Unfolding of the Phospholipase A 2 Receptor Story Laurence H. Beck, Jr., MD, PhD Renal Section,...

Unfolding of the Phospholipase A2 Receptor

Story

Unfolding of the Phospholipase A2 Receptor

Story

Laurence H. Beck, Jr., MD, PhDRenal Section, Department of MedicineBoston University School of Medicine

23rd European Congress of Pathology

August 30, 2011

Primary membranous nephropathy

• A leading cause of adult nephrotic syndrome

• Rare; incidence 1/100,000

• Organ-specific, autoimmune disease

• Variable clinical courseo Spontaneous remissiono Persistent proteinuriao Progression to ESRD

• Treated with non-selective, often TOXIC, immuno-suppressive agents

Is there an intrinsic glomerular antigen in adult primary MN?

Is there an intrinsic glomerular antigen in adult primary MN?

Circulating anti-podocyte Ag

antibody

+ =?

Podocyte Ag

Experimental technique

Experimental technique

Normal Human Kidney

Normal Glomeruli

Patient Serum

Immunoglobulin

Separate proteins by SDS-PAGE

Western blot to look for reactive bands

What is the antigen?

• Took advantage of its heavy glycosylation• Partial purification on wheat germ agglutinin• Separation by gel electrophoresis

Mass spectrometry Evaluate candidate proteins

M-type phospholipase A2 receptor

• 185 kDa type I transmembrane glycoprotein

• Expressed in human kidney, lung, placenta, WBC

• Member of the mannose receptor family– Mannose Receptor (CD206)– Endo180 (uPAR-associated protein or CD280)– DEC-205 (CD205), dendritic cell receptor

– M-type phospholipase A2 receptor

– FcRY = avian yolk sac IgY receptor

• Binds certain sPLA2s, but exact function is not known

• May play a role in cellular replicative senescence

IgG4 is the dominant anti-PLA2R subclass in human primary membranous

nephropathy

• Human glomerular extract in all lanes

• Primary Ab: Sera from 6 patients with MN (1 – 6)

• Secondary Abs specific to each human IgG subclass(IgG1, IgG2, IgG3, IgG4)

• Arrowhead: PLA2R

Beck et al. (2009) New Engl J Med 361:11-21

PLA2R in the normal glomerulusPLA2R in the normal glomerulus

PLA2R AGRIN NUCLEI

Ancian et al. (1995) J Biol Chem 270: 8963-70

PLA2R and IgG4 co-localize in human primary MN biopsy

specimens

IgG4 eluted from MN biopsy specimens recognizes PLA2R

Beck et al. (2009) New Engl J Med 361:11-21

1. Diagnosis and classification

2. Monitoring of disease activity

Clinical utility of anti-PLA2RClinical utility of anti-PLA2R

Membranous nephropathy

Primary(Idiopathic)

Secondary - Lupus - Hepatitis B - NSAIDs - Malignancy - Toxins (Hg) - Others

75% 25%

anti-PLA2Rassociated

??

Biopsy and clinical impression vs. anti-PLA2R

serology

SLEHBV

MCDFSGSIgANDN

Immunologicallyinactive?

Another antigen?

Breakdown of ‘indeterminate’ group

Anti-PLA2R-positive• Atypical IF or EM (8)• ANCA-positivity (1)• Sarcoidosis (1)• Malignant polyp (1)

Anti-PLA2R-negative• Atypical IF or EM (12)• ANCA-positivity (1)• NSAID associated (2)• CLL associated (2)• RA associated (2)• IgG4 RSD (2)• Malignancy (3)• Sjögren’s (1)• HIV (1)

Primary MN with atypical histopathology and/or coincidental disease?

True secondary causes of membranous nephropathy?

