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Anti-Factor B Antibodies and Acute Postinfectious

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GN in Children
Sophie Chauvet,1,2,3 Romain Berthaud,3,4 Magali Devriese,5 Morgane Mignotet,5
Paula Vieira Martins,5 Tania Robe-Rybkine,1 Maria A. Miteva,3,6 Aram Gyulkhandanyan,7
Amélie Ryckewaert,8 Ferielle Louillet,9 Elodie Merieau,10 Guillaume Mestrallet,11
Caroline Rousset-Rouvière,12 Eric Thervet,2,3 Julien Hogan,13 Tim Ulinski,14
Bruno O. Villoutreix,3,15 Lubka Roumenina,1 Olivia Boyer,3,4,16 and
Véronique Frémeaux-Bacchi1,5,3
Due to the number of contributing authors, the affiliations are listed at the end of this article.

ABSTRACT
Background The pathophysiology of the leading cause of pediatric acute nephritis, acute postinfectious
GN, including mechanisms of the pathognomonic transient complement activation, remains uncertain. It
shares clinicopathologic features with C3 glomerulopathy, a complement-mediated glomerulopathy that,
unlike acute postinfectious GN, has a poor prognosis.
Methods This retrospective study investigated mechanisms of complement activation in 34 children
with acute postinfectious GN and low C3 level at onset. We screened a panel of anticomplement
protein autoantibodies, carried out related functional characterization, and compared results with those
of 60 children from the National French Registry who had C3 glomerulopathy and persistent
hypocomplementemia.
Results All children with acute postinfectious GN had activation of the alternative pathway of the com-
plement system. At onset, autoantibodies targeting factor B (a component of the alternative pathway C3
convertase) were found in a significantly higher proportion of children with the disorder versus children
with hypocomplementemic C3 glomerulopathy (31 of 34 [91%] versus 4 of 28 [14%], respectively). In acute
postinfectious GN, anti-factor B autoantibodies were transient and correlated with plasma C3 and soluble
C5b-9 levels. We demonstrated that anti-factor B antibodies enhance alternative pathway convertase
activity in vitro, confirming their pathogenic effect. We also identified crucial antibody binding sites on
factor B, including one correlated to disease severity.
Conclusions These findings elucidate the pathophysiologic mechanisms underlying acute postinfectious

CLINICAL RESEARCH
GN by identifying anti-factor B autoantibodies as contributing factors in alternative complement pathway
activation. At onset of a nephritic syndrome with low C3 level, screening for anti-factor B antibodies
might help guide indications for kidney biopsy to avoid misdiagnosed chronic glomerulopathy, such as
C3 glomerulopathy, and to help determine therapy.

JASN 31: 829–840, 2020. doi: https://doi.org/10.1681/ASN.2019080851

Acute postinfectious GN (APIGN) is the leading
cause of acute nephritis in children.1,2 Streptococcal
skin or throat infections are still the main triggers of
the disease in children. After a latency period after
infection, the typical presentation is an acute ne-
phritic syndrome with low C3 levels sometimes
associated to a nephrotic syndrome, and rarely to
a rapidly progressive GN. The diagnosis is mainly
Received August 28, 2019. Accepted December 26, 2019.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Veronique Frémeaux-Bacchi, Department
of Immunology, European Hospital Georges Pompidou, 20 rue
Leblanc 75015 Paris, France. Email: veronique.fremeaux-bacchi@
aphp.fr
Copyright © 2020 by the American Society of Nephrology

