Original Clinical ScienceçGeneral

Antiphospholipase A2 Receptor Antibody Levels Predict the Risk of Posttransplantation Recurrence of Membranous Nephropathy Luis F. Quintana,1 Miquel Blasco,1 Miguel Seras,2 Nuria S. Pérez,1 Marcos López-Hoyos,3 Patricia Villarroel,4 Emilio Rodrigo,2 Odette Viñas,4 Guadalupe Ercilla,4 Fritz Diekmann,1 José J. Gómez-Roman,5 Gema Fernandez-Fresnedo,2 Federico Oppenheimer,1 Manuel Arias,2 and Josep M. Campistol1 Background. Secretory phospholipase A2 receptor (PLA2R) is the target antigen of the auto-antibodies produced in most

(~70%) patients with primary membranous nephropathy (pMN). The applicability of anti-PLA2R1 antibody monitoring for the prediction of MN recurrence in kidney transplant recipients still is a matter of debate. Methods. We sought to characterize the presence and concentration of anti-PLA2R antibodies by enzyme-linked immunosorbent assay (ELISA) in a cohort of 21 patients with pMN before and after transplantation to evaluate whether anti-PLA2R concentrations could predict pMN recurrence. Results. The presence of pMN recurrence was significantly correlated with the existence of a positive ELISA assay at graft biopsy or with high level of anti-PLA2R1 activity before transplantation (P = 0.03). In the receiver operating characteristic analysis, anti-PLA2R levels (cut-off of 45 U/mL) during the pretransplantation period accurately predicted pMN recurrence, with a sensitivity of 85.3%, specificity of 85.1%, negative predictive value of 92%, and an area under the curve of 90.8%. This finding supports the hypothesis that anti-PLA2R cause pMN recurrence in humans and indicates the need to prove in an experimental model. Furthermore, 6 of 7 patients with recurrence were carriers of HLA DQA1* 05:01/05 and DQB1* 02:01, confirming these DQ alleles as those associated with higher anti-PLA2R levels. Conclusions. This study is the first to demonstrate pretransplantation circulating anti-PLA2R antibodies in a cohort of renal transplant recipients who prospectively developed recurrent disease. Currently, antiPLA2R levels measured by ELISA may be a rational tool to establish the risk of MN recurrence in renal allograft recipients. (Transplantation 2015;99: 1709–1714)

P

rimary membranous nephropathy (pMN) remains the most common cause of adult onset nephrotic syndrome. The clinical course of patients with pMN varies but around a third of patients remains nephrotic even with immunosuppressive treatment and progress to end-stage renal disease (ESRD)1; this glomerular disease is estimated to cause ESRD in 5% of patients.2 The preferred renal replacement therapy is transplantation, but this nephropathy recurs in 30% to 50% of recipients,3,4 with graft loss occurring in 50% of patients by 10 years, usually during the first 24 months.5 More than 50 years ago, the Heymann experimental nephritis model provided an understanding of the mechanisms

of in situ immune complex formation in pMN and, even more importantly, showed that transplantation of an affected kidney to a naive host results in a significant decrease in proteinuria but not in its complete elimination.6 Subsequently, Debiec et al7 provided the first evidence of this kind of complement deposit mechanism in a rare form of human neonatal MN. In recent years, an autoimmune basis for pMN has been established by the identification of the M-type phospholipase A2 receptor (PLA2R) as the major antigen after prospective serum screening by Western blotting8 and by the finding that the antigen in the deposits can be used to confirm PLA2R-associated MN when circulating anti-PLA2R antibody is undetectable.9 However, in situ PLA2R-mediated immune complex formation has not yet been confirmed in human pMN.

Received 22 July 2014. Revision requested 16 October 2014. Accepted 28 November 2014. 1 Servicio de Nefrología y Trasplante Renal, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universidad de Barcelona, Barcelona, Spain. 2

Servicio de Nefrología, Hospital Universitario Marqués de Valdecilla-IDIVAL, Universidad de Cantabria, Santander, Spain.

3 Servicio de Inmunología, Hospital Universitario Marqués de Valdecilla-IDIVAL, Universidad de Cantabria, Santander, Spain. 4

Servicio de Inmunología (CDB), Hospital Clinic, Barcelona, Spain.

