Comparison of the efficacy of rituximab monotherapy and combined immunotherapy for primary membranous nephropathy: a real-world cohort study

Comparison of the efficacy of rituximab monotherapy and combined immunotherapy for primary membranous nephropathy: a real-world cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparison of the efficacy of rituximab monotherapy and combined immunotherapy for primary membranous nephropathy: a real-world cohort study Ji Dong, Mingrui Fan, Jianping Si, Lipu Shi, Yue Ying, Yingying Ren, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8713007/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Rituximab (RTX) has been established as a first-line treatment for primary membranous nephropathy (pMN). However, the efficacy difference between RTX monotherapy and combined regimens incorporating additional immunosuppressants (e.g., glucocorticoids or calcineurin inhibitors) remains unclear, particularly in patient subgroups with distinct clinical and immunological characteristics. Therefore, clarification of individualized treatment strategies is urgently needed. Methods A total of 98 pMN patients receiving RTX-based treatment were enrolled and divided into three groups: monotherapy (RTX), doublet therapy (RTX in combination with tacrolimus), and triplet therapy (RTX, tacrolimus, and glucocorticoids concurrently). We compared the 12-month total remission rate, changes in renal function, proteinuria levels, and B cell depletion across groups, with subgroup analyses stratified by age and anti-PLA2R antibody titer. Results The monotherapy group achieved a significantly higher total remission rate than the doublet and triplet therapy groups. Despite poorer baseline renal function in the combined treatment groups, their improvements in renal function and proteinuria reduction at 12 months were inferior to those of the monotherapy group. Subgroup analyses revealed that monotherapy advantages were particularly prominent in patients with anti-PLA2R antibody titer ≤ 200 RU/mL and those aged > 60 years. Additionally, the monotherapy group exhibited unique kinetic characteristics of B cell depletion. Conclusion This real-world study demonstrates that RTX monotherapy provides superior clinical remission and renal protection compared to combined regimens, challenging the necessity of routine combined immunosuppressive treatment. The significant benefits observed in elderly patients and those with low anti-PLA2R antibody titers support RTX monotherapy as the preferred initial treatment strategy for most pMN patients. Primary membranous nephropathy Rituximab Tacrolimus Glucocorticoids Anti-PLA2R B cell depletion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults, characterized by immune complex deposition along the subepithelial surface of the glomerular basement membrane. The landmark 2009 discovery of the M-type phospholipase A2 receptor (PLA2R) as a key target antigen transformed our understanding of pMN, establishing it as an organ-specific autoimmune disorder [ 1 ]. In this disease, circulating autoantibodies—predominantly of the IgG4 subclass—bind to endogenous podocyte antigens such as PLA2R, driving in situ immune complex formation [ 2 ]. Complement activation, particularly via the lectin or alternative pathways, culminates in the assembly of the membrane attack complex (C5b-9), which induces podocyte injury, cytoskeletal disruption, and subsequent proteinuria [ 3 ]. Beyond PLA2R (accounting for ~ 70–80% of cases), multiple other target antigens have been identified, including thrombospondin type-1 domain-containing 7A (THSD7A), neural epidermal growth factor-like 1 (NELL1), and protocadherin 7 (PCDH7) [ 1 , 4 ]. These findings have challenged the traditional primary-secondary classification paradigm and support an antigen-based framework that more accurately reflects the underlying immunopathology [ 5 ]. The pathogenesis of pMN also involves genetic predisposition, environmental triggers, and dysregulated autophagy, which collectively promote the exposure of native podocyte proteins and the breakdown of immune tolerance[ 3 , 6 ]. These interconnected mechanisms confirm pMN as a prototypical antibody-mediated glomerular disease. The treatment paradigm for membranous nephropathy (MN) has evolved substantially over recent decades, shifting from non-specific immunosuppressive regimens to targeted B-cell therapies. Historically, cyclical regimens combining corticosteroids with alkylating agents (e.g., cyclophosphamide) served as the cornerstone of immunosuppression for high-risk patients, demonstrating efficacy in inducing remission but often burdened by substantial toxicity and high relapse rates upon discontinuation[ 7 , 8 ]. The advent of calcineurin inhibitors (CNIs) offered an alternative with favorable short-term remission rates; however, their utility was limited by nephrotoxicity and frequent relapses following withdrawal [ 9 ]. A transformative shift occurred with the introduction of rituximab (RTX), an anti-CD20 monoclonal antibody that selectively depletes B cells—central mediators in the production of pathogenic autoantibodies[ 15 , 16 ]. The pivotal MENTOR trial established RTX as a first-line therapy by demonstrating non-inferiority to cyclosporine in achieving remission at 12 months and superior response durability at 24 months, coupled with a significantly lower relapse rate and a more favorable safety profile [ 10 ]. Subsequent studies, including the RI-CYCLO and STARMEN trials, reinforced RTX’s efficacy, showing comparable or superior outcomes to cyclophosphamide-based regimens while underscoring the clinical value of anti-PLA2R antibody monitoring to guide retreatment and predict clinical response[ 11 , 12 ]. This evolution toward targeted B-cell depletion has redefined the therapeutic landscape of MN, emphasizing precision medicine strategies that prioritize efficacy, safety, and long-term remission. Despite established guidelines and robust trial evidence, a substantial proportion of clinicians in real-world practice continue to combine RTX with corticosteroids or CNIs, aiming to accelerate therapeutic response or enhance efficacy—particularly in high-risk patients. However, this approach lacks consistent evidence-based support, especially regarding long-term renal outcomes and safety. The rationale for combination therapy often centers on achieving rapid proteinuria reduction, yet its impact on sustained renal protection remains unclear. Critically, concerns persist about potential trade-offs, including heightened risks of infections, metabolic disturbances, and CNI-associated nephrotoxicity, which may offset long-term benefits [ 13 ]. The heterogeneity in MN pathogenesis, driven by diverse target antigens, further complicates standardized treatment protocols. For instance, antigen-specific responses may differentially influence treatment efficacy, but evidence linking combination therapy benefits to specific antigen subtypes remains sparse[ 5 , 14 ]. Thus, a critical knowledge gap exists: whether empirical combination regimens truly outperform RTX monotherapy in preserving renal function over the long term without amplifying adverse events. To address these uncertainties, we conducted a real-world, single-center cohort study to directly compare the efficacy and safety of RTX monotherapy versus combination strategies (dual or triple therapy) in the management of pMN. Primary endpoints included renal survival, quantified by sustained estimated glomerular filtration rate (eGFR) stability and composite kidney outcomes. Secondary analyses assessed antigen-specific serological responses and infection-related complications. By leveraging detailed clinical data, we aimed to delineate the risk-benefit profiles across different therapeutic regimens, with subgroup analyses stratified by antigen status and baseline clinical features. This study seeks to provide high-grade evidence to inform individualized treatment decisions, aligning with the emerging paradigm of antigen-driven classification while prioritizing long-term renal protection over short-term surrogate outcomes. 2. Methods 2.1. Study Design and Patient Selection This retrospective cohort study included 98 patients diagnosed with pMN who received treatment at our institution between January 2019 and December 2023. The diagnosis of pMN was confirmed by renal biopsy in all cases, with pathological examination revealing typical membranous nephropathy features—including diffuse thickening of the glomerular basement membrane, subepithelial immune complex deposition on electron microscopy, and granular staining for IgG along capillary loops on immunofluorescence—in the absence of pathological changes suggestive of other renal diseases. Inclusion criteria were as follows: (1) availability of anti-PLA2R antibody test results (measured by enzyme-linked immunosorbent assay [ELISA]; positive cutoff ≥ 2 RU/mL); (2) completion of at least 12 months of regular follow-up after immunosuppressive therapy; and (3) availability of complete electronic medical record data, including demographic characteristics, clinical symptoms, laboratory results, and treatment regimens. Exclusion criteria comprised: (1) secondary membranous nephropathy (e.g., associated with diabetes mellitus, systemic lupus erythematosus, hepatitis B virus infection, or drug-induced MN); (2) presence of life-threatening complications such as severe infection, malignancy, or acute cardio-cerebrovascular events; (3) loss to follow-up or follow-up duration < 12 months; and (4) prior renal replacement therapy (hemodialysis or peritoneal dialysis) or history of kidney transplantation. 2.2. Treatment Regimens and Grouping 2.2.1 Treatment Regimens and Dosing The dosing protocols for all immunosuppressive agents were in accordance with current clinical guidelines for the management of membranous nephropathy. Specific regimens were as follows: Rituximab (RTX):​ All patients received a standardized RTX induction regimen of 375 mg/m² body surface area, administered intravenously once weekly for 4 consecutive weeks. Premedication with antihistamines and glucocorticoids was administered prior to each infusion to prevent infusion-related reactions. Tacrolimus (TAC):​ For patients in the doublet and triplet therapy groups, TAC was initiated at a dose of 0.05–0.1 mg/kg per day, administered orally in two divided doses. The dose was subsequently adjusted to maintain a trough blood concentration within the target range of 5–8 ng/mL. Regular monitoring of TAC levels, renal function, and potential adverse effects was performed. For patients achieving complete or partial remission after 6–12 months of treatment, TAC was tapered, typically by reducing the dose by 25%–50% every 3–6 months, with the total treatment duration guided by clinical response. Glucocorticoids:​ In the triplet therapy group, glucocorticoids were initiated as prednisone (or equivalent) at a dose of 0.5–1.0 mg/kg per day (maximum 60 mg/day). This dose was maintained for 4–8 weeks, followed by a gradual taper by approximately 5 mg every 1–2 weeks, aiming for a low maintenance dose (e.g., 5–10 mg/day) or discontinuation by 6–12 months, contingent upon the patient's clinical response and tolerance. 2.2.2. Treatment Grouping Based on the initial immunosuppressive therapy, patients were categorized into three groups: Monotherapy group: Received RTX alone. Doublet therapy group: Received RTX in combination with TAC Triplet therapy group: Received RTX, TAC and glucocorticoids concurrently. 2.3. Data Collection and Outcome Measures Baseline data were retrieved from the hospital electronic medical record system, encompassing four core categories: demographic characteristics (age, sex, and comorbidities including hypertension, diabetes, and coronary artery disease); renal function and biochemical parameters (24-hour urinary protein excretion (24h UTP), estimated glomerular filtration rate (eGFR, computed via the CKD-EPI equation), serum creatinine (Scr), and serum albumin (Alb)); immunological markers (anti-PLA2R antibody titer, quantified by ELISA with a positive threshold of ≥ 2 RU/mL, and B cell count, detected via flow cytometry); and initial treatment regimens, specifically the types of immunosuppressive agents and combination therapy strategies. All enrolled patients underwent a 12-month follow-up, with scheduled evaluations at 3, 6, and 12 months post-treatment. Follow-up data collection focused on four dimensions of clinical outcomes: efficacy parameters (24h UTP and serum Alb levels at each timepoint, utilized for clinical response assessment); renal function indicators (eGFR and Scr at each timepoint); immunological measures (B cell count at month 6, from which B cell depletion rate and clearance rate were derived); and safety profiles, defined as the incidence of adverse events such as infections, gastrointestinal reactions, and hematological abnormalities. 2.4. Outcome Definitions The primary outcome was the 12-month clinical remission rate, which included three categories: complete remission (CR), defined as 24h UTP 35 g/L; partial remission (PR), characterized by a ≥ 50% reduction in 24h UTP from baseline, 24h UTP < 3.5 g, and concurrent significant improvement in serum Alb; and overall response (OR), referring to the proportion of patients achieving either CR or PR. Secondary outcomes encompassed dynamic efficacy trends (changes in 24h UTP and serum Alb from baseline to 3, 6, and 12 months), renal protective effects (temporal changes in eGFR and Scr), immunological effects [B cell depletion rate, calculated as (baseline B cell count – post-treatment B cell count)/baseline B cell count × 100%, and mean B cell clearance rate], as well as safety profile (incidence, severity, and outcomes of adverse events). 2.5. Subgroup Analyses Two predefined subgroup analyses were performed based on key clinical factors of pMN: anti-PLA2R antibody titer (high titer: >200 RU/mL vs. low titer: ≤200 RU/mL) and age (elderly: >60 years vs. non-elderly: ≤60 years). Within each subgroup, inter-group differences in 12-month eGFR, 24h UTP, and OR rate were compared between the monotherapy group and the combined doublet/triplet therapy groups. 