There were two cases with recurrent MG that showed negative PLA2R staining. One was from a patient who was 71 months after transplantation at the time of the biopsy. The deposits in this case were stage 2 by electron microscopy. No tissue was available from the native kidney biopsy to assess for PLA2R staining. The other recurrent case with negative PLA2R staining was from a patient 1 month after transplantation. The MG was “stage 0” with granular IgG by immunofluorescence but no subepithelial deposits visible by electron microscopy at the time of the first biopsy. The patient had a follow-up biopsy at 16 months after transplantation that showed persistent MG. At this time, there was 3+ staining for PLA2R in a pattern similar to the positive IgG (Fig. 1).
There was only one biopsy with de novo MG that had positive PLA2R staining. This patient was 4 months after transplantation and had concurrent BK nephritis at the time of biopsy. The patient was a 34-year-old female who progressed to end-stage renal disease secondary to congenital renal abnormalities. This biopsy showed staining for IgG and C4d within the deposits. C1q and all other immunoglobulins were negative. Immunofluorescent staining for IgG subtypes 1 to 4 was performed in this case. IgG1 was positive in a pattern similar to IgG, whereas IgG2, IgG3, and IgG4 were negative. IgG subtype analysis is used in many renal pathology laboratories for the purpose of determining primary versus secondary MG. IgG4-predominant staining is thought to be associated with primary MG, whereas IgG1, IgG2, and IgG3 predominate in the deposits of secondary MG (8, 11–14). However, the sensitivity and specificity of various IgG subtype staining patterns for primary MG has not been well documented.
The transplant-related findings are presented in Table 3. Banff staging for transplant rejection was similar in the two groups. Fifty-seven percent of the patients with recurrent MG had received a living-related renal transplant compared with only 13% in the de novo group. None of the patients had any known history of previous AMR. All but two cases of recurrent and two cases of de novo had no evidence of acute cellular rejection. There were two cases with recurrent MG that had acute Banff scores greater than 0: i2 t3 g0 v0 ptc0 and i2 t1 g1 v2 ptc2. Three cases with de novo MG had Banff scores greater than 0: i3 t3 g0 v0 ptc0, i3 t3 g2 v2 ptc1, and i0 t0 g0 v0 ptc1.
PLA2R is a useful marker for differentiation of de novo and recurrent MG after transplantation with a sensitivity and specificity 83% and 92%, respectively, in this series. We are aware of only one other case series on this topic (9). The findings in the present series differ somewhat in that, whereas both studies show a very high specificity, the sensitivity in the previous series was lower at 50% for detection of recurrent MG compared with 83% in the current study. The sensitivity in the present study is similar to that seen for the detection of primary MG in native biopsies (8, 15). Whereas most cases of recurrent MG appear to be PLA2R associated, this study and the previous show that there is some variability to the pathogenesis and not all cases of recurrent MG are driven by autoantibodies to PLA2R.
A recent genome-wide association study showed idiopathic MG to be strongly associated with two risk alleles in patients of white ancestry, HLA-DQA1 and PLA2R1 (16). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was a remarkable 78.5. This study raises interesting questions about the interaction between genetic variants of immune-system proteins and PLA2R in the pathogenesis of MG and could eventually have implications in renal transplantation. One recent small case series shows that MG is more likely to recur in living-donor transplants (17). If this finding is verified in other studies, this could possibly be explained by the fact that these grafts are considerably more likely to have risk alleles in common with recipients. More work is needed in this area; however, it is conceivable that, in the future, donor and recipient genotyping before transplantation could potentially decrease the incidence of recurrent MG. Interestingly, in the present study, there were more related donors in the recurrent group than in the de novo group, although the numbers were very small.
The etiology of de novo MG is currently unknown, although it is apparently not PLA2R associated in the vast majority of cases. Recent case reports and case series have suggested the involvement of AMR in the pathogenesis of this entity (4–6). Comparison of the morphologic features within the recurrent and de novo MG cases in this series showed no significant correlation with morphologic evidence of AMR in the de novo group and thus do not support AMR as the etiology of de novo MG. One limitation to this conclusion, however, is the lack of knowledge of the donor-specific antibody status in these patients, because morphologic evidence of AMR is not entirely sensitive or specific for AMR.