Debiec and Ronco (2011). New Engl J Med 364: 689-90

Biopsy may reveal “history” of recently-active disease

PLA2R

Association of primary MN with (anti-)PLA2R:

Sensitivity and specificity

Modified from Martas, Ravani, and Ghiggeri (2011) Nephrol Dial Transplant 26: 2428-30

1. Diagnosis and classification

2. Monitoring of disease activity

Clinical utility of anti-PLA2RClinical utility of anti-PLA2R

Association of anti-PLA2R with clinical status

Anti-PLA2R level correlates with

proteinuria

Hofstra JM, Beck LH et al. (2011) Clin J Am Soc Nephrol 6: 1286-91

Time following treatment with RTXH

um

an a

nti

-PL

A2R

, Ig

G4

sub

clas

s

Disappearance

Persistence

Relapse

Beck LH, Fervenza FC et al. (2011) J Am Soc Nephrol 22: 1543-50

Immunological remission in primary MN precedes clinical

remission

Beck LH, Fervenza FC et al. (2011) J Am Soc Nephrol 22: 1543-50

Time

100%

0%

Anti-PLA2R

Proteinuria

Partial remission

Complete remission

Clinical disease

Immunologic disease

Treatment?

Can we show efficacy for novel (or not-so-novel)

agents?

ACTH Gel 80 IU sc twice weekly

IgG4 subclass of anti-PLA2R

Recurrent MN vs. de novo MN in the kidney allograft:

Are they different diseases?

Primary MN

PLA2R-Cy3 IgG4-FITC

Merged

Recurrent MN (6d post-transplant)

De Novo MN

PLA2R-Cy3 IgG4-FITC

Merged

Detection of PLA2R in immune deposits of the biopsy specimen

Study Primary MN Recurrent MN De novo MN

Collins (unpubl) 9/9 2/3 0/5 *

Debiec (2011a) 31/42 ND ND

Debiec (2011b) ND 5/10 0/9

Total 40/51 (78%) 7/13 (54%) 0/14 (0%)

* 0/17 samples negative for circulating anti-PLA2R as well

aDebiec H and Ronco P (2011) New Engl J Med 364: 689-90bDebiec H et al. Am J Transplant (epub Aug 2011)

MN in nativekidneys

Progressionto ESKD

Kidneytransplant

Recurrenceof MN?

Anti-PLA2R positive?

Immunosuppression

78% (14/18) - recurred22% (4/18) - no recurrence

YES:

Median time to recurrence 4 mo (1-108)

NO: 56% (5/9) - recurred44% (4/9) - no recurrence

Median time to recurrence 4 mo (2-24)Are there autoantibodies other than anti-PLA2R in

these patients?

Expanded cohort from Mayo Clinic

4 ‘late’ recurrences (36, 48, 60, 108 mo)• disappearance (n=2) and reoccurrence of anti-PLA2R?

70% of patients with recurrent MN were anti-PLA2R positive

Clinical implications

• The majority of patients with primary MN have circulating autoantibodies against PLA2R, an intrinsic podocyte antigen

• Anti-PLA2R is highly specific for primary MN

• Clear association of anti-PLA2R with disease activity Positive in nephrotic state Declines prior to decrease in proteinuria Absent in remission Returns with relapse of disease Associated with recurrent MN (and not with de novo MN)

• Role in diagnosis and monitoring of immunologic disease activity during treatment

Pathologic mechanisms: Questions

• Is anti-PLA2R directly pathogenic?

• If so, how does it cause podocyte injury? Classical complement pathway(IgG1, IgG3) Mannan-binding lectin pathway? Direct cytotoxicity (IgG4?)

• Do genetic variations in PLA2R explain susceptibility to MN?

Complement C3 deposition on cultured differentiated human podocytes

Anti-PLA2R+ IgG4 fraction IgG4-depleted IgG fraction

normalratserum

heatinactivatedratserum

4Man1 4GlcNAc1 4GlcNAc 4GlcNAc1 Asn2976

3

6

Fuc16Gal1 4GlcNAc1 2Man1

6Gal1 4GlcNAc1 2Man1

VL

VH

CL

CH1

CH2

CH3

}Fc

-S-S--S-S--S

-S- -S-S-

Galactose-deficient IgG binds mannose-binding lectin

Malhotra R, et al Nat Med 237-243, 1995.

MN-derived IgG4 allows increased C4 deposition

Membranous nephropathy Normal control sera

MBL binds to affinity purified anti-PLA2R IgG4 heavy chain

Could genetic polymorphisms in PLA2R determine

susceptibility for developing disease?

