Urine PLA2R Antibody Detection in Hazard Stratification of PLA2R-Associated Membranous Nephropathy Open Access

Urine and serum samples were obtained from 20 healthy individuals, 34 patients with PLA2R-positive MN (P⁺), known as PMN; 6 patients with non-PLA2R–associated MN (P^–); 6 patients with minimal change disease (MCD); and 10 patients with IgA nephropathy (IgAN). Samples from patients with pathologic diagnosis were obtained from the Hangzhou Hospital of Traditional Chinese Medicine (Hangzhou, China). All patients were diagnosed by using renal puncture for light microscopy, immunohistochemistry, and immunofluorescence. Patients with MN were clinically excluded from secondary causes of MN, such as hepatitis viruses (hepatitis B virus), syphilis, neoplasms, systemic lupus erythematosus, infections, and drugs. After the diagnosis of MN was confirmed, the laboratory performed the MN-specific target antigen test, which ruled out positive results for the known target antigens THSD7A, NELL-1, SEMA3B, EXT1/2, and NEP/MME, and determined that the samples were representative of PMN. Supplemental Figure 1 (see supplemental digital content containing 3 figures and 2 tables at https://meridian.allenpress.com/aplm in the July 2025 table of contents) shows the typical fluorescence immunohistochemistry of PMN. All samples were documented with serial follow-up, which ruled out the non–PLA2R-associated MN group as being in the early stages of PLA2R-positive MN. The samples and clinical data used in this study were obtained from the initial pretreatment diagnosis. P⁺ patients were classified into the remission group if they met the following criteria after 12 months of treatment: more than 50% decrease in urinary protein after 24 hours of treatment; urinary protein less than 3.5 g; and serum albumin concentration greater than 35 g/L. Those who did not meet one of the abovementioned indicators after 12 months of treatment were classified into the nonremission group. This study was approved by the ethics committee of Hangzhou Hospital of Traditional Chinese Medicine (ethics approval number: 2017LH001) and conducted in accordance with the ethical principles stated in the Declaration of Helsinki.

PLA2R-IgG and PLA2R-IgG4 detection kits were purchased from Zhejiang Boshi Biotechnology Company (Hangzhou, Zhejiang, China), including Eu³⁺-labeled anti-human IgG, Eu³⁺-labeled anti-human IgG4, recombinant PLA2R antigens, PLA2R-Ab standard solution, coating buffer, blocking buffer, enhancement solution, elution buffer, and analysis buffer. The time-resolved fluorescence immunoassay (TRFIA) analyzer HG-1000 was purchased from Shanghai Huguo Science Instrument Co, Ltd (Shanghai, China). All other chemicals and reagents used were of analytical grade.

First, the PLA2R-Ab standard solution or urine sample was added to the PLA2R-coated plate, and the plate was incubated on a micro-oscillator at 25°C. After washing the plate, Eu³⁺-labeled anti-human IgG or Eu³⁺-labeled anti-human IgG4 was added to each well and incubated again. After filling the wells with the enhancement solution, the fluorescence of each well was detected with the TRFIA analyzer HG-1000.

Considering that the applicability of urine and serum samples to commercial kits was different, the appropriate urine dilution was selected. Strongly positive samples, which contain high levels of uPLA2R-Ab, and samples from healthy individuals were diluted with analysis buffer at different proportions, and their concentrations were determined.

SPSS 15.0 (SPSS Inc, Chicago, Illinois) was used for data analysis, and GraphPad Prism 8 (GraphPad, San Diego, California) was used to generate graphs. Data are expressed as median (interquartile range) and mean ± standard deviation (⁠ ± SD). Distributions between the 2 groups were assessed by using the χ² and t tests. Correlation analysis between uPLA2R-Ab and sPLA2R-Ab, urinary protein, and other data was performed by using Spearman rank correlation coefficients. The cutoff value for PLA2R-Ab detection was defined as ± 2SD from P⁻, MCD, IgAN, and healthy individuals. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The best cutoff value of the hazard stratification grouping was defined from the Youden index. All statistical analyses were 2-tailed, and differences were considered statistically significant at P < .05.

As shown in Figure 1, A and B, the contrast ratio represents the ratio between the strongly positive and healthy groups; 1:10 was selected as the appropriate dilution for urine PLA2R-IgG (uPLA2R-IgG) detection. The original solution was selected as the optimal experimental solution for urine PLA2R-IgG4 (uPLA2R-IgG4) detection.

The dilution ratios for sPLA2R-IgG and sPLA2R-IgG4 detection were 1:100 and 1:20, respectively, as previously reported.¹⁸  To ensure that the final concentrations of serum and urine were at the same dilution, the final results of uPLA2R-IgG and uPLA2R-IgG4 were divided by 10 and 20, respectively.

