Abstract
Neural epidermal growth factor-like protein 1 (NELL-1) and the exostosin 1/2 complex (EXT1/2) are recently identified target antigens in membranous nephropathy (MN), yet their prevalence and clinical features in Chinese populations remain poorly characterized. This study enrolled 197 consecutive patients with biopsy-proven MN, including 186 with idiopathic membranous nephropathy (IMN) and 11 with secondary membranous nephropathy (SMN). Serum levels of NELL-1 and EXT1/2 antigens were detected via enzyme-linked immunosorbent assay, with pre-biopsy baseline and at least one-year follow-up data collected for analysis. Among the participants, 152 were anti-phospholipase A2 receptor antibody-positive, while 8.6% (17/197) were positive for NELL-1 antigen and 6.5% (13/197) for EXT1/2 antigen. Following biopsy, 95.4% (188/197) of patients received treatment: 61.9% (122/188) underwent immunosuppressive therapy, and the remainder received RAAS-blockade alone. IgG subclass distributions varied significantly, with IgG4 predominating in 93.4% (142/152) of anti-PLA2R antibody-positive cases, whereas IgG1 was dominant in 58.9% (10/17) of NELL-1 antigen-positive and 84.6% (11/13) of EXT1/2 antigen-positive cases. After a median follow-up of 13.0 months, 67.5% (133/197) achieved proteinuria remission. In conclusion, NELL-1 and EXT1/2 antigens were detected in 8.63% and 6.60% of patients, respectively; however, antigen status did not predict treatment response or clinical outcomes such as proteinuria remission.
Keywords: Idiopathic membranous nephropathy, membranous lupus nephritis, undetermined atpical membranous nephropathy, neural epidermal growth factor-like 1 protein, exostosin 1/2 complex
Introduction
Membranous nephropathy (MN) is characterized by the presence of antigen–antibody complexes in the glomerular basement membrane (GBM), which result in complement activation, podocyte injury, and thickening and remodeling of the GBM. Previously, MN was classified as primary if there were no associated conditions and as secondary if there was a systemic disease (such as autoimmune disease, infections), drugs, or malignancy. Over the past 20 years, our understanding of the pathophysiology of MN has significantly advanced with the discovery of new target antigens expressed in podocytes and GBM, as well as the detection of their associated circulating antibodies that are correlated with disease activity. A novel classification method has been proposed [1]. In this classification, the antigen associated with MN is first detected by serology, immunofluorescence, immunohistochemistry and/or mass spectrometry. Based on the identified target antigen, patients should undergo clinical evaluation to determine the presence of a systemic disease related to the antigen.
In 2009, Beck et al. [2] discovered that phospholipase A2 receptor (PLA2R) colocalized with immunoglobulin G 4 (IgG4) in kidney tissues of adult patients with primary MN. Serum anti-PLA2R antibodies have currently been used for the diagnosis of primary MN and prediction of treatment efficacy [3–5].
In 2019, the neural epidermal growth factor-like protein 1 (NELL-1) was identified as a target antigen for MN and seems to be the second most common antigen in MN, accounting for approximately 10% of MN cases [6]. Anti-NELL-1 antibodies, primarily of the IgG1 subclass, can be detected in the serum [7]. Several currently known etiologies related to NELL1-associated MN include malignancies [6,8], use of alpha-lipoic acid [9,10] and mercury exposure due to the use of indigenous medicines from India [11]. However, no definite associated diseases were identified in most cases. Exostosin 1/2 complex (EXT1/2) were also identified in 2019 as potential antigens in MN associated with autoimmune diseases, such as lupus. In a cohort of 374 biopsy-proven membranous lupus nephritis (MLN) cases, 122 (32.6%) were EXT1/2-positive and 252 (67.4%) were EXT1/2-negative [12]. It is considered that EXT1/2-associated MN is related to a favorable clinical prognosis and relatively mild degree of chronic lesions on kidney biopsy [12,13].
Although PLA2R is the predominant antigen in primary MN, the detection of NELL-1 antigen and EXT1/2 antigen serves crucial diagnostic purposes. These biomarkers help identify PLA2R-negative cases requiring alternative management approaches, reveal potential underlying malignancies (particularly with NELL-1 antigen positivity) or autoimmune disorders, and prevent misclassification of secondary forms as primary MN when these antigens are detected.
Notably, the clinicopathological information for most novel antigens presently stems from a relatively limited patient cohort. Incorporating the benefits of progress can be aided by proper measures of skepticism. This limitation prompted us to evaluate the prevalence of these antigens in our MN cohort derived from a kidney biopsy database and correlate these findings with clinical outcomes. Between January 2000 and December 2019, 4,550 kidney biopsies were performed in our clinic. MN, the second most common type of primary glomerular disease after IgA nephropathy (39.9%), accounted for 32.6% of cases and served as the leading cause of nephrotic syndrome and proteinuria [14]. We established a clinicopathologic database based on the kidney biopsies performed in August 2015.
We conducted a retrospective cohort study to investigate the clinicopathological characteristics, follow-up data, treatment strategies, and outcomes in patients with MN based on the specific types of three positive antigens: PLA2R, NELL-1 and EXT1/2.
Materials and methods
Study patients and follow-up cohort
This retrospective single-center cohort study included all consecutive patients who underwent kidney biopsy and were diagnosed with MN at the nephrology department of the Second Hospital of Shanxi Medical University from January 2019 to December 2022. The pathological specimens were evaluated by the kidney pathology center of Shanxi Province. Based on the results of kidney biopsies and the clinical characteristics of the included patients, the clinical diagnoses of MN were classified as idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy (SMN), with SMN further including MLN and undetermined atypical membranous nephropathy (UAMN). Data on clinical diagnoses were extracted from medical records coded according to the International Classification of Diseases 10th edition (ICD-10).
