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Anti-glomerular basement membrane (anti-GBM) disease is an aggressive form of glomerulonephritis, usually mediated by immunoglobulin G (IgG) autoantibodies to the noncollagenous (NC1) domain of alpha 3(IV) collagen. Less is known about the target antigen(s) in patients with atypical anti-GBM disease involving IgA autoantibodies. We report a new case of IgA anti-GBM disease in a patient with a history of proliferative lupus nephritis who presented with increasing creatinine levels, proteinuria, and hematuria, but no clinical or serological evidence of lupus recurrence. Renal biopsy showed focal and segmental necrotizing glomerulonephritis with strong linear capillary loop IgA staining by means of immunofluorescence. Serological test results were negative for IgG or IgA autoantibodies against the alpha 3NC1 domain. By means of immunoblotting, IgA from patient serum bound to 38- to 48-kd antigens collagenase-solubilized from human GBM, but not to purified NC1 domains of GBM collagen IV. The target of patient's IgA autoantibodies thus was identified as a novel GBM antigen, distinct from the alpha 3NC1 domain or other known targets of anti-GBM IgA autoantibodies. Clinical resolution was attained by means of conventional treatment with steroids and cyclophosphamide. The diversity of antigens recognized by anti-GBM IgA autoantibodies highlights the importance of renal biopsy for the reliable diagnosis of this rare condition because conventional serological immunoassays likely would yield false-negative results. [ABSTRACT FROM AUTHOR]

Perinatal mood and anxiety disorders (PMADs) profoundly impact maternal and infant health, affecting women worldwide during pregnancy and postpartum. This review synthesizes current research on the neurobiological effects of PMADs, particularly their influence on brain structure, function, and corresponding cognitive, behavioral, and mental health outcomes in mothers. A literature search across PubMed, PsycINFO, and Google Scholar yielded studies utilizing neuroimaging (MRI, fMRI) and cognitive assessments to explore brain changes in PMADs. The key findings indicate significant neurobiological alterations in PMADs, such as glutamatergic dysfunction, neuronal damage, and altered neural connectivity, particularly in postpartum depression (PPD). Functional MRI studies reveal distinct patterns of brain function alteration, including amygdala non-responsivity in PPD, differing from traditional major depressive disorder (MDD). These neurobiological changes are connected with cognitive impairments and behavioral modifications, impacting maternal caregiving. Understanding these alterations is fundamental for developing effective treatments. The findings emphasize the importance of focusing on maternal mental health, advocating for early detection, and personalized treatment strategies to improve maternal and child outcomes.

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, β, 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., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare a potential conflict of interest and state it below. LB reports being a co-inventor on the U.S. patent “Diagnostics for Membranous Nephropathy” and receives royalty income through Boston University. LB has served on advisory boards on the topic of MN and other glomerular diseases for Visterra, Ionis, Alexion, and Novartis, and receives royalties from UpToDate for topics related to MN. TC is employed by Arkana Laboratories, Little Rock, AR. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Manral, Caza, Storey, Beck and Borza.)

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., (Copyright © 2021 by the American Society of Nephrology.)

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., (© 2021 The Authors.)

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., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

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.

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.

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-β-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.

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., (Copyright © 2014 by the American Society of Nephrology.)

Purpose: To evaluate the repeatability of MRI-derived relative blood volume (RBV) measurements in mouse kidneys across subjects and days and to evaluate sensitivity of this approach to renal pathology., Materials and Methods: A 7 Tesla MRI system and an intravascular iron-oxide contrast agent were used to acquire spin-echo-based renal RBV maps in 10 healthy mice on 2 consecutive days. Renal RBV maps were also acquired in the Alport and unilateral ureteral obstruction mouse models of renal disease., Results: The average renal RBV measured on consecutive days was 19.97 ± 1.50 and 19.86 ± 1.62, yielding a concordance correlation coefficient of 0.94, indicating that this approach is highly repeatable. In the disease models, the RBV values were regionally dissimilar and substantially lower than those found in control mice., Conclusion: In vivo renal iron-oxide-based RBV mapping in mice complements the physiological information obtained from conventional assays of kidney function and could shed new insights into the pathological mechanisms of kidney disease., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

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.