JASN 31: 829–840, 2020 ISSN : 1046-6673/3104-829 829

 CLINICAL RESEARCH www.jasn.org
based on clinical and biologic features and confirmed by nor-
malization of C3 levels within a few days or weeks and by the
resolution of nephritic syndrome either with supportive care
only, or with a short corticosteroid therapy.3 Kidney biopsy is
not required in most children.3 It is only indicated in case of
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atypical presentation or course (i.e., persistent hypocomple-
mentemia beyond 3 months) to rule out differential diagnosis
or to assess prognosis in severe forms. Using light micros-
copy, APIGN is characterized by glomerular cell proliferation
with endocapillary hypercellularity due to varying degrees of
neutrophil recruitment. Characterization by immunofluo-
rescence microscopy involves bright glomerular C3 staining
with typical subepithelial deposits associated or not with
Ig staining. Mechanisms underlying the glomerular lesions
remain unclear. It was first suggested that the formation
of immune complexes, containing specific antigens (i.e.,
streptococcal antigens), and their deposition within the glo-
merular tuft drive complement activation and immune cell
infiltration.4 Later, it was proposed that the alternative pathway
of the complement system has a major contribution in APIGN
as assessed by decreased plasma C3 and normal plasma C4
levels in a large majority of patients and the demonstration
that streptococcal antigens were able to directly activate the
complement alternative pathway.5 The main differential diag-
nosis in a child with postinfectious acute nephritic syndrome
and low C3 levels is C3 glomerulopathy (C3G), a glomerul-
opathy of poor prognosis that warrants prompt specific
treatment.6,7 C3G is mediated by acquired or inherited dys-
regulation of the complement alternative pathway, resulting
in prolonged low plasma C3 levels in half of the patients
and predominant C3 deposition in glomeruli.8,9 Consider-
able overlapping in the clinical, biologic, and histopatho-
logic features of APIGN with C3G hampers appropriate
management.7
This study was designed to investigate the mechanism of
complement pathway activation in a cohort of patients with
APIGN. We discovered pathogenic anti-factor B (anti-FB)
autoantibodies that drive complement overactivation in
APIGN in contrast to C3G. The screening of anti-FB anti-
bodies may become a useful tool for guiding diagnosis and
therapeutic strategies in patients with nephritic syndrome
and low C3 levels.
METHODS
Study Population
We conducted a multicentric, retrospective study on 34 chil-
dren diagnosed with APIGN between April 2008 and August
2017. The children’s plasma was sent to the French Reference
Laboratory in the Georges Pompidou European Hospital
(Paris) for complement analyses. Patients were eligible if
they were ,18 years of age, with clinical and/or biopsy-
proven diagnosis of APIGN with low plasma C3 levels followed
by C3 normalization within 90 days (Figure 1, Supplemental
830 JASN
Significance Statement
Acute postinfectious GN, the leading cause of acute nephritis in
children, associates with transient complement activation of un-
determined mechanism. Its clinical features overlap considerably
with those of C3 glomerulopathy, a severe chronic condition. In this
retrospective study, the authors demonstrated that in more than
90% of children with acute postinfectious GN, complement over-
activation results from activation of the alternative pathway of the
complement system, driven by transient presence of autoanti-
bodies targeting factor B, a component of the alternative C3 con-
vertase. They also identified crucial antibody binding sites on factor
B, including one correlated to disease severity at onset. The pres-
ence of anti-factor B antibodies was highly specific to acute post-
infectious GN, suggesting that screening for these antibodies might
help clinicians distinguish the disorder during its acute phase from
C3 glomerulopathy.
Methods). For comparison, we used clinical and biologic
data of 60 children from the National French Registry with
biopsy-proven C3G and persistent hypocomplementemia
defined by low C3 levels for at least 1 year. In this group,
only plasma samples collected at the acute phase, available in
28 of 60 hypocomplementemic C3G (H-C3G) patients, were
used for antibody screening (Supplemental Figure 1). His-
tologic criteria used for diagnosis of APIGN and C3G are
provided in the Supplemental Methods. The local ethical
committee approved the study and written informed consent
was obtained from all patients’ legal guardians for genetic
analysis.
Assays for Complement Component and Complement
Gene Analysis
C3, C4, and soluble C5b-9 (sC5b-9) were measured in patients’
EDTA plasma samples. Screening for anti-factor H, anti-C3b,
anti-C3bBb, anti-FB autoantibodies in patients’ plasma
and study of antibody isotype specificity were performed
by ELISA (Supplemental Methods).10–12 Genetic variation
screening of complement factor H (CFH), complement factor I,
C3, complement FB (CFB), and complement factor H related
5 (CFHR5) genes and complex rearrangement between
CFH/CFHRs were performed as previously described.12 Results
of genetic analysis were compared with that of 45 of 60 patients
with H-C3G with available DNA, 80 French volunteers
who were healthy, 13 and European individuals from the
1000 Genomes Project.14
Epitope Mapping and Functional Characterization of
Anti-FB Antibodies
By ELISA, we tested the ability of anti-FB autoantibodies to
bind to 32 linear peptides of FB or 15 recombinant FB proteins
carrying selected amino acid changes generated by site-
directed mutagenesis as previously described (Supplemental
Methods).15 Peptides and amino acid changes were mapped
on the structure of FB, C3bB, and C3bBb using the Pymol
software and the atomic coordinates of FB,16 C3bBD,17 and
C3bBb18 (Supplemental Methods).
JASN 31: 829–840, 2020

To determine the functional consequences of anti-FB au-
toantibodies on complement activation, total IgG purified
from EDTA plasma of patients with APIGN were tested
for their capacity to enhance fluid-phase and cell-surface
alternative pathway convertase activity. In fluid phase, IgG
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from patients with APIGN were incubated in normal
human serum as a source of complement in the presence
of EGTA-magnesium to block the classic/lectin pathways.
The release of cleavage fragments C3a, Bb, C5a, and sC5b-9 was
then measured by ELISA (Supplemental Methods). We studied
the surface activation of the complement alternative pathway
by ELISA and hemolytic assays. We tested both the ability of
anti-FB autoantibodies to enhance formation of C3bB and
C3bBb complexes on plates, and we tested the ability of anti-
FB antibodies to enhance formation of C3 convertase or to
stabilize a preformed C3 convertase on sensitized sheep
erythrocytes. Lysis of erythrocytes measured by spectrome-
try (OD 414 nm) directly reflects the convertase activity
(Supplemental Methods).
Statistical Analyses
Data are expressed as number and percentage for categoric
variables and as median and interquartile range for continu-
ous variables. Differences among the APIGN and C3G groups
were evaluated using the Mann–Whitney and Kruskal–Wallis
tests, as appropriate, for comparison of continuous variables.
Chi-squared or Fisher exact tests were used for comparison of
categoric variables. The correlation coefficient between two
normally or non-normally distributed variables was calculated
using the Pearson method and the Spearman correlation co-
efficient, respectively. Statistical analyses were generated using
Prism 5 GraphPad software. P values ,0.05 were considered
significant.
RESULTS
Clinical Features of Patients at the Acute Phase and
during Follow-Up
A total of 34 children with APIGN were included in the study
(Figure 1). Main clinical data at diagnosis are detailed in
Table 1. The median (interquartile range) age at diagnosis
was 5.6 years (4.7–7.6). All 34 children had nephritic syn-
drome. A total of 15 of 34 patients had criteria of severe
disease with nephrotic syndrome and/or dialysis requirement.
Of the 34 patients, 82% had a clear history of recent infection
and renal disease occurred after a median latency period of
5 (1–10) days. This result is consistent with data from a large
pediatric study reporting that 16% of children had no recent
history of infection at the time of APIGN diagnosis.19 At
the time of renal diagnosis, the infection site was available for
23 patients and Streptococcus was the likely responsible patho-
gen in 20 of 23 (87%) patients. Kidney biopsy performed in
20 of 34 patients (59%) was compatible with APIGN in all cases
(Supplemental Table 1). Endocapillary proliferation with
JASN 31: 829–840, 2020
www.jasn.org CLINICAL RESEARCH
Suspected APIGN
N=62
Incomplete clinical data
N=5
APIGN diagnosis ruled out
N=10
Clinically confirmed APIGN
N=47
Persistent low C3 level after
90 days
N=6
No available control sample
before 90 days of follow up
N=7
APIGN with transient low C3
N=34
Biopsy-proven APIGN
N=20/34
Figure 1. Thirty-four children with APIGN were included in the
study. Among the 62 patients with suspected APIGN, five pa-
tients had incomplete data for making a final diagnosis and in ten
patients APIGN diagnosis was ruled out. Six patients had anam-
nestic, clinical, and biologic data that supported the diagnosis of
APIGN but plasma C3 levels remained low for .90 days. In seven
other cases with APIGN, normalization of C3 levels was not as-
sessed within 90 days after the diagnosis.
polymorphonuclear cell infiltration and subepithelial deposits
by light microscopy or C3 deposition by immunofluorescence
were also identified in patients with H-C3G. Chronic lesions
(i.e., sclerotic glomeruli, interstitial fibrosis, or tubular atro-
phy) were only found in patients with C3G (Supplemental
Results, Supplemental Table 1). A total of 13 children (39%)
received corticosteroids. After a median follow-up of 316 days
(154–496), recovery from nephritic syndrome was observed in
all 32 children with complete available data. Notably, none had
chronic renal insufficiency. One child had persistent micro-
scopic hematuria and proteinuria at a follow-up of 57 days,
another patient had normal urinary sediment while receiving
enalapril after a follow-up of 864 days, and one had persistent
hypertension 127 days after the onset. In all three patients, a
biopsy had been performed and kidney histology was com-
patible with the diagnosis of APIGN (Table 1, Supplemental
Results).
Anti-Factor B and Postinfectious GN 831
 CLINICAL RESEARCH www.jasn.org