A web-based database of the kidney biopsy cohort was established in August 2015 (http://tdiga.edc-china.com.cn). The method of data collection and patient follow-up has been previously reported [14,15]. Briefly, data were gathered prospectively, periodically controlled, and entered into an electronic database. The study nurses were responsible for providing follow-up reminders (every 1–3 months) to the patients. The clinical and pathological information of patients with MN at the time of biopsy (index day) and during follow-up were used in this analysis. The final date for data collection was from December 20 to 31, 2023, during which all enrolled patients were subjected to follow-up and outcome confirmation, either on-site or through telephone. We included only patients who had complete serum samples and at least 6 months of follow-up data. On the day of kidney biopsy or the day before, blood samples were routinely collected, centrifuged, and then frozen at −80 °C for storage.
In accordance with the current Kidney Disease: Improving Global Outcomes (KDIGO) guidelines [16], MN is classified into four risk groups—low-risk, moderate-risk, high-risk, and very high-risk, based on 24-h urinary total protein (24h-UTP) excretion and estimate glomerular filtration rate (eGFR). Patients in the low-risk group may undergo follow-up observation or be treated with reninangiotensin-aldosterone system (RAAS)-blockade. For those in the moderate-risk, high-risk, and very high-risk groups, treatment with corticosteroids and/or immunosuppressive agents/biologic agents is chosen based on the judgment of clinicians. Detailed descriptions of the treatment regimen for corticosteroids and immunosuppressive in MN are provided in Item S1.
The kidney biopsy cohort was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (No. 2015-KY-006). The study was conducted in accordance with the Declaration of Helsinki and all patients provided written informed consent.
Detection of three target biomarkers
Since January 2019, serum anti-PLA2R antibody testing has been implemented as a standard diagnostic procedure for all patients primarily presenting with proteinuria. Therefore, data on anti-PLA2R antibodies were retrospectively collected from medical records. The detection method used was an enzyme-linked immunosorbent assay (ELISA, Euroimmun). ELISA findings were interpreted as follows: <14RU/ml: negative; 14–20 RU/ml: borderline; and >20RU/ml: positive [17]. The detection of NELL-1 and EXT1/2 antigen was performed using blood samples frozen prior to the patient’s kidney biopsies, as mentioned above. ELISA kits (J&L Biological, China) were used in accordance utilized with the manufacturer’s instructions. ELISA findings for NELL-1antigen were interpreted as follows: >1.25 ng/ml: positive; and ELISA findings for anti-EXT1/2 antigen were interpreted as follows: >1ng/ml: positive. Detailed experimental procedures and supporting materials are provided in the Supplementary Materials (Item S2–S3, Tables S1 and S2).
Patients were grouped according to the type of the biomarkers in the serum and three clinical diagnoses: IMN, MLN, and UAMN.
Outcomes and definitions
The clinical outcome was the incidence of proteinuria remission, including complete remission (CR) or partial remission (PR), non-response, and relapse. CR was characterized by a decrease in 24h-UTP excretion to ≤0.3 g/day, along with normalized serum albumin levels and stable renal function. PR was defined as ≥ 50% reduction in the 24h-UTP ratio and a final excretion of ≤3.5 g/day. Non-response was defined as 24h-UTP remaining above 3.5 g/day throughout follow-up, with a urinary protein reduction of less than 50%. Relapse was defined as new development of the 24h-UTP >3.5 g/d after CR or PR. Remission of proteinuria was referred to as CR and PR. The glomerular filtration rate (GFR) was calculated using the CKD Epidemiology Collaboration (CKD-EPI) creatinine equation [18]. The mean arterial pressure (MAP) was determined by taking two-thirds of the diastolic blood pressure and adding one-third of the systolic blood pressure. All pathologic sections were reevaluated independently by one of two experienced nephropathologists to verify the previous diagnoses.
Statistical analysis
All the patients with a follow-up period of at least 12 months were included in the analyses. Continuous data were reported as mean ± standard deviation, or median (interquartile range [IQR]) as appropriate, and comparisons between two groups were made using the two-independent samples t-test. For comparisons among multiple groups, one-way ANOVA was employed, followed by the Kruskal-Wallis H test for pairwise comparisons when appropriate; otherwise, non-parametric rank sum tests were used. Categorical data are presented as number (percentage), and group comparisons were made using the chi-square test or Fisher’s exact test. Spearman correlation analysis was performed to assess the relationships between baseline serum NELL-1/EXT1/2 titers and changes in urine protein/serum albumin levels. Data analysis was performed using SPSS Statistics (version 26.0, IBM) and GraphPad Prism (version 9.5), and the significance level for all tests was set at p < 0.05 (two-tailed).
Results
Study cohort
From January 2019 to December 2022, 1651 consecutive patients underwent kidney biopsy at the Second Hospital of Shanxi Medical University. After screening based on the inclusion and exclusion criteria 197 patients were finally included in the study (Figure 1). Of the 197 patients, 152 (77.2%) were positive for anti-PLA2R antibodies, 17 (8.6%) for NELL-1 antigen, 13 (6.6%) for EXT1/2 antigen, and the remaining 15 patients (7.6%) were negative for all three biomarkers: anti-PLA2R antibody, NELL-1 antigen, and EXT1/2 antigen. Baseline clinical characteristics of all enrolled patients stratified by different types of biomarkers and etiological diagnoses are presented in Tables 1 and S3.
Figure 1.
Flowchart of patient selection.
EXT1/2, exostosin 1/2; IgAN, immunoglobulin A nephropathy; IMN, idiopathic membranous nephropathy; MCD, minimal change disease; MLN, membranous lupus nephritis; MN, membranous nephropathy; NELL-1, epidermal growth factor-like protein 1; PLA2R, phospholipase A2 receptor; SMN, secondary membranous nephropathy; UAMN, undetermined atpical membranous nephropathy.
Table 1.
