Transcript PPT / 13904 KB - Nephropathology Working Group

Unfolding of the Phospholipase
A2 Receptor Story
Laurence H. Beck, Jr., MD, PhD
Renal Section, Department of Medicine
Boston 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 course
o
o
o
Spontaneous remission
Persistent proteinuria
Progression to ESRD
• Treated with non-selective,
often TOXIC, immunosuppressive agents
Is there an intrinsic glomerular
antigen in adult primary MN?
Podocyte Ag
+
Circulating antipodocyte Ag
antibody
?
=
Experimental
technique
Normal Human
Kidney
Patient Serum
Separate proteins
by SDS-PAGE
Normal Glomeruli
Western blot to look
for reactive bands
Immunoglobulin
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 glomerulus
Ancian et al. (1995) J Biol Chem 270: 8963-70
PLA2R
AGRIN
NUCLEI
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
Clinical utility of anti-PLA2R
1. Diagnosis and classification
2. Monitoring of disease activity
Membranous nephropathy
Primary
(Idiopathic)
75%
anti-PLA2R
associated
25%
??
Secondary
- Lupus
- Hepatitis B
- NSAIDs
- Malignancy
- Toxins (Hg)
- Others
Biopsy and clinical impression
vs. anti-PLA2R serology
Immunologically
inactive?
Another antigen?
SLE
HBV
MCD
FSGS
IgAN
DN
Breakdown of ‘indeterminate’ group
Anti-PLA2R-positive
•
•
•
•
Atypical IF or EM (8)
ANCA-positivity (1)
Sarcoidosis (1)
Malignant polyp (1)
Primary MN with atypical
histopathology and/or
coincidental disease?
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)
True secondary causes of
membranous nephropathy?
Biopsy may reveal “history” of recently-active disease
PLA2R
Debiec and Ronco (2011). New Engl J Med 364: 689-90
Association of primary MN with (anti-)PLA2R:
Sensitivity and specificity
Cases (n)
aPLA2R-Positive
CASES (%)
“Enhanced”
positivity
Controls
(n)
aPLA2R-Positive
CONTROLS (%)
Beck (2009)
37
26 (70%)
26 (70%)
60
0 (0%)
Debiec (2011)
42
24 (57%)
34 (81%)
ND
ND
Hofstra (2011)
18
14 (78%)
14 (78%)
ND
ND
Qin (2011)
60
47 (87%)
59 (98%)
46
5 (11%)
Hoxha (2011)
100
52 (52%)
23/35 (66%)
260
0 (0%)
Beck (2011)
35
25 (71%)
27 (77%)
ND
ND
292
190 (65%)
183/227 (81%)
366
5 (1%)
Study
Total
Modified from Martas, Ravani, and Ghiggeri (2011) Nephrol Dial Transplant 26: 2428-30
Clinical utility of anti-PLA2R
1. Diagnosis and classification
2. Monitoring of disease activity
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
Human anti-PLA2R, IgG4 subclass
Time following treatment with RTX
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
Clinical disease
Immunologic disease
Treatment
?
100%
Proteinuria
Anti-PLA2R
Partial remission
0%
Time
Complete remission
Can we show efficacy for novel
(or not-so-novel) agents?
IgG4 subclass
of anti-PLA2R
ACTH Gel 80 IU sc twice weekly
Recurrent MN vs. de novo MN
in the kidney allograft:
Are they different diseases?
Primary MN
Merged
PLA2R-Cy3
IgG4-FITC
Recurrent MN (6d post-transplant)
De Novo MN
Merged
PLA2R-Cy3
IgG4-FITC
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
40/51 (78%)
7/13 (54%)
0/14 (0%)
Total
* 0/17 samples negative for circulating anti-PLA2R as well
aDebiec
bDebiec
H and Ronco P (2011) New Engl J Med 364: 689-90
H et al. Am J Transplant (epub Aug 2011)
Expanded cohort from Mayo Clinic
Anti-PLA2R
positive?
Immunosuppression
MN in native
kidneys
Progression
to ESKD
Kidney
transplant
4 ‘late’ recurrences
(36, 48, 60, 108 mo)
• disappearance (n=2) and
reoccurrence of anti-PLA2R?
Are there autoantibodies
other than anti-PLA2R in
these patients?