5

Servicio de Anatomía Patológica, Hospital Universitario Marqués de Valdecilla-IDIVAL, Universidad de Cantabria, Santander, Spain. L.F.Q. is supported by the professional association of doctors of Hospital Clinic in Barcelona with a sabbatical year. N.S.P. is supported by the Fundación SENEFRO, Sociedad Española de Nefrología.

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The authors declare no conflicts of interest. L.F.Q., N.S.P., M.L.-H., F.D., F.O., J.M.C., M.A. participated in research design. L.F.Q., M.B., G.F.-F., E.R., J.M.C., M.A. participated in the writing of the paper. M.S., E.R., P.V., N.S.P., G.F.-F., L.F.Q., O.V., J.J.G.-R. participated in the performance of the research. O.V., G.E., J.J.G.-R., M.L.-H., P.V. contributed new reagents or analytic tools. L.F.Q., G.E., M.B., F.D., M.S., G.F.-F., F.O., J.M.C., M.A. participated in data analysis. Correspondence: Luis F. Quintana, MD, PhD, Servicio de Nefrología y Trasplante Renal, Hospital Clinic, Villarroel 170, 08036, Barcelona, Spain. ([email protected]) Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0041-1337/15/9908-1709 DOI: 10.1097/TP.0000000000000630

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From a clinical point of view, it is difficult to use Western blotting to determine circulating anti-PLA2R outside of an expert laboratory, and the method is not suitable to evaluate large samples. These limitations led to the development of a recombinant cell-based indirect immunofluorescence assay that uses the human cell line, HEK293, which overexpresses the full-length human PLA2R1 as substrate.10 The assay can be used as a diagnostic tool but is not the test of choice to approximate antibody concentrations during patient monitoring. More recently, an enzyme-linked immunosorbent assay (ELISA) protocol has been developed as the most robust test system that can be used in a routine environment to determine and quantify autoantibodies against PLA2R.11 A strong association has been reported between anti-PLA2R and immunologic activity in this autoimmune disease, suggesting that these autoantibodies have inherent pathogenicity. Nevertheless, proteinuria, the main clinical marker of disease activity, and the presence of anti-PLA2R are intimately, but not completely, correlated.12 Sclerotic changes that occur in renal tissue after established pMN can also cause persistent proteinuria unconnected to immunologic activity, as demonstrated by renal allograft surveillance by protocol biopsies, which also revealed that the earliest deposits occurring in recurrent MN can be associated with minimal proteinuria.2 Certain PLA2R1 gene variants have been strongly associated with pMN, revealing a genetic component in this disease. However, these variants are common in the general population and, given the low frequency of pMN, are unlikely to explain its pathogenesis.13 A possible explanation may lie in the previously reported interaction between PLA2R1 variants and specific HLA class II molecules as an alternative mechanism of genetic predisposition to autoimmunity in this glomerular disease.11 The applicability of anti-PLA2R1 antibody monitoring for the prediction of MN recurrence in kidney transplant recipients still is a matter of debate. Several case reports14,15 have demonstrated, even in the first post-transplantation week, an association between recurrent MN in transplant recipients and the qualitative detection of circulating anti-PLA2R antibodies before surgery; conversely, a study by Debiec et al16 in 10 patients with recurrent MN suggested that anti-PLA2R1 antibodies were implicated in only 5 of 10 patients, showing no simple association between anti-PLA2R1 activity at transplantation and the risk of MN recurrence. Nonetheless they used a qualitative method of detection and some patients had incomplete sampling before and after transplantation. Finally, a very recent paper by Seitz-Polski and colleagues,17 showed that a persistent anti-PLA2R1 activity after transplantation was associated with MN recurrence in a cohort of 15 patients with primary or secondary cases of MN, who underwent deceased donor transplantation. It should be noted that 4 patients of this last cohort never had a kidney graft biopsy for rule out the presence of MN recurrence. Despite the significant burden of MN worldwide, many questions on recurrent disease remain to be answered; therefore, the development of a non-invasive predictive model for disease recurrence in renal transplantation would be invaluable not only to alleviate the health and economic burden caused by this condition but also to explore the consequences of anti-PLA2R in renal allografts before the disease becomes clinically evident. We sought to characterize the presence and concentration of anti-PLA2R antibodies by ELISA in a Spanish cohort of

21 patients with pMN before and after transplantation to evaluate whether anti-PLA2R concentrations could predict pMN recurrence. Additionally, we sought to address whether the type of donor, the HLA alleles of both the recipient and donor, or immunosuppressive therapy could promote the development of anti-PLA2R antibodies and pMN recurrence.

