Caspase-11/GSDMD contributes to the progression of hyperuricemic nephropathy by promoting NETs formation

Abstract

Hyperuricemia is an independent risk factor for chronic kidney disease (CKD) and promotes renal fibrosis, but the underlying mechanism remains largely unknown. Unresolved inflammation is strongly associated with renal fibrosis and is a well-known significant contributor to the progression of CKD, including hyperuricemia nephropathy. In the current study, we elucidated the impact of Caspase-11/Gasdermin D (GSDMD)-dependent neutrophil extracellular traps (NETs) on progressive hyperuricemic nephropathy. We found that the Caspase-11/GSDMD signaling were markedly activated in the kidneys of hyperuricemic nephropathy. Deletion of Gsdmd or Caspase-11 protects against the progression of hyperuricemic nephropathy by reducing kidney inflammation, proinflammatory and profibrogenic factors expression, NETs generation, α-smooth muscle actin expression, and fibrosis. Furthermore, specific deletion of Gsdmd or Caspase-11 in hematopoietic cells showed a protective effect on renal fibrosis in hyperuricemic nephropathy. Additionally, in vitro studies unveiled the capability of uric acid in inducing Caspase-11/GSDMD-dependent NETs formation, consequently enhancing α-smooth muscle actin production in macrophages. In summary, this study demonstrated the contributory role of Caspase-11/GSDMD in the progression of hyperuricemic nephropathy by promoting NETs formation, which may shed new light on the therapeutic approach to treating and reversing hyperuricemic nephropathy.

Keywords: Caspase-11; Gasdermin D; Hyperuricemic nephropathy; Neutrophil extracellular traps.

Fig. 1

Gsdmd deficiency alleviated renal fibrogenesis in the hyperuricemic nephropathy model. A Representative microphotographs of Masson trichrome staining in mice kidney cortical tissue after daily adenine feeding for 0, 14, and 28 days. Scale bar = 100 μm. B Renal fibrosis scores assessed by Masson trichrome staining. n = 6. C, E Representative immunofluorescence images stained with α-SMA (C) and Col I (E) in mice kidney cortical tissue. Scale bar = 100 μm. D, F Quantitative analysis of α-SMA (D) and Col I (F) expression. n = 6. G Representative Western blot images, showing the expression of α-SMA and Col I expression in kidney tissue. Kidney tissue lysates were isolated on day 0, 7, 14, 21, and 28 after daily adenine feeding. n = 4. H Representative Western blot images, showing the expression of GSDMD and its cleaved form, GSDMD-N, in kidney tissue. n = 4. ***P < 0.001

Fig. 2

Caspase-11 deficiency alleviated renal fibrogenesis in the hyperuricemic nephropathy model. A Representative microphotographs of Masson trichrome staining in mice kidney cortical tissue. Kidneys were harvested from wild-type mice (Caspase-11^+/+) and Caspase-11-deficient mice (Caspase-11^−/−) after daily adenine feeding for 0, 14, and 28 days. Scale bar = 100 μm. B Renal fibrosis scores assessed by Masson trichrome staining. n = 6. C, E Representative immunofluorescence images stained with α-SMA (C) and Col I (E) in mice kidney cortical tissue. Scale bar = 100 μm. D, F Quantitative analysis of α-SMA (D) and Col I (F) expression. n = 6. G Representative Western blot images, showing the expression of α-SMA and Col I in kidney tissue, which were isolated on days 0, 14, and 28 after daily adenine feeding. n = 4. H Representative Western blot images, showing the expression of Caspase-11 in kidney tissue. Kidney tissue lysates were isolated on day 0, 7, and 14 after daily adenine feeding. n = 4. ***P < 0.001

Fig. 3

Gsdmd deficiency diminished inflammatory cell infiltration in the hyperuricemic nephropathy model. A, C Representative immunofluorescence images stained with Ly6G A and F4/80 C in mice kidney cortical tissue. Scale bar = 100 μm. B, D Positive staining cells of Ly6G (B) and positive staining cells of F4/80 (D) were calculated and graphed. n = 6. E, G Representative flow cytometry results of Ly6G (E) and F4/80 (G) in the kidney. F, H Quantitative analysis of the percentage of neutrophils (Ly6G⁺) and macrophages (F4/80⁺) infiltration. n = 6. ***P < 0.001

