PPT / 13904 KB - Nephropathology Working Group

January 29, 2018 | Author: Anonymous | Category: Science, Health Science, Immunology
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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)

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