Visual Abstract
Keywords: membranous nephropathy, neural epidermal growth factor-like 1 protein, phospholipaseA2 receptor, thrombospondintype 1 domain-containing7A
Abstract
Background and objectives
The neural EGF-like 1 (NELL-1) protein is a novel antigen in primary membranous nephropathy. The prevalence and clinical characteristics of NELL-1–positive membranous nephropathy in Chinese individuals with primary membranous nephropathy are unclear.
Design, setting, participants, & measurements
A total of 832 consecutive patients with biopsy-proven primary membranous nephropathy were enrolled. The glomerular expression of phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A (THSD7A) was screened. Glomerular immunohistochemistry staining for NELL-1 was performed in 43 patients with PLA2R- and THSD7A-negative membranous nephropathy, 31 patients with PLA2R-positive membranous nephropathy, and two patients with PLA2R and THSD7A double positivity. The NELL-1 antibody was also detected in the sera of patients with NELL-1–positive membranous nephropathy by western blot. Clinical and pathologic features were comparable between patients with isolated NELL-1–positive, isolated PLA2R/THSD7A-positive, and triple antigen–negative membranous nephropathy.
Results
Among the 832 patients with primary membranous nephropathy, 11 of 54 (20%) patients with PLA2R-negative membranous nephropathy had THSD7A-positive membranous nephropathy. NELL-1–positive membranous nephropathy accounted for 35% (15 of 43) of all patients with PLA2R- and THSD7A-negative membranous nephropathy. One patient was double positive for NELL-1 and PLA2R in glomerular deposits and positive for only the PLA2R antibody in the serum. Most patients with NELL-1–positive membranous nephropathy were women. No tumors were found. There were significant differences in the prevalence of IgG subtypes between patients with different antigen positivity. Among patients with isolated NELL-1–positive membranous nephropathy, although 80% (12 of 15) were IgG4 staining positive, the proportion of IgG4 dominance was only 67% (ten of 15).
Conclusions
About one third of patients who were PLA2R and THSD7A negative were NELL-1 positive in Chinese patients with primary membranous nephropathy. NELL-1–positive membranous nephropathy was more common than THSD7A-positive membranous nephropathy in PLA2R-negative membranous nephropathy.
Introduction
Primary membranous nephropathy, a common cause of nephrotic syndrome in adults and the second most common primary glomerular disease in China (1,2), is characterized by immune complex deposition in the subepithelial space of glomerular capillaries. In 2009, Beck et al. (3) first reported that the phospholipase A2 receptor (PLA2R) was a major target antigen involved in the pathogenesis of primary membranous nephropathy, and thrombospondin type 1 domain-containing 7A (THSD7A) was identified as an additional antigen in 2014 (4). It is now known that 70%–80% of cases of primary membranous nephropathy are related to PLA2R, while an additional 1%–5% are associated with THSD7A (5). The antigens and antibodies underlying the disease remain unknown for up to 20% of patients. Sethi et al. (6) recently described a new antigen, neural EGF-like 1 (NELL-1) protein, as a distinct cause of primary membranous nephropathy in patients with PLA2R- and THSD7A-negative membranous nephropathy.
NELL-1 contains 810 amino acids, has a molecular mass of 90, and has potent osteoinductive activity both in vivo and in vitro (7,8). NELL-1 is found in the kidney tubules and less so in the glomeruli (9). In some patients with PLA2R- and THSD7A-negative primary membranous nephropathy, NELL-1 staining is positive along the glomerular basement membrane (GBM) and subepithelium (6). Sethi et al. (6) also suggested that serum NELL-1 antibody titers may be used for clinical follow-up.
To date, there are few clinical studies on the proportion and clinical characteristics of NELL-1–positive membranous nephropathy. Therefore, in this study, glomerular immunohistochemistry staining for NELL-1 was performed in patients who were PLA2R and THSD7A negative and patients with positive glomerular PLA2R from China, aiming to observe the clinical and pathologic features of NELL-1–positive membranous nephropathy.