Fig. 4

Caspase-11 deficiency diminished inflammatory cell infiltration in the hyperuricemic nephropathy model. A, C Representative immunofluorescence images stained with Ly6G (A) and F4/80 (C) in mice kidney cortical tissue. Scale bar = 100 μm. B, D Positive staining cells of Ly6G (B) and positive staining cells of F4/80 (D) were calculated and graphed. n = 6. E, G Representative flow cytometry results of Ly6G (E) and F4/80 (G) in the kidneys. F, H Quantification of the percentage of neutrophils (Ly6G⁺) and macrophages (F4/80⁺) infiltration. n = 6. ***P < 0.001

Fig. 5

Deletion of Caspase-11/Gsdmd reduced the expression of proinflammatory and profibrogenic factors, and the production of α-SMA in macrophages in the hyperuricemic nephropathy model. A–H Expression level of IL-1β (A, E), TNFα (B, F), HMGB1 (C, G), and TNF-β1 (D, H) production in kidneys were evaluated by ELISA. Kidneys were harvested from Gsdmd^+/+ mice, Gsdmd^−/− mice, Capase-11^+/+ mice, and Caspase-11^−/− mice after daily adenine feeding for 0, 7, and 14 days. n = 6. I–P Real-time quantitative polymerase chain reaction analysis revealed the mRNA expression level of IL-1β (I, M), TNFα (J, N), HMGB1 (K, O), and TNF-β1 (L, P) in immune cells isolated from kidneys. n = 6. Q, S Representative images of immunofluorescence staining showing macrophage-to-myofibroblast transition cells that coexpress F4/80 and α-SMA in mice kidneys on day 28 after daily adenine feeding. Scale bar = 100 μm. R, T Quantification of cells coexpressing F4/80 and α-SMA by immunofluorescence. n = 6. *P < 0.05. **P < 0.01. ***P < 0.001

Fig. 6

Specific deletion of Caspase-11/Gsdmd protected against the progression of hyperuricemic nephropathy by reduce fibrosis. A Representative images of Masson trichrome staining in mice kidney cortical tissue. Kidneys were harvested from Vav-Cre mice, Vav-Cre Gsdmd^fl/fl mice, and Vav-Cre Caspase-11^fl/fl mice after daily adenine feeding for 0, 14, and 28 days. Scale bar = 100 μm. B Renal fibrosis scores assessed by Masson trichrome staining. n = 6. C, E Representative immunofluorescence images stained with α-SMA (C) and Col I (E) in mice kidney cortical tissue. Scale bar = 100 μm. D, F Quantitative analysis of α-SMA (D) and Col I (F) expression. n = 6. ***P < 0.001

Fig. 7

Deletion of Caspase-11/Gsdmd reduced NETs formation in vivo and in vitro. A Representative immunofluorescence images stained with Histone-H3 and MPO in mice kidney cortical tissue. Kidneys were harvested from WT mice, Gsdmd^−/− mice, and Caspase-11^−/− mice after daily adenine feeding for 14 days. Scale bar = 100 μm. B Quantification of neutrophils with Histone-H3 and MPO double-positive staining. n = 6. C Representative immunofluorescence images stained with Histone-H3 and MPO in neutrophils isolated from Gsdmed^+/+ mice and Gsdmd^−/− mice and then were incubated with or without UA for 3 h. Scale bar = 50 μm. D Quantification of the percentage of Histone-H3 and MPO double-positive area. E Representative Western blot images showing GSDMD cleavage in neutrophils after incubation with UA for 1 h and 2 h. The knockdown efficiency of Gsdmd was confirmed. n = 6. F Representative images of immunofluorescence staining of Histone-H3 and MPO in neutrophils isolated from Caspase-11^+/+ mice and Caspase-11^−/− mice and then were incubated with or without UA for 3 h. Scale bar = 50 μm. G Quantification of the percentage of Histone-H3 and MPO double-positive area. H Western blot showing Caspase-11 activation in neutrophils after incubation with UA for 1 and 2 h. The knockout efficiency of Caspase-11 was confirmed. n = 6. ***P < 0.001

Fig. 8

NETs promoted α-SMA production in macrophages in vitro. A, B Representative immunofluorescence images showing the expression of α-SMA in microphages. Microphages were isolated form Gsdmed^+/+ mice, Gsdmd^−/−mice, Caspse-11^+/+ mice, and Caspase-11^−/−mice, and were then treated with NETs, NETs plus DNase I, NETs plus NE inhibitor or NETs plus anti-TGF-β1 antibody for 96 h. Scale bar = 50 μm. C, D Representative Western blot results showing the expression levels of α-SMA and its regulator, pSmad3, in macrophages. n = 4. E Schematic model: GSDMD/Caspase-11 contributes to the progression of hyperuricemic nephropathy by promoting NETs formation