Baseline clinical characteristics based on different types of positive antibodies.
| Clinical characteristics | Total (n = 197) | Anti-PLA2R (n = 152) | NELL-1 (n = 17) | EXT1/2 (n = 13) | Negative (n = 15) |
|---|---|---|---|---|---|
| Male sex | 122 (61.9) | 96 (63.2) | 10 (58.8) | 7 (53.8) | 9 (60.0) |
| Duration of illness, months | 3.0 (1.0,12.0) | 3.0 (1.0,12.0) | 3.0 (1.0,11.5) | 12.0 (2.5,18.0) | 3.0 (1.0,10.0) |
| Age, years | 49.0 (43.0,56.5) | 50 (43,56) | 48.0 (34.0,57.5) | 46.0 (34.0,59.0) | 46 (41,55) |
| Smoking history | 65 (33.0) | 52 (34.2) | 4 (23.5) | 9 (69.2) | 5 (33.3) |
| Drinking history | 42 (21.3) | 37 (24.3) | 2 (11.8) | 1 (7.7) | 2 (13.3) |
| Hepatitis B virus infection | 6 (3.05) | 0 | 4 (23.5) | 2 (15.4) | 0 |
| Proteinuria, g/24 h | 3.6 (2.6,5.1) | 3.7 (2.7,5.0) | 4.0 (3.0,6.0) | 3.4 (2.3,4.8) | 2.6 (1.7,5.8) |
| Albumin, g/L | 26.7 (22.7,30.9) | 26.3 (22.5,30.5) | 23.5 (19.0,31.9) | 30.7 (25.7,32.2) | 28.3 (24.2,32.7) |
| Scr, μmol/L | 67.0 (56.0,78.5) | 66.3 (56.0,77.0) | 62.0 (53.5,69.0) | 71.0 (60.0,88.0) | 74.0 (65.0,91.0) |
| Total cholesterol, mmol/L | 6.5 (5.4,8.1) | 6.60 (5.6,8.4) | 6.1 (5.1,7.1) | 6.1 (5.0,8.1) | 7.2 (5.8,8.1) |
| Uric acid, μmol/L | 362.0 (308.0,423.5) | 362.00 (307.5,413.8) | 342.0 (293.0,419.5) | 382.0 (291.0,464.0) | 439.0 (331.0,480.0) |
| Urea, mmol/L | 5.1 (4.0,6.2) | 5.10 (4.2,6.2) | 4.6 (3.4,6.9) | 4.4 (3.7,5.2) | 5.1 (4.1,6.5) |
| eGFR, mL/min/1.73 m2 | 106.2 (94.6,119.3) | 106.9 (96.2,120.5) | 106.2 (99.2,118.0) | 93.8 (76.1,115.7) | 99.1 (67.9,111.7) |
| C3, g/L | 1.1 (0.9,1.2) | 1.1 (0.9,1.3) | 0.9 (0.8,1.1) | 1.2 (0.9,1.3) | 0.9 (0.6,1.2) |
| C4, g/L | 0.3 (0.2,0.3) | 0.2 (0.2,0.3) | 0.3 (0.2,0.4) | 0.27 (0.2,0.3) | 0.2 (0.1,0.3) |
| Systolic BP, mmHg | 130 (122,143) | 130 (122,143) | 129 (121,143) | 128 (118,139) | 130 (114,155) |
| Diastolic BP, mmHg | 82 (73,89) | 81 (74,89) | 79 (70,90) | 86 (78,93) | 85 (70,96) |
| MAP, mmHg | 97.0 (90.0,106.0) | 97.5 (91.3,105.9) | 95.3 (88.3,104.3) | 99.3 (91.3,105.7) | 98.0 (88.0,117.0) |
| BMI, Kg/m2 | 25.0 (22.9,27.9) | 25.3 (23.3,28.0) | 24.3 (21.4,26.0) | 25.6 (24.3,29.1) | 22.6 (21.5,25.4) |
| Concomitant disease | |||||
| None | 102 (51.8) | 80 (52.63) | 13 (76.5) | 8 (61.5) | 11 (73.3) |
| Hypertension | 71 (36.0) | 61 (40.1) | 4 (23.5) | 3 (23.1) | 3 (20.0) |
| Diabetes | 22 (11.2) | 19 (12.5) | 0 | 2 (15.4) | 1 (6.7) |
| Pathological Pattern | |||||
| IMN | |||||
| Stage I ∼ II MN | 168 (85.3) | 136 (89.5) | 14 (82.3) | 12 (92.3) | 10 (66.7) |
| Stage III MN | 17 (8.6) | 11 (7.2) | 1 (5.9) | 0 | 1 (6.7) |
| Stage IV MN | 1 (0.5) | 0 | 1 (5.9) | 0 | 0 |
| MLN | 4 (2.0) | 1 (0.7) | 0 | 1 (7.7) | 2 (13.3) |
| UAMN | 7 (3.6) | 4 (2.6) | 1 (5.9) | 0 | 2 (13.3) |
| Use anticoagulants | 46 (23.4) | 39 (25.7) | 3 (17.6) | 2 (15.4) | 2 (13.3) |
| Enoxaparin | 21 (10.7) | 15 (9.9) | 2 (11.8) | 2 (15.4) | 2 (13.3) |
| LMWH | 25 (12.7) | 24 (15.8) | 1 (5.9) | 0 | 0 |
Values for categorical variables are presented as number (percentage); values for continuous variables presented as mean ± standard deviation or median (interquartile range).
BMI, body mass index; BP, blood pressure; EXT1/2, exostosin 1/2; eGFR, estimated glomerular fltration rate; LMWH, low molecular weight heparin; MAP, mean arterial pressure; MLN, membranous lupus nephritis; MN, membranous nephropathy; NELL-1, epidermal growth factor-like protein 1; PLA2R, phospholipase A2 receptor; UAMN, undetermined atpical membranous nephropathy.
The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.
Table 3.
