Predictive value of the domain specific PLA2R antibodies for clinical remission in patients with primary membranous nephropathy: A retrospective study

Background M-type phospholipase A2 receptor (PLA2R) is a major auto-antigen of primary membranous nephropathy(PMN). Anti-PLA2R antibody levels are closely associated with disease severity and therapeutic effectiveness. Analysis of PLA2R antigen epitope reactivity may have a greater predictive value for remission compared with total PLA2R-antibody level. This study aims to elucidate the relationship between domain-specific antibody levels and clinical outcomes of PMN. Methods This retrospective analysis included 87 patients with PLA2R-associated PMN. Among them, 40 and 47 were treated with rituximab (RTX) and cyclophosphamide (CTX) regimen, respectively. The quantitative detection of -immunoglobulin G (IgG)/-IgG4 targeting PLA2R and its epitope levels in the serum of patients with PMN were obtained through time-resolved fluorescence immunoassays and served as biomarkers in evaluating the treatment effectiveness. A predictive PMN remission possibility nomogram was developed using multivariate logistic regression analysis. Discrimination in the prediction model was assessed using the area under the receiver operating characteristic curve (AUC-ROC).Bootstrap ROC was used to evaluate the performance of the prediction model. Results After a 6-month treatment period, the remission rates of proteinuria, including complete remission and partial remission in the RTX and CTX groups, were 70% and 70.21% (P = 0.983), respectively. However, there was a significant difference in immunological remission in the PLA2R-IgG4 between the RTX and CTX groups (21.43% vs. 61.90%, P = 0.019). Furthermore, we found differences in PLA2R-CysR-IgG4(P = 0.030), PLA2R-CTLD1-IgG4(P = 0.005), PLA2R-CTLD678-IgG4(P = 0.003), and epitope spreading (P = 0.023) between responders and non-responders in the CTX group. Multivariate logistic analysis showed that higher levels of urinary protein (odds ratio [OR], 0.49; 95% confidence interval [CI], 0.26–0.95; P = 0.035) and higher levels of PLA2R-CTLD1-IgG4 (OR, 0.79; 95%CI,0.62–0.99; P = 0.041) were independent risk factors for early remission. A multivariate model for estimating the possibility of early remission in patients with PMN is presented as a nomogram. The AUC-ROC of our model was 0.721 (95%CI, 0.601–0.840), in consistency with the results obtained with internal validation, for which the AUC-ROC was 0.711 (95%CI, 0.587–0.824), thus, demonstrating robustness. Conclusions Cyclophosphamide can induce immunological remission earlier than rituximab at the span of 6 months. The PLA2R-CTLD1-IgG4 has a better predict value than total PLA2R-IgG for remission of proteinuria at the 6th month.


