Abstract
Membranoproliferative glomerulonephritis (MPGN) was recently classified as C3 glomerulopathies (C3G), and immune-complex (IC) mediated MPGN. Dysregulation of the complement alternative pathway, driven by acquired and/or genetic defects, plays a pathogenetic role in C3G. However, alternative pathway abnormalities were also found in IC-MPGN. The most common acquired drivers are the C3 nephritic factors (C3NeFs), heterogeneous autoantibodies that stabilize the C3 convertase, C3bBb. C3NeFs are traditionally detected by hemolytic assays based on sheep erythrocyte lysis, which however do not provide a direct molecular estimation of C3bBb formation and decay. We set up a microplate/western blot assay that specifically detects and quantifies C3bBb, and its precursor, the C3 proconvertase C3bB, to investigate the complex mechanistic effects of C3NeFs from patients with primary IC-MPGN (n = 13) and C3G (n = 13). In the absence of properdin, 9/26 patients had C3NeF IgGs stabilizing C3bBb against spontaneous and FH-accelerated decay. In the presence of properdin the IgGs of all but one patient had C3bBb-stabilizing activity. Properdin-independent C3NeFs were identified mostly in DDD patients, while properdin-dependent C3NeFs associated with either C3GN or IC-MPGN and with higher incidence of nephrotic syndrome. When we grouped patients based on our recent cluster analysis, patients in cluster 3, with highly electron-dense intramembranous deposits, low C3, and mostly normal sC5b-9 levels, had a higher prevalence of properdin-independent C3NeFs than patients in clusters 1 and 2. Conversely, about 70% of cluster 1 and 2 patients, with subendothelial, subepithelial, and mesangial deposits, low C3 levels and high sC5b-9 levels, had properdin-dependent C3NeFs. The flexibility of the assay allowed us to get deep insights into C3NeF mechanisms of action, showing that: (1) most C3NeFs bind strongly and irreversibly to C3 convertase; (2) C3NeFs and FH recognize different epitopes in C3 convertase; (3) C3NeFs bind rapidly to C3 convertase and antagonize the decay accelerating activity of FH on newly formed complexes; (4) C3NeFs do not affect formation and stability of the C3 proconvertase. Thus, our study provides a molecular approach to detecting and characterizing C3NeFs. The results highlight different mechanisms of complement dysregulation resulting in different complement profiles and patterns of glomerular injury, and this may have therapeutic implications.
Highlights
Membranoproliferative glomerulonephritis (MPGN) is a rare chronic kidney disorder associated with thickening of the glomerular capillary wall and mesangial expansion, which are the result of the deposition of immune-complexes and complement factors [1, 2]
An advanced approach toward an etiology-based diagnosis of this disease arose from the more recent immunofluorescence (IF)-based classification that distinguishes between immunecomplex-mediated MPGN (IC-MPGN), with glomerular IgG and C3 deposits, and C3 glomerulopathy (C3G) with predominant C3 deposits [8, 9]
C3 levels at onset were low and did not differ between histology groups (C3GN: 40 ± 11; Dense-Deposit Disease (DDD): 19 ± 19; IC-MPGN: 21 ± 15 mg/dl), while plasma sC5b-9 levels were higher in C3 glomerulonephritis (C3GN) (1249 ± 736 ng/ml) and ICMPGN (1895 ± 1291 ng/ml) than in DDD (416 ± 374 ng/ml, P < 0.01) (Table 1)
Summary
Membranoproliferative glomerulonephritis (MPGN) is a rare chronic kidney disorder associated with thickening of the glomerular capillary wall and mesangial expansion, which are the result of the deposition of immune-complexes and complement factors [1, 2]. On the basis of electron microscopy localization of electron-dense deposits relative to the glomerular basement membrane, MPGN was divided into type I, with subendothelial deposits [5], type II or Dense-Deposit Disease (DDD), with intramembranous highly electron-dense deposits [6], and type III, with subendothelial and subepithelial deposits [7]. C3G is further divided into DDD, with distinctive highly electron-dense deposits within the glomerular basement membrane; and C3 glomerulonephritis (C3GN), with mesangial, intramembranous, subendothelial and sometimes subepithelial deposits [1, 6, 10, 11]. According to the current classification, IC-MPGN derives from the deposition of immune-complexes that form in the context of infections, autoimmune diseases and malignancies, and trigger the classical complement pathway [8]. Genetic and acquired alternative pathway abnormalities have been found as frequently in patients with IC-MPGN as in those with C3G [14,15,16], suggesting that dysregulation of the complement alternative pathway may play a role in both conditions
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