Home About us Current issue Back issues Submission Instructions Advertise Contact Login   

Search Article 
  
Advanced search 
 
Saudi Journal of Kidney Diseases and Transplantation
Users online: 1204 Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size 
 


 
Table of Contents   
ORIGINAL ARTICLE  
Year : 2018  |  Volume : 29  |  Issue : 3  |  Page : 524-530
Correlation and discrepancy of phospholipase A2 receptor staining in membranous nephropathy in paraffin-embedded kidney biopsies


1 Department of Allied Health Sciences, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Sultanate of Oman
2 Department of Pathology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Sultanate of Oman

Click here for correspondence address and email

Date of Submission06-Jun-2017
Date of Decision10-Jul-2017
Date of Acceptance10-Jul-2017
Date of Web Publication28-Jun-2018
 

   Abstract 

Distinguishing primary from secondary forms of membranous nephropathy (MN) in paraffin-embedded kidney biopsies is challenging. Previous studies have measured the accuracy, sensitivity, and specificity of phospholipase A2 receptor (PLA2R) on MN. However, due to the absence of a gold standard method for diagnosis, further studies are necessary. Here, we measure the correlation and discrepancy of PLA2R on MN in paraffin-embedded kidney biopsies by correlating PLA2R findings with immunofluorescence (IF), light microscopy (LM), and electron microscopy (EM) results. A total of 116 kidney biopsies were investigated including 87 MN and 29 nonmembranous kidney diseases. PLA2R was examined by indirect IF assay. A total of 79 MN cases were subjected to PLA2R staining and histopathological analyses. The remaining eight cases were excluded due to the lack of the glomeruli in the sections. The correlation and discrepancy between PLA2R and IF findings were seen in 65.45% and 34.55%, respectively. PLA2R and EM findings showed a 65.52% correlation and 34.48% discrepancy. LM features and PLA2R findings showed a correlation of 58.44% and discrepancy of 41.56%. Overall correlation between histopathology and PLA2R was seen in 65.82% and 34.18% showed discordant cases. All 29 non-membranous MN cases were negative for PLA2R. Staining for PLA2R could be a useful adjunctive tool in the stratification of cases of MN. The findings of this study strongly recommend the use of PLA2R IF staining method as a routine test for all MN cases.

How to cite this article:
Alwahaibi NY, Alrawahi UY, Alriyami MA. Correlation and discrepancy of phospholipase A2 receptor staining in membranous nephropathy in paraffin-embedded kidney biopsies. Saudi J Kidney Dis Transpl 2018;29:524-30

How to cite this URL:
Alwahaibi NY, Alrawahi UY, Alriyami MA. Correlation and discrepancy of phospholipase A2 receptor staining in membranous nephropathy in paraffin-embedded kidney biopsies. Saudi J Kidney Dis Transpl [serial online] 2018 [cited 2018 Sep 23];29:524-30. Available from: http://www.sjkdt.org/text.asp?2018/29/3/524/235181

   Introduction Top


Worldwide, kidney diseases contribute greatly to morbidity and mortality.[1] The incidence of membranous nephropathy (MN) is one case per 100,000 people per year.[2] In developed countries, 80% of MN cases are primary MN, while 20% are secondary MN.[3] MN is an autoimmune disease that attacks the renal glome-ruli.[4] The current diagnosis of MN is based on the clinical history, physical examination, and renal biopsy findings including immunofluo-rescence (IF), light microscopy (LM), and electron microscopy (EM).[5] Secondary MN is usually associated with autoimmune disorders such as systemic lupus erythematous, rheumatoid arthritis, connective tissue diseases, sarcoidosis, neoplasms such as lung, colon, breast, kidney, stomach and non-Hodgkin's lymphoma, infections, and medications.[6] If these conditions are absent, then it is termed primary MN. However, these tests cannot accurately distinguish between primary and secondary forms of MN. It is clinically important to discriminate between these two entities as different treatments apply. The treatment for patients with secondary MN is directed toward the underlying etiology, whereas patients with primary MN are usually treated with immunosuppressive therapy.