Comparative analysis of baseline clinical features across different biomarker-positive groups.
| Clinical characteristics | Anti-PLA2R (n = 152) | NELL-1 (n = 17) | EXT1/2 (n = 13) | p Value |
|---|---|---|---|---|
| Age, years | 50.0 (43.0,56.0) | 48.0 (34.0,57.5) | 46.0 (34.0,59.0) | 0.738 |
| Hepatitis B virus infection | 0 | 4 (23.5) | 2 (15.4) | <0.001 |
| Proteinuria, g/24 h | 3.7 (2.7,5.0) | 4.0 (3.0,6.0) | 3.4 (2.3,4.8) | 0.522 |
| Albumin, g/L | 26.3 (22.5,30.5) | 23.5 (19.0,31.9) | 30.7 (25.7,32.2) | 0.091 |
| Scr, μmol/L | 66.3 (56.0,77.0) | 62.0 (53.5,69.0) | 71.0 (60.0,88.0) | 0.184 |
| eGFR, mL/min/1.73 m2 | 106.9 (96.2,120.5) | 106.2 (99.2,118.0) | 93.8 (76.1,115.7) | 0.147 |
| Hypertension | 61 (40.1) | 4 (23.5) | 3 (23.1) | 0.277 |
| BMI, Kg/m2 | 25.3 (23.3,28.0) | 24.3 (21.4,26.0) | 25.6 (24.3,29.1) | 0.131 |
| Outcomes | 0.673 | |||
| CR | 53 (34.9) | 8 (47.1) | 6 (46.1) | |
| PR | 49 (32.2) | 3 (17.6) | 3 (23.1) | |
| Non-response | 38 (25.0) | 6 (35.3) | 3 (23.1) | |
| Relapse after remission | 12 (7.9) | 0 | 1 (7.7) |
CR, complete remission; EXT1/2, exostosin 1/2; NELL-1, epidermal growth factor-like protein 1; PLA2R, phospholipase A2 receptor; PR, partial remission.
Of the 197 patients, 122 were males and the median age was 49.0 years. The median 24h-UTP excretion at baseline was 3.6 g/24h, and the median eGFR level was 106.2 mL/min/1.73m2. Seventy-one (36.0%) patients had hypertension, 22 (11.2%) had diabetes, and 102 (51.8%) were free of any concomitant disease. Pathological examination revealed that 186 patients were diagnosed with IMN, accounting for 94.4% of the entire cohort. The others included four patients (2.0%) with V-type MLN and seven patients (3.6%) with UAMN. The staging of IMN was mainly stages 1 to 2, with a proportion of 85.3%. A total of 46 patients with nephrotic IMN received anticoagulant therapy, including enoxaparin and low-molecular-weight heparin (LMWH). Of these, 41 patients (89.1%) were treated for thrombosis prophylaxis, while the remaining 5 patients (10.9%) received therapeutic anticoagulation for established thromboembolism.
Therapy
According to the treatment guidelines for MN, enrolled patients were categorized into treatment and non-treatment groups. The treatment group was further divided into: RAAS-blockade therapy and immunosuppressive therapy (IST). The IST included corticosteroids and/or immunosuppressants and/or biologics. The immunosuppressants comprised cyclophosphamide (CYC), calcineurin inhibitors (CNIs) such as cyclosporine A and tacrolimus, hydroxychloroquine, Tripterygium glycosides, mycophenolate mofetil, and leflunomide, while the biologic agent used was rituximab (RTX).
Following kidney biopsy, 188/197 (95.4%) patients were enrolled in our treatment protocols (Table 2). The median follow-up duration is 13.0 (IQR, 12.0–18.0) months. Among them, 122/188 (61.9%) patients underwent IST, while the remaining 66/188 (33.5%) patients opted for treatment with RAAS-blockade alone. Focusing on exposure to IST, 110 (55.8%) patients were exposed to glucocorticosteroids (GC). Twenty percent of the patients (22/110) received oral GC alone, and another 80% received GC in combination with other IST. CNIs, RTX and CYC were the most frequently prescribed ISTs (23.4%, 11.7% and 22.3%, respectively). The treatments within the first six months after kidney biopsy are also presented in Table 2. During the follow-up period, 31 patients (15.8%) failed to achieve proteinuria remission with RAAS-blockade alone therapy. Of these, 8 patients (4.1%) were non-responsive to initial 6-month RAAS-blockade therapy and subsequently required conversion to IST.
Table 2.
The therapeutic regimen for the patient post-kidney biopsy.
