1. Unfolding of the Phospholipase A 2 Receptor Story Laurence H. Beck, Jr., MD, PhD Renal Section, Department of Medicine Boston University School of Medicine 23 rd European Congress of Pathology August 30, 2011

2. Primary membranous nephropathy A leading cause of adult nephrotic syndrome Rare; incidence 1/100,000 Organ-specific, autoimmune disease Variable clinical course o Spontaneous remission o Persistent proteinuria o Progression to ESRD Treated with non-selective, often TOXIC, immuno- suppressive agents

3. Is there an intrinsic glomerular antigen in adult primary MN? Circulating anti- podocyte Ag antibody += ? Podocyte Ag

4. Experimental technique Normal Human Kidney Normal Glomeruli Patient Serum Immunoglobulin Separate proteins by SDS-PAGE Western blot to look for reactive bands

5. 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

6. M-type phospholipase A 2 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 A 2 receptor – FcRY = avian yolk sac IgY receptor Binds certain sPLA 2 s, but exact function is not known May play a role in cellular replicative senescence

7. IgG4 is the dominant anti-PLA 2 R 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: PLA 2 R Beck et al. (2009) New Engl J Med 361:11-21

8. PLA 2 R in the normal glomerulus PLA 2 RAGRINNUCLEI Ancian et al. (1995) J Biol Chem 270: 8963-70

9. PLA 2 R and IgG4 co-localize in human primary MN biopsy specimens

10. IgG4 eluted from MN biopsy specimens recognizes PLA 2 R Beck et al. (2009) New Engl J Med 361:11-21

11. 1. Diagnosis and classification 2. Monitoring of disease activity Clinical utility of anti-PLA 2 R

12. Membranous nephropathy Primary (Idiopathic) Secondary - Lupus - Hepatitis B - NSAIDs - Malignancy - Toxins (Hg) - Others 75%25% anti-PLA 2 R associated ??

13. Biopsy and clinical impression vs. anti-PLA 2 R serology SLE HBV MCD FSGS IgAN DN Immunologically inactive? Another antigen?

14. Breakdown of ‘indeterminate’ group Anti-PLA 2 R-positive Atypical IF or EM (8) ANCA-positivity (1) Sarcoidosis (1) Malignant polyp (1) Anti-PLA 2 R-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?

15. Debiec and Ronco (2011). New Engl J Med 364: 689-90 Biopsy may reveal “history” of recently-active disease PLA 2 R

16. Association of primary MN with (anti-)PLA 2 R: Sensitivity and specificity Modified from Martas, Ravani, and Ghiggeri (2011) Nephrol Dial Transplant 26: 2428-30

17. 1. Diagnosis and classification 2. Monitoring of disease activity Clinical utility of anti-PLA 2 R

18. Association of anti-PLA 2 R with clinical status Anti-PLA 2 R level correlates with proteinuria Hofstra JM, Beck LH et al. (2011) Clin J Am Soc Nephrol 6: 1286-91

19. Time following treatment with RTX Human anti-PLA 2 R, IgG4 subclass Disappearance Persistence Relapse Beck LH, Fervenza FC et al. (2011) J Am Soc Nephrol 22: 1543-50

20. Immunological remission in primary MN precedes clinical remission Beck LH, Fervenza FC et al. (2011) J Am Soc Nephrol 22: 1543-50

21. Time 100% 0% Anti-PLA 2 R Proteinuria Partial remission Complete remission Clinical disease Immunologic disease Treatment ?

22. Can we show efficacy for novel (or not-so-novel) agents? ACTH Gel 80 IU sc twice weekly IgG4 subclass of anti-PLA 2 R

23. Recurrent MN vs. de novo MN in the kidney allograft: Are they different diseases?

24. Primary MN PLA2R-Cy3 IgG4-FITC

25. Recurrent MN (6d post-transplant)

26. De Novo MN PLA2R-Cy3 IgG4-FITC

27. Detection of PLA 2 R in immune deposits of the biopsy specimen StudyPrimary MNRecurrent MNDe novo MN Collins (unpubl)9/92/30/5 * Debiec (2011 a )31/42ND Debiec (2011 b )ND5/100/9 Total40/51 (78%)7/13 (54%)0/14 (0%) * 0/17 samples negative for circulating anti-PLA 2 R as well a Debiec H and Ronco P (2011) New Engl J Med 364: 689-90 b Debiec H et al. Am J Transplant (epub Aug 2011)