Table 1. Clinical data for treatment of children with APIGN at admission and than in the H-C3G group (22 of 27 [81%]
last follow-up versus 31 of 55 [56%], P50.02) (Table 2).
Number At the acute phase, anti-FB autoanti-
Characteristic Value
(n534) bodies were identified in 31 of 34 (91%)
Median age, yr (IQR) [min–max] 34 5.6 (4.7–7.7) children with APIGN and in four of
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[3.2–13.2] 28 (14%) children with H-C3G (P50.001;

Sex ratio, n, male/female 34 23/11 odds ratio, 62.00; 95% confidence interval
Anamnesis [CI], 12.65 to 303.8; relative risk, 7.97)
History of recent infection, n (%) 33 27 (82%) (Figure 2A, Table 2). Sensitivity and speci-
Median time between infection and first renal 27 5 (1–10) ficity of anti-FB antibodies for APIGN
symptom, d (IQR)
diagnosis were 95% (95% CI, 85% to
Type of infection, n (%) 23
99%) and 82% (95% CI, 66% to 93%)
Tonsillitis 4 (17%)
Peritonsilar abscess 1 (4%)
(Figure 2B). Frequency of anti-FB anti-
Retropharyngeal abscess 1 (4%) bodies remained significantly higher
Acute otitis media 2 (9%) even when comparing subgroups of
Pneumonia 1 (4%) biopsy-proven APIGN (19 of 20 [95%])
Cutaneous infections 0 (0%) and H-C3G with infectious trigger (0 of
Other 14 (61%) 10 [0%], P,0.001) (Supplemental
Likely responsible pathogens, n (%) 28 22 (79%) Figure 2A, Supplemental Table 2). Anti-
Streptococcus 20 (71%) FB antibodies were not found in 15 pa-
Mycoplasm pneumoniae 2 (7%) tients with IgA nephropathy and 26 with
Other 4 (14%)
lupus nephritis used as control groups
No pathogen identified 28 6 (21.4%)
(Supplemental Figure 2B). A total of
No screening for infectious pathogen 34 6 (18%)
Clinical presentation at diagnosis, n (%)
24 of 31 patients with APIGN were
Macroscopic hematuria 34 24 (71%) tested for anti-FB IgG subclasses. In
Microscopic hematuria 34 5 (15%) 18 of 24 (75%) samples, anti-FB antibodies
Edema 32 14 (44%) were of the IgG1 isotype (Supplemental
Hypertension 34 18 (53%) Results, Supplemental Table 3).
Biology at diagnosis At disease onset, plasma C3 levels were
Proteinuria, n (%) 34 34 (100%) inversely correlated with anti-FB autoanti-
Nephrotic syndrome, n (%) 33 11 (33%) body titer, and plasma sC5b-9 levels were
ARF, n (%) 34 27 (79%) positively correlated with them (P50.01,
Median serum creatinine, mmol/L (IQR) [min–max] 32 70 (48–106) [31–609]
R 2
50.21 and P50.003, R250.41, respec-
Median eGFR, ml/min per 1.73 m2 (IQR) [min–max] 29 74 (43–96) [7–122]
tively) (Figure 2, C and D). Correlation be-
RRT, n (%) 33 5 (15%)
Kidney biopsy, n (%) 34 20 (59%)
tween C3 level and anti-FB titer tend to be
Evolution, n (%) significant and correlation between sC5b-9
Treatment 33 13 (39%) level and antibody titer remained significant
Corticosteroids 13 (39%) in patients with isolated anti-FB antibodies
Nephritic syndrome recovery 32 32 (100%) (Supplemental Figure 3, A and B). In con-
Persistent isolated microscopic hematuria 31 4 (13%) trast, C3 and sC5b-9 levels did not correlate
Renal abnormalities at last follow-up 32 4 (12%) with anti-C3bBb antibody titer in patients
Persistent proteinuria 32 2 (6%) with both anti-FB and anti-C3bBb anti-
No proteinuria under ACEI 32 1 (3%) bodies (Supplemental Figure 3, C and D).
Persistent hypertension 32 1 (3%)
During the follow-up, the anti-FB an-
Persistent renal failure 32 0 (0%)
tibody levels became negative in 23 of 31
Death 34 0 (0%)
Median follow-up duration, d (IQR) [min–max] 34 316 (154–496)
or decreased in six of 31 patients with
[90–1671] APIGN (Figure 3, A and B, Supplemental
IQR, interquartile range; ACEI, angiotensin-converting enzyme inhibitor.
Table 4). At the last anti-FB antibody
screening, all patients except three had nor-
mal C3 levels. None of the 23 tested patients
Complement Activation Biomarkers and Identification with APIGN had any anti-FH autoantibodies, whereas ten of
of Anti-FB Antibodies in Children with APIGN 48 (21%) children with H-C3G did have anti-FH autoantibodies
At diagnosis, all patients had low C3 levels and normal C4 (P50.02). Ten of 31 (32%) children from the APIGN group had
levels. The proportion of tested children with sC5b-9 above anti-C3bBb autoantibodies versus 24 of 41 (59%) in the H-C3G
the normal range was significantly higher in the APIGN group group (P50.033) (Table 2).

