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1.
Kidney Int ; 104(6): 1124-1134, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37598856

RESUMEN

Anti-glomerular basement membrane (anti-GBM) disease is an organ-specific autoimmune disorder characterized by autoantibodies against GBM components. Evidence from human inherited kidney diseases and animal models suggests that the α, ß, and γ chains of laminin-521 are all essential for maintaining the glomerular filtration barrier. We previously demonstrated that laminin-521 is a novel autoantigen within the GBM and that autoantibodies to laminin-521 are present in about one-third of patients. In the present study, we investigated the pathogenicity of autoantibodies against laminin-521 with clinical and animal studies. Herein, a rare case of anti-GBM disease was reported with circulating autoantibodies binding to laminin-521 but not to the NC1 domains of α1-α5(IV) collagen. Immunoblot identified circulating IgG from this patient bound laminin α5 and γ1 chains. A decrease in antibody levels was associated with improved clinical presentation after plasmapheresis and immunosuppressive treatments. Furthermore, immunization with laminin-521 in female Wistar-Kyoto rats induced crescentic glomerulonephritis with linear IgG deposits along the GBM, complement activation along with infiltration of T cells and macrophages. Lung hemorrhage occurred in 75.0% of the rats and was identified by the presence of erythrocyte infiltrates and hemosiderin-laden macrophages in the lung tissue. Sera and kidney-eluted antibodies from rats immunized with laminin-521 demonstrated specific IgG binding to laminin-521 but not to human α3(IV)NC1, while the opposite was observed in human α3(IV)NC1-immunized rats. Thus, our patient data and animal studies imply a possible independent pathogenic role of autoantibodies against laminin-521 in the development of anti-GBM disease.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular , Humanos , Femenino , Animales , Ratas , Ratas Endogámicas WKY , Autoanticuerpos , Laminina , Inmunoglobulina G
2.
J Am Soc Nephrol ; 32(8): 1887-1897, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33893224

RESUMEN

BACKGROUND: Antiglomerular basement membrane (anti-GBM) disease is characterized by GN and often pulmonary hemorrhage, mediated by autoantibodies that typically recognize cryptic epitopes within α345(IV) collagen-a major component of the glomerular and alveolar basement membranes. Laminin-521 is another major GBM component and a proven target of pathogenic antibodies mediating GN in animal models. Whether laminin-521 is a target of autoimmunity in human anti-GBM disease is not yet known. METHODS: A retrospective study of circulating autoantibodies from 101 patients with anti-GBM/Goodpasture's disease and 85 controls used a solid-phase immunoassay to measure IgG binding to human recombinant laminin-521 with native-like structure and activity. RESULTS: Circulating IgG autoantibodies binding to laminin-521 were found in about one third of patients with anti-GBM antibody GN, but were not detected in healthy controls or in patients with other glomerular diseases. Autoreactivity toward laminin-521 was significantly more common in patients with anti-GBM GN and lung hemorrhage, compared with those with kidney-limited disease (51.5% versus 23.5%, P=0.005). Antilaminin-521 autoantibodies were predominantly of IgG1 and IgG4 subclasses and significantly associated with lung hemorrhage (P=0.005), hemoptysis (P=0.008), and smoking (P=0.01), although not with proteinuria or serum creatinine at diagnosis. CONCLUSIONS: Besides α345(IV) collagen, laminin-521 is another major autoantigen targeted in anti-GBM disease. Autoantibodies to laminin-521 may have the potential to promote lung injury in anti-GBM disease by increasing the total amount of IgG bound to the alveolar basement membranes.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/sangre , Autoanticuerpos/sangre , Hemoptisis/sangre , Inmunoglobulina G/sangre , Laminina/inmunología , Adulto , Anciano , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/complicaciones , Autoantígenos/inmunología , Estudios de Casos y Controles , Colágeno Tipo IV/inmunología , Colágeno Tipo IV/metabolismo , Creatinina/sangre , Progresión de la Enfermedad , Epítopos/inmunología , Femenino , Hemoptisis/complicaciones , Humanos , Riñón/metabolismo , Fallo Renal Crónico/etiología , Pulmón/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Pronóstico , Proteinuria/etiología , Estudios Retrospectivos , Saimiri , Fumar/sangre
3.
Kidney Int ; 96(6): 1320-1331, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31530475