Since PLA2R discovery in 2009, there is an increasing interest in the study of PLA2R as a target antigen in the diagnosis of primary MN.[7] Serologic tests for the diagnosis of PLA2R have a sensitivity and specificity of 70%–82% and 57%–89%, respectively.[8] However, there are limited data available on the accuracy of PLA2R staining in kidney paraffin sections, in particular, in Arab countries, where and according to the best of our knowledge, no study has been performed on PLA2R staining in cases of biopsy-proven MN. Furthermore, we report a detailed comparison between IF, LM, and EM and PLA2R findings of MN. Therefore, the aim of this study was to detect the presence of PLA2R in kidney paraffin sections of MN and correlate this with other histo-pathological features.


   Materials and Methods Top


During the period from 2006 to 2015, a total of 116 cases were investigated including 87 cases of MN and 29 cases of nonmembranous kidney diseases. These nonmembranous kidney diseases included eight cases of IgA nephropathy, six nephritis, six focal segmental glomerulonephritis, seven minimal change disease, and two lupus nephritis. PLA2R was examined by indirect IF assay using comercial antibodies, rabbit polyclonal anti-PLA2R antibodies (atlas anti-bodies), and fluorescein isothiocyanate-conjugated monoclonal goat anti-rabbit IgG antibodies (sigma antibodies). Several optimization experiments were performed till the ideal method was reached [Table 1]. Known positive and negative controls were run with each set. The stain was examined by an IF microscope (Olympus, BX50F4, Japan). A positive reaction was seen as granular staining with green fluorescence along glomerular capillary loops. All slides were evaluated by two bio-medical scientists and confirmed by a renal pathologist.
Table 1: Phospholipase A2 receptor immunofluorescence staining method.

Click here to view


The findings of PLA2R were correlated with other three histopathological parameters: LM, mainly for the presence of mesangial hyper-cellularity; IF, mainly for the presence of IgA, IgG, IgM, C3, fibrin, and C1q markers; and finally EM, mainly for the presence of mesangial and subendothelial deposits.


   Statistical Analysis Top


The data were analyzed by the Statistical Package for the Social Sciences (SPSS) version 19.0 for Windows (SPSS Inc., Chicago, IL, USA). For comparison analysis, Chi-square test was used. Ethical approval was obtained from the Medical Research Committee and Ethics Committee at Sultan Qaboos University, College of Medicine and Health Sciences, Oman (MREC# 925).


   Results Top


There were a total of 87 MN patients ranging in age from two to 76 years. The average age was 33.71 years. A total of 54 of 87 (62.1%) cases were in the age between 20 and 40 years, whereas only nine (10.3%) cases were <20 years old. Most cases of <20 years and between 20 and 40 years were favored to have a secondary MN. In 34 of 87 (39.1%) cases were found in males, while the remaining 53 (60.9%) cases were found in females with a ratio of 1:1.56. All cases of nonmembranous kidney diseases showed a negative reaction for PLA2R.

Phospholipase A2 receptor and LM findings

The presence of mesangial hypercellularity in renal glomeruli highly favors secondary MN. A total of ten cases were excluded due to the lack of the glomeruli in the paraffin sections. A total of 51 cases were found to favor secondary MN, of which 33 of 51 (84.7%) cases showed negative reaction for PLA2R. A total of 26 cases favored primary MN, of which 12 of 26 (46.2%) cases showed a positive reaction for PLA2R [Table 2]. Correlation was seen in 58.44% while discrepancy was seen in 41.56%.
Table 2: Comparison between phospholipase A2 receptor and hyper-cellularity proliferation of membranous nephropathy cases.

Click here to view


Phospholipase AZ receptor and EM findings

The presence of mesangial and subendo-thelial deposits in the kidney tissues favors secondary MN. A total of 29 cases were excluded due to the lack of the glomeruli in the resin sections. A total of 41 cases were found to favor secondary MN, of which 28 of 41 (68.3%) cases showed negative reaction for PLA2R. A total of 17 cases favored primary MN, of which10 of 17 cases (58.8%) showed positive reaction for PLA2R [Table 3]. Correlation was seen in 65.52%, while discrepancy was seen in 34.48%.
Table 3: Comparison between phospholipase A2 receptor and mesangial and sub-endothelial deposits of membranous nephropathy cases.

