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Patterns of IgG Subclass Deposits in Membranous Glomerulonephritis in Renal Allografts
Patterns of IgG Subclass Deposits in Membranous Glomerulonephritis in Renal Allografts N. Kearney, J. Podolak, L. Matsumura, D. Houghton, and M. Troxell ABSTRACT Background. Membranous glomerulonephritis (MGN) may develop in the renal allograft either de novo or as a recurrence. These 2 forms of MGN may have different pathogenic mechanisms, with different IgG subclass profiles in the immune deposits. This study examined IgG subclass distributions in recurrent and de novo MGN in allograft kidneys. Methods. We identified allograft kidneys with MGN, including 7 with recurrent MGN, 2 with de novo MGN, and 2 atypical/indeterminate, and determined the relative intensity of IgG1, IgG2, IgG3, and IgG4 staining in capillary wall deposits by immunofluorescence microscopy. Results. IgG4 was the dominant or codominant IgG subclass in capillary loop deposits in all 7 cases of recurrent MGN. IgG1 staining was dominant in 3 of 4 de novo or atypical MGN cases and codominant with IgG4 in the fourth. Conclusions. Although pretransplantation kidney biopsies were not available for comparisons, these findings suggest that all allograft recurrences represent idiopathic MGN and that de novo MGN cases had a different pathogenic mechanism. embranous glomerulonephritis (MGN) is characterized by deposits along the subepithelial aspect of the glomerular basement membrane, comprised of IgG and C3 (⫾ other immunoglobins and complement factors). MGN occurs in 2 clinical scenarios: 75% are so-called idiopathic (or primary) MGN, and the remainder are secondary MGN, associated with settings that include malignancy, lupus, infection, drugs, and possibly allograft rejection.1 IgG4 predominates in the subepithelial deposits of idiopathic MGN and was recently shown to have specificity for podocyte M-type phospholipase A2 receptor (PLA2R) in most of these patients.1–7 In contrast, IgG1, IgG2, or IgG3 predominates in the deposits in native kidneys with secondary MGN.2– 4,7– 8 Membranous glomerulonephritis accounts for ⬃5% of end-stage kidney diseases requiring renal transplantation9 and recurs in up to 50% of the grafts.9 –13 Presumably, most recurrences are instances of idiopathic MGN. In addition, MGN occurs de novo in ⬃2% of renal allografts in patients with other forms of end-stage kidney disease.10 The recent characterization of the immunologic basis of idiopathic MGN5–7 offer opportunities to better define the nature of recurrent and de novo MGN in the renal allograft. We
M
studied the IgG subclass distributions in a series of MGN in renal allografts, both recurrent and de novo, reasoning that consistent differences between these 2 forms might reflect differences in pathogenesis.
METHODS Selection and Description of Participants This study was approved by the Institutional Review Board of the Oregon Health & Science University. Department of Pathology files were searched for allograft kidney biopsies with MGN from 2003 to 2010. Thirty-two specimens from 23 patients were initially identified; however, archival frozen tissue containing glomeruli was available in biopsies from 11 patients. Suitable tissue was not available from the preceding native kidney biopsies. From the School of Medicine, Oregon Health & Science University (N. K.), Portland, Oregon; and Department of Pathology, Oregon Health & Science University (N.K., J.P., L.M., D. H., M.T.), Portland, Oregon. Address correspondence to Megan L. Troxell, MD, PhD, Department of Pathology, L471, 3181 SW Sam Jackson Park Rd, Portland, OR 97239. E-mail: [email protected]; mltroxell@ yahoo.com (can be published)
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2011.10.042
Transplantation Proceedings, 43, 3743–3746 (2011)
3743
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KEARNEY, PODOLAK, MATSUMURA ET AL Table 1. Clinicopathologic Data
Case #
Primary Diagnosis
Patient Age (y)
Group 1: recurrent MGN 1 MGN 73 2 MGN 53 3 MGN 72 4 MGN 46 5 MGN 51 6 MGN 75 7 MGN 61 Group 2: de novo MGN 8 Bardet-Biedl 35 9 Obstructive 8 Group 3: atypical MGN or indeterminate native kidney disease 10 Unknown1 39 11 Atypical MGN with crescents2 17
Graft Age (y)
Type of Graft
Acute Cellular Rejection
6 13 0.056 0.24 na 15 15
DD na LRD LUD na DD LRD
No na Yes6 No No No No
No No No No No No No
17 7
LRD LUD
No Yes7
No Yes7
na LRD
na Yes2
Yes1 No2
0.251 3.32
Antibody-Mediated Rejection/C4d⫹