2.6. Statistical Analysis All statistical analyses were performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test was used to assess normality of continuous variables. Normally distributed data are presented as mean ± standard deviation and compared using one-way ANOVA (with Bonferroni post-hoc test for pairwise comparisons). Non-normally distributed data are expressed as median (interquartile range) and compared using the Kruskal–Wallis H test. Categorical variables are presented as number (percentage) and compared using the chi-square test or Fisher’s exact test when expected frequencies were < 5. A two-sided P-value < 0.05 was considered statistically significant. 2.7. Sample Size Justification and Post-hoc Power Analysis As a retrospective real-world cohort study, a prospective power calculation was not performed prior to patient enrollment. The sample size was determined by the availability of all eligible patients meeting the inclusion criteria at our institution during the study period (January 2019 to December 2023), which amounted to 98 patients. To address the robustness of our findings, a post-hoc power analysis​ was conducted using the observed effect sizes from our primary outcome (the difference in 12-month overall response rate, ORR). Given the observed ORR of 84.0% in the monotherapy group (n = 25) versus 46.7% in the doublet therapy group (n = 15), yielding an absolute difference of 37.3%, and with an alpha level set at 0.05, the achieved post-hoc statistical power for this comparison exceeded 90%​ (using a chi-square test for proportions). This indicates that the present sample size provided ample power to detect the clinically significant differences in efficacy observed among the treatment groups. 3. Results 3.1. Baseline Characteristics of the Study Population The study included 98 patients with biopsy-proven pMN, stratified into two groups based on the presence of comorbidities: with comorbidities (n = 67) and without comorbidities (n = 31). The distribution of treatment regimens (monotherapy, doublet therapy, triplet therapy) was balanced between groups ( P = 0.555 ), with detailed comparative results of baseline characteristics presented in Table 1 . Demographic analysis revealed that patients with comorbidities were significantly older than those without [median (IQR): 60.0 (51.5–67.0) years vs 39.0 (21.0–55.5) years, P < 0.001 ], while gender distribution showed no significant difference (males: 53.7% [36/67] vs 51.6% [16/31], P = 0.785 ), indicating that comorbidities are more prevalent in middle-aged and elderly patients with membranous nephropathy. Comparative analysis of renal function and biochemical parameters demonstrated that the comorbidity group had significantly lower baseline eGFR [91.6 (59.12–101.22) mL/min/1.73m² vs 100.0 (91.15–120.8) mL/min/1.73m², P = 0.007 ]. Serum creatinine levels showed an increasing trend, though not statistically significant [76.0 (60.4–102.4) µmol/L vs 69.4 (53.62–83.65) µmol/L, P = 0.086 ]. Both groups had similar median 24h UTP [4.31g (1.75–7.28) g vs (2.55–7.04) g, P = 0.73 ], and serum albumin levels were comparable [28.1 ± 7.0 g/L vs 26.82 ± 9.92 g/L, P = 0.526 ], suggesting that comorbidities primarily affect glomerular filtration function without significantly influencing proteinuria severity or hypoalbuminemia status. Immunological and treatment-related parameters showed no significant differences in anti-PLA2R antibody titers [5.36 (2.0–101.77) RU/mL vs 2.0 (2.0–20.04) RU/mL, P = 0.081 ] or B-lymphocyte counts [214.0 (77.0–348.0) cells/µl vs 233.0 (99.5–292.0) cells/µl, P = 0.884 ] between groups. The balanced distribution of treatment regimens across groups (monotherapy: 58.2% vs 54.8%; doublet therapy: 29.9% vs 38.7%; triplet therapy: 11.9% vs 6.5%; P = 0.555 ) indicates that comorbidities did not significantly alter immune status, and treatment strategies were consistent between groups, thereby eliminating potential confounding from baseline immunological characteristics or treatment selection bias in subsequent efficacy analyses. Table 1 Baseline Demographic and Clinical Characteristics of the Study Population Stratified by Comorbidity Status. Demographics Characteristics With Comorbidities(n = 67) No Comorbidities(n = 31) P-value Age (years) 60.0(51.5–67.0) 39.0(21.0-55.5) 0 Sex (Male) 36/67(53.7%) 16/31(51.6%) 0.785 Key Laboratory Values (Pre-treatment) Characteristics With Comorbidities(n = 67) No Comorbidities(n = 31) P-value 24h Urine Protein (g/24h) 4.31(1.75–7.28) 4.31(2.55–7.04) 0.73 eGFR(mL/min/1.73m²) 91.6(59.12-101.22) 100.0(91.15–120.8) 0.007 Serum Creatinine (µmol/L) 76.0(60.4-102.4) 69.4(53.62–83.65) 0.086 Serum Albumin (g/L) 28.1 ± 7.0 26.82 ± 9.92 0.526 Anti-PLA2R Ab Titer (RU/mL) 5.36(2.0-101.77) 2.0(2.0-20.04) 0.081 B-cell Count (cells/µL) 214.0(77.0-348.0) 233.0(99.5–292.0) 0.884 Monotherapy 39/67(58.2%) 17/31(54.8%) 0.555 Doublet Therapy 20/67(29.9%) 12/31(38.7%) 0.555 Triplet Therapy 8/67(11.9%) 2/31(6.5%) 0.555 3.2. One-Year Clinical Response Rates by Treatment Regimen Comparative results of CR, PR, and OR rates at one-year post-treatment are presented in Fig. 1 . The monotherapy group demonstrated significantly higher OR rates compared to the doublet and triplet therapy groups [84.0% (21/25) vs 46.7% (7/15) vs 50.0% (3/6), respectively; P < 0.05 ], confirming the superior clinical benefit of monotherapy in treating pMN. While no significant differences were observed in CR rates among the three groups [28.0% (7/25) vs 33.3% (5/15) vs 33.3% (2/6), respectively; P > 0.05 ], the superiority in OR rates for the monotherapy group primarily stemmed from significantly higher PR rates [56.0% (14/25) vs 13.3% (2/15) vs 16.7% (1/6), respectively; P < 0.05 ], indicating that monotherapy has a more pronounced effect in achieving partial remission. Notably, the doublet and triplet therapy regimens, despite being more intensive, failed to demonstrate expected efficacy advantages, with OR rates below 50%, significantly lower than the monotherapy group. This suggests that combination therapies may be associated with increased adverse effects, reduced patient tolerance, or clinical selection bias, highlighting potential risks of overtreatment. 3.3. Dynamic Changes in 24-hour Urinary Protein and Serum Albumin 3.3.1. 24-hour Urinary Protein Changes Significant differences were observed in the trends of 24h UTP at various timepoints (baseline, 3, 6, and 12 months) across the three treatment groups (Fig. 2 ). The monotherapy group exhibited a consistent and sustained decline in proteinuria, decreasing from 4.78 ± 3.41 g at baseline to 3.48 ± 2.95 g at 3 months, 2.85 ± 2.62 g at 6 months, and reaching the lowest value of 1.75 ± 2.50 g at 12 months. The relatively small standard deviations at each timepoint suggest stable and durable proteinuria-reducing efficacy with monotherapy. In contrast, the doublet therapy group showed fluctuating proteinuria levels, with a sharp decrease to 0.88 ± 1.01 g at 3 months, followed by a significant rebound to 3.36 ± 5.05 g at 6 months, and further increasing to 3.45 ± 4.91 g at 12 months, indicating substantial short-term efficacy but poor long-term sustainability. The triplet therapy group, with limited sample sizes at various timepoints, exhibited considerable variability in proteinuria measurements, decreasing from 7.82 ± 8.61 g at baseline to 3.30 ± 0.85 g at 3 months, 2.77 ± 2.14 g at 6 months, and 2.05 ± 3.32 g at 12 months, without statistical significance to confirm stable proteinuria-reducing advantages. 3.3.2. Serum Albumin Changes All three groups demonstrated gradual increases in serum albumin levels over time, approaching the normal reference range (39–41 g/L) by 12 months (Fig. 3 ). The monotherapy group showed the most significant and steady improvement, increasing from 30.15 ± 6.25 g/L at baseline to 33.58 ± 6.58 g/L at 3 months, 36.80 ± 5.75 g/L at 6 months, and reaching 39.51 ± 5.11 g/L at 12 months, with progressively decreasing standard deviations, indicating stable improvement in hypoalbuminemia with good consistency in individual patient responses. The doublet and triplet therapy groups showed similar increasing trends but without significant advantages: doublet therapy increased from 30.22 ± 6.33 g/L to 39.37 ± 5.18 g/L; triplet therapy increased from 31.50 ± 7.65 g/L to 41.33 ± 4.74 g/L. Combined with proteinuria results, these findings confirm that monotherapy is more effective, consistent with clinical response rates. 3.4. Impact of Different Treatment Regimens on Renal Function (eGFR) The three treatment groups exhibited significantly different trends in eGFR changes at various post-treatment timepoints (Fig. 4 ). The monotherapy group demonstrated a consistent and sustained increase in eGFR, rising from 91.82 ± 27.65 mL/min/1.73m² at baseline to 95.93 ± 20.47 mL/min/1.73m² at 3 months, 96.65 ± 23.45 mL/min/1.73m² at 6 months, and reaching 101.68 ± 17.4 mL/min/1.73m² at 12 months, with progressively decreasing standard deviations, indicating that monotherapy not only improves clinical symptoms but also continuously protects and enhances renal function with improving safety and treatment stability. The doublet therapy group showed a slow increasing trend in eGFR, from 82.55 ± 38.38 mL/min/1.73m² at baseline to 84.26 ± 38.1 mL/min/1.73m² at 3 months, 85.74 ± 36.93 mL/min/1.73m² at 6 months, and 89.24 ± 37.47 mL/min/1.73m² at 12 months, but remained below monotherapy levels at all timepoints with persistently large standard deviations, suggesting limited renal functional improvement with significant interindividual variability. The triplet therapy group exhibited an unfavorable pattern of initial increase followed by decrease in eGFR: 77.07 ± 29.44 mL/min/1.73m² at baseline, increasing to 79.6 ± 23.1 mL/min/1.73m² at 3 months, peaking at 88.3 ± 20.77 mL/min/1.73m² at 6 months, but declining to 71.79 ± 27.57 mL/min/1.73m² at 12 months, below baseline levels, suggesting potential renal function impairment risk with long-term triplet therapy, possibly related to excessive immunosuppression. 3.5. B-cell Depletion Efficacy of Different Treatment Regimens Significant differences were observed in B-cell depletion effects among the treatment regimens (Fig. 5 ). While the triplet therapy group showed the highest B-cell depletion rate (71.40%), followed by doublet therapy (63.60%) and monotherapy (37.00%), indicating stronger immunosuppressive effects with combination therapies, analysis of mean B-cell clearance rate revealed an opposite trend: monotherapy had the highest mean clearance rate (57.30%), followed by triplet therapy (37.70%) and doublet therapy (32.60%). This suggests that although monotherapy resulted in lower overall B-cell depletion, it achieved superior clearance efficiency of surviving B-cells, possibly related to immune microenvironment interference caused by combination therapies. Combined with the superior one-year clinical response rates in the monotherapy group, these findings indicate that B-cell clearance efficiency (rather than mere depletion rate) is a key predictor of clinical efficacy in membranous nephropathy immunotherapy, providing important insights for mechanism research and treatment monitoring. 3.6. Subgroup Analysis Results 3.6.1. Anti-PLA2R Antibody Titer Subgroup Analysis Subgroup analysis based on anti-PLA2R antibody titers (Table 2 ) revealed that in the high-titer subgroup (> 200 RU/mL), the monotherapy group had significantly higher 12-month eGFR (106.17 ± 12.15 mL/min/1.73m²) compared to the combination therapy group (62.5 ± 34.22 mL/min/1.73m²), lower 24h UTP (1.02 ± 1.58 g vs 3.38 ± 4.57 g), and higher OR rate (100.0% [3/3] vs 50.0% [1/2]), with statistical significance ( P < 0.05 ). In the low-titer subgroup (≤ 200 RU/mL), the monotherapy group maintained advantages: 12-month eGFR (102.72 ± 17.6 mL/min/1.73m² vs 80.38 ± 32.93 mL/min/1.73m²), 24h UTP (1.85 ± 2.69 g vs 3.63 ± 4.96 g), and OR rate (81.0% [17/21] vs 46.7% [7/15]), confirming the superiority of monotherapy regardless of anti-PLA2R antibody titer levels. Analysis based on anti-PLA2R antibody status (Fig. 6 ) showed that among antibody-positive patients (titer ≥ 2 RU/mL), the monotherapy group had significantly higher OR rates (80.80%) compared to doublet (46.70%, n = 30) and triplet therapy (55.60%) groups ( P < 0.05 ). Among antibody-negative patients (titer < 2 RU/mL), despite smaller sample sizes, the monotherapy group maintained higher OR rates (75.00% vs 50.00%), suggesting therapeutic value even in non-PLA2R antibody-mediated membranous nephropathy. The high proportion of anti-PLA2R antibody-positive patients across all groups (monotherapy: 92.9%; doublet therapy: 93.8%; triplet therapy: 90.0%) further supports monotherapy as first-line treatment for membranous nephropathy. Table 2 Subgroup Analysis of Treatment Efficacy at 12 Months Subgroup Characteristic​ Treatment Group​ n 12-month eGFR (mean ± SD, mL/min/1.73m²)​ 12-month 24h UTP (mean ± SD, g)​ 12-month Overall Response Rate, ORR (%)​ anti-PLA2R Antibody > 200 RU/mL Monotherapy 3 106.17 ± 12.15 1.02 ± 1.58 100.0%(3/3) Combination Therapy* 2 62.5 ± 34.22 3.38 ± 4.57 50.0%(1/2) anti-PLA2R Antibody ≤ 200 RU/mL Monotherapy 21 102.72 ± 17.6 1.85 ± 2.69 81.0%(17/21) Combination Therapy* 15 80.38 ± 32.93 3.63 ± 4.96 46.7%(7/15) Age > 60 years (Elderly)​ Monotherapy 10 89.15 ± 16.84 1.28 ± 1.57 80.0%(8/10) Combination Therapy* 7 62.65 ± 23.12 1.74 ± 1.36 28.6%(2/7) Age ≤ 60 years (Non-elderly)​ Monotherapy 18 106.7 ± 15.19 1.98 ± 2.87 83.3%(15/18) Combination Therapy* 9 107.78 ± 31.92 4.83 ± 5.96 44.4%(4/9) *Combination therapy group includes patients receiving doublet therapy (RTX + TAC) and triplet therapy (RTX + TAC+ glucocorticoids). 