Materials and Methods
Patients
A total of 832 consecutive patients with biopsy-proven primary membranous nephropathy in Beijing Anzhen Hospital were enrolled in this study from 2010 to 2019. Patient medical records were reviewed, and all patients had complete baseline clinical, laboratory, and pathologic data. 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. Serum samples were collected on the day of kidney biopsy and stored at −80°C until use. Ten patients with IgA nephropathy and ten patients with diabetic nephropathy diagnosed by kidney biopsy were included as a control group for the detection of anti–NELL-1 antibody positivity by western blot. The research complied with the Declaration of Helsinki and was approved by the ethics committee of Beijing Anzhen Hospital. Written informed consent was obtained for sampling tissue and blood.
Membranous nephropathy was diagnosed on the basis of pathologic parameters, including light microscopy, immunofluorescence, and electron microscopy. Patients with secondary causes, such as autoimmune diseases (e.g., lupus), hepatitis virus infection (hepatitis B or hepatitis C), and toxicants (e.g., mercury), detected at onset or during follow-up were excluded. The definitions of remission and relapse complied with the 2012 Kidney Disease Improving Global Outcomes guideline for glomerular nephropathy (10). 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. Kidney dysfunction was defined as a 30% decrease in the initial eGFR and below 60 ml/min per 1.73 m2.
Detection of Glomerular Neural Epidermal Growth Factor–Like 1 Expression
Glomerular NELL-1 deposition was detected by immunohistochemistry staining. Paraffin-embedded sections (4 μm) of formalin-fixed kidney tissues were deparaffinized and hydrated. Antigen retrieval involved autoclave heating–induced epitope retrieval (in citrate solution, pH 6.0). A rabbit polyclonal anti–NELL-1 antibody (1:800; Sigma-Aldrich) was added as the primary antibody at 4°C overnight. An alkaline phosphatase-labeled goat anti-rabbit IgG antibody was used as the secondary antibody and incubated for 60 minutes at room temperature. Color was developed with a diaminobenzine chromogenic solution, and nuclei were stained with hematoxylin. A positive NELL-1 immunohistochemical result was defined as strong diffuse global granular staining along capillary loops.
Circulating Antineural Epidermal Growth Factor–Like 1 Antibody Measurement by Western Blot
Serum anti–NELL-1 antibodies were detected by western blot. Recombinant NELL-1 (Abnova) was electrophoresed under nonreducing conditions, and proteins were transferred to nitrocellulose membranes and blocked in Odyssey blocking buffer (Li-Cor Biosciences) for 60 minutes at room temperature. The membranes were incubated with patient sera (1:50), control (1:50; sera from ten patients with IgA nephropathy and ten patients with diabetic nephropathy), and a rabbit polyclonal primary antibody against NELL-1 (1:500; Abcam) overnight at 4°C and with the IRDye 800 CW goat anti-human IgG secondary antibody (1:10,000; Li-Cor) for 60 minutes at room temperature; then, they were imaged using a Li-Cor Odyssey scanner (Li-Cor).
Detection of Glomerular Phospholipase A2 Receptor and Thrombospondin Type 1 Domain-Containing 7A Expression and Circulating Antiphospholipase A2 Receptor and Antithrombospondin Type 1 Domain-Containing 7A Antibodies
Immunohistochemistry staining for PLA2R and THSD7A was performed as previously described (11,12), and the protocol is provided in Supplemental Material. The rabbit anti-human PLA2R polyclonal antibody (1:800; Sigma-Aldrich) and the rabbit anti-human THSD7A polyclonal antibody (1:1500; Sigma-Aldrich) were the primary antibodies.
The serum anti-PLA2R antibody levels were detected by ELISA (EUROIMMUN, Lübeck, Germany). The circulating anti-THSD7A antibody was detected by an indirect immunofluorescence assay kit (EUROIMMUN) according to standard instructions. Further details are provided in Supplemental Material.
Statistical Analyses
Statistical analyses were performed using SPSS 21.0 (SPSS Inc., Chicago, IL). Parametric data are presented as the means ± SDs. Nonparametric data are presented as the median values with intervals from the 25th to 75th percentiles. Comparisons between groups of continuous variables were performed by one-way ANOVA or the Mann–Whitney U test (Kruskal–Wallis), and categorical variables were compared by the chi-squared test. All P values were two tailed, and P=0.05 indicated statistical significance.
