In situ assessment of glomerular C3/C5 convertases indicates ongoing complement activation in IgA nephropathy

BACKGROUND AND HYPOTHESIS: The complement system contributes to the progression of immunoglobulin A nephropathy (IgAN) and offers promising therapeutic targets currently under investigation. Identifying appropriate candidates for these treatments is now critical. Currently, complement activation in IgAN is assessed using immunostaining of kidney tissues, a method that lacks precision in differentiating ongoing and past complement activation. METHODS: Complement deposition was assessed in diagnostic kidney biopsies from 53 patients with primary IgAN and positive C3c staining. Immunohistochemistry was used to assess glomerular deposition of C4d and C5b-9, while immunofluorescence was employed for C3b/iC3b and C1q. In situ detection of C3 and C5 convertase complexes from the classical/lectin (C4b2b) and alternative (C3bBb) complement pathways was performed on paraffin-embedded kidney biopsies from 33 of these patients using a proximity ligation assay (Duolink®). Biopsies from patients with lupus nephritis and C3 glomerulopathy (C3G) served as positive controls. Convertase signals were quantified using QuPath® Software and correlated with MEST-C score, C5b-9 staining and clinical outcomes. RESULTS: C3bBb convertases were identified in 20/33 C3c-positive (60.6%) patients, with a median positive area of 15.5 (3.4-24.8) ‰ of the glomerular surface. C4b2b convertases were detected in 22/33 (66.7%) patients, covering a median positive area of 8.3 (4.1-11.8) ‰. Both convertases were present in 13/33 (39.4%) patients, while 4/33 (12.1%) patients had no convertases despite positive C3c staining. Double-negative patients had better renal survival (P = .0387). C3bBb-staining positive area significantly correlated with mesangial C5b-9 deposition intensity (P = .0011). Patients with MEST-C scores E1 and S1 showed greater C3bBb intensity at diagnosis (P = .0251 and P = .0004, respectively). CONCLUSION: Our study demonstrates ongoing complement activation in 29/33 (87.9%) C3c-positive IgAN patients. Notably, activation of the alternative pathway correlated with E1 and S1 lesions, C5b-9 deposition and poorer renal survival. In situ convertase detection may help identify patients most likely to benefit from complement-targeted therapies.