RESUMEN

Mammalian immune systems are not mature until well after birth. However, transfer of maternal IgG to the fetus and newborn usually provides immunoprotection from infectious diseases. IgG transfer occurs before birth in humans across the placenta and continues after birth across the intestine in many mammalian species, including rodents. Transfer, which is mediated by the neonatal IgG Fc receptor, occurs by transcytosis across placental syncytiotrophoblasts and intestinal epithelium. Although maternal IgG is generally beneficial, harmful maternal allo- and autoantibodies can also be transferred to the fetus/infant, resulting in serious disease. To test this we generated transgenic mice that widely express human laminin α5 in their basement membranes. When huLAMA5 transgenic males were crossed with wild-type females, there was a maternal anti-human laminin α5 immune response. Maternal IgG alloantibody crossed the yolk sac and post-natal intestine invivo and bound in bright, linear patterns to kidney glomerular basement membranes of transgenic fetuses/neonates but not those of wild-type siblings. By postnatal day 18, most transgenic mice were proteinuric, had glomerular C3 deposits and inflammatory cell infiltrates, thickened and split glomerular basement membranes, and podocyte foot process effacement. Thus, our novel model of perinatal anti-glomerular basement membrane disease may prove useful for studying pediatric glomerulopathies, formation of the fetomaternal interface, and maternal alloimmunization.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Membrana Basal Glomerular/inmunología , Inmunoglobulina G/inmunología , Laminina/inmunología , Animales , Animales Recién Nacidos , Femenino , Membrana Basal Glomerular/ultraestructura , Humanos , Inmunidad Humoral , Masculino , Ratones Transgénicos , Embarazo
5.
Proc Natl Acad Sci U S A ; 111(1): 331-6, 2014 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-24344311

RESUMEN

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution.


Asunto(s)
Membrana Basal/metabolismo , Evolución Biológica , Iminas/química , Compuestos de Azufre/química , Secuencia de Aminoácidos , Animales , Colágeno Tipo IV/química , Reactivos de Enlaces Cruzados/química , Drosophila melanogaster , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Hemo/química , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos/química , Peroxidasa/química , Peroxidasas/química , Estructura Terciaria de Proteína , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido , Pez Cebra , Peroxidasina
7.
Kidney Int ; 90(1): 109-22, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27165610

RESUMEN

Mutations in the complement regulatory proteins are associated with several different diseases. Although these mutations cause dysregulated alternative pathway activation throughout the body, the kidneys are the most common site of injury. The susceptibility of the kidney to alternative pathway-mediated injury may be due to limited expression of complement regulatory proteins on several tissue surfaces within the kidney. To examine the roles of the complement regulatory proteins factor H and Crry in protecting distinct renal surfaces from alternative pathway mediated injury, we generated mice with targeted deletions of the genes for both proteins. Surprisingly, mice with combined genetic deletions of factor H and Crry developed significantly milder renal injury than mice deficient in only factor H. Deficiency of both factor H and Crry was associated with C3 deposition at multiple locations within the kidney, but glomerular C3 deposition was lower than that in factor H alone deficient mice. Thus, factor H and Crry are critical for regulating complement activation at distinct anatomic sites within the kidney. However, widespread activation of the alternative pathway reduces injury by depleting the pool of C3 available at any 1 location.


Asunto(s)
Complemento C3/metabolismo , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento/inmunología , Glomerulonefritis/inmunología , Glomérulos Renales/inmunología , Receptores de Complemento/metabolismo , Animales , Factor H de Complemento/genética , Glomerulonefritis/genética , Glomerulonefritis/patología , Glomérulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Receptores de Complemento/genética , Receptores de Complemento 3b
8.
J Immunol ; 190(4): 1424-32, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23303673

