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Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.
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Glomerulonefritis Membranoproliferativa , Enfermedades Renales , Animales , Complemento C3/genética , Complemento C3/metabolismo , Factor B del Complemento/metabolismo , Factor H de Complemento/genética , Vía Alternativa del Complemento/genética , Glomerulonefritis Membranoproliferativa/patología , Humanos , Ratones , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Between 5 and 50% of atypical hemolytic uremic syndrome (aHUS) cases in children are caused by autoantibodies against complement factor H (CFH). Given the acquired autoimmune nature of the disease, plasma exchange (PE) and various immunosuppressive treatments have been used. More recently, eculizumab has been proposed. METHODS: In this multicenter, retrospective study, we report outcomes of 12 children with anti-FH antibody-associated HUS treated with eculizumab associated with various immunosuppressive regimens. RESULTS: Patients were treated with eculizumab for 15.5 [9.5;23.0] months and 3 received PE or IgG adsorption. Three patients received mycophenolate mofetil (MMF) alone, 1 patient received MMF and steroids, 1 patient received MMF and rituximab, 3 patients received MMF/steroids and rituximab, and 4 patients did not receive any immunosuppression. Anti-FH antibody levels significantly decreased but no difference was observed based on the immunosuppressive regimen. Eculizumab was discontinued in 7/10 patients after 11 [7.5;15.5] months and MMF in 6/8 patients after 36 [35;40] months. Anti-FH titers at MMF discontinuation ranged from 257 to 3425 UI/L. None of these patients relapsed and eGFR at last follow-up was above 70 mL/min/1.73 m2 in all patients. CONCLUSIONS: Eculizumab is effective and safe in inducing and maintaining remission in aHUS secondary to anti-FH antibodies and renders reduction of anti-FH titers less urgent. Anti-FH antibody titers decreased in most patients irrespective of the immunosuppressive treatment chosen, so that a strategy consisting of combining eculizumab with MMF monotherapy seems sufficient at least in non-Indian or less severe forms of anti-FH antibody-associated HUS.
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RATIONALE & OBJECTIVE: Although primary atypical hemolytic uremic syndrome (aHUS) is associated with abnormalities in complement genes and antibodies to complement factor H, the role of complement in secondary aHUS remains debatable. We evaluated the usefulness of an ex vivo test to: (1) detect complement activation within the endothelium in primary and secondary aHUS, (2) differentiate active disease from remission, (3) monitor the effectiveness of eculizumab therapy, and (4) identify relapses during eculizumab dosage tapering and after discontinuation of treatment. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 121 patients with primary aHUS and 28 with secondary aHUS. Serum samples were collected during acute episodes, following remission, and during eculizumab treatment and were assessed using a serum-induced ex vivo C5b-9 endothelial deposition test. RESULTS: Serum-induced C5b-9 deposition on cultured microvascular endothelium was quantified by calculating the endothelial area covered by C5b-9 staining; values were expressed as percentage of C5b-9 deposits induced by a serum pool from healthy controls. Testing with adenosine diphosphate-activated endothelium demonstrated elevated C5b-9 deposits for all untreated patients with aHUS independent of disease activity, while testing with unstimulated endothelium demonstrated deposits only in active disease. Similar findings were observed in secondary aHUS. Serum-induced C5b-9 deposits on activated and unstimulated endothelium normalized during eculizumab treatment. 96% (22/23) of patients receiving eculizumab at extended 3- or 4-week dosing intervals demonstrated normal C5b-9 deposits on activated endothelium, despite most patients having CH50Eq (serum complement activity) > 20 UEq/mL, indicating that adequate complement control was achieved even with incomplete blockade of circulating C5. During eculizumab dosage tapering or after treatment discontinuation, all patients experiencing relapses versus only 6% (1/17) of those in stable remission had elevated C5b-9 deposits on unstimulated endothelium. LIMITATIONS: The C5b-9 endothelial deposition test can be performed in only specialized laboratories. Findings on eculizumab dosage tapering need to be confirmed with longitudinal monitoring of C5b-9 deposition. CONCLUSIONS: The C5b-9 endothelial deposition assay may represent an advance in our ability to monitor aHUS activity and individualize therapy.
