Serum anti-phospholipase A2 receptor antibody in pathological diagnosis of type 2 diabetes mellitus patients with proteinuria

Patients with diabetes mellitus complicated with proteinuria can be diabetic nephropathy (DN), diabetic complicated with non-diabetic kidney disease (NDKD), or DN with NDKD. Among these membranous nephropathy accounted for a large proportion of DN with NDRD. At present, serum anti-phospholipase A2 receptor (PLA2R) antibody is widely used in the diagnosis and evaluation of therapy in idiopathic membranous nephropathy, our study aimed to investigate the diagnostic significance of anti-PLA2R antibody in type 2 diabetes mellitus (T2DM) patients with proteinuria, providing a method for patients with contraindications of kidney biopsy. Eighty-seven T2DM patients with proteinuria who went on kidney biopsy were divided into the DN group, idiopathic membranous nephropathy (IMN) group, and others group according to their pathological results. In our study, 52.87% and 28.74% of patients were found to have IMN and diabetic nephropathy respectively. The levels of anti-PLA2R antibody, total cholesterol, triglyceride, and estimated glomerular filtration rate (eGFR) were higher in the IMN group, while the prevalence of diabetic retinopathy (DR), systolic blood pressure (SBP) and HbA1c were higher in the DN group. For T2DM patients with proteinuria, anti-PLA2R antibody (AUC = 0.904, 95%CI 0.838–0.970) has a high diagnostic value for IMN. The duration of diabetes (OR = 0.798, P = 0.030), eGFR level (OR = 1.030, P = 0.024), and positive anti-PLA2R antibody (OR = 72.727, P < 0.001) favor the diagnosis of IMN, while DR (OR = 50.234, P < 0.001), SBP (OR = 1.041, P = 0.030), and negative anti-PLA2R antibody (OR = 0.008, P  = 0.001) is beneficial to the diagnosis of DN. Our study found that NDKD is not uncommon in patients with T2DM and proteinuria, and IMN was the main pathological type. Positive anti-PLA2R antibody has a strong accuracy in the diagnosis of IMN in patients with T2DM and proteinuria.

In 2009, Beck detected an antigen normally expressed on the podocyte cell, the M-type phospholipase A2 receptor (PLA2R) by using western blotting 12 , and the PLA2R antibody may cause IMN in up to 80% of patients 13 .Anti-PLA2R antibody is a type I transmembrane receptor and a member of the mammalian mannose receptor family.Anti-PLA2R antibody and PLA2R form an antigen/antibody complex that is deposited on the glomerular basement membrane, activate the complement system, causes damage to podocytes and the glomerular filtration barrier, and eventually results in proteinuria, which offers clinicians and scientists a powerful tool to differentiate primary from secondary forms and monitor disease activity and response to therapy 14 .The anti-PLA2R antibody has a high specificity in comparing IMN with other glomerular diseases.Zhang showed that the positive rate of anti-PLA2R antibody was 33% in 36 patients with tumor-associated membranous nephropathy.However, due to the possibility of the coexistence of membranous nephropathy and malignant tumors, it is difficult to distinguish IMN with malignant tumors from tumor-associated membranous nephropathy 15 .It still can not be called reliable evidence in distinguishing tumor-associated membranous nephropathy but performing routine screening for anti-PLA2R antibody in patients with MN and malignancy is reasonable 16 .However, this kind of error can be reduced by medical history and laboratory examination.In addition, IMN can be treated with cyclophosphamide and corticosteroids, immunosuppressive therapy, rituximab, or supportive therapy 17 .It was demonstrated that reductions in anti-PLA2R antibody titers predicted disease remission regardless of the method used 18,19 .The increase in antibody titer indicates the development of the disease 20 .
However, there are limited studies on serum anti-PLA2R antibodies in diabetic patients with proteinuria, and the diagnostic effect of anti-PLA2R antibody in this population is still unclear.This study aimed to find the expression of serum anti-PLA2R antibody in patients with T2DM proteinuria and to explore the diagnostic value of antibodies for IMN in this population.

Study population
A total of 107 T2DM patients with 24-h urine protein > 0.5g/d who went on kidney biopsy were recruited in Hebei General Hospital from January 2017 to December 2019.Twenty patients were excluded because of lack of sufficient clinical data, type 1 diabetes, use of steroids, immunosuppressants before kidney biopsy, no anti-PLA2R antibody result, no fundus examination, and no peripheral neuropathy.The remaining 87 patients were divided into the IMN group (n = 46), DN group (n = 25), and T2DM with other glomerulopathy group (n = 16) according to kidney pathological patterns.DN was diagnosed according to the pathological diagnostic criteria established by Tervaert 21 .Obtain informed consent from all recruited patients (Fig. 1).The study was approved by the Institutional Ethics Committee of Hebei General Hospital.The approval number is NO.2021259.We confirm that all analyses were performed according to relevant guidelines and regulations.And confirm the informed consent of all participants.