Introduction
Membranous nephropathy (MN) is the main type of adult nephrotic syndrome, which is caused by binding of auto-antibodies to antigens expressed on the podocyte surface.These auto-antibodies are directed against PLA2R and other antigens in 70-80% and 10-15% of cases, respectively [1][2][3].As most cases of MN are associated with PLA2R, the total anti-PLA2R antibody levels are useful for disease risk stratification, treatment response, and recurrence [4,5].However, cases that call for additional biomarkers still exist.Most patients with high anti-PLA2R antibody titers have poor renal outcomes [4,6,7], but some exhibit different disease prognosis.Furthermore, although PLA2R is highly expressed in the kidney podocytes [1], alveolar type II epithelial cells [8], and neutrophils [9], anti-PLA2R autoantibodies only induce autoimmune diseases limited to the kidney.These factors indicate the importance of researching conformational antigenic epitopes of PLA2R.Further research the clinic role of these antigen epitopes can help to refine patients subgroups and explain diverse disease outcomes.
In the last decade, various immune-dominant epitopes of the PLA2R antigen, including the CysR, CTLD1, and CTLD7 domains, have been reported [10][11][12].In 2016, Polski et al. found that proteinuria remission rate may be associated with the number of epitope domains involved in the immune reaction in patients with PMN, leading to the hypothesis of 'epitope spreading'.The GEMRITUX study demonstrated that epitope spreading at baseline was an independent risk factor for adverse prognosis [13].However, different perspectives on the role of epitope spreading exist.Reinhard et al. observed that clinical outcomes were related to total anti-PLA2R antibody levels, but not the epitope-recognition profiles at the time of diagnosis Foundation of Jiangsu Province (grant no.BK20210067), the National Natural Science Foundation of China(No.82172336),theNatural Science Foundation of Zhejiang Province (LQ23H050005), the Scientific research project of Zhejiang Provincial Education Department (Y202250731), the Scientific research project of Zhejiang Provincial Education Department (Y202250731) and the Scientific Research Projects of Wuxi Municipal Health Commissio(Youth Program Q202053).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:
The authors have declared that no competing interests exist.[14].The research recommendations in the 2021 Kidney Disease Improving Global Outcomes (KDIGO) guideline note the importance of understanding the mechanisms of epitope spreading and immunodominance and determining whether analysis of epitope reactivity has a predictive value greater than that of PLA2R-antibody level [15].
Concerning the treatment of PMN, according to the 2021 KDIGO guideline, immunosuppressive therapy should be based on the disease risk stratification.In detail, rituximab(RTX) is the first-line therapy, and cyclophosphamide (CTX) remains the first choice for patients at a very high risk [15].However, the precise treatment for high risk patients is still controversial.Epitope spreading has been reported to be associated with a decreased remission rate at six months [13,16].Also, the percentage of epitope spreaders tends to be lower after 6 months of RTX [13,17].It merits additional study to determine if it improves outcomes compared with those in patients who fail to experience spreading reversal.
Based on our previous studies [18][19][20][21], we have discovered the clinical significance of PLA2R-IgG and -IgG4, using time-resolved fluorescence immunoassays (TRFIA), for disease diagnosis, risk stratification, and prediction of proteinuria remission.Furthermore, we have established the TRFIA method to detect specific domain antibodies targeting PLA2R antigen in 2022 [22].In this study, we retrospectively analyzed the efficacy of both RTX and CTX in combination with steroids in the treatment of PLA2R-associated PMN.We also aimed to evaluate the clinical value of the PLA2R domain-specific antibodies and epitope spreading as sensitive laboratory biomarkers for proteinuria remission and prognostic prediction.

Study cohort
From January 2020 to August 2022, a total of 97 patients were screened.Ten had received calcineurin inhibitors and were excluded to prevent statistical bias.The remaining 87 consecutive patients treated at Affiliated Wuxi People's Hospital of Nanjing Medical University were retrospectively reviewed and met the following criteria: (i) had biopsy-proven PLA2R-associated PMN; (ii) accepted RTX or CTX regimen after serum sample collection; and (iii) had no chronic infectious diseases that affect immunosuppressive therapy, such as tuberculosis.All renal tissue specimens were examined using light microscope, immunofluorescence, and electron microscope with routinely PLA2R and IgG subclass staining.Pathological grading was performed by Ehrenreich and Churg standards.Clinical data were extracted from the patients' medical records.All enrolled patients provided written informed consent for their participation in the study.The study protocol was approved by the Institutional Review Board of the Affiliated Wuxi People's Hospital of Nanjing Medical University (ethical approval no.kyl2016001).Additionally, access to information that could potentially identify individual participants post data collection was secured.

Patient sera
Blood samples were collected before the immunosuppressive therapy and after 3, 6, and 12 months of treatment and were stored at −80˚C after centrifuging at 3,000 rpm/min for 5 min and tested in batches in our laboratory.