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Anticuerpos Monoclonales Humanizados , Síndrome Hemolítico Urémico Atípico , Activación de Complemento/efectos de los fármacos , Complejo de Ataque a Membrana del Sistema Complemento/análisis , Monitoreo de Drogas/métodos , Adulto , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacocinética , Síndrome Hemolítico Urémico Atípico/sangre , Síndrome Hemolítico Urémico Atípico/tratamiento farmacológico , Factor H de Complemento/análisis , Factor H de Complemento/genética , Inactivadores del Complemento/administración & dosificación , Inactivadores del Complemento/farmacocinética , Relación Dosis-Respuesta a Droga , Endotelio Vascular/metabolismo , Femenino , Humanos , Técnicas In Vitro/métodos , Masculino , Reproducibilidad de los Resultados , Prevención Secundaria/métodos , Prevención Secundaria/estadística & datos numéricosRESUMEN
von Willebrand factor (VWF), a multimeric protein with a central role in hemostasis, has been shown to interact with complement components. However, results are contrasting and inconclusive. By studying 20 patients with congenital thrombotic thrombocytopenic purpura (cTTP) who cannot cleave VWF multimers because of genetic ADAMTS13 deficiency, we investigated the mechanism through which VWF modulates complement and its pathophysiological implications for human diseases. Using assays of ex vivo serum-induced C3 and C5b-9 deposits on endothelial cells, we documented that in cTTP, complement is activated via the alternative pathway (AP) on the cell surface. This abnormality was corrected by restoring ADAMTS13 activity in cTTP serum, which prevented VWF multimer accumulation on endothelial cells, or by an anti-VWF Ab. In mechanistic studies we found that VWF interacts with C3b through its three type A domains and initiates AP activation, although assembly of active C5 convertase and formation of the terminal complement products C5a and C5b-9 occur only on the VWF-A2 domain. Finally, we documented that in the condition of ADAMTS13 deficiency, VWF-mediated formation of terminal complement products, particularly C5a, alters the endothelial antithrombogenic properties and induces microvascular thrombosis in a perfusion system. Altogether, the results demonstrated that VWF provides a platform for the activation of the AP of complement, which profoundly alters the phenotype of microvascular endothelial cells. These findings link hemostasis-thrombosis with the AP of complement and open new therapeutic perspectives in cTTP and in general in thrombotic and inflammatory disorders associated with endothelium perturbation, VWF release, and complement activation.
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Complemento C3b/metabolismo , Vía Alternativa del Complemento , Células Endoteliales/inmunología , Microvasos/patología , Trombosis/fisiopatología , Factor de von Willebrand/metabolismo , Proteína ADAMTS13/sangre , Proteína ADAMTS13/deficiencia , Proteína ADAMTS13/inmunología , Proteína ADAMTS13/metabolismo , Adolescente , Adulto , Niño , Preescolar , Convertasas de Complemento C3-C5/metabolismo , Complemento C3b/inmunología , Complemento C5a/inmunología , Complemento C5a/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Humanos , Recién Nacido , Masculino , Microvasos/inmunología , Púrpura Trombocitopénica Trombótica/congénito , Púrpura Trombocitopénica Trombótica/inmunología , Púrpura Trombocitopénica Trombótica/fisiopatología , Trombosis/inmunología , Adulto Joven , Factor de von Willebrand/inmunologíaRESUMEN
Membranoproliferative glomerulonephritis (MPGN) is a rare chronic kidney disease associated with complement activation. Recent immunofluorescence-based classification distinguishes between immune complex (IC)-mediated MPGN, with glomerular IgG and C3 deposits, and C3 glomerulopathies (C3G), with predominant C3 deposits. Genetic and autoimmune abnormalities causing hyperactivation of the complement alternative pathway have been found as frequently in patients with immune complex-associated MPGN (IC-MPGN) as in those with C3G. In the last decade, there have been great advances in research into the autoimmune causes of IC-MPGN and C3G. The complement-activating autoantibodies called C3-nephritic factors (C3NeFs), which are present in 40-80% of patients, form a heterogeneous group of autoantibodies that stabilise the C3 convertase or the C5 convertase of the alternative pathway or both. A few patients, mainly with IC-MPGN, carry autoantibodies directed against the two components of the alternative pathway C3 convertase, factors B and C3b. Finally, autoantibodies against factor H, the main regulator of the alternative pathway, have been reported in a small proportion of patients with IC-MPGN or C3G. The identification of distinct pathogenetic patterns leading to kidney injury and of targets in the complement cascade may pave the way for tailored therapies for IC-MPGN and C3G, with specific complement inhibitors in the development pipeline.