RESUMEN

Goodpasture disease is an autoimmune kidney disease mediated by autoantibodies against noncollagenous domain 1 (NC1) monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis (GN). We identified a novel type of human IgG4-restricted anti-GBM autoantibodies associated with mild nonprogressive GN, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoantibodies were investigated in mouse models recapitulating this phenotype. Wild-type and FcγRIIB(-/-) mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoantibodies specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause GN. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3(-/-) Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG Abs specific for α345NC1 hexamers, which were not subclass restricted. As heterologous Ag in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a, and IgG2b autoantibodies specific for α345NC1 hexamers and induced anti-GBM Ab GN. These findings indicate that tolerance toward autologous intact α345(IV) collagen is established in hosts expressing this Ag, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α345NC1 hexamers. This provides a mechanism eliciting autoantibodies specific for α345NC1 hexamers, which are restricted to noninflammatory IgG subclasses and are nonnephritogenic. In Alport syndrome, lack of tolerance toward α345(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including proinflammatory IgG subclasses that mediate posttransplant anti-GBM nephritis.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Especificidad de Anticuerpos , Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Colágeno Tipo IV/inmunología , Tolerancia Inmunológica , Proteolisis , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/genética , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Células HEK293 , Humanos , Tolerancia Inmunológica/genética , Inmunoglobulina G/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos
9.
Acta Derm Venereol ; 95(7): 826-9, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25633161

RESUMEN

We report a 68-year-old Japanese female patient with subepidermal blistering disease with autoantibodies to multiple laminins, who subsequently developed membranous glomerulonephropathy. At skin disease stage, immunofluorescence demonstrated IgG anti-basement membrane zone antibodies reactive with dermal side of NaCl-split skin. Immunoblotting of human dermal extract, purified laminin-332, hemidesmosome-rich fraction and laminin-521 trimer recombinant protein (RP) detected laminin γ-1 and α-3 and γ-2 subunits of laminin-332. Three years after skin lesions disappeared, nephrotic symptoms developed. Antibodies to α-3 chain of type IV collagen (COL4A3) were negative, thus excluding the diagnosis of Goodpasture syndrome. All anti-laminin antibodies disappeared. Additional IB and ELISA studies of RPs of various COL4 chains revealed reactivity with COL4A5, but not with COL4A6 or COL4A3. Although diagnosis of anti-laminin γ-1 (p200) pemphigoid or anti-laminin-332-type mucous membrane pemphigoid could not be made, this case was similar to previous cases with autoantibodies to COL4A5 and/or COL4A6.


Asunto(s)
Autoanticuerpos/análisis , Enfermedades Autoinmunes/inmunología , Vesícula/inmunología , Colágeno Tipo IV/inmunología , Glomerulonefritis Membranosa/inmunología , Riñón/inmunología , Laminina/inmunología , Piel/inmunología , Anciano , Autoanticuerpos/sangre , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/terapia , Biopsia , Vesícula/sangre , Vesícula/diagnóstico , Vesícula/terapia , Femenino , Técnica del Anticuerpo Fluorescente , Glomerulonefritis Membranosa/sangre , Glomerulonefritis Membranosa/diagnóstico , Glucocorticoides/uso terapéutico , Humanos , Riñón/ultraestructura , Intercambio Plasmático , Valor Predictivo de las Pruebas , Subunidades de Proteína , Piel/efectos de los fármacos , Piel/patología , Factores de Tiempo
10.
Proc Natl Acad Sci U S A ; 109(39): E2615-24, 2012 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-22955884

RESUMEN

Microscopic polyangiitis is an autoimmune small-vessel vasculitis that often manifests as focal and necrotizing glomerulonephritis and renal failure. Antineutrophil cytoplasmic Abs (ANCAs) specific for myeloperoxidase (MPO) play a role in this disease, but the role of autoreactive MPO-specific CD4(+) T cells is uncertain. By screening overlapping peptides of 20 amino acids spanning the MPO molecule, we identified an immunodominant MPO CD4(+) T-cell epitope (MPO(409-428)). Immunizing C57BL/6 mice with MPO(409-428) induced focal necrotizing glomerulonephritis similar to that seen after whole MPO immunization, when MPO was deposited in glomeruli. Transfer of an MPO(409-428)-specific CD4(+) T-cell clone to Rag1(-/-) mice induced focal necrotizing glomerulonephritis when glomerular MPO deposition was induced either by passive transfer of MPO-ANCA and LPS or by planting MPO(409-428) conjugated to a murine antiglomerular basement membrane mAb. MPO(409-428) also induced biologically active anti-MPO Abs in mice. The MPO(409-428) epitope has a minimum immunogenic core region of 11 amino acids, MPO(415-426), with several critical residues. ANCA-activated neutrophils not only induce injury but lodged the autoantigen MPO in glomeruli, allowing autoreactive anti-MPO CD4(+) cells to induce delayed type hypersensitivity-like necrotizing glomerular lesions. These studies identify an immunodominant MPO T-cell epitope and redefine how effector responses can induce injury in MPO-ANCA-associated microscopic polyangiitis.


Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/inmunología , Glomerulonefritis/inmunología , Inmunidad Celular , Poliangitis Microscópica/inmunología , Peroxidasa/inmunología , Animales , Anticuerpos Anticitoplasma de Neutrófilos/genética , Anticuerpos Anticitoplasma de Neutrófilos/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Mapeo Epitopo , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/metabolismo , Glomerulonefritis/enzimología , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Ratones , Ratones Noqueados , Poliangitis Microscópica/enzimología , Poliangitis Microscópica/genética , Poliangitis Microscópica/patología , Activación Neutrófila/genética , Activación Neutrófila/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/patología , Peroxidasa/genética , Peroxidasa/metabolismo
11.
J Am Soc Nephrol ; 25(5): 918-25, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24357670

RESUMEN

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.


Asunto(s)
Complejo Antígeno-Anticuerpo/fisiología , Glomerulonefritis/etiología , Antígenos de Histocompatibilidad Clase I/fisiología , Receptores Fc/fisiología , Albuminuria/etiología , Albuminuria/metabolismo , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/etiología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/metabolismo , Complejo Antígeno-Anticuerpo/efectos adversos , Autoantígenos/fisiología , Colágeno Tipo IV/fisiología , Glomerulonefritis/inmunología , Glomerulonefritis/metabolismo , Células HEK293 , Humanos , Inmunoglobulina G/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL
12.
J Immunol ; 188(7): 3268-77, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22371398

RESUMEN

Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults and a significant cause of end-stage renal disease, yet current therapies are nonspecific, toxic, and often ineffective. The development of novel targeted therapies requires a detailed understanding of the pathogenic mechanisms, but progress is hampered by the lack of a robust mouse model of disease. We report that DBA/1 mice as well as congenic FcγRIII(-/-) and FcRγ(-/-) mice immunized with a fragment of α3(IV) collagen developed massive albuminuria and nephrotic syndrome, because of subepithelial deposits of mouse IgG and C3 with corresponding basement membrane reaction and podocyte foot process effacement. The clinical presentation and histopathologic findings were characteristic of MN. Although immunized mice produced genuine anti-α3NC1 autoantibodies that bound to kidney and lung basement membranes, neither crescentic glomerulonephritis nor alveolitis ensued, likely because of the predominance of mouse IgG1 over IgG2a and IgG2b autoantibodies. The ablation of activating IgG Fc receptors did not ameliorate injury, implicating subepithelial deposition of immune complexes and consequent complement activation as a major effector pathway. We have thus established an active model of murine MN. This model, leveraged by the availability of genetically engineered mice and mouse-specific reagents, will be instrumental in studying the pathogenesis of MN and evaluating the efficacy of novel experimental therapies.


Asunto(s)
Autoantígenos/toxicidad , Colágeno Tipo IV/toxicidad , Modelos Animales de Enfermedad , Glomerulonefritis Membranosa/inmunología , Síndrome Nefrótico/etiología , Albuminuria/etiología , Albuminuria/inmunología , Animales , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Membrana Basal/inmunología , Colágeno Tipo IV/inmunología , Complemento C3/inmunología , Glomerulonefritis Membranosa/complicaciones , Inmunización , Inmunoglobulina G/inmunología , Riñón/inmunología , Riñón/patología , Riñón/fisiopatología , Ratones , Ratones Congénicos , Ratones Endogámicos DBA , Síndrome Nefrótico/inmunología , Alveolos Pulmonares/inmunología , Receptores de IgG , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/toxicidad
13.
J Am Soc Nephrol ; 24(6): 889-95, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23620401

