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Alport syndrome is caused by mutations in collagen IV that alter the morphology of renal glomerular basement membrane. Mutations result in proteinuria, tubulointerstitial fibrosis, and renal failure but the pathogenic mechanisms are not fully understood. Using imaging mass spectrometry, we aimed to determine whether the spatial and/or temporal patterns of renal lipids are perturbed during the development of Alport syndrome in the mouse model. Our results show that most sulfatides are present at similar levels in both the wild-type (WT) and the Alport kidneys, with the exception of two specific sulfatide species, SulfoHex-Cer(d18:2/24:0) and SulfoHex-Cer(d18:2/16:0). In the Alport but not in WT kidneys, the levels of these species mirror the previously described abnormal laminin expression in Alport syndrome. The presence of these sulfatides in renal tubules but not in glomeruli suggests that this specific aberrant lipid pattern may be related to the development of tubulointerstitial fibrosis in Alport disease.
© 2019 AOCS.
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.
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.
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 α3α4α5(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 α3α4α5(IV). This animal model shares many features with human Alport patients, including the retention of collagen α1α2α1(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 α1 expression in Alport mesangial cells and an increase in integrin α3 in Alport podocytes. We conclude that overexpression of mesangial integrin α1 and podocyte vimentin and integrin α3 may be important features of glomerular Alport disease, possibly affecting cell-signaling, cell shape and cellular adhesion to the GBM.
Collagen IV is the major protein found in basement membranes. It comprises three heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity.We constructed linear maps of the collagen IV heterotrimers ("interactomes") that indicated major structural landmarks, known and predicted ligand-binding sites, and missense mutations, in order to identify functional and disease-associated domains, potential interactions between ligands, and genotype–phenotype relationships. The maps documented more than 30 known ligand-binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin, and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection, and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases, and heparin. The maps indicated that mutations affecting major ligand-binding sites, for example, for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms, and muscle Cramps [HANAC] syndrome, and early-onset Alport syndrome, respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets.
© 2011 Wiley-Liss, Inc.
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
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.