Dysregulated extracellular matrix is the hallmark of fibrosis, and it has a profound impact on kidney function in disease. Furthermore, perturbation of matrix homeostasis is a feature of aging and is ...associated with declining kidney function. Understanding these dynamic processes, in the hope of developing therapies to combat matrix dysregulation, requires the integration of data acquired by both well-established and novel technologies. Owing to its complexity, the extracellular proteome, or matrisome, still holds many secrets and has great potential for the identification of clinical biomarkers and drug targets. The molecular resolution of matrix composition during aging and disease has been illuminated by cutting-edge mass spectrometry–based proteomics in recent years, but there remain key questions about the mechanisms that drive altered matrix composition. Basement membrane components are particularly important in the context of kidney function; and data from proteomic studies suggest that switches between basement membrane and interstitial matrix proteins are likely to contribute to organ dysfunction during aging and disease. Understanding the impact of such changes on physical properties of the matrix, and the subsequent cellular response to altered stiffness and viscoelasticity, is of critical importance. Likewise, the comparison of proteomic data sets from multiple organs is required to identify common matrix biomarkers and shared pathways for therapeutic intervention. Coupled with single-cell transcriptomics, there is the potential to identify the cellular origin of matrix changes, which could enable cell-targeted therapy. This review provides a contemporary perspective of the complex kidney matrisome and draws comparison to altered matrix in heart and liver disease.
The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build ...understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456.
Podocytes are specialized epithelial cells that cover the outer surfaces of glomerular capillaries. Unique cell junctions, known as slit diaphragms, which feature nephrin and Neph family proteins in ...addition to components of adherens, tight, and gap junctions, connect adjacent podocyte foot processes. Single gene disorders affecting the slit diaphragm result in nephrotic syndrome in humans, characterized by massive loss of protein across the capillary wall. In addition to specialized cell junctions, interconnecting podocytes also adhere to the glomerular basement membrane (GBM) of the capillary wall. The GBM is a dense network of secreted, extracellular matrix (ECM) components and contains tissue-restricted isoforms of collagen IV and laminin in addition to other structural proteins and ECM regulators such as proteases and growth factors. The specialized niche of the GBM provides a scaffold for endothelial cells and podocytes to support their unique functions and human genetic mutations in GBM components lead to renal failure, thus highlighting the importance of cell-matrix interactions in the glomerulus. Cells adhere to ECM via adhesion receptors, including integrins, syndecans, and dystroglycan and in particular the integrin heterodimer α3β1 is required to maintain barrier integrity. Therefore, the sophisticated function of glomerular filtration relies on podocyte adhesion both at cell junctions and at the interface with the ECM. In health, the podocyte coordinates signals from cell junctions and cell-matrix interactions, in response to environmental cues in order to regulate filtration and as our understanding of mechanisms that control cell adhesion in the glomerulus develops, then insight into the effects of disease will improve. The ultimate goal will be to develop targeted therapies to prevent or repair defects in the filtration barrier and to restore glomerular function.
Accumulation of extracellular matrix in organs and tissues is a feature of both aging and disease. In the kidney, glomerulosclerosis and tubulointerstitial fibrosis accompany the decline in function, ...which current therapies cannot address, leading to organ failure. Although histologic and ultrastructural patterns of excess matrix form the basis of human disease classifications, a comprehensive molecular resolution of abnormal matrix is lacking.
Using mass spectrometry-based proteomics, we resolved matrix composition over age in mouse models of kidney disease. We compared the changes in mice with a global characterization of human kidneymatrix during aging and to existing kidney disease datasets to identify common molecular features.
Ultrastructural changes in basement membranes are associated with altered cell adhesion and metabolic processes and with distinct matrix proteomes during aging and kidney disease progression in mice. Within the altered matrix, basement membrane components (laminins, type IV collagen, type XVIII collagen) were reduced and interstitial matrix proteins (collagens I, III, VI, and XV; fibrinogens; and nephronectin) were increased, a pattern also seen in human kidney aging. Indeed, this signature of matrix proteins was consistently modulated across all age and disease comparisons, and the increase in interstitial matrix was also observed in human kidney disease datasets.
This study provides deep molecular resolution of matrix accumulation in kidney aging and disease, and identifies a common signature of proteins that provides insight into mechanisms of response to kidney injury and repair.
Cells have evolved mechanisms to sense the composition of their adhesive microenvironment. Although much is known about general mechanisms employed by adhesion receptors to relay signals between the ...extracellular environment and the cytoskeleton, the nuances of ligand-specific signalling remain undefined. Here, we investigated how glomerular podocytes, and four other basement membrane-associated cell types, respond morphologically to different basement membrane ligands. We defined the composition of the respective adhesion complexes using mass spectrometry-based proteomics. On type IV collagen, all epithelial cell types adopted a round morphology, with a single lamellipodium and large adhesion complexes rich in actin-binding proteins. On laminin (511 or 521), all cell types attached to a similar degree but were polygonal in shape with small adhesion complexes enriched in endocytic and microtubule-binding proteins. Consistent with their distinctive morphologies, cells on type IV collagen exhibited high Rac1 activity, while those on laminin had elevated PKCα. Perturbation of PKCα was able to interchange morphology consistent with a key role for this pathway in matrix ligand-specific signalling. Therefore, this study defines the switchable basement membrane adhesome and highlights two key signalling pathways within the systems that determine distinct cell morphologies. Proteomic data are availableviaProteomeXchange with identifier PXD017913.