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Factor Nefrítico del Complemento 3/inmunología , Complemento C3/inmunología , Vía Alternativa del Complemento/genética , Glomerulonefritis Membranoproliferativa/inmunología , Complemento C3/genética , Convertasas de Complemento C3-C5 de la Vía Alternativa/inmunología , Factor B del Complemento/inmunología , Factor H de Complemento/inmunología , Membrana Basal Glomerular/inmunología , Membrana Basal Glomerular/patología , Mesangio Glomerular/inmunología , Mesangio Glomerular/patología , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/patología , HumanosRESUMEN
VOLUME 291 (2016) PAGES 82148230 This article has been withdrawn by the authors. Lanes 1 and 7 of Fig. 4B were duplicated.
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The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies (Conrad, D. H., Carlo, J. R., and Ruddy, S. (1978)J. Exp. Med.147, 1792-1805; Pangburn, M. K., and Müller-Eberhard, H. J. (1978)Proc. Natl. Acad. Sci. U.S.A.75, 2416-2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979)J. Immunol.122, 75-81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings.
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C3 Convertasa de la Vía Alternativa del Complemento/inmunología , Convertasas de Complemento C3-C5/inmunología , Factor H de Complemento/inmunología , Complemento C3/inmunología , C3 Convertasa de la Vía Alternativa del Complemento/análisis , Convertasas de Complemento C3-C5/análisis , Complemento C3b/inmunología , Factor B del Complemento/inmunología , Factor H de Complemento/análisis , Ensayo de Inmunoadsorción Enzimática , Humanos , Manganeso/análisis , Manganeso/inmunología , Properdina/inmunologíaRESUMEN
Atypical hemolytic-uremic syndrome (aHUS) is associated with genetic complement abnormalities/anti-complement factor H antibodies, which paved the way to treatment with eculizumab. We studied 44 aHUS patients and their relatives to (1) test new assays of complement activation, (2) verify whether such abnormality occurs also in unaffected mutation carriers, and (3) search for a tool for eculizumab titration. An abnormal circulating complement profile (low C3, high C5a, or SC5b-9) was found in 47% to 64% of patients, irrespective of disease phase. Acute aHUS serum, but not serum from remission, caused wider C3 and C5b-9 deposits than control serum on unstimulated human microvascular endothelial cells (HMEC-1). In adenosine 5'-diphosphate-activated HMEC-1, also sera from 84% and 100% of patients in remission, and from all unaffected mutation carriers, induced excessive C3 and C5b-9 deposits. At variance, in most patients with C3 glomerulopathies/immune complex-associated membranoproliferative glomerulonephritis, serum-induced endothelial C5b-9 deposits were normal. In 8 eculizumab-treated aHUS patients, C3/SC5b-9 circulating levels did not change posteculizumab, whereas serum-induced endothelial C5b-9 deposits normalized after treatment, paralleled or even preceded remission, and guided drug dosing and timing. These results point to efficient complement inhibition on endothelium for aHUS treatment. C5b-9 endothelial deposits might help monitor eculizumab effectiveness, avoid drug overexposure, and save money considering the extremely high cost of the drug.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Activación de Complemento/efectos de los fármacos , Síndrome Hemolítico-Urémico/sangre , Síndrome Hemolítico-Urémico/tratamiento farmacológico , Monitoreo Fisiológico , Adenosina Difosfato Ribosa/farmacología , Síndrome Hemolítico Urémico Atípico , Complemento C3/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Glomerulonefritis Membranoproliferativa/sangre , Glomerulonefritis Membranoproliferativa/tratamiento farmacológico , Glomerulonefritis Membranoproliferativa/patología , Síndrome Hemolítico-Urémico/patología , Humanos , Masculino , Inducción de Remisión , Factores de TiempoRESUMEN
Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association.
