Key Points Activation of the alternative and lectin complement pathways contributes to disease severity and progression in IgA nephropathy. In this French cohort with severe IgA nephropathy, complement factor H CFH-H2 was underrepresented, and rare complement factor H missense variants were more frequent, affecting regulatory domains. Homozygosity for the membrane cofactor protein CD46 ggaac haplotype was associated with progression to ESKD, warranting confirmation in larger cohorts. Background Complement activation plays a role in IgA nephropathy. Indeed, genome-wide association studies have identified a susceptibility locus for IgA nephropathy complement factor H ( CHF ) and its related proteins genes. However, the relationship between complement genetics and IgA nephropathy severity is not established. Methods We sequenced 260 adults with a primary IgA nephropathy by next-generation sequencing of the CFH , complement factor I, complement factor B, membrane cofactor protein (encoded by CD46 ), C3 , and CFH related protein 5 genes. The frequencies of rare variants (minor allele frequency <0.1%) and CFH , membrane cofactor protein, and complement factor B haplotypes were compared with the European population from the 1000 Genomes Project. Functional in vitro studies were performed on recombinant full-length CFH variants. Results Our genetic analysis revealed a higher prevalence of rare CFH nonsynonymous variants in patients with IgA nephropathy compared with controls (5% versus 2%, P = 0.03). In vitro studies showed impaired complement regulatory activity for five variants. The CFH-H2 haplotype was associated with lower IgA nephropathy susceptibility (odds ratio, 0.52 [0.34 to 0.79], P = 0.03, dominant model). Moreover, multivariable analysis revealed that the homozygous CD46 ggaaac haplotype was associated with ESKD 15 years after diagnosis (hazard ratio, 2.83; 95% confidence interval, 1.20 to 6.68; P = 0.02). Conclusions Our study shed light on the dual role of complement genetics in IgA nephropathy. Our exploratory findings strengthen the evidence for a role of complement genetics in IgA nephropathy but will need confirmation in larger and independent studies.

Functional approaches to properly examine individual's susceptibility to complement dysregulation are limited. We assessed ex vivo complement activation induced by sera from 38 healthy donors on resting microvascular endothelial cells with or without complement dysregulation (OX-24 monoclonal antibody). Following incubation, immunofluorescence was used to quantify membrane-bound C3b/iC3b and C5b-9 with a computer-assisted method (H-score). C3a and C5a anaphylatoxins were quantified in supernatants by ELISA. Genetic sequencing of 32 donors was also performed to identify variants in alternative pathway genes. Elevated complement deposition was defined by H-scoreC3c > 50 and/or H-scoreC5b-9 > 30. Combined analysis of C3b/iC3b and C5b-9 deposition in 35 donors showed that one donor (2.8%) had an isolated increase in C3b/iC3b deposits, three (8.6%) had an isolated increase in C5b-9 deposits, and one (2.8%) had both increased C3b/iC3b and C5b-9 deposition. Genetic analysis revealed three heterozygous rare/low frequency missense variants in CFH (p.N1050Y, p.R1210C) and CFI (p.A76G) in 3/5 donors with increased complement deposition. This model revealed distinct patterns of complement activation among healthy individuals and identified a genetic basis for dysregulation in three cases. This assay offers a promising tool to study complement activity and its mechanisms in research.

