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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 16  |  Issue : 4  |  Page : 119-123

Role of anti-phospholipase A2 receptor antibodies in monitoring of the clinical status in idiopathic membranous nephropathy in Egyptian patients


1 Department of Internal Medicine and Nephrology, Kasr Al-Aini School of Medicine, Cairo University, Cairo, Egypt
2 Department of Clinical Pathology, Kasr Al-Aini School of Medicine, Cairo University, Cairo, Egypt

Date of Submission07-Dec-2016
Date of Acceptance25-Dec-2016
Date of Web Publication20-Feb-2017

Correspondence Address:
Rabab Mahmoud Ahmed Mahmoud
Department of Internal Medicine and Nephrology, Kasr Al-Aini School of Medicine, Cairo University, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-9165.200356

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  Abstract 

Aim
Phospholipase A2 receptor (PLA2R) in the past few years has been identified as an antigenic target in idiopathic membranous nephropathy (IMN). The question remains, however, whether the diagnostic and prognostic values of anti-phospholipase A2 receptor (APLA2R) antibodies apply to patients with IMN who are of different ethnicities. The aim of this research was to assess the prevalence of APLA2R antibodies in Egyptian patients with IMN and to describe the clinical importance of measuring APLA2R antibodies in those patients.
Patients and methods
Using an indirect immunofluorescence (IF) assay, we measured APLA2R antibodies level in 30 patients with IMN in Egypt (three samples/patient). Patients were divided in two groups: group 1 included 15 consecutive patients at the time of diagnosis and group 2 included 15 consecutive patients during their remission or relapse period.
Results
APLA2R antibodies were detected in 40% of the patients in both groups equally. Overall, 68% of them had a nephrotic-range proteinuria (P=0.002). The titer ranged from 1 : 10 to 1 : 40. The reactive patients had significantly lower serum albumin levels at the presentation (P=0.049), and the average time to remission for them was longer in comparison with the nonreactive patients.
Conclusion
In our study, APLA2R antibodies were found in 40% of the patients. It correlates with the disease activity regarding remission and relapse, and its reactivity was higher in more severe disease.

Keywords: anti-phospholipase a2 receptor antibodies, idiopathic membranous nephropathy, nephrotic syndrome, proteinuria


How to cite this article:
Mahmoud RM, El-din Saadi MG, Fayad TM, Hassaballa MA, Sadek KM, Shaker AM, El-Ansari MM. Role of anti-phospholipase A2 receptor antibodies in monitoring of the clinical status in idiopathic membranous nephropathy in Egyptian patients. J Egypt Soc Nephrol Transplant 2016;16:119-23

How to cite this URL:
Mahmoud RM, El-din Saadi MG, Fayad TM, Hassaballa MA, Sadek KM, Shaker AM, El-Ansari MM. Role of anti-phospholipase A2 receptor antibodies in monitoring of the clinical status in idiopathic membranous nephropathy in Egyptian patients. J Egypt Soc Nephrol Transplant [serial online] 2016 [cited 2017 Jun 29];16:119-23. Available from: http://www.jesnt.eg.net/text.asp?2016/16/4/119/200356


  Introduction Top


Membranous nephropathy is one of the major causes of nephrotic syndrome in nondiabetic adult patients. One of the most important recent advances in the field of clinical nephrology was the discovery of podocyte M-type phospholipase A2 receptor (PLA2R) as an antigenic target in idiopathic membranous nephropathy (IMN) [1]. Since its discovery in 2009, there has been an abundance of literature regarding the use of anti‐phospholipase A2 receptor (APLA2R) antibodies as a diagnostic marker for IMN. The question remains, however, whether the diagnostic and prognostic values of APLA2R antibodies apply to patients with IMN who are of different ethnicities [2].

The objectives of this research are to (a) evaluate the use of APLA2R antibodies in the diagnosis and monitoring of IMN and (b) clarify the role of APLA2R antibodies in prediction of remission or relapse in Egyptian patients.