Transplantation

PATIENTS AND METHODS Study Design

The study was approved by the local ethics committees in Spain and was performed in accordance with the declarations of Helsinki and Istanbul. All patients enrolled in this study gave their consent. The study population consisted of 21 adults with underlying pMN and was based on an observational, post-transplantation design. Primary MN was defined for patients, where no secondary cause was evident. Secondary MN was ruled out after confirm the absence of anti-nuclear antibodies, hepatitis B or C serologies or neoplasia during more than 10 years of follow up, in the group of patients with no evidence of APLA2R activity. We included consecutive patients with pMN who underwent kidney (living and deceased donors) transplantation from January 1994 to April 2013 at the Departments of Nephrology and Renal Transplantation, Hospital Marquez de Valdecilla in Santander and Hospital Clinic in Barcelona, Spain. There were no other specific inclusion or exclusion criteria. Allograft biopsies were performed based on clinical indication in all the patients included. Prior to renal transplantation and allograft biopsy, serum samples were prospectively collected. A-PLA2R levels were investigated retrospectively in this cohort of recipients with underlying pMN Evaluation of Clinical Parameters

The patients' age, sex, renal function, 24-hour proteinuria, donor-recipient HLA typing, and immunosuppressive therapy were recorded at the time of renal transplantation. The primary outcome for this analysis was the appearance of biopsy-proven pMN recurrence. Histopathology

A minimum of 6 glomeruli was considered adequate for a biopsy to be included. Standard processing of renal biopsies included light microscopy and immunofluorescence. All specimens were stained with hematoxylin and eosin, periodic acidSchiff, Masson's trichrome and Jones methenamine silver. For immunofluorescence 3 lm cryostat sections were stained with polyclonal fluorescein isothiocyanate–conjugated antibodies to immunoglobulin (Ig)G, IgM, IgA, C3, C1q, kappa, lambda, fibrinogen, albumin and C4d , as per routine clinical testing. Detection of Anti-PLA2R Antibodies

All serum samples were tested for the presence of antiPLA2R IgG antibodies using an anti-PLA2R IgG quantitative ELISA (Euroimmun, Luebeck, Germany), according to the manufacturer's instructions. Sera were diluted 1:101 in sample buffer and dispensed to the wells of the polystyrene microplate strips already coated with PLA2R and incubated for 30 minutes. Following a 3-step wash procedure with wash buffer, bound antibodies were detected by a second, 30-minute incubation with peroxidase-labeled anti-humanIgG (rabbit). After a second 3-step wash procedure, the substrate solution (chromogen TMB/H2O2) was added and

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TABLE 1.

Main characteristics of the clinical cohort Male/female, n

19/2

Age, median/mean (SD) Donor type, n Deceased Living related CKD Pre-RT status, n CKD 5 CKD 5D HD/PD Median time to allograft biopsy (SD), mo Previous Transplants, 0/1, n Proteinuria, median (SD), g/d Pre-RT/RB Serum Cr, median (SD), mg/dL at RB Pre-RT APLA2R levels, median/mean (SD), U/mL

44/47(13.81) 14 7 5 14/2 51 (257) 13/8 5.46 (5.6)/4.05(6.8) 1.80 (1.2) 17.58/192.38(384)

Cr, creatinine; RT, renal transplantation; RB, renal biopsy.

incubated for 15 minutes protected from direct sunlight. Finally, 100 μL of stop solution were added to each well and the photometric measurement of the color intensity was made at a 450-nm wavelength and a reference wavelength between 620 nm and 650 nm, within 30 minutes of adding the stop solution. All incubation steps were carried out at room temperature. All calibrators, controls, and a blank were always processed in parallel to the assayed samples. In positive samples, specific IgG antibodies present in the sera of the patients bound to the PLA2R-coated bottom of the wells, and the color intensity (or extinction), measured after the addition of stop solution, was proportional to the human IgG captured in the corresponding well. The final concentrations for each unknown sample, in RU/mL, were finally calculated from the calibration curve extinction values plotted against the concentration for each calibrator. According to the manufacturer, the results were interpreted as negative for less than 14 RU/mL, borderline from 14 or higher to less than 20 RU/mL, and positive for 20 RU/mL or higher. HLA Typing

