C3 glomerulopathy (C3G) encompasses a group of severe renal diseases characterized by “dominant C3” deposition in the renal glomerulus. Patients typically present as nephritic nephrotics, with hematuria, hypertension, heavy proteinuria and edema. Within ten years of diagnosis, 50% of affected patients progress to end-stage renal disease and require dialysis or renal transplantation. No treatment is available to halt disease progression and thus both disease recurrence and allograft loss are common after transplantation. Genetic studies of C3G have firmly implicated dysregulation of the alternative pathway (AP) of complement in disease pathogenesis. In addition to genetic factors, acquired factors like autoantibodies can also exaggerate AP activity in the circulation to cause C3G. Although AP dysregulation in the circulation (i.e. fluid-phase dysregulation) has been well studied in these patients, AP activity in the glomerular microenvironment is not well understood. In this body of work, we used MaxGel, an ex-vivo surrogate for the glomerular extracellular matrix, to study AP activity and regulation. We showed that C3 convertase can be assembled on MaxGel and elucidated the dynamics of its formation and decay in the presence of complement regulators. We confirm that on MaxGel factor H (fH) inhibits C3 convertase formation and accelerates its decay, while properdin has a stabilizing effect. We also show that the complement factor H-related proteins (FHRs) are vital to the regulation of AP activity. Consistent with our MaxGel data, CFHR gene-fusion events have been reported as genetic drivers of disease in a few familial cases of C3G. One such familial case in which we identified and characterized the rearrangement event results from a novel CFHR5-CFHR2 fusion gene. The fusion gene is translated into a circulating FHR-5/-2 protein that consists of the first two SCRs of FHR-5 followed by all four SCRs of FHR-2. The structural repetition of SCR1-2 followed by another SCR1-2 motif facilitates the formation of complex FHR-1, FHR-2 and FHR-5 multimers, which have enhanced affinity for C3b and by out-competing fH, lead to impaired C3 convertase regulation in the glomerular microenvironment. Finally, we tested gene therapy as a tool to rescue the disease phenotype and restore fluid-phase AP complement control in a mouse model of C3G (Cfh-/-/huCR1-Tg mice). Using the piggyBac transposon system, we introduced a construct derived from complement regulator 1 (CR1) into Cfh-/-/huCR1-Tg mice. Delivery of sCR1-AC via hydrodynamic tail vein injection provided constitutive circulatory expression of sCR1-AC, and in animals followed for 6 months, we found that long-term expression of this complement regulator rescued the renal phenotype. These results suggest that sCR1 may be a potential therapy for patients with this disease.

De façon physiologique, la voie alterne (VA) du complément est activée en permanence. Elle doit donc être finement régulée à tous les niveaux de la cascade afin de ne pas être délétère pour l'hôte. Chez l'homme, deux pathologies rénales sont associées à une activation non contrôlée de la VA du complément: le syndrome hémolytique et urémique atypique et la glomérulopathie à dépôts de C3 (GP-C3). Pathologie du sujet jeune essentiellement, la GP-C3 regroupe deux entités, la maladie des dépôts denses (GN-DD) et la glomérulonéphrite à dépôts de C3 (GN-C3), d'expressions clinique et histologique hétérogènes et toutes les deux de pronostic rénal réservé en l’absence de traitement efficace disponible (médiane de survie rénale de l’ordre de 8 à 10 ans). Les anomalies de la VA du complément mises en évidence dans la GP-C3 sont essentiellement acquises en rapport avec la présence d'auto anticorps (Ac) dirigés contre la C3 convertase alterne, le C3NeF, ou contre le FH, une protéine régulatrice essentielle de la VA. Dans 15 à 20% des cas seulement, les anomalies sont génétiques en rapport avec des mutations du FH, du FI. Les approches in silico de modélisation moléculaire des protéines mutées apportent des éléments de réponse quant à la responsabilité potentielle d'une mutation dans l’activation non contrôlée de la VA, néanmoins, l'étude des conséquences fonctionnelles est indispensable pour relier l'anomalie à la survenue de la pathologie rénale mais aussi pour comprendre les mécanismes précis impliqués dans le déterminisme des lésions rénales, de GN-DD ou de GN-C3. J'ai dans un premier temps étudié les conséquences fonctionnelles de la première mutation de C3 