YES:
NO:
Recurrence
of MN?
Median time to recurrence 4 mo (1-108)
78% (14/18) - recurred
22% (4/18) - no recurrence
Median time to recurrence 4 mo (2-24)
56% (5/9) - recurred
44% (4/9) - no recurrence
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
normal
rat
serum
heat
inactivated
rat
serum
IgG4-depleted IgG fraction
Galactose-deficient IgG binds mannose-binding lectin
VH
CH1
-S-S-S-S-
6Gal1 4GlcNAc1 2Man1
}
Fc
VL
CL
CH2
CH3
Fuc1
6
6
4Man1 4GlcNAc1 4GlcNAc 4GlcNAc1 Asn297
3
6Gal1 4GlcNAc1 2Man1
Malhotra R, et al Nat Med 237-243, 1995.
MN-derived IgG4 allows increased
C4 deposition
Membranous nephropathy
Normal control sera
0.7
0.6
C4 deposition
0.5
0.4
0.3
0.2
0.1
0
MN 09-23
MN 09-24
MN 09-45
MN 09-56
NHS-RS
NHS-RG
NHS-YG
NHS-RA
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 mannose
receptor 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)
linkage
r2 = 0.70
dysequilibrium
Coding SNP M292V (exon 5)
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?
Immunologic
initiation
Complementmediated
cytotoxicity
(?)
PLA2R1
ESRD
Genetics (?)
?
HLA-DQA1
?
α-PLA2R
α-NEP
α-SOD
α-AR
Persistent
proteinuria
Progression
factors
α-Enolase
Relapse
Remission
Acknowledgments
Boston University
David Salant
Ramon Bonegio
Rivka Ayalon
Tep Chongkrairatanakul
Fahim Malik
Hong Ma
Neetika Garg
University of Iowa
Christie Thomas
Christopher Blosser
Mayo Clinic, Rochester, MN
Fernando Fervenza
Fernando Cosio
Nanjing University School of Medicine
Weisong Qin
Columbia University, New York, NY
Andy Bomback
Jerry Appel
University of Louisville, KY
David Powell
Jon Klein
CNRS; Université de Nice Sophia Antipolis
Gérard Lambeau
Radboud Univ. Nijmegen Medical Center
Julia Hofstra
Jack Wetzels
With special thanks to:
The New England Organ Bank
Families of the deceased kidney donors
Patients and volunteers
This work was supported by:
The Halpin Foundation – ASN
National Institutes of Health/NIDDK
Questcor Pharmaceuticals
Sensitivity and specificity of anti-PLA2R
for primary MN
Sensitivity 83%
Primary MN
Specificity 96%
Can we distinguish MN that truly a secondary
process from MN that occurs coincidentally?
Serum anti-PLA2R
Negative
Positive
IgG4
LN-MN
HBV-MN
Ca-MN
Qin W-S, Beck LH et al. J Am
Soc Nephrol 2011 (in press)
How do the deposits form?
PODOCYTE
GBM
A
Intrinsic podocyte Ag
+
Circulating Ab
B
Preformed IC
(Ag + Ab)
C
Planted Ag
+
Circulating Ab
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
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)
Polanco et al. J Am Soc Nephrol 21: 697-704, 2010
Return of anti-PLA2R precedes relapse of
nephrotic syndrome
IgG1 (387)
IgG3 (387)
IgG4 (387)
Proteinuria (387)
30000
25000
Arbitrary units
20000
15000
10000
5000
0
0
20
40
60
80
100
Weeks
120
140
160
180
Complement-mediated cytotoxicity assay
Anti-PLA2R
+ complement factors
ETHIDIUM
Complement components identified in primary MN
Classical Pathway
Ag-Ab complexes
Lectin pathway
Microbial surfaces, agalactosyl IgG
C1q C1r C1s
Alternative Pathway
Spontaneous, foreign surfaces
C3
MBL MASPs
C3a
C4C2
C3b
C4bC2a
(C3 convertase)
C3bBbP
(C3 convertase)
C3
C3
C3a
C3a
C3b
C3b
C4bC2aC3b
(C5 convertase)
C3bC3bBbP
(C5 convertase)
C5
C5b-9
(MAC)
C5b
C6+C7+C8+C9
C5a
Debiec H et al. American Journal of Transplantation 2011 (epub ahead of print)