•Cells have evolved mechanisms to sense the composition of their adhesive microenvironment and although much is known about general mechanisms employed by adhesion receptors to relay signals between the extracellular environment and the cytoskeleton, the nuances of ligand-specific signalling remain undefined.•We investigated cellular responses to different basement membrane ligands and defined the composition of their respective adhesion complexes using mass spectrometry-based proteomics. On type IV collagen, multiple human epithelial cell types adopted a round shape with large adhesion complexes rich in actin-binding proteins.•By contrast cells on laminin were elongated with small adhesion complexes enriched in endocytic and microtubule-binding proteins.•Consistent with their distinctive shapes, cells on type IV collagen exhibited high Rac1 activity, while those on laminin had elevated PKCα and perturbation of either Rac1 or PKCα caused cell shape interchange.•Overall this study defines ligand-specific adhesomes and highlights two key signalling hubs that influence cell shape.
Glomeruli are highly sophisticated filters and glomerular disease is the leading cause of kidney failure. Morphological change in glomerular podocytes and the underlying basement membrane are ...frequently observed in disease, irrespective of the underlying molecular etiology. Standard electron microscopy techniques have enabled the identification and classification of glomerular diseases based on two-dimensional information, however complex three-dimensional ultrastructural relationships between cells and their extracellular matrix cannot be easily resolved with this approach. We employed serial block face-scanning electron microscopy to investigate Alport syndrome, the commonest monogenic glomerular disease, and compared findings to other genetic mouse models of glomerular disease (Myo1e-/-, Ptpro-/-). These analyses revealed the evolution of basement membrane and cellular defects through the progression of glomerular injury. Specifically we identified sub-podocyte expansions of the basement membrane with both cellular and matrix gene defects and found a corresponding reduction in podocyte foot process number. Furthermore, we discovered novel podocyte protrusions invading into the glomerular basement membrane in disease and these occurred frequently in expanded regions of basement membrane. These findings provide new insights into mechanisms of glomerular barrier dysfunction and suggest that common cell-matrix-adhesion pathways are involved in the progression of disease regardless of the primary insult.
Nephrotic syndrome is characterized by severe proteinuria, hypoalbuminaemia, edema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a ...crucial role in slit diaphragm signaling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin β2 and laminin γ1, is a major component of the glomerular basement membrane, a critical component of the glomerular filtration barrier. Mutations in LAMA5 were recently identified in children with nephrotic syndrome. Here, we have identified a novel missense mutation (E884G) in the uncharacterized L4a domain of LAMA5 where homozygous mice develop nephrotic syndrome with severe proteinuria with histological and ultrastructural changes in the glomerulus mimicking the progression seen in most patients. The levels of LAMA5 are reduced in vivo and the assembly of the laminin 521 heterotrimer significantly reduced in vitro. Proteomic analysis of the glomerular extracellular fraction revealed changes in the matrix composition. Importantly, the genetic background of the mice had a significant effect on aspects of disease progression from proteinuria to changes in podocyte morphology. Thus, our novel model will provide insights into pathologic mechanisms of nephrotic syndrome and pathways that influence the response to a dysfunctional glomerular basement membrane that may be important in a range of kidney diseases.
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Glomerular disease often features altered histologic patterns of extracellular matrix (ECM). Despite this, the potential complexities of the glomerular ECM in both health and disease are poorly ...understood. To explore whether genetic background and sex determine glomerular ECM composition, we investigated two mouse strains, FVB and B6, using RNA microarrays of isolated glomeruli combined with proteomic glomerular ECM analyses. These studies, undertaken in healthy young adult animals, revealed unique strain- and sex-dependent glomerular ECM signatures, which correlated with variations in levels of albuminuria and known predisposition to progressive nephropathy. Among the variation, we observed changes in netrin 4, fibroblast growth factor 2, tenascin C, collagen 1, meprin 1-α, and meprin 1-β. Differences in protein abundance were validated by quantitative immunohistochemistry and Western blot analysis, and the collective differences were not explained by mutations in known ECM or glomerular disease genes. Within the distinct signatures, we discovered a core set of structural ECM proteins that form multiple protein-protein interactions and are conserved from mouse to man. Furthermore, we found striking ultrastructural changes in glomerular basement membranes in FVB mice. Pathway analysis of merged transcriptomic and proteomic datasets identified potential ECM regulatory pathways involving inhibition of matrix metalloproteases, liver X receptor/retinoid X receptor, nuclear factor erythroid 2-related factor 2, notch, and cyclin-dependent kinase 5. These pathways may therefore alter ECM and confer susceptibility to disease.
Abstract
Phospholipase A
2
receptor (PLA
2
R) is a member of the mannose receptor family found in podocytes in human kidney. PLA
2
R is the target of the autoimmune disease, membranous nephropathy, ...characterised by production of anti-PLA
2
R autoantibodies which bind to the podocyte. However the function of PLA
2
R in health and in disease remains unclear. To gain insight into the molecular mechanisms of PLA
2
R function, we searched for its endogenous binding partners. Proteomic analysis identified annexinA2 as a potential interactor with the extracellular domains of PLA
2
R. We confirmed that PLA
2
R binds to annexinA2-S100A10 (A2t) complex with specific high affinity to the S100A10 component. The binding occured within the PLA
2
R NC3 fragment and was increased in acidic pH. Furthermore Ca
2+
promoted the association of the PLA
2
R-A2t complex with phospholipid membranes
in vitro
. Within the podocyte, all three proteins were enriched in the plasma membrane and organelle membrane compartments. PLA
2
R co-localised with S100A10 at the cell surface and in extracellular vesicles. This novel interaction between PLA
2
R and the A2t complex offers insights into the role of PLA
2
R in podocytes and how autoantibodies might disrupt PLA
2
R function. The ability of podocytes to secrete vesicles containing PLA
2
R provides a route for engagement of PLA
2
R with the immune system.
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