Click here to view


Phospholipase A2 receptor and immunofluorescence findings

The presence of IgA, IgG, IgM, C3c, and C1q IF markers in the kidney tissues favor secondary MN. A total of 32 cases were excluded due to the lack of the glomeruli in the frozen sections. A total of 35 cases were found to favor secondary MN, of which 25 of 35 (71.4%) showed negative reaction for PLA2R. A total of 20 cases found to favor primary MN, Of which 11 of 20 (55%) showed a positive reaction for PLA2R [Table 4]. Correlation was seen in 65.45%, while discrepancy was seen in 34.55% cases.
Table 4: Comparison between phospholipase A2 receptor and immunofluorescence findings of membranous nephropathy cases.

Click here to view


Phospholipase A2 receptor and overall histo-pathological findings

Histopathological findings, including LM, EM, and IF as well as the clinical history, were used to classify MN into primary and secondary. The terminology used in the histopathological archival reports was either favor primary or favor secondary MN. A total of eight cases were excluded due to the lack of the glomeruli in the sections. A total of 47 cases were found to favor secondary MN, of which 72.3% showed negative reaction for PLA2R [Figure 1]a. A total of 32 cases favored primary MN. In 18 of 32 (56.3%) cases showed positive reaction for PLA2R [Table 5] [Figure 1]b. Overall correlation between histo-pathology that takes into account the LM, IF, and EM findings together and the presence or absence of PLA2R findings staining was seen in 52 (18 + 34) of 79 cases (65.82%), whereas discrepancy was seen in 27 (13 + 14) of 79 cases (34.18%).
Figure 1: PLA2R staining in MN. (a) Negative staining for PLA2R (indirect immunofluorescence method, x40), (b) Positive staining for PLA2R in glomerular capillary loops of primary MN (indirect immunofluorescence method, x40).
PLA2R: Phospholipase A2 receptor, MN: Membranous nephropathy.


Click here to view
Table 5: Correlation between histopathology and phospholipase A2 receptor findings of membranous nephropathy cases.

Click here to view



   Discussion Top


We report a detailed correlation between histo-pathological findings including LM, IF, and EM, and PLA2R results in paraffin-embedded kidney biopsies of MN cases. The accuracy, sensitivity, and specificity cannot be measured due to the absence of gold standard method. As mentioned earlier, histopathological findings with clinical history of the patients can only indicate the probability of being primary or secondary. Thus, correlation and discrepancy are used for an accurate measurement. This study shows that PLA2R staining has a correlation of 65.82% with primary MN and a discre-pancy of 34.18 % with secondary MN.

Light microscopic features of MN do not correlate well with PLA2R findings as only 58.44% showed correlation. This might indicate that the diagnosis of MN by LM only is not reliable to distinguish primary from secondary. This finding is consistent with other study.[9] However, ultrastructural features of MN show a better correlation with PLA2R findings as 65.5% of cases were correlated. We report that mesangial and subendothelial deposits in the kidney tissues are good indicators of secondary MN but are not specific. Another study has also used the same criteria.[6] Discordant cases between EM and PLA2R of MN were seen in only 34.48% cases.

The current study shows that IF markers, including IgG, IgM, IgA, C3, and C1q, correlate well with PLA2R findings in MN cases. The percentage (65.45%) is almost similar to that of EM findings. The presence of all IF markers and mesangial and subendothelial deposits in the kidney tissues of MN cases favors secondary MN. This finding has also been reported by several studies.[6],[10],[11] However, those studies reported the sensitivity and specificity rather than the correlation and discrepancy. In fact, some studies have reported higher correlations between all IF markers and mesangial and subendothelial deposits with secondary MN.[5],[12],[14]

The discrepancy of PLA2R and overall histo-pathological findings in MN cases could be due to several reasons. First and most likely, misdiagnoses of MN cases, cases were reported secondary, but they were actually primary and vice versa. This is inline with other studies.[14],[15] Second, rapid clearance of PLA2R in kidney tissues.[16] Third, the levels of PLA2R antigen are too low to be detected by this method. Lastly its less likely there is a presence of two diseases at the same time.[12] Despite the discordant cases and suggestive explanations between histopathology and PLA2R findings, the absence of PLA2R in paraffin-embedded kidney biopsies of MN does not rule out the possible diagnosis of primary MN. This finding is consistent with many reported similar studies.[14],[15],[16],[17] This finding enhances our proposed theory that the relation between histopathology and PLA2R findings should be measured by correlation and discrepancy rather than the sensitivity and specificity.

