Podocyte and food antigens in membranous nephropathy

Pierre RoncoINSERM Unit 702

and Division of Nephrology,Tenon hospital, Paris, France

ECP, Helsinki, 2011August 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 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

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?

YGBM

Proteinuria

Activation ofcomplement

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, 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

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 - IgG

red - NEP

Profile 9

0.00

50.00

100.00

150.00

200.00

250.00

300.00

0

1.02

2.03

3.05 4.0

7

5.08 6.1

7.12 8.1

3

9.15

10.17

11.18 12

.2

13.22

14.23

15.25

16.27

17.28 18

.3

XY-Position [µm]

Inten

sity

Channel 1

Channel 2

Channel 3

Length [µm]: Sum: Mean: Std. dev.: Minimum: Maximum:

18.64 2685 48.82 43.99 4.00 180.00

18.64 2780 50.55 47.20 0.00 186.00

18.64 3570 64.91 77.64 0.00 255.00

Green IgG

Red NEP

Blue C5b-9

Human IgG NEP

NEWBORN RABBIT KIDNEY

Induction of neonatal renal disease in pregnant rabbit by injection of human

maternal IgG

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

YY

Y Y YYY

Y

Podocytes

FETUS

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

• 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

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

Western blot

analysis

1 2 3 4 5 6 7 8 9

11080

4732

24

Different autoantibody profiles

Patients’ sera

kDa

Identification of antigens

Human podocyte lysate

Differential extraction

Ion exchange chromatography

Silver stainingWestern immunoblottingwith patient’s serum

2D-electrophoresis

Identification

MALDI-TOF MS

MALDI-TOF MS

LC-MS/MS

Candidate autoantigens recognized by autoantibodies in sera from MN patients

« Metabolic » proteins

Cytoskeletal proteinsMolecular chaperones

> 10 Cytoplasm/cytoskeletalproteins

J Am Soc Nephrol 21 : 507-519, 2010

Mechanisms of immune complex formation and podocyte injury in MN

Y

Y

YY

YYY

Y

Activation ofcomplementGBM

Podocytes

C5b-9 C5b-9

ROSProteases

Proteinuria

Cytoskeletalchanges

degradation Detachment

DNAdamage

Apoptosis

Lack ofproliferation

Podocytopenia

EndotheliumYY

Initiation Progression

Stress proteins

Altered intracellular

transport

PLA2R

Renal failure

Enolase (Wakui et al, 1999)

Cytoskeletal proteins

NEPProgression

SOD2Aldose reductase

A role for nonnative antigens?

Glassock , New Engl J Med 2009, 361:81

Serum Sickness Heymann nephritis « Planted » antigen

Anti-BSA antibodies in patients with MN

Debiec et al, NEJM 2011, 364 : 2101

IgG binding of BSA synthetic peptides

CDEFKADEKKFWGKYLY

CTAFHDNEETFLKKYLY

BSA

HSA

A BMNM

BSA HSA

PeptidePeptide

MN patients Controls

Debiec et al, NEJM 2011, 364 : 2101

Debiec et al, NEJM 2011, 364 : 2101

ELISA BSA

B

2D electrophoresis and immunoblot

Circulating BSA in patients with MN

Colocalization of BSA and IgG in immune deposits

BSA IgG

Green 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

Debiec et al, NEJM 2011, 364 : 2101

Y

APC

B cell

YYY

Y YYY Y

Anti-BSAAntibodies

Y YYY

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

Pro

tein

uri

a g

/l

IgG subclass

2004

2006

2008

initial disease

relapse

remission

1 2 3 4

Circulating BSA1000

500

2004 2006

ng

/ml

2008

Association with disease activity

2005

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 » MNAgs to be identified

• Graft MN- Recurrent : PLA2R (and other antigens)

- de novo : allo-immune

Acknowledgments

TENON CENTER O

F NEPHROLO

GY

Bergamo (I)G. RemuzziM. AbbateNijmegen (NL)J. WetzelsJ. HofstraUKR. Kleta (London)P. Mathieson (Bristol)A.Rees (Vienna)CNG (Evry)D. Bacq-Daian

PaediatriciansV. 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)

iMN LN-MN CancerHBV GvH DC HC

N=46664.5%

Europe + AsiaP

osit

ive f

or

an

ti-P

LA

2R

(%

)

100

75

50

25

N=454.4%

N=1428%

N=1817%

N=1315%

N=900%

N=1530%

Secondary MN

Anti-PLA2R antibody in membranous nephropathy

Debiec ,Tesar and Ronco; Hoxha et al, NDT 2011; Qin et al, JASN 2011

100

50

75

25

iMNItaly

N=1643%

iMNJapan

N=14265%

iMNDutch

iMNFrance

N=4257%

N=6080%

iMNChina

iMNGermany

N=10052%

N=8172%

iMNCzech

IFA

N=2568%

Posit

ive f

or

an

ti-P

LA

2R

(%

)

Anti-PLA2R antibody in idiopathic membranous nephropathy

Debiec ,Tesar and Ronco; Hoxha et al, NDT 2011; Qin et al, JASN 2011