| Total (n = 197) |
Anti-PLA2R (n = 152) |
NELL-1 (n = 17) |
EXT1/2 (n = 13) |
Negative (n = 15) |
||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Entire follow-up period | Follow-up in the first six months | Entire follow-up period | Follow-up in the first six months | Entire follow-up period | Follow-up in the first six months | Entire follow-up period | Follow-up in the first six months | Entire follow-up period | Follow-up in the first six months | |
| Treated | 188 (95.4) | 186 (94.4) | 146 (96.1) | 144 (94.7) | 16 (94.1) | 16 (94.1) | 13 (1.00) | 13 (1.00) | 13 (86.7) | 13 (86.7) |
| Any IST | 122 (61.9) | 111 (56.3) | 97 (63.8) | 88 (57.9) | 11 (64.7) | 11 (64.7) | 4 (30.8) | 3 (23.1) | 10 (66.7) | 9 (60.0) |
| Oral glucocorticoids | 110 (55.8) | 101 (51.3) | 87 (57.1) | 79 (52.0) | 10 (58.8) | 10 (58.8) | 4 (30.8) | 3 (23.1) | 9 (60.0) | 9 (60.0) |
| Calcineurin inhibitors | 46 (23.4) | 33 (16.8) | 39 (25.7) | 30 (19.7) | 1 (5.9) | 0 | 3 (23.1) | 1 (7.7) | 3 (20.0) | 2 (13.3) |
| Tacrolimus | 26 (13.2) | 18 (9.1) | 24 (15.8) | 17 (11.2) | 0 | 0 | 2 (15.4) | 1 (7.7) | 0 | 0 |
| Cyclosporine A | 20 (10.2) | 15 (7.6) | 15 (9.9) | 13 (8.6) | 1 (5.9) | 0 | 1 (7.7) | 0 | 3 (20.0) | 2 (13.3) |
| Cyclophosphamide | 44 (22.3) | 39 (19.8) | 38 (25.0) | 33 (21.7) | 3 (176) | 3 (17.6) | 1 (7.7) | 1 (7.7) | 2 (13.3) | 2 (13.3) |
| Oral administration | 31 (15.7) | 26 (13.2) | 28 (18.4) | 23 (15.1) | 2 (11.8) | 2 (11.8) | 1 (7.7) | 1 (7.7) | 0 | 0 |
| Intravenous administration | 13 (6.6) | 13 (6.6) | 10 (6.6) | 10 (6.6) | 1 (5.9) | 1 (5.9) | 0 | 0 | 2 (13.3) | 2 (13.3) |
| Rituximab | 23 (11.7) | 15 (7.6) | 21 (13.8) | 14 (9.2) | 1 (5.9) | 1 (5.9) | 1 (7.7) | 0 | 0 | 0 |
| Hydroxychloroquine | 9 (4.6) | 9 (4.6) | 4 (2.6) | 4 (2.6) | 1 (5.9) | 1 (5.9) | 1 (7.7) | 1 (7.7) | 3 (20.0) | 3 (20.0) |
| Tripterygium glycosides | 7 (3.6) | 5 (2.5) | 6 (3.9) | 4 (2.6) | 0 | 0 | 0 | 0 | 1 (6.7) | 1 (6.7) |
| Mycophenolate mofetil | 2 (1.0) | 1 (0.5) | 0 | 0 | 0 | 0 | 1 (7.7) | 1 (7.7) | 1 (6.7) | 0 |
| Leflunomide | 1 (0.5) | 1 (0.5) | 0 | 0 | 1 (5.9) | 1 (5.9) | 0 | 0 | 0 | 0 |
| RAAS-blockadea | 115 (58.4) | 112 (56.9) | 89 (58.6) | 86 (56.6) | 10 (58.8) | 10 (58.8) | 10 (76.9) | 10 (76.9) | 6 (40.0) | 6 (40.0) |
| Alone | 66 (33.5) | 74 (37.60) | 49 (32.2) | 56 (36.8) | 5 (29.4) | 5 (29.4) | 9 (69.2) | 10 (76.9) | 3 (20.0) | 4 (26.7) |
| Combination with IST | 49 (24.9) | 38 (19.3) | 40 (26.3) | 30 (19.7) | 5 (29.4) | 5 (29.4) | 1 (7.7) | 0 | 3 (20.0) | 2 (13.3) |
| ACE-inhibitors | 35 (17.8) | 34 (17.3) | 29 (19.1) | 28 (18.4) | 4 (23.5) | 4 (23.5) | 1 (7.7) | 1 (7.7) | 1 (6.7) | 1 (6.7) |
| ARB | 81 (41.1) | 79 (40.1) | 61 (40.1) | 59 (38.8) | 6 (35.3) | 6 (35.3) | 9 (69.2) | 9 (69.2) | 5 (33.3) | 5 (33.3) |
| None | 9 (4.6) | 11 (5.6) | 6 (3.9) | 8 (5.3) | 1 (5.9) | 1 (5.9) | 0 | 0 | 2 (13.3) | 2 (13.3) |
Categorical variables were expressed as number (percentage).
ACE, angiotensin converting enzyme; ARB, angiotensin receptor blockers; EXT1/2, exostosin 1/2; MN, membranous nephropathy; NELL-1, epidermal growth factor-like protein 1; PLA2R, phospholipase A2 receptor; IST, immunosuppressive therapy; RAAS, renin-aldosterone-angiotensin system.
One anti-PLA2R-positive patient out of 197 used both ACE-inhibitors and ARBs throughout the entire treatment process.
Positive biomarkers and clinical outcomes
Among the 197 enrolled patients, 152 were anti-PLA2R antibody-positive, 17 were NELL-1 antigen-positive, and 13 were EXT1/2 antigen-positive (Tables S4 and S5, Figure S1, Item S4). We compared the baseline clinical characteristics and outcomes among the three groups, revealing significant differences in hepatitis B virus (HBV) infection rates (Table 3). Pathological examination further demonstrated prominent nodular mesangial sclerosis and arteriolar hyalinosis across all groups (Table 4).
Table 4.
Comparison of kidney pathological characteristics among different biomarker groups.
| Pathological characteristics | Anti-PLA2R (n = 152) | NELL-1 (n = 17) | EXT1/2 (n = 13) | p Value |
|---|---|---|---|---|
| IgG subclass | ||||
| IgG1 | 39 (25.7) | 10 (58.8) | 11 (84.6) | |
| IgG2 | 60 (39.5) | 6 (35.3) | 8 (61.5) | |
| IgG3 | 66 (43.4) | 5 (29.4) | 4 (30.8) | |
| IgG4 | 142 (93.4) | 7 (41.2) | 9 (69.2) | |
| Mesangial deposition of immune complexes | 17 (11.2) | 1 (5.9) | 3 (23.1) | 0.364 |
| Crescent | 4 (2.6) | 0 | 2 (15.4) | 0.093 |
| Nodular mesangial sclerosis present | 87 (57.2) | 3 (17.6) | 7 (53.8) | 0.008 |
| Arteriolar hyalinosis | 63 (41.4) | 3 (17.6) | 2 (15.4) | 0.033 |
| IFTAa | 0.460 | |||
| Absence | 126 (82.9) | 17 (100.0) | 10 (76.9) | |
| Mild | 21 (13.8) | 0 | 3 (23.1) | |
| Moderate | 3 (2.0) | 0 | 0 | |
| Diffuse | 2 (1.3) | 0 | 0 |
EXT1/2, exostosin 1/2; IgG, immunoglobulin G; NELL-1, epidermal growth factor-like protein 1; PLA2R, phospholipase A2 receptor.