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.

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.

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.

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.

A new transgenic mouse model for global increases in the Sodium Dependent Vitamin C transporter 2 (SVCT2) has been generated. The SVCT2-Tg mouse shows increased SVCT2 mRNA levels in all organs tested and correspondingly increased ascorbic acid (ASC) levels in all organs except liver. The extent of the increase in transporter mRNA expression differed among mice and among organs. The increased ASC levels did not have any adverse effects on behavior in the SVCT2-Tg mice, which did not differ from wild-type mice on tests of locomotor activity, anxiety, sensorimotor or cognitive ability. High levels of SVCT2 and ASC were found in the kidneys of SVCT2-Tg mice and urinary albumin excretion was lower in these mice than in wild-types. No gross pathological changes were noted in kidneys from SVCT2-Tg mice. SVCT2 immunoreactivity was detected in both SVCT2 and wild-type mice, and a stronger signal was seen in tubules than in glomeruli. Six treatments with Paraquat (3x10 and 3x15 mg/kg i.p.) were used to induce oxidative stress in mice. SVCT2-Tg mice showed a clear attenuation of Paraquat-induced oxidative stress in lung, as measured by F(2)-isoprostanes. Paraquat also decreased SVCT2 mRNA signal in liver, lung and kidney in SVCT2-Tg mice.

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.

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.

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.), (2010 Massachusetts Medical Society)

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.

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.

Defective assembly of alpha 3 alpha 4 alpha 5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha1/alpha 5 chimeras containing alpha 5 NC1 residues 188-227 replicated the ability of alpha 5 NC1 to bind to alpha3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5 NC1 residues 188-227 by alpha1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha3NC1 encoded binding to alpha 5 NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5 NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5 NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5 NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients.

The detailed structural basis for the cryptic nature (crypticity) of a B cell epitope harbored by an autoantigen is unknown. Because the immune system may be ignorant of the existence of such "cryptic" epitopes, their exposure could be an important feature in autoimmunity. Here we investigated the structural basis for the crypticity of the epitopes of the Goodpasture autoantigen, the alpha3alpha4alpha5 noncollagenous-1 (NC1) hexamer, a globular domain that connects two triple-helical molecules of the alpha3alpha4alpha5 collagen IV network. The NC1 hexamer occurs in two isoforms as follows: the M-isoform composed of monomer subunits in which the epitopes are accessible to autoantibodies, and the D-isoform composed of both monomer and dimer subunits in which the epitopes are cryptic. The D-isoform was characterized with respect to quaternary structure, as revealed by mass spectrometry of dimer subunits, homology modeling, and molecular dynamics simulation. The results revealed that the D-isoform contains two kinds of cross-links as follows: S-hydroxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the alpha3alpha5-heterodimers and alpha4alpha4-homodimers, respectively. Construction and analysis of a three-dimensional model of the D-isoform of the alpha3alpha4alpha5 NC1 hexamer revealed that crypticity is a consequence of the following: (a) sequestration of key residues between neighboring subunits that are stabilized by domain-swapping interactions, and (b) by cross-linking of subunits at the trimer-trimer interface, which stabilizes the structural integrity of the NC1 hexamer and protects against binding of autoantibodies. The sequestrated epitopes and cross-linked subunits represent a novel structural mechanism for conferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.