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Síndrome Hemolítico Urémico Atípico/genética , Síndrome Hemolítico Urémico Atípico/inmunología , Factor B del Complemento/genética , Mutación , Sustitución de Aminoácidos , Sitios de Unión/genética , C3 Convertasa de la Vía Alternativa del Complemento/química , C3 Convertasa de la Vía Alternativa del Complemento/genética , C3 Convertasa de la Vía Alternativa del Complemento/metabolismo , Complemento C3b/metabolismo , C5 Convertasa de la Vía Alternativa del Complemento/química , C5 Convertasa de la Vía Alternativa del Complemento/genética , C5 Convertasa de la Vía Alternativa del Complemento/metabolismo , Factor B del Complemento/química , Factor B del Complemento/metabolismo , Vía Alternativa del Complemento/genética , Simulación por Computador , Frecuencia de los Genes , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ligandos , Modelos Moleculares , Complejos Multiproteicos/química , Polimorfismo Genético , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Focal segmental glomerulosclerosis is a kidney disease that is manifested as the nephrotic syndrome. It is often resistant to glucocorticoid therapy and progresses to end-stage renal disease in 50 to 70% of patients. Genetic studies have shown that familial focal segmental glomerulosclerosis is a disease of the podocytes, which are major components of the glomerular filtration barrier. However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive. METHODS: We performed whole-genome linkage analysis followed by high-throughput sequencing of the positive-linkage area in a family with autosomal recessive focal segmental glomerulosclerosis (index family) and sequenced a newly discovered gene in 52 unrelated patients with focal segmental glomerulosclerosis. Immunohistochemical studies were performed on human kidney-biopsy specimens and cultured podocytes. Expression studies in vitro were performed to characterize the functional consequences of the mutations identified. RESULTS: We identified two mutations (A159P and Y695X) in MYO1E, which encodes a nonmuscle class I myosin, myosin 1E (Myo1E). The mutations in MYO1E segregated with focal segmental glomerulosclerosis in two independent pedigrees (the index family and Family 2). Patients were homozygous for the mutations and did not have a response to glucocorticoid therapy. Electron microscopy showed thickening and disorganization of the glomerular basement membrane. Normal expression of Myo1E was documented in control human kidney-biopsy specimens in vivo and in glomerular podocytes in vitro. Transfection studies revealed abnormal subcellular localization and function of the A159P-Myo1E mutant. The Y695X mutation causes loss of calmodulin binding and of the tail domains of Myo1E. CONCLUSIONS: MYO1E mutations are associated with childhood-onset, glucocorticoid-resistant focal segmental glomerulosclerosis. Our data provide evidence of a role of Myo1E in podocyte function and the consequent integrity of the glomerular filtration barrier.
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Glomeruloesclerosis Focal y Segmentaria/genética , Mutación , Miosina Tipo I/genética , Edad de Inicio , Animales , Niño , Preescolar , Resistencia a Medicamentos , Femenino , Genes Recesivos , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Glomeruloesclerosis Focal y Segmentaria/patología , Glucocorticoides/uso terapéutico , Humanos , Lactante , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/ultraestructura , Masculino , Ratones , Microscopía Fluorescente , Mutación Missense , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Linaje , Podocitos/metabolismo , Podocitos/ultraestructura , Alineación de SecuenciaRESUMEN
The quantification of residual plasmatic ADAMTS13 activity in congenital thrombotic thrombocytopenic purpura (TTP) patients is constrained by limitations in sensitivity and reproducibility of commonly used assays at low levels of ADAMTS13 activity, blunting efforts to establish genotype-phenotype correlations. In the present study, the residual plasmatic activity of ADAMTS13 was measured centrally by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (limit of detection = 0.5%) in 29 congenital TTP patients. The results were used to study correlations among ADAMTS13 genotype, residual plasmatic activity, and clinical phenotype severity. An ADAMTS13 activity above 0.5% was measured in 26 (90%) patients and lower levels of activity were associated with earlier age at first TTP episode requiring plasma infusion, more frequent recurrences, and prescription of fresh-frozen plasma prophylaxis. Receiver operating characteristic curve analysis showed that activity levels of less than 2.74% and 1.61% were discriminative of age at first TTP episode requiring plasma infusion < 18 years, annual rate of TTP episodes > 1, and use of prophylaxis. Mutations affecting the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity and a more severe phenotype in an allelic-dose dependent manner. The results of the present study show that residual ADAMTS13 activity is associated with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.