There was one case of very early recurrent MG in which PLA2R was negative on the initial biopsy at 1 month but positive on a follow-up biopsy at 16 months. The initial biopsy displayed the earliest morphologic form of disease in which there are no deposits visible by electron microscopy (Fig. 1). This very early form of MG has been referred to as “stage 0” (18). We hypothesize that this first biopsy represents a false-negative result rather than the involvement of another antigen–antibody system in the earliest form of disease. Very early recurrent MG without appreciable ultrastructural deposits likely has PLA2R below the threshold of visibility. This is similar to the lack of detection of PLA2R known to be present in normal podocytes. It is thought that PLA2R is present at such low levels in normal glomeruli that it is undetectable with this fluorescent method.
We present the largest series of posttransplantation MG with PLA2R results to date and detail the sensitivity and specificity of this immunofluorescence assay for the detection of recurrent MG. Recurrent MG is strongly correlated with PLA2R positivity with a sensitivity of 83% and specificity of 92% for recurrent MG. There was no morphologic evidence of an association between AMR and de novo MG, because both groups had a similar degree of microcirculation inflammation and peritubular capillary C4d staining. Most interestingly, PLA2R staining was almost always negative in de novo MG, suggesting a different mechanism in this unique form of MG.
MATERIALS AND METHODS
There were 105 cases of MG in the setting of renal transplant identified in our renal biopsy database between January 2006 and August 2012. Only 22 of the 105 cases had a tissue diagnosis of the primary native renal disease and were used for this study, including 11 cases with MG, 3 with diabetes mellitus, 3 with focal segmental glomerulosclerosis, 2 with polycystic kidney disease, 1 with IgA nephropathy, 1 with antiglomerular basement membrane antibody disease, and 1 with renal congenital abnormalities. There were 12 biopsies from 11 patients with recurrent MG and 12 biopsies from 11 patients with de novo MG.
Renal Biopsy Processing Techniques
Standard renal biopsy processing techniques were used, including light, immunofluorescence, and electron microscopies (19, 20). All light microscopy samples were stained with hematoxylin-eosin, Jones methenamine silver, and Masson trichrome and reacted with the periodic acid–Schiff reagent. All direct immunofluorescence sections were cut at 5 μm and reacted with fluorescein-tagged polyclonal rabbit anti-human antibodies to IgG, IgA, IgM, C3, C1q, fibrinogen, and κ- and λ-light chains (Dako, Carpinteria, CA) for 1 h, rinsed, and a coverslip applied using aqueous mounting media. One case was stained for fluorescein-tagged polyclonal mouse anti-human antibodies to IgG1, IgG2, IgG3, and IgG4 (Sigma-Aldrich, St. Louis, MO). For electron microscopy, thin sections were examined in a Jeol JEM-1011 electron microscope (Jeol, Tokyo, Japan). Photomicrographs were routinely taken at ×5000, ×12,000, and ×20,000 magnifications. Electron photomicrographs were used to stage the cases of MG according to the classification of Ehrenreich and Churg (21). Electron photomicrographs were also used to identify mesangial and subendothelial deposits. For this report, the term “mesangial deposits” refers to deep mesangial deposits within the mesangial matrix and internal to an identifiable paramesangial basement membrane (22).
PLA2R was detected in paraffin-embedded sections using rabbit polyclonal anti-PLA2R antibodies (Sigma-Aldrich) at a dilution of 1:50 followed by highly cross-adsorbed Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies, Carlsbad, CA) at a dilution of 1:100 (10). Each case was run with a positive and negative (secondary antibody only) control. The stain was evaluated by standard immunofluorescence microscopy using a Leica L5 filter cube. It was judged to be positive if there was positive granular capillary loop staining in the glomeruli and negative if there was no staining in glomeruli. Each stain was given a score on a scale of 0 to 3+.
Banff Staging for Transplant Rejection
Each biopsy was staged for transplant rejection using the Banff classification scheme (23, 24). Biopsies were evaluated for evidence of AMR, including C4d staining of peritubular capillaries, as well as microcirculation inflammation, including glomerulitis, peritubular capillaritis, and transplant glomerulopathy.