DNA extracted from peripheral blood was available in 17 of the 21 patients. Sequence-specific oligonucleotide polymerase chain reaction (PCR-SSO) (Lifecodes, Immucor, Stamford, CT) and /or the sequence-specific primer (PCR-SSP, Pleasanton, CA) (ALLSet + Gold SSP; Invitrogen) techniques were used for typing the HLA-DQA1 and -DQB1 alleles. HLA specificities were assigned according the World Health Organization's nomenclature (http://www.ebi.ac.uk/ipd/imgt/hla/align.html) using the Mach-IT DNA and Unimatch software, respectively. Statistics

Descriptive data are presented as the median or mean (SD) for continuous variables and as frequency (%) for categorical data. The Mann-Whitney, Kruskal-Wallis, and Fisher exact test were used for comparisons between groups. Correlation was assessed with Spearman coefficient and outcome data, including the associated risk of recurrence of APLA2R titers, were analyzed with Cox regression analysis. Coefficients are expressed as hazard ratios with 95% confidence interval. A receiver operating characteristic (ROC) analysis was performed to evaluate whether APLA2R levels could serve as a

predictive marker of post-transplantation pMN with adequate sensitivity and specificity, positive predictive value and negative predictive value. All P values were 2-tailed and were considered significant below 0.05. The software used for the statistical analyses was SPSS 22 version for windows (SPSS, Inc., Chicago, IL). RESULTS Twenty-one patients (Santander = 12, Barcelona = 9) were identified with underlying pMN before renal transplantation from January 1994 to April 2013. In all these recipients, native kidney biopsies showed pMN. Baseline characteristics are presented in Table 1. Ninety-one percent of the recipients were men, with a median age of 44 years. The median length of posttransplantation follow-up was 141 months. Induction therapy consisted of basiliximab in 5 patients, antithymocyte globulin in 2, alefacept in 1, and daclizumab in 1; no induction therapy was provided in 12. Maintenance triple immunosuppressive therapy with steroids, mycophenolic acid derivative (MAD) and a calcineurin inhibitor was administered in 17 patients, triple therapy with steroids, and a mammalian target of rapamycin inhibitor and MAD in 3, and steroid-free protocols with MAD + calcineurin inhibitor after early steroid discontinuation in one. Transplants consisted of deceased donor kidneys in 14 patients and living-related donor kidneys in 7. The HLA DQA1 05 haplotype was present in 88% of the recipients. DQ A1 05:01(05) and DQB1 02:01 were present in 70% of the recipients; being 5 of them DQA*05 homozygous. DQA * 5:01 is identical to DQA * 5:05 at the region of peptides combination, which validates that are considered functionally equivalent alleles. Information on anti-HLA antibodies determined by complementdependent techniques was available in 11 of the 21 patients. None of these 11 patients showed anti-HLA antibodies at any time point, as determined by complement-dependent cytotoxicity. In 5 of these 11 patients, Luminex-based antibody determinations were available. Only one showed a positive Luminex screening test for anti-HLA antibodies. This patient was transplanted twice within less than 1 year, without evidence of recurrent disease or acute rejection at the allograft TABLE 2.

Characteristics of the clinical cohort and distribution of patients with and without recurrence of membranous nephropathy Cases (N = 7) Controls (N = 14)

Male/female, n Previous transplants 0/1, n Donor Type D/LR Age, mean (SD) Proteinuria, median (SD), g/d Pre-RT 3 moa Pre-RB SCr, mean (SD), mg/dL at RB Mean time to RB (SD), mo Mean pre-RT APLA2R levels, (SD) U/mLb a

7/0 5/2 3/4 53.4 (12.7)

12/2 8/6 11/3 44 (13.6)

10.05 (8.1) 3.04 (3.49) 9.17 (7.2) 2.07 (0.92) 99 (122) 485 (553)

4.53 (2.89) 0.51 (0.62) 3.84 (5.7) 1.7 (0.33) 162 (306) 34 (67)

P = 0.018. P = 0.008. RB, renal biopsy; RT, renal transplantation; SCr, serum creatinine.