Germinal matrix is selectively vulnerable to hemorrhage in premature infants, and use of prenatal betamethasone is associated with a lower occurrence of germinal matrix hemorrhage. Because the major components of extracellular matrix of the cerebral vasculature-laminin, fibronectin, collagen IV, and perlecan-provide structural stability to blood vessels, we examined whether the expression of these molecules was decreased in the germinal matrix and affected by betamethasone. In both human fetuses and premature infants, fibronectin was significantly lower in the germinal matrix than in the cortical mantle or white matter anlagen. Conversely, laminin alpha1 gene expression was greater in the human germinal matrix compared with the cortical mantle or white matter. Expression of alpha1- and alpha2(IV) collagen chains increased with advancing gestational age. Low-dose prenatal betamethasone treatment enhanced fibronectin level by 1.5-2-fold whereas a high dose reduced fibronectin expression by 2-fold in rabbit pups. Because fibronectin provides structural stability to the blood vessels, its reduced expression in the germinal matrix may contribute to the fragility of germinal matrix vasculature and the propensity to hemorrhage in premature neonates.

Integrins are transmembrane heteromeric receptors that mediate interactions between cells and extracellular matrix (ECM). beta1, the most abundantly expressed integrin subunit, binds at least 12 alpha subunits. beta1 containing integrins are highly expressed in the glomerulus of the kidney; however their role in glomerular morphogenesis and maintenance of glomerular filtration barrier integrity is poorly understood. To study these questions we selectively deleted beta1 integrin in the podocyte by crossing beta1(flox/flox) mice with podocyte specific podocin-cre mice (pod-Cre), which express cre at the time of glomerular capillary formation. We demonstrate that podocyte abnormalities are visualized during glomerulogenesis of the pod-Cre;beta1(flox/flox) mice and proteinuria is present at birth, despite a grossly normal glomerular basement membrane. Following the advent of glomerular filtration there is progressive podocyte loss and the mice develop capillary loop and mesangium degeneration with little evidence of glomerulosclerosis. By 3 weeks of age the mice develop severe end stage renal failure characterized by both tubulointerstitial and glomerular pathology. Thus, expression of beta1 containing integrins by the podocyte is critical for maintaining the structural integrity of the glomerulus.

Podocyte adhesion to the glomerular basement membrane is required for proper function of the glomerular filtration barrier. However, the mechanism whereby podocytes adhere to collagen IV networks, a major component of the glomerular basement membrane, is poorly understood. The predominant collagen IV network is composed of triple helical protomers containing the alpha3alpha4alpha5 chains. The protomers connect via the trimeric noncollagenous (NC1) domains to form hexamers at the interface. Because the NC1 domains of this network can potentially support integrin-dependent cell adhesion, it was determined whether individual NC1 monomers or alpha3alpha4alpha5 hexamers support podocyte adhesion. It was found that, although human podocytes did not adhere to NC1 domains proper, they did adhere via integrin alphavbeta3 to a KRGDS motif located adjacent to alpha3NC1 domains. Because the KRGDS motif is a site of phosphorylation, its interactions with integrin alphavbeta3 may play a critical role in cell signaling in physiologic and pathologic states.

Hydra, as an early diploblastic metazoan, has a well-defined extracellular matrix (ECM) called mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and scanning electron microscopy (SEM). Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesoglea is porous with multiple trans-mesoglea pores ranging from 0.5 to 1 microm in diameter and about six pores per 100 microm(2) in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell-cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