Interventions and follow-up
Risk stratification and choice of therapeutic regimens were performed in accordance with the 2021 KDIGO guidelines or its draft after 2020.After diagnosis, all patients received renal-support therapy using angiotensin-converting enzyme inhibitors or angiotensin receptor blockade.The doses administered ranged from normal dose to the maximally tolerated dose.
Patients who were assigned to the RTX group received 1,000 mg of intravenous medication on days 1 and 15.A second course of RTX was administered if the patient did not achieve complete remission or if the CD19+ B-cell count reached >5 cells/mL at 6 months.Patients who were assigned to the CTX group received weight-adjusted intravenous CTX once a month, combined with oral dose-adjusted prednisone(0.6−1.0 g/m 2 ).
All patients underwent a series of follow-up appointments after the initiation of treatment at 0, 3, 6, and 12 months, and, then, at six-month intervals until reaching our endpoints.The following laboratory evaluations were performed at baseline and at every visit: 24-hour proteinuria, serum albumin, creatinine, estimated glomerular filtration rate (eGFR), anti-PLA2R antibody, and epitope levels.

Endpoints and definitions
The endpoint was the composite of complete or partial remission at our follow-up.Complete remission(CR) was defined as proteinuria<0.3g/24h, and partial remission was defined as proteinuria <3.5g/24h or a reduction of >50% from baseline, with improvement or normalization of serum albumin concentration, and stable or elevated <30% from baseline of serum creatinine [23].

Detection of antibodies for PLA2R and its epitopes by TRFIA
The purified PLA2R and domain proteins (CysR, CTLD1, and CTLD678) were coated in 96-well plates (2 μg/mL, 100 μL/well).Then, we added diluted serum samples in quadruplicate to the antigen-coated plates and placed them on the incubator shaker at 25˚C for 1 h.After washing three times with wash buffer, Eu3+-labeled anti-human IgG/G4 antibody (diluted 1:100 in assay buffer) was added (100 μL/well) in the plates, which were managed as mentioned before.After rinsing for six times, 200 μL enhancement solution was added to the 96-well plates and the plates were agitated for 5 min.Finally, a TRFIA analyzer (excitation and emission wavelengths were 340 and 613 nm, respectively) was used to measure fluorescence.More specific details are available in a previous study [22].

Epitope spreading
Epitope spreading was defined as PLA2R-CysR being the primary dominant epitope with evidence for epitope spreading toward CTLD1 and CTLD7 [13].

Statistical analyses
Statistical analyses were performed using R statistical software (version 4.2.3;R Software for Statistical Computing, Vienna, Austria) and IBM SPSS Statistics (version 26; IBM Corp., Armonk, NY, USA).To decrease the variability of data, PLA2R and domain-specific IgG/G4 titers were logarithmically transformed based on the natural logarithm, and data following a non-normal distribution are presented as medians (interquartile ranges [IQRs]).Regarding data following a normal distribution, quantitative data were expressed as mean±SDs.A t-test and the Mann-Whitney U-test were used to assess differences between quantitative data, which were expressed as mean values (percentages).Finally, a chi-squared test or one-way variation analysis (ANOVA) was used to assess the differences.All probabilities were two-sided and P<0.05 was considered statistically significant.Logistic regression analyses were performed to confirm potential risk or protection factors of treatment responses.Variables with P <0.25 were chosen to be included in the binary logistic regression model.This model was constructed by using backward elimination.The nomogram and bootstrap receiver operating characteristic (ROC) curve were drawn using the R packages "rms" and"pROC," respectively.

Development of an individualized prediction model
Multivariate logistic regression analysis demonstrated that two variables urinary protein and PLA2R-CTLD1-IgG4 were independently associated with disease early remission.A model incorporating the two potential predictors is presented as a nomogram (Fig 4).The nomogram was assigned a specific score, and the total score was used to obtain the probability of disease early remission possibility.The ratios of the calculated beta were used to evaluate the proportional predictive effects of these variables.The projections from total points on the scales The nomogram was developed in the entire cohort based on urinary protein and PLA2R--IgG4 level.Points for each variable were acquired by drawing a straight line upward from the corresponding value to the 'Points' line.Then sum the points received from each variable and locate the number on the 'Total Points' axis.To conclude the patient's sort probability of proteinuria remission at the 6th month, draw a straight line down to the corresponding 'Proteinuria Remission Possibility' axis.