b

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Patient 3 had positive anti-PLA2R at 3 and 6 months after transplantation confirming APLA2R MN recurrence Patient 7 had positive anti-PLA2R at first month after transplantation confirming APLA2R MN recurrence, NR, no remission; NA, not available, LR, living related; DD, deceased donor; PE, plasma exchange. All the cases had nephrotic syndrome at presentation, which was defined by a proteinuria value > 3.5 g/d along with hypoalbuminemia (serum albumin < 3 g/dL). CR, complete remission was defined by proteinuria value < 0.5 g/d in the least 3 consecutive visits in absence of concomitant renal function worsening. PR, partial remission was defined by proteinuria < 3.5 g/d along with normal serum albumin in the absence of concomitant renal function worsening. All the recipients received maintenance triple immunosuppressive therapy with steroids, mycophenolic acid derivative, and a calcineurin inhibitor except case 5 that received a free steroid protocol with MMF and CNI. b

Rituximab + PE Rituximab + PE Rituximab

CR/PR NR/NR NR/PR PR/PR NR/PR CR/NA PR/CR Rituximab Rituximab + PE Rituximab

4200 10,000 3875 4050 22,000 16,000 4100 1.2 2.3 1.3 1.1 1.8 4.8 1.5 23 51 10a 55 77 793 10b 12.00 3.00 152.00 182.00 3.00 0.23 (1 week) 3.00 46 799 93 11 216 1500 734 LR DD DD DD LR LR LR Basiliximab Basiliximab

37 60 49 77 61 61 42

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Disease duration, mo Anti-PLA2R level at RT, UI/mL Donor type Induction therapy Age, y Case

TABLE 3.

Follow-up of patients with MN recurrence and response to therapy

Anti-PLA2R level at RB (UI/mL)

Creatinine at RB, mg/dL

Proteinuria at RB, mg/d

Therapy

Outcome 12/24 mo

Comments

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biopsy during the first transplantation. At the time of the first transplantation, he showed nondonor-specific anti-HLA antibodies. At the second transplantation, this patient showed a donor-specific anti DRB1*01 antibody with a mean fluorescence intensity of 4552, which was probably acquired during the course of the previous transplantation. Biopsy-proven recurrence occurred in 7 recipients (33.3%) at an average of 22 months after transplantation (0.23-73 months). The indication for obtaining a biopsy in the control group was a rise in serum creatinine in 100% of the cases, combining with proteinuria greater than 1 g/24 hours in 4 patients. The biopsy result was unspecific interstitial fibrosis with tubular atrophy (IFTA) in the whole group of 14 control patients. Eight patients had IFTA grade 1 and 6 had IFTA grade 2 combining with nephroangiosclerosis in 2 cases. There was no evidence of C4d deposits on peritubular capillaries in patients with recurrence or in the group with no evidence of MN. The cumulative recurrence rate was 57.1% in the recipients of living-related donor kidneys and 21.4% in recipients of deceased-donor kidneys (log-rank test, P = 0.03). There were no differences in renal status or proteinuria pretransplantation, sex, induction, or maintenance immunosuppressive therapy between recipients with and without pMN recurrence. Six recipients received treatment for recurrence; 3 with Rituximab monotherapy and 3 with a combination of plasma exchange with Rituximab. Patients were treated with 4 weekly doses of Rituximab given at 375 mg/m2. Plasma exchange consisted of 7 exchanges over 14 days of 60 mL/kg per session using 3% to 5% albumin as a replacement solution. In this study, treatment received after diagnosis was not taken into account in the analysis. The main clinical and laboratory features in cases and controls are summarized in Table 2, and a description of followup and response to therapy in recipients with pMN recurrence has been provided in Table 3. The APLA2R antibodies were measured by ELISA before transplantation and before allograft biopsy. The pregrafting test was positive in 85.7% and 35.7% of the recipients with and without pMN recurrence, respectively. At the time of allograft biopsy, the test was positive in 71.4% and 14.3% of cases and controls, respectively. The association between APLA2R levels and pMN recurrence after transplantation has been confirmed by biopsy in all the cases included, and subclinical recurrence has ruled out allograft biopsy in all the control cases after a mean time of 162 months (Table 2). There was no significant correlation with proteinuria before transplantation but APLA2R levels and protein excretion were significantly correlated at the time of allograft biopsy (r = 0.66, P = 0.026). The presence of pMN recurrence after transplantation was significantly correlated with the existence of a positive ELISA assay at graft biopsy (P = 0.017) or with high level of antiPLA2R1 activity before transplantation ( P = 0.03). In the ROC analysis, anti-PLA2R levels (cutoff level of 45 U/mL) during the pretransplantation period accurately predicted pMN recurrence after transplantation, with a sensitivity of 85.3%, specificity of 85.1%, negative predictive value of 92%, and an area under the curve of 90.8% (P = 0.03; 95% confidence interval, 0.78-1.00; SE, 0.06) (Figure 1). There were no differences in the frequency of HLA DQB1 or DQA1 donor- recipient mismatch between patients with and without biopsy-proven recurrence. However, an association between HLA DQA1 05:01(05) and HLA DQB1 02:01

