C4 Nephritic Factors in C3 Glomerulopathy: A Case Series

Abstract

Background: C3 glomerulopathy (C3G) defines a group of rare complement-mediated kidney diseases with a shared underlying pathophysiology: dysregulation of complement in the fluid phase and glomerular microenvironment. Dysregulation can be driven by autoantibodies to C3 and C5 convertases.

Study design: Case series.

Setting & participants: 168 patients with C3G (dense deposit disease, 68; C3 glumerulonephritis, 100) selected from our C3G biobank.

Outcomes: Patient-purified immunoglobulin Gs were tested for C4 nephritic factors (C4NeFs). These autoantibodies recognize C4b2a, the C3 convertase of the classical pathway of complement.

Measurements: C4NeFs were detected using a modified hemolytic assay.

Results: C4NeFs were identified in 5 patients, 4 of whom had C3 glomerulonephritis. C4NeFs were associated with dysregulation of C3 and C5 convertases, and they appear to stabilize these convertases in a dose-dependent manner. C4NeFs also appear to protect C4b2a from decay mediated by soluble CR1 and C4 binding protein. The stabilizing activity of the autoantibodies was further demonstrated by using heat treatment to inactivate complement. C4NeFs were not detected in 150 patients with another complement-mediated kidney disease, atypical hemolytic uremic syndrome. They were also absent in 300 apparently healthy controls.

Limitations: In addition to C4NeFs, 2 patients had positive findings for other autoantibodies: one patient also had autoantibodies to factor H; the other patient also had autoantibodies to C3bBb (C3NeFs).

Conclusions: The finding of C4NeFs in a small percentage of patients with C3G highlights the challenge in identifying autoantibodies that drive complement dysregulation and underscores the complexity of the autoantibody repertoire that can be identified in these patients.

Keywords: C3 glomerulonephritis; C3 glomerulopathy (C3G); C3 nephritic factors (C3NeFs); C4 nephritic factors (C4NeFs); autoantibodies; case series; complement dysregulation; complement-mediated renal disease; dense deposit disease (DDD); immune deposits; kidney biopsy.

Figure 1

The complement cascade. A simplified schematic of the alternative, lectin and classical pathways of complement. Each of these initiating pathways leads to the generation of C3 convertases (C3bBb or C4b2a) and C5 convertases (C3bBbC3b or C4b2aC3b). C5 convertases cleave C5 and to trigger the generation of membrane attack complex or C5b-9. The presence of C4Nef and/or C3Nef stabilizes C3 convertases, resulting in hyperactivity of the alternative and terminal pathways.(MASP, mannose-associated serine protease; P, properdin; MAC, membrane attack complex; C3Nef, C3 nephritic factor; C4Nef, C4 nephritic factor; FHAA, Factor H autoantibody; FBAA, Factor B autoantibody).

Figure 2

C4Nef assay. Antibody-sensitized sheep erythrocytes (EA) are used as index cells to generate EAC1C4b by incubating pooled normal human serum in presence of TTHA (a strong magnesium chelator) and calcium. After washes, EAC1C4b2a (C3 convertase of the classical pathway) is made by adding purified C2. To test for the presence of C4b2a autoantibodies (C4Nefs), patient-purified IgG is added and cells are allowed to decay for different periods of time (7.5,15, 22.5 and 30 min). After each time point, 50μl of the cell mixture is removed. An equal volume of rat-EDTA serum (1:19 dilution) is added as a source of complement proteins C3 through C9. To stop the reaction after 1hr, 150 μl of cooled EDTA-GVB buffer is added. Non-lysed cells are removed by centrifugation and hemoglobin in supernatant (200 μl) is measured at OD415.

Figure 3

Kidney biopsy from Patient 2. A) PAS staining shows a membranoproliferative pattern with mesangial and capillary loop hypercellularity. B) GBM duplication with deposition of hyaline material is seen on the Masson trichrome stain (×40). C–E) IF findings are positive for C3 (C) but negative for C4d (D) and all other immunoreactants (IgG is shown). F) EM shows diffuse effacement of podocyte foot processes and thickening of the GBM by a combination of subendothelial deposits, basement membrane duplication and mesangial cell interposition.

Figure 4

C4 nephritic factors. A) In each of 5 patients, purified IgGs (500μg) had varying ability to prolong the half-life of C4b2a and cause hemolysis. B) The effect was dose dependent (50μg to 500μg of IgG) confirming the ability of these autoantibodies to stabilize C4b2a (green line, pooled normal IgG; patient-purified IgGs: dark blue, patient 1; red, patient 2; purple, patient 3; light blue, patient 4; orange, patient 5). C) and D) In addition, the C4Nefs protected C4b2a from decay by soluble CR1 (C) and C4 Binding Protein (C4BP) (D) (red, patient 1; blue, control).