Quaternary epitopes of ?345(IV) collagen initiate Alport post-transplant anti-GBM nephritis.
ABSTRACT: 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 ?5(IV) collagen, which occurs in normal kidneys, including renal allografts, forming distinct ?345(IV) and ?1256(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 ?5NC1 monomers, shared by ?345NC1 and ?1256NC1 hexamers. Other alloantibodies specifically targeted alloepitopes that depended on the quaternary structure of ?345NC1 hexamers. In Col4a5-null mice, immunization with native forms of allogeneic collagen IV exclusively elicited antibodies to quaternary ?345NC1 alloepitopes, whereas alloimmunogens lacking native quaternary structure elicited antibodies to shared ?5NC1 alloepitopes. These results imply that quaternary epitopes within ?345NC1 hexamers may initiate alloimmune responses after transplant in X-linked Alport patients. Thus, ?345NC1 hexamers are the culprit alloantigen and primary target of all alloantibodies mediating APTN, whereas ?1256NC1 hexamers become secondary targets of anti-?5NC1 alloantibodies. Reliable detection of alloantibodies by immunoassays using ?345NC1 hexamers may improve outcomes by facilitating early, accurate diagnosis.
Project description: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.
Project description: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.
Project description:Background: Goodpasture's disease (GP) is mediated by autoantibodies that bind the glomerular and alveolar basement membrane, causing rapidly progressive glomerulonephritis with or without pulmonary hemorrhage. The autoantibodies bind neoepitopes formed upon disruption of the quaternary structure of ?345NC1 hexamer, a critical structural domain of ?345 collagen IV scaffolds. Hexamer disruption leads to a conformational changes that transitions ?3 and ?5NC1 subunits into immunogens, however, the trigger remains unknown. This contrasts with another anti-GBM disease, Alports' post-transplant nephritis (APTN), where the pathogenic alloantibody binds directly to native NC1 hexamer. The current report includes the first study of antigenic specificity and allo-incompatability in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). Results: The anti-GBM antibodies were found to be directed predominantly against the EA epitope of the ?3 NC1 monomer of collagen IV and developed rapidly in patient serum reaching peak level within 5 weeks. Autoantibody binding to native ?345NC1 hexamer was minimal; however, binding was greatly increased upon dissociation of the native hexamer. There were no polymorphic genetic differences between donor and recipient collagen IV genes which would be predicted to cause a significant NC1 conformational change or to provide a target for antibody binding. Both patient and donor possessed the Goodpasture's susceptibility HLA-allele DRB1 * 1501. Conclusions: The current report includes the first in-depth study of allo-incompatability and antigenic specificity in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). No polymorphic genetic differences were identified between donor and recipient collagen IV genes which would be predicted to provide a target for antibody binding. Furthermore, autoantibody binding to native ?345NC1 hexamer was minimal, increasing greatly upon dissociation of the native hexamer, resembling wild-type GP diseases and marking this as the first example of a post-HSCT conformeropathy.
Project description:Alport syndrome is an inherited disorder of basement membrane collagen IV that frequently results in end-stage renal disease. Patients with Alport syndrome who undergo renal transplantation have generally excellent outcomes. Posttransplant antiglomerular basement membrane nephritis is a rare complication of renal transplantation for Alport syndrome. Because Alport syndrome is a genetic disorder, potential related donors must be carefully evaluated in order to minimize harm.
Project description: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.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.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.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.)
Project description:AIM:Type IV collagen nephropathies include Alport Syndrome and thin basement membrane nephropathy (TBMN), which are caused by mutations in COL4A3/A4/A5 genes. Recently, reports of patients with heterozygous mutations in COL4A3/A4 have been increasing. The clinical course of these patients has a wide variety, and they are diagnosed as TBMN, autosomal dominant Alport syndrome (ADAS), or familial focal segmental glomerular sclerosis. However, diagnosis, frequency and clinicopathological manifestation of them remains unclear. We tested COL4A3/A4/A5 genes in patients with hereditary nephritis that was difficult to diagnose clinicopathologically, and investigated who should undergo such testing. METHODS:We performed immunostaining for α5 chain of type IV collagen [α5 (IV)] in 27 patients from 21 families who fitted the following criteria: (i) haematuria and proteinuria (± renal dysfunction); (ii) family history of haematuria, proteinuria, and/or renal dysfunction (autosomal dominant inheritance); (iii) no specific glomerulonephritis; and (iv) thinning, splitting, or lamellation of the glomerular basement membrane (GBM) on electron microscopy. Then we performed genetic testing in 19 patients from 16 families who showed normal α5 (IV) patterns. We conducted a retrospective analysis of their clinicopathological findings. RESULTS:Among 16 families, 69% were detected heterozygous mutations in COL4A3/A4, suggesting the diagnosis of TBMN/ADAS. Twenty-one percent of patients developed end stage renal disease. All patients showed thinning of GBM, which was accompanied by splitting or lamellation in seven patients. CONCLUSION:A considerable fraction of patients with hereditary nephritis that is difficult to diagnose clinicopathologically have TBMN/ADAS. It is important to recognize TBMN/ADAS and perform genetic testing in appropriate patients.