Pathological examination further demonstrated prominent nodular mesangial sclerosis and arteriolar hyalinosis across all groups. In the manuscript, bold type indicates significant differences in nodular mesangial sclerosis present and arteriolar hyalinosis among the three groups stratified by positivity for different biomarkers.
IFTA was graded as absence, mild, moderate, and diffuse according to the percentage of glomeruli or cortical tubulointerstitial area involved (absence: ≤10%; mild: 10–30%; moderate: 31–60%; diffuse: >60%).
In the cohort of 17 NELL-1 antigen positive MN patients (Table S6), there were 7 women and 10 men, with a median age of 48 years (range: 23–63). IgG1 immunostaining was positive in 10 patients (58.8%), IgG4 in 7 patients (41.2%), IgG2 in 6 patients (35.3%) and IgG3 in 5 patients (29.4%). With a median follow-up time of 12.0 (IQR 12.0–21.0) months, 11/17 patients (64.7%) underwent IST, and 8 patients (47.1%) achieved CR or PR. None of the patients reported a relapse. We did not study the differences in the treatment efficacies between groups due to the small patient numbers.
Among the 13 patients with EXT1/2 antigen positivity, 11 (84.6%) were positive for IgG1, and 9 patients (69.2%) were positive IgG4. As shown in Table 1, the median 24h-UTP excretion at baseline was 3.4 g/24h, and the median eGFR was 93.8 mL/min/1.73m2. Only four (30.8%) were administered IST, and the remaining nine patients received RAAS-blockade therapy alone. After a median follow-up time of 21.0 (IQR 16.5–25.5) months, six (46.2%) patients achieved either CR or PR, while one patient experienced a relapse (Table S6). One female patient with EXT1/2 antigen positivity was diagnosed with MLN and failed to achieve clinical remission 15 months after IST.
Among the 197 patients included in this study, with a median follow-up time of 13.0 (IQR 12.0 –18.0) months, 133 (67.5%) individuals achieved clinical remission of proteinuria. Specifically, 75 (38.1%) patients achieved CR and 58 (29.4%) patients achieved PR. In contrast, 64 (32.5%) patients did not achieve clinical remission, including 51 (25.9%) with non-response and 13 (6.6%) with relapse (Table 5). The baseline clinical data of the remission and non-remission groups are presented in Table S7.
Table 5.
Clinical outcomes of patients stratified by specific target antigens and therapeutic regimens.
| Total (n = 197) | Anti-PLA2R (+) |
Anti-PLA2R (-) |
Other |
||||||
|---|---|---|---|---|---|---|---|---|---|
| Anti-NELL (+) |
Anti-EXT1/2 (+) |
||||||||
| Treateda (n = 97) | Untreatedb (n = 55) | Treated (n = 11) | Untreated (n = 6) | Treated (n = 4) | Untreated (n = 9) | Treated (n = 10) | Untreated (n = 5) | ||
| Follow-up time | 13.0 (12.0,18.0) | 12.0 (12.0,18.0) | 15.0 (12.0,21.0) | 15.0 (12.0,21.0) | 12.0 (12.0,18.8) | 19.5 (15.8,30.0) | 21.0 (16.5,25.5) | 12.5 (12.0,24.0) | 12.0 (12.0,22.5) |
| Outcomes | |||||||||
| CR | 75 (38.1) | 33 (34.0) | 20 (36.4) | 6 (54.5) | 2 (33.3) | 1 (25.0) | 5 (55.6) | 6 (60.0) | 2 (40.0) |
| PR | 58 (29.4) | 33 (34.0) | 16 (29.1) | 2 (18.2) | 1 (16.7) | 0 | 3 (33.3) | 1 (10.0) | 2 (40.0) |
| Non-response | 51 (25.9) | 20 (20.6) | 18 (32.7) | 3 (27.3) | 3 (50.0) | 2 (50.0) | 1 (11.1) | 3 (30.0) | 1 (20.0) |
| Relapse | 13 (6.6) | 11 (11.4) | 1 (1.8) | 0 | 0 | 1 (25.0) | 0 | 0 | 0 |
Values for categorical variables are given as number (percentage); values for continuous variables are given as median (interquartile range).
CR, complete remission; EXT1/2, exostosin 1/2; MN, membranous nephropathy; NELL-1, epidermal growth factor-like protein 1; PLA2R, phospholipase A2 receptor; PR, partial remission.
Treated group refers to patients on any one or several immunosuppressive agents (glucocorticoids, calcineurin inhibitors, cyclophosphamide, rituximab, hydroxychloroquine, tripterygium glycosides, mycophenolate mofetil, and leflunomide) as part of their treatment regimen during follow-up after kidney biopsy.
Untreated group was defined as patients who were not treated with immunosuppressive agents (glucocorticoids, calcineurin inhibitors, cyclophosphamide, rituximab, hydroxychloroquine, Tripterygium glycosides, mycophenolate mofetil, and leflunomide) during follow-up after kidney biopsy.
We assessed the correlation between baseline serum titers of NELL-1 and EXT1/2 and the longitudinal changes in urinary protein and serum albumin levels. The analysis revealed no significant correlations between the baseline antibody titers and changes in either proteinuria or serum albumin (Figures S2 and S3).
In the cohort with positive NELL-1 antigen and EXT1/2 antigen results, we did not find prominent and distinct clinical and pathological features.