The linear relationship between sPLA2R-IgG and uPLA2R-IgG yielded an R value of 0.7893 and Spearman rank correlation coefficient of ρ = 0.693 (Figure 2, A). Figure 2, B, shows the linear relationship between sPLA2R-IgG4 and uPLA2R-IgG4, which yielded an R value of 0.8081 and a Spearman correlation coefficient of ρ = 0.781. Figure 2, C and D, shows the correlation between uPLA2R-Ab after correction for urinary creatinine and sPLA2R-Ab. It can be seen that detection of PLA2R-IgG has an improved correlation value (R = 0.8001) after urine creatinine correction, whereas PLA2R-IgG4 detection was not improved.

Figure 3, A through D, shows the correlation between PLA2R-Ab and urinary protein. There was a significant correlation between uPLA2R-IgG and urinary protein (P = .01), sPLA2R-IgG4 and urinary protein (P = .03), and uPLA2R-IgG4 and urinary protein (P = .001). As shown in Supplemental Figure 1, A through F, normalized uPLA2R-Ab was used as the x-axis to exclude the effect of urine creatinine. A significant positive correlation was found between uPLA2R-IgG and urinary protein (ρ = 0.546, P = .001). A significant negative correlation was observed between uPLA2R-IgG and serum albumin (ρ = −0.621, P = .001), as well as the estimated glomerular filtration rate (eGFR; ρ = −0.569, P = .001). In PLA2R-IgG4 detection, a positive correlation was found between uPLA2R-IgG4 and urinary protein (ρ = 0.411, P = .02). uPLA2R-Ab was negatively correlated with serum albumin (ρ = −0.466, P = .005) and eGFR (ρ = −0.429, P = .01).

The cutoff values for uPLA2R-IgG and uPLA2R-IgG4 detection in distinguishing between patients with PMN and healthy individuals were 1.26 relative units (RU)/mL and 11.01 ng/mL, respectively. Based on the cutoff value, the sensitivity and specificity of uPLA2R-IgG detection were 41.18% and 100%, respectively. PPV and NPV for uPLA2R-IgG detection were 100% and 67.74%, respectively. Meanwhile, the sensitivity and specificity of uPLA2R-IgG4 detection were 64.71% and 100%, and the PPV and NPV of uPLA2R-IgG4 detection were 100% and 77.36% The result of uPLA2R-IgG4 detection was better than that of uPLA2R-IgG with regard to sensitivity and specificity (Table 1), and uPLA2R-IgG4 detection was found to have a higher sensitivity than the current standard reference item sPLA2R-IgG. Supplemental Table 1 shows the detection values of P⁺, P⁻, MCD, IgAN, and healthy individuals for serum and urine PLA2R-Ab. These results indicate that uPLA2R-Ab can specifically distinguish PMN from other glomerular diseases and from healthy individuals.

This study included 34 patients diagnosed with pathologic PLA2R-positive MN. The initial samples were grouped according to the mitigated results after treatment. The clinical characteristics and detection values of the remission and nonremission groups are presented in Table 2. Renal electron microscope stage was based on the pathologic morphology of renal biopsy specimens under electron microscopy, with tissue fluorescence staining showing positivity for IgG subtypes. The initially detected values of sPLA2R-Ab and uPLA2R-Ab in the nonremission group were significantly higher than in the remission group. In addition, the serum creatinine level in the nonremission group was significantly different from that in the remission group.

One patient in the remission group had a quick recovery after 12 months of treatment (Supplemental Table 2). Her initial sPLA2R-IgG detection value was 506.16 RU/mL, and the sPLA2R-IgG4 value was 40 233.66 ng/mL, which were both higher than the average of the nonremission group. Her initial uPLA2R-IgG detection value was 2.93 RU/mL, and the uPLA2R-IgG4 value was 122.99 ng/mL, which were both lower than the average of the nonremission group and were close to those of the remission group. Another patient had the same results for PLA2R-IgG4 detection (Supplemental Table 2). His initial sPLA2R-IgG4 detection value was 17 516.34 ng/mL, which was higher than that in the nonremission group, and his uPLA2R-IgG4 value was 44.64 ng/mL, which was close to the median of the remission group.

Between the remission and nonremission groups, the area under the receiver operating characteristic curve (AUC) of sPLA2R-IgG, uPLA2R-IgG, sPLA2R-IgG4, and uPLA2R-IgG4 was 0.9702, 0.9821, 0.9583, and 0.9940, respectively (Supplemental Figure 2). The AUC of the direct combined detection of serum and urine PLA2R-IgG and PLA2R-IgG4 was 0.9821 and 0.9940, respectively (Supplemental Figure 3, A and B). Direct serum-urine combined detection does not provide a significant boost to the already high AUC.

