Anti-phospholipase A 2 receptor antibody in membranous nephropathy ; an Indian experience

Implication for health policy/practice/research/medical education: In an observational study,a group of patients with membranous nephropathy showed correlation of anti-PLA2R antibodies with disease activity and presence of primary MN. Please cite this paper as: Yachha M, Sharma RK, Mehrotra S, Prasad N, Gupta A, Kaul A, Bhadauria DS. Anti-phospholipase A2 receptor antibody in membranous nephropathy; an Indian experience. J Renal Inj Prev. 2018;7(1):16-21. DOI: 10.15171/jrip.2018.04. Introduction: Autoantibodies against phospholipase-A2 Receptor (PLA2R), a normally occurring antigen presented on podocyte membranes, have recently been implicated in the pathology of membranous nephropathy (MN). Objectives: In this observational study, we have evaluated the correlation of patient’s disease activity against serum levels of anti-PLA2R in patients with primary and secondary MN in a North Indian cohort. Patients and Methods: We measured serum anti-PLA2R antibody by ELISA in 63 adult patients with MN. Out of these 63 patients, the majority (58) had primary MN (pMN) and the remaining five had secondary MN (sMN). Results: Around 55.2% had detectable anti-PLA2R autoantibodies (63% in those with newonset pMN, 67% in patients with relapse and 3% in patients in remission). However, all patients with refractory pMN (n = 4) were anti-PLA2R negative. There was significantly higher anti-PLA2R positivity in active stage of disease in contrast to those in remission (P < 0.001). Autoantibody level was proportional to the disease activity, with a trend towards significance (P = 0.052). Hypoalbuminemia and proteinuria were also significantly worse in the antiPLA2R positive patients. All patients with sMN tested negative for anti-PLA2R autoantibodies. Conclusion: PLA2R is a major target antigen in Indian population with idiopathic MN. AntiPLA2R positivity is a specific tool to detect idiopathic MN, and its levels correspond well with other disease activity markers. In addition, anti-PLA2R antibody was negative in refractory pMN, which suggests the existence of additional autoantibodies in this subgroup, with a different target antigen. These autoantibodies may be resistant to the currently recommended immunosuppressants for the disease, thus the refractory nature of the disease. A R T I C L E I N F O


Introduction
Membranous nephropathy (MN) is amongst the most common aetiologies of nephrotic syndrome in adults, particularly in the non-diabetic population (1)(2)(3).In majority, almost three-quarter cases, MN is idiopathic or primary (pMN), however in remaining patients, it occurs secondary to a variety of conditions including hepatitis B, systemic lupus erythematosus (SLE), thyroiditis, malignancies and drugs like gold, penicillamine and captopril (4,5).Based on the rat model of Heymann's nephritis, autoimmune mechanisms were implicated in the genesis of MN (6,7).In 2009, Beck et al in their landmark study reported M-type phospholipase A 2 receptor (PLA 2 R), on glomerular podocytes, to be the target antigen in 26 of their 37 (70%) patients having idiopathic MN (iMN).The circulating anti-PLA 2 R antibodies target a specific region of the PLA 2 R protein and are predominantly of the IgG4-type (8)(9)(10)(11).Similar results with prevalence of anti-PLA 2 R antibodies ranging from 60%-80% in idiopathic MN have been observed by different researchers worldwide (12)(13)(14)(15)(16)(17).The anti-PLA 2 R antibodies have been associated with high specificity and sensitivity of approximately 95% and 75% respectively for diagnosis of pMN.Few recent studies have also reported correlation of anti-PLA 2 R levels with the clinical severity, with higher spontaneous remission rates observed in patients and with lower antibody titres.They can therefore be considered as a non-invasive marker for both diagnosis and prognosis in iMN (14)(15)(16)(17)(18)(19).The US Food and Drug Administration (FDA) in 2014, accepted two commercially available tests for anti-PLA 2 R autoantibodies, including an enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IIFA).

Objectives
In this observational study from a tertiary institute in North India, we have evaluated the correlation of patient's disease activity with serum anti-PLA 2 R levels in primary and secondary forms of MN with variable levels of disease activity.

