Pediatric Nephrology

, Volume 34, Issue 2, pp 269–281 | Cite as

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

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



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.


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.


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.


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.


Hemolytic uremic syndrome Anti-CFH autoantibody CFH Biofunction Immunological feature 


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)

467_2018_4074_MOESM1_ESM.tif (2 mb)
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)

467_2018_4074_MOESM2_ESM.tiff (103 kb)
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)

467_2018_4074_MOESM3_ESM.tif (2.2 mb)
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)


  1. 1.
    Hofer J, Giner T, Safouh H (2014) Diagnosis and treatment of the hemolytic uremic syndrome disease spectrum in developing regions. Semin Thromb Hemost 40(4):478–486CrossRefGoogle Scholar
  2. 2.
    Noris M, Remuzzi G (2009) Atypical hemolytic-uremic syndrome. N Engl J Med 361(17):1676–1687CrossRefGoogle Scholar
  3. 3.
    Sanchez-Corral P, Gonzalez-Rubio C, Rodriguez de Cordoba S, Lopez-Trascasa M (2004) Functional analysis in serum from atypical hemolytic uremic syndrome patients reveals impaired protection of host cells associated with mutations in factor H. Mol Immunol 41(1):81–84CrossRefGoogle Scholar
  4. 4.
    Strobel S, Abarrategui-Garrido C, Fariza-Requejo E, Seeberger H, Sanchez-Corral P, Jozsi M (2011) Factor H-related protein 1 neutralizes anti-factor H autoantibodies in autoimmune hemolytic uremic syndrome. Kidney Int 80(4):397–404CrossRefGoogle Scholar
  5. 5.
    Jozsi M, Strobel S, Dahse HM, Liu WS, Hoyer PF, Oppermann M, Skerka C, Zipfel PF (2007) Anti factor H autoantibodies block C-terminal recognition function of factor H in hemolytic uremic syndrome. Blood 110(5):1516–1518CrossRefGoogle Scholar
  6. 6.
    Strobel S, Hoyer PF, Mache CJ, Sulyok E, Liu WS, Richter H, Oppermann M, Zipfel PF, Jozsi M (2010) Functional analyses indicate a pathogenic role of factor H autoantibodies in atypical haemolytic uraemic syndrome. Nephrol Dial Transplant 25(1):136–144CrossRefGoogle Scholar
  7. 7.
    Blanc C, Roumenina LT, Ashraf Y, Hyvarinen S, Sethi SK, Ranchin B, Niaudet P, Loirat C, Gulati A, Bagga A, Fridman WH, Sautes-Fridman C, Jokiranta TS, Fremeaux-Bacchi V, Dragon-Durey MA (2012) Overall neutralization of complement factor H by autoantibodies in the acute phase of the autoimmune form of atypical hemolytic uremic syndrome. J Immunol 189(7):3528–3537CrossRefGoogle Scholar
  8. 8.
    Dragon-Durey MA, Loirat C, Cloarec S, Macher MA, Blouin J, Nivet H, Weiss L, Fridman WH, Fremeaux-Bacchi V (2005) Anti-factor H autoantibodies associated with atypical hemolytic uremic syndrome. J Am Soc Nephrol 16(2):555–563CrossRefGoogle Scholar
  9. 9.
    Krishnappa V, Gupta M, Elrifai M, Moftakhar B, Ensley MJ, Vachharajani TJ, Sethi SK, Raina R (2018) Atypical hemolytic uremic syndrome: a meta-analysis of case reports confirms the prevalence of genetic mutations and the shift of treatment regimens. Ther Apher Dial 22(2):178–188CrossRefGoogle Scholar
  10. 10.
    Lee BH, Kwak SH, Shin JI, Lee SH, Choi HJ, Kang HG, Ha IS, Lee JS, Dragon-Durey MA, Choi Y, Cheong HI (2009) Atypical hemolytic uremic syndrome associated with complement factor H autoantibodies and CFHR1/CFHR3 deficiency. Pediatr Res 66(3):336–340CrossRefGoogle Scholar
  11. 11.
    Hofer J, Janecke AR, Zimmerhackl LB, Riedl M, Rosales A, Giner T, Cortina G, Haindl CJ, Petzelberger B, Pawlik M, Jeller V, Vester U, Gadner B, van Husen M, Moritz ML, Würzner R, Jungraithmayr T, German-Austrian HUS Study Group (2013) Complement factor H-related protein 1 deficiency and factor H antibodies in pediatric patients with atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol 8(3):407–415CrossRefGoogle Scholar