Results
Prevalence of Neural Epidermal Growth Factor–Like 1–Positive, Phospholipase A2 Receptor–Positive, Thrombospondin Type 1 Domain-Containing 7A–Positive, and Triple Antigen–Negative Membranous Nephropathy
We performed immunohistochemistry analysis of PLA2R in all 832 patients. A total of 778 patients (94%; 778 of 832) were PLA2R positive, and 54 patients (7%; 54 of 832) were PLA2R negative. Serologic testing for antibodies against PLA2R was performed in 818 patients; 535 patients (65%; including 14 patients who were serum PLA2R antibody negative at the time of kidney biopsy and found to be positive for PLA2R antibody at least once during follow-up) were positive (Figure 1). Eleven of 54 (20%) patients with PLA2R-negative membranous nephropathy had strong positive staining for THSD7A. Immunohistochemistry analysis of THSD7A was performed on 319 patients who were PLA2R positive; one patient had both PLA2R and THSD7A in glomerular deposits, and one patient was positive for glomerular THSD7A and positive for serum PLA2R antibody (Supplemental Table 1). Serum anti-THSD7A antibody testing was performed in 124 patients (including 13 with positive glomerular THSD7A). Four patients were positive (also glomerular THSD7A positive) (Figure 1). Immunohistochemistry staining for NELL-1 was performed in 43 patients who were PLA2R and THSD7A negative, revealing that 15 (35%) were positive for NELL-1 and 28 (65%) were negative. Additionally, among 31 patients who were PLA2R positive and two patients with PLA2R and THSD7A double positivity, one patient stained positive for both PLA2R and NELL-1. Western blot screening of the serum anti–NELL-1 antibody in 15 patients with positive NELL-1 staining (except for one patient without serum sample) led to the identification of two serum NELL-1 antibody–positive patients (Figures 1 –3). Ten patients with IgA nephropathy and ten patients with diabetic nephropathy were negative for anti–NELL-1 antibody. The patient with glomerular dual PLA2R and NELL-1 positivity was negative for the serum anti–NELL-1 antibody but positive for the anti-PLA2R antibody (Figure 4, Supplemental Table 2). Therefore, in patients with PLA2R- and THSD7A-negative membranous nephropathy, 35% (15 of 43) were NELL-1 positive (Figure 1).
Figure 1.
Flow chart of the patients enrolled in the study. MN, membranous nephropathy; NELL-1, neural EGF-like 1; PLA2R, phospholipase A2 receptor; THSD7A, thrombospondin type 1 domain-containing 7A.
Figure 2.
G lomerular NELL-1 staining by immunohistochemistry and immunofluorescence staining for IgG1 and IgG4 were positive in one patient with NELL-1–positive MN. (A) presents NELL-1 strong granular glomerular capillary loop staining (×400). (B, C, and E) show bright capillary wall staining for IgG, IgG1, and IgG4 (×200). (D) shows negative capillary wall staining for IgG3. (F) shows the deposition of granular electron dense in the subepithelial of glomerular capillary wall under electron microscopy (×5000).
Figure 3.
Two patients were serum positive for the anti–NELL-1 antibody detected by western blot analysis in 15 patients with NELL-1-positive staining. Serum anti–NELL-1 antibodies were detected by western blot under nonreducing conditions. NELL-1 is detected as 280-kD homodimers (arrow). Lane 1: positive control (rabbit polyclonal antibodies against NELL-1). Lanes 2 and 3: the two patients who were positive for anti–NELL-1 antibody. Lane 4: the patient who was double positive for PLA2R and NELL-1 staining and negative for anti–NELL-1 antibody. Lanes 5–16: the 12 patients who were negative for anti–NELL-1 antibody.
Figure 4.
Segmental enhancement of NELL-1 staining and diffuse enhancement of PLA2R staining in glomeruli in the patient who was dual positive for NELL-1 and PLA2R. This patient showed bright granular staining for NELL-1 and PLA2R along the glomerular basement membrane and negative staining for THSD7A. However, NELL-1 staining showed only segmental granular enhancement. Immunofluorescence shows capillary wall staining for IgG, IgG1, and IgG4. Immunohistochemistry staining, ×400; immunofluorescence, ×200.