3.6.2. Age Subgroup Analysis Age-based subgroup analysis (Table 2 ) demonstrated that in elderly patients (> 60 years), the monotherapy group had significantly higher 12-month eGFR (89.15 ± 16.84 mL/min/1.73m², n = 16 vs 62.65 ± 23.12 mL/min/1.73m², n = 22), lower 24h UTP (1.28 ± 1.57 g vs 1.74 ± 1.36 g), and higher OR rates (80.0% [8/10] vs 28.6% [2/7], P < 0.05 ), with significant eGFR decline in the combination therapy group, suggesting that elderly patients with membranous nephropathy should avoid combination therapies in favor of safer and more effective monotherapy. In non-elderly patients (≤ 60 years), the monotherapy group maintains advantages: OR rate (83.3% [15/18], n = 40 vs 44.4% [4/9], n = 18), 24h UTP (1.98 ± 2.87 g vs 4.83 ± 5.96 g, P < 0.05 ), with no significant eGFR difference between groups (106.7 ± 15.19 mL/min/1.73m² vs 107.78 ± 31.92 mL/min/1.73m²), confirming the significant efficacy advantages of monotherapy across all age groups of membranous nephropathy patients. 4. Discussion This study analyzed 98 patients with pMN following 12 months of follow-up, with the core finding that RTX monotherapy exhibited significantly superior clinical overall response rate (84.0%) and renal function protection compared to doublet (46.7%) and triplet (50.0%) combination regimens. The eGFR in the monotherapy group showed a sustained and stable increasing trend, reaching 101.68 ± 17.4 mL/min/1.73m² at 1 year. In contrast, the triplet therapy group demonstrated potential long-term nephrotoxic risks, with eGFR declining to 71.79 ± 27.57 mL/min/1.73m² at 1 year, which was below baseline levels. The doublet therapy group, meanwhile, showed limited improvement in renal function accompanied by significant individual variability. The core reasons for the optimal long-term outcomes of RTX monotherapy can be attributed to three aspects. Firstly, RTX specifically targets CD20 to selectively eliminate pathogenic B cells, exerting a durable immunomodulatory effect by blocking the source of antibody production and avoiding the non-specific interference of combination therapy on the immune microenvironment. This is consistent with the superior B-cell depletion efficiency (57.30%) observed in this study[ 17 , 18 ]. Secondly, monotherapy avoids the metabolic disorders and infection risks associated with glucocorticoids, as well as the direct nephrotoxicity of CNIs, reducing secondary renal damage caused by drug side effects. It can even improve renal function in patients with poor baseline renal function and comorbidities [ 18 , 19 ]. Thirdly, RTX monotherapy is associated with relatively mild adverse reactions, leading to better patient tolerance and treatment adherence. This is evidenced by the stability of urinary protein reduction and serum albumin recovery with a small standard deviation, laying a foundation for long-term efficacy[ 18 , 20 ]. The combination regimens exhibited a pattern of high initial response followed by declining efficacy, which is supported by clear clinical rationale. The doublet therapy showed a significant short-term effect, with urinary protein dropping sharply to 0.88 ± 1.01g at 3 months, but it began to rebound after 6 months, reaching 3.45 ± 4.91g at 1 year, with a long-term remission rate of less than 50%. This may be due to patients' inability to sustain treatment tolerance due to side effects of glucocorticoids or CNIs, or the interference of combination therapy with the immunomodulatory process of RTX [ 21 , 22 ]. More notably, despite the small sample size of the triplet therapy group, the finding that eGFR was lower than baseline at 1 year is of important warning significance. This is closely related to the chronic nephrotoxicity of CNIs. Excessive immunosuppression not only fails to enhance efficacy but also disrupts the homeostasis of the renal microenvironment and increases the risk of renal function damage, indicating that unnecessary intensive triplet therapy should be strictly avoided in clinical practice [ 18 , 23 ]. An intriguing finding was the dissociation between B-cell depletion rate and clinical outcome. Although triplet therapy achieved the highest B-cell depletion rate (71.40%), it correlated with inferior renal function preservation. In contrast, monotherapy, with the lowest depletion rate (37.00%), demonstrated the highest B-cell clearance rate (57.30%) and the best clinical outcomes.​ This suggests that the efficacy of RTX in pMN may not solely depend on the depth of B-cell reduction, but rather on the quality of immune reconstitution and the efficient clearance of pathogenic B-cell clones, a process that might be disrupted by concurrent non-specific immunosuppressants. The overall homeostasis of the immune environment, the precise clearance efficiency of pathogenic B-cell subsets, and drug safety may play more critical roles. Although combination therapy can enhance B-cell depletion, it may simultaneously disrupt the balanced regulation of the immune system, and the risk of treatment interruption caused by side effects further offsets potential benefits, ultimately leading to poor clinical outcomes[ 17 , 24 ]. Based on the results of this study, we put forward clear clinical practice recommendations: RTX monotherapy should be the preferred initial treatment for most pMN patients, especially for anti-PLA2R antibody-positive patients (overall remission rate of 80.80% with monotherapy) and elderly patients (> 60 years old, overall remission rate of 80.0% with monotherapy), who derive more significant benefits from monotherapy [ 25 , 26 ]. Combination therapy should be strictly limited to specific scenarios, such as critically ill patients requiring extremely rapid control of severe nephrotic syndrome (e.g., massive proteinuria with severe hypoalbuminemia). During treatment, close monitoring of renal function, infection, and metabolic indicators is necessary. Once the condition stabilizes, timely evaluation should be conducted to determine whether conversion to monotherapy maintenance is feasible, avoiding the cumulative risks of long-term combination therapy [ 23 , 27 ]. This study has certain limitations. Firstly, as a single-center retrospective cohort study, the treatment regimens were selected by clinicians based on patient conditions, inevitably leading to potential selection bias. Although the baseline data were well-balanced, confounding factors cannot be completely excluded. Secondly, the sample sizes of each group were uneven, especially the triplet therapy group and the antibody-negative subgroup, which may affect the statistical power of the results, and the relevant conclusions should be interpreted with caution. Finally, the follow-up period was 1 year, failing to evaluate the long-term efficacy stability, disease recurrence rate, and the risk of end-stage renal disease progression. Future multi-center, large-sample prospective randomized controlled trials with extended follow-up are needed to further verify the long-term differences between monotherapy and combination therapy, and to explore individualized treatment strategies based on B-cell depletion efficiency and dynamic changes in antibody titers, providing more solid evidence-based medical evidence for the precise treatment of pMN [ 27 , 28 ]. 5. Conclusion In summary, this real-world study demonstrates that RTX monotherapy achieves a superior overall response rate​ and better renal function protection compared to combination immunosuppressive regimens over a 1-year period in patients with pMN. This advantage is particularly evident in elderly patients and those with low anti-PLA2R antibody titers. Therefore, RTX monotherapy should be regarded as an efficient and safe preferred first-line treatment strategy for the majority of pMN patients, while the routine use of combination therapies involving glucocorticoids and/or CNIs requires critical re-evaluation regarding their appropriate target population. Abbreviations RTX, Rituximab TAC,Tacrolimus pMN, primary membranous nephropathy MN, membranous nephropathy PLA2R, phospholipase A2 receptor THSD7A, thrombospondin type-1 domain-containing 7A NELL1, neural epidermal growth factor-like 1 PCDH7, protocadherin 7 CNIs, calcineurin inhibitors eGFR, estimated glomerular filtration rate 24h UTP, 24-hour urinary protein excretion Scr, serum creatinine Alb, serum albumin CR, complete remission PR, partial remission OR, overall response Declarations Supplementary Material The Supplementary Material for this article can be found online at: XX Data Availability Statement The requirement for individual patient informed consent was waived for both participation and publication because this retrospective study utilized only fully anonymized data, posing no risk to patient privacy. The datasets generated and analyzed during the current study are not publicly available due to hospital policy and patient privacy regulations. However, the de-identified data supporting the main findings of this study are available from the corresponding author upon reasonable request. Acknowledgments Not applicable. Author Contribution Wei Xian (Xian W) provided the concept and designed the study. Ji Dong (Ji D) and Mingrui Fan performed the experiments, analyzed the data, and wrote the manuscript. Lipu Shi contributed to the data curation. Han Li and Jianping Si participated in the experimental verification and result discussion. All authors discussed the results and revised the manuscript critically for important intellectual content.. Funding No funding Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Henan Provincial People's Hospital (Approval Number: (2021) Ethics Review No. (78)). The ethics committee waived the requirement for informed consent. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Department of Clinical Medicine, Henan Medical College, Zhengzhou 451191, China. 2 Department of Public Health and Health Management, Henan Medical College, Zhengzhou 451191, China. 3 Department of Nephrology, Henan Provincial People's Hospital, Zhengzhou 450000, China 4 Department of Immunology, Henan Provincial People's Hospital, Zhengzhou 450000, China 5 Henan University of Chinese Medicine, Zhengzhou 450000, China 6 Department of Clinical Laboratory, Henan Second Provincial People’s Hospital, Zhengzhou 450000, China References Beck LH Jr, Bonegio RGB, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med. 2009;361:11–21. https://doi.org/10.1056/NEJMoa0810457. Ronco P, Debiec H. Molecular pathogenesis of membranous nephropathy. Annu Rev Pathol. 2020;15:287–313. https://doi.org/10.1146/annurev-pathol-020117-043811. Sethi S, Madden B, Casal Moura M, Nasr SH, Klomjit N, Gross L, et al. Hematopoietic stem cell transplant-membranous nephropathy is associated with protocadherin FAT1. J Am Soc Nephrol. 2022;33:1033–44. https://doi.org/10.1681/ASN.2021111488. Tomas NM, Beck LH Jr, Meyer-Schwesinger C, Seitz-Polski B, Ma H, Zahner G, et al. Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy. N Engl J Med. 2014;371:2277–87. https://doi.org/10.1056/NEJMoa1409354. Sethi S, Beck LH Jr, Glassock RJ, Haas M, De Vriese AS, Caza TN, et al. Mayo Clinic consensus report on membranous nephropathy: Proposal for a novel classification. Mayo Clin Proc. 2023;98:1671–84. https://doi.org/10.1016/j.mayocp.2023.08.006. Ponticelli C. Membranous nephropathy. J Clin Med. 2025;14:761. https://doi.org/10.3390/jcm14030761. Ponticelli C, Zucchelli P, Imbasciati E, Cagnoli L, Pozzi C, Passerini P, et al. Controlled trial of methylprednisolone and chlorambucil in idiopathic membranous nephropathy. N Engl J Med. 1984;310:946–50. https://doi.org/10.1056/NEJM198404123101503. Jha V, Ganguli A, Saha TK, Kohli HS, Sud K, Gupta KL, et al. A randomized, controlled trial of steroids and cyclophosphamide in adults with nephrotic syndrome caused by idiopathic membranous nephropathy. J Am Soc Nephrol. 2007;18:1899–904. https://doi.org/10.1681/ASN.2007020166. Praga M, Barrio V, Juárez GF, Luño J, Grupo Español de Estudio de la Nefropatía Membranosa. Tacrolimus monotherapy in membranous nephropathy: a randomized controlled trial. Kidney Int. 2007;71:924–30. https://doi.org/10.1038/sj.ki.5002215. Fervenza FC, Appel GB, Barbour SJ, Rovin BH, Lafayette RA, Aslam N, et al. Rituximab or cyclosporine in the treatment of membranous nephropathy. N Engl J Med. 2019;381:36–46. https://doi.org/10.1056/NEJMoa1814427. Scolari F, Delbarba E, Santoro D, Gesualdo L, Pani A, Dallera N, et al. Rituximab or cyclophosphamide in the treatment of membranous nephropathy: The RI-CYCLO randomized trial. J Am Soc Nephrol. 2021;32:972–82. https://doi.org/10.1681/ASN.2020071091. Fernández-Juárez G, Rojas-Rivera J, Logt A-E van de, Justino J, Sevillano A, Caravaca-Fontán F, et al. The STARMEN trial indicates that alternating treatment with corticosteroids and cyclophosphamide is superior to sequential treatment with tacrolimus and rituximab in primary membranous nephropathy. Kidney Int. 2021;99:986–98. https://doi.org/10.1016/j.kint.2020.10.014. Gauckler P, Shin JI, Alberici F, Audard V, Bruchfeld A, Busch M, et al. Rituximab in membranous nephropathy. Kidney Int Rep. 2021;6:881–93. https://doi.org/10.1016/j.ekir.2020.12.035. Andeen NK, Kung VL, Avasare RS, Barbour S, Griffith M, Bissonnette MLZ, et al. Questions and caveats in antigen-defined membranous nephropathy. J Am Soc Nephrol. 2025;36:1639–51. https://doi.org/10.1681/ASN.0000000769. Kattah AG, Fervenza FC. Rituximab: emerging treatment strategies of immune-mediated glomerular disease. Expert Rev Clin Immunol. 2012;8:413–21. https://doi.org/10.1586/eci.12.26. Rosenzwajg M, Languille E, Debiec H, Hygino J, Dahan K, Simon T, et al. B- and T-cell subpopulations in patients with severe idiopathic membranous nephropathy may predict an early response to rituximab. Kidney Int. 2017;92:227–37. https://doi.org/10.1016/j.kint.2017.01.012. Dahan K, Debiec H, Plaisier E, Cachanado M, Rousseau A, Wakselman L, et al. Rituximab for severe membranous nephropathy: A 6-month trial with extended follow-up. J Am Soc Nephrol. 2017;28:348–58. https://doi.org/10.1681/ASN.2016040449. Fervenza FC, Appel GB, Barbour SJ, Rovin BH, Lafayette RA, Aslam N, et al. Rituximab or cyclosporine in the treatment of membranous nephropathy. N Engl J Med. 2019;381:36–46. https://doi.org/10.1056/NEJMoa1814427. Seitz-Polski B, Dahan K, Debiec H, Rousseau A, Andreani M, Zaghrini C, et al. High-dose rituximab and early remission in PLA2R1-related Membranous Nephropathy. Clin J Am Soc Nephrol. 2019;14:1173–82. https://doi.org/10.2215/CJN.11791018. Ruggenenti P, Cravedi P, Chianca A, Perna A, Ruggiero B, Gaspari F, et al. Rituximab in idiopathic membranous nephropathy. J Am Soc Nephrol. 