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Table 2. Immunologic findings of patients with APIGN and amount of C3a, Ba, C5a, and sC5b9 released in normal human
H-C3G at onset of disease serum by ELISA after incubation with IgG from patients or
Measure APIGN (n534) H-C3G (n560) P Value healthy donors. After incubation, Bb and C3a were signifi-
C3 level, mg/L (660–1250) 217 (138–310) 257 (138–449) 0.12 cantly increased by four of 13 tested patients’ IgG, and C5a
Low C3 level (,660 mg/L) 100% 100% 1 and sC5b-9 levels were significantly increased by five of
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C4 level, mg/L (93–380) 247 (189–316) 176 (138–231)
Low C4 level, mg/L 0 (0%) 3/60 (5%)
sC5b-9 level (,460 ng/ml) 877 (659–1700) 634 (368–1565)
Elevated sC5b-9 22 of 27 (81%) 31 of 55 (56%)
(.460 ng/ml)
Anti-FB Abs, n (%)a 31 of 34 (91%) 4 of 28 (14%)b
Anti-C3bBb Abs, n (%)a 10 of 31 (32%) 24 of 41 (59%)
Anti-FH Abs, n (%)a 0 of 23 (0%) 10 of 48 (21%)
Anti-C3b Abs, n (%)a 4 of 34 (12%)c 3 of 41 (9%)d
Results are expressed as median (interquartile range), unless otherwise
specified. C3, C4 and sC5b-9 levels are reference values obtained from 100
french healthy donors. Abs, antibodies; FH, factor H.
a
Autoantibodies were screened by ELISA.
b
Anti-FB antibodies were screened at acute phase of H-C3G.
c
Anti-C3b antibodies were combined with anti-FB antibodies in four out of
four cases.
d
Anti-C3b antibodies were isolated in three out of three cases.
Epitope Mapping of Anti-FB Antibodies in APIGN
Reactivity against 32 linear peptides of FB (Supplemental
Table 5) in plasma from 29 of 34 patients with APIGN was
tested by ELISA. Nine of these 32 peptides were recognized
in more than four patient samples and were considered hot-
spot sequences (Figure 4A, Supplemental Figure 4). Peptide
624–637, located on the serine protease (SP) domain, was
more frequently recognized by IgG from children with non-
severe disease onset (as defined by absence of nephrotic syn-
drome and/or dialysis requirement) (five of 15 [33%] versus
zero of 13 [0%]; P50.04) (Supplemental Figures 5 and 6). In
the same area of peptide 624–637, two peptides (591–606
and 660–674) were only recognized by IgG from patients
with nonsevere renal presentation, suggesting that the bind-
ing of anti-FB antibodies in the vicinity of the catalytic triad,
in the SP domain, could modulate the pathogenic effect of
anti-FB antibodies.
To confirm the importance of these hotspots for anti-FB
antibody binding, we produced 15 recombinant FB mutants,
including nine recombinant FBs bearing amino acid changes
on the identified hotspots. By ELISA, we showed that six of
these nine recombinant FBs were associated with significant
modification in anti-FB antibody binding compared with that
with wild-type recombinant FB (Figure 4B). Among the six
recombinant FB mutants with mutations outside the hotspot
regions, only one showed modified IgG binding (Supplemental
Figure 7). Positions of the seven amino acid changes of interest
and the nine hotspot peptides are indicated on the molecular
structure of FB (Figure 4C) and FB within C3bB and C3bBb
complexes (Supplemental Figure 8).
Functional Consequences of Anti-FB Autoantibodies
To test the capacity of patients’ IgG to induce activation of the
fluid-phase alternative pathway convertase, we measured the
0.003 13 tested patients’ IgG compared with IgG from healthy donors
0.55 (above mean12 SD of healthy donors), suggesting that anti-
0.1 FB antibodies are able to induce overactivation of fluid-phase
0.02 C3 and C5 convertases (Figure 5, A–D).
By ELISA assays, we found that C3b binding to FB and C3bBb
,0.001
formation on plates were significantly enhanced in the presence
0.033
of purified IgG from 15 of 28 (54%) and from four of 20 (20%)
0.02
0.7
patients with APIGN compared with IgG from healthy donors,
respectively (Supplemental Figure 9, A and B).
On sheep erythrocytes, anti-FB antibodies enhance C3
convertase activity, resulting in a significant increase of eryth-
rocyte lysis compared with lysis obtained with Ig from healthy
donors (P50.02) (Figure 5E, Supplemental Figure 10). To test
the capacity of anti-FB antibodies to stabilize a preformed C3
convertase, sheep erythrocytes bearing C3bBb were incubated
in EDTA, in the presence of total purified IgG from patients
with APIGN. We used as positive control Ig from patients with
H-C3G who were positive for anti-C3bBb antibodies and Ig
from healthy donors was used as negative control. The lysis
change between baseline and after 20 minutes of incubation,
reflecting the dissociation of C3 convertase, was significantly in-
creased in patients with APIGN compared with patients with
H-C3G (P50.002). These results suggest that anti-FB antibodies
are not able to stabilize a preformed C3 convertase (Figure 5F).
Complement Gene Analysis in Patients with APIGN
and H-C3G
Genetic analysis was performed in 23 of 34 patients with
APIGN and 45 of 60 patients with H-C3G. Rare complement
gene variants (defined by a minor allele frequency ,0.1%)
were detected in 22% (five of 23) of patients with APIGN,
which was significantly higher than in healthy donors (6%,
P50.04) but similar to the control group of patients with
H-C3G (six of 45, 13%) (Supplemental Table 6). Among the
five rare variants identified in patients with APIGN, one is
located in the C-terminal portion of CFH which is involved
in the binding to surface-bound C3b and to negatively charged
cellular structures and is classified as probably damaging by in
silico prediction scores. Two variants in CFH and two in CFB
(without published functional study or without any demon-
strated functional effect) are classified as variants of undeter-
mined significance.24,25 The three variants in the CFH gene
identified in the H-C3G cohort are classified as pathogenic
and/or previously published (Supplemental Table 7).26,27
DISCUSSION
We demonstrated that the pathophysiology of APIGN in
children implies an activation of the complement alternative