  12. 12.
    Abarrategui-Garrido C, Martinez-Barricarte R, Lopez-Trascasa M, de Cordoba SR, Sanchez-Corral P (2009) Characterization of complement factor H-related (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome. Blood 114(19):4261–4271CrossRefGoogle Scholar
  13. 13.
    Moore I, Strain L, Pappworth I, Kavanagh D, Barlow PN, Herbert AP, Schmidt CQ, Staniforth SJ, Holmes LV, Ward R, Morgan L, Goodship TH, Marchbank KJ (2010) Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome. Blood 115(2):379–387CrossRefGoogle Scholar
  14. 14.
    Ripoche J, Day AJ, Harris TJ, Sim RB (1988) The complete amino acid sequence of human complement factor H. Biochem J 249(2):593–602CrossRefGoogle Scholar
  15. 15.
    DiScipio RG (1992) Ultrastructures and interactions of complement factors H and I. J Immunol 149(8):2592–2599PubMedGoogle Scholar
  16. 16.
    Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, Lopez-Trascasa M, Sanchez-Corral P (2004) The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol 41(4):355–367CrossRefGoogle Scholar
  17. 17.
    Bhattacharjee A, Lehtinen MJ, Kajander T, Goldman A, Jokiranta TS (2010) Both domain 19 and domain 20 of factor H are involved in binding to complement C3b and C3d. Mol Immunol 47(9):1686–1691CrossRefGoogle Scholar
  18. 18.
    Kajander T, Lehtinen MJ, Hyvarinen S, Bhattacharjee A, Leung E, Isenman DE, Meri S, Goldman A, Jokiranta TS (2011) Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost discrimination by complement. Proc Natl Acad Sci U S A 108(7):2897–2902CrossRefGoogle Scholar
  19. 19.
    Morgan HP, Schmidt CQ, Guariento M, Blaum BS, Gillespie D, Herbert AP, Kavanagh D, Mertens HD, Svergun DI, Johansson CM, Uhrín D, Barlow PN, Hannan JP (2011) Structural basis for engagement by complement factor H of C3b on a self surface. Nat Struct Mol Biol 18(4):463–470CrossRefGoogle Scholar
  20. 20.
    Giannakis E, Male DA, Ormsby RJ, Mold C, Jokiranta TS, Ranganathan S, Gordon DL (2001) Multiple ligand binding sites on domain seven of human complement factor H. Int Immunopharmacol 1(3):433–443CrossRefGoogle Scholar
  21. 21.
    Song D, Liu XR, Chen Z, Xiao HJ, Ding J, Sun SZ, Liu HY, Guo WY, Wang SX, Yu F, Zhao MH, Chinese Renal-TMA Network Institutes (2017) The clinical and laboratory features of Chinese Han anti-factor H autoantibody-associated hemolytic uremic syndrome. Pediatr Nephrol 32(5):811–822CrossRefGoogle Scholar
  22. 22.
    Geerdink LM, Westra D, van Wijk JA, Dorresteijn EM, Lilien MR, Davin JC, Komhoff M, Van Hoesck K, van der Vlugt A, van den Heuvel LP, van de Kar NC (2012) Atypical hemolytic uremic syndrome in children: complement mutations and clinical characteristics. Pediatr Nephrol 27(8):1283–1291CrossRefGoogle Scholar
  23. 23.
    Watson R, Lindner S, Bordereau P, Hunze EM, Tak F, Ngo S, Zipfel PF, Skerka C, Dragon-Durey MA, Marchbank KJ (2014) Standardisation of the factor H autoantibody assay. Immunobiology 219(1):9–16CrossRefGoogle Scholar
  24. 24.
    Dragon-Durey MA, Sethi SK, Bagga A, Blanc C, Blouin J, Ranchin B, André JL, Takagi N, Cheong HI, Hari P, Le Quintrec M, Niaudet P, Loirat C, Fridman WH, Frémeaux-Bacchi V (2010) Clinical features of anti-factor H autoantibody- associated hemolytic uremic syndrome. J Am Soc Nephrol 21(12):2180–2187CrossRefGoogle Scholar
  25. 25.
    Hellwage J, Jokiranta TS, Friese MA, Wolk TU, Kampen E, Zipfel PF, Meri S (2002) Complement C3b/C3d and cell surface polyanions are recognized by overlapping binding sites on the most carboxyl-terminal domain of complement factor H. J Immunol 169(12):6935–6944CrossRefGoogle Scholar
  26. 26.
