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Phospholipase A2 Receptor (PLA2R) Staining Is Useful in the Determination of De Novo Versus Recurrent Membranous Glomerulopathy

Larsen, Christopher P.1,2,3; Walker, Patrick D.1,2

doi: 10.1097/TP.0b013e31828a947b
Clinical and Translational Research
Free

Background Membranous glomerulopathy (MG) is one of the most common glomerulonephritides involving the renal transplant. We sought to determine the utility of phospholipase A2 receptor (PLA2R) staining for the detection of recurrent MG. We also evaluated for increased evidence of antibody-mediated rejection in the de novo group, as some have reported.

Methods Twenty-two cases of MG occurring in renal transplant biopsies were identified, who had a tissue diagnosis documenting the primary native renal disease. There were 12 biopsies from 11 patients with recurrent MG and 12 biopsies from 11 patients with de novo MG. Morphologic evaluation and PLA2R staining was performed in all cases.

Results Ten of 12 (83%) recurrent MG and 1 of 12 (8%) de novo MG biopsies showed positive glomerular staining for PLA2R, giving PLA2R a sensitivity of 83% (95% confidence interval, 51%–97%) and specificity of 92% (95% confidence interval, 60%–100%) for recurrent MG. There were 2 of 12 (17%) de novo and 1 of 12 (8%) recurrent biopsies showing the presence of microcirculation inflammation. Peritubular capillary C4d staining was negative in all cases.

Conclusion 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 antibody-mediated rejection 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.

1 Nephropath, Little Rock, AR.

2 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR.

3 Address correspondence to: Christopher P. Larsen, M.D., Nephropath, 10810 Executive Center Drive, Suite 100 Little Rock, AR 72211.

The authors declare no funding or conflicts of interest.

E-mail: chris.larsen@nephropath.com

C.P.L. and P.D.W. participated in the research design, data analysis, and writing of the article. C.P.L. searched the database for cases and performed the immunostains.

Received 18 December 2012. Revision requested 9 January 2013.

Accepted 29 January 2013.

Glomerulonephritis in the renal allograft is an important clinical challenge. Recurrent glomerulonephritis is the third most common cause of graft loss (1). Membranous glomerulopathy (MG) is one of the most frequent glomerulonephritides in the transplant setting, and recurrent MG occurs as frequently as 44% (2). In addition to recurrent MG, many patients develop de novo MG that is morphologically indistinguishable from recurrent MG. Both recurrent and de novo MG have a poor prognosis and frequently result in graft loss (1, 3). The etiology of de novo MG is currently unknown, although some have recently raised the possibility that antibody-mediated rejection (AMR) might play some role in the disease (4–6).

In recent years, our understanding of primary MG has been transformed through the discovery of phospholipase A2 receptor (PLA2R) as the target antigen in most cases of primary MG (7). This discovery has made it possible to design serologic assays that hold promise for the diagnosis and monitoring of MG (8). A recent report has shown that PLA2R was involved in recurrent but not de novo MG (9). This is an important finding considering the fact that many patients never have a tissue diagnosis of their native kidney disease, making it impossible to determine with certainty whether MG in the transplant setting is a recurrent or de novo process. We sought to confirm the presence of PLA2R involvement in posttransplantation MG using PLA2R staining of renal biopsies in the largest case series to date. This technique is easy to institute in renal pathology laboratories and thus potentially widely available (10). We also compared the morphologic features of the two groups to determine if there is increased evidence of AMR in the de novo group.

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RESULTS

The clinical and morphologic details are shown in Tables 1 and 2. Cases of MG showed either granular positive staining for PLA2R in the same pattern as the IgG or were completely negative. The sensitivity and specificity of PLA2R for detection of recurrent MG was 83% (95% confidence interval [CI], 51%–97%) and 92% (95% CI, 60%–100%), respectively, with a positive likelihood ratio of 10.0 (95% CI, 1.5–66.4). There were a total of 11 cases that stained positive for PLA2R, including 9 that were scored 3+ and 2 that scored 2+ (scale of 0–3+).

TABLE 1

TABLE 1

TABLE 2

TABLE 2

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).

FIGURE 1

FIGURE 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.

TABLE 3

TABLE 3

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DISCUSSION

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.

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MATERIALS AND METHODS

Patient Selection

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.

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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).

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PLA2R Immunofluorescence

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+.

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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.

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Keywords:

De novo glomerulonephritis; Kidney transplant; Membranous glomerulopathy; PLA2R; Recurrent glomerulonephritis

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