RATIONALE & OBJECTIVE:Autoantibodies targeting complement factor B (anti-FB) are among the most recently described alternative pathway anomalies. Although they have been strongly associated with post-infectious glomerulonephritis in children, the clinical phenotypes associated with such antibodies, along with their effects on the alternative pathway in adults, remain elusive and require further characterization, which was the goal of this study. STUDY DESIGN:Retrospective case series. SETTING & PARTICIPANTS:Patients for whom anti-FB IgG was systematically detected by the French reference center for complement abnormalities between October 2017 and June 2020 and who underwent concurrent kidney biopsy confirming kidney disease were included as cases. FINDINGS:Seventy-one patients tested positive for anti-FB antibodies using ELISA and single-bead antigen assays in the setting of newly diagnosed biopsy-proven kidney disease. Detection of both anti-FB and anti-C3b autoantibodies occurred for 36/71 (51%) patients. In vitro, total purified IgG from these patients enhanced alternative pathway activity, with anti-FB titers being correlated with C3bB proconvertase formation (R2 = 0.40, p < 0.001) and anti-C3b titers being correlated with C3bBb convertase stabilization (R2 = 0.46, p < 0.001), suggesting distinct and potentially synergistic functional effects. Clinically, the most frequent diagnosis associated with anti-FB detection was infection-related glomerulonephritis (IR-GN) (46/71 - 65%), with higher anti-FB titers in patients with IR-GN compared to those with other diagnoses (median 791 [IQR 287-2000] vs. 326 [173-790] AU/mL; Hodges-Lehmann difference 354 AU/mL [95% CI: 58-1020]; p = 0.01). No difference was detected in anti-C3b titers between IR-GN and other diagnoses. Anti-FB antibodies became undetectable in 21 of 36 retested patients (58%); among the 10 patients with persistent detection beyond 3 months, 8 (80%) had uncontrolled infection. After a median follow-up of 12.5 (5-24) months, 18/67 (27%) patients experienced a major adverse kidney event (kidney failure or a sustained >50 decline in eGFR below the baseline value). LIMITATIONS:Retrospective design, the series was enriched in alternative pathway anomalies due to reference center recruitment. CONCLUSIONS:Detection of anti-FB antibodies is strongly associated with infection-related glomerulonephritis in adult patients, highlighting an important mechanism of alternative pathway deregulation in such diseases. PLAIN LANGUAGE SUMMARY:Antibodies targeting factor B, a key protein of the complement cascade, have been shown to activate the complement system and promote kidney inflammation. However, the clinical characteristics and outcomes of patients with such autoantibodies remain largely understudied, particularly in adults. This study systematically screened over 700 adults referred for complement investigation and identified 71 patients with anti-factor B antibodies and concurrent biopsy-proven kidney disease. These antibodies were predominantly associated with infection-related glomerulonephritis, activated the alternative complement pathway, and tended to disappear once the underlying infection was controlled. These findings highlight the importance of screening for anti-factor B antibodies in patients with complement-mediated kidney disease, as their detection may prompt the search for an underlying infection and guide therapeutic decisions.

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.

Dysregulation of the alternative pathway of complement plays a central role in the pathophysiology of C3 glomerulopathy (C3G). Various autoimmune and genetic factors targeting the alternative pathway have been associated with both C3G and primary immunoglobulin-associated membranoproliferative glomerulonephritis (Ig-MPGN), suggesting shared pathophysiological mechanisms. This review highlights the wide range of disease drivers identified that mainly target components or protein complexes of the alternative pathway, both in C3G and Ig-MPGN. Nephritic factors, which constitute a heterogeneous group of autoantibodies targeting the C3 or the C5 convertase, are the most common abnormalities. Monoclonal gammopathies are frequent in aging adults. They may promote complement activation and have in some cases also been found to target alternative pathway regulatory proteins. Additionally, some patients with C3G and Ig-MPGN carry rare variants in genes encoding complement activating or regulating proteins of the alternative pathway. This review provides an informative overview of pathogenetic mechanisms associated with each abnormality, acting at different steps in the complement cascade. The diversity of targets involved in the C3G pathophysiology suggests the potential benefit of therapeutical approaches tailored to the underlying disease drivers, with a pivotal impact upstream or at the level of the C3 or C5 convertase activity.

Introduction: Antineutrophil cytoplasmic autoantibody (ANCA)-associated renal vasculitis (AAV) glomerulonephritis (GN) (AAV-GN) frequently leads to end-stage kidney disease (ESKD). Optimal management for patients with AAV-GN receiving a kidney transplantation (KT) remains poorly characterized. We compared posttransplant outcomes between patients with AAV-GN and controls in the modern era of immunosuppressive therapy. Methods: This multicenter retrospective study included 206 adult patients with AAV-GN and 412 matched controls who underwent KT between 2005 and 2023 in 12 French transplant centers. We compared the incidence of delayed graft function (DGF), graft failure, AAV relapses, acute rejection, and mortality between groups; and analyzed risk factors using multivariable models. Results: DGF incidence and kidney function up to 5 years after KT were similar between groups. Patients with AAV-GN showed a trend toward poorer graft survival (hazard ratio [HR] = 1.55, P = 0.077) and significantly lower overall survival (HR = 1.48, P = 0.034). AAV relapses occurred in 15 patients with AAV-GN (7.3%), significantly impacting graft survival (P = 0.008). ANCA positivity at KT tended to be associated with higher relapse risk (HR = 4.17, P = 0.065) and was associated with lower rejection risk (HR = 0.31, P = 0.016). Acute rejection incidence was comparable between groups. Azathioprine (AZA) maintenance therapy was associated with increased rejection (HR = 3.733, P = 0.012) and graft failure (HR = 3.73, P = 0.007 < 0.001). Although patients with AAV-GN were waitlisted later than controls, no specific transplant timing was associated with improved outcomes. Conclusion: Although KT offers patients with AAV-GN short-term outcomes similar to controls, they face higher long-term risk of graft failure and mortality. ANCA status at transplantation may help predict immunological events, emphasizing the need for careful evaluation and monitoring, but without delaying the process. As for other nephropathies, AZA should be avoided as maintenance therapy in patients with AAV-GN. These findings highlight the need for tailored posttransplant management in patients with AAV-GN.