Patients with Isolated Neural Epidermal Growth Factor–Like 1–Positive Membranous Nephropathy
Among 15 patients with isolated NELL-1–positive membranous nephropathy, 11 women and four men (women-men ratio of three), with a median age of 49 (44–50) years, were analyzed. Twelve patients (80%) presented with nephrotic syndrome, and the eGFR was <60 ml/min per 1.73 m2 for three patients at the time of kidney biopsy. NELL-1 staining showed bright, granular GBM staining in all 15 patients. However, the staining of NELL-1 was not uniform and showed segmental deposition in some glomeruli. Among these 15 patients, IgG4 staining was positive on the GBM in 12 patients (80%), whereas IgG1 staining was positive in 11 patients (73%), including three with IgG4 negative staining. Three of 15 patients (20%) were IgG2 positive. However, IgG4 was still predominant in patients with IgG2 positivity. Three patients were lost to follow-up. Of the 12 patients with follow-up data, ten (83%) received immunosuppressive therapy. Eleven (92%) patients achieved complete or partial remission, and no patient experienced relapse (Supplemental Table 2). Because the number of men with NELL-1 positivity was significantly lower than that of women, we compared the characteristics of men and women. Although the serum albumin level of women was significantly higher than that of men (2.9 [2.6–3.3] g/dl versus 1.7 [1.6–2.7] g/dl, respectively; P=0.04), no significant differences were observed in other clinical and pathologic features and the treatment response (Supplemental Tables 3 and 4).
Comparison of the Clinical and Pathologic Characteristics of Patients with Isolated Neural Epidermal Growth Factor–Like 1–Positive, Isolated Phospholipase A2 Receptor–Positive, Isolated Thrombospondin Type 1 Domain-Containing 7A–Positive, and Triple Antigen–Negative Membranous Nephropathy
The clinical and pathologic features were compared among patients with isolated NELL-1–positive, isolated PLA2R-positive, isolated THSD7A-positive, and triple antigen–negative membranous nephropathy (Table 1). Interestingly, the proportion of women with isolated NELL-1–positive membranous nephropathy was higher than that in the other groups. No patient with isolated NELL-1–positive membranous nephropathy had tumors at the time of diagnosis or during the follow-up (Table 2). There were significant differences in the prevalence and staining intensity of IgG subtypes. Although IgG4 positivity was predominant in the four groups, the proportion of IgG4 dominance in patients with triple antigen–negative membranous nephropathy was only 54% (15 of 28), and that in patients with isolated PLA2R-positive membranous nephropathy was 91% (702 of 775). The positive rate of glomerular IgG1 staining in patients with isolated NELL-1–positive and isolated THSD7A-positive membranous nephropathy was 73%, which was higher than 43% in patients with PLA2R-positive and triple antigen–negative membranous nephropathy. (Table 1).
Table 1.
Comparison of clinical and pathologic characteristics among patients with neural EGF-like 1–positive, phospholipase A2 receptor–positive, thrombospondin type 1 domain-containing 7A–positive, and triple antigen–negative membranous nephropathy
| Characteristics | Neural EGF-Like 1–Positive Membranous Nephropathy, n=15 | Phospholipase A2 Receptor–Positive Membranous Nephropathy, n=775 | Thrombospondin Type 1 Domain-Containing 7A–Positive Membranous Nephropathy, n=11 | Triple Antigen–Negative Membranous Nephropathy, n=28 |