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A B
<0.0001
<0.0001 0.61 100
2800
2200
1600
Anti-FB antibodies UA/ml 1400 75
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1200

Sensitivity
1000
50
800
600
25
400
200 AUC 0.94 [95% CI 0.88-1]
0 0
APIGN H-C3G HD 0 25 50 75 100
n=34 n=28 n=85 100 - Specificity

C D
2500
500

2000
400

sC5b-9 level (ng/ml)

1500
C3 (mg/L)

300

200 1000

100 500

0 0
0 1000 2000 3000 0 500 1000 1500 2000
Anti-FB Antibodies (UA) Anti-FB Antibodies (UA)
N=34/34 children with APIGN N=27/34 children with APIGN

Figure 2. Anti-FB antibodies at acute phase of the disease are specific of APIGN and correlate with C3 and sC5b-9 levels. (A) Reactivity
of autoantibodies against FB was tested by ELISA in plasma samples from 34 patients with APIGN, 28 patients with H-C3G (collected at
acute phase of disease), and 84 healthy donors. Results of antibodies are expressed in arbitrary units (UA). The cutoff of positivity was
determined with plasma samples from the 84 healthy donors (HD). (B) Receiving operater characteristic curve of the positivity of anti-FB
autoantibodies in APIGN. The area under the receiving operater characteristic curve for the use of anti-FB antibodies titer to dis-
criminate APIGN from H-C3G patients was 0.93 (95% CI, 0.88 to 0.99). The optimal cutoff point was 88.5, which had a sensitivity of
0.91 (95% CI, 0.76 to 0.98) and a specificity of 0.85 (95% CI, 0.71 to 0.94). (C and D) Correlation between titers of anti-FB antibodies
and C3 levels and sC5b9 investigated in 34 and 27 tested patients, respectively. (C) Plasma C3 levels inversely correlate with titers of
anti-FB antibodies (P50.01; 95% CI, 20.68 to 20.142; R520.46). (D) Soluble C5b9 levels in plasma correlate with anti-FB antibody
titers (P50.001; 95% CI, 0.35 to 0.82; R50.64). AUC, area under the curve.

pathway resulting from the presence of transient autoanti-
bodies targeting FB, a component of the alternative C3 con-
vertase. This is a major breakthrough in understanding the
mechanisms underlying this frequent disease.
Of the patients in this study who were clinically diagnosed
with APIGN, 90% had IgG autoantibodies that reacted with
FB. Upon activation of the alternative pathway, FB is cleaved
by factor D, yielding the noncatalytic portion Ba and the active
fragment Bb. The Bb fragment comprises two protein do-
mains: a von Willebrand (vW) type A domain, which associates
to C3b to form the C3 convertase C3bBb, and an SP domain
that cleaves C3. In APIGN, we found that anti-FB autoanti-
bodies are transient, mainly of the IgG1 subclass and drive
activation of the complement alternative pathway. Autoanti-
body titers correlated with the level of hypocomplementemia
and their disappearance was associated with normalization of
C3 and sC5b-9 in patients’ plasma. In vitro, we demonstrated
that anti-FB IgG enhances C3 convertase formation.
Pathogenic variants in CFB have been described in atypical
hemolytic uremic syndrome, another complement-mediated
disorder.16,20–23 These variants, located on the FB vW domain,
result in C3 convertase overactivation. Interestingly, we
identified hotspots for anti-FB autoantibody binding that
encompass the majority of the FB variants identified in atypical
hemolytic uremic syndrome, suggesting that anti-FB anti-
bodies binding on vW-domain hotspots could increase the
formation of the C3 convertase. We also identified hotspots
in the SP domain of FB that are in immediate proximity to
the catalytic triad, including one hotspot associated with
nonsevere disease onset (i.e., with no nephrotic syndrome
nor dialysis requirement). Binding of anti-FB antibodies on
the SP domain could modulate the C3 convertase activity

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A B 3000
34
33
32
31
30
2500
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29
28
27
26
25
24 2000

Anti-FB antibodies UA/ml

23
22
21
P7
20
19
18 1500
17
16
15
14
13
12 1000
11
10
9 P24
8 P28
7
6 500
5 P6
4
P32
3 P22
2 P29 P25
1
0
0 30 60 90 120 150 180 210 240 270 300 500 600 700 800 900 At acute phase last sample
days after first renal symptoms

Figure 3. Anti-FB antibodies in APIGN patients are transient. (A) Kinetics of anti-FB antibody positivity, C3 levels, and renal symptoms
in patient with APIGN according to renal symptoms. Bars in gray indicate the presence of renal symptoms (high BP and/or hematuria
and/or proteinuria and/or renal failure). Samples positive for anti-FB antibodies and low C3 levels are indicated by a red asterisk.
Samples positive for anti-FB antibodies with normal C3 levels are indicated by a blue asterisk. Samples negative for anti-FB antibodies
with low C3 levels are indicated by a yellow asterisk. Samples negative for anti-FB antibodies and/or normal C3 levels are indicated by a
green asterisk. In patients 2, 9, 11, 20, 21, and 27, measurements of C3 levels confirming normalization of C3 within 90 days after renal
diagnosis were performed in an external biologic center and were not indicated on the figure. (B) Titer of anti-FB autoantibodies at
acute phase and on the last available sample. In 31 of 34 patients with anti-FB antibodies at diagnosis, control of anti-FB reactivity was
available in 29 patients. For two patients (P7 and P24), no sample was available during follow-up. In six of 29 patients, antibody titer
remained positive during the follow-up in patient P28 (titers 622 UA at day 852), P6 (titer 364 UA at day 28), P22 (titer 189 UA at day 30),
P29 (titer 123 UA at day 75), P32 (titer 260 at day 32), and P25 (titer 103 at day 31). Dotted line indicates the positivity threshold
(.88 UA/ml).