identifiée dans une forme familiale de GN-C3. In silico, cette mutation I734T est située au niveau d'un site impliqué dans la liaison de C3 avec deux protéines régulatrices, le FH et CR1 et à proximité du site d’interaction avec le FB. In vitro, j'ai pu confirmer que la mutation était responsable d'une activation de la VA au niveau tissulaire en rapport avec un défaut induit de régulation par le FH mais surtout CR1, une protéine régulatrice membranaire, identifié pour la première fois comme acteur dans la physiopathologie de la GP-C3. Par ailleurs, en raison de l'expression exclusivement podocytaire de CR1 au niveau glomérulaire, nous avons émis l'hypothèse que les régulateurs pouvaient jouer un rôle déterminant dans la localisation préférentielle des dépôts de C3 au niveau glomérulaire dans cette pathologie. Dans une deuxième partie, à la fois clinique et expérimentale, j'ai travaillé sur les formes acquises de GP-C3 en étudiant un sous-groupe particulier de patients de la cohorte française de GP-C3, âgés de plus de 50 ans et présentant une gammapathie monoclonale. La partie clinique de ce travail a permis de démontrer que la fréquence des gammapathies monoclonales est très nettement augmentée dans la GP-C3 chez les patients âgés de plus de 50 ans comparée à la population générale, que le pronostic rénal est particulièrement sombre avec une médiane de survie rénale de l’ordre de 2 ans et demi mais que le traitement efficace du clone B sous-jacent permet d'améliorer significativement la survie rénale. Les données de l’étude clinique suggèrent indirectement qu’il existe un lien entre l'immunoglobuline (Ig) monoclonale et l'activation de la VA responsable de l'apparition des lésions rénales. Dans la partie expérimentale, j'ai étudié les mécanismes d'activation de la VA dans ce contexte de gammapathie afin de confronter les données de l’étude clinique. L'étude des biomarqueurs d'activation de la C3/C5 convertase et le démembrement des mécanismes d'activation ont permis de conclure que la GP-C3 associée aux gammapathies monoclonales est caractérisée par une activation tissulaire de la VA impliquant la C3 convertase mais aussi et surtout la C5 convertase. (...) / Complement alternative pathway is physiologically activated. It need to be tightly regulated to avaoid uncontrolled deleterious overactivation on host cell surface. In human, two renal diseaes are associated with uncontrolled AP activation, hemolytic uremic syndrom atypical (aHUS) and C3 glomerulopathy (C3G). C3G occures mainly in children and young adults and regoups two distinct histopathological entities, dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). Renal outcomes in C3G is poor since up to 50% of patients reach end stage renal disease 8 to 10 years after diagnosis. Complement abnormalities in C3G are mainly acquired induced by the presence of C3 Nephritic Factor (C3NeF): an autoantibdy targeting the AP C3 convertase. Less frequently C3G patients have anti-FH autoantibodies (Ref) or genetic abnormalities (variants in the FH, FI or CFHR5 genes. In silico analysis of mutated proteins give information about the role of mutation on AP overactivation. However, characterization of functional consequences of these mutations is required to proved the direct link between the abnormality and the occurrence of C3G. I first studied the functional consequences of the first C3 mutation, C3I734T, identified in a familial C3GN. In silico analysis revealed that the mutated residue, T734, is located on the C3 and C3b protein surface and that the substitution I734T may not be associated with major structural changes. The mutated amino acid is located on interaction site between C3b and complement regulatory proteins, FH and CR1. In vitro, the major defect of C3I734T was a decrease in binding to CR1, resulting in lower CR1 dependent cleavage of C3b by FI. These results provide evidence for a CR1 functional deficiency being responsible for deficient complement regulation. Binding of C3I734T to Factor H (FH) was normal, but C3I734T was less efficiently cleaved by Factor I, leading to enhanced C3 fragments binding on glomerular cells. In the second part of my work, I studied acquired C3G in patients with concomitant monoclonal gammopathy. In the clinical part of this study, we demonstrated The high prevalence of monoclonal gammopathy in C3G patients aged over 50, reaching 65% in the French C3G national cohort, strongly suggests a pathogenic link between the two conditions. Next, we demonstrated that renal