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FIGURE 1. The ROC curve for the prediction of recurrence of primary membranous nephropathy after renal transplantation, based on pretransplant levels of APLA2R antibody. The curve was generated using logistic regression scores, with an area under the curve of 0.908.

was identified in 6 of the 7 patients with recurrence, and this combination of DQ alleles was associated with the highest pretransplantation anti-PLA2R levels (Figure 2). DISCUSSION Primary membranous nephropathy is an organ-specific, antibody-mediated, autoimmune glomerular disease characterized by IgG-containing subepithelial deposits in the glomerular capillary wall. One of the main achievements of molecular medicine in glomerular diseases has been the identification of secretory phospholipase A2 receptor (PLA2R) as the target antigen of the autoantibodies produced in most (~70%) patients with pMN.8 We sought to characterize the presence and concentration of anti-PLA2R antibodies by ELISA in a Spanish cohort of 21 patients with pMN before and after transplantation to evaluate whether anti-PLA2R levels before surgery could predict pMN recurrence after renal transplantation. In common with the case reports by Stahl et al and Blosser et al14,15 and in contrast to the Seitz-Polski study17, we have confirmed that a high level of anti-PLA2R1 antibodies (over 45 RU/mL) at kidney transplantation is associated with MN recurrence. This quantitative result, which was not found previously in a clinical cohort, contributes to the stratification of patients into more consistent disease groups for therapeutic, epidemiological, and basic research. Moreover, this finding supports the hypothesis that anti-PLA2R causes pMN recurrence in humans and highlights the need to demonstrate, in an experimental model, that these antibodies target the podocytes of the donor kidney and lead to the progressive formation of subepithelial immune complexes. In this cohort, the earliest case was detected 1 week after grafting and the last was identified after 6 years, confirming the variable course of pMN recurrence in the context of routine antirejection therapy and the urgent need for a noninvasive diagnostic tool for early detection of pMN recurrence; such a tool is needed not only for anti-PLA2R detection but also to quantify the antibody titer. This need is highlighted by the apparent absence of recurrent disease in 35% of cases

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in this cohort who were known to be anti-PLA2R-positive but who had low antibody levels at the time of transplantation, and by the presence of 2 control patients that had borderline anti-PLA2R1 activity at the time of allograft biopsy without evidence of MN recurrence after processing of renal biopsies, including light microscopy and immunofluorescence. Especially striking was the presence of 1 patient that had MN recurrence without anti-PLA2R1 activity before transplantation but with high level of anti PLA2R at graft biopsy, in relation with a low level of immunosuppression during the posttransplant period (see patient 4 in the Table 3). This case, in common with the observations by Seitz-Polski et al,17 highlights the importance of the immunosuppressive therapy in the development of MN recurrence after transplantation. From another point of view, the presence of pMN recurrence after transplantation was significantly correlated with the existence of a positive ELISA assay at graft biopsy but the levels of antibodies poorly predicted MN recurrence (area under the curve, 0.79) and could be biased by the immunosuppressive therapy (see patients 3 and 7 in Table 3). In part, differences between results in this cohort and Seitz-Polski study could be related with the fact that 42% of our patients had induction therapy, whereas it was 60% in the French cohort.17 However, in this cohort, there were no differences in induction or maintenance immunosuppressive therapy between cases and controls, and currently, we cannot establish the prophylactic role of a specific immunosuppression therapy on the incidence of MN recurrence. In this study, the behavior of APLA2R measured by ELISA was explored by an ROC analysis showing that this test is accurate as a noninvasive pretransplantation diagnostic tool to quantify the risk of pMN recurrence in the allograft. Enzymelinked immunosorbent assay could be used in future prospective clinical trials to indicate preemptive induction therapy aimed at removing circulating anti-PLA2R in the renal transplant before allografting. Many factors, based on observations in animal models, have been implicated in the timing and severity of recurrence,18 but the data described in the present study draw attention to the main role of antibody concentrations in clinical expression of pMN recurrence after renal transplantation. Furthermore, the higher cumulative incidence of recurrence in living-related donor kidneys than in deceased-donor kidneys in this cohort and