|---|---|---|---|---|
| Women, n (%) | 11 (73) | 294 (38) | 4 (36) | 13 (46) |
| Age, yr | 49 (44–50) | 51 (39–59) | 51 (30–60) | 48 (35–60) |
| Urinary protein, g/d | 4.6 (3.4–5.5) | 5.2 (3.5–8.0) | 4.8 (3.3–8.5) | 3.5 (1.7–7.2) |
| Albumin, g/dl | 2.9 (2.0–3.0) | 2.6 (2.1–3.0) | 2.2 (1.7–2.6) | 2.9 (2.5–3.6) |
| Nephrotic syndrome, n (%) | 12 (80) | 589 (76) | 10 (91) | 18 (64) |
| eGFR, ml/min per 1.73 m2 | 96±23 | 104±21 | 99±23 | 93±33 |
| Cholesterol, mg/dl | 348±77 | 309±116 | 309±77 | 232±77 |
| Malignancy, n (%) | 0 | 4 (0.5) | 1 (9) | 1 (4) |
| Interstitial fibrosis, %, n (%) | ||||
| <25 | 0 | 190 (25) | 3 (27) | 10 (36) |
| 25–50 | 12 (80) | 474 (61) | 8 (73) | 14 (50) |
| 50–75 | 2 (13) | 89 (12) | 0 | 3 (11) |
| >75 | 1 (7) | 22 (3) | 0 | 1 (4) |
| IgG1 positive, n (%) | 11 (73) | 331 (43) | 8 (73) | 12 (43) |
| IgG2 positive, n (%) | 3 (20) | 46 (6) | 1 (9) | 7 (25) |
| IgG3 positive, n (%) | 0 | 98 (13) | 0 | 5 (18) |
| IgG4 positive, n (%) | 12 (80) | 709 (92) | 8 (73) | 16 (57) |
| IgG4 predominant positive, n (%) | 10 (67) | 702 (91) | 7 (64) | 15 (54) |
| IgA positive, n (%) | 1 (7) | 146 (19) | 2 (18) | 5 (20) |
| Segmental sclerosis, n (%) | 0 | 29 (4) | 0 | 1 (4) |
Immunofluorescence values 2+ and above are positive. eGFR of patients was calculated by the Chronic Kidney Disease Epidemiology Collaboration equation.
Table 2.
Comparison of treatment and prognosis among patients with neural EGF-like 1–positive, phospholipase A2 receptor–positive, thrombospondin type 1 domain-containing 7A–positive and triple antigen–negative membranous nephropathy
| Characteristics | Neural EGF-Like 1–Positive Membranous Nephropathy, n=12 | Phospholipase A2 Receptor–Positive Membranous Nephropathy, n=382 | Thrombospondin Type 1 Domain-Containing 7A–Positive Membranous Nephropathy, n=6 | Triple Antigen–Negative Membranous Nephropathy, n=12 |
|---|---|---|---|---|
| Immunosuppressive therapy (%) | ||||
| Pred and cyclosporin A | 4 (33) | 83 (22) | 2 (33) | 0 |
| Cyclosporin A | 4 (33) | 203 (53) | 1 (17) | 5 (42) |
| Pred and cyclophosphamide | 2 (17) | 34 (9) | 2 (33) | 3 (25) |
| Remission (%) | 11 (92) | 238 (62) | 6 (100) | 11 (92) |
| Complete remission (%) | 4 (33) | 103 (26) | 5 (83) | 7 (58) |
| Partial remission (%) | 7 (58) | 135 (35) | 1 (17) | 4 (33) |
The definitions of partial remission and complete remission are the same as described above. Pred, prednisone.
Because of greater patient mobility and a change of contact, some patients were lost to follow-up. Among the 15 patients with isolated NELL-1–positive membranous nephropathy, 12 were followed up for an average of 25±21 months. In total, 382 patients who were isolated PLA2R positive, six patients who were THSD7A positive, and 12 patients with triple antigen–negative membranous nephropathy had follow-up data. The average follow-up times were 18±18, 19±8, and 31±23 months, respectively. The total remission rate of patients who were isolated NELL-1 positive (92%) was higher than that of patients who were isolated PLA2R positive (62%). The complete remission rate of patients who were isolated NELL-1 positive was 33%, and that of patients who had isolated THSD7A-positive membranous nephropathy was 83% (Table 2). However, we did not study the differences in the treatment efficacies between groups due to the small patient numbers.