Abbreviations: MGN, membranous glomerulopathy; LRD, living related donor; LUD, living unrelated donor; DD, deceased donor; na, not available. 1 Patient presented with preeclampsia and hypertension; cause of native kidney disease was not determined. First renal transplant at age 30; allograft failed 7 years after transplantation with allograft glomerulopathy and nongranular IgG deposition along capillary walls. In second allograft, positive peritubular capillary C4d staining was concurrent with diagnosis of MGN 2.5 months after transplantation. 2 Native renal biopsy showed cellular crescents in all open glomeruli, with subepithelial basement membrane spikes and electron dense deposits; however, deposits did not contain IgG by immunofluorescence microscopy (C3 dominant, also staining for IgM, kappa, lambda; essentially negative for C1q; negative for IgA). MGN in graft first documented 2.5 years after transplantation; acute cellular tubulointerstitial rejection (Banff type I) diagnosed 3.2 years after transplantation, along with MGN and FSGS. 3 Second graft. First failed with recurrent MGN after 17 years. Recurrent MGN in second graft documented at 5 months after transplantation. 4 Second graft. First failed with recurrent MGN after 6 years. 5 MGN documented at 1.5 months after transplantation. 6 Acute cellular vascular rejection (Banff type 2) diagnosed concurrently with MGN 17 days after transplantation. 7 Chronic rejection and positive peritubular capillary staining concurrent with diagnosis of MGN 8 years after transplantation; no prior biopsies.
Technical Information Frozen sections were stained using standard techniques with fluorescein-conjugated antibodies to IgG1, IgG2, IgG3, and IgG4 (dilutions: IgG1 and IgG4, 1:20; IgG2 and IgG3, 1:10; The Binding Site, San Diego, CA). In addition, indirect stains for IgG4 antibody used mouse anti-IgG4 primary antibody (1:1,500; The Binding Site) and secondary fluorescein-conjugated horse antimouse secondary antibody (1:100; Vector Laboratories, Burlingame, CA). Granular capillary loop immunofluorescence under ultraviolet illumination was scored independently by 3 observers on a scale of 0-3⫹ while blinded to the specifics of the clinical history. Differences in scoring were resolved by consensus.
RESULTS
The 11 allograft biopsy specimens consisted of 7 cases of recurrent MGN, 2 cases of de novo MGN, and 2 cases with atypical/indeterminate (A/I) native kidney disease (Table 1). No patients in either group had a known malignancy, lupus, or viral hepatitis. Only 1 of the 7 recurrent MGN patients had concurrent cellular rejection; none had experienced antibody-mediated rejection (Table 1). One each of the de novo and A/I patients had concurrent acute cellular rejection; both of the A/I patients had positive peritubular capillary staining for C4d. The results of IgG subclass immunofluorescence examination are detailed in Fig 1, and representative examples are provided in Fig 2. All 7 recurrent MGN cases demonstrated dominant or codominant IgG4 deposits. IgG4 was also codominant in 1 case of de novo MGN in a patient whose renal failure was secondary to Bardet-Biedl syn-
drome. IgG4 staining intensity averaged 2.1 in recurrent MGN and 1.0 in de novo and A/I MGN. The average intensity of IgG1 staining was similar across groups (1.6 – 1.8). IgG1 was dominant or codominant in both de novo MGN cases, dominant in both atypical cases, and codominant in 3 of the recurrent MGN cases. DISCUSSION
The finding of dominance or codominance of IgG4 in the capillary loop deposits in all of the recurrent MGN cases in our series supports the hypothesis that these are recurrences of idiopathic MGN because the IgG subclass profile is similar. The recent report of a patient with recurrent MGN with elevated PLA2R serum antibody titers also supports this hypothesis.14 In contrast, IgG4 was not clearly dominant in the de novo or atypical MGN cases, consistent with the theory that these differ pathogenically from primary MGN. These studies help to substantiate the hypothesis that recurrent and de novo MGN are fundamentally different diseases in allografts. Larger studies are indicated to confirm these findings, to better establish their diagnostic importance, and to correlate them with PLA2R antibody assays as they become available. ACKNOWLEDGMENTS This study was presented in abstract form at the United States and Canadian Academy of Pathology Meeting, March 2, 2011, San Antonio, Texas. The authors thank Alex Bolinder for assistance with Fig 1.