  Patients and methods Top


Study population and inclusion criteria

A prospective study was done on 30 patients with IMN, who were included in this study from the Nephrology Unit in Cairo University Hospitals and were divided in two groups:

Group 1 included 15 consecutive patients at the time of diagnosis.

Group 2 is a heterogonous group that included 15 consecutive patients with established diagnosis of IMN on treatment and were in either remission or relapse.

Each of these groups was divided into two subgroups: APLA2R reactive patients and APLA2R nonreactive patients.

Diagnosis of membranous nephropathy in all cases was based on renal biopsy.

Light microscopy

Light microscopy showed diffuse thickening of the glomerular basement membrane throughout the glomeruli in the absence of significant hypercellularity.

Immunofluorescence

IF showed diffuse granular pattern of immunoglobulin G and C3 staining along the glomerular basement membrane.

All of our patients received immunosuppression therapy at one point of time during their treatment courses.

These patients were followed up for 9 months.

The local ethics committee approved the study.

Anti-phospholipase A2 receptor antibodies detection

Serum levels of APLA2R antibodies were measured at baseline and two other time points during the disease course by indirect IF test.

PLA2R reactivity indicates any positive sample at any stage of follow-up period.

Association with disease activity was analyzed in correlation with serum albumin (g/dl) and urinary protein excretion (g/day). Other biomarkers of disease activity such as serum creatinine (mg/dl), serum cholesterol (mg/dl), serum triglycerides (mg/dl) were also analyzed.

Exclusion criteria

Causes of secondary membranous nephropathy were excluded by detailed medical history and examination. Serological markers of systemic autoimmunity such as C3, C4, antinuclear antibodies, anti-DNA, and antineutrophil cytoplasmic antibodies; viral hepatitis B and C and HIV infections; fasting blood sugar; postprandial blood sugar; neoplastic conditions; and exposure to toxic agents were also ruled out.

Endpoints definitions

  1. Nephrotic syndrome: serum albumin less than 3.0 g/dl; proteinuria more than 3.5 g/day.
  2. Partial remission: proteinuria reduction of more than 50% with proteinuria less than 3.5 g/day.
  3. Complete remission: proteinuria value less than 0.3 g/24 h in at least three consecutive visits.
  4. Persistent proteinuria: proteinuria more than 3.5 g/day or proteinuria reduction less than 50%.
  5. Relapse: proteinuria more than 3.5 g/d after a period of remission.


Test principle

Indirect immunofluorescence test

The slide contained a mosaic of two different biochips in one incubation field. The first biochip is coated with transfected cells with PLA2R. The second biochip contained nontransfected cells as substrates. For measuring APLA2R antibodies, serum samples were diluted 1/10 in PBS containing 0.2% Tween and incubated for 30 min using the recombinant cell-based assay. A fluorescein isothiocyanate-conjugated antihuman IgG antibody from goat was used to detect bound antibodies using an IF microscope. A specific cytoplasmic fluorescence of transfected cells at a dilution of 1 : 10 or higher was considered as positive.

Statistical analysis

Precoded data were entered on the computer using ‘Microsoft Office Excel Software’ program for windows (2010). Data were then transferred to the statistical package for the social sciences software program, version 21 (SPSS; SPSS Inc., Chicago, Illinois, USA), to be statistically analyzed. Data were summarized using mean and SD for quantitative variables and frequency and percentage for qualitative ones. Comparison between groups was performed using independent sample t-test and χ2 or Fisher’s exact test for qualitative ones. Pearson’s correlation coefficients were calculated to get the association between different quantitative variables. Repeated related measures were tested using Friedman’s test in each group separately. P values less than 0.05 were considered statistically significant and less than 0.01 were considered highly significant.


  Results Top


Sera from 30 patients who met the inclusion criteria were analyzed. We measured the level of APLA2R antibodies in 90 samples from those 30 patients (three samples for every patient).

Group 1 included 15 consecutive patients at the time of diagnosis and group 2 included another 15 consecutive patients during their remission or relapse periods.

