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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)