Discussion
This retrospective single-center study included 197 patients with MN confirmed by kidney biopsy. We found that 152 (77.2%) patients were positive for anti-PLA2R antibody, 17 (8.6%) for NELL-1 antigen, 13 (6.6%) for EXT1/2 antigen, and the remaining 15 (7.6%) were negative for all three biomarkers. The results of this study are consistent with those of a study by Pierre Ronco [19], which found that NELL-1 is associated with approximately 8% of IMN cases, and a study by Sanjeev Sethi [1], which suggests that EXT1/2 may account for 5–10% of MN cases. First, after 13.0 (IQR 12.0 –18.0) months of follow-up, a total of 133 (67.5%) of 197 patients achieved clinical remission (including 75 (38.1%) CR and 58 (29.4%) PR), and 64 (32.5%) did not achieve remission (including 51 (25.9%) non-response and 13 (6.6%) relapsed after remission). Second, logistic regression revealed that a history of hypertension and 24h-UTP were independent risk factors that influenced whether patients had CR of proteinuria. Finally, in the specific analysis of NELL-1 antigen-positive and EXT1/2 antigen-positive patients, IgG1 was the major IgG subclass in the kidney biopsy tissue of NELL-1 antigen-positive patients and EXT1/2 antigen-positive patients. Among NELL-1 antigen-positive patients, the median follow-up was 12.0 (IQR 12.0–21.0) months, with 8 (47.1%) achieving CR or PR, and no patients relapsed. In EXT1/2 antigen-positive patients, the median follow-up was 21.0 (IQR 16.5–25.5) months, 6 (46.2%) achieved CR or PR, and 1 (7.7%) relapsed.
IMN is a common cause of nephrotic syndrome in adults, and approximately one-third of patients may experience spontaneous remission, while others may experience long-term kidney deterioration. The conventional approach to IMN initially involves standalone supportive treatment that is maintained throughout the patient’s treatment journey. This includes controlling blood pressure, using angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) to reduce proteinuria, enhancing the likelihood of spontaneous remission, restricting salt intake, and employing diuretics to manage edema [20]. In this study, 58.4% of the patients were treated with ACEIs or ARBs, which is consistent with this principle. The prognosis of patients with MN has significantly improved with the widespread use of immunosuppressive therapies in patients with IMN. CYC in combination with GC has long been recognized as the gold standard treatment for IMN, with remission rates of approximately 72–93% [21,22]. Prior to the issuance of the 2021 KDIGO Guidelines, RTX was generally considered to be available for patients with IMN who did not respond to immunologic therapy, but based on its mechanism of directly inducing apoptosis in B-cells, RTX has become more widely available, and the 2021 KDIGO Guidelines [16] formally list RTX as a first-line agent for the treatment of IMN. Guidelines recommend that RTX, CNI ± GC or CTX+GC or RTX+CNI can be used as the initial treatment for intermediate and high-risk patients. In our study, with a median follow-up of 13.0 (IQR, 12.0–18.0) months, 188 (95.4%) patients were treated with RAAS-blockade or IST. Considering the severity of the disease, 122 (61.9%) patients received IST, and 66 (33.5%) chose to be treated with RAAS-blockade alone. Among patients treated with IST, 110 (55.8%) were exposed to GC. Twenty percent of the patients received oral GC alone, and another 80% in combination with other ISTs. The CNIs, RTX, and CYC were the most commonly used ISTs (23.4%,11.7% and 22.3%). In recent years, the use of RTX in MN has increased. The relatively low proportion of RTX use in our study may be attributed to its initial application and high cost. The administration of RTX during the tapering or withdrawal of CNIs has a beneficial effect in preventing relapses in IMN patients who respond to cyclosporine or tacrolimus [23]. These findings suggest that future treatment strategies may need to combine different drugs to increase remission and reduce relapse rates.
A total of 56.3% of patients initiated IST within 6 months after kidney biopsy, which deviates from the KDIGO guidelines’ [16] recommendation to observe and wait for at least six months to monitor for spontaneous remission. After analyzing the baseline data of the patients on IST, the reasons can be considered as follows. First, many patients exhibit clinical manifestations of kidney disease, such as proteinuria and hypoalbuminemia, well before these conditions are detected in laboratory tests, with the disease course potentially lasting for over six months. Consequently, clinicians often believe that these patients are unlikely to achieve spontaneous remission. Second, numerous patient develop complications such as thromboembolism prior to kidney biopsy, indicating an immediate need for IST. In addition, many patients show high titers of anti-PLA2R antibodies, leading clinicians to believe that the likelihood of remission in such patients is low. Therefore, many patients begin treatment with GC and/or immunosuppressants without meeting the indications outlined in the KDIGO guidelines. This deviation from the guideline-based treatment plan for MN is also evident in the ‘Cure Glomerular Disease Network (CureGN)’ cohort study, where 55% of adults initiated IST within six months after kidney biopsy, including those with a urine protein-to-creatinine ratio of less than 4 g/g [24].
With a deeper understanding of the molecular mechanisms of MN, diagnostic and therapeutic strategies have entered the molecular era, increasingly relying on molecular markers and targeted therapies. The core goal of treatment is to reduce antibody production and subepithelial immune complex deposition. Currently, in clinical practice, targeted drugs available for B-cell therapy in MN include anti-CD20 monoclonal antibodies (RTX [25], ofatumumab [26], and obinutuzumab [27]), plasma cell-depleting agents (daratumumab [28], and B-cell activating factor inhibitors (belimumab [29]). The proliferation of new medications has opened up more possibilities for alleviating proteinuria in MN, thereby better controlling the progression of kidney disease in patients.
IST significantly improved proteinuria remission in patients [30]. In this study, clinical remission of proteinuria was achieved in 133 patients (67.5%). Conversely, 64 (32.5%) patients did not achieve clinical remission. Based on grouping patients with different biomarkers and with or without immunosuppression, the number of patients in remission was greater in those using immunosuppression than in those not using immunosuppression. Wang et al. [31] reported that some NELL1-positive patients achieved proteinuria remission after IST, but the remission rate was relatively low. Long-term follow-up showed that NELL1-positive patients had a higher risk of progression to end-stage renal disease (ESRD), approximately 20–30%. Meanwhile, our study found that a history of hypertension and 24h-UTP were independent risk factors influencing whether patients had complete proteinuria remission. This is consistent with a foreign study that included 132 patients with IMN and found that hypertension was a risk factor for remission of clinical proteinuria [32]. Of the 17 NELL-1 antigen-positive patients, 11 (64.7%) received IST during follow-up, and 8 (72.7%) achieved CR or PR. Among the 13 EXT1/2 antigen-positive patients, 4 (30.8%) were treated with immunosuppressive agents, with 1 (25.0%) achieving CR. Due to the small sample size, it is difficult to determine the optimal treatment strategy for patients positive for NELL-1 antigen or EXT1/2 antigen. Future studies with larger sample sizes are needed to further investigate this area.