Introduction: The diagnosis of thrombotic microangiopathy (TMA) relies on common biological parameters, the diagnostic value of which are unknown. Methods: The presence of common biological parameters was assessed in 967 patients with TMA from 2009 to 2023 (ClinicalTrials.gov: NCT05991245). Results: The median age was 49 (36–64) years and 53.2% were male. All TMA causes were represented (atypical hemolytic uremic syndrome [aHUS]: 41.6%, drugs: 24.9%, malignancy: 21.4%, autoimmune disease: 18.8%, infection: 7.6%, complement-mediated HUS: 6.8%, organ transplantation: 5.8%, pregnancy: 3.8%, bone marrow transplantation [BMT]: 2.9%, Shiga toxin Escherichia coli hemolytic uremic syndrome [STEC-HUS]: 0.6%, and thrombotic thrombocytopenic purpura [TTP]: 0.6%). The presence of TMA-related parameters concerned virtually all patients with TTP but varied widely for the other patients as follows: anemia: 81.7%, high lactate dehydrogenase (LDH) (75.4%), low haptoglobin (53.7%), and thrombocytopenia (40.3%). Their diagnostic performance was accurate only for TTP. Eleven distinct ways were used for schistocyte metrics and reporting. Relying on schistocyte presence as the single diagnostic criterion would lead to missed diagnosis in 23.8% (STEC-HUS) to 86.4% (BMT) of patients (for anemia: 8.2%–22.3%; thrombocytopenia: 31.8%–67.9%; high LDH: 10.0%–40.7%, low haptoglobin: 0%–70.4%, according to the causes of TMA). The overall risk of missed diagnosis using these parameters was ≥ 50% in all TMA, except in TTP. The best diagnostic performances were obtained when fibrinogen levels were < 5 g/l, creatinine ≥ 300 μmol/l, prothrombin time (PT) < 90%; and when TMA causes were TTP, STEC-HUS, infection, or complement-mediated HUS. Conclusion: Common biological parameters miss the diagnosis in more than 50% of TMA except when fibrinogen is < 5 g/l, creatinine ≥ 300 μmol/l, and PT < 90%. Schistocyte reporting is heterogenous, and its results are usually deceptive in TMA.

Complement inhibition has revolutionized the management of hemolytic diseases by targeting the underlying drivers of red blood cell destruction in disorders such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and cold agglutinin disease. Recent advances have expanded the therapeutic landscape beyond terminal C5 inhibition to proximal strategies targeting C3, factor B, and factor D. These newer agents - such as pegcetacoplan, iptacopan, and danicopan - not only control intravascular hemolysis but also address extravascular hemolysis and offer oral or subcutaneous alternatives. In cold agglutinin disease, the efficacy of the classical pathway inhibitor sutimlimab provides compelling evidence for the therapeutic value of targeting early initiating molecules of the complement cascade. For clinicians, these innovations provide personalized treatment choices based on disease phenotype, hemolysis profile, patient preference, and risk stratification. The evolving complement inhibitor arsenal enables optimized, targeted care and pathway-specific interventions in hemolytic conditions.

