Background
Membranous glomerulonephritis (MGN) is a leading cause of the nephrotic syndrome in adults and occurs as a primary disorder (primary membranous nephropathy – PMN) or secondary to other disorders like lupus nephritis, infections and certain drugs.[
1‐
6] Primary membranous nephropathy is a kidney-specific autoimmune disorder in which autoantibodies develop against self, podocyte antigens.[
5] Phospholipase A2 receptor 1 (PLA2R) and thrombospondin type-1 domain containing 7A (THSD7A) are two podocyte specific antigens that have been identified in PMN.[
7,
8] Determining the presence or absence of PLA2R and anti-THSD7A antibodies in serum and renal biopsy specimen, has significantly reshaped the diagnostic approach to PMN by providing important immunologic information which aid in discriminating between primary and secondary MGN, as well as predicting clinical outcomes in PMN.[
9] Clinically, PLA2R and anti-THSD7A antigens may be identified in sera or by immunostaining methods in renal biopsy samples. While quantitative and semi-quantitative serologic assays are now commercially available for PLA2R and THSD7A antibodies, it has been noted that a significant number of individuals with histologically confirmed antigens to PLA2R in glomerular deposits (and hence a biopsy diagnosis of PMN) have negative PLA2R serology results.[
10] It has also been shown that early in the time course of PMN, due to delayed seroconversion, PLA2R may be negative in serum but demonstrable in histologic specimen.[
11] Kidney biopsy tissue-based testing may thus be a veritable diagnostic tool in appropriately identifying PMN.
The sensitivity and specificity of these tissue-based antibody tests in identifying PMN have largely varied across studies. However, one meta-analysis reported a pooled sensitivity and specificity of 65% (63–67%) and 97% (97–98%) for serum PLA2R antibodies and 79% (76–81%) and 90% (88–92%), respectively, for glomerular PLA2R antigens.[
12] Hoxha et al.,[
13] in a prospective cohort of 345 patients with MN reported the prevalence of THSD7A-associated MN to be 2.6% with 92% sensitivity and a 100% specificity using immunofluorescence techniques. Given that MGN (both primary and secondary types due to hepatitis B and lupus nephritis) has been shown to be a common cause of nephrotic syndrome in adults in African studies of kidney biopsies, differentiating primary from secondary type is important for making therapeutic decisions.[
3,
6,
14] There are currently no studies in Africa assessing the utility of serum or glomerular PLA2R or THSD7A for accurately identifying patients with PMN. The aim of this study is to assess the sensitivity and specificity of PLA2R and THSD7A for identifying PMN in South African patients using kidney biopsy tissue-based antibody tests.
Discussion
With the identification of candidate podocyte antigen targets such as PLA2R and THSD7A and their respective PLA2R and anti-THSD7A autoantibodies, the autoimmune basis of MGN has effectively been established. Our study aimed to determine the occurrence of novel antigens associated with MGN among South African patients previously defined as PMN by standard microscopic techniques. We found a respective frequency of occurrence of positive glomerular staining for PLA2R and THSD7A in 51.2% (21/41 patients) and 4.9% (2/41 patients) of biopsies initially categorized as PMN. None of the biopsies of LN-related MGN or DN stained positively for either PLA2R or THSD7A.
Since the canonical work of Beck et al. [
7] in which the autoimmune target podocyte antigen PLA2R was discovered, the prevalence of PLA2R antibody in serum and/or PLA2R antigen in kidney tissue deposits in PMN have been variously described among different populations. In our cohort of predominantly non-black South Africans, the frequency of occurrence of histologic autoantibody positivity was relatively low at 51.2% compared to data from European cohorts in whom rates from 69.2–73.8% have been observed.[
10,
19] Among Japanese patients however, lower rates as seen in our study have also been reported.[
20] Evidence continues to accrue for a genetic component in the pathogenesis of PMN and a genetic role in the varying prevalence observed particularly with respect to PLA2R autoantibody positivity across different ethnicities.
HLA-DQA1 and
PLA2R1 are risk alleles for PMN that have been identified and demonstrated to confer different disease risk in different populations with a strong association shown among European, Indian, Chinese but not African Americans patients.[
21‐
23].
Approximately 1 in 2 patient histologic samples initially identified as PMN by standard microscopic techniques did not demonstrate enhanced glomerular immune deposits staining for PLA2R antigen. This PLA2R immune deposit negative state could be accounted for by either of two possibilities. It is probable that these are indeed PMN cases with yet to be identified target podocyte antigen(s) as research continues to identify other candidate glomerular antigens that are autoimmune targets in PMN. Indeed 1 of our 20 PLA2R negative patients had a positive glomerular immune deposit staining for THSD7A, an antigenic target that was identified 6 years after PLA2R. Other novel proteins including neural epidermal growth factor-like 1 protein (NELL-1) [
24] and Semaphorin 3B [
25] have recently been identified to be associated with membranous nephropathy. NELL-1 was present in 23% [29/126] of European PLA2R and THSD7A negative PMN patients while Semaphorin 3B was predominantly present in paediatric patients with MGN.[
24,
25].
