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Kidney Week

Abstract: FR-OR023

Neutrophil Extracellular Traps Are Triggered by C3 and Contribute to AKI

Session Information

Category: Acute Kidney Injury

  • 103 AKI: Mechanisms

Author

  • Wan, Jian-xin, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
Background

Infiltration neutrophils can be stimulated to formed neutrophil extracellular trap (NETs), which may lead to renal injury. Complement C3 play a prominent role in inflammatory processes, its activation exacerbates AKI. But the relationship between the formation NETs and activation C3 in IR injury-induced AKI was not clear.

Methods

C57BL/6 mice (WT) were subjected to renal IR injury-induced AKI model by clamping both renal pedicle for 45 min. To deplete neutrophil, mice were treated with intraperitoneal injection of anti-Ly6G IgG (1A8) or control IgG 24h and 2h before bilateral IR injury. C3KO mice were subjected to renal IR injury. The level of renal neutrophils were evaluated by MPO, LY6G and ICAM-1 immunohistochemistry. NETs formation was defined by the colocalization of diffused DAPI, Ly6G and CitH3 signal by immunofluorescence and protein level of CitH3 by western blot. The expression of C3 in renal were estimated by immunofluorescence, qPCR and ELISA. In vitro, neutrophil were isolated from normal individuals and were assessed by Wright-Giemsa stain. Neutrophils in RPMI were simulated with PMA and C3a.

Results

The expression of neutrophil, NETs and C3 of each temporal point after IR increased obviously. At 24h, post-ischemic kidneys represent positivity for DAPI, CitH3 and Ly6G colocalizing of NETs in the outer medulla. Injection of 1A8 suppressed the increase in BUN and Scr 24h after renal IR injury, with a concomitant reduction of neutrophils infiltration and NETs formation, while there were no significant differences in the expression of C3 in mice with and without neutrophil depletion. Compared with WT-sham group, C3KO can ameliorate the accumulation of neutrophil and protect renal against IR injury. Kidney sections from C3KO mice contained less NETs formation with WT mice, corroborating the CitH3 western blot. In vitro, neutrophils were assessed to be >90% pure. After 4h of incubation, 0.1 μM C3a simulated the formation of NETs which reflected by amorphous extracellular DNA structures that colocalized with CitH3 and MPO. PMA altered neutrophil phenotype with decondensed chromatin, while C3a remained intact neutrophil phenotype with lobulated nuclei.

Conclusion

C3 activation can stimulate neutrophil motivation and lead to the formation of NETs in renal IR injury.