Internal validation of the model
Given the small sample size, we used the bootstrap method to validate our prediction model.After 1000 repeated samplings, the AUC-ROC is 0.711 (95% CI: 0.587-0.824)(Fig 5B).

Discussion
MN is an autoimmune disease in which autoantibodies combine with antigens expressed on the glomerular podocytes.The 2021 KDIGO guideline recommends RTX as the first-line therapy, while CTX remains the preferred option for very high-risk cases [10].There is no precise evidence for the treatment of moderate and high risk PMN patients.This retrospective study enrolled 87 patients who was diagnosed with PLA2R-associated PMN from 2020 to 2022 with a moderate or high risk of progressive loss of kidney function and compared the effectiveness of RTX and CTX regimen in those patients.
Both RTX and CTX therapy showed good efficacy in achieving clinical remission in PMN patients(70%vs.70.21%,P = 0.983).When RTX was administrated as first-line therapy, patients' clinical remission(70%) was comparable to the remission rate of 69.1% reported by Ruggenenti et al. [24], and even higher than those reported in the MENTOR [23] and GEMRITUX study [14].(60.0% and 64.9%, respectively) The clinical remission rate for CTX therapy was 70.21%, which was similar to rates reported in other observational studies.A retrospective analysis of 32 cases with PMN treated with intravenous CTX (500-750 mg/m2 every month for 6 months) plus steroids reported that 81% of the patients achieved clinical remission [25].A long-term observational study of 55 PMN patients found that 70.9% of patients achieved clinical response with CTX regimen [26].Another retrospective analysis studied 40 patients treated with intravenous CTX (600 mg/m2 every 4 weeks for up to 6 months) plus steroids.In this study, 67% of patients reached some form of remission [27].However, the RTX regimen could induce more rapid normalization of  serum albumin compared with the CTX regimen.Supported by the GEMRITUX study, the serum albumin increase preceded that of proteinuria [14].RTX directly targets CD20-B cells to reduce the production of circulating antibodies and prevent the formation of immune  deposits thus maintaining the integrity of podocytes [28].In addition, it has a potential effect on T-cells, including reducing the release of cytokines (e.g., interleukin-13,) helping recover the function of regulatory T-cells, and stabilizing the cytoskeleton [29,30].
Consistent with the RI-CYCLO study [31], our study also indicated that the CTX regimen may induce immunological remission earlier than the RTX regimen at the 6-months observation.The aforementioned findings were supported by those of a small study conducted in the United Kingdom that included new-incident and relapse patients, the anti-PLA2R levels decreased from 244 U/L to <14 U/L at 6 months, and 44% of the new-incident patients achieved complete response after a median follow-up period of 32 months [32].CTX induces a generalized leukocyte and mature plasma cell ablation, resulting in a more drastic reduction in antibody production than RTX which is a more specifically targeted drugs.Early immunological response appears to be a strong predictor of clinical remission [4,6,[33][34][35].Stefan et al reported that the negativity of anti-PLA2R antibodies at 3 months after diagnosis was associated with a 60% increase in the chances of later remission [33].In a study by Ruggenenti et al., a 50% reduction in anti-PLA2R titer preceded an equivalent reduction in proteinuria by 10 months [34].However, there were still other studies found only a weak or no relationship between the remission rate and anti-PLA2R antibodies titer [2,[36][37][38].Moreover, the high sensitivity of PLA2R-IgG4 should be considered in the future disease monitoring instead of total PLA2R-IgG level.In our previous study, we have quantitatively detected PLA2R-IgG4 antibody level and confirmed their clinical values in risk stratification and treatment effectiveness analysis [20,21].