1. Briganti EM, Russ GR, McNeil JJ, et al. Risk of renal allograft loss from recurrent glomerulonephritis
. N Engl J Med
2002; 347: 103.
2. Sprangers B, Lefkowitz GI, Cohen SD, et al. Beneficial effect of rituximab in the treatment of recurrent idiopathic membranous nephropathy after kidney transplantation. Clin J Am Soc Nephrol
2010; 5: 790.
3. Pirson Y, Ghysen J, Cosyns JP, et al. Aetiology and prognosis of de novo graft membranous nephropathy. Proc Eur Dial Transplant Eur Ren Assoc
1985; 21: 672.
4. El Kossi M, Harmer A, Goodwin J, et al. De novo membranous nephropathy associated with donor-specific alloantibody. Clin Transplant
2008; 22: 124.
5. Lim BJ, Kim MS, Kim YS, et al. C4d deposition and multilayering of peritubular capillary basement membrane in posttransplantation membranous nephropathy indicate its association with antibody-mediated injury. Transplant Proc
2012; 44: 619.
6. Honda K, Horita S, Toki D, et al. De novo membranous nephropathy and antibody-mediated rejection in transplanted kidney. Clin Transplant
2011; 25: 191.
7. Beck LH, Bonegio RG, Lambeau G, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med
2009; 361: 11.
8. Qin W, Beck LH Jr, Zeng C, et al. Anti-phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol
2011; 22: 1137.
9. Debiec H, Martin L, Jouanneau C, et al. Autoantibodies specific for the phospholipase A2 receptor in recurrent and de novo membranous nephropathy. Am J Trans
2011; 11: 2144.
10. Larsen CP, Messias NC, Silva FG, et al. Determination of primary versus secondary membranous glomerulopathy
utilizing phospholipase A2 receptor (PLA2R1) staining in renal biopsies. Mod Pathol
2012. doi: 10.1038/modpathol.2012.207 [Epub ahead of print].
11. Doi T, Mayumi M, Kanatsu K, et al. Distribution of IgG subclasses in membranous nephropathy. Clin Exp Immunol
1984; 58: 57.
12. Ohtani H, Wakui H, Komatsuda A, et al. Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy. Nephrol Dial Transplant
2004; 19: 574.
13. Haas M. IgG subclass deposits in glomeruli of lupus and nonlupus membranous nephropathies. Am J Kidney Dis
1994; 23: 358.
14. Kearney N, Podolak J, Matsumura L, et al. Patterns of IgG subclass deposits in membranous glomerulonephritis in renal allografts. Transplant Proc
2011; 43: 3743.
15. Hoxha E, Kneissler U, Stege G, et al. Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy. Kidney Int
2012; 82: 797.
16. Stanescu HC, Arcos-Burgos M, Medlar A, et al. Risk HLA-DQA1 and PLA2R1 alleles in idiopathic membranous nephropathy. N Engl J Med
2011; 364: 616.
17. Andresdottir MB, Wetzels JF. Increased risk of recurrence of membranous nephropathy after related donor kidney transplantation. Am J Transplant
2012; 12: 265.
18. Rodriguez EF, Cosio FG, Nasr SH, et al. The pathology and clinical features of early recurrent membranous glomerulonephritis. Am J Transplant
2012; 12: 1029.
19. Walker PD. The renal biopsy. Arch Pathol Lab Med
2009; 133: 181.
20. Walker PD, Cavallo T, Bonsib SM. Practice guidelines for the renal biopsy. Mod Pathol
2004; 17: 1555.
21. Ehrenreich T, Churg J. Pathology of membranous nephropathy. Pathol Annu
1968; 2: 145.
22. Jennette JC, Iskandar SS, Dalldorf FG. Pathologic differentiation between lupus and nonlupus membranous glomerulopathy
. Kidney Int
1983; 24: 377.
23. Mengel M, Sis B, Haas M, et al. Banff 2011 meeting report: new concepts in antibody-mediated rejection. Am J Transplant
2012; 12: 563.
24. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft pathology: updates and future directions. Am J Transplant
2008; 8: 753.
Keywords:© 2013 Lippincott Williams & Wilkins, Inc.
De novo glomerulonephritis; Kidney transplant; Membranous glomerulopathy; PLA2R; Recurrent glomerulonephritis