In our study, the sex ratio of NELL-1 antigen-positive patients was essentially the same; this result was similar to that of Sethi et al. [6]. In a study by Sethi [6], cancer was detected in four of the five positive patients in the validation cohort. However, these tumors were not detected in our cohort. Other studies [6,31] have also found that NELL-1 positivity is associated with higher proteinuria levels and disease activity. No significant predictive value of serum NELL-1 and EXT1/2 was found for treatment response in our cohort. This may be explained by four key factors. First, unclear mechanistic links limit predictivity: despite being established podocyte antigens, pathways connecting their serum levels to immune modulation, podocyte repair, or renal recovery remain uncharacterized. So their ability to serve as surrogate markers was weakened. Second, disease heterogeneity may impair their utility: NELL-1-associated MN encompasses both primary cases and secondary forms linked to thiol-containing drugs, malignancies, or infections [6,8,9], while EXT1/2-related MN is predominantly associated with autoimmune disorders (e.g., systemic lupus erythematosus) in young women [12]. Undiagnosed etiological differences (via routine clinical tests) in our cohort might cause variable subgroup treatment responses, making antigen titers alone insufficient to reflect differential efficacy to standard therapies. Third, confounding clinical/pathological factors (age, baseline renal function, proteinuria, comorbidities, glomerulosclerosis) exert a more dominant effect on outcomes than these antigens in our cohort. Finally, interindividual variability (e.g., genetic polymorphisms, differences in pharmacokinetics of immunosuppressive agents) can lead to divergent responses even with similar titers, underscoring the limitations of relying solely on these markers.
In primary MN, IgG1 and IgG4 are highly expressed along the GBM [33,34], and PLA2R and thrombospondin type 1 domain-containing 7 A (THSD7A) colocalize with IgG4 along the GBM [2,35]. In Wang’s study [31], the glomerular IgG1 staining positivity rate in patients with isolated NELL-1-positive MN was 73%, compared with 67% IgG4 dominance; in our study, IgG1 had the highest percentage of all types of IgG typing in NELL-1 antigen-positive patients and EXT1/2 antigen-positive patients. However, there were fewer patients with relevant NELL-1 antigen and EXT1/2 antigen positivity at our center, and the expression of IgG subclasses should be further observed in more cases.
This study has some limitations that must be recognized. Initially, our research did not consider variations in the treatment choices made by different clinicians. Additionally, the relatively short follow-up period was insufficient to fully observe the progression of glomerular diseases in patients. Prolonging the follow-up duration for longitudinal observation is essential to gain a comprehensive understanding of the dynamics of proteinuria and its impact on the progression of renal diseases. Thirdly, our study did not conduct staining classification of the target antigens in the glomeruli. The current classification is based on the positivity of serological antigen, and the significance of these two aspects cannot be completely equated. For example, the patients who have auto-antibodies against PLA2R, NELL1, EXT1/2 as well as glomerular antigens may have a different clinical course compared with those who only have circulating antibodies, and there may be differences in the therapeutic responses to RAAS-blockade and immunological drugs. These issues need to be verified by future studies. Fourthly, we only analyzed IgG subclasses in renal tissues but did not perform corresponding serum IgG subclass testing. This precludes direct comparison between circulating and tissue-deposited IgG subclasses, this question merit dedicated investigation in future cohort studies with paired serum-kidney profiling. Fifthly, our study detected relatively low serum antigen levels, but we did not further analyze the potential biological effects of these low-titer antigens in vivo. Owing to the retrospective nature of our research, it was not feasible to evaluate the association of serum titer changes with clinical data. This aspect will be refined in subsequent studies. Finally, potential confounders due to unmeasured variables were always present and could not be addressed using statistical modeling. Our findings may provide valuable insights for clinicians but must be interpreted and applied with caution.
In conclusion, of the 197 patients who underwent kidney biopsy, 152 (77.2%) were positive for anti-PLA2R antibody, 17 (8.6%) were positive for NELL-1antigen, 13 (6.6%) were positive for EXT1/2 antigen, and the remaining 15 (7.6%) were negative for all three biomarkers. After a median of 13.0 (12.0, 18.0) months of follow-up, a total of 75 (38.1%) patients showed CR of proteinuria, 58 (29.4%) had PR, 51 (25.9%) did not achieve proteinuria remission, and 13 (6.6%) relapsed after remission. The use of immunosuppressive agents was initiated earlier than that recommended by the KDIGO guidelines. Multifactorial logistic analysis showed that the baseline 24h-UTP level and a history of hypertension were independent risk factors for clinical proteinuria remission. More information on the prognostic factors associated with NELL-1 and EXT1/2 positivity needs to be confirmed using larger sample sizes and multicenter studies.
Supplementary Material
Acknowledgments
The authors thank the participants for their efforts during this study.
Funding Statement
There were no funds focused to this study.
Ethics statement
This study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University (approval number: No. 2015-KY-006). Written informed consent was obtained from all the patients.
Disclosure statement
The authors declare no potential conflicts of interest with respect to the investigation, authorship, or publication of this article.
Data availability statement
The datasets generated and/or analyzed during the current study are not publicly available due to ethical/legal restrictions. Further inquiries can be directed to the corresponding author.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets generated and/or analyzed during the current study are not publicly available due to ethical/legal restrictions. Further inquiries can be directed to the corresponding author.