Abstract
Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study, we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cutoff values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin (Ig) fraction, demonstrating the autoantibody nature. By contrast, the Igs of a familial aHUS patient carrying the complement factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.
Highlights
The complement system, a cornerstone of innate immunity, provides the body with protection against invading pathogens and dangerous host cells [1, 2]
Hydrolyzed C3 can bind to complement factor B (FB), which is subsequently cleaved by complement factor D (FD) to form an initial, fluid phase alternative pathway (AP) C3 convertase, C3(H2O)Bb, that is capable of converting native C3 into the active C3a and C3b fragments
Subdivision of C3 glomerulopathy (C3G) into C3 glomerulonephritis (C3GN) and dense deposit disease (DDD) was solely based on electron microscopy appearance
Summary
The complement system, a cornerstone of innate immunity, provides the body with protection against invading pathogens and dangerous host cells [1, 2]. All pathways converge at the central event of complement activation: the cleavage of C3 by C3 convertases This enzymatic reaction supports further activation of the complement cascade with the release of various anaphylatoxins and opsonins, and eventually the formation of the membrane attack complex (MAC). Hydrolyzed C3 can bind to complement factor B (FB), which is subsequently cleaved by complement factor D (FD) to form an initial, fluid phase AP C3 convertase, C3(H2O)Bb, that is capable of converting native C3 into the active C3a (anaphylatoxin) and C3b (opsonin) fragments This mechanism providing a persistent low level of active C3 in the blood is known as “tick-over” and allows constant responsiveness to potential danger. C3 may be cleaved into C3b by certain non-specific proteases, especially at sites of inflammation, coagulation, and infection [6]
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