Statistical analysis
All data were analyzed by using IBM SPSS Statistics software version 25.0.Quantitative variables with normal distribution were expressed as χ ± s , with abnormally distributed data were expressed as [M(P25, P75)].Categorical variables were expressed as percentages.Multiple groups of quantitative variables were compared using ANOVA or Kruskal-WallisH test.Two group differences in quantitative variables were analyzed using the independent samples t-test or Mann-Whitney U test.The Chi-square test was used to compare categorical variables.The receiver operating characteristics (ROC) curve was used to analyze the predictive value of serum anti-PLA2R antibody for IMN.Multivariate logistic regression analysis was performed to identify risk factors of IMN and DN.P value < 0.05 was set to statistically significant.
The comparison of clinical baseline and biochemical characteristics among the three groups is shown in Table 1.Compared with the IMN group, the DN group had a longer duration of diabetes, more patients with DR, DPN, and hypertension, higher levels of SBP, ALB, BUN, Scr, HbA1c, and lower positive rate of anti-PLA2R antibody (P < 0.05).However, there was no significant difference in UTP between the two groups.Compared with other glomerulopathy groups, the DN group had a higher prevalence of DR and SBP, while the IMN group had a higher UTP level and anti-PLA2R antibody positive rate, but lower ALB, BUN, Scr, and eGFR (P < 0.05).In addition, there was no statistically significant difference in sex, age, BMI, family history of diabetes, DBP, UA, and FPG among the three groups (P > 0.05).Moreover, in this study population, the anti-PLA2R (AUC = 0.904, sensitivity: 0.804%, specificity:0.951%,95%CI 0.838-0.970,P < 0.001) antibody has a high diagnostic accuracy for IMN (Fig. 3).
To explore the risk factors for pathological diagnosis of IMN in T2DM patients with proteinuria, the indicators with statistical significance between IMN and DN groups in Table 1 were included in the multivariate logistic regression analysis.The results are shown in Tables 2 and 3. Shorter duration of diabetes (OR = 0.798, P = 0.030), higher eGFR levels (OR = 1.030,P = 0.024), and positive anti-PLA2R antibody (OR = 72.727,P < 0.001) are more likely to be diagnosed as IMN.Meanwhile, patients with higher SBP (OR = 1.041,P = 0.030), DR (OR = 50.234,P < 0.001), or negative anti-PLA2R antibody (OR = 0.008, P = 0.001) were more likely to be diagnosed as DN.

Discussion
The prevalence of T2DM has gradually increased in recent years.DN has surpassed primary glomerular disease as the main cause of chronic kidney disease (CKD) and end-stage kidney disease (ESKD) 23,24 .However, many diabetic patients with proteinuria are classified as NDKD in pathology.Kidney biopsy is the gold standard for pathological diagnosis up to now.But in some undeveloped areas, there are limited conditions to perform kidney biopsy.How to identify the pathological types by non-invasive examination for such patients is becoming an urgent problem.
In our study, 71.26% of patients with T2DM combined with proteinuria were NDKD, among which IMN accounted for 52.87%.The IMN group had an anti-PLA2R antibody positivity rate as high as 80.43%.It suggested that the pathological type of these patients was more likely to be NDKD or DN combined with NDKD when the anti-PLA2R antibody was positive.This is consistent with previous studies.For IMN, anti-PLA2R antibody has higher diagnostic accuracy (sensitivity: 0.804%, specificity: 0.951%) 7,25,26 .
NDKD may be considered when diabetic patients have proteinuria but a short duration of disease.Our study showed the same results as the others [27][28][29] .In addition, DR had a close correlation with the presence of DN, which means that patients without DR were more likely to be diagnosed with NDKD 10 .However, there are still some severe DR patients without DN 30 .Nevertheless, this does not deny the diagnostic value of DR.In this study, the frequency of hypertension in the DN group was higher than in the other two groups, which was consistent with the results of Dong et al 8 .Hypertension not only accelerates the progression of DN but is also an important risk factor for DN.Moreover, it is associated with macrovascular and microvascular complications in T2DM patients 31 .Overall, the kidney prognosis of patients with NDKD is better than that of patients with DN 32 .In our study, patients with higher eGFR levels are more likely to be diagnosed with IMN.However, this prediction is unstable and needs long-term follow-up to prove it.Compared with NDKD patients, DN patients are more prone to suffer from anemia 33 , monitoring of hemoglobin levels may help to identify DN or NDKD 34 .There is no significant difference in FPG between the DN group and the other two groups, but the level of HbA1c is higher than those two groups, which is different from the results of Liu et al 35 .
T2DM patients with proteinuria who have short DM duration, higher eGFR levels, and positive anti-PLA2R antibody tend to be diagnosed as NDKD, and its pathological type is IMN.By contrast, DR patients with negative anti-PLA2R antibody were more likely to be diagnosed as DN.Nonetheless, for the latter, kidney biopsy is still recommended.Anti-PLA2R antibody play an important role in distinguishing DN and NDKD, but they still can not replace kidney biopsy as a diagnostic criterion.
There are several limitations to this study.First, nonalbuminuric diabetic kidney disease (NADKD) was not included in this study.This clinical type of DN usually has a short course of diabetes, and it is rarely complicated with DR 36 .Additionally, the underlying pathology of NADKD might be different from that of the classic phenotype of DN.However, the recruitment of such patients may have a subtle impact on the current outcomes, as the number of them is small.Additionally, although anti-PLA2R antibody has shown high diagnostic accuracy due to the small number of samples in this study, large-scale studies are still needed in the future to verify the conclusion.

Figure 1 .
Figure 1.Flow diagram of the study participants.

Figure 2 .
Figure 2. The distribution of pathological types in T2DM patients with proteinuria.

Table 1 .
Comparison of baselines characteristics among three groups.*Compared with IMN group, P < 0.05, # compared with DN group, P < 0.05.

Table 2 .
Multivariate logistic regression analysis of IMN predictors.

Table 3 .
Multivariate logistic regression analysis of DN predictors.