The prevalence of APLA2R antibodies in our patients was 40% as shown in [Figure 1]. The titer range from 1/10 to 1/40 at different stages of follow-up.
Figure 1: The prevalence of anti-phospholipase A2 receptor antibodies in both groups.

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There was no statistically significant difference between reactive and nonreactive patients in terms of age and sex distribution in both groups.

In group 2, APLA2R reactive patients had higher prevalence of hypertension, higher serum triglyceride levels, and high frequency of diuretic use. Most of the patients in both groups received prednisone or equivalent, cyclosporine, and mycophenylate mofetil (MMF), but reactive patients used MMF more than the nonreactive group (P=0.01) ([Table 1]).
Table 1: Clinical and laboratory characteristics of the study population

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In group 1, we found that the reactive patients had lower serum albumin at the presentation (P=0.049). After 9 months of follow-up, APLA2R nonreactive patients had a higher serum albumin and less proteinuria than the reactive patients did, but this was statistically insignificant. In group 2 also, serum albumin level in APLA2R reactive patients did not show significant improvement at the end of the follow-up period; the proteinuria for this group was worsened by time but was much improved in APLA2R nonreactive patients, and this was statistically insignificant as shown in [Table 2].
Table 2: Mean serum albumin level and proteinuria (g/day) at different stages of follow-up in both groups

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During the follow-up of group 1, five patients had a positive initial test result for APLA2R. Of them, three with low titers of APLA2R went into remission within 9 months of treatment and developed no relapse until the end of the follow-up period. The other two patients with higher titer of APLA2R antibodies had a persistent proteinuria during follow-up and had positive test results in all the three samples.

Of the 15 patients in group 1, 10 had initially negative samples for PLA2R antibodies; one of them was found to be reactive upon subsequent retesting during the follow-up after increasing of his proteinuria, whereas the other nine patient’s test results remained negative (six of them enter remission during the follow-up period).

In group 2, two patients had a positive initial test result for APLA2R antibodies. Both patients had a persistent proteinuria during our follow-up, and they had positive tests in all the three samples. The remaining 13 patients initially had negative samples for APLA2R antibodies (three of them were in complete remission at the time of first sample). Of these 13 patients, four were found to be reactive upon subsequent retesting at the time of relapse whereas the other nine patient’s test results remained negative (six of them entered remission during the follow-up period).

APLA2R antibodies were associated with nephrotic-range proteinuria in 68.1%, and this was statistically significant (P=0.002).

Another important finding as shown in [Table 3] was that the number of patients who entered remission was double in nonreactive patient group than in reactive patient group, and time to enter a remission was shorter in nonreactive patients. These results suggested that patients who had negative test result for APLA2R antibodies enter remission earlier, but this was statistically insignificant.
Table 3: Comparison between number and time of remission in reactive and nonreactive patients

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  Discussion Top


A number of studies have reported that prevalence of APLA2R antibodies varies between 52% in a German study and 82% in a Chinese cohort [3],[4]. These differences may be explained by differences in ethnicity, technique of the assay, and clinical characteristics of the included patients. This study sets out with the aim of assessing the prevalence of APLA2R antibodies in Egyptian patients with IMN and to clarify the role of APLA2R antibodies in prediction of remission or relapse.

The results of our study show that 12 (40%) patients of 30 had detectable APLA2R antibodies. Our results come close to the results of the work of Hoxha et al. [3] in Germany.

A possible explanation for our result might be the smaller number of patients in our study or there may be target antigens other than phospholipase A2, which is unknown yet, leading to comparable clinical disease [5].

As the second autoantigen is thrombospondin type 1 domain-containing A, and circulating autoantibodies against this protein were detected in 5–10% of patients negative for APLA2R [2]. In addition, we did not assess APLA2 in the biopsies from our patients. Also it is possible that our patients with negative APLA2R antibodies at the end of follow-up but had a persistent proteinuria had actually remittent immunologically but the residual proteinuria is present on basis of secondary focal segmental lesions or tubulointerstitial damage as explained by the work of Beck and Salant [6]. Another explanation is rapid clearance of the antibody from the blood because of high affinity for PLA2R [2].

