Autoantibodies against laminin-521 are pathogenic in anti-glomerular basement membrane disease.

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.

The Alternative Pathway Is Necessary and Sufficient for Complement Activation by Anti-THSD7A Autoantibodies, Which Are Predominantly IgG4 in Membranous Nephropathy.

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.

Laminin-521 is a Novel Target of Autoantibodies Associated with Lung Hemorrhage in Anti-GBM Disease.

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.

A glycine substitution in the collagenous domain of Col4a3 in mice recapitulates late onset Alport syndrome.

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.

Maternal alloimmune IgG causes anti-glomerular basement membrane disease in perinatal transgenic mice that express human laminin α5.

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.

Alternative Pathway Is Essential for Glomerular Complement Activation and Proteinuria in a Mouse Model of Membranous Nephropathy.

Membranous nephropathy is an immune kidney disease caused by IgG antibodies that form glomerular subepithelial immune complexes. Proteinuria is mediated by complement activation, as a result of podocyte injury by C5b-9, but the role of specific complement pathways is not known. Autoantibodies-mediating primary membranous nephropathy are predominantly of IgG4 subclass, which cannot activate the classical pathway. Histologic evidence from kidney biopsies suggests that the lectin and the alternative pathways may be activated in membranous nephropathy, but the pathogenic relevance of these pathways remains unclear. In this study, we evaluated the role of the alternative pathway in a mouse model of membranous nephropathy. After inducing the formation of subepithelial immune complexes, we found similar glomerular IgG deposition in wild-type mice and in factor B-null mice, which lack a functional alternative pathway. Unlike wild-type mice, mice lacking factor B did not develop albuminuria nor exhibit glomerular deposition of C3c and C5b-9. Albuminuria was also reduced but not completely abolished in C5-deficient mice. Our results provide the first direct evidence that the alternative pathway is necessary for pathogenic complement activation by glomerular subepithelial immune complexes and is, therefore, a key mediator of proteinuria in experimental membranous nephropathy. This knowledge is important for the rational design of new therapies for membranous nephropathy.

Glomerular basement membrane heparan sulfate in health and disease: A regulator of local complement activation.

The glomerular basement membrane (GBM) is an essential component of the glomerular filtration barrier. Heparan sulfate proteoglycans such as agrin are major components of the GBM, along with α345(IV) collagen, laminin-521 and nidogen. A loss of GBM heparan sulfate chains is associated with proteinuria in several glomerular diseases and may contribute to the underlying pathology. As the major determinants of the anionic charge of the GBM, heparan sulfate chains have been thought to impart charge selectivity to the glomerular filtration, a view challenged by the negligible albuminuria in mice that lack heparan sulfate in the GBM. Recent studies provide increasing evidence that heparan sulfate chains modulate local complement activation by recruiting complement regulatory protein factor H, the major inhibitor of the alternative pathway in plasma. Factor H selectively inactivates C3b bound to surfaces bearing host-specific polyanions such as heparan sulfate, thus limiting complement activation on self surfaces such as the GBM, which are not protected by cell-bound complement regulators. We discuss mechanisms whereby the acquired loss of GBM heparan sulfate can impair the local regulation of the alternative pathway, exacerbating complement activation and glomerular injury in immune-mediated kidney diseases such as membranous nephropathy and lupus nephritis.

Distinct roles for the complement regulators factor H and Crry in protection of the kidney from injury.

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.

Alternative Pathway Dysregulation and the Conundrum of Complement Activation by IgG4 Immune Complexes in Membranous Nephropathy.

Membranous nephropathy (MN), a major cause of nephrotic syndrome, is a non-inflammatory immune kidney disease mediated by IgG antibodies that form glomerular subepithelial immune complexes. In primary MN, autoantibodies target proteins expressed on the podocyte surface, often phospholipase A2 receptor (PLA2R1). Pathology is driven by complement activation, leading to podocyte injury and proteinuria. This article overviews the mechanisms of complement activation and regulation in MN, addressing the paradox that anti-PLA2R1 and other antibodies causing primary MN are predominantly (but not exclusively) IgG4, an IgG subclass that does not fix complement. Besides immune complexes, alterations of the glomerular basement membrane (GBM) in MN may lead to impaired regulation of the alternative pathway (AP). The AP amplifies complement activation on surfaces insufficiently protected by complement regulatory proteins. Whereas podocytes are protected by cell-bound regulators, the GBM must recruit plasma factor H, which inhibits the AP on host surfaces carrying certain polyanions, such as heparan sulfate (HS) chains. Because HS chains present in the normal GBM are lost in MN, we posit that the local complement regulation by factor H may be impaired as a result. Thus, the loss of GBM HS in MN creates a micro-environment that promotes local amplification of complement activation, which in turn may be initiated via the classical or lectin pathways by subsets of IgG in immune complexes. A detailed understanding of the mechanisms of complement activation and dysregulation in MN is important for designing more effective therapies.

A case of subepidermal blistering disease with autoantibodies to multiple laminin subunits who developed later autoantibodies to alpha-5 chain of type IV collagen associated with membranous glomerulonephropathy.

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.

Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways.

MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti-miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti-miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-ß-induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy.

Neonatal Fc receptor promotes immune complex-mediated glomerular disease.

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.

A unique covalent bond in basement membrane is a primordial innovation for tissue evolution.

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.

Mouse models of membranous nephropathy: the road less travelled by.

Membranous nephropathy (MN) is a major cause of idiopathic nephrotic syndrome in adults, often progressing to end-stage kidney disease. The disease is mediated by IgG antibodies that form subepithelial immune complexes upon binding to antigens expressed by podocytes or planted in the subepithelial space. Subsequent activation of the complement cascade, podocyte injury by the membrane attack complex and the expansion of the glomerular basement membrane cause proteinuria and nephrotic syndrome. The blueprint for our current understanding of the pathogenic mechanisms of MN has largely been provided by studies in rat Heymann nephritis, an excellent animal model that closely replicates human disease. However, further progress in this area has been hindered by the lack of robust mouse models of MN that can leverage the power of genetic approaches for mechanistic studies. This critical barrier has recently been overcome by the development of new mouse models that faithfully recapitulate the clinical and morphologic hallmarks of human MN. In these mouse models, subepithelial ICs mediating proteinuria and nephrotic syndrome are induced by injection of cationized bovine serum albumin, by passive transfer of heterologous anti-podocyte antibodies, or by active immunization with the NC1 domain of α3(IV) collagen. These mouse models of MN will be instrumental for addressing unsolved questions about the basic pathomechanisms of MN and also for preclinical studies of novel therapeutics. We anticipate that the new knowledge to be gained from these studies will eventually translate into much needed novel mechanism-based therapies for MN, more effective, more specific, and less toxic.

Quaternary epitopes of α345(IV) collagen initiate Alport post-transplant anti-GBM nephritis.

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.