2012;23:1416–25. https://doi.org/10.1681/ASN.2012020181. Praga M, Barrio V, Juárez GF, Luño J, Grupo Español de Estudio de la Nefropatía Membranosa. Tacrolimus monotherapy in membranous nephropathy: a randomized controlled trial. Kidney Int. 2007;71:924–30. https://doi.org/10.1038/sj.ki.5002215. Qiu TT, Zhang C, Zhao HW. Calcineurin Inhibitors Versus Cyclophosphamide for Idiopathic Membranous Nephropathy: A Systematic Review and Meta-Analysis of 21 Clinical Trials. Autoimmun Rev. 2017;16:136–45. https://doi.org/10.1016/j.autrev.2016.10.009. Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol. 2009;4:481–508. https://doi.org/10.2215/CJN.04800908. Zou P, Li H, Cai J. Therapy of Rituximab in Idiopathic Membranous Nephropathy With Nephrotic Syndrome: A Systematic Review and Meta-Analysis. Chin Med Sci J. 2018;33:9–19. https://doi.org/10.4236/cmsj.2018.331002. Rovin BH, Adler SG, Barratt J. Executive Summary of the KDIGO 2021 Guideline for the Management of Glomerular Diseases. Kidney Int. 2021;100:753–79. https://doi.org/10.1016/j.kint.2021.05.01. Beck LH Jr, Bonegio RGB, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med. 2009;361:11–21. https://doi.org/10.1056/NEJMoa0810457. Rojas-Rivera JE, Carriazo S, Ortiz A. Treatment of idiopathic membranous nephropathy in adults: KDIGO 2012, cyclophosphamide and cyclosporine A are out, rituximab is the new normal. Clin Kidney J. 2019 Sep 30;12(5):629-638. doi: 10.1093/ckj/sfz127. Ford I, Norrie J. Pragmatic Trials. N Engl J Med. 2016;375:454–63. https://doi.org/10.1056/NEJMp1601564. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8713007","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601246095,"identity":"0ef6fbaf-7bbb-4aea-b998-68f5992c9b71","order_by":0,"name":"Ji Dong","email":"","orcid":"","institution":"Henan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"","lastName":"Dong","suffix":""},{"id":601246096,"identity":"e2ec0777-3590-43e0-afc4-b2696a10d414","order_by":1,"name":"Mingrui Fan","email":"","orcid":"","institution":"Henan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Mingrui","middleName":"","lastName":"Fan","suffix":""},{"id":601246097,"identity":"0ae622fe-d6a8-4fd6-888b-332fc2e73d67","order_by":2,"name":"Jianping Si","email":"","orcid":"","institution":"Henan University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jianping","middleName":"","lastName":"Si","suffix":""},{"id":601246098,"identity":"d105ead0-9c79-450f-8093-4ea7e31410ff","order_by":3,"name":"Lipu Shi","email":"","orcid":"","institution":"Henan Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lipu","middleName":"","lastName":"Shi","suffix":""},{"id":601246099,"identity":"b907555c-3c43-4c70-ad04-5e702536f16e","order_by":4,"name":"Yue Ying","email":"","orcid":"","institution":"Henan Second Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Ying","suffix":""},{"id":601246100,"identity":"a5aaddea-1609-41a9-be35-882d3d807852","order_by":5,"name":"Yingying Ren","email":"","orcid":"","institution":"Henan Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Ren","suffix":""},{"id":601246101,"identity":"d7f42935-6991-4ab3-bd44-9a3e8d09285c","order_by":6,"name":"Li Han","email":"","orcid":"","institution":"Henan Provincial People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Han","suffix":""},{"id":601246102,"identity":"23876afb-a8c2-4a47-a822-cf5711d9ca53","order_by":7,"name":"Wei Xian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYBACAxiDn4GxgUQtkg0kazE4QJwGBgZz6fZnn24wHE7cfP5w24MfDHZyuoQss5xzxnh2DkNa4rYbie2GPQzJxmaErDO4kcPMnMNgk7vtBmObBA/DgcRthLWkPwZqkcjd3H+wTfIPcVoSjMG2bGBIbJMm0pYckJa0+hk3gFpkDIjyC9hhh435+48/k3xTYSdHUAsYMP6Dm0CM8lEwCkbBKBgFBAEAKgI/DB66ziwAAAAASUVORK5CYII=","orcid":"","institution":"Henan Provincial People's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xian","suffix":""}],"badges":[],"createdAt":"2026-01-27 17:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8713007/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8713007/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104182768,"identity":"496d0259-f9f7-41d2-b7a5-fffef2642491","added_by":"auto","created_at":"2026-03-08 17:39:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110845,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Clinical Response Rates Across Treatment Regimens. Bar graph depicting the CR, PR, and OR rates achieved at 12 months in patients with pMN treated with RTX Monotherapy (black bars), Doublet Therapy (RTX plus calcineurin inhibitor; pink bars), or Triplet Therapy(RTX plus both glucocorticoids and a calcineurin inhibitor; teal bars). Statistical significance for differences in OR rates between the monotherapy and combination therapy groups is indicated (\u003cem\u003eP = 0.032\u003c/em\u003e for monotherapy vs. doublet therapy; \u003cem\u003eP = 0.030\u003c/em\u003e for monotherapy vs. triplet therapy). The data demonstrate the superior overall response rate of the RTX monotherapy regimen. CR: Complete remission; PR: Partial remission; OR: Overall response.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/8c36ff0d254e5fd9835da1ca.jpg"},{"id":104182767,"identity":"7f90db01-5cba-40e5-841f-0750105b81e8","added_by":"auto","created_at":"2026-03-08 17:39:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":664242,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal Changes in 24-hour Urinary Protein Excretion Across Treatment Regimens.Line chart illustrating the mean 24h UTP levels from baseline to 12 months of follow-up in patients with pMN treated with RTX Monotherapy (black circles), Doublet Therapy (pink squares), or Triplet Therapy (teal triangles). Error bars represent the standard deviation. All three treatment groups demonstrated a reduction in proteinuria over time. A rapid decline is evident at 3 months, particularly in the doublet therapy group, followed by a more gradual decrease thereafter. The trajectories highlight the temporal dynamics of treatment response, with initial proteinuria reduction not necessarily predicting the long-term level achieved.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/b501ce75e269c40f5114918d.jpg"},{"id":104182759,"identity":"b7876a5d-8c3d-4ec1-8527-64cfa748a85e","added_by":"auto","created_at":"2026-03-08 17:39:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":651964,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal Trajectory of Serum Albumin Recovery Following Immunosuppressive Treatment. Line chart depicting the mean serum albumin levels at serial timepoints (baseline, 3, 6, and 12 months) in patients with pMN treated with RTX Monotherapy (black circles), Doublet Therapy (pink squares), or Triplet Therapy (teal triangles). Error bars represent standard deviation. All three regimens led to a progressive increase in serum albumin over 12 months, reflecting the reversal of nephrotic hypoalbuminemia. The trajectories demonstrate a sustained recovery pattern, culminating in mean levels approaching the lower limit of the normal reference range (\u0026gt;35 g/L) by 12 months. The similar slopes and endpoints of the three lines suggest that the rate and degree of albumin restoration were comparable across the different immunosuppressive strategies, independent of the specific treatment regimen.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/73d4aa5c39c0773e36c0b251.jpg"},{"id":104182738,"identity":"aef3acba-c483-415e-b43a-be607e357218","added_by":"auto","created_at":"2026-03-08 17:39:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130490,"visible":true,"origin":"","legend":"\u003cp\u003eDynamic Changes in Estimated Glomerular Filtration Rate (eGFR) Following Treatment Initiation. Bar graph illustrating the mean eGFR at baseline, 3 months, 6 months, and 12 months in patients with pMN treated with RTX Monotherapy (black bars), Doublet Therapy (pink bars), or Triplet Therapy (teal bars). Error bars represent standard deviation. All three treatment regimens were associated with an increase in mean eGFR from baseline to 12 months. RTX Monotherapy demonstrated a progressive improvement throughout the follow-up period. The doublet and triplet therapy groups showed a more pronounced early increase at 3 months, with subsequent stabilization. The changes in eGFR over time did not reach statistical significance between the treatment groups at any assessed timepoint (\u003cem\u003eP \u0026gt; 0.05\u003c/em\u003e for all intergroup comparisons). eGFR: Estimated glomerular filtration rate.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/938197c18596f29f75616d65.jpg"},{"id":104182757,"identity":"57569228-9121-4afe-9c3b-4fd4b907e65a","added_by":"auto","created_at":"2026-03-08 17:39:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":113324,"visible":true,"origin":"","legend":"\u003cp\u003eComparative Analysis of B-Cell Pharmacodynamics Across Treatment Regimens. Bar graph illustrating the differential effects of RTX Monotherapy, Doublet Therapy and Triplet Therapy on B-cell depletion. The graph presents three metrics: the average post-treatment B-cell count in cells/μL (black bars, left y-axis), the B-cell depletion rate (magenta bars, right y-axis), and the B-cell clearance rate (teal bars, left y-axis). A significant difference among the three treatment groups is indicated (\u003cem\u003eP\u0026lt; 0.01\u003c/em\u003e). RTX Monotherapy resulted in the highest average B-cell count (43.5 cells/μL) and the highest clearance rate (57.3%), despite having the lowest depletion rate (37.0%). In contrast, combination therapies achieved lower average B-cell counts (21.4 and 27.3 cells/μL) and higher depletion rates (63.6% and 71.4%), but substantially lower clearance rates (32.6% and 37.7%). This dissociation highlights that combination regimens induce more profound quantitative B-cell depletion, whereas monotherapy is associated with superior efficiency in clearing the residual B-cell pool.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/1172843ed202f877732a15cc.jpg"},{"id":104182743,"identity":"c4970dd2-beaf-4e1b-a741-c4d162a52f58","added_by":"auto","created_at":"2026-03-08 17:39:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":122895,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation Between Antibody Status, Treatment Regimen, and Clinical Response. Bar graph comparing the 12-month ORR in patients with pMN, stratified by anti-PLA2R antibody status (negative: black bars; positive: pink bars) and treatment regimen (RTX monotherapy, doublet therapy, triplet therapy). A statistically significant difference in response rates was observed among the treatment groups (\u003cem\u003eP=0.005\u003c/em\u003e). RTX monotherapy demonstrated high and comparable efficacy in both antibody-negative (75.0%) and antibody-positive (80.0%) patients. While triplet therapy achieved a 100% response rate in the small antibody-negative subgroup, its efficacy in antibody-positive patients (55.6%) was notably lower than that of monotherapy. Doublet therapy showed intermediate efficacy. The data suggest that treatment response is influenced by both the immunosuppressive regimen and the underlying serological profile, with RTX monotherapy maintaining robust efficacy irrespective of antibody status.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/a086d52e24aeb03c62e908d0.jpg"},{"id":108965373,"identity":"d77333b7-af6c-4027-898f-f451ab9046f6","added_by":"auto","created_at":"2026-05-11 09:32:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2123286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/64df88ef-5c8f-4ac4-83ab-651f1154139b.pdf"},{"id":104182725,"identity":"dbf6c58a-9faf-4bd7-8110-ded26018a710","added_by":"auto","created_at":"2026-03-08 17:39:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19819,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8713007/v1/219f672c539769d4120387d5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison of the efficacy of rituximab monotherapy and combined immunotherapy for primary membranous nephropathy: a real-world cohort study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePrimary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults, characterized by immune complex deposition along the subepithelial surface of the glomerular basement membrane. The landmark 2009 discovery of the M-type phospholipase A2 receptor (PLA2R) as a key target antigen transformed our understanding of pMN, establishing it as an organ-specific autoimmune disorder [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In this disease, circulating autoantibodies\u0026mdash;predominantly of the IgG4 subclass\u0026mdash;bind to endogenous podocyte antigens such as PLA2R, driving in situ immune complex formation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Complement activation, particularly via the lectin or alternative pathways, culminates in the assembly of the membrane attack complex (C5b-9), which induces podocyte injury, cytoskeletal disruption, and subsequent proteinuria [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Beyond PLA2R (accounting for ~\u0026thinsp;70\u0026ndash;80% of cases), multiple other target antigens have been identified, including thrombospondin type-1 domain-containing 7A (THSD7A), neural epidermal growth factor-like 1 (NELL1), and protocadherin 7 (PCDH7) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These findings have challenged the traditional primary-secondary classification paradigm and support an antigen-based framework that more accurately reflects the underlying immunopathology [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The pathogenesis of pMN also involves genetic predisposition, environmental triggers, and dysregulated autophagy, which collectively promote the exposure of native podocyte proteins and the breakdown of immune tolerance[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These interconnected mechanisms confirm pMN as a prototypical antibody-mediated glomerular disease.