    Chua JS, Baelde HJ, Zandbergen M, Wilhelmus S, van Es LA, de Fijter JW, Bruijn JA, Bajema IM, Cohen D (2015) Complement factor C4d is a common denominator in thrombotic microangiopathy. J Am Soc Nephrol 26:2239–2247CrossRefGoogle Scholar
  27. 27.
    Roux KH, Strelets L, Michaelsen TE (1997) Flexibility of human IgG subclasses. J Immunol 159(7):3372–3382PubMedGoogle Scholar
  28. 28.
    Hamilton RG (1987) Human IgG subclass measurements in the clinical laboratory. Clin Chem 33(10):1707–1725PubMedGoogle Scholar
  29. 29.
    Nayak A, Pednekar L, Reid KB, Kishore U (2012) Complement and non-complement activating functions of C1q: a prototypical innate immune molecule. Innate Immun 18(2):350–363CrossRefGoogle Scholar
  30. 30.
    Manuelian T, Hellwage J, Meri S, Caprioli J, Noris M, Heinen S, Jozsi M, Neumann HP, Remuzzi G, Zipfel PF (2003) Mutations in factor H reduce binding affinity to C3b and heparin and surface attachment to endothelial cells in hemolytic uremic syndrome. J Clin Invest 111(8):1181–1190CrossRefGoogle Scholar
  31. 31.
    Yamanaka N, Shimizu A (1999) Role of glomerular endothelial damage in progressive renal disease. Kidney Blood Press Res 22(1–2):13–20CrossRefGoogle Scholar
  32. 32.
    Jozsi M, Heinen S, Hartmann A, Ostrowicz CW, Halbich S, Richter H, Kunert A, Licht C, Saunders RE, Perkins SJ, Zipfel PF, Skerka C (2006) Factor H and atypical hemolytic uremic syndrome: mutations in the C-terminus cause structural changes and defective recognition functions. J Am Soc Nephrol 17(1):170–177CrossRefGoogle Scholar
  33. 33.
    Ueda Y, Mohammed I, Song D, Gullipalli D, Zhou L, Sato S, Wang Y, Gupta S, Cheng Z, Wang H, Bao J, Mao Y, Brass L, Zheng XL, Miwa T, Palmer M, Dunaief J, Song WC (2017) Murine systemic thrombophilia and hemolytic uremic syndrome from a factor H point mutation. Blood 129(9):1184–1196CrossRefGoogle Scholar
  34. 34.
    Jokiranta TS, Hellwage J, Koistinen V, Zipfel PF, Meri S (2000) Each of the three binding sites on complement factor H interacts with a distinct site on C3b. J Biol Chem 275(36):27657–27662PubMedGoogle Scholar
  35. 35.
    Schmidt CQ, Herbert AP, Kavanagh D, Gandy C, Fenton CJ, Blaum BS, Lyon M, Uhrin D, Barlow PN (2008) A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol 181(4):2610–2619CrossRefGoogle Scholar
  36. 36.
    Clark SJ, Ridge LA, Herbert AP, Hakobyan S, Mulloy B, Lennon R, Wurzner R, Morgan BP, Uhrin D, Bishop PN, Day AJ (2013) Tissue-specific host recognition by complement factor H is mediated by differential activities of its glycosaminoglycan-binding regions. J Immunol 190(5):2049–2057CrossRefGoogle Scholar
  37. 37.
    Fedarko NS, Fohr B, Robey PG, Young MF, Fisher LW (2000) Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack. J Biol Chem 275(22):16666–16672CrossRefGoogle Scholar
  38. 38.
    Dragon-Durey MA, Blanc C, Garnier A, Hofer J, Sethi SK, Zimmerhackl LB (2010) Anti-factor H autoantibody-associated hemolytic uremic syndrome: review of literature of the autoimmune form of HUS. Semin Thromb Hemost 36(6):633–640CrossRefGoogle Scholar
  39. 39.
    Bhattacharjee A, Reuter S, Trojnar E, Kolodziejczyk R, Seeberger H, Hyvarinen S, Uzonyi B, Szilagyi A, Prohaszka Z, Goldman A, Jozsi M, Jokiranta TS (2015) The major autoantibody epitope on factor H in atypical hemolytic uremic syndrome is structurally different from its homologous site in factor H-related protein 1, supporting a novel model for induction of autoimmunity in this disease. J Biol Chem 290(15):9500–9510CrossRefGoogle Scholar

Copyright information

© IPNA 2018

Authors and Affiliations

  • Wei-yi Guo
    • 1
    • 2
    • 3
    • 4
  • Di Song
    • 1
    • 2
    • 3
    • 4
  • Xiao-rong Liu
    • 5
    Email author
  • 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
    Email author
  • 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

Personalised recommendations