Project description:Alport syndrome is a hereditary glomerulopathy with proteinuria and nephritis caused by defects in genes encoding type IV collagen in the glomerular basement membrane. All male and most female patients develop end-stage renal disease. Effective treatment to stop or decelerate the progression of proteinuria and nephritis is still under investigation. Here we showed that combination treatment of mild electrical stress (MES) and heat stress (HS) ameliorated progressive proteinuria and renal injury in mouse model of Alport syndrome. The expressions of kidney injury marker neutrophil gelatinase-associated lipocalin and pro-inflammatory cytokines interleukin-6, tumor necrosis factor-? and interleukin-1? were suppressed by MES+HS treatment. The anti-proteinuric effect of MES+HS treatment is mediated by podocytic activation of phosphatidylinositol 3-OH kinase (PI3K)-Akt and heat shock protein 72 (Hsp72)-dependent pathways in vitro and in vivo. The anti-inflammatory effect of MES+HS was mediated by glomerular activation of c-jun NH(2)-terminal kinase 1/2 (JNK1/2) and p38-dependent pathways ex vivo. Collectively, our studies show that combination treatment of MES and HS confers anti-proteinuric and anti-inflammatory effects on Alport mice likely through the activation of multiple signaling pathways including PI3K-Akt, Hsp72, JNK1/2, and p38 pathways, providing a novel candidate therapeutic strategy to decelerate the progression of patho-phenotypes in Alport syndrome.
Project description:Alport disease in humans, which usually results in proteinuria and kidney failure, is caused by mutations to the COL4A3, COL4A4, or COL4A5 genes, and absence of collagen ?3?4?5(IV) networks found in mature kidney glomerular basement membrane (GBM). The Alport mouse harbors a deletion of the Col4a3 gene, which also results in the lack of GBM collagen ?3?4?5(IV). This animal model shares many features with human Alport patients, including the retention of collagen ?1?2?1(IV) in GBMs, effacement of podocyte foot processes, gradual loss of glomerular barrier properties, and progression to renal failure. To learn more about the pathogenesis of Alport disease, we undertook a discovery proteomics approach to identify proteins that were differentially expressed in glomeruli purified from Alport and wild-type mouse kidneys. Pairs of cy3- and cy5-labeled extracts from 5-week old Alport and wild-type glomeruli, respectively, underwent 2-dimensional difference gel electrophoresis. Differentially expressed proteins were digested with trypsin and prepared for mass spectrometry, peptide ion mapping/fingerprinting, and protein identification through database searching. The intermediate filament protein, vimentin, was upregulated ?2.5 fold in Alport glomeruli compared to wild-type. Upregulation was confirmed by quantitative real time RT-PCR of isolated Alport glomeruli (5.4 fold over wild-type), and quantitative confocal immunofluorescence microscopy localized over-expressed vimentin specifically to Alport podocytes. We next hypothesized that increases in vimentin abundance might affect the basement membrane protein receptors, integrins, and screened Alport and wild-type glomeruli for expression of integrins likely to be the main receptors for GBM type IV collagen and laminin. Quantitative immunofluorescence showed an increase in integrin ?1 expression in Alport mesangial cells and an increase in integrin ?3 in Alport podocytes. We conclude that overexpression of mesangial integrin ?1 and podocyte vimentin and integrin ?3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.
Project description:Alport syndrome is a hereditary glomerular disease that leads to kidney failure. It is caused by mutations affecting one of three chains of the collagen ?3?4?5(IV) heterotrimer, which forms the major collagen IV network of the glomerular basement membrane (GBM). In the absence of the ?3?4?5(IV) network, the ?1?1?2(IV) network substitutes, but it is insufficient to maintain normal kidney function. Inhibition of angiotensin-converting enzyme slows progression to kidney failure in patients with Alport syndrome but is not a cure. Restoration of the normal collagen ?3?4?5(IV) network in the GBM, by either cell- or gene-based therapy, is an attractive and logical approach toward a cure, but whether or not the abnormal GBM can be repaired once it has formed and is functioning is unknown. Using a mouse model of Alport syndrome and an inducible transgene system, we found that secretion of ?3?4?5(IV) heterotrimers by podocytes into a preformed, abnormal, filtering Alport GBM is effective at restoring the missing collagen IV network, slowing kidney disease progression, and extending life span. This proof-of-principle study demonstrates the plasticity of the mature GBM and validates the pursuit of therapeutic approaches aimed at normalizing the GBM to prolong kidney function.
Project description:X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the alpha 5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant alpha 5(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine alpha 5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the alpha1(IV) and alpha2(IV), but not the alpha 3(IV) to alpha 6(IV) collagen chains. By Northern blotting, an alpha 5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the alpha1(IV) and alpha2(IV) chains but the recombinant alpha 5(IV) chain was not incorporated. These findings are consistent with the concept that the alpha 5(IV) chain requires one or more of the alpha 3(IV), alpha 4(IV), or alpha 6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the alpha 5(IV) transgene was injected into bladder smooth muscle that lacks both the alpha 5(IV) and alpha 6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the alpha 5(IV) and alpha 6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant alpha 5(IV) chain is capable of restoring expression of a second alpha(IV) chain that requires the presence of the alpha 5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome.