We continuously reviewed patients diagnosed with IMN by renal biopsy in the Department of Nephrology, Beijing Anzhen Hospital, Capital Medical University from 2015 to 2019. By staining of PLA2R, THSD7A, NELL-1 antigen in kidney tissue and detection of corresponding serum antibodies, we screened the dual antigen- positive IMN and PLA2R single-positive IMN, diagnosed by positive tissue staining or positive serum antibodies. Their baseline clinical, pathological and prognostic information were retrospectively collected. Follow-up data were obtained by reviewing the medical records and/or from telephone interviews of patients, and they were available on some patients. The treatment and prognostic analyses were performed in patients with complete information on follow-up. The definitions of remission complied with the 2012 Kidney Disease Improving Global Outcomes guideline for glomerular nephropathy (9). Complete remission was defined as urinary protein excretion <0.3 g/d, confirmed by two values at least 1 week apart, accompanied by normal serum albumin and creatinine levels. Partial remission was defined as urinary protein excretion <3.5 g/d and at least a 50% reduction from peak values accompanied by an improvement or normalization of serum albumin and stable serum creatinine levels. Complete remission and partial remission are collectively referred to as remission. Worsening of renal function was defined as a doubling of creatinine.

Paraffin-embedded kidney tissue (4 μm) was obtained. PLA2R and NELL-1 antigens were retrieved with pH 6 citrate solution combined with trypsin; THSD7A was retrieved with pH 9 EDTA solution. The primary antibodies used were monoclonal rabbit anti-human PLA2R1 antibody (Sigma, HPAO12657) (1:800 dilution), monoclonal rabbit anti-human NELL-1 antibody (Sigma, HPAO51535) (1:400 dilution), and monoclonal rabbit anti-human THSD7A antibody (Sigma, HPAO00923) (1:3000 dilution), which were incubated overnight at 4°C. The secondary antibody, alkaline phosphatase-labeled immunohistochemistry reagent (Max Vision, KIT-5103) or horseradish peroxidase-labeled immunohistochemistry reagent (Max Vision, KIT-5004), was added and incubated at room temperature for 30 minutes. Fast-Red reagent (Zhongshan Jinqiao, ZLI-9042) or DAB reagent (Zhongshan Jinqiao, ZLI-9018) was used for color development. Antigen positive is judged as granular brown staining in the glomerular capillary loops, while the renal tubule and other background staining linear reduction (10).

The routine fluorescent staining and histochemical staining are carried out in accordance with the standard procedures and the recommended procedures of the reagent specification. Fluorescence intensity criteria: there is no light at both low and high magnification as “-”; negative at low magnification, seemed to be visible at high magnification as “±”; it seems to be visible at low magnification, and blurred at high magnification as “+”; it is obviously visible at low magnification and clearly visible at high magnification as “++”; clearly visible at low magnification and dazzling fluorescence at high magnification as “+++”: dazzling at low magnification and dazzling fluorescence at high magnification as “++++”. Electron microscope examination of renal tissue was delivered to the testing center.

Serum samples were collected on the day of kidney biopsy and stored at -80°C until use. Serum anti-PLA2R antibodies were detected using the ELISA (EUROIMMUN, Germany) method. Serum anti-THSD7A and anti-NELL-1 antibodies were detected using an indirect immunofluorescence assay kit (EUROIMMUN, Germany) according to the standard protocol.

We searched for “M-type anti-phospholipase A2 receptor”, “thrombospondin type 1 domain containing 7A”, and “neuroepidermal growth factor-like type 1 protein” in the three major English databases Medline, Embase, and Cochrane Library and the two major Chinese databases Wan fang and CNKI (the specific search strategies are provided in Supplementary Material ). All literature related to dual antigen-positive IMN was screened as of June 6, 2023, and literature information and clinicopathological and prognostic data of dual antigen-positive patients were extracted.

The continuous variables of normal distribution were represented by mean ± standard deviation, and the difference between groups was compared by independent sample t test. Continuous variables with non-normal distributions were expressed as median and quartile distances (P25, P75), and non-parametric tests were used to compare group differences. The number and percentage of categorical variables were expressed, and Chi-square test and Fisher exact test were used to compare the differences between groups. SPSS 23.0 software was used for statistical analysis, and bilateral P value <0.05 was considered statistically significant.

A total of 827 patients diagnosed with IMN by renal biopsy at our center from 2015 to 2019, with an average age of 48 years old, accounted for 63.6% of males. Through tissue antigen staining and serum antibody detection, 6 patients with dual antigen positivity were screened, including 3 PLA2R- and NELL-1-positive patients and 3 PLA2R- and THSD7A-positive patients. Detection of tissue antigens and serum antibodies in the 6 patients is shown in Table 1 .

The clinical data of the 6 patients are shown in Table 1 . The median age was 55.5 years old, and 4 patients were older than 45 years. Four patients (66.7%) had significant proteinuria, of whom only 3 (50%) presented with nephrotic syndrome (NS); 1 patient (MN4) had a slight increase in blood creatinine. To date, no concomitant tumor diseases have been detected during follow-up of the 6 patients, but the tumor markers of patient MN1 continued to increase during the course of the disease ( Supplementary Figure 1 ).

Typical IMN manifestations under light microscopy were observed for these six patients, with all cases showing subepithelial deposition of fuchsinophilic protein. Only one IMN patient with PLA2R and THSD7A positivity (MN4) exhibited mild mesangial cell proliferation and increased mesangial matrix in the glomerulus. Immunofluorescence showed IgG particles deposited along the basement membrane in all 6 patients, without any other immunoglobulin deposits. IgG subclass staining for all 6 patients mainly showed IgG4; only one PLA2R- and NELL-1-positive patient (MN1) showed the same fluorescence intensity as for IgG1 and IgG4. The results of electron microscopy were consistent with those of light microscopy. In all 6 patients, electronic dense matter was found to be deposited in the subepithelial area of basement membrane area of the glomerulus ( Table 1 , Figure 1 ).

The median follow-up time of the 6 patients was 40 months. Except for patient 5, whose treatment regimen we did not track, all patients received immunosuppressive therapy. Four of the patients achieved disease remission after treatment, but only 1 patient (MN1) achieved it within half a year. Of the 2 patients (MN1, MN2) with follow-up data after remission, 1 patient experienced relapse, whereas remission was maintained in the other patient. None of the patients had worsening of renal function during follow-up ( Table 1 ).

We conducted a systematic review of the literature on previous studies involving double antigen-positive IMN. By June 6, 2023, a total of 11 studies (7, 11–20) involving 43 dual antigen-positive IMN patients, were included ( Supplementary Table 1 , Supplementary Figure 2 ). Of the 43 patients, highly detailed information was available for only 11, and we extracted clinical, pathological, and prognostic information for these 11 patients ( Table 2 ) and then performed comparisons with the 6 dual antigen-positive IMN patients at our center ( Supplementary Table 2 ).

To explore the difference between dual antigen-positive IMN and PLA2R single-positive IMN, we compared the two groups of patients. However, because the number of PLA2R single-positive IMN at our center is much larger than the number of dual antigen-positive IMN in the same time period, we only continuously enrolled 141 patients with PLA2R single-positive IMN within one year (2018-2019)of this time period. The comparison of clinicopathology and prognosis of the two groups at our center is shown in Supplementary Tables 3, 4 . There was no significant difference in clinicopathology and prognosis between the two groups. Since the number of IMN patients with dual antigen positivity is small, meanwhile, we compared the baseline clinical and pathological data of all known dual antigen-positive IMN patients, including the 6 cases from our center and the 11 cases in the literature with complete clinical and pathological data, with 141 PLA2R single-positive patients at our center ( Table 3 ). The results showed that the dual antigen-positive patients had a higher IgG1-positive rate in renal tissue than the PLA2R single-positive patients (58.3% vs. 22.3%, p=0.016). We also conducted a meta-analysis of baseline urine protein and serum albumin comparison results between 17 dual antigen-positive patients and the 141 PLA2R single-positive patients ( Supplementary Figures 3, 4 ), with no significant difference in baseline urine protein and blood albumin between the two groups. Prognosis comparison between the two groups showed a longer time to achieve remission in the patients with dual antigen positivity than in those with PLA2R single positivity, even though this difference did not reach statistical significance (p=0.052) ( Table 4 ).

Serum antibody levels were measured at multiple nodes of disease change for three of the patients at our center (MN1, MN2, and MN4) ( Figure 2 ). The condition of MN1 was protracted, starting with NS. After 4 months of treatment, partial remission was achieved, and the serum anti-PLA2R antibody titer gradually decreased. After 4 years, relapse of NS occurred, and the serum anti-PLA2R antibody titer continued to increase by >1500 RU/ml. Urinary protein remained stable, but his serum anti-NELL-1 antibody remained negative throughout the disease course. MN2 did not achieve complete remission until 21 months of immunosuppressive therapy, and there was no recurrence thereafter. During the course of the disease, the patient’s serum anti-PLA2R antibodies and anti-NELL-1 antibodies remained negative. After 4 months of immunosuppressive therapy, the condition of MN4 did not improve. The serum anti-PLA2R antibody titer decreased only slightly from 41.49 RU/ml to 30 RU/ml, and serum was negative for anti-THSD7A antibody.