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Proteínas ADAM/sangre , Proteínas ADAM/deficiencia , Púrpura Trombocitopénica Trombótica/sangre , Púrpura Trombocitopénica Trombótica/congénito , Proteínas ADAM/genética , Proteína ADAMTS13 , Adolescente , Adulto , Factores de Edad , Anciano , Análisis Químico de la Sangre , Transfusión Sanguínea , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Plasma , Púrpura Trombocitopénica Trombótica/genética , Púrpura Trombocitopénica Trombótica/terapia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adulto JovenRESUMEN
Introduction: Complement factor H (FH) is a major regulator of the complement alternative pathway, its mutations predispose to an uncontrolled activation in the kidney and on blood cells and to secondary C3 deficiency. Plasma exchange has been used to correct for FH deficiency and although the therapeutic potential of purified FH has been suggested by in vivo experiments in animal models, a clinical approved FH concentrate is not yet available. We aimed to develop a purification process of FH from a waste fraction rather than whole plasma allowing a more efficient and ethical use of blood and plasma donations. Methods: Waste fractions from industrial plasma fractionation (pooled human plasma) were analyzed for FH content by ELISA. FH was purified from unused fraction III and its decay acceleration, cofactor, and C3 binding capacity were characterized in vitro. Biodistribution was assessed by high-resolution dynamic PET imaging. Finally, the efficacy of the purified FH preparation was tested in the mouse model of C3 glomerulopathy (Cfh-/- mice). Results: Our purification method resulted in a high yield of highly purified (92,07%), pathogen-safe FH. FH concentrate is intact and fully functional as demonstrated by in vitro functional assays. The biodistribution revealed lower renal and liver clearance of human FH in Cfh-/- mice than in wt mice. Treatment of Cfh-/- mice documented its efficacy in limiting C3 activation and promoting the clearance of C3 glomerular deposits. Conclusion: We developed an efficient and economical system for purifying intact and functional FH, starting from waste material of industrial plasma fractionation. The FH concentrate could therefore constitute possible treatments options of patients with C3 glomerulopathy, particularly for those with FH deficiency, but also for patients with other diseases associated with alternative pathway activation.
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Complemento C3 , Factor H de Complemento , Ratones Noqueados , Factor H de Complemento/metabolismo , Factor H de Complemento/genética , Animales , Humanos , Ratones , Modelos Animales de Enfermedad , Prueba de Estudio Conceptual , Ratones Endogámicos C57BLRESUMEN
Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenia purpura (TTP) are rare diseases sharing a common pathological feature, thrombotic microangiopathy (TMA). TMA is characterized by microvascular thrombosis with consequent thrombocytopenia, microangiopathic hemolytic anemia and/or multiorgan dysfunction. In the past, the distinction between HUS and TTP was predominantly based on clinical grounds. However, clinical presentation of the two syndromes often overlaps and, the differential diagnosis is broad. Identification of underlying pathogenic mechanisms has enabled the classification of these syndromes on a molecular basis: typical HUS caused by Shiga toxin-producing Escherichia coli (STEC-HUS); atypical HUS or complement-mediated TMA (aHUS/CM-TMA) associated with genetic or acquired defects leading to dysregulation of the alternative pathway (AP) of complement; and TTP that results from a severe deficiency of the von Willebrand Factor (VWF)-cleaving protease, ADAMTS13. The etiology of TMA differs between pediatric and adult patients. Childhood TMA is chiefly caused by STEC-HUS, followed by CM-TMA and pneumococcal HUS (Sp-HUS). Rare conditions such as congenital TTP (cTTP), vitamin B12 metabolism defects, and coagulation disorders (diacylglycerol epsilon mutation) present as TMA chiefly in children under 2 years of age. In contrast secondary causes and acquired ADAMT13 deficiency are more common in adults. In adults, compared to children, diagnostic delays are more frequent due to the wide range of differential diagnoses. In this review we focus on the three major forms of TMA, STEC-HUS, aHUS and TTP, outlining the clinical presentation, diagnosis and management of the affected patients, to help highlight the salient features and the differences between adult and pediatric patients which are relevant for management.