Introduction:Goodpasture disease, or antiglomerular basement membrane (anti-GBM) disease, is a rare autoimmune disorder that often leads to end-stage kidney disease (ESKD). Although kidney transplantation (KT) is the preferred treatment, concerns exist about disease recurrence and graft outcomes in patients with GBM-associated glomerulonephritis (GBM-GN). This study aimed to evaluate posttransplant outcomes in patients with GBM-GN compared with matched controls. Methods:This retrospective, multicenter study included 100 patients with anti-GBM who received KT between 2005 and 2023 in 13 French transplant centers, matched with 200 control recipients. We compared the incidence of delayed graft function (DGF), graft failure, relapse, acute rejection, and death between groups and analyzed risk factors using multivariable models. Results:No significant differences in DGF incidence (22% vs. 19%, P = 0.5), graft survival (87% vs. 88% at 5 years, P = 0.4), or patient survival (93% vs. 89% at 5 years, P = 0.4) were found between patients with GBM-GN and controls. Patients with GBM-GN tended to have lower risk of acute rejection (hazard ratio [HR] = 0.51, 95% confidence interval: 0.25-1.02, P = 0.055). Only 1 patient with GBM-GN (1%) experienced disease relapse. Although patients with GBM-GN were waitlisted and transplanted later than controls, specific transplant timing was not associated with improved outcomes. Conclusion:KT in patients with GBM-GN offers comparable outcomes to other nephropathies in the current era. Disease relapse is rare, even in the few patients with detectable antibodies pretransplantation. The lower incidence of acute rejection in the GBM-GN group warrants further investigation. These findings support KT as a viable option in patients with GBM-GN, though specific pre- and posttransplant monitoring is advised.

Neutralizing antibodies (NAbs) have been proposed as biomarkers for stratifying BK polyomavirus (BKPyV) replication risk, while torque teno virus (TTV) viral load is recognized as a global immunomarker in kidney transplantation. This study investigates their combined utility to improve BKPyV DNAemia risk assessment in 187 patients within 12 months posttransplant. BKPyV DNAemia occurred in 19% of recipients. Donor seronegativity was protective, whereas in case of seropositive donor, replication risk varied according to recipient NAb titers. Indeed, patients with low NAbs faced a high risk of BKPyV DNAemia regardless of TTV load, whereas in those with high NAbs, replication was influenced by TTV load at 1 month posttransplant. These findings provide a deeper understanding of BKPyV infection in kidney transplant recipients. In patients with low humoral immunity, the risk of early replication is high, whereas in those with adequate humoral immunity, replication appears to be primarily driven by the overall burden of immunosuppression. Donor status and cold ischemia time were also associated with BKPyV DNAemia, suggesting an influence of transplant characteristics and related injury. Our findings support a multifactorial BKPyV risk assessment integrating immune markers to enable personalized prevention and potentially reduce BKPyV risk.

INTRODUCTION:The complement system is involved in numerous diseases, through diverse mechanisms and degree of activation. With the emergence of complement targeting therapeutic, simple and accessible tools to evaluate the extent of complement activation are strongly needed.METHODS:We evaluated two multiplex panels, measuring complement activation fragments (C4a, C3a, C5a, Bb, Ba, sC5b9) and intact components or regulators (C1q, C2, C3, C4, C5, FD, FP, FH, FI). The specificity of each measurement was assessed by using complement proteins depleted sera and plasma collected from patients with complement deficiencies. Normal values distribution was estimated using 124 plasma samples from healthy donors and complement activation profile was assessed in plasma collected from 31 patients with various complement-mediated disorders.RESULTS:We observed good inter-assay variation. All tested protein deficiencies were accurately detected. We established assay-specific reference values for each analyte. Except for C3, C4 and C4a, the majority of the measurements were in good agreement with references methods or published data.CONCLUSION:Our study substantiates the utility of the Complement Multiplex assay as a tool for measuring complement activation and deficiencies. Quantifying complement cleavage fragments in patients exhibiting classical or alternative pathway activation allowed evaluating the activation state of the whole cascade.