\u003c/p\u003e \u003cp\u003eThe treatment paradigm for membranous nephropathy (MN) has evolved substantially over recent decades, shifting from non-specific immunosuppressive regimens to targeted B-cell therapies. Historically, cyclical regimens combining corticosteroids with alkylating agents (e.g., cyclophosphamide) served as the cornerstone of immunosuppression for high-risk patients, demonstrating efficacy in inducing remission but often burdened by substantial toxicity and high relapse rates upon discontinuation[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The advent of calcineurin inhibitors (CNIs) offered an alternative with favorable short-term remission rates; however, their utility was limited by nephrotoxicity and frequent relapses following withdrawal [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A transformative shift occurred with the introduction of rituximab (RTX), an anti-CD20 monoclonal antibody that selectively depletes B cells\u0026mdash;central mediators in the production of pathogenic autoantibodies[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The pivotal MENTOR trial established RTX as a first-line therapy by demonstrating non-inferiority to cyclosporine in achieving remission at 12 months and superior response durability at 24 months, coupled with a significantly lower relapse rate and a more favorable safety profile [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Subsequent studies, including the RI-CYCLO and STARMEN trials, reinforced RTX\u0026rsquo;s efficacy, showing comparable or superior outcomes to cyclophosphamide-based regimens while underscoring the clinical value of anti-PLA2R antibody monitoring to guide retreatment and predict clinical response[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This evolution toward targeted B-cell depletion has redefined the therapeutic landscape of MN, emphasizing precision medicine strategies that prioritize efficacy, safety, and long-term remission.\u003c/p\u003e \u003cp\u003e Despite established guidelines and robust trial evidence, a substantial proportion of clinicians in real-world practice continue to combine RTX with corticosteroids or CNIs, aiming to accelerate therapeutic response or enhance efficacy\u0026mdash;particularly in high-risk patients. However, this approach lacks consistent evidence-based support, especially regarding long-term renal outcomes and safety. The rationale for combination therapy often centers on achieving rapid proteinuria reduction, yet its impact on sustained renal protection remains unclear. Critically, concerns persist about potential trade-offs, including heightened risks of infections, metabolic disturbances, and CNI-associated nephrotoxicity, which may offset long-term benefits [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The heterogeneity in MN pathogenesis, driven by diverse target antigens, further complicates standardized treatment protocols. For instance, antigen-specific responses may differentially influence treatment efficacy, but evidence linking combination therapy benefits to specific antigen subtypes remains sparse[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Thus, a critical knowledge gap exists: whether empirical combination regimens truly outperform RTX monotherapy in preserving renal function over the long term without amplifying adverse events.\u003c/p\u003e \u003cp\u003eTo address these uncertainties, we conducted a real-world, single-center cohort study to directly compare the efficacy and safety of RTX monotherapy versus combination strategies (dual or triple therapy) in the management of pMN. Primary endpoints included renal survival, quantified by sustained estimated glomerular filtration rate (eGFR) stability and composite kidney outcomes. Secondary analyses assessed antigen-specific serological responses and infection-related complications. By leveraging detailed clinical data, we aimed to delineate the risk-benefit profiles across different therapeutic regimens, with subgroup analyses stratified by antigen status and baseline clinical features. This study seeks to provide high-grade evidence to inform individualized treatment decisions, aligning with the emerging paradigm of antigen-driven classification while prioritizing long-term renal protection over short-term surrogate outcomes.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Study Design and Patient Selection\u003c/h2\u003e \u003cp\u003eThis retrospective cohort study included 98 patients diagnosed with pMN who received treatment at our institution between January 2019 and December 2023. The diagnosis of pMN was confirmed by renal biopsy in all cases, with pathological examination revealing typical membranous nephropathy features\u0026mdash;including diffuse thickening of the glomerular basement membrane, subepithelial immune complex deposition on electron microscopy, and granular staining for IgG along capillary loops on immunofluorescence\u0026mdash;in the absence of pathological changes suggestive of other renal diseases.\u003c/p\u003e \u003cp\u003eInclusion criteria were as follows: (1) availability of anti-PLA2R antibody test results (measured by enzyme-linked immunosorbent assay [ELISA]; positive cutoff\u0026thinsp;\u0026ge;\u0026thinsp;2 RU/mL); (2) completion of at least 12 months of regular follow-up after immunosuppressive therapy; and (3) availability of complete electronic medical record data, including demographic characteristics, clinical symptoms, laboratory results, and treatment regimens.\u003c/p\u003e \u003cp\u003eExclusion criteria comprised: (1) secondary membranous nephropathy (e.g., associated with diabetes mellitus, systemic lupus erythematosus, hepatitis B virus infection, or drug-induced MN); (2) presence of life-threatening complications such as severe infection, malignancy, or acute cardio-cerebrovascular events; (3) loss to follow-up or follow-up duration\u0026thinsp;\u0026lt;\u0026thinsp;12 months; and (4) prior renal replacement therapy (hemodialysis or peritoneal dialysis) or history of kidney transplantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Treatment Regimens and Grouping\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Treatment Regimens and Dosing\u003c/h2\u003e \u003cp\u003e The dosing protocols for all immunosuppressive agents were in accordance with current clinical guidelines for the management of membranous nephropathy. Specific regimens were as follows:\u003c/p\u003e \u003cp\u003eRituximab (RTX):​ All patients received a standardized RTX induction regimen of 375 mg/m\u0026sup2; body surface area, administered intravenously once weekly for 4 consecutive weeks. Premedication with antihistamines and glucocorticoids was administered prior to each infusion to prevent infusion-related reactions.\u003c/p\u003e \u003cp\u003e Tacrolimus (TAC):​ For patients in the doublet and triplet therapy groups, TAC was initiated at a dose of 0.05\u0026ndash;0.1 mg/kg per day, administered orally in two divided doses. The dose was subsequently adjusted to maintain a trough blood concentration within the target range of 5\u0026ndash;8 ng/mL. Regular monitoring of TAC levels, renal function, and potential adverse effects was performed. For patients achieving complete or partial remission after 6\u0026ndash;12 months of treatment, TAC was tapered, typically by reducing the dose by 25%\u0026ndash;50% every 3\u0026ndash;6 months, with the total treatment duration guided by clinical response.\u003c/p\u003e \u003cp\u003eGlucocorticoids:​ In the triplet therapy group, glucocorticoids were initiated as prednisone (or equivalent) at a dose of 0.5\u0026ndash;1.0 mg/kg per day (maximum 60 mg/day). This dose was maintained for 4\u0026ndash;8 weeks, followed by a gradual taper by approximately 5 mg every 1\u0026ndash;2 weeks, aiming for a low maintenance dose (e.g., 5\u0026ndash;10 mg/day) or discontinuation by 6\u0026ndash;12 months, contingent upon the patient's clinical response and tolerance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Treatment Grouping\u003c/h2\u003e \u003cp\u003eBased on the initial immunosuppressive therapy, patients were categorized into three groups:\u003c/p\u003e \u003cp\u003eMonotherapy group: Received RTX alone.\u003c/p\u003e \u003cp\u003eDoublet therapy group: Received RTX in combination with TAC\u003c/p\u003e \u003cp\u003eTriplet therapy group: Received RTX, TAC and glucocorticoids concurrently.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Data Collection and Outcome Measures\u003c/h2\u003e \u003cp\u003eBaseline data were retrieved from the hospital electronic medical record system, encompassing four core categories: demographic characteristics (age, sex, and comorbidities including hypertension, diabetes, and coronary artery disease); renal function and biochemical parameters (24-hour urinary protein excretion (24h UTP), estimated glomerular filtration rate (eGFR, computed via the CKD-EPI equation), serum creatinine (Scr), and serum albumin (Alb)); immunological markers (anti-PLA2R antibody titer, quantified by ELISA with a positive threshold of \u0026ge;\u0026thinsp;2 RU/mL, and B cell count, detected via flow cytometry); and initial treatment regimens, specifically the types of immunosuppressive agents and combination therapy strategies. All enrolled patients underwent a 12-month follow-up, with scheduled evaluations at 3, 6, and 12 months post-treatment. Follow-up data collection focused on four dimensions of clinical outcomes: efficacy parameters (24h UTP and serum Alb levels at each timepoint, utilized for clinical response assessment); renal function indicators (eGFR and Scr at each timepoint); immunological measures (B cell count at month 6, from which B cell depletion rate and clearance rate were derived); and safety profiles, defined as the incidence of adverse events such as infections, gastrointestinal reactions, and hematological abnormalities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Outcome Definitions\u003c/h2\u003e \u003cp\u003eThe primary outcome was the 12-month clinical remission rate, which included three categories: complete remission (CR), defined as 24h UTP\u0026thinsp;\u0026lt;\u0026thinsp;0.3 g with serum Alb\u0026thinsp;\u0026gt;\u0026thinsp;35 g/L; partial remission (PR), characterized by a\u0026thinsp;\u0026ge;\u0026thinsp;50% reduction in 24h UTP from baseline, 24h UTP\u0026thinsp;\u0026lt;\u0026thinsp;3.5 g, and concurrent significant improvement in serum Alb; and overall response (OR), referring to the proportion of patients achieving either CR or PR. Secondary outcomes encompassed dynamic efficacy trends (changes in 24h UTP and serum Alb from baseline to 3, 6, and 12 months), renal protective effects (temporal changes in eGFR and Scr), immunological effects [B cell depletion rate, calculated as (baseline B cell count \u0026ndash; post-treatment B cell count)/baseline B cell count \u0026times; 100%, and mean B cell clearance rate], as well as safety profile (incidence, severity, and outcomes of adverse events).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Subgroup Analyses\u003c/h2\u003e \u003cp\u003eTwo predefined subgroup analyses were performed based on key clinical factors of pMN: anti-PLA2R antibody titer (high titer: \u0026gt;200 RU/mL vs. low titer: \u0026le;200 RU/mL) and age (elderly: \u0026gt;60 years vs. non-elderly: \u0026le;60 years). Within each subgroup, inter-group differences in 12-month eGFR, 24h UTP, and OR rate were compared between the monotherapy group and the combined doublet/triplet therapy groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using SPSS version 26.0 (IBM Corp., Armonk, NY, USA). The Shapiro\u0026ndash;Wilk test was used to assess normality of continuous variables. Normally distributed data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and compared using one-way ANOVA (with Bonferroni post-hoc test for pairwise comparisons). Non-normally distributed data are expressed as median (interquartile range) and compared using the Kruskal\u0026ndash;Wallis H test. Categorical variables are presented as number (percentage) and compared using the chi-square test or Fisher\u0026rsquo;s exact test when expected frequencies were \u0026lt;\u0026thinsp;5. A two-sided P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Sample Size Justification and Post-hoc Power Analysis\u003c/h2\u003e \u003cp\u003eAs a retrospective real-world cohort study, a prospective power calculation was not performed prior to patient enrollment. The sample size was determined by the availability of all eligible patients meeting the inclusion criteria at our institution during the study period (January 2019 to December 2023), which amounted to 98 patients. To address the robustness of our findings, a post-hoc power analysis​ was conducted using the observed effect sizes from our primary outcome (the difference in 12-month overall response rate, ORR). Given the observed ORR of 84.0% in the monotherapy group (n\u0026thinsp;=\u0026thinsp;25) versus 46.7% in the doublet therapy group (n\u0026thinsp;=\u0026thinsp;15), yielding an absolute difference of 37.3%, and with an alpha level set at 0.05, the achieved post-hoc statistical power for this comparison exceeded 90%​ (using a chi-square test for proportions). This indicates that the present sample size provided ample power to detect the clinically significant differences in efficacy observed among the treatment groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Baseline Characteristics of the Study Population\u003c/h2\u003e \u003cp\u003eThe study included 98 patients with biopsy-proven pMN, stratified into two groups based on the presence of comorbidities: with comorbidities (n\u0026thinsp;=\u0026thinsp;67) and without comorbidities (n\u0026thinsp;=\u0026thinsp;31). The distribution of treatment regimens (monotherapy, doublet therapy, triplet therapy) was balanced between groups (\u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.555\u003c/em\u003e), with detailed comparative results of baseline characteristics presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Demographic analysis revealed that patients with comorbidities were significantly older than those without [median (IQR): 60.0 (51.5\u0026ndash;67.0) years vs 39.0 (21.0\u0026ndash;55.5) years, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e], while gender distribution showed no significant difference (males: 53.7% [36/67] vs 