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Síndrome Hemolítico Urémico Atípico , Púrpura Trombocitopénica Trombótica , Microangiopatías Trombóticas , Adulto , Humanos , Niño , Lactante , Púrpura Trombocitopénica Trombótica/diagnóstico , Púrpura Trombocitopénica Trombótica/terapia , Microangiopatías Trombóticas/diagnóstico , Microangiopatías Trombóticas/etiología , Síndrome Hemolítico Urémico Atípico/diagnóstico , Diagnóstico Diferencial , MutaciónRESUMEN
Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease. Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely. Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.
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Glomerulonefritis Membranoproliferativa , Enfermedades Renales , Humanos , Animales , Ratones , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Complemento C3 , Homocigoto , Eliminación de Secuencia , Factor H de Complemento/genética , Hígado/metabolismo , Vía Alternativa del Complemento/genética , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/terapia , Glomerulonefritis Membranoproliferativa/metabolismoRESUMEN
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited disease of the kidney. It occurs in adulthood but is also rarely diagnosed in early childhood. The majority of the disease-causing variants observed in ADPKD patients are in two genes: PKD1 and PKD2. METHODS: 237 patients from 198 families with a clinical diagnosis of ADPKD were screened for PKD1 and PKD2 genetic variants using Sanger sequencing and Multiple Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: Disease-causing (diagnostic) variants were identified in 173 families (211 patients), 156 on PKD1 and 17 on PKD2. Variants of unknown significance (VUS) were detected in 6 additional families, while no mutations were found in the remaining 19 families. Among the diagnostic variants detected, 51 were novel. In ten families, seven large rearrangements were found and the molecular breakpoints of 3 rearrangements were identified. Renal survival was significantly worse for PKD1 mutated patients, particularly those carrying truncating mutations. In patients with PKD1 truncating ( PKD1-T) mutations, disease onset was significantly earlier than in patients with PKD1 non-truncating (PKD1-NT) variants or PKD2 mutated patients. CONCLUSIONS: Comprehensive genetic testing confirms its utility in diagnosing patients with ADPKD and contributes to explaining the clinical heterogeneity observed in this disease. Moreover, the genotype-phenotype correlation can allow a more accurate disease prognosis.
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Hemolytic uremic syndrome (HUS) is a rare disease characterized by hemolytic anemia, thrombocytopenia, and renal impairment mostly triggered by strains of Shiga-like toxin-producing Escherichia coli (STEC-HUS). A rarer form of HUS, defined as atypical HUS (aHUS), is associated with genetic or acquired dysregulation of the alternative pathway of the complement system and presents a poorer prognosis than STEC-HUS. Factor H autoantibodies (anti-FHs) have been reported in aHUS in 5-11% of cases and are strongly associated with the homozygous deletion of CFHR3-CFHR1 genes. In the large majority of patients, anti-FH-associated aHUS is commonly preceded by gastrointestinal or respiratory tract infections. Here, we described the clinical case of a 3-year-old boy who was hospitalized for aHUS preceded by Mycoplasma pneumoniae (MP) infection. He resulted positive for anti-FHs and carried the homozygous deletion of CFHR3-CFHR1. Of relevance, he also showed a variant of unknown significance in the C5 gene. The patient was successfully treated with eculizumab and achieved hematological and renal remission. The anti-FH titer decreased, became negative after 6 months of mycophenolate mofetil (MMF) treatment, and remained negative for 21-month follow-up indicating that immunosuppression was effective and could prevent the reappearance of anti-FHs. We hypothesized that MP, likely through an evasion strategy of immunosurveillance based on binding of pathogen to FH, triggers anti-FH antibody generation and aHUS in a subject genetically predisposed. In conclusion, to the best of our knowledge, here, we reported the first case of anti-FH-mediated aHUS after an MP infection who benefited from eculizumab and immunosuppressive therapy based on MMF. Hence, monitoring of anti-FHs in patients with post-MP infection glomerulonephritis could be recommended, especially in those with low C3 plasma levels.