In the current study, the prevalence of APLA2R reactivity was higher in patients with nephrotic-range proteinuria and when there is more use of diuretics and MMF. This comes in agreement with the study by Oh et al. [7], which demonstrates that the prevalence of APLA2R reactivity was 80% in patients with IMN and nephrotic-range proteinuria.

Of the six APLA2 positive patients in group 1, five were positive at the diagnosis, and only one case had negative result in the first sample that turned positive; this may be because of too early sampling when the disease was still starting, as evident clinically by progression of proteinuria and hypoalbuminemia in this patient. Of these six reactive patients in the same group, three turned negative in the second sample and continued to be negative in the third sample also. This denotes remission and a good response to therapy evident by the clinical remission, which was observed in the follow-up.

In group 2, two of six patients were APLA2 reactive and persistent positive in the second and third samples, and this was correlated with the clinical activity. In another case, the proteinuria started to decrease and serum albumin became increased, and actually this correlates with the APLA2R antibodies titer which started to decrease to 1/10 as well.

The other four were negative at the beginning but turned positive in the second sample in one patient and in the third sample in the remaining three patients, and this was correlated clinically, as it predicts the relapse, and the decrease in the titer was associated by a remission in the same patient.

On contrary, APLA2R antibodies may be found in remission because of the following:

  1. The positive antibodies may disappear later.
  2. The remission should not be based on proteinuria alone because variability in proteinuria caused by dosing and compliance with inhibitors of the renin–angiotensin system and/or declining renal function may also lead to spurious conclusions about remission status [6].



  Conclusion Top


Our study concluded that 40% of Egyptian patients had detectable anti-PLA2 antibodies in there serum samples. Antibodies against PLA2R were present during clinically significant disease activity, with a decline or disappearance of APLA2R antibodies before or during the remission periods. These findings suggest that in general, APLA2R antibodies can be used as a biomarker in IMN, and it correlates with the disease activity and can be used for monitoring of the clinical status of those patients. Further work needs to be done to establish whether APLA2R antibodies can affect the treatment strategy in IMN or not.

Limitations

Small sample size, short duration of follow-up, and lack of biopsy PLA2R data were the limitations of the study.

Acknowledgements

The authors thank Dr Maher Fouad for his support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Beck L, Bonegio R, Lambeau G, Beck DM, Powell DW, Cummins TD et al. M-type phospholipase A2 receptor as target antigen in idiopathic MN. N Engl J Med 2009; 361:11–21.  Back to cited text no. 1
    
2.
Ronco P, Debiec H. Pathophysiological advances in membranous nephropathy: time for a shift in patient’s care. Lancet 2015; 385:1983–1992.  Back to cited text no. 2
    
3.
Hoxha E, Harendza S, Zahner G, Panzer U, Steinmetz O, Fechner K et al. An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis. Nephrol Dial Transplant. 2011 26:2526–2532.  Back to cited text no. 3
    
4.
Qin W, Beck LH Jr, Zeng C, Chen Z, Li S, Zuo K et al. Anti-phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol 2011; 22:1137–1143.  Back to cited text no. 4
    
5.
Hofstra J, Wetzels J. Anti-PLA2R antibodies in membranous nephropathy: ready for routine clinical practice? Neth J Med 2012; 70:109–113.  Back to cited text no. 5
    
6.
Beck L, Salant DJ. Membranous nephropathy: recent travels and new roads ahead. Kidney Int 2010; 77:765–770.  Back to cited text no. 6
    
7.
Oh YJ, Yang SH, Kim DK, Kang S-W, Kim YS. Autoantibodies against phospholipase A2 receptor in Korean patients with membranous nephropathy. PLoS One 2013; 8:e62151.  Back to cited text no. 7
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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