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Immunological features and functional analysis of anti-CFH autoantibodies in patients with atypical hemolytic uremic syndrome

  • Wei-yi Guo
  • Di Song
  • Xiao-rong Liu
  • Zhi Chen
  • Hui-jie Xiao
  • Jie Ding
  • Shu-zhen Sun
  • Hong-yan Liu
  • Su-xia Wang
  • Feng Yu
  • Ming-hui Zhao
  • On behalf of the Chinese Renal-TMA Network
Original Article

Abstract

Objective

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Anti-complement factor H (CFH) antibodies were thought to participate in the pathogenesis of aHUS. The aim of this study was to address the functions and properties of CFH autoantibodies in a Chinese Han cohort of aHUS patients.

Methods

Thirty-six anti-CFH antibody-positive aHUS patients at the acute phase of the disease were involved in this study. Clinical data of the patients were collected. Anti-CFH immunoglobulin G (IgG) subclasses and antibody isotypes were detected by ELISA. Epitope mapping was performed using recombinant CFH fragments (SCRs 1–4, SCR 7, SCRs 11–14, and SCRs 19–20). Purified IgG from plasma from seven patients were used for functional analyses.

Results

All patients presented with the classic triad of HUS. The anti-CFH autoantibodies mostly bound to the SCRs 19–20 domains of CFH but not the SCRs 1–4 domains. CFI cofactor activity was not disturbed by the anti-CFH antibody in any of the seven patients. Purified IgG interfered with the binding of CFH to C3b and CFH-mediated sheep erythrocyte protection in all seven patients. IgG from 4/5 (80%) patients tested inhibited the binding of CFH to glomerular endothelial cells.

Conclusions

Our study suggests that the properties of CFH antibodies from patients with aHUS, including the recognition of SCRs and IgG subclasses, can influence and impair the biological role of CFH and therefore contribute to aHUS susceptibility.

Keywords

Hemolytic uremic syndrome Anti-CFH autoantibody CFH Biofunction Immunological feature 

Notes

Funding information

This work was supported by grants of the National Natural Science Foundation of China to Innovation Research Group (No. 81621092), National Natural Science Foundation of China (No. 81470932, No. 81500526, No. 81670640, and No. 81670639), Beijing Natural Science Foundation (7172215), The Capital Health Research and Development of Special (No. 2016-2-2094), and the Research on the Application of Capital Clinical Characteristics Program of Beijing Municipal Science and Technology Commission (No. Z161100000516106).

Compliance with ethical standards

The research complied with the principles of the Declaration of Helsinki and was approved by the local ethical committees. Informed consent for blood sampling was obtained from all participants or their parents.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

467_2018_4074_Fig7_ESM.png (1.1 mb)
Supplementary Figure 1

The SDS-PAGE analysis with CFH fragments. (A) CFH SCRs 1–4 (B) CFH SCR 7 (C) CFH SCRs 11–14 (D) CFH SCRs 19–20. (PNG 1135 kb)

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High Resolution Image (TIF 2047 kb)
467_2018_4074_Fig8_ESM.png (75 kb)
Supplementary Figure 2

Anti-CFH IgG subclasses. The cut-off value of anti-CFH IgG1 was 0.017; anti-CFH IgG2 was 0.236; anti-CFH IgG3 was 0.022; anti-CFH IgG4 was 0.028; The titers above the cut-off value were considered to be positive. (PNG 74 kb)

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High Resolution Image (TIFF 102 kb)
467_2018_4074_Fig9_ESM.png (340 kb)
Supplementary Figure 3

Cofactor activity for C3b was tested in kinetic assays at each 1-, 2-, 5-, 10-, 15-, 20-, 30-, 60-minute time point. Quantification of the C3 α43-chain generation was made by densitometry quantification of scanned band (ImageJ software) and calculation of the α43/β’-chain ratio. (A) Purified IgG from patient 15 did not affect the cofactor activity of CFH on CFI at each time point. (B) Purified IgG from patient 21 did not affect the cofactor activity of CFH on CFI at each time point. (C) The α43/β’-chain ratio at each time point of Figure A. (D) The α43/β’-chain ratio at each time point of Figure B. (PNG 339 kb)

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High Resolution Image (TIF 2245 kb)
467_2018_4074_Fig10_ESM.png (497 kb)
Supplementary Figure 4

IgG from P34 dose-dependently decreased CFH binding to GenCs. Commercial CFH (5 μg/ml) was incubated with purified IgG from P34 (10 μg/ml, 100 μg/ml and 500 μg/ml). The average fluorescence intensity of P34 at different IgG concentration were 81.50 ± 4.02 (10 μg/ml), 62.07 ± 4.02 (100 μg/ml) and 31.4 ± 5.65 (500 μg/ml). While IgG from P25 could not decrease the CFH binding to GenCs. The average fluorescence intensity of P25 at different IgG concentration were 80.87 ± 5.36 (10 μg/ml), 79.61 ± 2.82 (100 μg/ml) and 81.2 ± 6.32 (500 μg/ml). (PNG 497 kb)

467_2018_4074_MOESM4_ESM.tif (1.2 mb)
High Resolution Image (TIF 1253 kb)
467_2018_4074_MOESM5_ESM.docx (25 kb)
Table S1 (DOCX 25 kb)

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Copyright information

© IPNA 2018

Authors and Affiliations

  • Wei-yi Guo
    • 1
    • 2
    • 3
    • 4
  • Di Song
    • 1
    • 2
    • 3
    • 4
  • Xiao-rong Liu
    • 5
  • Zhi Chen
    • 5
  • Hui-jie Xiao
    • 6
  • Jie Ding
    • 6
  • Shu-zhen Sun
    • 7
  • Hong-yan Liu
    • 8
  • Su-xia Wang
    • 1
    • 2
    • 3
    • 4
  • Feng Yu
    • 1
    • 2
    • 3
    • 4
    • 9
  • Ming-hui Zhao
    • 1
    • 2
    • 3
    • 4
    • 10
  • On behalf of the Chinese Renal-TMA Network
  1. 1.Renal Division, Department of MedicinePeking University First HospitalBeijingPeople’s Republic of China
  2. 2.Peking University Institute of NephrologyBeijingPeople’s Republic of China
  3. 3.Key laboratory of Renal DiseaseMinistry of Health of ChinaBeijingPeople’s Republic of China
  4. 4.Key Laboratory of Chronic Kidney Disease Prevention and TreatmentMinistry of Education of ChinaBeijingPeople’s Republic of China
  5. 5.Department of NephrologyBeijing Children’s Hospital affiliated to Capital Medical UniversityBeijingPeople’s Republic of China
  6. 6.Department of PediatricsPeking University First HospitalBeijingPeople’s Republic of China
  7. 7.Department of PediatricsShandong Provincial Hospital affiliated with Shandong UniversityJinanPeople’s Republic of China
  8. 8.Department of NephrologyRenmin Hospital of Wuhan UniversityWuhanPeople’s Republic of China
  9. 9.Department of NephrologyPeking University International HospitalBeijingPeople’s Republic of China
  10. 10.Peking-Tsinghua Center for Life SciencesBeijingPeople’s Republic of China

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