IgG SUBCLASSES IN ALLOGRAFT MGN
3745
REFERENCES
Fig 1. Distribution of IgG subclass staining in allograft biopsies with membranous nephropathy. (A) Recurrent MGN (B) De novo and atypical/indeterminate MGN.
1. Markowitz GS, Stokes B, Kambham N, et al: Membranous nephropathy. Nephrol Self Assess Program 4:273, 2011 2. Doi T, Mayumi M, Kanatsu K, et al: Distribution of IgG subclasses in membranous nephropathy. Clin Exp Immunol 58:57, 1984 3. Noel LH, Aucouturier P, Monteiro RC, et al: Glomerular and serum immunoglobulin G subclasses in membranous nephropathy and anti-glomerular basement membrane nephritis. Clin Immunol Immunopathol 46:186, 1988 4. Kuroki A, Shibata T, Honda H, et al: Glomerular and serum IgG subclass in diffuse proliferative lupus nephritis, membranous lupus nephritis, and idiopathic membranous nephropathy Intern Med 41:936, 2002 5. Beck LH, Bonegio RG, Lambeau G, et al: M-Type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 361:11, 2009 6. Beck LH, Salant DJ: Membranous nephropathy: recent travels and new roads ahead. Kidney Int 77:765, 2010. 7. Qin W, Beck LH, Zeng C, et al: Anti–phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol 22:1137, 2011 8. Ohtani H, Wakui H, Komatsuda A, et al: Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy. Nephrol Dial Transplant 19:574, 2004 9. Marcen R, Mampaso F, Teruel JL, et al: Membranous nephropathy: recurrence after kidney transplantation. Nephrol Dial Transplant 11:1129, 1996 10. Ivanyi B: A primer on recurrent and de novo glomerulonephritis in renal allografts. Nat Clin Pract Nephrol 4:446, 2008 11. Ponticelli C, Glassock RJ: Posttransplant recurrence of primary glomerulonephritis. Clin J Am Soc Nephrol 5:2363, 2010 12. Dabade TS, Grande JP, Norby SM, et al: Recurrent idiopathic membranous nephropathy after kidney transplantation: a surveillance biopsy study. Am J Transplant 8:1318, 2008 13. El-Zoughby ZM, Grande JP, Fraile MG, et al: Recurrent idiopathic membranous nephropathy: early diagnosis by protocol biopsies and treatment with anti-CD20 antibodies. Am J Transplant 9; 2800, 2009 14. Stahl R, Hoxha E, Fechner K: PLA2R autoantibodies and recurrent membranous nephropathy after transplantation. N Engl J Med 363:496, 2010.
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KEARNEY, PODOLAK, MATSUMURA ET AL
Fig 2. Representative immunofluorescent staining. A-D: recurrent MGN with dominant IgG4 staining (case # 6); E-H: recurrent MGN with dominant IgG4 staining (case # 5); I-M: de novo MGN with codominant IgG1 & IgG3 staining (case # 9); N-Q: allograft MGN in a patient with atypical native renal MGN, with dominant IgG1 staining (case # 11) (IgG1⫽A, E, I, M; IgG2⫽B, F, J, N; IgG3⫽C, G, K, O; IgG4⫽D, H, L, P).
M
studied the IgG subclass distributions in a series of MGN in renal allografts, both recurrent and de novo, reasoning that consistent differences between these 2 forms might reflect differences in pathogenesis.