Anti-glomerular basement membrane (GBM) antibody nephritis is caused by an autoimmune or alloimmune reaction to the NC1 domains of alpha3alpha4alpha5(IV) collagen. Some patients with X-linked Alport syndrome (XLAS) develop post-transplant nephritis mediated by pathogenic anti-GBM alloantibodies to collagen IV chains present in the renal allograft but absent from the tissues of the patient. In this work, the epitopes targeted by alloantibodies from these patients were identified and characterized. All XLAS alloantibodies recognized conformational epitopes in the NC1 domain of alpha5(IV) collagen, which were mapped using chimeric alpha1/alpha5 NC1 domains expressed in mammalian cells. Allograft-eluted alloantibodies mainly targeted two conformational alloepitopes mapping to alpha5NC1 residues 1-45 and 114-168. These regions also encompassed the major epitopes of circulating XLAS alloantibodies, which in some patients additionally targeted alpha5NC1 residues 169-229. Both kidney-eluted and circulating alloantibodies to alpha5NC1 distinctively targeted epitopes accessible in the alpha3alpha4alpha5NC1 hexamers of human GBM, unlike anti-GBM autoantibodies, which targeted sequestered alpha3NC1 epitopes. The results identify two immunodominant alpha5NC1 epitopes as major alloantigenic sites of alpha3alpha4alpha5(IV) collagen specifically implicated in the pathogenesis of post-transplant nephritis in XLAS patients. The contrast between the accessibility of these alloepitopes and the crypticity of autoepitopes indicates that distinct molecular forms of antigen may initiate the immunopathogenic processes in the two forms of anti-GBM disease.

Alport syndrome, caused by mutations that interfere with the normal assembly of the alpha3alpha4alpha5(IV) collagen network in the glomerular basement membrane (GBM), is the most common inherited glomerular disease leading to renal failure. A detailed knowledge of the underlying pathogenic mechanisms is necessary for developing new, more specific, and effective therapeutic strategies aimed at delaying the onset and slowing disease progression. Studies of several dog and mouse models of Alport syndrome have significantly enhanced our understanding of the disease mechanisms and provided systems for testing potential therapies. In the most widely used Col4a3-/- mouse models of autosomal-recessive Alport syndrome (ARAS), the genetic background strongly affects renal survival. One contributing factor may be the strong ectopic deposition of alpha5alpha6(IV) collagen in the GBM of Col4a3-/- mice on the C57BL/6J background, which is almost undetectable on the 129/Sv background. This isoform 'switch' has not been observed in human ARAS, although it had been reported in the dog model of ARAS. In human patients as well as dog and mouse models of X-linked Alport syndrome, the alpha3-alpha6(IV) collagen chains are absent from the GBM. These biochemical differences among Alport animal models provide an opportunity to determine how the molecular makeup of the GBM affects the glomerular function. At the same time, potentially confounding influences of characteristics unique to a particular strain or model should be carefully considered in the design of studies aiming to define key events underlying the pathobiology of Alport glomerular disease.

The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2alpha and HIF1beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2alpha/LacZ transgenic mouse to define patterns of HIF2alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2alpha mutants. To examine responsiveness of HIF2alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O(2)) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1alpha or -3alpha. Our results show that, although HIF2alpha was widely transcribed by kidney endothelium and vascular smooth muscle, knockouts displayed no detectable deficits in vessel development or VEGF or Flk-1 expression.

Anti-glomerular basement membrane (anti-GBM) antibodies elicited by autoimmune or alloimmune mechanisms are associated with aggressive forms of rapid progressive glomerulonephritis. Pathogenic anti-GBM autoantibodies and alloantibodies target the noncollagenous (NC1) domains of the alpha3alpha4alpha5(IV) collagen, a major GBM component. In autoimmune anti-GBM glomerulonephritis, a breakdown of immune self-tolerance leads to the activation of autoreactive B and T cells recognizing epitopes within the alpha3NC1 subunit. In the GBM, the conformational epitopes targeted by anti-GBM autoantibodies are structurally sequestered within the alpha3alpha4alpha5NC1 hexamer complex formed upon assembly of collagen IV chains into trimeric molecules and networks. Autoantibodies selectively bind to and dissociate a subset of alpha3alpha4alpha5NC1 hexamers composed of monomer subunits, whereas hexamers containing NC1 dimer subunits are resistant to dissociation by autoantibodies. The crypticity of alpha3NC1 autoepitopes suggests that self-tolerance to alpha3(IV) collagen is broken by structural alterations of the native alpha3alpha4alpha5NC1 hexamer that unmask normally sequestered epitopes, triggering an autoimmune reaction. Post-transplant anti-GBM nephritis in the renal allograft of transplanted Alport patients is mediated by an alloimmune reaction to the NC1 domains of alpha3alpha4alpha5(IV) collagen, present in the allograft GBM but absent from Alport basement membranes. Alloantibodies from patients with autosomal-recessive Alport syndrome predominantly bind to the alpha3NC1 domain, whereas alloantibodies from X-linked Alport patients target preferentially, though not exclusively, epitopes within the alpha5NC1 subunit. The accessibility of the alloantigenic sites within the alpha3alpha4alpha5NC1 hexamers, contrasting with the crypticity of autoantigenic sites, suggest that different molecular forms of alpha3alpha4alpha5(IV) collagen initiate the immunopathogenic responses in the two forms of anti-GBM disease. Advances in elucidating the structure of the GBM antigen and the identification of the pathogenic B and T cell epitopes, along with new insights into the pathogenic mechanisms at cellular and molecular level will facilitate the development of targeted strategies for prevention, detection, and treatment of human anti-GBM antibody glomerulonephritis., (Copyright 2007 S. Karger AG, Basel.)