enhanced by anti-FB antibodies binding to the vW domain.
Given the usually favorable outcome of APIGN, our findings
are unlikely to modify the classic management of APIGN.
Indeed, a strategy based on removal of anti-FB antibodies by
plasma exchange seems unnecessary in most patients but
may be discussed in the rare cases requiring RRT. Our find-
ings also improve our understanding of the mechanisms of
action of immunosuppressive therapies. Corticosteroids
could not only act as antiproliferative agents but could also
decrease the production of anti-FB antibodies when the use
of long-acting immunosuppressors seems less appropriate
in the setting of APIGN. Moreover, one can assume that a
transient inhibition of C5 may improve the outcome in se-
vere cases of APIGN that require dialysis, although further
confirmation is required.28
The mechanism of anti-FB autoantibody emergence re-
mains unclear. Streptococcal infection was identified in a large
majority of children with APIGN. We can hypothesize two
different mechanisms: (1) because streptococcal surface is
able to directly activate the alternative pathway,29 it may
lead to the formation of FB neoepitopes; and (2) streptococcal
proteins and FB may share common conformational and/or
cryptic epitopes. In both cases, streptococcal infection may
promote the emergence of transient anti-FB autoantibodies,
contributing to complement overactivation and deposition of
complement C3 breakdown products in the glomeruli. In this
hypothesis, the control of infection and clearance of strepto-
coccal antigens from glomeruli may result in anti-FB autoan-
tibody disappearance, leading to the decrease of complement
activation followed by resolution of nephritic syndrome. Anti-
FB antibodies are exceptional in C3G, identified in 3% of the
French cohort of adult patients with C3G11,30 and in 14% of
children with H-C3G in this study where they remain detect-
able over years.30 Anti-FB antibodies have also been described

JASN 31: 829–840, 2020 Anti-Factor B and Postinfectious GN 835

 CLINICAL RESEARCH www.jasn.org

A 10 *
8
* * *

patients (Nb)
*
6 * * * *
4
2
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251-265
261-277
268-283
290-307
300-314
310-326
331-347
342-356
366-380
376-391
386-397
407-421
417-431
429-443
439-451
456-468
471-485
498-511
509-523
520-534
532-546
556-569
575-589
584-597
591-606
603-619
624-637
634-654
660-674
680-686
693-705
719-739
vW domain SP domain

Catalytic triad H501 D551 S674

D251-
MIDAS S253- T328 D364
S255

B 1.5
OD(450nm)

1.0
m+2SD

0.5

m-2SD
0.0
T

8A

1A

6A

8A

6A

A
W

12

07

09
79
31

32

34

37

50
K3

P4

E5
E3
N

-D

D
5A
34
D

vW domain SP domain

C E379D
D378A

376-391

D254G K298E

F261L
342-356
300-314
P407A
310-326

M433I 429-443

K704A

624-637 K701A

719-739 520-534

498-511 E509A
K508R
E541A D506A

Figure 4. Anti-Fb antibodies recognize hotspots on serine protease and von Willebrand type A domains of FB. (A) Reactivity of au-
toantibodies against 16 linear peptides of vW type A domain and 16 linear peptides of SP domain of FB was tested by ELISA in plasma
samples from 29 patients with APIGN. A total of 27 of 32 peptides were recognized by at least one patient sample positive for anti-FB
antibodies. Five hotspot peptides of the vW type A domain and four hotspot peptides of the SP domain recognized by more than four
patient samples (indicated by an asterisk) were identified. Location of the amino acids forming the catalytic triads and MIDAS site are
indicated below the graph. (B) Reactivity against recombinant FB (wild-type and nine recombinant FBs bearing mutations on hotspot
peptide sequences [K312A, N318A, D321A, D378E, E379D P407A, D506A, E509A, and the double mutant D345A-D346A]) in plasma
samples from 13 patients with APIGN was tested by ELISA. Results are expressed in OD. Means62 SD (m12SD, m-2SD) of binding to
the wild type are indicated in dotted lines. Five mutations were associated with a significant decrease of anti-FB antibody binding to
recombinant FB (D321A, D378E, E379D, P407A, and D506A), whereas one mutation was associated with a significant increase (E509A).
(C) Localization of the hotspots on the FB structure. The five hotspot peptides of the vW domain are indicated by red ribbon, and the four
hotspot peptides of SP domain are indicated in green. The hotspot amino acid changes are in dark blue. Amino acids of the MIDAS are
indicated in magenta and amino acids of the catalytic triads are indicated in yellow. Cyan indicates amino acids referred to in the literature
as being associated with hyperfunctional C3 convertase.20–23 MIDAS, magnesium ion–dependent, metal ion–dependent adhesion site.

836 JASN JASN 31: 829–840, 2020

 www.jasn.org CLINICAL RESEARCH

A B
600 100
80

C3a (ng/ml)

Bb (μg/ml)
400 60
40
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200
20
0 0
HD-Ig APIGN-Ig anti-C3bBb HD-Ig APIGN-Ig anti-C3bBb
Ab/C3G Ab/C3G
n=7 n=13 n=8 n=7 n=13 n=8

C D
50
400
40

sC5b9 (μg/ml)
C5a (ng/ml)

300
30
200
20
100 10
0 0
HD-Ig APIGN-Ig anti-C3bBb HD-Ig APIGN-Ig anti-C3bBb
Ab/C3G Ab/C3G
n=7 n=13 n=8 n=7 n=13 n=8

E F
0.02 0.001
ΔLysis between T0 and T20 (%)