Patients
Adult patients ≥18 years of age, with biopsy-proven MN, including newly diagnosed secondary MN were prospectively enrolled over a period of 12 months, from January 2015 to December 2015.Majority of the patients were started on angiotensinconverting-enzyme inhibitor (ACE inhibitor) or angiotensin II receptor blockers (ARBs).Decision to initiate immunosuppressive therapy (IST) was made in the follow-up, depending on the trending levels of 24hour urinary-protein and serum creatinine (according to KDIGO guidelines 2012).Baseline characteristics of all the patients were recorded at the beginning of the study.All patients were worked up for any underlying secondary cause of MN, which included assessment of viral markers, ANA, anti-dsDNA, C3, C4, chest X-ray, PSA in men, breast examination in women, stool for occult blood, history of any implicated drugs.Patients were accordingly labelled as having pMN or sMN.'New-onset disease' was defined as patients with having <6 months of diagnosis and no previous exposure to immunosuppressive therapy.Remission, relapse and refractory disease were defined according to the KDIGO 2012 definitions (20).Clinical data was prospectively collected.

Quantification of circulating anti-PLA 2 R
ELISA kit (EUROIMMUN AG, Lubick, Germany) containing PLA 2 R1-coated microplates was used to determine the circulating anti-PLA 2 R antibody levels.After incubation with human sera, diluted 1:100 in sample for 30 minutes, antibodies were detected with anti-human IgG horse-radish peroxidase conjugate (EUROIMMUN AG) diluted 1:1000 in sample buffer for 30 minutes.Chromogen substrate solution (EUROIMMUN AG) was added for 15 minutes after washing.Stopping solution was thereafter used to stop the reaction.An automated microplate, absorbance reader (iMark, Bio-Rad, Veenendaal, the Netherlands) was used to read optical density.PLA 2 R levels of >20 RU/mL were considered to be positive, <14 were taken as negative and levels between 14 and 20 RU/mL were taken as borderline as per the manufacturer specifications.

Ethical consideration
The study has been approved by the Institute Ethical Committee of Sanjay Gandhi Post Graduate Institute of Medical Sciences.Written informed consent was obtained from all patients.All participating patients gave written informed consent prior to inclusion in the study.

Statistical analysis
Continuous variables were presented as the mean ± SD.Frequencies or percentages were used to describe categorical variables.Differences in continuous and categorical variables were assessed using the independent sample t test/Mann-Whitney U test and chi-square / Fisher's exact test, respectively.SPSS version 20 for Windows (IBM, Chicago, Ill., USA) was used for statistical analysis.

Primary versus secondary MN
Amongst 63 patients with MN in our study, 58 patients (92%) had pMN, while 5 patients (8%) had sMN.Amongst patients with sMN, 4 patients had SLE and 1 had hepatitis B infection.Majority of patients with pMN were males (M:F = 2.4), whereas all the patients with sMN were females (SLE being the predominant cause).Mean age of patients with iMN and sMN were 39 years and 32 years respectively.Nearly 57% of patients of iMN (n = 33) had already been initiated on immunosuppressive therapy (IST) at the time of inclusion in the study (modified Ponticelli regimen or calcineurin inhibitors or both), whereas only 1 patient (20%) with sMN had already been put on IST for lupus nephritis.All patients with sMN tested negative for anti-PLA 2 R despite having slightly more severe disease, with higher degree of proteinuria (3656 mg/day versus 3484 mg/day, respectively) and more severe hypoalbuminemia (2.52 g/dL versus 3.37 g/dL in sMN and pMN respectively (Table 1).

Patient characteristics in primary membranous nephropathy
Half (n = 29 (50%)) of the patients with pMN had active disease at the start of study.One-third of our study population were newly diagnosed and had not received any treatment.Six patients (10%) were with disease relapse and 4 (7%) with refractory disease.The remaining 29 (50%) had responded to treatment and were under remission.Amongst the newly diagnosed cases of pMN, 12 cases (63%) were anti-PLA 2 R positive.Similarly, 4 (67%) with Yachha M et al relapse had positive values of PLA 2 R antibodies (Table 2).However, none of the patients who were refractory to treatment had PLA 2 R reactivity (Tables 3 and 4).Only 4 % of those in remission were PLA 2 R positive (Table 2).For further analysis, we grouped the pMN patients with active disease together (those with persistent nephrotic proteinuria, relapse of disease after achieving remission and refractory disease) and compared them to the cohort in remission.The anti-PLA 2 R was significantly more positive in the active pMN group (55.2% in pMN versus 3% in sMN) (P < 0.001) and the anti-PLA 2 R levels also had a trend towards significance (103 ± 275 RU/mL in pMN versus 2 ± 5 RU/ml in sMN) (P = 0.05).