Abstract Background and Aims Thrombotic microangiopathies (TMA) are usually associated with hematological features (RH-TMA). The epidemiology of TMA limited to kidneys (RL-TMA) is unclear. Method Patients with TMA and native kidney biopsies were identified during the 2009-2022 period in 20 French hospitals. Results RL-TMA was present in 341/757 (45%) patients and associated with lower creatinine levels (184 [115-297] vs 346 [206-626] µmol/L) than RH-TMA. RL-TMA resulted from virtually all KDIGO causes (more frequently from anti-VEGF treatment and hematological malignancy, less frequently from shiga toxin-associated hemolytic uremic syndrome (HUS), systemic sclerosis, gemcitabine and bacterial infection) but less frequently combined (≥3 causes/triggers: RL-TMA: 5%; RH-TMA: 12%). RL-TMA were associated with significantly lower major cardiovascular events (10% vs 20%), kidney replacement therapy (23% vs 43%) and death (12% vs 20%) than RH-TMA during follow-up (median: 28 (interquartile range: 7-72) months. Atypical HUS (aHUS) was found in 326 patients (RL-TMA: 43%, RH-TMA: 46%). Among the 69 patients with complement-mediated aHUS, 17 (25%) had RL-TMA and eculizumab was used in 43 (62%) (RL-TMA: 35%; RH-TMA: 71%). Among the 257 other aHUS patients, including 51% with RL-TMA, complete complement evaluation was performed in 85 (33%) (RL-TMA: 33%, RH-TMA: 33%) and eculizumab was used in 29 (11%). The effects of eculizumab were unclear. Conclusion RL-TMA represent a very high proportion of TMA patients, result from virtually all causes, including the 25% of patients with complement-mediated aHUS, and have a poor prognosis. Anti-C5 therapy is rarely used in RL-TMA, even in proven complement-mediated aHUS, and thus its effects remain to be assessed.

Abstract Background and Aims Complement is involved in numerous kidney diseases. However, functional approaches to properly examine individual's susceptibility to complement dysregulation are limited. Method We assessed ex vivo complement activation induced by sera from 38 healthy donors on resting microvascular endothelial cells (HMEC-1) with or without complement dysregulation by an anti-FH monoclonal antibody (OX-24 mAb). Following incubation, immunofluorescence was used to reveal and quantify membrane-bound C3c (evaluating C3b/iC3b) and C5b-9 with a computer-assisted method (H-score). Additionally, complement anaphylatoxins (C3a and C5a) were quantified in cell supernatants by ELISA. Further exploration of ex vivo complement activation profiles were conducted by next generation sequencing of a panel of alternative pathway genes in 32 donors. Results Increase in complement deposition on HMEC-1 surface was defined for a H-scoreC3c > 50 and/or a H-scoreC5b-9 > 30. Combined analysis of C3b/iC3b and C5b-9 deposition in 35 donors showed that one donor (2.8%) had an isolated increase in C3b/iC3b deposits, 3 (8.6%) had an isolated increase in C5b-9 deposits and one other (2.8%) had both increased C3b/iC3b and C5b-9 deposition. Genetic analysis of 32/38 donors revealed 3 heterozygous rare or low frequency missense variants in CFH (p.N1050Y and p.R1210C) and CFI (p.A76G) in 3/5 donors (60%) with increased complement deposition. Conclusion Our ex vivo model for measuring complement activation allowed the identification of distinct complement activation profiles among healthy donors, revealing genetic susceptibility in 3 donors. This new dynamic and functional model provides an exciting avenue for exploring the mechanisms governing complement dysregulation in various kidney diseases in research.

Background C3 glomerulopathy is a rare disease resulting from an overactivation of the complement alternative pathway. Although there is also evidence of terminal pathway activation, its occurrence and consequences on the disease have been poorly studied. Methods We retrospectively studied a cohort of 42 patients diagnosed with C3 glomerulopathy. We performed centralized extensive characterization of histological parameters. Kidney C5b-9 staining was performed as a marker of terminal pathway activation; intrarenal immune response was characterized through transcriptomic analysis. Results Eighty-eight percent of biopsies showed C5b-9 deposits in glomeruli. Biopsies were grouped according to the amount of C5b-9 deposits (no or low n=15/42, 36%; intermediate n=15/42, 36%; and high n=12/42, 28%). Patients with high C5b-9 deposits significantly differed from the two other groups of patients and were characterized by a significant higher histological chronicity score (P = 0.005) and lower outcome-free survival (P = 0.001). In multivariable analysis, higher glomerular C5b-9 remained associated with poor kidney prognosis after adjustment. One third of the 847 studied immune genes were upregulated in C3 glomerulopathy biopsies compared with controls. Unsupervised clustering on differentially expressed genes identified a group of kidney biopsies enriched in high glomerular C5b-9 with high immune and fibroblastic signature and showed high chronicity scores on histological examination. Conclusions In a cohort of patients with C3 glomerulopathy, intrarenal terminal pathway activation was associated with specific histological phenotype and disease prognosis.