The discrepancies between the clinical outcomes and anti-PLA2R titers have motivated researchers to find other biomarkers to assist in the evaluation of disease prognosis.Three important studies have shown that at least three PLA2R epitope regions are targeted by autoantibodies in MN [11][12][13].In a study conducted by Seitz-Polski et al, they stratified patients positive for PLA2R by serum reactivity to one or more of these domains and reported that epitope spreading beyond the CysR epitope was an independent risk factors of poor renal prognosis [12].Whether the level of antibodies specific to a certain PLA2R epitope region may have a better prediction value than anti-PLA2R level is worth researching.To address this question we used TRFIA assay to quantify the titer of the individual PLA2R domain-specific antibody.Using multivariate logistic analysis, we discovered that it was PLA2R-CTLD1-IgG4 antibody level(P = 0.041) be independently associated with disease proteinuria remission at the 6 th month instead of PLA2R-IgG(P = 0.063).As with the GEMRITUX study [13],we have also found that epitope spreading beyond CysR domain at baseline was associated with a decreased remission rate at month 6.The question of why PLA2R-CTLD1-IgG4 holds the potential to be a new biomarker for predicting disease remission to be addressed.PMN is an autoimmune disease, and in most cases antibodies typically first recognize the immunodominant epitope (CysR for PLA2R).When epitope spreading occurs, it initially extends to non-cross reactive epitopes on the same protein(CTLD1 for PLA2R).Interestingly, one patient, whose epitope reactivity was negative at baseline and became an epitope spreader at the 6 th month, suffered from persistent nephrotic syndrome and treatment resistance, despite a relatively low anti-PLA2R antibody titer.This may explain the discrepancies observed between clinical features and the anti-PLA2R titers, which suggests that the domain-specific antibody level can be a more potent predictor of disease remission than the PLA2R antibody titer.
Furthermore, we established an individualized model to predict disease proteinuria remission possibilities at 6 months.In our study, two independent risk factors (i.e., proteinuria and PLA2R-CTLD1-IgG4) were used to construct an easy-to-use nomogram model.The predictive ability of a new prognostic score is critical for decision making regarding treatment strategies and for predicting outcomes in patients with PMN.The nomogram we constructed showed superior predictive ability (all AUC-ROCs were > 0.70) in both the training cohorts and internal validation.Thus, the nomograms we have established are accurate, widely beneficial and user-friendly in clinical practice.For example, the total score of a patient diagnosed with PLA2R-associated PMN with 24h urine protein of 4.5 g and LnPLA2R-CTLD1-IgG4 of 3.97 is 94.Therefore the probability of early proteinuria remission is 70%.There is a possibility of delayed immunosuppression therapy for this patient.Under these circumstances, our established nomogram might be utilized as a more powerful and conventional tool to predict disease proteinuria remission and help the decision of early therapeutic intervention.
This study had some limitations.First, it was a retrospective, single-center study with a small sample size, limiting the significance of the statistical analysis of therapeutic evaluation.Additionally, many values of PLA2R-CysR/CTLD1/CTLD678-IgG/-IgG4 were missing because of the retrospective nature of the study.Second, the endpoint of our study was the remission rate of proteinuria after immunosuppressive therapy for 6 months, and the followup time should be prolonged because RTX may require a longer time to respond, as indicated by previous studies.More prospective multi-center studies are needed to validate our findings.

Conclusions
In conclusion, the CTX regimen can induce immunological remission earlier than RTX regimen at the span of 6 months, while the overall clinical remission rate is comparable between regimes.The PLA2R-CTLD1-IgG4 level has a better predict value at proteinuria remission at the 6 th month than total anti-PLA2R-IgG antibody level.

Table 2 .
(Continued) SD, the interquartile range (IQR) or n (%).Domain specific antibody levels were detected by TRFIA and transfored with logarithmic transformation based on natural logarithm.