According to the definition given in the 2021 Kidney Disease: Improving Global Outcomes (KDIGO) guidelines,¹⁹  the cutoff value of sPLA2R-IgG in the remission and nonremission groups is 50 RU/mL. According to the Youden index, the cutoff values of uPLA2R-IgG, sPLA2R-IgG4, and uPLA2R-IgG4 between the remission and nonremission groups are 3.51 RU/mL, 6835 ng/mL, and 143.4 ng/mL, respectively.

Patients with PLA2R-Ab greater than 50 RU/mL were selected. In Figure 4, the nonremission group was drawn as blue, and the remission group was defined as white. The portion of cutoff value of uPLA2R-IgG greater than 3.51 RU/mL, sPLA2R-IgG4 greater than 6835 ng/mL, and uPLA2R-IgG4 greater than 143.4 ng/mL was drawn as blue, and insufficiency was defined as white. Meanwhile, patients with values above the indexed high-risk determination value were drawn as blue: urinary protein greater than 3.5 g/24 h, serum albumin concentration below 25 g/L, serum creatinine level above 1.24 mg/dL, eGFR below 90 mL/min; and those not reaching the threshold were defined as white. In the nonremission population classified by the KDIGO guidelines, in addition to the actual 6 nonremission cases, 5 patients in the remission group were included; the accuracy of sPLA2R-IgG in judging the risk stratification of PMN was only 54.5%. Compared with the actual group pattern, the accuracy of uPLA2R-Ab in secondary judgment of the prognosis of patients with PMN reached 100%. The prognosis of patients with suspected high-risk PMN (11 cases of sPLA2R-IgG >50 RU/mL), using uPLA2R-IgG and uPLA2R-IgG4, was the most accurate in determining risk stratification in patients with PMN, in comparison with actual outcomes, and reached 100%; the hazard stratification accuracy of sPLA2R-IgG4, urinary protein, serum albumin, serum creatinine, and eGFR below 90 mL/min was 90.9%, 72.7%, 63.6%, 63.6%, and 53.6%, respectively.

MN is an immune-mediated disease. When the autoantibody IgG (mainly IgG4)–antigen complex is deposited on the subepithelial aspect of the GBM, it leads to the disruption of glomerular filtration,²⁰  renal filtration impairment, urinary protein, and hypoproteinemia²¹ ; PLA2R-Ab as a molecular protein would then enter the urine, and therefore uPLA2R-Ab can also serve as a biomarker in PMN detection.

Urinary protein is a clinical monitor marker of renal injury,²  and there was a significant correlation between uPLA2R-Ab and urinary protein; compared to the urinary protein, PLA2R-Ab was more specific in PMN, indicating that uPLA2R-Ab may be useful as a monitoring indicator for PMN. However, no significant correlation was found between urinary protein and risk stratification of PMN in this trial (P = .81). The significant correlation between uPLA2R-Ab and PMN risk stratification (uPLA2R-IgG, P < .001; uPLA2R-IgG4, P < .001) indicated their potential application as a predictor of PMN disease and a condition for classification. The detection of uPLA2R-Ab from damaged glomerular filtration membranes has higher sensitivity and disease relevance than does urinary protein. This is not only because of the large difference between the average molecular weights of urinary protein and uPLA2R-Ab, but also because changes in the concentration of uPLA2R-Ab surpass changes in urinary proteins. When a large amount of uPLA2R-Ab leaks from the glomerular filtration membrane into the urine, it represents a high-risk PMN.

There have been several reports on urine MN biomarkers, including the direct detection of uPLA2R-Ab by enzyme-linked immunosorbent assay (ELISA)¹⁷  and the detection of urine IgG.¹³  The study by Wang et al¹⁷  showed that the correlation coefficient between uPLA2R-Ab and sPLA2R-Ab was R = 0.459 (ELISA) and R = 0.508 (indirect immunofluorescence). In our study, a highly sensitive TRFIA method was applied, and the correlation coefficient between uPLA2R-Ab and sPLA2R-Ab reached an R value of 0.7893 (uPLA2R-IgG) and R = 0.8081 (uPLA2R-IgG4). Compared with ELISA,^11,22  the TRFIA method improved the detection sensitivity of PLA2R-IgG and PLA2R-IgG4 to 74% and 90%, respectively.

