) Representative Western blots and (I) quantification of IL-1β in atrial tissue. Data are expressed as the mean ± SEM. *P < 0.05 and ***P < 0.001, by Welch’s ANOVA and Dunnett’s T3 multiple-comparison test (B and C) and ordinary 1-way ANOVA with Šidák’s multiple-comparison test (D and I).

Figure 3. Increased AF susceptibility in CKD mice.

(A) Representative recordings of lead 2 surface ECG and intracardiac atrial and ventricular electrograms in WT and Nlrp3^–/– mice subjected to sham or CKD procedures. (B) Incidence of pacing-induced reproducible AF. (C) Duration of pacing-induced AF. Data are expressed as a percentage in B and as the mean ± SEM in C. *P < 0.05 and **P < 0.01, by Fisher’s exact test (B) and Kruskal-Wallis test with Dunn’s multiple-comparison test (C).

Figure 4. CKD promotes electrical remodeling for AF.

(A) Representative activation maps from an optical mapping study. (B) Incidence of abnormal activation patterns in each treatment group. (C and D) Summary of CV (C) in the RA and AERP (D) in atria of WT and Nlrp3^–/– mice subjected to sham or CKD procedures. (E) APD at 20%, 50%, 70%, and 90% repolarization. (F) Kv1.5 protein levels in atria of WT and Nlrp3^–/– mice subjected to sham or CKD procedures. Data are expressed as a percentage in B and as the mean ± SEM in C–F. *P < 0.05 and **P < 0.01, by Fisher’s exact test (B), ordinary 1-way ANOVA with Šidák’s multiple-comparison test (D and F), and 2-way ANOVA with Tukey’s multiple-comparison test (E).

Figure 5. CKD promotes structural remodeling.

(A) Representative echocardiographic images of the long-axis view of hearts. (B) Quantification LA areas in WT and Nlrp3^–/– mice that were subjected to sham or CKD procedures. (C) SBP measurement. (D) Representative pulsed-wave Doppler images. (E) Quantification of E/A ratios. Data are expressed as the mean ± SEM. *P < 0.05 and ***P < 0.001, by ordinary 1-way ANOVA with Šidák’s multiple-comparison test (B and C).

Figure 6. CKD promotes atrial fibrosis.

(A) Representative images of Masson’s trichrome staining of heart sections from WT and Nlrp3^–/– mice subjected to sham or CKD procedures. Scale bar: 1 mm. (B) Quantification of fibrosis in the LA and RA of WT and Nlrp3^–/– mice subjected to sham or CKD procedures. (C–G) Representative Western blots and quantification of collagen I (C and D), vimentin (E and F), and α-SMA (E and G). Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01, by Welch’s ANOVA and Dunnett’s T3 multiple-comparison test (B) and ordinary 1-way ANOVA with Šidák’s multiple-comparison test (D, F, and G).

Figure 7. Neutralization of circulating IL-1β attenuates CKD-induced atrial arrhythmogenesis.

(A) Serum levels of IL-1β in WT mice 2 and 3 weeks after sham or CKD surgeries. (B) Timeline of anti–IL-1β antibody (5 mg/kg, i.p.) or IgG (as a control) injections into WT-CKD mice. 2/3 Nx, two-thirds nephrectomy. Panel B was created with BioRender.com. (C and D) Representative echocardiographic long-axis views of the heart (C) and quantification of the LA area (D) in WT-CKD mice that received IgG or anti–IL-1β antibody injections. Blue outlined areas indicate the LA. Scale bar: 3 mm. (E) Serum levels of IL-1β in WT-CKD mice that received IgG or anti–IL-1β antibody injections following the CKD procedure. (F and G) Representative Western blots (F) and quantification (G) of IL-1β in atrial tissue of WT-CKD mice after 3 weekly injections of IgG or anti–IL-1β antibody. (H and I) Representative images (H) of Masson’s trichrome staining in atria and quantification of atrial fibrosis (I) in WT-CKD mice after 3 weekly injections of IgG or anti–IL-1β antibody. Scale bar: 200 μm. (J–L) Representative lead 2 surface ECG and intracardial ECG recordings (J), incidence (K), and duration (L) of pacing-induced AF in WT-CKD mice after 3 injections of IgG or anti–IL-1β antibody. Scale bar: 200 ms (J). Data are expressed as the mean ± SEM in A, D, E, G, I, and L, and as a percentage in K. *P < 0.05 and ***P < 0.001, by unpaired, 2-tailed Student’s t test (A, D, E, G, and I), Fisher’s exact test (K), and Mann-Whitney U test (L).