Podocyte and food antigens in membranous nephropathy (PPT

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