FIGURE 2. Pretransplantation PLA2R antibody levels in relation to possession of DQA1 *0501 and DQB1 *0201 in renal recipients (P = 0.025).

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in previous observations19 supports the idea that genetic factors may determine the class of antibody and contribute to pMN recurrence; conversely, tissue damage due to cold perfusion and reperfusion on reimplantation of deceased-donor kidneys seems to have no influence. Although pMN is the most common cause of nephrotic syndrome in nondiabetic adults, it is still considered a rare disease, perhaps because, genetically, it represents a typical multifactorial disease. The PLA2R gene is a logical candidate. However, the most strongly associated PLA2R1 variants in pMN are common in a wide range of people, most of whom will not develop the disease.13 The infrequent concurrence of common PLA2R1 variants in individuals predisposed to autoimmunity and positive for an HLA-DQA1*0501 has been suggested by a genome-wide association study, reporting that anti-PLA2R levels were significantly correlated with the gene dose of HLA-DQA1*0501.11 Another genomewide association study in pMN conducted by 3 European consortia revealed strong associations with a noncoding SNP in PLA2R1 (rs4664308) and another in HLA-DQA1 (rs2187668), a member of HLA class II.20 To prove this hypothesis, HLA DQA1 and DQB1 allele identification of recipients was performed combined with measurements of circulating antibodies against PLA2R at the time of renal transplantation, showing that 6 of 7 patients with recurrence were carriers of DQA1 05:01(05) and DQB1 02:01 and confirming these DQ alleles as those associated with highest anti-PLA2R levels (Figure 2). This finding is extremely interesting because it confirms, in the context of renal transplantation, previous and initial observations in native kidney, suggesting that susceptibility loci may predict the severity of this primary glomerular disease11,20; however, it also raises questions about environmental and/or donor factors that could lead to the development of the disease in genetically susceptible individuals. Even though the analysis of HLADQ in pairs of recipient/ donor with or without recurrence of PLA2R1-related pMN showed no differences in this series, future prospective studies might find it worthwhile to investigate the possibility of modulating the donor haplotype combination as a control mechanism for T-helper cells and for the quality and quantity of antigen-specific B-cell responses to protein immunization in the recipient to avoid APLA2R antibody synthesis and pMN recurrence.21 However, although PLA2R1 and HLA genotypes may serve as a method for risk stratification in pMN, it is necessary to bear in mind that proteins—and not DNA—are the main biological targets in the genesis of pMN recurrence after renal transplantation and that the different structural effects on PLA2R conformation could play a role in the development of recurrent disease in most renal transplant patients. Characterization of posttranslational PLA2R modifications would improve analysis of disease onset and remission and could potentially explain why pMN recurs in some kidney transplant recipients and not in others and elucidate the influence of current anti-rejection therapy. This study is limited by its retrospective design, despite prospective serum collection from 2 centers. Very little is known about the earliest stages of human MN because biopsies are typically taken once the disease is more established. Protocol biopsies would likely provide further insight into the mechanisms of this disease.

Despite the major and rapid progress made in elucidating the mechanisms of pMN and recurrent disease, much work remains to be done before we can identify the biological factors that trigger pMN after renal transplantation. Currently, ELISA measurement of antiphospholipase A2 receptor antibody levels may be a rational tool to establish the risk of MN recurrence in renal allograft recipients and to establish the optimal therapeutic approach in this clinical context.

Transplantation

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Antiphospholipase A2 Receptor Antibody Levels Predict the Risk of Posttransplantation Recurrence of Membranous Nephropathy.

Secretory phospholipase A2 receptor (PLA2R) is the target antigen of the auto-antibodies produced in most (∼ 70%) patients with primary membranous nep...
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