Correlation of clinical activity in pMN patients with anti-PLA2R levels
The anti-PLA 2 R levels correlated with the disease activity.
Those with anti-PLA 2 R positivity, had significantly lower levels of serum albumin and higher levels of proteinuria (P < 0.01) (Figure 1).However there was no significant difference in serum creatinine and cholesterol levels among the groups.Anti-PLA 2 R positivity was also directly proportional to age, with the mean being 45 years in the PLA 2 R positive group as compared to 36 years in the other group (P = 0.01; Table 5).

Discussion
This study was designed to validate PLA 2 R antibody trends in North Indian patients with MN.Amongst the iMN patients, we found anti-PLA 2 R positivity in 55.2% of patients with active disease, in 63% of the newly diagnosed untreated patients, and in 67% with full-blown relapse of disease.This prevalence of anti-PLA 2 R is in concordance with other studies which have  Abbreviations: pMN, primary membranous nephropathy.reported seropositivity ranging from 52%-82% (19)(20)(21)(22)(23).
In another study of 114 newly diagnosed pMN patients from India, Ramachandran et al found, 66.7% anti-PLA 2 R positivity (by ELISA) (24).Lower prevalence in our study may be explained by the fact that we included patients with relapse and refractory disease in our study group.The anti-PLA 2 R negativity in these patients can be explained by the possible presence of other target antigens implicated in pathogenesis of idiopathic MN such as non-PLA 2 R podocyte antigens (25).Anti-PLA 2 R was negative in five patients with secondary MN, majority with SLE as the secondary cause.This is consistent with the current data of anti-PLA 2 R antibody's specificity for idiopathic MN (8,(12)(13)(14)(15)(16)(17)26).Amongst subjects in clinical remission, PLA 2 R was detectable in just one of 29 patients (3%).In contrast, 4 of 6 patients (67%) with relapse had positive PLA 2 R antibodies, thus further corroborating the fact that the levels of PLA 2 R reflect disease activity.Previous studies have also  shown that, the antibody titres disappear with remission and reappear with disease relapses (8,13,14,17,18,(27)(28)(29)(30)(31)(32).Moreover, anti-PLA 2 R levels were in direct proportion to proteinuria and serum albumin levels.Absence of the PLA 2 R antibodies in the four treatmentrefractory patients is suggestive of presence of antibodies to some unidentified antigens, leading to a more resistant variant of the disease.Thus, the aforementioned subgroup requires further genetic and autoimmune evaluation to look for these yet unidentified autoantibodies.No significant difference was observed in the serum creatinine levels between PLA 2 R-positive patients and those with undetectable levels of these antibodies.Males were affected more than females (M: F = 2.4).Majority of our patients were in their third or fourth decade of life.These findings are in consistent with those reported in the literature (33).

Conclusion
To conclude, our data adds weight to the fact that anti-PLA 2 R is a relatively sensitive and specific marker of idiopathic MN in Indian population.Furthermore anti-PLA 2 R positivity and levels correlate with disease activity, with more severe proteinuria and lower serum albumin levels in antibody positive patients.Absence of anti-PLA 2 R in patients with refractory pMN needs further validation and could be suggestive of more severe form of disease with different immune mechanism.Thus, anti-PLA 2 R positivity by ELISA could possibly replace kidney biopsy in patients with suspected MN, and therefore may help to diagnose and predict the disease activity.

Limitations of the study
Our study had certain limitations.We had a relatively small sample size.Our study was confined to patients from Northern Indian belt.In patients in remission, we did not have the baseline anti-PLA 2 R levels when they had proteinuria, and there was lack of follow-up levels of anti-PLA 2 R antibody

Table 1 .
Primary versus secondary membranous nephropathy

Table 2 .
Baseline characteristics of patients with primary membranous nephropathy

Table 3 .
Comparison of pMN patients as per disease activity Abbreviations: pMN, primary membranous nephropathy.

Table 4 .
Characteristics of primary membranous nephropathy patients with refractory disease

Table 5 .
Clinical characteristics of patients with idiopathic membranous nephropathy according to anti-PLA 2 R reactivity PLA2R+Figure1.Prevalence of nephrotic-proteinuria in idiopathic membranous nephropathy according to PLA2R activity.