51.6% [16/31], \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.785\u003c/em\u003e), indicating that comorbidities are more prevalent in middle-aged and elderly patients with membranous nephropathy. Comparative analysis of renal function and biochemical parameters demonstrated that the comorbidity group had significantly lower baseline eGFR [91.6 (59.12\u0026ndash;101.22) mL/min/1.73m\u0026sup2; vs 100.0 (91.15\u0026ndash;120.8) mL/min/1.73m\u0026sup2;, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.007\u003c/em\u003e]. Serum creatinine levels showed an increasing trend, though not statistically significant [76.0 (60.4\u0026ndash;102.4) \u0026micro;mol/L vs 69.4 (53.62\u0026ndash;83.65) \u0026micro;mol/L, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.086\u003c/em\u003e]. Both groups had similar median 24h UTP [4.31g (1.75\u0026ndash;7.28) g vs (2.55\u0026ndash;7.04) g, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.73\u003c/em\u003e], and serum albumin levels were comparable [28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0 g/L vs 26.82\u0026thinsp;\u0026plusmn;\u0026thinsp;9.92 g/L, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.526\u003c/em\u003e], suggesting that comorbidities primarily affect glomerular filtration function without significantly influencing proteinuria severity or hypoalbuminemia status. Immunological and treatment-related parameters showed no significant differences in anti-PLA2R antibody titers [5.36 (2.0\u0026ndash;101.77) RU/mL vs 2.0 (2.0\u0026ndash;20.04) RU/mL, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.081\u003c/em\u003e] or B-lymphocyte counts [214.0 (77.0\u0026ndash;348.0) cells/\u0026micro;l vs 233.0 (99.5\u0026ndash;292.0) cells/\u0026micro;l, \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.884\u003c/em\u003e] between groups. The balanced distribution of treatment regimens across groups (monotherapy: 58.2% vs 54.8%; doublet therapy: 29.9% vs 38.7%; triplet therapy: 11.9% vs 6.5%; \u003cem\u003eP\u0026thinsp;=\u0026thinsp;0.555\u003c/em\u003e) indicates that comorbidities did not significantly alter immune status, and treatment strategies were consistent between groups, thereby eliminating potential confounding from baseline immunological characteristics or treatment selection bias in subsequent efficacy analyses.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline Demographic and Clinical Characteristics of the Study Population Stratified by Comorbidity Status.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eDemographics\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWith Comorbidities(n\u0026thinsp;=\u0026thinsp;67)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo Comorbidities(n\u0026thinsp;=\u0026thinsp;31)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.0(51.5\u0026ndash;67.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.0(21.0-55.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (Male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36/67(53.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16/31(51.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.785\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKey Laboratory Values (Pre-treatment)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCharacteristics\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eWith Comorbidities(n\u0026thinsp;=\u0026thinsp;67)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eNo Comorbidities(n\u0026thinsp;=\u0026thinsp;31)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eP-value\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24h Urine Protein (g/24h)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.31(1.75\u0026ndash;7.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.31(2.55\u0026ndash;7.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eeGFR(mL/min/1.73m\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e91.6(59.12-101.22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.0(91.15\u0026ndash;120.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum Creatinine (\u0026micro;mol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.0(60.4-102.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.4(53.62\u0026ndash;83.65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.086\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum Albumin (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.1\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.82\u0026thinsp;\u0026plusmn;\u0026thinsp;9.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.526\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnti-PLA2R Ab Titer (RU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.36(2.0-101.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0(2.0-20.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB-cell Count (cells/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e214.0(77.0-348.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e233.0(99.5\u0026ndash;292.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.884\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39/67(58.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17/31(54.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.555\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoublet Therapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20/67(29.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12/31(38.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.555\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTriplet Therapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8/67(11.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2/31(6.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.555\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. One-Year Clinical Response Rates by Treatment Regimen\u003c/h2\u003e \u003cp\u003eComparative results of CR, PR, and OR rates at one-year post-treatment are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The monotherapy group demonstrated significantly higher OR rates compared to the doublet and triplet therapy groups [84.0% (21/25) vs 46.7% (7/15) vs 50.0% (3/6), respectively; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e], confirming the superior clinical benefit of monotherapy in treating pMN. While no significant differences were observed in CR rates among the three groups [28.0% (7/25) vs 33.3% (5/15) vs 33.3% (2/6), respectively; \u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e], the superiority in OR rates for the monotherapy group primarily stemmed from significantly higher PR rates [56.0% (14/25) vs 13.3% (2/15) vs 16.7% (1/6), respectively; \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e], indicating that monotherapy has a more pronounced effect in achieving partial remission. Notably, the doublet and triplet therapy regimens, despite being more intensive, failed to demonstrate expected efficacy advantages, with OR rates below 50%, significantly lower than the monotherapy group. This suggests that combination therapies may be associated with increased adverse effects, reduced patient tolerance, or clinical selection bias, highlighting potential risks of overtreatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Dynamic Changes in 24-hour Urinary Protein and Serum Albumin\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. 24-hour Urinary Protein Changes\u003c/h2\u003e \u003cp\u003eSignificant differences were observed in the trends of 24h UTP at various timepoints (baseline, 3, 6, and 12 months) across the three treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The monotherapy group exhibited a consistent and sustained decline in proteinuria, decreasing from 4.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41 g at baseline to 3.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95 g at 3 months, 2.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62 g at 6 months, and reaching the lowest value of 1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50 g at 12 months. The relatively small standard deviations at each timepoint suggest stable and durable proteinuria-reducing efficacy with monotherapy. In contrast, the doublet therapy group showed fluctuating proteinuria levels, with a sharp decrease to 0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01 g at 3 months, followed by a significant rebound to 3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.05 g at 6 months, and further increasing to 3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91 g at 12 months, indicating substantial short-term efficacy but poor long-term sustainability. The triplet therapy group, with limited sample sizes at various timepoints, exhibited considerable variability in proteinuria measurements, decreasing from 7.82\u0026thinsp;\u0026plusmn;\u0026thinsp;8.61 g at baseline to 3.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85 g at 3 months, 2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14 g at 6 months, and 2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;3.32 g at 12 months, without statistical significance to confirm stable proteinuria-reducing advantages.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Serum Albumin Changes\u003c/h2\u003e \u003cp\u003eAll three groups demonstrated gradual increases in serum albumin levels over time, approaching the normal reference range (39\u0026ndash;41 g/L) by 12 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The monotherapy group showed the most significant and steady improvement, increasing from 30.15\u0026thinsp;\u0026plusmn;\u0026thinsp;6.25 g/L at baseline to 33.58\u0026thinsp;\u0026plusmn;\u0026thinsp;6.58 g/L at 3 months, 36.80\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75 g/L at 6 months, and reaching 39.51\u0026thinsp;\u0026plusmn;\u0026thinsp;5.11 g/L at 12 months, with progressively decreasing standard deviations, indicating stable improvement in hypoalbuminemia with good consistency in individual patient responses. The doublet and triplet therapy groups showed similar increasing trends but without significant advantages: doublet therapy increased from 30.22\u0026thinsp;\u0026plusmn;\u0026thinsp;6.33 g/L to 39.37\u0026thinsp;\u0026plusmn;\u0026thinsp;5.18 g/L; triplet therapy increased from 31.50\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65 g/L to 41.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.74 g/L. Combined with proteinuria results, these findings confirm that monotherapy is more effective, consistent with clinical response rates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Impact of Different Treatment Regimens on Renal Function (eGFR)\u003c/h2\u003e \u003cp\u003eThe three treatment groups exhibited significantly different trends in eGFR changes at various post-treatment timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The monotherapy group demonstrated a consistent and sustained increase in eGFR, rising from 91.82\u0026thinsp;\u0026plusmn;\u0026thinsp;27.65 mL/min/1.73m\u0026sup2; at baseline to 95.93\u0026thinsp;\u0026plusmn;\u0026thinsp;20.47 mL/min/1.73m\u0026sup2; at 3 months, 96.65\u0026thinsp;\u0026plusmn;\u0026thinsp;23.45 mL/min/1.73m\u0026sup2; at 6 months, and reaching 101.68\u0026thinsp;\u0026plusmn;\u0026thinsp;17.4 mL/min/1.73m\u0026sup2; at 12 months, with progressively decreasing standard deviations, indicating that monotherapy not only improves clinical symptoms but also continuously protects and enhances renal function with improving safety and treatment stability. The doublet therapy group showed a slow increasing trend in eGFR, from 82.55\u0026thinsp;\u0026plusmn;\u0026thinsp;38.38 mL/min/1.73m\u0026sup2; at baseline to 84.26\u0026thinsp;\u0026plusmn;\u0026thinsp;38.1 mL/min/1.73m\u0026sup2; at 3 months, 85.74\u0026thinsp;\u0026plusmn;\u0026thinsp;36.93 mL/min/1.73m\u0026sup2; at 6 months, and 89.24\u0026thinsp;\u0026plusmn;\u0026thinsp;37.47 mL/min/1.73m\u0026sup2; at 12 months, but remained below monotherapy levels at all timepoints with persistently large standard deviations, suggesting limited renal functional improvement with significant interindividual variability. The triplet therapy group exhibited an unfavorable pattern of initial increase followed by decrease in eGFR: 77.07\u0026thinsp;\u0026plusmn;\u0026thinsp;29.44 mL/min/1.73m\u0026sup2; at baseline, increasing to 79.6\u0026thinsp;\u0026plusmn;\u0026thinsp;23.1 mL/min/1.73m\u0026sup2; at 3 months, peaking at 88.3\u0026thinsp;\u0026plusmn;\u0026thinsp;20.77 mL/min/1.73m\u0026sup2; at 6 months, but declining to 71.79\u0026thinsp;\u0026plusmn;\u0026thinsp;27.57 mL/min/1.73m\u0026sup2; at 12 months, below baseline levels, suggesting potential renal function impairment risk with long-term triplet therapy, possibly related to excessive immunosuppression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5. B-cell Depletion Efficacy of Different Treatment Regimens\u003c/h2\u003e \u003cp\u003eSignificant differences were observed in B-cell depletion effects among the treatment regimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). While the triplet therapy group showed the highest B-cell depletion rate (71.40%), followed by doublet therapy (63.60%) and monotherapy (37.00%), indicating stronger immunosuppressive effects with combination therapies, analysis of mean B-cell clearance rate revealed an opposite trend: monotherapy had the highest mean clearance rate (57.30%), followed by triplet therapy (37.70%) and doublet therapy (32.60%). This suggests that although monotherapy resulted in lower overall B-cell depletion, it achieved superior clearance efficiency of surviving B-cells, possibly related to immune microenvironment interference caused by combination therapies. Combined with the superior one-year clinical response rates in the monotherapy group, these findings indicate that B-cell clearance efficiency (rather than mere depletion rate) is a key predictor of clinical efficacy in membranous nephropathy immunotherapy, providing important insights for mechanism research and treatment monitoring.