These discoveries lend credence to our hypothesis that other glomerular basement membrane antigens may well underly the pathogenesis of PMN in this African cohort, especially with our relatively younger group of patients. An alternate explanation could be that these are secondary cases of MGN currently misidentified as PMN as predisposing factors such as in chronic viral infections like chronic hepatitis B, autoimmune disorders such as LN and solid organ malignancies were not clinically apparent and detectable by routine checks at the time of renal biopsy. Hepatitis-B associated glomerular disease and LN constitute the two commonest causes of secondary GNs in Africa and in both entities glomerular disease has been known to occur in the absence of systemic evidence of chronic hepatitis B antigenemia and lupus serologies respectively.[
3,
26] We however could not demonstrate any significant clinical or histologic differences between our PLA2R positive and PLA2R negative patients to suggest that they represent two distinct pathogenetic groups of patients (Table
3). Unfortunately, we were unable to stain the biopsies for exostosin which can detect cases labelled as PMN but related to LN. Sethi et al.[
27] detected exostosin 1 (EXT1) and exostosin 2 (EXT2) in 21 cases of PLA2R-negative MN, but not in PLA2R-associated MN and control cases, suggesting that subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Thus, a yet to be identified glomerular antigen in this African group of PMN patient may be a more probable theory underlying PLA2R negative immune deposit staining. It is also possible, although we used an IHC method that has been previously described, that methodological differences in IHC techniques could have contributed to the proportion of PLA2R positive biopsies in our group.
Distinguishing between PMN and LN-V histologically has traditionally rested upon the additional demonstration of subendothelial deposits, mesangial hypercellularity and positive IHC/IF staining for C3, C1q, IgM, IgA and IgG1/IgG3 subclass in LN-V biopsies. Positive staining for glomerular PLA2R antigen by IF/IHC has now been added to the armamentarium of diagnostic tools in differentiating between PMN and LN-V with the latter typically, but not always, demonstrating negative serum PLA2R antibodies and/or glomerular staining for the PLA2R antigen.[
28] Although we were not able to subtype IgG class or stain for C1q, we could not broadly exemplify this differential IHC staining pattern between our PMN patients and LN-V patients (Table
4). Although immunofluorescence examination can aid in differentiating LN-V from PMN given the absence of IgM, IgA, and C1q in the latter, it has been noted that these differences can be challenging due to the overlap that can occur.[
20,
29] This may explain the presence, in a few PMN patients, of positive IgA, IgM and C1q despite these patients testing negative for all common secondary causes of MGN. Based on our findings there were notable clinical differences between PLA2R positive patients and LN-V patients with PLA2R patients having 1.7 fold heavier proteinuria and more than 2-fold higher total serum cholesterol levels than lupus MN patients; as is typical for the demographic epidemiology of lupus, there was a female predisposition for lupus. Dual positivity for the glomerular PLA2R and THSD7A antigens in patients with PMN is being increasingly reported from some studies.[
30,
31] Only one patient in our cohort demonstrating dual positivity was demonstrated. It remains unclear the clinical implications of this dual staining pattern.
Our study is not without its limitations. Firstly, we were unable to stain the PLA2R negative samples for EXT1 and EXT 2, especially considering the younger age of patients in our study. This may have shown, as others have done in recent studies,[
27] that some PLA2R negative patients treated as PMN could be patients with LN. Additionally, there was no outcome data reported for the patients to evaluate the role of glomerular PLA2R or THSD7A reactivity with response to therapy and disease progression. Our study was not designed to do this as we wanted to assess the relevance of these tests in an African population given that there are no previous studies. Furthermore, we were also unable to include the biopsies of patients with cancers and secondary MN to assess if THSD7A shows any association in such patients in our population. Lastly, we did not have serum samples at the time of diagnosis of MGN to assess serum PLA2R antibodies and determine if there existed any correlation between serum PLA2R antibodies and glomerular staining in our PLA2R positive patients. Despite these limitations and given the high specificity and PPV of our study results, it shows that glomerular PLA2R and THSD7A staining can be used for identifying PMN in African patients with MGN. We also encourage outcome studies using these tests in patients with biopsy proven PMN as this will improve the use of appropriate therapies for treating patients.
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