Patients with Membranous Nephropathy and Double Neural Epidermal Growth Factor–Like 1 and Phospholipase A2 Receptor Positivity
The patient with double positivity for PLA2R and NELL-1 had low serum anti-PLA2R antibody titers (41 RU/ml) at the time of kidney biopsy. After cyclosporin treatment, the patient achieved complete remission and negative conversion of the anti-PLA2R antibody. However, the patient experienced relapse, with the antibody concentration increasing significantly and remaining at a high titer (>1500 RU/ml); the patient did not achieve remission after additional cyclosporin treatment. The patient was still negative for the serum anti–NELL-1 antibody at the time of kidney biopsy and relapse. Bright granular staining for IgG, IgG1, IgG4, and PLA2R along the GBM and diffuse subepithelial deposits were observed by electron microscopy. However, NELL-1 staining showed only segmental granular enhancement in the GBM (Figure 4).
Discussion
In 2020, Sethi et al. (6) identified NELL-1 as another pathogenic antigen of primary membranous nephropathy in addition to PLA2R and THSD7A. In this study, the prevalence of NELL-1–positive membranous nephropathy was 35% (15 of 43) in patients with PLA2R- and THSD7A-negative membranous nephropathy. One patient with double positivity for PLA2R and NELL-1 was also revealed herein by immunohistochemistry. In our study, only two of 15 patients with NELL-1–positive staining were serum positive for the anti–NELL-1 antibody detected by western blot analysis, suggesting that the sensitivity of NELL-1 immunohistochemistry staining is much higher than that of serum antibody detection, consistent with the sensitivity of glomerular PLA2R and THSD7A staining and serum antibodies (3,4). In this study, 94% of patients had PLA2R-positive staining, which was higher than the seropositive rate of anti-PLA2R antibody; this may have led to a decreased number of patients who were PLA2R negative and overestimated the proportion of NELL-1–positive membranous nephropathy. We considered that double repair used in PLA2R immunochemistry staining can better expose the PLA2R antigen, thus increasing the positive rate of staining. The reasons for the low seroprevalence of anti-PLA2R antibody were that the high cutoff (20 RU/ml) value affected the sensitivity of ELISA (13) and that patients received immunosuppressive therapy before collection of serum. Pang et al. (14) also reported that positive serum anti-PLA2R antibody and glomerular PLA2R deposition were seen in 58.8% and 95.6% of patients, respectively, in a cohort of Chinese patients with membranous nephropathy, which were close to our study. Similarly, in 13 patients negative for the serum anti–NELL-1 antibody, three had received immunosuppressive therapy, and three additional patients did not have nephrotic syndrome when samples were collected. We hypothesized that pretreatment and non-nephrotic syndrome may affect the serum testing results. In the study by Sethi et al. (6), only five patients with NELL-1–positive membranous nephropathy were evaluated by western blot and were positive for the serum anti–NELL-1 antibody. At present, there is no currently available ELISA or immunofluorescence test for serum NELL-1 antibody detection. Therefore, the sensitivity of serum antibody detection for diagnosis needs to be further studied.
In this study, most patients with isolated NELL-1–positive membranous nephropathy were women, unlike in the study by Sethi et al. (6). We compared the clinical and pathologic features between women and men, and the serum albumin level of women was significantly higher than that of men. However, only four men were included, and the statistical results may be biased. Previous studies reported that the majority of patients with THSD7A-positive membranous nephropathy were women (4,15,16), but this was not observed in other cohorts (17,18). Therefore, the factors underlying this sex difference and its significance in patients with NELL-1–positive membranous nephropathy need to be examined in more samples. Previously, THSD7A-positive membranous nephropathy was thought to be associated with tumors (16,19). In the study by Sethi et al. (6), cancer was detected in four of the five positive patients in the validation cohort. However, tumors were not detected in the discovery or pilot cohort. In our study, none of the 15 patients with isolated NELL-1–positive membranous nephropathy had tumors at the time of diagnosis. Tumors were also not detected in 12 patients with NELL-1–positive membranous nephropathy who were followed up for 25±21 months. However, there are few cases, and it is unknown whether NELL-1 positivity is found in tumor cells like THSD7A antigen (20,21). The relationship between NELL-1 and tumors needs further confirmation.