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Subgroup Analysis Results\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1. Anti-PLA2R Antibody Titer Subgroup Analysis\u003c/h2\u003e \u003cp\u003eSubgroup analysis based on anti-PLA2R antibody titers (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed that in the high-titer subgroup (\u0026gt;\u0026thinsp;200 RU/mL), the monotherapy group had significantly higher 12-month eGFR (106.17\u0026thinsp;\u0026plusmn;\u0026thinsp;12.15 mL/min/1.73m\u0026sup2;) compared to the combination therapy group (62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;34.22 mL/min/1.73m\u0026sup2;), lower 24h UTP (1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58 g vs 3.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57 g), and higher OR rate (100.0% [3/3] vs 50.0% [1/2]), with statistical significance (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). In the low-titer subgroup (\u0026le;\u0026thinsp;200 RU/mL), the monotherapy group maintained advantages: 12-month eGFR (102.72\u0026thinsp;\u0026plusmn;\u0026thinsp;17.6 mL/min/1.73m\u0026sup2; vs 80.38\u0026thinsp;\u0026plusmn;\u0026thinsp;32.93 mL/min/1.73m\u0026sup2;), 24h UTP (1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69 g vs 3.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.96 g), and OR rate (81.0% [17/21] vs 46.7% [7/15]), confirming the superiority of monotherapy regardless of anti-PLA2R antibody titer levels. Analysis based on anti-PLA2R antibody status (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) showed that among antibody-positive patients (titer\u0026thinsp;\u0026ge;\u0026thinsp;2 RU/mL), the monotherapy group had significantly higher OR rates (80.80%) compared to doublet (46.70%, n\u0026thinsp;=\u0026thinsp;30) and triplet therapy (55.60%) groups (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). Among antibody-negative patients (titer\u0026thinsp;\u0026lt;\u0026thinsp;2 RU/mL), despite smaller sample sizes, the monotherapy group maintained higher OR rates (75.00% vs 50.00%), suggesting therapeutic value even in non-PLA2R antibody-mediated membranous nephropathy. The high proportion of anti-PLA2R antibody-positive patients across all groups (monotherapy: 92.9%; doublet therapy: 93.8%; triplet therapy: 90.0%) further supports monotherapy as first-line treatment for membranous nephropathy.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSubgroup Analysis of Treatment Efficacy at 12 Months\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubgroup Characteristic​\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment Group​\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12-month eGFR\u003c/p\u003e \u003cp\u003e(mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, mL/min/1.73m\u0026sup2;)​\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12-month 24h UTP (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, g)​\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12-month Overall Response Rate, ORR (%)​\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eanti-PLA2R Antibody\u0026thinsp;\u0026gt;\u0026thinsp;200 RU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e106.17\u0026thinsp;\u0026plusmn;\u0026thinsp;12.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e100.0%(3/3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCombination Therapy*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;34.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e50.0%(1/2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eanti-PLA2R Antibody\u0026thinsp;\u0026le;\u0026thinsp;200 RU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e102.72\u0026thinsp;\u0026plusmn;\u0026thinsp;17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e81.0%(17/21)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCombination Therapy*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e80.38\u0026thinsp;\u0026plusmn;\u0026thinsp;32.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.63\u0026thinsp;\u0026plusmn;\u0026thinsp;4.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46.7%(7/15)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAge\u0026thinsp;\u0026gt;\u0026thinsp;60 years (Elderly)​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e89.15\u0026thinsp;\u0026plusmn;\u0026thinsp;16.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e80.0%(8/10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCombination Therapy*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e62.65\u0026thinsp;\u0026plusmn;\u0026thinsp;23.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e28.6%(2/7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAge\u0026thinsp;\u0026le;\u0026thinsp;60 years (Non-elderly)​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e106.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e83.3%(15/18)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCombination Therapy*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e107.78\u0026thinsp;\u0026plusmn;\u0026thinsp;31.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;5.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.4%(4/9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Combination therapy group includes patients receiving doublet therapy (RTX\u0026thinsp;+\u0026thinsp;TAC) and triplet therapy (RTX\u0026thinsp;+\u0026thinsp;TAC+ glucocorticoids).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2. Age Subgroup Analysis\u003c/h2\u003e \u003cp\u003eAge-based subgroup analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) demonstrated that in elderly patients (\u0026gt;\u0026thinsp;60 years), the monotherapy group had significantly higher 12-month eGFR (89.15\u0026thinsp;\u0026plusmn;\u0026thinsp;16.84 mL/min/1.73m\u0026sup2;, n\u0026thinsp;=\u0026thinsp;16 vs 62.65\u0026thinsp;\u0026plusmn;\u0026thinsp;23.12 mL/min/1.73m\u0026sup2;, n\u0026thinsp;=\u0026thinsp;22), lower 24h UTP (1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57 g vs 1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 g), and higher OR rates (80.0% [8/10] vs 28.6% [2/7], \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), with significant eGFR decline in the combination therapy group, suggesting that elderly patients with membranous nephropathy should avoid combination therapies in favor of safer and more effective monotherapy. In non-elderly patients (\u0026le;\u0026thinsp;60 years), the monotherapy group maintains advantages: OR rate (83.3% [15/18], n\u0026thinsp;=\u0026thinsp;40 vs 44.4% [4/9], n\u0026thinsp;=\u0026thinsp;18), 24h UTP (1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87 g vs 4.83\u0026thinsp;\u0026plusmn;\u0026thinsp;5.96 g, \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), with no significant eGFR difference between groups (106.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.19 mL/min/1.73m\u0026sup2; vs 107.78\u0026thinsp;\u0026plusmn;\u0026thinsp;31.92 mL/min/1.73m\u0026sup2;), confirming the significant efficacy advantages of monotherapy across all age groups of membranous nephropathy patients.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study analyzed 98 patients with pMN following 12 months of follow-up, with the core finding that RTX monotherapy exhibited significantly superior clinical overall response rate (84.0%) and renal function protection compared to doublet (46.7%) and triplet (50.0%) combination regimens. The eGFR in the monotherapy group showed a sustained and stable increasing trend, reaching 101.68\u0026thinsp;\u0026plusmn;\u0026thinsp;17.4 mL/min/1.73m\u0026sup2; at 1 year. In contrast, the triplet therapy group demonstrated potential long-term nephrotoxic risks, with eGFR declining to 71.79\u0026thinsp;\u0026plusmn;\u0026thinsp;27.57 mL/min/1.73m\u0026sup2; at 1 year, which was below baseline levels. The doublet therapy group, meanwhile, showed limited improvement in renal function accompanied by significant individual variability.\u003c/p\u003e \u003cp\u003eThe core reasons for the optimal long-term outcomes of RTX monotherapy can be attributed to three aspects. Firstly, RTX specifically targets CD20 to selectively eliminate pathogenic B cells, exerting a durable immunomodulatory effect by blocking the source of antibody production and avoiding the non-specific interference of combination therapy on the immune microenvironment. This is consistent with the superior B-cell depletion efficiency (57.30%) observed in this study[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Secondly, monotherapy avoids the metabolic disorders and infection risks associated with glucocorticoids, as well as the direct nephrotoxicity of CNIs, reducing secondary renal damage caused by drug side effects. It can even improve renal function in patients with poor baseline renal function and comorbidities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thirdly, RTX monotherapy is associated with relatively mild adverse reactions, leading to better patient tolerance and treatment adherence. This is evidenced by the stability of urinary protein reduction and serum albumin recovery with a small standard deviation, laying a foundation for long-term efficacy[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe combination regimens exhibited a pattern of high initial response followed by declining efficacy, which is supported by clear clinical rationale. The doublet therapy showed a significant short-term effect, with urinary protein dropping sharply to 0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01g at 3 months, but it began to rebound after 6 months, reaching 3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;4.91g at 1 year, with a long-term remission rate of less than 50%. This may be due to patients' inability to sustain treatment tolerance due to side effects of glucocorticoids or CNIs, or the interference of combination therapy with the immunomodulatory process of RTX [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. More notably, despite the small sample size of the triplet therapy group, the finding that eGFR was lower than baseline at 1 year is of important warning significance. This is closely related to the chronic nephrotoxicity of CNIs. Excessive immunosuppression not only fails to enhance efficacy but also disrupts the homeostasis of the renal microenvironment and increases the risk of renal function damage, indicating that unnecessary intensive triplet therapy should be strictly avoided in clinical practice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAn intriguing finding was the dissociation between B-cell depletion rate and clinical outcome. Although triplet therapy achieved the highest B-cell depletion rate (71.40%), it correlated with inferior renal function preservation. In contrast, monotherapy, with the lowest depletion rate (37.00%), demonstrated the highest B-cell clearance rate (57.30%) and the best clinical outcomes.​ This suggests that the efficacy of RTX in pMN may not solely depend on the depth of B-cell reduction, but rather on the quality of immune reconstitution and the efficient clearance of pathogenic B-cell clones, a process that might be disrupted by concurrent non-specific immunosuppressants. The overall homeostasis of the immune environment, the precise clearance efficiency of pathogenic B-cell subsets, and drug safety may play more critical roles. Although combination therapy can enhance B-cell depletion, it may simultaneously disrupt the balanced regulation of the immune system, and the risk of treatment interruption caused by side effects further offsets potential benefits, ultimately leading to poor clinical outcomes[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on the results of this study, we put forward clear clinical practice recommendations: RTX monotherapy should be the preferred initial treatment for most pMN patients, especially for anti-PLA2R antibody-positive patients (overall remission rate of 80.80% with monotherapy) and elderly patients (\u0026gt;\u0026thinsp;60 years old, overall remission rate of 80.0% with monotherapy), who derive more significant benefits from monotherapy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Combination therapy should be strictly limited to specific scenarios, such as critically ill patients requiring extremely rapid control of severe nephrotic syndrome (e.g., massive proteinuria with severe hypoalbuminemia). During treatment, close monitoring of renal function, infection, and metabolic indicators is necessary. Once the condition stabilizes, timely evaluation should be conducted to determine whether conversion to monotherapy maintenance is feasible, avoiding the cumulative risks of long-term combination therapy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study has certain limitations. Firstly, as a single-center retrospective cohort study, the treatment regimens were selected by clinicians based on patient conditions, inevitably leading to potential selection bias. Although the baseline data were well-balanced, confounding factors cannot be completely excluded. Secondly, the sample sizes of each group were uneven, especially the triplet therapy group and the antibody-negative subgroup, which may affect the statistical power of the results, and the relevant conclusions should be interpreted with caution. Finally, the follow-up period was 1 year, failing to evaluate the long-term efficacy stability, disease recurrence rate, and the risk of end-stage renal disease progression. Future multi-center, large-sample prospective randomized controlled trials with extended follow-up are needed to further verify the long-term differences between monotherapy and combination therapy, and to explore individualized treatment strategies based on B-cell depletion efficiency and dynamic changes in antibody titers, providing more solid evidence-based medical evidence for the precise treatment of pMN [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, this real-world study demonstrates that RTX monotherapy achieves a superior overall response rate​ and better renal function protection compared to combination immunosuppressive regimens over a 1-year period in patients with pMN. This advantage is particularly evident in elderly patients and those with low anti-PLA2R antibody titers. Therefore, RTX monotherapy should be regarded as an efficient and safe preferred first-line treatment strategy for the majority of pMN patients, while the routine use of combination therapies involving glucocorticoids and/or CNIs requires critical re-evaluation regarding their appropriate target population.