50 0.31 0.002
100

80 40
% of lysis

60 30

40 20

20 10

0 0
HD-Ig APIGN HD-Ig APIGN H-GC3
n=16 n=17 n=7 n=6 n=12

Figure 5. Anti-FB antibodies from children with APIGN enhance activity of fluid-phase and cell-surface C3 convertase but do not
stabilize solid-phase C3 convertase. Activation of the fluid-phase complement alternative pathway in the presence of anti-FB anti-
bodies. After incubation of normal human serum (EGTA-magnesium) in the presence of total purified IgG from 13 patients with APIGN,
eight patients with C3G positive for anti-C3bBb antibodies, and seven healthy controls, C3a, Bb, C5a, and sC5b9 generation was
quantified by ELISA. In the experimental condition, at baseline, median C3a, Bb, C5a, and sC5b9 levels in normal human serum are
6 ng/ml, 18 mg/ml, 9 ng/ml, and 1.8 mg/ml, respectively. After 30 minutes of incubation, median levels obtained with IgG from healthy
donors (HD-Ig) are 253.9, 40.6, 159 ng/ml, and 18.6 mg/ml, respectively. (A-B) C3a and Bb releases were significantly increased in
patients with APIGN (four of 13 and four of 13, respectively) and in patients with C3G positive for anti-C3bBb antibodies (four of eight
and four of eight patients, respectively) compared with IgG from healthy donors (upper mean12 SD). (C-D) C5a and sC5b9 were
significantly increased in patients with APIGN but not in the presence of anti-C3bBb antibodies from patients with C3G. (E-F) Activation
of the solid-phase complement alternative pathway in the presence of anti-FB antibodies. The capacity of total purified IgG from
patients and controls (healthy donors and patients with C3G positive for anti-C3bBb antibodies) to enhance C3/C5 convertase for-
mation and stabilization activity was tested using hemolytic assays. Results are expressed as the percentage of the maximum lysis of
erythrocytes (osmotic lysis with water). (E) To evaluate the capacity of anti-FB antibodies to enhance cell-surface C3 convertase formation,
C3 convertase was formed on sensitized sheep erythrocytes bearing C3b in the presence of Ig from patients with APIGN or controls. In the
presence of Ig from healthy donors, the median of the maximum lysis is 20%. Lysis was significantly increased in presence of Ig from patients
with APIGN. (F) To evaluate the capacity of anti-FB antibodies to stabilize a preformed surface-bound C3 convertase, total purified IgG from
patients with APIGN, diluted in gelatin veronal buffer-EDTA, were incubated on sheep erythrocyte bearing preformed C3bBb. IgG from
patients with H-C3G positive for anti-C3bBb antibodies were used as positive control and Ig from healthy donors as negative controls. At
baseline (T0), the median percentage of maximum lysis is 43% (40%–53%), 79% (43%–100%), and 75% (42%–100%) in the presence of Ig
from those with HD-Ig, APIGN, or H-C3G, respectively. At 20 minutes (T20), median percentage of maximum lysis is 13% (15%–19%),
48% (15%–65%) and 65% (31%–100%) in the presence of Ig from those with HD-Ig, APIGN, or H-C3G, respectively. The change in lysis (D)
between T0 and T20, reflecting dissociation of C3 convertase, is significantly increased in patients with APIGN compared with those
with H-C3G. Results are expressed as DLysis, representing the difference of lysis between baseline and after 20 minutes of incubation. An
increase of DLysis suggests a decay of the enzyme. Ranges are the extrems. HD-Ig, Ig from healthy donors.

JASN 31: 829–840, 2020 Anti-Factor B and Postinfectious GN 837

 CLINICAL RESEARCH www.jasn.org
in rare adult cases with immune complex GN in the setting of
concomitant infection of nonstreptococcal origin, suggesting
the potential role of other microorganisms in the emergence
of such autoantibodies.11
The contribution of rare variants in CFH and CFB genes
Downloaded from https://jasn.asnjournals.org at UPPSALA MEDICINSKA on May 12, 2020. Copyright 2020 The American Society of Nephrology
significantly enriched in children with APIGN needs to be
determined. It may signify that a specific genetic background
predisposes children to a complement-mediated disease or to
the emergence of anti-FB antibodies after a streptococcal
infection.
At the acute phase of the disease, anti-FB autoantibodies
may be of crucial help to distinguish APIGN from H-C3G, in
particular when H-C3G is triggered by infection. Both diseases
may share similar clinical features— i.e., present as acute
nephritic syndrome with low C3 levels—and cannot be dif-
ferentiated by renal biopsy alone.31 The distinction of both,
based on a bundle of arguments, remains crucial because the
two diseases have completely different outcomes. Complete
remission in APIGN is the rule, whereas H-C3G has poor renal
outcome despite intensified immunosuppression.32–34 In vitro,
we demonstrated that anti-FB autoantibodies are not able to
stabilize a C3 convertase formed on red blood cells. Therefore,
anti-FB antibodies cannot play the role of C3 nephritic factor
that per definition are antibodies that recognize a neoepitope
of C3bBb without binding FB or C3b alone and result in
stabilization of C3 convertase.35 C3 nephritic factors are
found in most patients with C3G and are correlated with
complement biomarkers. 12 In APIGN, anti-C3bBb anti-
bodies were found in less than a third of patients, were mainly
of the IgG3 subclass, and did not correlate with C3 and sC5b-
9 levels. Taken together, these findings suggest they are not
relevant in the APIGN process and, unlike anti-FB antibodies,
cannot help clinicians to distinguish between APIGN and
H-C3G.
The limitations of this study are the following: (1) pa-
tients with APIGN were recruited from hospitals and this
may have led to recruitment bias toward more severe cases of
APIGN36; (2) its retrospective nature has hampered exten-
sive/standardized complement exploration—we are now de-
signing a prospective study that will include incident cases of
both APIGN and H-C3G to confirm the specificity of the
anti-FB antibody assay; and (3) the role of Streptococcus
or other microorganisms on the emergence of anti-FB
antibodies requires further investigation. First, compari-
son of the amino acid sequence of identified peptides with
Streptococcus sequences did not identify any sequence ho-
mology; second, APIGN occurs after a latent period, render-
ing the identification of a specific bacterial strain at onset of
renal symptoms difficult.
In conclusion, our results provide new insights into the
pathophysiologic mechanisms underlying complement acti-
vation in APIGN in children. We identified anti-FB antibodies
as new players in the disease contributing to overactivation of
the complement alternative pathway. Therefore, anti-FB anti-
bodies may be of great help at the acute phase of nephritic
838 JASN
syndrome with low C3 levels to differentiate children with
anti-FB antibodies, who would likely have a good renal
prognosis without major support, from children without
any detectable anti-FB antibody, who would be at risk for
H-C3G requiring long-term immunosuppression and may
have worse renal prognosis.
ACKNOWLEDGMENTS
We thank clinicians who referred patients: Prof. Bernard Boudaillez
(Department of Pediatrics, University Hospital Center of Amiens,
Amiens, France), Dr. Pascal Saunier (Department of Pediatrics,
Fontainebleau Hospital Center, Fontainebleau, France), Dr. Gwenaelle
Roussey-Kesler (Department of Pediatric Nephrology, University
Hospital Center of Nantes, Nantes, France), Dr. Sophie Taque (De-
partment of Pediatrics, University Hospital Center of Rennes, Rennes,
France), and Dr. Robert Novo (Department of Pediatric Nephrology,
University Hospital Center–Jeanne de Flandre University, Lille,
France). We thank Prof. Chantal Loirat for her expert advice.
DISCLOSURES
Dr. Frémeaux-Bacchi received fees from Alexion Pharmaceuticals, Apellis,
and Roche for invited lectures and/or board membership and is the recipient
of a research grant from Alexion Pharmaceuticals. All remaining authors have
nothing to disclose.
FUNDING
This work was supported by a European Union Seventh Framework Pro-
gramme grant 2012-305608 (EURenOmics), Kidneeds research grant 2015,
Agence Nationale de la Recherche research grant ANR-16-CE18-0015-01,
CompC3, and the Fondation pour la Recherche Médicale (FDM
20130727355).
SUPPLEMENTAL MATERIAL
This article contains the following supplemental material online at
http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019080851/-/
DCSupplemental.
Supplemental Methods.
Supplemental Results.
Supplemental Table 1. Histopathological data of APIGN patients
and hypocomplementemic C3 glomerulopathy (with or without in-
fectious trigger).
Supplemental Table 2. Immunological findings of patients
with acute post-infectious GN and hypocomplementemic-C3
glomerulopathy.
Supplemental Table 3. Isotype specificity of anti-Factor B and anti-
C3bBb antibodies by ELISA in patients with acute post-infectious
GN and related C3 and sC5b-9 levels.
JASN 31: 829–840, 2020