• age of onset• aggressiveness of disease• recurrence in allograft

“Bent” (vs. extended) conformations of mannosereceptor family members

from Llorca, O (2008) Cell Mol Life Sci 65: 1302-10

Human anti-PLA2R antibodies recognize an epitope in the N-terminal

part of the protein

PLA2R contains several SNPs in the region of the anti-PLA2R epitope

GWAS: rs4664308 (intron 1)

Coding SNP M292V (exon 5)

linkage dysequilibriumr2 = 0.70

Detailed genotyping and sequencing of PLA2R1 in

cases of anti-PLA2R associated MN vs. controls

The pathogenesis of MN:How does it all fit together?

ESRDESRD

PersistentproteinuriaPersistent

proteinuria

RemissionRemission

Relapse

α-PLA2Rα-AR

α-SOD

α-NEP

Progression factors

Complement-mediated

cytotoxicity (?)

?

?

Genetics (?)

Immunologic initiation

PLA2R1

HLA-DQA1

α-Enolase

Acknowledgments

Boston UniversityDavid SalantRamon BonegioRivka AyalonTep ChongkrairatanakulFahim MalikHong MaNeetika Garg

Mayo Clinic, Rochester, MNFernando FervenzaFernando Cosio

Columbia University, New York, NYAndy BombackJerry Appel

University of Louisville, KYDavid PowellJon Klein

University of IowaChristie ThomasChristopher Blosser

CNRS; Université de Nice Sophia AntipolisGérard Lambeau

Radboud Univ. Nijmegen Medical CenterJulia HofstraJack Wetzels

Nanjing University School of MedicineWeisong Qin

With special thanks to:The New England Organ BankFamilies of the deceased kidney donorsPatients and volunteers

This work was supported by:The Halpin Foundation – ASNNational Institutes of Health/NIDDKQuestcor Pharmaceuticals

Sensitivity and specificity of anti-PLA2R for primary MN

Specificity 96%

Sensitivity 83%

Primary MN

Serum anti-PLA2R

Negative Positive

LN-MN

HBV-MN

Ca-MN

Can we distinguish MN that truly a secondary process from MN that occurs

coincidentally?

Qin W-S, Beck LH et al. J Am Soc Nephrol 2011 (in press)

IgG4

PODOCYTE

GBM

A B C

Intrinsic podocyte Ag+

Circulating Ab

Preformed IC

(Ag + Ab)

Planted Ag+

Circulating Ab

How do the deposits form?

From Llorca, O (2008) Cell Mol Life Sci 65: 1302-10

The PLA2R epitope identified by MN autoantibodies is sensitive to

reduction

Beck et al. (2009) New Engl J Med 361: 11-21

Identification of the 185 kDa MN-Ag as the M-type phospholipase A2

receptor

Beck et al. (2009) New Engl J Med 361: 11-21

Cultured immortalized human podocytes express PLA2R mRNA and

protein

Anti-PLA2R Anti-PLA2R +

blocking peptide

Polanco et al. J Am Soc Nephrol 21: 697-704, 2010

Remission of proteinuria takes time

104 of 328 (32%) conservatively treated patients with primary MN achieved spontaneous remission (CR or PR)

Mean time to PR = 14.7 ± 11.4 months - 50% persisted with PR - 50% progressed to CR (38.5 ± 25.2 months)

Return of anti-PLA2R precedes relapse of nephrotic syndrome

Complement-mediated cytotoxicity assay

ETHIDIUM

Anti-PLA2R+ complement factors

C1q C1r C1s MBL

C4C2

C4bC2a(C3 convertase)

C4bC2aC3b(C5 convertase)

C3

C3a

C5

C5a

C3bBbP(C3 convertase)

C3bC3bBbP(C5 convertase)

C5b-9 (MAC)

Classical Pathway

Ag-Ab complexes

Lectin pathway

Microbial surfaces, agalactosyl IgG

Alternative Pathway

Spontaneous, foreign surfaces

MASPs

C3b

C3

C3a

C3b

C3

C3a

C3b

C5b

C6+C7+C8+C9

Complement components identified in primary MN

Debiec H et al. American Journal of Transplantation 2011 (epub ahead of print)