RESUMEN

Alport post-transplant nephritis (APTN) is an aggressive form of anti-glomerular basement membrane disease that targets the allograft in transplanted patients with X-linked Alport syndrome. Alloantibodies develop against the NC1 domain of α5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct α345(IV) and α1256(IV) networks. Here, we studied the roles of these networks as antigens inciting alloimmunity and as targets of nephritogenic alloantibodies in APTN. We found that patients with APTN, but not those without nephritis, produce two kinds of alloantibodies against allogeneic collagen IV. Some alloantibodies targeted alloepitopes within α5NC1 monomers, shared by α345NC1 and α1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of α345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary α345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared α5NC1 alloepitopes. These results imply that quaternary epitopes within α345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, α345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas α1256NC1 hexamers become secondary targets of anti-α5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using α345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Autoantígenos/inmunología , Colágeno Tipo IV/inmunología , Mapeo Epitopo , Trasplante de Riñón/inmunología , Nefritis Hereditaria/inmunología , Nefritis Hereditaria/cirugía , Animales , Autoantígenos/química , Membrana Basal/inmunología , Bovinos , Colágeno Tipo IV/química , Haplorrinos , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Glomérulos Renales/inmunología , Ratones , Ratones Transgénicos , Complicaciones Posoperatorias/inmunología , Dominios y Motivos de Interacción de Proteínas/inmunología , Estructura Cuaternaria de Proteína , Trasplante Homólogo
14.
N Engl J Med ; 363(4): 343-54, 2010 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-20660402

RESUMEN

BACKGROUND: In Goodpasture's disease, circulating autoantibodies bind to the noncollagenous-1 (NC1) domain of type IV collagen in the glomerular basement membrane (GBM). The specificity and molecular architecture of epitopes of tissue-bound autoantibodies are unknown. Alport's post-transplantation nephritis, which is mediated by alloantibodies against the GBM, occurs after kidney transplantation in some patients with Alport's syndrome. We compared the conformations of the antibody epitopes in Goodpasture's disease and Alport's post-transplantation nephritis with the intention of finding clues to the pathogenesis of anti-GBM glomerulonephritis. METHODS: We used an enzyme-linked immunosorbent assay to determine the specificity of circulating autoantibodies and kidney-bound antibodies to NC1 domains. Circulating antibodies were analyzed in 57 patients with Goodpasture's disease, and kidney-bound antibodies were analyzed in 14 patients with Goodpasture's disease and 2 patients with Alport's post-transplantation nephritis. The molecular architecture of key epitope regions was deduced with the use of chimeric molecules and a three-dimensional model of the alpha345NC1 hexamer. RESULTS: In patients with Goodpasture's disease, both autoantibodies to the alpha3NC1 monomer and antibodies to the alpha5NC1 monomer (and fewer to the alpha4NC1 monomer) were bound in the kidneys and lungs, indicating roles for the alpha3NC1 and alpha5NC1 monomers as autoantigens. High antibody titers at diagnosis of anti-GBM disease were associated with ultimate loss of renal function. The antibodies bound to distinct epitopes encompassing region E(A) in the alpha5NC1 monomer and regions E(A) and E(B) in the alpha3NC1 monomer, but they did not bind to the native cross-linked alpha345NC1 hexamer. In contrast, in patients with Alport's post-transplantation nephritis, alloantibodies bound to the E(A) region of the alpha5NC1 subunit in the intact hexamer, and binding decreased on dissociation. CONCLUSIONS: The development of Goodpasture's disease may be considered an autoimmune "conformeropathy" that involves perturbation of the quaternary structure of the alpha345NC1 hexamer, inducing a pathogenic conformational change in the alpha3NC1 and alpha5NC1 subunits, which in turn elicits an autoimmune response. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases.)