outcomes is significantly worser in patients with monoclonal gammopathy compared to patients without monoclonal gammopathy but that efficient chemotherapy resulted in higher renal response rate and longer renal survival than conservative or immunosuppressive therapy. Results of the clinical part of the study strongly suggest a link betwwen the monoclonal gammopathy and the occurrence of C3G. In the experimental part of this work, I studied the mechanisms of complement AP activation in these population. Biomarkers of C3 and C5 convertase activation were present 40% and 81% of patients respectively. Anti-complement protein antibodies were found in 23/41 (56%) patients, including in most of patients, anti-FH and anti-CR1 antibodies. I found new antigenic target, C5 and properdin in few cases. The anti-FH and anti-CR1 antibodies were associated with clear functional consequences. Nevertheless, the anti-complement proteins reactivity was not carried out by the MIg in 75% of the cases. I discovered that the MIg induced a direct AP C3 convertase overactivation in 23/34 (67%) patients responsaible for a C5 convertase overactivation in presence of patients’ Ig, in a properdin dependant manner. Our results suggest that MIg and polyclonal autoantibodies could act in synergy: AP overactivation induced by the MIg could be amplified by the inefficient complement regulation, caused by the polyclonal anti-complement autoantibodies. All my results allow to better understand the pathophysiological mechanisms involved in C3G and open up reflection on therapeutic approaches for C3G associated with monoclonal gammopathy.

Normalt sett skyddar komplementsystemet kroppen mot infektioner och patogener. Vid vissa typer av njursjukdomar, framför allt vid C3-glomerulopati, förekommer autoantikroppar som kallas ”nephritic factors” (NeF). Sådana antikroppar stabiliserar enzymkomplex (konvertas) i komplementsystemet, vilket leder till destruktiv komplementaktivering via den alternativa vägen. Syftet med studien var att utveckla minst en metod för att analysera C5NeF på kliniska prover.  C5NeF In-House ELISA analyserade bindning av C5NeF till C5-konvertas. Analys av C5-klyvning i löslig fas kvantifierade mängden C5a som bildats vid stabilisering av C5-konvertas. Cut-off för analyserna bestämdes genom analys av prover från 20 friska blodgivare. Tolv patientprover med möjlig förekomst av C5NeF analyserades. För att utesluta falskt positiv reaktion i C5NeF in-house ELISA analyserades även förekomst av antikroppar mot specifika enskilda komplementproteiner. Åtta patientprover var positiva i C5NeF In-House ELISA, fem patientprover uppvisade positivt resultat för C3NeF, vilket inte var oväntat utifrån tidigare publikationer som visat att det är vanligt att patienter med C5NeF också ofta är positiva för C3NeF. Tre patientprover erhöll positivt resultat i endast C5NeF In-House ELISA och två av dessa var positiva i analys av C5-klyvning i löslig fas. Studien resulterade i etablering av en metod för analys av C5NeF. / Normally the complement system protects the body from infections and pathogens. In certain types of kidney diseases, mainly C3-glomerulopathy, autoantibodies called ”Nephritic Factors” (NeF) are found. NeFs stabilize enzyme complexes (convertases) in the complement system, an event which leads to destructive complement activation via the alternative pathway. The purpose of this study was to develop at least one method to analyse C5NeF on clinical samples.  C5NeF In-House ELISA analysed binding of C5NeF to C5 convertases. Analysis of C5-cleavage in the soluble phase measured the amount of C5a formed when C5-convertase was stabilized. Cut-off for the analyses was determined through analysis of 20 blood donor samples from healthy individuals. Twelve patient samples with possible C5NeF were analysed. To exclude false positive results in C5NeF In-House ELISA analysis of antibodies against specific single complement factors was performed.  Eight patient samples were positive in C5NeF In-House ELISA, five patient samples showed positive result for C3NeF, a finding which was not unexpected as previous publications have shown that concomitant presence of C3NeF and C5NeF is common in C3-glomerulopathy. Where most patients are positive for both C3NeF and C5NeF. Three patient samples received positive result in only C5NeF In-House ELISA and two of these samples were positive in the analysis of C5-cleavage in soluble phase. In conclusion, in this study a method to examine C5NeF was developed.