Podocyte and food antigens in membranous
nephropathy
Pierre Ronco
INSERM Unit 702
and Division of Nephrology,
Tenon hospital, Paris, France
ECP, Helsinki, 2011
August 30th
Membranous Nephropathy
Major cause of nephrotic syndrome and chronic renal failure
Aetiologies of membranous nephropathy
• 30% associated with :
- infections
- cancers
- autoimmune diseases
- drugs
• 70% « idiopathic forms »
• Proteinuria is complement-dependent, and may
involve production of oxygen free radicals and
metalloproteases
Membranous Nephropathy:
IgG Subclass Distribution
IgG1
IgG2
IgG3
IgG4
Idiopathic
+
+
±
++++
Lupus
+++
+++
++
±
Neoplasia
+++
+++
+
+
Ohtani et al, Nephrol Dial Transplant, 2004, 19:574
Possible mechanisms of the formation of
subepithelial deposits
Serum Sickness
Heymann nephritis
« Planted » antigen
Glassock , New Engl J Med 2009, 361:81
Heymann nephritis
Renal BB
IgG deposits
Megalin, the target
antigen of HN
Podocytes
Activation of
complement
Rat: megalin
Human?
GBM
Proteinuria
Endothelium
In situ formation of immune deposits
Kerjaschki and Farquhar
From rats to men :
Allo-immune neonatal MN
1982-2002
The case : Hugo D. (male gender)
• 34 Wks of gestation : oligohydramnios and enlarged
kidneys
• 38 Wks : birth
• 1st Days of life : respiratory distress and oligoanuria,
followed by nephrotic range proteinuria and increased
blood pressure
• 4 Wks : CT-guided kidney biopsy
• Negative tests for syphilis, toxoplasmosis, cytomegalovirus
and hepatitis -B virus infections
• Negative Coombs’ test. Normal levels of complement
components at day 35
PLACENTA
MOTHER
before
FETUS
after
NEP
NEP
NEP
mother
1
2
control
1
NEP
2
175
IgG
C5b-9
83
Blot: anti-NEP mAb
anti-NEP IgG
Infant born with MN and
nephrotic syndrome
Debiec et al. N Engl J Med 2002, 346:2053
Potential mechanisms of albuminuria
Green IgG
green
Red
NEP -
IgG
red C5b-9- NEP
Blue
Induction of neonatal renal disease in pregnant
rabbit by injection of human maternal IgG
NEWBORN RABBIT KIDNEY
Human IgG
NEP
Why did the mother become immunized
without developing the renal disease ?
PLACENTA
MOTHER
FETUS
Genetic defect
Podocytes
NEP
NEP deficient
M
C
NEP
GBM
Y
NEP
Y
Endothelium
IgG
C5b-9
anti-NEP IgG
IgG
(Debiec et al. Lancet. 2004)
Infant born with MN
and nephrotic syndrome
 Feto-Maternal Allo-Immunization with antenatal Glomerulopathies (FMAIG)
Western blotting of glomerular proteins with
serum from patients with iMN
Beck et al, New Engl J Med, 2009, 361:11
Expression of PLA2R in normal kidney
and glomeruli
Beck et al, New Engl J Med, 2009, 361:11
GWAS analysis of patients
with idiopathic MN
• Three cohorts
Patients
Controls
French
75
157
Dutch
146
1,832
British
335
349
Total
556
2,338
• Controls : ethnically matched
• Illumina platform : 300,000 SNPs
• Bioinformatics analysis
Stanescu et al, New Engl J Med, 2011, 364: 616
Single Nucleotide Polymorphisms (SNPs)
•
•
•
•
•
•
•
Single base mutation in DNA
Most simple form of genetic polymorphism
90% of all human DNA polymorphisms
Occur 0.5-10 per every 1 000 base pairs
Not uniformly distributed
> 1, 000, 000 SNPs identified
SNP in a coding region can be
– Synonymous (silent mutation)
– Non-synonymous (missense or nonsense mutation)
Principle of pangenomic (GWAS) studies
A risk HLA-DQA1 allele is associated with iMN and
may interact with PLA2R alleles
French (n=75 ; c=157)
British (n=335 ; c=349)
Dutch (n=146 ; c=1832)
All patients (n=556; c=2338)
Stanescu et al, New Engl J Med, 2011, 364: 616
Conclusions
• An HLA-DQA1 allele on chromosome 6p21 is most
closely associated with idiopathic membranous
nephropathy in persons of white ancestry
• This allele may facilitate an autoimmune response
against targets such as variants of PLA2R1
• Our findings suggest a basis for understanding this
disease and illuminate how adaptive immunity is
regulated by HLA
• The risk of iMN is higher with the HLA-DQA1 allele
than with the PLA2R1 allele, suggesting that the
HLA-DQ1 allele might favor autoantibody targeting
also other antigens
PLA2R autoantibodies and PLA2R glomerular
deposits in membranous nephropathy
Debiec and Ronco, New Engl J Med, 2011, 364 :689
Are there other antigens on the
horizon in patients with idiopathic MN ?