28. MN in native kidneys Progression to ESKD Kidney transplant Recurrence of MN? Anti-PLA 2 R positive? Immunosuppression 78% (14/18) - recurred 22% (4/18) - no recurrence YES: Median time to recurrence 4 mo (1-108) NO: 56% (5/9) - recurred 44% (4/9) - no recurrence Median time to recurrence 4 mo (2-24) Are there autoantibodies other than anti-PLA 2 R in these patients? Expanded cohort from Mayo Clinic 4 ‘late’ recurrences (36, 48, 60, 108 mo) disappearance (n=2) and reoccurrence of anti-PLA 2 R? 70% of patients with recurrent MN were anti-PLA 2 R positive

29. Clinical implications The majority of patients with primary MN have circulating autoantibodies against PLA 2 R, an intrinsic podocyte antigen Anti-PLA 2 R is highly specific for primary MN Clear association of anti-PLA 2 R 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

30. Pathologic mechanisms: Questions Is anti-PLA 2 R 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 PLA 2 R explain susceptibility to MN?

31. Complement C3 deposition on cultured differentiated human podocytes

32. Anti-PLA 2 R + IgG4 fractionIgG4-depleted IgG fraction normal rat serum heat inactivated rat serum

33. 4Man  14GlcNAc  1 4GlcNAc 4GlcNAc  1 Asn297 6 3 6 Fuc  1 6Gal  14GlcNAc  12Man  1 6Gal  14GlcNAc  12Man  1 VL VH CL CH1 CH2 CH3 } Fc -S-S- Galactose-deficient IgG binds mannose-binding lectin Malhotra R, et al Nat Med 237-243, 1995.

34. MN-derived IgG4 allows increased C4 deposition Membranous nephropathy Normal control sera

35. MBL binds to affinity purified anti-PLA 2 R IgG4 heavy chain

36. Could genetic polymorphisms in PLA 2 R determine susceptibility for developing disease? age of onset aggressiveness of disease recurrence in allograft

37. “Bent” ( vs. extended) conformations of mannose receptor family members from Llorca, O (2008) Cell Mol Life Sci 65: 1302-10

38. Human anti-PLA 2 R antibodies recognize an epitope in the N-terminal part of the protein

39. PLA 2 R contains several SNPs in the region of the anti-PLA 2 R epitope GWAS: rs4664308 (intron 1) Coding SNP M292V (exon 5) linkage dysequilibrium r 2 = 0.70 Detailed genotyping and sequencing of PLA2R1 in cases of anti-PLA 2 R associated MN vs. controls

40. The pathogenesis of MN: How does it all fit together? ESRD Persistent proteinuria Persistent proteinuria Remission Relapse α-PLA 2 R α-AR α-SOD α-NEP Progression factors Complement- mediated cytotoxicity (?) ? ? Genetics (?) Immunologic initiation PLA2R1 HLA-DQA1 α-Enolase

41. Acknowledgments Boston University David Salant Ramon Bonegio Rivka Ayalon Tep Chongkrairatanakul Fahim Malik Hong Ma Neetika Garg Mayo Clinic, Rochester, MN Fernando Fervenza Fernando Cosio Columbia University, New York, NY Andy Bomback Jerry Appel University of Louisville, KY David Powell Jon Klein University of Iowa Christie Thomas Christopher Blosser CNRS; Université de Nice Sophia Antipolis Gérard Lambeau Radboud Univ. Nijmegen Medical Center Julia Hofstra Jack Wetzels Nanjing University School of Medicine Weisong Qin 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

43. Sensitivity and specificity of anti-PLA 2 R for primary MN Specificity 96% Sensitivity 83% Primary MN

44. Serum anti-PLA 2 R 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

45. PODOCYTE GBM A B C Intrinsic podocyte Ag + Circulating Ab Preformed IC (Ag + Ab) Planted Ag + Circulating Ab How do the deposits form?

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

48. The PLA 2 R epitope identified by MN autoantibodies is sensitive to reduction Beck et al. (2009) New Engl J Med 361: 11-21

49. Identification of the 185 kDa MN-Ag as the M-type phospholipase A 2 receptor Beck et al. (2009) New Engl J Med 361: 11-21

50. Cultured immortalized human podocytes express PLA 2 R mRNA and protein Anti-PLA 2 R + blocking peptide

51. 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)

52. Return of anti-PLA 2 R precedes relapse of nephrotic syndrome

53. Complement-mediated cytotoxicity assay ETHIDIUM Anti-PLA 2 R + complement factors

54. 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

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