METHODS Selection and Description of Participants This study was approved by the Institutional Review Board of the Oregon Health & Science University. Department of Pathology files were searched for allograft kidney biopsies with MGN from 2003 to 2010. Thirty-two specimens from 23 patients were initially identified; however, archival frozen tissue containing glomeruli was available in biopsies from 11 patients. Suitable tissue was not available from the preceding native kidney biopsies. From the School of Medicine, Oregon Health & Science University (N. K.), Portland, Oregon; and Department of Pathology, Oregon Health & Science University (N.K., J.P., L.M., D. H., M.T.), Portland, Oregon. Address correspondence to Megan L. Troxell, MD, PhD, Department of Pathology, L471, 3181 SW Sam Jackson Park Rd, Portland, OR 97239. E-mail: [email protected]; mltroxell@ yahoo.com (can be published)
© 2011 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/–see front matter doi:10.1016/j.transproceed.2011.10.042
Transplantation Proceedings, 43, 3743–3746 (2011)
3743
3744
KEARNEY, PODOLAK, MATSUMURA ET AL Table 1. Clinicopathologic Data
Case #
Primary Diagnosis
Patient Age (y)
Group 1: recurrent MGN 1 MGN 73 2 MGN 53 3 MGN 72 4 MGN 46 5 MGN 51 6 MGN 75 7 MGN 61 Group 2: de novo MGN 8 Bardet-Biedl 35 9 Obstructive 8 Group 3: atypical MGN or indeterminate native kidney disease 10 Unknown1 39 11 Atypical MGN with crescents2 17
Graft Age (y)
Type of Graft
Acute Cellular Rejection
6 13 0.056 0.24 na 15 15
DD na LRD LUD na DD LRD
No na Yes6 No No No No
No No No No No No No
17 7
LRD LUD
No Yes7
No Yes7
na LRD
na Yes2
Yes1 No2
0.251 3.32
Antibody-Mediated Rejection/C4d⫹
Abbreviations: MGN, membranous glomerulopathy; LRD, living related donor; LUD, living unrelated donor; DD, deceased donor; na, not available. 1 Patient presented with preeclampsia and hypertension; cause of native kidney disease was not determined. First renal transplant at age 30; allograft failed 7 years after transplantation with allograft glomerulopathy and nongranular IgG deposition along capillary walls. In second allograft, positive peritubular capillary C4d staining was concurrent with diagnosis of MGN 2.5 months after transplantation. 2 Native renal biopsy showed cellular crescents in all open glomeruli, with subepithelial basement membrane spikes and electron dense deposits; however, deposits did not contain IgG by immunofluorescence microscopy (C3 dominant, also staining for IgM, kappa, lambda; essentially negative for C1q; negative for IgA). MGN in graft first documented 2.5 years after transplantation; acute cellular tubulointerstitial rejection (Banff type I) diagnosed 3.2 years after transplantation, along with MGN and FSGS. 3 Second graft. First failed with recurrent MGN after 17 years. Recurrent MGN in second graft documented at 5 months after transplantation. 4 Second graft. First failed with recurrent MGN after 6 years. 5 MGN documented at 1.5 months after transplantation. 6 Acute cellular vascular rejection (Banff type 2) diagnosed concurrently with MGN 17 days after transplantation. 7 Chronic rejection and positive peritubular capillary staining concurrent with diagnosis of MGN 8 years after transplantation; no prior biopsies.
Technical Information Frozen sections were stained using standard techniques with fluorescein-conjugated antibodies to IgG1, IgG2, IgG3, and IgG4 (dilutions: IgG1 and IgG4, 1:20; IgG2 and IgG3, 1:10; The Binding Site, San Diego, CA). In addition, indirect stains for IgG4 antibody used mouse anti-IgG4 primary antibody (1:1,500; The Binding Site) and secondary fluorescein-conjugated horse antimouse secondary antibody (1:100; Vector Laboratories, Burlingame, CA). Granular capillary loop immunofluorescence under ultraviolet illumination was scored independently by 3 observers on a scale of 0-3⫹ while blinded to the specifics of the clinical history. Differences in scoring were resolved by consensus.