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Síndrome Hemolítico Urémico Atípico , Neumonía por Mycoplasma , Humanos , Preescolar , Autoanticuerpos , Homocigoto , Eliminación de SecuenciaRESUMEN
Microvascular thrombosis is associated with multiorgan failure and mortality in coronavirus disease 2019 (COVID-19). Although thrombotic complications may be ascribed to the ability of SARS-CoV-2 to infect and replicate in endothelial cells, it has been poorly investigated whether, in the complexity of viral infection in the human host, specific viral elements alone can induce endothelial damage. Detection of circulating spike protein in the sera of severe COVID-19 patients was evaluated by ELISA. In vitro experiments were performed on human microvascular endothelial cells from the derma and lung exposed to SARS-CoV-2-derived spike protein 1 (S1). The expression of adhesive molecules was studied by immunofluorescence and leukocyte adhesion and platelet aggregation were assessed under flow conditions. Angiotensin converting enzyme 2 (ACE2) and AMPK expression were investigated by Western Blot analysis. In addition, S1-treated endothelial cells were incubated with anti-ACE2 blocking antibody, AMPK agonist, or complement inhibitors. Our results show that significant levels of spike protein were found in the 30.4% of severe COVID-19 patients. In vitro, the activation of endothelial cells with S1 protein, via ACE2, impaired AMPK signalling, leading to robust leukocyte recruitment due to increased adhesive molecule expression and thrombomodulin loss. This S1-induced pro-inflammatory phenotype led to exuberant C3 and C5b-9 deposition on endothelial cells, along with C3a and C5a generation that further amplified S1-induced complement activation. Functional blockade of ACE2 or complement inhibition halted S1-induced platelet aggregates by limiting von Willebrand factor and P-selectin exocytosis and expression on endothelial cells. Overall, we demonstrate that SARS-CoV-2-derived S1 is sufficient in itself to propagate inflammatory and thrombogenic processes in the microvasculature, amplified by the complement system, recapitulating the thromboembolic complications of COVID-19.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Proteínas Quinasas Activadas por AMP/metabolismo , Enzima Convertidora de Angiotensina 2 , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , Humanos , Agregación Plaquetaria , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare disease that manifests with microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, and is associated with dysregulation of the alternative complement pathway. The chromosomal region including CFH and CFHR1-5 is rich in repeated sequences, favoring genomic rearrangements that have been reported in several patients with aHUS. However, there are limited data on the prevalence of uncommon CFH-CFHR genomic rearrangements in aHUS and their impact on disease onset and outcomes. Methods: In this study, we report the results of CFH-CFHR Copy Number Variation (CNV) analysis and the characterization of resulting structural variants (SVs) in a large cohort of patients, including 258 patients with primary aHUS and 92 with secondary forms. Results: We found uncommon SVs in 8% of patients with primary aHUS: 70% carried rearrangements involving CFH alone or CFH and CFHR (group A; n=14), while 30% exhibited rearrangements including only CFHRs (group B; n=6). In group A, 6 patients presented CFH::CFHR1 hybrid genes, 7 patients carried duplications in the CFH-CFHR region that resulted either in the substitution of the last CFHR1 exon(s) with those of CFH (CFHR1::CFH reverse hybrid gene) or in an internal CFH duplication. In group A, the large majority of aHUS acute episodes not treated with eculizumab (12/13) resulted in chronic ESRD; in contrast, anti-complement therapy induced remission in 4/4 acute episodes. aHUS relapse occurred in 6/7 grafts without eculizumab prophylaxis and in 0/3 grafts with eculizumab prophylaxis. In group B, 5 subjects had the CFHR31-5::CFHR410 hybrid gene and one had 4 copies of CFHR1 and CFHR4. Compared with group A, patients in group B exhibited a higher prevalence of additional complement abnormalities and earlier disease onset. However, 4/6 patients in this group underwent complete remission without eculizumab treatment. In secondary forms we identified uncommon SVs in 2 out of 92 patients: the CFHR31-5::CFHR410 hybrid and a new internal duplication of CFH. Discussion: In conclusion, these data highlight that uncommon CFH-CFHR SVs are frequent in primary aHUS and quite rare in secondary forms. Notably, genomic rearrangements involving the CFH are associated with a poor prognosis but carriers respond to anti-complement therapy.