According to our knowledge, there is no study performed in the Arab countries on PLA2R of MN cases. Previous studies in Asian countries reported mainly the sensitivity in serum PLA2R antibodies of MN cases. In Iran, anti-PLA2R antibodies were detected in 74% of patients with primary MN.[17] In Japan, serum anti-PLA2R antibodies and glomerular PLA2R deposits in renal biopsies were detected in 55% and 64% of patients with primary MN, respectively.[18] In China, serum anti-PLA2R antibodies were seen in 82% of primary MN.[4] In Korea, serum anti-PLA2R antibodies were demonstrated in 69% of primary MN.[19]

The demonstration of PLA2R in paraffin-embedded kidney biopsies of MN cases by indirect IF method is quite straightforward. The method consists of two steps. The inter-pretation is easy where green fluorescence against black background is well seen. In this study, we used a standard IF microscope, which usually used to examine other IF markers. Intensity was also measured using similar approach to that used for IF markers. Most of the cases show strong reaction (three plus) for PLA2R. Our biomedical scientists and renal pathologists found that the evaluation of PLA2R is easy. Nonspecific reaction was not observed in this method. The PLA2R IF staining was negative in all non-membranous 29 cases, which shows that PLA2R is only found in membranous cases. This finding is consistent with other studies.[12],[20]

When compared with males, both primary and secondary MN forms are more common in females. The reason for the predominance of secondary MN in females could be due to that autoimmune diseases such as systemic lupus erythematosus and mixed connective tissue diseases which are more common in females while the reason of predominance of primary MN in females is unknown. Furthermore, it was noticed that our study had a male-to-female ratio of 1:1.56. This was in agreement with some studies[2] but not others which reported higher male-to-female ratio.[21]

In conclusion, staining for PLA2R could be a useful adjunctive tool in the stratification of cases of MN. Cases showing strong positive staining for PLA2R are most likely to be of a primary form that would be treated by immuno-suppression, particularly in cases where LM, IF, and EM findings are either absent or inconclusive. Whereas, in similar situations, the absence of PLA2R staining should prompt a careful search for a secondary cause, albeit recognizing that negative PLA2R staining does not exclude a primary MN. The findings of this study strongly recommend the use of PLA2R IF staining method as a routine test for all MN cases.

Conflict of interest: None declared.

 
   References Top

1.
Levey AS, Atkins R, Coresh J, et al. Chronic kidney disease as a global public health problem: Approaches and initiatives - A position statement from Kidney Disease Improving Global Outcomes. Kidney Int 2007;72:247-59.  Back to cited text no. 1
[PUBMED]    
2.
Stanescu HC, Arcos-Burgos M, Medlar A, et al. Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy. N Engl J Med 2011;364:616-26.  Back to cited text no. 2
[PUBMED]    
3.
Ronco P, Debiec H. Pathogenesis of membranous nephropathy: Recent advances and future challenges. Nat Rev Nephrol 2012;8:203-13.  Back to cited text no. 3
[PUBMED]    
4.
Qin W, Beck LH Jr., Zeng C, et al. Anti-phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol 2011; 22:1137-43.  Back to cited text no. 4
    
5.
Hoxha E, KneiBler U, Stege G, et al. Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy. Kidney Int 2012;82:797-804.  Back to cited text no. 5
    
6.
Markowitz GS. Membranous glomerulopathy: Emphasis on secondary forms and disease variants. Adv Anat Pathol 2001;8:119-25.  Back to cited text no. 6
[PUBMED]    
7.
Beck LH Jr., Bonegio RG, Lambeau G, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephro-pathy. N Engl J Med 2009;361:11-21.  Back to cited text no. 7
    