Excessive glomerular collagen IV and reactive oxygen species (ROS) production are key factors in the development of diabetic nephropathy. Integrin alpha1beta1, the major collagen IV receptor, dowregulates collagen IV and ROS production, suggesting this integrin might determine the severity of diabetic nephropathy. To test this possibility, wild-type and integrin alpha1-null mice were rendered diabetic with streptozotocin (STZ) (100 mg/kg single intraperitoneal injection), after which glomerular filtration rate (GFR), glomerular collagen deposition, and glomerular basement membrane (GBM) thickening were evaluated. In addition, ROS and collagen IV production by mesangial cells as well as their proliferation was measured in vitro. Diabetic alpha1-null mice developed worse renal disease than diabetic wild-type mice. A significant increase in GFR was evident in the alpha1-null mice at 6 weeks after the STZ injection; it started to decrease by week 24 and reached levels of non-diabetic mice by week 36. In contrast, GFR only increased in wild-type mice at week 12 and its elevation persisted throughout the study. Diabetic mutant mice also showed increased glomerular deposition of collagen IV and GBM thickening compared to diabetic wild-type mice. Primary alpha1-null mesangial cells exposed to high glucose produced more ROS than wild-type cells, which led to decreased proliferation and increased collagen IV synthesis, thus mimicking the in vivo finding. In conclusion, this study suggests that lack of integrin alpha1beta1 exacerbates the glomerular injury in a mouse model of diabetes by modulating GFR, ROS production, cell proliferation, and collagen deposition.

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.

Mutations in COL4A3/4/5 genes that affect the normal assembly of the alpha3/4/5(IV) collagen network in the glomerular basement membrane (GBM) cause Alport syndrome. Patients progress to renal failure at variable rates that are determined by the underlying mutation and putative modifier genes. Col4a3(-/-) mice, a model for autosomal recessive Alport syndrome, progress to renal failure significantly slower on the C57BL/6 than on the 129X1/Sv background. Reported here is a novel strain-specific alternative collagen IV isoform switch that is associated with the differential renal survival in Col4a3(-/-) Alport mice. The downregulation or the absence of alpha3/4(IV) collagen chains in the GBM of Lmx1b(-/-) and Col4a3(-/-) mice was found to induce ectopic deposition of alpha5/6(IV) collagen. The GBM deposition of alpha5/6(IV) collagen was abundant in C57BL/6 Col4a3(-/-) mice but almost undetectable in 129X1/Sv Col4a3(-/-) mice. This strain difference was due to overall low expression of alpha6(IV) chain and alpha5/6(IV) protomers in the tissues of 129X1/SvJ mice, a natural Col4a6 knockdown. In (129 x B6)F1 Col4a3(-/-) mice, the amount of alpha5/6(IV) collagen in the GBM was inherited in a mother-to-son manner, suggesting that it is controlled by one or more X-linked loci, possibly Col4a6 itself. Importantly, high levels of ectopic alpha5/6(IV) collagen in the GBM were associated with approximately 46% longer renal survival. These findings suggest that alpha5/6(IV) collagen, the biologic role of which has been hitherto unknown, may partially substitute for alpha3/4/5(IV) collagen. Therapeutically induced GBM deposition of alpha5/6(IV) collagen may provide a novel strategy for delaying renal failure in patients with autosomal recessive Alport syndrome.