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Síndrome Hemolítico Urémico Atípico , Humanos , Síndrome Hemolítico Urémico Atípico/epidemiología , Síndrome Hemolítico Urémico Atípico/genética , Síndrome Hemolítico Urémico Atípico/tratamiento farmacológico , Factor H de Complemento/genética , Prevalencia , Variaciones en el Número de Copia de ADN , Recurrencia Local de Neoplasia , GenómicaRESUMEN
Glomerulopathy with fibronectin (FN) deposits (GFND) is an autosomal dominant disease with age-related penetrance, characterized by proteinuria, microscopic hematuria, hypertension, and massive glomerular deposits of FN that lead to end-stage renal failure. The genetic abnormality underlying GFND was still unknown. We hypothesized that mutations in FN1, which encodes FN, were the cause of GFND. In a large Italian pedigree with eight affected subjects, we found linkage with GFND at the FN1 locus at 2q32. We sequenced the FN1 in 15 unrelated pedigrees and found three heterozygous missense mutations, the W1925R, L1974R, and Y973C, that cosegregated with the disease in six pedigrees. The mutations affected two domains of FN (Hep-II domain for the W1925R and the L1974R, and Hep-III domain for the Y973C) that play key roles in FN-cell interaction and in FN fibrillogenesis. Mutant recombinant Hep-II fragments were expressed, and functional studies revealed a lower binding to heparin and to endothelial cells and podocytes compared with wild-type Hep-II and an impaired capability to induce endothelial cell spreading and cytoskeletal reorganization. Overall dominant mutations in FN1 accounted for 40% of cases of GFND in our study group. These findings may help understanding the pathogenesis of proteinuria and glomerular FN deposits in GFND and possibly in more common renal diseases such as diabetic nephropathy, IgA nephropathy, and lupus nephritis. To our knowledge no FN1 mutation causing a human disease was previously reported.
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Fibronectinas/genética , Fibronectinas/metabolismo , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Mutación/genética , Adolescente , Adulto , Niño , Femenino , Fibronectinas/química , Fibronectinas/clasificación , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Linaje , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Complement factor H (FH) is the main plasma regulator of the alternative pathway of complement. Genetic and acquired abnormalities in FH cause uncontrolled complement activation amplifying, with the consequent accumulation of complement components on the renal glomeruli. This leads to conditions such as C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS). There is no effective therapy for these diseases. Half of the patients progress to end-stage renal disease and the condition recurs frequently in transplanted kidneys. Combined liver/kidney transplantation is a valid option for these patients, but the risks of the procedure and donor organ shortages hamper its clinical application. Therefore, there is an urgent need for alternative strategies for providing a normal FH supply. Human amnion epithelial cells (hAEC) have stem cell characteristics, including the capability to differentiate into hepatocyte-like cells in vivo.Here, we administered hAEC into the livers of newborn Cfh-/- mice, which spontaneously developed glomerular complement deposition and renal lesions resembling human C3G. hAEC engrafted at low levels in the livers of Cfh-/- mice and produced sufficient human FH to prevent complement activation and glomerular C3 and C9 deposition. However, long-term engraftment was not achieved, and eventually hAEC elicited a humoral immune response in immunocompetent Cfh-/- mice.hAEC cell therapy could be a valuable therapeutic option for patients undergoing kidney transplantation in whom post-transplant immunosuppression may protect allogeneic hAEC from rejection, while allogeneic cells provide normal FH to prevent disease recurrence.