In primary membranous nephropathy, IgG1 and IgG4 tend to be highly expressed along the GBM (22,23). PLA2R and THSD7A colocalized with IgG4 along the GBM (3,4,24). If IgG1, IgG2, and IgG3 exhibit positive staining but IgG4 exhibits negative or nondominant positive staining, it is recommended to search for secondary causes (22,25). In this study, the proportion of IgG4 dominance in patients with isolated NELL-1–positive membranous nephropathy was only 67%, whereas the positive rate of glomerular IgG1 staining was 73%. In addition, 20% of patients with isolated NELL-1–positive membranous nephropathy had positive staining for IgG2, whereas IgG4 was still predominant in those patients. Therefore, IgG subclass expression should be further observed, and secondary causes may need to be screened in patients with NELL-1–positive membranous nephropathy.
In this study, we present the first report of a patient with dual positivity for NELL-1 and PLA2R. At present, the serum anti-PLA2R antibody level is thought to be a useful marker of clinical activity (26,27). In this patient, the remission and relapse of disease were accompanied by anti-PLA2R antibody seroconversion to negative and high titers, respectively. However, because the anti–NELL-1 antibody was not detected, the relationship between the anti–NELL-1 antibody and disease activity could not be examined. Whether the poor treatment responses and abnormally high titers of the anti-PLA2R antibody are related to the presence of NELL-1 remains unknown. Therefore, the role of autoimmunity against NELL-1 and PLA2R in double-positive patients should be examined in further studies.
Our study has some limitations. First, we performed NELL-1 staining in 43 patients who were PLA2R and THSD7A negative and 31 patients who were PLA2R positive, and therefore, this was a small patient cohort study. There is a possible patient selection bias because NELL-1 staining was not performed in all 832 patients, which may have led to the omission of NELL-1 and PLA2R/THSD7A double-positive patients. Second, because of the small numbers of patients who were NELL-1 positive and THSD7A positive, the statistical analyses deviated in some instances. Therefore, we did not study the differences in treatment efficacy. Third, this is a retrospective study, and 16% of patients had received immunosuppressive therapy at baseline, which may have affected the serum detection results. In addition, follow-up data were only available from 412 patients in this retrospective study of 832 patients with membranous nephropathy, which may have led to an underestimation of the incidence of tumors. Therefore, more patients and a longer length of follow-up data need to be observed in further studies.
In conclusion, this is the first study to observe the prevalence and characteristics of NELL-1–positive membranous nephropathy in Chinese patients with primary membranous nephropathy. In our study, about one third of patients who were PLA2R and THSD7A negative were NELL-1 positive. Most patients who were NELL-1 positive were women. The glomerular deposition of IgG subclasses was different between patients with different antigen positivity. We also reported for the first time one patient with double NELL-1 and PLA2R positivity. The roles of the immune response against different antigens in the pathogenesis of membranous nephropathy need to be further investigated.
Disclosures
All authors have nothing to disclose.
Funding
This work is supported by National Natural Science Foundation of Beijing grant 7192050.
Supplementary Material
Acknowledgments
The authors acknowledge six participating hospitals for their contributions to this study, including Division of Nephrology, Beijing Jishuitan Hospital; Division of Nephrology, Shunyi Hospital of Beijing Hospital Traditional Chinese Medicine; Division of Nephrology, Fenyang People’s Hospital; Division of Nephrology, The Central Hospital of Jiamusi City; Division of Nephrology, Qinghai University Affiliated Hospital; and the Division of Nephrology, Qinghai Hospital of Traditional Chinese Medicine. The authors also thank AJE (www.AJE.com) for providing English language editing for this manuscript.
Footnotes
Published online ahead of print. Publication date available at www.cjasn.org.
See related editorial, “Growing Understanding of the Antigenic Basis for Membranous Nephropathy,” on pages 677–678.
Supplemental Material
This article contains the following supplemental material online at http://cjasn.asnjournals.org/lookup/suppl/doi:10.2215/CJN.11860720/-/DCSupplemental.
Supplemental Material. Materials and methods.
Supplemental Table 1. Clinical and pathologic characteristics of dual PLA2R- and THSD7A-positive patients.
Supplemental Table 2. Clinical and pathologic data of the 16 patients with primary membranous nephropathy and glomerular staining for NELL-1.
Supplemental Table 3. Comparison of clinical and pathologic data between women and men with NELL-1–positive membranous nephropathy.
Supplemental Table 4. Comparison of treatment and prognosis between women and men with NELL-1–positive membranous nephropathy.
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