Alport posttransplantation anti-glomerular basement membrane (GBM) nephritis is mediated by alloantibodies against the noncollagenous (NC1) domains of the alpha3alpha4alpha5(IV) collagen network, which is present in the GBM of the allograft but absent from Alport kidneys. The specificity of kidney-bound anti-GBM alloantibodies from a patient who had autosomal recessive Alport syndrome (ARAS) and developed posttransplantation nephritis was compared with that of Goodpasture autoantibodies from patients with autoimmune anti-GBM disease. Allograft-eluted alloantibodies reacted specifically with alpha3alpha4alpha5 NC1 hexamers, targeting their alpha3NC1 and alpha4NC1 subunits, and recognized a noncontiguous alloepitope formed jointly by the E(A) and E(B) regions of alpha3NC1 domain. In contrast, human Goodpasture autoantibodies recognized the separate E(A) and E(B) autoepitopes of alpha3NC1 but not the composite alloepitope. Molecular modeling of alpha3NC1 revealed that the alloepitope is more accessible within the NC1 hexamers than the partially sequestered Goodpasture autoepitopes. Overall, the specificity of alloantibodies indicated a selective lack of immune tolerance toward the alpha3 and alpha4(IV) collagen chains not expressed in patients with ARAS. Using COL4A3 knockout mice, a model of ARAS, it was shown further that acid-dissociated rather than native alpha3alpha4alpha5 NC1 hexamers elicited murine anti-GBM antibodies most closely resembling human ARAS alloantibodies. In contrast, alpha3NC1 monomers elicited Goodpasture-like murine antibodies, targeting the E(A) and E(B) autoepitopes. Thus, the identity of alpha3NC1 epitopes targeted by anti-GBM antibodies is strongly influenced by the molecular organization of the immunogen. These findings suggest that different isoforms of alpha3(IV) collagen may be implicated in the pathogenesis of ARAS posttransplantation anti-GBM nephritis and Goodpasture disease.

Rapidly progressive glomerulonephritis in Goodpasture disease is mediated by autoantibodies binding to the non-collagenous NC1 domain of alpha3(IV) collagen in the glomerular basement membrane. Goodpasture epitopes in the native autoantigen are cryptic (sequestered) within the NC1 hexamers of the alpha3alpha4alpha5(IV) collagen network. The biochemical mechanism for crypticity and exposure for autoantibody binding is not known. We now report that crypticity is a feature of the quaternary structure of two distinct subsets of alpha3alpha4alpha5(IV) NC1 hexamers: autoantibody-reactive M-hexamers containing only monomer subunits and autoantibody-impenetrable D-hexamers composed of both dimer and monomer subunits. Goodpasture antibodies only breach the quaternary structure of M-hexamers, unmasking the cryptic epitopes, whereas D-hexamers are resistant to autoantibodies under native conditions. The epitopes of D-hexamers are structurally sequestered by dimer reinforcement of the quaternary complex, which represents a new molecular solution for conferring immunologic privilege to a potential autoantigen. Dissociation of non-reinforced M-alpha3alpha4alpha5(IV) hexamers by Goodpasture antibodies is a novel mechanism whereby pathogenic autoantibodies gain access to cryptic B cell epitopes. These findings provide fundamental new insights into immune privilege and the molecular mechanisms underlying the pathogenesis of human autoimmune Goodpasture disease.