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Knockout of the C3a receptor protects against renal ischemia reperfusion injury by reduction of NETs formation

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Abstract

Renal ischemia/reperfusion (I/R) injury is a local sterile inflammatory response driven by innate immunity. Emerging data have revealed that complement and neutrophils contribute to hyperinflammation and oxidative stress in I/R induced acute kidney injury (AKI). However, the interplay between the C3a/C3aR axis and neutrophil extracellular traps (NETs) is imcompletelyunderstood. Here, we utilize genetically engineered mouse models and pharmacological inhibitors to investigate this association. The C3a/C3aR axis is found to promote neutrophil recruitment and NETs formation, thereby accelerating renal damage and dysfunction. Knockout of C3aR restores NETs release and improves renal function after I/R injury. Antibody-mediated blockade of NETs can also significantly ameliorate renal tubular injury and inflammation. Consistently, under stimulation by C3a, neutrophils are activated to promote NETs formation and subsequent renal tubular epithelial cell damage, and blocking C3aR rescued the injury. Interfering with reactive oxygen species (ROS) accumulation in neutrophils by antioxidant treatment significantly attenuates NETs formation. Our findings demonstrate that the C3a/C3aR-ROS-NETs axis constitutes a promising target for prevention or treatment of renal I/R injury.

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Data availability

All the data used for this study are presented in the paper or the Supplementary materials.

Abbreviations

I/R:

Ischemia/reperfusion

AKI:

Acute kidney injury

NETs:

Neutrophil extracellular traps

PAD4:

Ptidyl arginine deiminase 4

TUNEL:

TdT-mediated dUTP nick-end labeling

C3aR KO:

C3aR knockout

C3aRA:

C3aR antagonist

SCr:

Serum creatinine

BUN:

Blood urea nitrogen

MPO:

Myeloperoxidase

NE:

Neutrophil elastase

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Acknowledgements

We are grateful to Liangdi Xie for providing experimental sites and instruments. Changsheng Xu and Guili Lian provided technical assistance and research resources. We thank the central laboratory for providing instruments.

Funding

This study was supported by grants from the Joint Funds for the Innovation of Science and Technology, Fujian Province (No. 2019Y9117). Fujian Provincial Health Technology Project (No. 2021CXA018). The Scientific Research Foundation for the Introduction of Talent of the First Affiliated Hospital of Fujian Medical University (YJRC3992). The authors declare that no other funds, grants, or support were received during the preparation of this manuscript.

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  1. Xiaoting Wu, Danyu You and Maoen Pan contributed equally to this works.

Authors and Affiliations

  1. Department of Nephrology, Blood Purification Research Center, the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350005, China

    Xiaoting Wu, Danyu You, Mengjie Weng, Jing Zheng, Songhua Lin, Xiaohong Zhang & Jianxin Wan

  2. Fujian Clinical Research Center for Metabolic Chronic Kidney Disease, the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350005, China

    Xiaoting Wu, Danyu You, Mengjie Weng, Jing Zheng, Songhua Lin, Xiaohong Zhang & Jianxin Wan

  3. Department of Nephrology, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, 350212, China

    Xiaoting Wu, Danyu You, Mengjie Weng, Jing Zheng, Songhua Lin, Xiaohong Zhang & Jianxin Wan

  4. Department of Nephrology, Huashan Hospital, Fudan University, Shanghai, 200040, China

    Qionghong Xie, Yi Guan & Chuanming Hao

  5. Department of General Surgery, Fujian Medical University Union Hospital, Fuzhou, 350001, China

    Maoen Pan

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Contributions

XW, CH and JW designed the research and wrote the manuscript. XW and MP did animal experiments and analyzed data. DY, MW and JZ performed in vitro experiments. QX and YG designed experimental and edited the manuscript. SL and XZ performed histological analysis. CH and JW supervised the whole study. We declare that all authors read, verified, and approved the final version of the manuscript.

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Correspondence to Chuanming Hao or Jianxin Wan.

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All animal experiments were approved by the the Institutional Animal Care and Use Committee at Fujian Medical University.

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18_2023_4967_MOESM1_ESM.tif

Figure S1. I/R leads to alterations of renal function and morphology. A. Serum levels of creatinine and urea were measured in mice at different times of I/R. B. Urinary protein was measured in mice at different times of I/R. C. Representative microphotographs of H&E stained histological sections from sham and I/R mice, and tubular damage was scored. Scale bars: left, 50 μm; right, 10 μm. D. Representative TUNEL assay images and quantification of TUNEL positive cells in the respective groups. Scale bar: 25 μm. * P＜0.5, ** P＜0.01, *** P＜0.001, **** P＜0.0001, ns, no significant. (TIF 1504 KB)

18_2023_4967_MOESM2_ESM.tif

Figure S2 C3aR is vital for renal injury after I/R. A. Western blot and quantitative analysis of C3aR protein expression levels in lysates of renal tissue. B. ELISA measurement of C3a levels in WT mouse renal tissue lysates in the respective groups. C. Representative immunofluorescence staining of MPO and NE in neutrophils treat with C3a stimulated. Scale bars: 25 μm. D. Representative immunofluorescence staining of C3aR and NE in neutrophils after C3a stimulated, with or without C3aRA. Scale bars: 50 μm. * P＜0.5, ** P＜0.01, *** P＜0.001, **** P＜0.0001, ns, no significant. (TIF 949 KB)

18_2023_4967_MOESM3_ESM.tif

Figure S3 C3a mediates NETs formation via the ERK and ROS pathways in neutrophils. A. Representative image of DCFH immunofluorescence in neutrophils after hypoxia exposure. Scale bars: 25 μm. B. ROS content in neutrophils as determined by flow cytometric analysis and statistical analysis of ROS level in neutrophils from each group. C. Representative western blots and quantitative analysis of p-p38 and p-ERK1/2 protein levels in mice treated with or without C3a. D. Representative western blots and quantitative analysis of p-ERK1/2 protein levels in mice with C3aR inhibition. E. Representative blots and quantitative analysis of p-ERK1/2 protein levels in mice with NETs inhibition. * P＜0.5, ** P＜0.01, *** P＜0.001, ns, no significant. (TIF 867 KB)

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Wu, X., You, D., Pan, M. et al. Knockout of the C3a receptor protects against renal ischemia reperfusion injury by reduction of NETs formation. Cell. Mol. Life Sci. 80, 322 (2023). https://doi.org/10.1007/s00018-023-04967-6

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