HMGB1-mediated formation of IL-33–abundant NETs drives lung-to-kidney injury in severe pneumonia–associated acute kidney injury
Mengqing Ma, Hao Zhang, Weijuan Deng, Xia Du, Mengxing Chen, Dawei Chen, Binbin Pan, Zhaowei Wang, Ting Chen, Caimei Chen, Xin Wan, Changchun Cao
Mengqing Ma, Hao Zhang, Weijuan Deng, Xia Du, Mengxing Chen, Dawei Chen, Binbin Pan, Zhaowei Wang, Ting Chen, Caimei Chen, Xin Wan, Changchun Cao
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Research Article Inflammation Nephrology Pulmonology

HMGB1-mediated formation of IL-33–abundant NETs drives lung-to-kidney injury in severe pneumonia–associated acute kidney injury

Abstract

Acute kidney injury (AKI) is a common and fatal complication of severe pneumonia, yet the mechanisms linking pulmonary inflammation to remote kidney injury remain poorly understood. Multicenter cohort data (n = 300) revealed that the incidence of severe pneumonia–associated AKI (SP-AKI) was 53.6%, with a mortality rate of 24.2%. SP-AKI was associated with elevated circulating levels of HMGB1, NETs, and IL-33. Murine experiments demonstrated that alveolar HMGB1 triggers the formation of IL-33–enriched NETs, which migrate to the kidney and activate tubular ST2/NF-κB signaling, driving inflammation and apoptosis. Genetic knockout of IL-33, ST2, or the NET-forming key enzyme PAD4, as well as pharmacological inhibition of HMGB1, IL-33, or NETs, all attenuated lung and kidney injury. Exogenous HMGB1 amplified NET-mediated IL-33 release, establishing a self-sustaining HMGB1/NET/IL-33 feed-forward loop. PAD4 deficiency completely blocked NET generation and disrupted HMGB1/IL-33 signaling. This study identified and validated a damage-associated molecular pattern–driven (DAMP-driven) HMGB1/NET/IL-33 signaling axis that mediates remote kidney injury in SP-AKI, redefining NETs from local effectors to cross-organ pathogenic carriers, thereby providing potential DAMP-targeted therapeutic avenues for SP-AKI.

Authors

Mengqing Ma, Hao Zhang, Weijuan Deng, Xia Du, Mengxing Chen, Dawei Chen, Binbin Pan, Zhaowei Wang, Ting Chen, Caimei Chen, Xin Wan, Changchun Cao

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Figure 9

HMGB1/IL-33 neutralization reduces NETs and renal tubular damage in vitro.

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HMGB1/IL-33 neutralization reduces NETs and renal tubular damage in vitr...
(A and B) Representative flow cytometry plots and quantification of NET formation in neutrophils from healthy donors, severe pneumonia patients without AKI, and SP-AKI patients, gated as CD66b+MPO+. (C) Immunofluorescence showing that IL-33 colocalized with MPO within NET structures. Pretreatment with anti–IL-33 (1 μg/mL) reduced the formation of NETs. Scale bars: 20 μm. (D and E) Quantification of mean fluorescence intensity of MPO (D) and IL-33 (E) in neutrophils under the indicated conditions. (F) Scanning electron microscopy showing the morphology of NETs after neutrophils were stimulated by serum from patients with SP-AKI. Scale bars: 50 μm (left) and 5 μm (right). (G) Schematic illustration of NET isolation from SP-AKI neutrophils and subsequent stimulation of HK-2 cells. (H) Flow cytometric analysis of apoptosis in HK-2 cells cocultured with healthy neutrophils, NETs, and anti-HMGB1–treated (glycyrrhizic acid, 200 μM) NETs. (I) Inhibition of NET-induced HK-2 cell apoptosis after anti-HMGB1 administration. (J) Flow cytometric analysis of apoptosis in HK-2 cells cocultured with healthy neutrophils, NETs, and anti–IL-33–treated NETs. (K) Inhibition of NET-induced HK-2 cell apoptosis after anti–IL-33 administration. (L and M) Western blot analysis and quantification of ST2 expression in HK-2 cells after stimulation with NETs from healthy donors, patients with SP-AKI, anti–IL-33–treated SP-AKI neutrophils, or PMA-treated neutrophils. (N and O) Immunofluorescence analysis of NF-κB activation in HK-2 cells following NET stimulation and quantification of fluorescence intensity. Scale bars: 20 μm. (P and Q) ELISA measurement of TNF-α and NGAL levels in cell culture supernatants following pretreatment with the NF-κB inhibitor Bay 11-7082 (2 μM). (R) Mechanisms of SP-AKI. Data are presented as mean ± SD. n = 5 per group. P values were calculated by 1-way ANOVA with post hoc Holm-Šídák test. *P < 0.05; **P < 0.01; ****P < 0.0001. NS, no statistically significant difference.