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Autoanticuerpos/química , Colágeno Tipo IV/química , Nefritis Hereditaria/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos/sangre , Anticuerpos/química , Autoanticuerpos/inmunología , Autoanticuerpos/metabolismo , Autoantígenos/sangre , Autoantígenos/química , Sitios de Unión de Anticuerpos , Colágeno Tipo IV/inmunología , Colágeno Tipo IV/metabolismo , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos/química , Epítopos/metabolismo , Femenino , Membrana Basal Glomerular/inmunología , Humanos , Isoanticuerpos/química , Isoanticuerpos/metabolismo , Glomérulos Renales/inmunología , Trasplante de Riñón/inmunología , Masculino , Persona de Mediana Edad , Nefritis/inmunología , Nefritis Hereditaria/cirugía , Complicaciones Posoperatorias/inmunología , Conformación Proteica , Isoformas de Proteínas , Estudios Retrospectivos , Adulto Joven
15.
J Immunol ; 185(6): 3520-8, 2010 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-20709951

RESUMEN

The noncollagenous (NC1) domains of alpha3alpha4alpha5(IV) collagen in the glomerular basement membrane (GBM) are targets of Goodpasture autoantibodies or Alport posttransplant nephritis alloantibodies mediating rapidly progressive glomerulonephritis. Because the autoepitopes but not the alloepitopes become cryptic upon assembly of alpha3alpha4alpha5NC1 hexamers, we investigated how the accessibility of B cell epitopes in vivo influences the development of glomerulonephritis in mice passively immunized with human anti-GBM Abs. Alport alloantibodies, which bound to native murine alpha3alpha4alpha5NC1 hexamers in vitro, deposited linearly along the mouse GBM in vivo, eliciting crescentic glomerulonephritis in Fcgr2b(-/-) mice susceptible to Ab-mediated inflammation. Goodpasture autoantibodies, which bound to murine alpha3NC1 monomer and dimer subunits but not to native alpha3alpha4alpha5NC1 hexamers in vitro, neither bound to the mouse GBM in vivo nor induced experimental glomerulonephritis. This was due to quinary NC1 crosslinks, recently identified as sulfilimine bonds, which comprehensively locked the cryptic Goodpasture autoepitopes in the mouse GBM. In contrast, non-crosslinked alpha3NC1 subunits were identified as a native target of Goodpasture autoantibodies in the GBM of squirrel monkeys, a species susceptible to Goodpasture autoantibody-mediated nephritis. Thus, crypticity of B cell autoepitopes in tissues uncouples potentially pathogenic autoantibodies from autoimmune disease. Crosslinking of alpha3alpha4alpha5NC1 hexamers represents a novel mechanism averting autoantibody binding and subsequent tissue injury by posttranslational modifications of an autoantigen.


Asunto(s)
Enfermedad por Anticuerpos Antimembrana Basal Glomerular/inmunología , Autoanticuerpos/fisiología , Autoantígenos/inmunología , Colágeno Tipo IV/inmunología , Epítopos/inmunología , Glomerulonefritis/inmunología , Isoanticuerpos/fisiología , Nefritis Hereditaria/inmunología , Animales , Autoanticuerpos/metabolismo , Autoantígenos/metabolismo , Sitios de Unión de Anticuerpos , Colágeno Tipo IV/metabolismo , Reacciones Cruzadas/inmunología , Epítopos/metabolismo , Glomerulonefritis/metabolismo , Glomerulonefritis/prevención & control , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estructura Terciaria de Proteína
16.
Front Immunol ; 13: 952235, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35874690

