Overactivity of Alternative Pathway Convertases in Patients With Complement-Mediated Renal Diseases

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.

Keywords: C3 glomerulopathy; C3 nephritic factor; alternative pathway; atypical hemolytic uremic syndrome; complement factor B mutation; complement system; convertase.

Figure 1

Principles of the convertase activity assay for detecting convertase-stabilizing factors in serum. In step 1, rabbit erythrocytes (RbE) are incubated in an alternative pathway (AP)-permissive buffer with test serum mixed 1:1 with pooled normal human serum (NHS) to compensate for possible C3 depletion in the test sample. The C5 inhibitor eculizumab is added to halt complement activation at the level of the C3/C5 convertases. Convertase assembly and decay are followed over time using different incubation times (5–60 min). Convertase-stabilizing factors present in the sample, e.g., C3 nephritic factor (C3NeF), may interfere at this point with convertase decay. Before proceeding to step 2, erythrocytes are washed to remove remaining complement factors and C5 inhibitor. Then, convertase-bearing erythrocytes are incubated for 60 min with guinea pig serum as a source of membrane attack complex (MAC) components. The presence of ethylenediaminetetraacetic acid (EDTA) disables de novo formation of convertases from guinea pig serum and assures that only preformed convertases of the first step may initiate MAC formation and subsequent hemolysis. The released hemoglobin is quantified by spectrophotometric measurement and reflects the activity of the preformed convertases in step 1 per experimental time point. These data are used to generate convertase activity profiles over time. *If desired, immunoglobulin fractions may be added to NHS to dissect the nature of the stabilizing factor (see Materials and Methods).

Figure 2

Convertase activity in serum samples of healthy controls. Samples collected from 15 healthy controls were mixed 1:1 with pooled normal human serum (NHS) to an end concentration of 3.75%. Heat-inactivated NHS (ΔNHS) and an NHS sample from which guinea pig serum was omitted during the second part of the assay (no step 2) served as negative controls for the first and second steps, respectively. Means are given of ≥2 independent experiments for each control. Error bars were omitted for better visibility of the graph. Hemolysis levels are presented as percentage of full lysis of erythrocytes in water.

Figure 3

Screening for convertase-stabilizing factors in patients with C3 glomerulopathy (C3G). (A) Convertase activity of 27 patients with C3G as measured in a single time point screening with 30 min of incubation time. Samples showing hemolysis above that of pooled normal human serum (NHS), represented by the dotted line, are indicated with colored symbols and were selected for further analysis of convertase activity over time. (B,C) Convertase activity profiles of the 21 patients selected from the t₃₀ screening in panel (A). Samples positive for convertase-stabilizing factors (top/t₃₀ ratio < 2.7) are given in panel (B); samples negative for the presence of convertase-stabilizing factors (top/t₃₀ ratio > 2.7) in panel (C). Results given are from a single assay set representative for at least two performed on each sample. (A–C) Serum samples were all tested mixed 1:1 with NHS to a final concentration of 3.75%. Colors and symbols correspond to the same patients in all panels. Hemolysis levels are given as percentage of full lysis of erythrocytes in water.

Figure 4

Convertase activity profiles of patients with C3 glomerulopathy over disease course. Patient serum samples of P24 (A), P27 (B), P25 (C), and P26 (D) from different collection dates associated with different disease states were tested mixed 1:1 with pooled normal human serum (NHS) to a final concentration of 3.75% in experiments shown in panels (C,D) or 5% in the experiments shown in panels (A,B), since they were performed using different batches of erythrocytes. Abbreviations: Acu, acute phase at first clinical presentation of disease; Act, active disease; Part Rem, partial remission; Rem, remission. Heat-inactivated NHS (ΔNHS) and an NHS sample from which guinea pig serum were omitted during the second part of the assay (no step 2) served as negative controls for the first and second steps, respectively. Representative data are given. Hemolysis levels are given as percentage of full lysis of erythrocytes in water.

Figure 5

Identification of convertase-stabilizing factors in immunoglobulin (Ig) fractions of patients with C3 glomerulopathy. Serum samples of P24 (A), P27 (B), P25 (C), and P26 (D) were tested mixed 1:1 with pooled normal human serum (NHS) to a final concentration of 5%. Alternatively, the Ig fractions of these patients or of pooled normal human plasma (NHP) were added to 5% NHS in an equal volume. Data were collected from three independent experiments; means are given with error bars showing SDs. Statistical analysis for test samples compared with NHP Ig from t = 20 to t = 60 as calculated using two-way analysis of variance is given for the patient Igs only: **P < 0.01, ***P < 0.001, ns not significant. Heat-inactivated NHS (ΔNHS) and an NHS sample from which guinea pig serum were omitted during the second part of the assay (no step 2) served as negative controls for the first and second steps, respectively. Hemolysis levels are given as percentage of full lysis of erythrocytes in water.

Figure 6

Assessment of convertase activity in a family with complement factor B (FB) mutation and atypical hemolytic uremic syndrome (aHUS). (A) Pedigree with carriers of the FB p.Lys323Glu mutation and/or aHUS. No genetic information of this variation was available for I. 2 who is deceased. (B) Analysis of convertase stability in available samples from seven family members. The sera were tested mixed 1:1 with normal human serum (NHS) to a final concentration of 5%. Data for each test sample were obtained from three independent experiments; means are given with error bars showing SDs. Statistical analysis for test sera compared with NHS from t = 20 to t = 60 as calculated using two-way analysis of variance is given: *P < 0.05, ***P < 0.001, ns not significant. (C) Assessment of the presence of convertase-stabilizing factors in the immunoglobulin (Ig) fraction of aHUS patient III. 6. Purified Igs from patient plasma or pooled normal human plasma (NHP) were added in an equal volume to 5% NHS. Data were collected from three independent experiments; means are given with error bars showing SDs. (B,C) Heat-inactivated NHS (ΔNHS) and an NHS sample from which guinea pig serum were omitted during the second part of the assay (no step 2) served as negative controls for the first and second steps, respectively. Hemolysis levels are given as percentage of full lysis of erythrocytes in water.