The identification of glomerular auto-antigens in idiopathic human membranous glomerulonephritis (MGN) is a crucial step towards the definition of the mechanisms of the disease. Recent 'in vivo' studies demonstrated a heterogeneous composition of glomerular immune-deposits in MGN biopsies only a part of which have been characterized. We studied with a proteomical approach IgGs eluted from laser capture microdissected glomeruli of 8 MGN patients and showed the existence of other three immune proteins in MGN glomeruli (α-enolase, elongation factor 2 and Glycyl Aminoacyl-tRNA Synthetase). One of these, i.e. α-enolase, fulfilled all criteria for being considered an auto-antigen. Specific IgG₁ and IgG₄ reacting with podocyte α-enolase were, in fact, eluted from microdissected glomeruli and Confocal- and Immuno Electron-Microscopy showed co-localization of α-enolase with IgG₄ and C5b-9 in immune-deposits. Serum levels of anti a-enolase IgG4 were determined in 131 MGN patients and were found elevated in 25% of cases. Overall, our data demonstrate that glomerular α-enolase is a target antigen of autoimmunity in human MGN. Circulating anti α-enolase auto-antibodies can be detected in sera of a significant quota of MGN patients. Like other auto-antigens, α-enolase may be implicated in the pathogenesis of human MGN.
"The observation that some patients with IMN do not have the PLA2R autoantibodies could be explained by limitations of current immunoassays, and/or the absence of these autoantibodies during treatment or inactive disease. In addition, autoantibodies to PLA2R may not be a universal feature of IMN because other autoantibodies, such as those directed against α-enolase or aldose reductase, have previously been reported in patients with IMN, albeit at a much lower frequency , . Proteinuria as a traditional marker of disease activity in IMN correlates with, but does not perfectly parallel, anti-PLA2R levels . "
[Show abstract][Hide abstract] ABSTRACT: The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.
PLoS ONE 04/2013; 8(4):e61669. DOI:10.1371/journal.pone.0061669 · 3.23 Impact Factor
"iMN is usually characterized by a strong IgG4 reaction detectable by immunofluorescence [9, 26]. It has recently been determined that these IgG4 antibodies may react with antigens normally expressed on the podocyte cell membrane (M-type phospholipase A2 receptor) , or only abnormally so (aldose reductase , superoxide dismutase , α-enolase ). The antigen-antibody complexes are then shed to form the characteristic subepithelial deposits seen on electron microscopy. "
[Show abstract][Hide abstract] ABSTRACT: Membranous nephropathy (MN) is one of the most common causes of nephrotic syndrome (NS) in adults. It may be primary, usually mediated by IgG4 anti-phospholipase A2 autoantibodies or secondary to various other conditions. Guillain- Barré syndrome (GBS) has been associated with MN, but a cause and effect relation has not been proven. We present a case of concurrent development of GBS and severe NS, with renal biopsy demonstrating MN. IgG4 stain was negative, indicating that most likely, the MN was secondary and probably caused by the underlying GBS.
"Furthermore, high titer circulating IgG4 against these antigens was found in over 50% of patients as well. Bruschi et al.  found circulating IgG4 antibodies against α-enolase in the serum of 25% of 131 iMN patients. Confocal-and immuno electron-microscopy showed co-localization of α-enolase with IgG4 and C5b-9 in immune deposits. "
[Show abstract][Hide abstract] ABSTRACT: Idiopathic membranous nephropathy (iMN) is a single-organ autoimmune disease characterized by subepithelial deposition of immune complexes containing IgG4 resulting in proteinuria, nephrotic syndrome, and, in some, end-stage renal disease. The pathogenesis involves a chronic IgG4 response against specific podocyte antigens which have now been at least partially defined in the neonatal, early childhood, and adult varieties. More has recently been learned about the genetic predisposition as well. This review discusses the pathophysiology of iMN in light of these discoveries and what is known about the genesis and potential clinical ramifications of an antigenspecific IgG4 response.
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