RESUMEN

Membranous nephropathy (MN) is an immune kidney disease characterized by glomerular subepithelial immune complexes (ICs) containing antigen, IgG, and products of complement activation. Whereas proteinuria is caused by complement-mediated podocyte injury, the pathways of complement activation remain controversial due to the predominance of IgG4 in ICs, an IgG subclass considered unable to activate complement. THSD7A, a transmembrane protein expressed on podocytes, is the target autoantigen in ~3% of cases of primary MN. In this study, we analyzed sera from 16 patients with THSD7A-associated MN with regard to the anti-THSD7A IgG subclasses and their ability to fix complement in vitro. The serum concentration of anti-THSD7A IgG varied over two orders of magnitude (1.3-243 µg/mL). As a relative proportion of all IgG anti-THSD7A, IgG4 was by far the most abundant subclass (median 79%), followed by IgG1 (median 11%). IgG4 was the dominant subclass of anti-THSD7A antibodies in 14 sera, while IgG1 was dominant in one and co-dominant in another. One quarter of MN sera additionally contained low levels of anti-THSD7A IgA1. ICs formed by predominantly IgG4 anti-THSD7A autoantibodies with immobilized THSD7A were relatively weak activators of complement in vitro, compared to human IgG1 and IgG3 mAbs used as positive control. Complement deposition on THSD7A ICs was dose-dependent and occurred to a significant extent only at relatively high concentration of anti-THSD7A IgG. C3b fixation by THSD7A ICs was completely abolished in factor B-depleted sera, partially inhibited in C4-depleted sera, unchanged in C1q-depleted sera, and also occurred in Mg-EGTA buffer. These results imply that THSD7A ICs predominantly containing IgG4 activate complement at high IgG4 density, which strictly requires a functional alternative pathway, whereas the classical and lectin pathways are dispensable. These findings advance our understanding of how IgG4 antibodies activate complement.


Asunto(s)
Glomerulonefritis Membranosa , Complejo Antígeno-Anticuerpo , Autoanticuerpos , Activación de Complemento , Proteínas del Sistema Complemento , Humanos , Inmunoglobulina G , Trombospondinas
17.
Kidney Int ; 79(1): 120-7, 2011 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20881942

RESUMEN

A novel COL4A5 mutation causes rapid progression to end-stage renal disease in males, despite the absence of clinical and biopsy findings associated with Alport syndrome. Affected males have proteinuria, variable hematuria, and an early progression to end-stage renal disease. Renal biopsy findings include global and segmental glomerulosclerosis, mesangial hypercellularity and basement membrane immune complex deposition. Exon sequencing of the COL4A5 locus identified a thymine to guanine transversion at nucleotide 665, resulting in a phenylalanine to cysteine missense mutation at codon 222. The phenylalanine at position 222 is absolutely conserved among vertebrates. This mutation was confirmed in 4 affected males and 4 female obligate carriers, but was absent in 6 asymptomatic male family members and 198 unrelated individuals. Immunostaining for α5(IV) collagen in renal biopsies from affected males was normal. This mutation, in a non-collagenous interruption associated with severe renal disease, provides evidence for the importance of this structural motif and suggests the range of phenotypes associated with COL4A5 mutations is more diverse than previously realized. Hence, COL4A5 mutation analysis should be considered when glomerulonephritis presents in an X-linked inheritance pattern, even with a presentation distinct from Alport syndrome.


Asunto(s)
Cromosomas Humanos X/genética , Colágeno Tipo IV/genética , Membrana Basal Glomerular/patología , Glomerulonefritis/genética , Fallo Renal Crónico/genética , Mutación Missense , Adolescente , Niño , Colágeno Tipo IV/metabolismo , Femenino , Heterocigoto , Humanos , Fallo Renal Crónico/patología , Masculino , Linaje
18.
Matrix Biol Plus ; 9: 100053, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33718859

RESUMEN

Alport syndrome (AS) is a severe inherited glomerulopathy caused by mutations in the genes encoding the α-chains of type-IV collagen, the most abundant component of the extracellular glomerular basement membrane (GBM). Currently most AS mouse models are knockout models for one of the collagen-IV genes. In contrast, about half of AS patients have missense mutations, with single aminoacid substitutions of glycine being the most common. The only mouse model for AS with a homozygous knockin missense mutation, Col4a3-p.Gly1332Glu, was partly described before by our group. Here, a detailed in-depth description of the same mouse is presented, along with another compound heterozygous mouse that carries the glycine substitution in trans with a knockout allele. Both mice recapitulate essential features of AS, including shorten lifespan by 30-35%, increased proteinuria, increased serum urea and creatinine, pathognomonic alternate GBM thinning and thickening, and podocyte foot process effacement. Notably, glomeruli and tubuli respond differently to mutant collagen-IV protomers, with reduced expression in tubules but apparently normal in glomeruli. However, equally important is the fact that in the glomeruli the mutant α3-chain as well as the normal α4/α5 chains seem to undergo a cleavage at, or near the point of the mutation, possibly by the metalloproteinase MMP-9, producing a 35 kDa C-terminal fragment. These mouse models represent a good tool for better understanding the spectrum of molecular mechanisms governing collagen-IV nephropathies and could be used for pre-clinical studies aimed at better treatments for AS.