Supplemental Table 4. Complement assays at disease onset and at
last anti-FB antibody screening.
Supplemental Table 5. List of the 32 linear peptides of FB.
Supplemental Table 6. Number and proportions (%) of children
with APIGN, of French controls and of controls from the 1000 Genomes
Downloaded from https://jasn.asnjournals.org at UPPSALA MEDICINSKA on May 12, 2020. Copyright 2020 The American Society of Nephrology
project database, who carried at least one rare variant (MAF ,0.1) in
one of the four tested complement genes (C3, CFB, CFH, CFI).
Supplemental Table 7. Rare variants identified in 5 of 23 (21.7%)
patients with acute post-infectious GN and in 6 of 45 (13%) patients
with hypocomplemntemic-C3 glomerulopathy among C3, CFB,
CFH, CFI genes.
Supplemental Figure 1. Population study of 60 patients with
hypocomplementemic-C3 glomerulopathy.
Supplemental Figure 2. Titers of anti-Factor B antibodies in
patients with acute post infectious GN and hypocomplementemic
C3 glomerulopathy.
Supplemental Figure 3. Correlation between biomarkers and anti-
complement protein antibody titers in patients with Acute post
infectious GN.
Supplemental Figure 4. Anti-Factor B antibodies binding to peptides
of Serin Protease and von Willebrand domains of Factor B by ELISA.
Supplemental Figure 5. Anti-Factor B antibody binding to pep-
tides of Factor B according to the severity of the disease at diagnosis.
Supplemental Figure 6. Position on C3bBb molecular structure of
peptide 624-637 associated with non severe renal presentation.
Supplemental Figure 7. Anti-Factor B antibodies binding to re-
combinant Factor B.
Supplemental Figure 8. Molecular structure of C3bB and C3bBb
with peptides and amino-acid changes of interest for anti-Factor B
antibody binding.
Supplemental Figure 9. Study of C3bB and C3bBb formation by
ELISA in presence of purified IgG from APIGN patients.
Supplemental Figure 10. C3 convertase formation on sheep
erythrocytes in presence of total IgG from patients with Acute post
infectious GN.
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AFFILIATIONS

1
Inflammation, Complement and Cancer Team, Cordeliers Research Center, Institut National de la Santé et de la Recherche Médicale
(INSERM) Unité Mixte de Recherche (UMR) S1138, Paris, France; Departments of 2Nephrology and 5Immunology, Assistance Publique-
Hôpitaux de Paris (AP-HP), Georges Pompidou European Hospital, Paris, France; 3Paris University, Paris, France; 4Department of Pediatric
Nephrology, AP-HP, Necker Hospital – Sick Children, Paris, France; 6INSERM U1268 Medicinal Chemistry and Translational Research, Cibles
Thérapeutiques et Conception du Médicament UMR8038 Centre National de la Recherche Scientifique (CNRS), Paris, France; 7University of
Paris Diderot, Sorbonne Paris Cité, Molécules Thérapeutiques In Silico, INSERM UMR S973, Paris, France; 8Department of Nephrology,
Rennes Hospital, Rennes, France; 9Department of Pediatric Nephrology, Rouen Hospital, Rouen, France; 10Department of Pediatric
Nephrology, Tours Hospital, Tours, France; 11Department of Pediatry, Villefranche sur Soane Hospital, Villefranche sur Soane, France;
12
Department of Pediatric Nephrology, Timone Hospital, Marseille, France; 13Department of Pediatric Nephrology, AP-HP, Robert Debré
Hospital, Paris, France; 14Department of Pediatric Nephrology, AP-HP, Trousseau Hospital, Paris, France; 15Laboratory of cristallography and
biological Nuclear magnetic resonance, UMR 8015 CNRS, Paris, France; and 16Reference Center for Hereditary Kidney and Childhood
Diseases (MARHEA), Imagine Institute, Paris, France

840 JASN JASN 31: 829–840, 2020