In serum tests, PLA2R-IgG4 detection has better specificity than the PLA2R-IgG assay.¹⁰  Considering that IgG4 is the only anionic IgG subtype, it is less likely to enter urine from normal renal tissue via GBM than the other subtypes.²³  However, in patients with PMN, after damage to glomerular podocytes, the renal filtration rate of IgG4 increases because of a change in its quantity and charge characteristics.^24,25  In PMN detection, uPLA2R-IgG4 has higher precision than uPLA2R-IgG.^12,26  In our study, the sensitivity of uPLA2R-IgG4 detection was 64.71%, which was higher than that of conventional sPLA2R-IgG detection (61.76%). In therapeutic monitoring of patients with MN, uPLA2R-IgG4 is more convenient and has greater accuracy than serum PLA2R-IgG.

Using an sPLA2R-IgG value greater than 50 RU/mL as a criterion to determine the risk stratification of patients with MN, 11 patients were classified into the nonremission group—more than the actual 6 patients. After using uPLA2R-IgG as an auxiliary criterion to exclude patients with values less than 3.51 RU/mL, the accuracy in patient risk stratification could reach 100%. The same sPLA2R-IgG4 value of 6835 ng/mL classified 8 people as belonging to the nonremission group, and after adjusting by using uPLA2R-IgG4 greater than 143.3 ng/mL, 2 cases in the remission group were excluded, allowing for 100% accuracy in risk stratification. The PPV of sPLA2R-IgG for distinguishing PMN prognosis was 54.55%, whereas PPV with uPLA2R-IgG as the adjunct reached 100%. The PPV of sPLA2R-IgG4 for distinguishing PMN prognosis was 75%, whereas PPV with uPLA2R-IgG4 as an adjunct reached 100%. The NPV was 100% for all 4 judgments.

In PLA2R-IgG detection, the initial sPLA2R-IgG level of one patient (red square in Figure 5, A and B) was 10.12 times higher than the standard provided by the KDIGO guidelines¹⁹ ; however, after a series of treatments, the patient’s metrics met the mitigation requirements. That is, when a patient’s sPLA2R-Ab level is high, rapid remission is still possible. This condition does not meet the risk classification of the KDIGO guidelines.¹⁹  Notably, the patient’s uPLA2R-IgG level was very low, close to the average level in the remission group. Similar results were observed for PLA2R-IgG4 detection. Two patients (red and blue squares in Figure 5, C and D) had high initial sPLA2R-IgG concentrations, much higher than the average value of the remission group, and close to those of the nonremission group, but their values returned to normal after treatment. Similarly, their uPLA2R-IgG4 levels were particularly low, almost as low as those of the remission group.

Disease risk stratification for MN includes the severity of MN and physical condition of the patient. Serum PLA2R-Ab could not adequately represent renal damage, so sPLA2R-Ab alone is not accurate for risk stratification of PMN. We attempted to establish a criterion for risk stratification of PMN by combining serum and urine PLA2R-Ab detection, with sPLA2R-Ab as the mainstay indicator and supplemented with uPLA2R-Ab, which can better predict the disease status of patients. When a patient’s sPLA2R-IgG level is higher than 50 RU/mL as a prerequisite and the uPLA2R-IgG level is also greater than 3.51 RU/mL, the patient’s prognosis is high risk; if uPLA2R-IgG is less than 3.51 RU/mL, the patient’s likelihood of a favorable outcome is greater. Similarly, when a patient’s sPLA2R-IgG4 level is greater than 6835 ng/mL and the value of uPLA2R-IgG4 is 143.4 ng/mL or higher, the patient is at high risk; otherwise, they are not.

The advantages of sPLA2R-Ab detection for assessing the severity of PMN have been confirmed by previous studies,²⁷  and the uPLA2R-Ab assay could be used as a painless option for the diagnosis and treatment of patients with PMN. Therefore, we report a mode of hazard stratification using sPLA2R-Ab as the main assessment criterion and uPLA2R-Ab as the auxiliary assessment criterion. The combined detection of sPLA2R-Ab and uPLA2R-Ab can optimize the risk assessment of existing PMN disease, leading to better and more prompt medication guidance.

This study applied a highly sensitive TRFIA method for uPLA2R-IgG and uPLA2R-IgG4 detection and confirmed that uPLA2R-IgG4 detection was more sensitive than sPLA2R-IgG detection. The detection of uPLA2R-Ab provides a simpler alternative for prognosis and follow-up of PMN. Combined detection of sPLA2R-Ab and uPLA2R-Ab can achieve better patient risk stratification and improve the monitoring of PMN prognosis. Furthermore, it can provide new possibilities for future exploration in the field of MN research.