19.
J Am Soc Nephrol ; 20(7): 1471-9, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19423686

RESUMEN

Laminin and type IV collagen composition of the glomerular basement membrane changes during glomerular development and maturation. Although it is known that both glomerular endothelial cells and podocytes produce different laminin isoforms at the appropriate stages of development, the cellular origins for the different type IV collagen heterotrimers that appear during development are unknown. Here, immunoelectron microscopy demonstrated that endothelial cells, mesangial cells, and podocytes of immature glomeruli synthesize collagen alpha 1 alpha 2 alpha1(IV). However, intracellular labeling revealed that podocytes, but not endothelial or mesangial cells, contain collagen alpha 3 alpha 4 alpha 5(IV). To evaluate the origins of collagen IV further, we transplanted embryonic kidneys from Col4a3-null mutants (Alport mice) into kidneys of newborn, wildtype mice. Hybrid glomeruli within grafts containing numerous host-derived, wildtype endothelial cells never expressed collagen alpha 3 alpha 4 alpha 5(IV). Finally, confocal microscopy of glomeruli from infant Alport mice that had been dually labeled with anti-collagen alpha 5(IV) and the podocyte marker anti-GLEPP1 showed immunolabeling exclusively within podocytes. Together, these results indicate that collagen alpha 3 alpha 4 alpha 5(IV) originates solely from podocytes; therefore, glomerular Alport disease is a genetic defect that manifests specifically within this cell type.


Asunto(s)
Colágeno Tipo IV/metabolismo , Glomérulos Renales/embriología , Podocitos/citología , Podocitos/metabolismo , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Modelos Animales de Enfermedad , Endotelio/citología , Endotelio/metabolismo , Glomérulos Renales/citología , Glomérulos Renales/metabolismo , Células Mesangiales/citología , Células Mesangiales/metabolismo , Ratones , Ratones Noqueados , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Nefritis Hereditaria/patología , Podocitos/ultraestructura
20.
J Am Soc Nephrol ; 20(12): 2518-24, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19820122

RESUMEN

Th1 effector CD4+ cells contribute to the pathogenesis of proliferative and crescentic glomerulonephritis, but whether effector Th17 cells also contribute is unknown. We compared the involvement of Th1 and Th17 cells in a mouse model of antigen-specific glomerulonephritis in which effector CD4+ cells are the only components of adaptive immunity that induce injury. We planted the antigen ovalbumin on the glomerular basement membrane of Rag1(-/-) mice using an ovalbumin-conjugated non-nephritogenic IgG1 monoclonal antibody against alpha3(IV) collagen. Subsequent injection of either Th1- or Th17-polarized ovalbumin-specific CD4+ effector cells induced proliferative glomerulonephritis. Mice injected with Th1 cells developed progressive albuminuria over 21 d, histologic injury including 5.5 +/- 0.9% crescent formation/segmental necrosis, elevated urinary nitrate, and increased renal NOS2, CCL2, and CCL5 mRNA. Mice injected with Th17 cells developed albuminuria by 3 d; compared with Th1-injected mice, their glomeruli contained more neutrophils and greater expression of renal CXCL1 mRNA. In conclusion, Th1 and Th17 effector cells can induce glomerular injury. Understanding how these two subsets mediate proliferative forms of glomerulonephritis may lead to targeted therapies.


Asunto(s)
Glomerulonefritis Membranoproliferativa/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Células TH1/inmunología , Inmunidad Adaptativa , Traslado Adoptivo , Animales , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Genes RAG-1 , Glomerulonefritis Membranoproliferativa/etiología , Glomerulonefritis Membranoproliferativa/genética , Inmunoglobulina G/metabolismo , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo
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