Screening of MN sera reactivity
with podocyte « proteome »
Human podocyte lysates
kDa
110
80
Western
blot analysis
47
32
24
1
2
3 4
5
6
7
8
9
Patients’ sera
Different autoantibody profiles
Identification of antigens
Human podocyte lysate
Differential extraction
Ion exchange
chromatography
MALDI-TOF MS
2D-electrophoresis
Identification
MALDI-TOF MS
LC-MS/MS
Western immunoblotting
with patient’s serum
Silver staining
Candidate autoantigens recognized by
autoantibodies in sera from MN patients
> 10 Cytoplasm/cytoskeletal
proteins
« Metabolic » proteins
Molecular chaperones
Cytoskeletal proteins
J Am Soc Nephrol 21 : 507-519, 2010
Mechanisms of immune complex formation and
podocyte injury in MN
Initiation
Progression
Proteinuria
Podocytes
Cytoskeletal
changes
DNA
damage
GBM
Activation of
complement
ROS
Proteases
C5b-9
NEP
Progression
PLA2R
Altered
intracellular
transport
Apoptosis
Lack of
proliferation
C5b-9
degradation
Endothelium
Podocytopenia
Detachment
Renal failure
SOD2
Aldose reductase
Enolase (Wakui et al, 1999)
Stress proteins
Cytoskeletal proteins
A role for nonnative antigens?
Serum Sickness
Heymann nephritis
« Planted » antigen
Glassock , New Engl J Med 2009, 361:81
Anti-BSA antibodies in patients with MN
Debiec et al, NEJM 2011, 364 : 2101
IgG binding of BSA synthetic peptides
M
MN patients
NA
M
1.4
1.2
1.4
1.2
0.8
0.8
OD
1
OD
1
Controls
B
0.6
0.6
0.4
0.4
0.2
0.2
0
0
0
1
2
3
4
5
6
7
8
9
Peptide
10
11
12
13
14
0
Peptide
CDEFKADEKKFWGKYLY
HSA
CTAFHDNEETFLKKYLY
Debiec et al, NEJM 2011, 364 : 2101
2
3
4
5
6
7
8
Peptide
9
10
11
Peptide
BSA
BSA
1
HSA
12
13
14
Circulating BSA in patients with MN
B
ELISA BSA
2D electrophoresis and
immunoblot
Debiec et al, NEJM 2011, 364 : 2101
Colocalization of BSA and IgG in immune deposits
BSA
IgG
Green BSA
Red IgG
Biopsy specimen stained for IgG
subclasses
Reactivity of eluted IgG
Patient with BSA in biopsy
IgG1
IgG2
1
4
1
4
BSA
IgG3
IgG4
HSA
Patients without BSA in biopsy
1
4
1
4 1
4
1
4
BSA
Debiec et al, NEJM 2011, 364 : 2101
Role of BSA in the pathophysiology of MN
BSA
Processing
Digestion
APC
T cell
B cell
YYY
Absorption of
antigen from
the intestinal
tract
Anti-BSA
Antibodies
Proteinuria g/l
Association with disease activity
10
IgG subclass
1
5
2004
2004 2005
2006
Circulating BSA
ng/ml
1000
500
2004 2006 2008
2008
2
3
4
initial
disease
2006
relapse
2008
remission
What do these results teach us?
• The repertoire of antigens involved in
« idiopathic » MN may include nonglomerular
antigens
• Food cationized BSA and/or abnormal processing
of BSA in the GI tract in young children may lead
to passage in the blood of cationic BSA,
immunization, and in situ formation of immune
complexes (Border et al, J Clin Invest, 1982,
69:451)
• These cases point to a role for environmental
factors in the pathogenesis of MN
The spectrum of human
membranous nephropathies
• Neonatal, alloimmune : NEP
• Early childhood MN : BSA
• « Idiopathic » MN
- 70-80% : PLA2R (+ other specificities:AR, SOD2, enolase..?)
- 20-30% : other Ags including food/environmental Ag (BSA)
• « Secondary » MN
Ags to be identified
• Graft MN
- Recurrent : PLA2R (and other antigens)
- de novo : allo-immune
Acknowledgments
Bergamo (I)
G. Remuzzi
M. Abbate
Nijmegen (NL)
J. Wetzels
J. Hofstra
UK
R. Kleta (London)
P. Mathieson (Bristol)
A.Rees (Vienna)
CNG (Evry)
D. Bacq-Daian
Paediatricians
V. Guigonis (Limoges, F)
A. Bensman (Paris, F)
G. Deschênes (Paris, F)
T. Ulinski (Paris, F)
J. Nauta (Rotterdam, NL)
F. Janssen (Brussels, B)
J. Nortier (Brussels, B)
D. Bockenhauer (London, UK)
M. Kemper (Hamburg, D)
B. Stengel (Paris, F)
Anti-PLA2R antibody in membranous nephropathy
Positive for anti-PLA2R (%)
Europe + Asia
100
N=466
64.5%
N=45 N=14 N=18 N=13
4.4% 28% 17% 15%
N=90 N=153
0%
0%
75
50
25
iMN
LN-MN Cancer HBV GvH
Secondary MN
DC
HC
Debiec ,Tesar and Ronco; Hoxha et al, NDT 2011; Qin et al, JASN 2011
Positive for anti-PLA2R (%)
Anti-PLA2R antibody in idiopathic membranous nephropathy
100
N=25
68%
N=142
65%
N=42
57%
N=100
52%
N=81
72%
N=16
43%
N=60
80%
iMN
Japan
iMN
China
75
50
25
iMN
iMN
Italy Dutch
iMN
iMN
iMN
France Germany Czech
IFA
Debiec ,Tesar and Ronco; Hoxha et al, NDT 2011; Qin et al, JASN 2011