RESULTS
The 11 allograft biopsy specimens consisted of 7 cases of recurrent MGN, 2 cases of de novo MGN, and 2 cases with atypical/indeterminate (A/I) native kidney disease (Table 1). No patients in either group had a known malignancy, lupus, or viral hepatitis. Only 1 of the 7 recurrent MGN patients had concurrent cellular rejection; none had experienced antibody-mediated rejection (Table 1). One each of the de novo and A/I patients had concurrent acute cellular rejection; both of the A/I patients had positive peritubular capillary staining for C4d. The results of IgG subclass immunofluorescence examination are detailed in Fig 1, and representative examples are provided in Fig 2. All 7 recurrent MGN cases demonstrated dominant or codominant IgG4 deposits. IgG4 was also codominant in 1 case of de novo MGN in a patient whose renal failure was secondary to Bardet-Biedl syn-
drome. IgG4 staining intensity averaged 2.1 in recurrent MGN and 1.0 in de novo and A/I MGN. The average intensity of IgG1 staining was similar across groups (1.6 – 1.8). IgG1 was dominant or codominant in both de novo MGN cases, dominant in both atypical cases, and codominant in 3 of the recurrent MGN cases. DISCUSSION
The finding of dominance or codominance of IgG4 in the capillary loop deposits in all of the recurrent MGN cases in our series supports the hypothesis that these are recurrences of idiopathic MGN because the IgG subclass profile is similar. The recent report of a patient with recurrent MGN with elevated PLA2R serum antibody titers also supports this hypothesis.14 In contrast, IgG4 was not clearly dominant in the de novo or atypical MGN cases, consistent with the theory that these differ pathogenically from primary MGN. These studies help to substantiate the hypothesis that recurrent and de novo MGN are fundamentally different diseases in allografts. Larger studies are indicated to confirm these findings, to better establish their diagnostic importance, and to correlate them with PLA2R antibody assays as they become available. ACKNOWLEDGMENTS This study was presented in abstract form at the United States and Canadian Academy of Pathology Meeting, March 2, 2011, San Antonio, Texas. The authors thank Alex Bolinder for assistance with Fig 1.
IgG SUBCLASSES IN ALLOGRAFT MGN
3745
REFERENCES
Fig 1. Distribution of IgG subclass staining in allograft biopsies with membranous nephropathy. (A) Recurrent MGN (B) De novo and atypical/indeterminate MGN.
1. Markowitz GS, Stokes B, Kambham N, et al: Membranous nephropathy. Nephrol Self Assess Program 4:273, 2011 2. Doi T, Mayumi M, Kanatsu K, et al: Distribution of IgG subclasses in membranous nephropathy. Clin Exp Immunol 58:57, 1984 3. Noel LH, Aucouturier P, Monteiro RC, et al: Glomerular and serum immunoglobulin G subclasses in membranous nephropathy and anti-glomerular basement membrane nephritis. Clin Immunol Immunopathol 46:186, 1988 4. Kuroki A, Shibata T, Honda H, et al: Glomerular and serum IgG subclass in diffuse proliferative lupus nephritis, membranous lupus nephritis, and idiopathic membranous nephropathy Intern Med 41:936, 2002 5. Beck LH, Bonegio RG, Lambeau G, et al: M-Type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med 361:11, 2009 6. Beck LH, Salant DJ: Membranous nephropathy: recent travels and new roads ahead. Kidney Int 77:765, 2010. 7. Qin W, Beck LH, Zeng C, et al: Anti–phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol 22:1137, 2011 8. Ohtani H, Wakui H, Komatsuda A, et al: Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy. Nephrol Dial Transplant 19:574, 2004 9. Marcen R, Mampaso F, Teruel JL, et al: Membranous nephropathy: recurrence after kidney transplantation. Nephrol Dial Transplant 11:1129, 1996 10. Ivanyi B: A primer on recurrent and de novo glomerulonephritis in renal allografts. Nat Clin Pract Nephrol 4:446, 2008 11. Ponticelli C, Glassock RJ: Posttransplant recurrence of primary glomerulonephritis. Clin J Am Soc Nephrol 5:2363, 2010 12. Dabade TS, Grande JP, Norby SM, et al: Recurrent idiopathic membranous nephropathy after kidney transplantation: a surveillance biopsy study. Am J Transplant 8:1318, 2008 13. El-Zoughby ZM, Grande JP, Fraile MG, et al: Recurrent idiopathic membranous nephropathy: early diagnosis by protocol biopsies and treatment with anti-CD20 antibodies. Am J Transplant 9; 2800, 2009 14. Stahl R, Hoxha E, Fechner K: PLA2R autoantibodies and recurrent membranous nephropathy after transplantation. N Engl J Med 363:496, 2010.
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KEARNEY, PODOLAK, MATSUMURA ET AL
Fig 2. Representative immunofluorescent staining. A-D: recurrent MGN with dominant IgG4 staining (case # 6); E-H: recurrent MGN with dominant IgG4 staining (case # 5); I-M: de novo MGN with codominant IgG1 & IgG3 staining (case # 9); N-Q: allograft MGN in a patient with atypical native renal MGN, with dominant IgG1 staining (case # 11) (IgG1⫽A, E, I, M; IgG2⫽B, F, J, N; IgG3⫽C, G, K, O; IgG4⫽D, H, L, P).