Experimental and human models of membranous nephropathy :
the story goes on
Pierre Ronco, Hanna DebiecUnité UMPC/IMSERM 702
HôpitalTenon
Actualités Néphrologiques19/04/2011
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 mayinvolve production of oxygen free radicals andmetalloproteases
•
Membranous Nephropathy: IgG Subclass Distribution
IgG1 IgG2 IgG3 IgG4
Idiopathic + + ± ++++
Lupus +++ +++ ++ ±
Neoplasia +++ +++ + +
Ohtani et al, Nephrol Dial Transplant, 2004, 19:574
Current treatments of membranous nephropathy
Glassock RJ, Am J Kidney Dis 2004, 44:562
The treatment of idiopathic membranous nephropathy : a dilemma or a conundrum ?
Why? • Heterogeneity of the disease• Lack of reliable biomarkers• Ignorance of the target antigen (s)
Possible mechanisms of the formation of subepithelial deposits
Glassock , New Engl J Med 2009, 361:81
Serum Sickness Heymann nephritis « Planted » antigen
Heymann nephritis
Renal BB IgG deposits Megalin, the target antigen of HN
In situ formation of immune deposits
Y YY
Y
YYY
Podocytes
Endothelium
YY
Rat: megalin
Human?
YGBMProteinuria
Activation ofcomplement
Kerjaschki and Farquhar
Glomerular immune depositsActivation of complement : variableProteinuria : variable
Passive Heymann nephritisDirect injection of heterologous
antibodies against rat megalin
Megalin
Glomerular immune depositsActivation of complement Heavy proteinuria nephrotic syndrome
Active Heymann nephritisIntradermal immunization with megalin
Ig deposits
Megalin pathogenic epitopes
Ligand-binding repeatEGF repeatYWTD spacer region
Transmembrane domain
Cytoplasmic tail
1 2 3 4
N C
Heterologous antibodies against all four ligand binding domains (LBD) can induce formation of glomerular immune deposits but not proteinuria (Yamakazi et al, JASN 1998, 9:1638).
Ronco and Debiec, JASN 2006, 17:1772
Megalin pathogenic epitopes
Ligand-binding repeatEGF repeatYWTD spacer region
Transmembrane domain
Cytoplasmic tail
1 2 3 4
N C
Heterologous antibodies against all four ligand binding domains (LBD) can induce formation of glomerular immune deposits but not proteinuria (Yamakazi et al, JASN 1998, 9:1638).
Ronco and Debiec, JASN 2006, 17:1772
B- and T-cell epitopes in the region spanning residues 157-236 can induce active HN and are critical for full expression of the disease(Tramontano et al, JASN 2006, 17:1979)
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, cytomegalovirusand hepatitis -B virus infections• Negative Coombs’ test. Normal levels of complement components at day 35
IgG
Infant born with MN and nephrotic syndrome
PLACENTA
C5b-9
MOTHER FETUS
Debiec et al. N Engl J Med 2002, 346:2053
IgG
Infant born with MN and nephrotic syndrome
PLACENTA
C5b-9
MOTHER FETUS
Debiec et al. N Engl J Med 2002, 346:2053
NEP
NEP
NEP
NEP
anti-NEP IgG
before after
Blot: anti-NEP mAb
mother control1 2
83
175
1 2
Potential mechanisms of albuminuria
green - IgGred - NEP
Profile
0.00
50.00
100.00
150.00
200.00
250.00
300.00
01.
022.
033.
05 4.07
5.08 6.
17.
12 8.13
9.15
10.1
711
.18
12.2
13.2
214
.23
15.2
516
.27
17.2
818
.3
XY-Positio
Inte
nsity Chann
Chann
Chann
Length [µ Sum: Mean: Std. dev Minimu Maximu
18.6 268 48.8 43.9 4.0 180.
18.6 278 50.5 47.2 0.0 186.
18.6 357 64.9 77.6 0.0 255.
Green IgG
Red NEP
Blue C5b-9
Why did the mother become immunized without developing the renal disease ?
IgG
(Debiec et al. Lancet. 2004)
NEP deficient
anti-NEP IgG
Genetic defect
M C
PLACENTA
MOTHER
NEP
NEP
NEPC5b-9IgG
Endothelium
GBM
YYY Y YY
YY
Podocytes
FETUS
Infant born with MN and nephrotic syndrome
Feto-Maternal Allo-Immunization with antenatal Glomerulopathies (FMAIG)
GWAS analysis of patients with idiopathic MN
• Three cohorts
• Controls : ethnically matched• Illumina platform : 300,000 SNPs• Bioinformatics analysis
Patients Controls
French 75 157
Dutch 146 1,832
British 335 349
Total 556 2,338
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) Dutch (n=146 ; c=1832)
British (n=335 ; c=349) All patients (n=556; c=2338)
Stanescu et al, New Engl J Med, 2011, 364: 616
A risk HLA-DQA1 allele is associated with iMN and may interact with PLA2R alleles
French (n=75 ; c=157) Dutch (n=146 ; c=1832)
British (n=335 ; c=349) All patients (n=556; c=2338)
Stanescu et al, New Engl J Med, 2011, 364: 616
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
A role for nonnative antigens?
Glassock , New Engl J Med 2009, 361:81
Serum Sickness Heymann nephritis « Planted » antigen
Border et al, JCI, 1982, 69 : 451
Localization of immune deposits after immunization with BSA
Lesions induced by immunization with BSA
Border et al, JCI, 1982, 69 : 451
Cati
onic
BSA
Ani
onic
BSA
Anti-BSA antibodies in patients with MN
Circulating BSA in patients with MN
B
Colocalization of BSA and IgG in immune deposits
BSA IgGGreen BSA
Red IgG
IgG1 IgG2
IgG3 IgG4
1 4 1 4
1 41 41 4 1 4
BSA
BSA
HSA
Biopsy specimen stained for IgG subclasses
Reactivity of eluted IgG
Patient with BSA in biopsy
Patients without BSA in biopsy
Y
APC
B cell
YYY
Y YYY Y
Anti-BSAAntibodies
Y YY Y
Processing
Digestion
BSA
T cell
Absorption of antigen from the intestinal
tract
Role of BSA in the pathophysiology of MN
2004 2006 2008
5
10
Prot
einu
ria
g/l
IgG subclass
2004
2006
2008
initial disease
relapse
remission
1 2 3 4
Circulating BSA1000
500
2004 2006
ng/m
l
2008
Association with disease activity
2005
A role for cytoplasmic antigens?
● α-enolase
● SOD2 : superoxide dismutase
● Aldose reductase
● Chaperonin CCT
Prunotto et al, JASN 2010, 21:507 ; Debiec unpublished
Antigens identified in MN
Glassock , New Engl J Med 2009, 361:81
Serum Sickness Heymann nephritis « Planted » antigenMegalin, NEP, PLA2R1Heymann nephritis BSA, others « Planted » antiLupus ???erum Sickne
Acknowledgments
PediatriciansV.Guigonis G.DeschênesA.BensmanT.UlinskiP.NiaudetM.KemperF.JanssenJ.Nauta
J.NortierB.Stengel
TENO
N CE
NTER
OF N
EPHR
OLOG
Y
Bergamo (I)G. RemuzziM. AbbateNijmegen (NL)J. WetzelsJ. HofstraUKR. Kleta (London)P. Mathieson (Bristol)A.Rees (Vienna)CNG (Evry)D. Bacq-Daian