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRTX, Rituximab\u003c/p\u003e\n\u003cp\u003eTAC,Tacrolimus\u0026nbsp;\u003c/p\u003e\n\u003cp\u003epMN, primary membranous nephropathy\u003c/p\u003e\n\u003cp\u003eMN, membranous nephropathy\u003c/p\u003e\n\u003cp\u003ePLA2R, phospholipase A2 receptor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTHSD7A, thrombospondin type-1 domain-containing 7A\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNELL1, neural epidermal growth factor-like 1\u003c/p\u003e\n\u003cp\u003ePCDH7, protocadherin 7\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCNIs, calcineurin inhibitors\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eeGFR, estimated glomerular filtration rate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e24h UTP, 24-hour urinary protein excretion\u003c/p\u003e\n\u003cp\u003eScr, serum creatinine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlb, serum albumin\u003c/p\u003e\n\u003cp\u003eCR, complete remission\u003c/p\u003e\n\u003cp\u003ePR, partial remission\u003c/p\u003e\n\u003cp\u003eOR, overall response\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Supplementary Material for this article can be found online at: XX\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe requirement for individual patient informed consent was waived for both participation and publication because this retrospective study utilized only fully anonymized data, posing no risk to patient privacy. The datasets generated and analyzed during the current study are not publicly available due to hospital policy and patient privacy regulations. However, the de-identified data supporting the main findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWei Xian (Xian W) provided the concept and designed the study. Ji Dong (Ji D) and Mingrui Fan performed the experiments, analyzed the data, and wrote the manuscript. Lipu Shi contributed to the data curation. Han Li and Jianping Si participated in the experimental verification and result discussion. All authors discussed the results and revised the manuscript critically for important intellectual content..\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Henan Provincial People\u0026apos;s Hospital (Approval Number: (2021) Ethics Review No. (78)). The ethics committee waived the requirement for informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Clinical Medicine, Henan Medical College, Zhengzhou 451191, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Public Health and Health Management, Henan Medical College, Zhengzhou 451191, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Department of Nephrology, Henan Provincial People\u0026apos;s Hospital, Zhengzhou 450000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eDepartment of Immunology,\u0026nbsp;Henan Provincial People\u0026apos;s Hospital, Zhengzhou 450000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u0026nbsp;\u003c/sup\u003eHenan University of Chinese Medicine, Zhengzhou 450000, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eDepartment of Clinical Laboratory, Henan Second Provincial People\u0026rsquo;s Hospital, Zhengzhou 450000, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBeck LH Jr, Bonegio RGB, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med. 2009;361:11\u0026ndash;21. https://doi.org/10.1056/NEJMoa0810457.\u003c/li\u003e\n\u003cli\u003eRonco P, Debiec H. Molecular pathogenesis of membranous nephropathy. Annu Rev Pathol. 2020;15:287\u0026ndash;313. https://doi.org/10.1146/annurev-pathol-020117-043811.\u003c/li\u003e\n\u003cli\u003eSethi S, Madden B, Casal Moura M, Nasr SH, Klomjit N, Gross L, et al. Hematopoietic stem cell transplant-membranous nephropathy is associated with protocadherin FAT1. J Am Soc Nephrol. 2022;33:1033\u0026ndash;44. https://doi.org/10.1681/ASN.2021111488.\u003c/li\u003e\n\u003cli\u003eTomas NM, Beck LH Jr, Meyer-Schwesinger C, Seitz-Polski B, Ma H, Zahner G, et al. Thrombospondin type-1 domain-containing 7A in idiopathic membranous nephropathy. N Engl J Med. 2014;371:2277\u0026ndash;87. https://doi.org/10.1056/NEJMoa1409354.\u003c/li\u003e\n\u003cli\u003eSethi S, Beck LH Jr, Glassock RJ, Haas M, De Vriese AS, Caza TN, et al. Mayo Clinic consensus report on membranous nephropathy: Proposal for a novel classification. Mayo Clin Proc. 2023;98:1671\u0026ndash;84. https://doi.org/10.1016/j.mayocp.2023.08.006.\u003c/li\u003e\n\u003cli\u003ePonticelli C. Membranous nephropathy. J Clin Med. 2025;14:761. https://doi.org/10.3390/jcm14030761.\u003c/li\u003e\n\u003cli\u003ePonticelli C, Zucchelli P, Imbasciati E, Cagnoli L, Pozzi C, Passerini P, et al. Controlled trial of methylprednisolone and chlorambucil in idiopathic membranous nephropathy. N Engl J Med. 1984;310:946\u0026ndash;50. https://doi.org/10.1056/NEJM198404123101503.\u003c/li\u003e\n\u003cli\u003eJha V, Ganguli A, Saha TK, Kohli HS, Sud K, Gupta KL, et al. A randomized, controlled trial of steroids and cyclophosphamide in adults with nephrotic syndrome caused by idiopathic membranous nephropathy. J Am Soc Nephrol. 2007;18:1899\u0026ndash;904. https://doi.org/10.1681/ASN.2007020166.\u003c/li\u003e\n\u003cli\u003ePraga M, Barrio V, Ju\u0026aacute;rez GF, Lu\u0026ntilde;o J, Grupo Espa\u0026ntilde;ol de Estudio de la Nefropat\u0026iacute;a Membranosa. Tacrolimus monotherapy in membranous nephropathy: a randomized controlled trial. Kidney Int. 2007;71:924\u0026ndash;30. https://doi.org/10.1038/sj.ki.5002215.\u003c/li\u003e\n\u003cli\u003eFervenza FC, Appel GB, Barbour SJ, Rovin BH, Lafayette RA, Aslam N, et al. Rituximab or cyclosporine in the treatment of membranous nephropathy. N Engl J Med. 2019;381:36\u0026ndash;46. https://doi.org/10.1056/NEJMoa1814427.\u003c/li\u003e\n\u003cli\u003eScolari F, Delbarba E, Santoro D, Gesualdo L, Pani A, Dallera N, et al. Rituximab or cyclophosphamide in the treatment of membranous nephropathy: The RI-CYCLO randomized trial. J Am Soc Nephrol. 2021;32:972\u0026ndash;82. https://doi.org/10.1681/ASN.2020071091.\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez-Ju\u0026aacute;rez G, Rojas-Rivera J, Logt A-E van de, Justino J, Sevillano A, Caravaca-Font\u0026aacute;n F, et al. The STARMEN trial indicates that alternating treatment with corticosteroids and cyclophosphamide is superior to sequential treatment with tacrolimus and rituximab in primary membranous nephropathy. Kidney Int. 2021;99:986\u0026ndash;98. https://doi.org/10.1016/j.kint.2020.10.014.\u003c/li\u003e\n\u003cli\u003eGauckler P, Shin JI, Alberici F, Audard V, Bruchfeld A, Busch M, et al. Rituximab in membranous nephropathy. Kidney Int Rep. 2021;6:881\u0026ndash;93. https://doi.org/10.1016/j.ekir.2020.12.035.\u003c/li\u003e\n\u003cli\u003eAndeen NK, Kung VL, Avasare RS, Barbour S, Griffith M, Bissonnette MLZ, et al. Questions and caveats in antigen-defined membranous nephropathy. J Am Soc Nephrol. 2025;36:1639\u0026ndash;51. https://doi.org/10.1681/ASN.0000000769.\u003c/li\u003e\n\u003cli\u003eKattah AG, Fervenza FC. Rituximab: emerging treatment strategies of immune-mediated glomerular disease. Expert Rev Clin Immunol. 2012;8:413\u0026ndash;21. https://doi.org/10.1586/eci.12.26.\u003c/li\u003e\n\u003cli\u003eRosenzwajg M, Languille E, Debiec H, Hygino J, Dahan K, Simon T, et al. B- and T-cell subpopulations in patients with severe idiopathic membranous nephropathy may predict an early response to rituximab. Kidney Int. 2017;92:227\u0026ndash;37. https://doi.org/10.1016/j.kint.2017.01.012.\u003c/li\u003e\n\u003cli\u003eDahan K, Debiec H, Plaisier E, Cachanado M, Rousseau A, Wakselman L, et al. Rituximab for severe membranous nephropathy: A 6-month trial with extended follow-up. J Am Soc Nephrol. 2017;28:348\u0026ndash;58. https://doi.org/10.1681/ASN.2016040449.\u003c/li\u003e\n\u003cli\u003eFervenza FC, Appel GB, Barbour SJ, Rovin BH, Lafayette RA, Aslam N, et al. Rituximab or cyclosporine in the treatment of membranous nephropathy. N Engl J Med. 2019;381:36\u0026ndash;46. https://doi.org/10.1056/NEJMoa1814427.\u003c/li\u003e\n\u003cli\u003eSeitz-Polski B, Dahan K, Debiec H, Rousseau A, Andreani M, Zaghrini C, et al. High-dose rituximab and early remission in PLA2R1-related Membranous Nephropathy. Clin J Am Soc Nephrol. 2019;14:1173\u0026ndash;82. https://doi.org/10.2215/CJN.11791018.\u003c/li\u003e\n\u003cli\u003eRuggenenti P, Cravedi P, Chianca A, Perna A, Ruggiero B, Gaspari F, et al. Rituximab in idiopathic membranous nephropathy. J Am Soc Nephrol. 2012;23:1416\u0026ndash;25. https://doi.org/10.1681/ASN.2012020181.\u003c/li\u003e\n\u003cli\u003ePraga M, Barrio V, Ju\u0026aacute;rez GF, Lu\u0026ntilde;o J, Grupo Espa\u0026ntilde;ol de Estudio de la Nefropat\u0026iacute;a Membranosa. Tacrolimus monotherapy in membranous nephropathy: a randomized controlled trial. Kidney Int. 2007;71:924\u0026ndash;30. https://doi.org/10.1038/sj.ki.5002215.\u003c/li\u003e\n\u003cli\u003eQiu TT, Zhang C, Zhao HW. Calcineurin Inhibitors Versus Cyclophosphamide for Idiopathic Membranous Nephropathy: A Systematic Review and Meta-Analysis of 21 Clinical Trials. Autoimmun Rev. 2017;16:136\u0026ndash;45. https://doi.org/10.1016/j.autrev.2016.10.009.\u003c/li\u003e\n\u003cli\u003eNaesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol. 2009;4:481\u0026ndash;508. https://doi.org/10.2215/CJN.04800908.\u003c/li\u003e\n\u003cli\u003eZou P, Li H, Cai J. Therapy of Rituximab in Idiopathic Membranous Nephropathy With Nephrotic Syndrome: A Systematic Review and Meta-Analysis. Chin Med Sci J. 2018;33:9\u0026ndash;19. https://doi.org/10.4236/cmsj.2018.331002.\u003c/li\u003e\n\u003cli\u003eRovin BH, Adler SG, Barratt J. Executive Summary of the KDIGO 2021 Guideline for the Management of Glomerular Diseases. Kidney Int. 2021;100:753\u0026ndash;79. https://doi.org/10.1016/j.kint.2021.05.01.\u003c/li\u003e\n\u003cli\u003eBeck LH Jr, Bonegio RGB, Lambeau G, Beck DM, Powell DW, Cummins TD, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med. 2009;361:11\u0026ndash;21. https://doi.org/10.1056/NEJMoa0810457.\u003c/li\u003e\n\u003cli\u003eRojas-Rivera JE, Carriazo S, Ortiz A. Treatment of idiopathic membranous nephropathy in adults: KDIGO 2012, cyclophosphamide and cyclosporine A are out, rituximab is the new normal. Clin Kidney J. 2019 Sep 30;12(5):629-638. doi: 10.1093/ckj/sfz127.\u003c/li\u003e\n\u003cli\u003eFord I, Norrie J. Pragmatic Trials. N Engl J Med. 2016;375:454\u0026ndash;63. https://doi.org/10.1056/NEJMp1601564.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Primary membranous nephropathy, Rituximab, Tacrolimus, Glucocorticoids, Anti-PLA2R, B cell depletion","lastPublishedDoi":"10.21203/rs.3.rs-8713007/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8713007/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRituximab (RTX) has been established as a first-line treatment for primary membranous nephropathy (pMN). However, the efficacy difference between RTX monotherapy and combined regimens incorporating additional immunosuppressants (e.g., glucocorticoids or calcineurin inhibitors) remains unclear, particularly in patient subgroups with distinct clinical and immunological characteristics. Therefore, clarification of individualized treatment strategies is urgently needed.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 98 pMN patients receiving RTX-based treatment were enrolled and divided into three groups: monotherapy (RTX), doublet therapy (RTX in combination with tacrolimus), and triplet therapy (RTX, tacrolimus, and glucocorticoids concurrently). We compared the 12-month total remission rate, changes in renal function, proteinuria levels, and B cell depletion across groups, with subgroup analyses stratified by age and anti-PLA2R antibody titer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe monotherapy group achieved a significantly higher total remission rate than the doublet and triplet therapy groups. Despite poorer baseline renal function in the combined treatment groups, their improvements in renal function and proteinuria reduction at 12 months were inferior to those of the monotherapy group. Subgroup analyses revealed that monotherapy advantages were particularly prominent in patients with anti-PLA2R antibody titer\u0026thinsp;\u0026le;\u0026thinsp;200 RU/mL and those aged\u0026thinsp;\u0026gt;\u0026thinsp;60 years. Additionally, the monotherapy group exhibited unique kinetic characteristics of B cell depletion.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis real-world study demonstrates that RTX monotherapy provides superior clinical remission and renal protection compared to combined regimens, challenging the necessity of routine combined immunosuppressive treatment. The significant benefits observed in elderly patients and those with low anti-PLA2R antibody titers support RTX monotherapy as the preferred initial treatment strategy for most pMN patients.\u003c/p\u003e","manuscriptTitle":"Comparison of the efficacy of rituximab monotherapy and combined immunotherapy for primary membranous nephropathy: a real-world cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 17:38:32","doi":"10.21203/rs.3.rs-8713007/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"655bed80-e784-4680-b416-6823a74afc61","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-11T09:15:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T05:23:42+00:00","index":280,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T16:39:03+00:00","index":279,"fulltext":""},{"type":"reviewerAgreed","content":"203467622202878948045809021369795494231","date":"2026-05-01T09:28:13+00:00","index":276,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T21:07:49+00:00","index":275,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T09:31:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 17:38:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8713007","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8713007","identity":"rs-8713007","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}