8.
Hoxha E, Harendza S, Zahner G, et al. An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulo-nephritis. Nephrol Dial Transplant 2011;26: 2526-32.  Back to cited text no. 8
[PUBMED]    
9.
Cossey LN, Walker PD, Larsen CP. Phospho-lipase A2 receptor staining in pediatric idio-pathic membranous glomerulopathy. Pediatr Nephrol 2013;28:2307-11.  Back to cited text no. 9
[PUBMED]    
10.
Jennette JC, Iskandar SS, Dalldorf FG. Pathologic differentiation between lupus and nonlupus membranous glomerulopathy. Kidney Int 1983;24:377-85.  Back to cited text no. 10
[PUBMED]    
11.
Glassock RJ. Secondary membranous glome-rulonephritis. Nephrol Dial Transplant 1992;7: 64-76.  Back to cited text no. 11
[PUBMED]    
12.
Larsen CP, Messias NC, Silva FG, Messias E, Walker PD. Determination of primary versus secondary membranous glomerulopathy utilizing phospholipase A2 receptor staining in renal biopsies. Mod Pathol 2013;26:709-15.  Back to cited text no. 12
[PUBMED]    
13.
Svobodova B, Honsova E, Ronco P, Tesar V, Debiec H. Kidney biopsy is a sensitive tool for retrospective diagnosis of PLA2R-related membranous nephropathy. Nephrol Dial Transplant 2013;28:1839-44.  Back to cited text no. 13
[PUBMED]    
14.
Hofstra JM, Beck LH Jr., Beck DM, Wetzels JF, Salant DJ. Anti-phospholipase A2 receptor antibodies correlate with clinical status in idiopathic membranous nephropathy. Clin J Am Soc Nephrol 2011;6:1286-91.  Back to cited text no. 14
    
15.
Cravedi P, Ruggenenti P, Remuzzi G. Circulating anti-PLA2R autoantibodies to monitor immunological activity in membranous nephropathy. J Am Soc Nephrol 2011;22: 1400-2.  Back to cited text no. 15
[PUBMED]    
16.
Debiec H, Ronco P. PLA2R autoantibodies and PLA2R glomerular deposits in membranous nephropathy. N Engl J Med 2011 ;364: 689-90.  Back to cited text no. 16
[PUBMED]    
17.
Ardalan M, Ghafari A, Hamzavi F, et al. Anti-phospholipase A2 receptor antibody in idio-pathic membranous nephropathy: A report from Iranian population. J Nephropathol 2013; 2:241-8.  Back to cited text no. 17
[PUBMED]    
18.
Hayashi N, Akiyama S, Okuyama H, et al. Clinicopathological characteristics of M-type phospholipase A2 receptor (PLA2R)-related membranous nephropathy in Japanese. Clin Exp Nephrol 2015;19:797-803.  Back to cited text no. 18
[PUBMED]    
19.
Oh YJ, Yang SH, Kim DK, Kang SW, Kim YS. Autoantibodies against phospholipase A2 receptor in Korean patients with membranous nephropathy. PLoS One 2013;8:e62151.  Back to cited text no. 19
[PUBMED]    
20.
Debiec H, Lefeu F, Kemper MJ, et al. Early-childhood membranous nephropathy due to cationic bovine serum albumin. N Engl J Med 2011;364:2101-10.  Back to cited text no. 20
[PUBMED]    
21.
Beck LH Jr., Salant DJ. Membranous nephropathy: From models to man. J Clin Invest 2014;124:2307-14.  Back to cited text no. 21
    

Top
Correspondence Address:
Dr. Nasar Yousuf Alwahaibi
Department of Allied Health Sciences, College of Medicine and Health Sciences, Sultan Qaboos University, P. O. Box 35 Postal Code 123, Muscat
Sultanate of Oman
Login to access the Email id


DOI: 10.4103/1319-2442.235181

PMID: 29970727

Rights and Permissions


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

Top
   
 
 
    Similar in PUBMED
    Search Pubmed for
    Search in Google Scholar for
    Email Alert *
    Add to My List *
* Registration required (free)  
 


 
    Abstract
   Introduction
    Materials and Me...
   Statistical Analysis
   Results
   Discussion
    References
    Article Figures
    Article Tables
 

 Article Access Statistics
    Viewed350    
    Printed21    
    Emailed0    
    PDF Downloaded111    
    Comments [Add]    

Recommend this journal