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