Collagen IV assembly is influenced by fluid flow in kidney cell-derived matrices.

Kidney podocytes and endothelial cells assemble a complex and dynamic basement membrane that is essential for kidney filtration. Whilst many components of this specialised matrix are known, the influence of fluid flow on its assembly and organisation remains poorly understood. Using the coculture of podocytes and glomerular endothelial cells in a low-shear stress, high-flow bioreactor, we investigated the effect of laminar fluid flow on the composition and assembly of cell-derived matrix. With immunofluorescence and matrix image analysis we found flow-mediated remodelling of collagen IV. Using proteomic analysis of the cell-derived matrix we identified changes in both abundance and composition of matrix proteins under flow, including the collagen-modifying enzyme, prolyl 4-hydroxylase (P4HA1). To track collagen IV assembly, we used CRISPR-Cas9 to knock in the luminescent marker HiBiT to the endogenous COL4A2 gene in podocytes. With this system, we found that collagen IV was secreted and accumulated consistently under both static and flow conditions. However knockdown of P4HA1 in podocytes led to a reduction in the secretion of collagen IV and this was more pronounced under flow. Together, this work demonstrates the effect of fluid flow on the composition, modification, and organisation of kidney cell-derived matrix and provides an in vitro system for investigating flow-induced matrix alteration in the context of kidney development and disease.

Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis.

Aberrant expansion of KRT5+ basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+ cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5+ cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5+ cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry- based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5+ cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5+ cell behaviour and function contributing to remodelling events in the fibrotic niche.

Integrating basic science with translational research: the 13th International Podocyte Conference 2021.

The 13th International Podocyte Conference was held in Manchester, UK, and online from July 28 to 30, 2021. Originally planned for 2020, this biannual meeting was postponed by a year because of the coronavirus disease 2019 (COVID-19) pandemic and proceeded as an innovative hybrid meeting. In addition to in-person attendance, online registration was offered, and this attracted 490 conference registrations in total. As a Podocyte Conference first, a day for early-career researchers was introduced. This premeeting included talks from graduate students and postdoctoral researchers. It gave early career researchers the opportunity to ask a panel, comprising academic leaders and journal editors, about career pathways and the future for podocyte research. The main meeting over 3 days included a keynote talk and 4 focused sessions each day incorporating invited talks, followed by selected abstract presentations, and an open panel discussion. The conference concluded with a Patient Day, which brought together patients, clinicians, researchers, and industry representatives. The Patient Day was an interactive and diverse day. As well as updates on improving diagnosis and potential new therapies, the Patient Day included a PodoArt competition, exercise and cooking classes with practical nutrition advice, and inspirational stories from patients and family members. This review summarizes the exciting science presented during the 13th International Podocyte Conference and demonstrates the resilience of researchers during a global pandemic.

Structure of PLA2R reveals presentation of the dominant membranous nephropathy epitope and an immunogenic patch.

Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations.

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins and their animal orthologs localized to BMs. Network analysis and screening in C. elegans and zebrafish uncovered BM regulators, including ADAMTS, ROBO, and TGFß. More than 100 BM network genes associate with human phenotypes, and by screening 63,039 genomes from families with rare disorders, we found loss-of-function variants in LAMA5, MPZL2, and MATN2 and show that they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity of BMs and their impact on in human health.

A Point-of-Care Immunosensor Based on a Quartz Crystal Microbalance with Graphene Biointerface for Antibody Assay.

We present a sensitive and low-cost immunoassay, based on a customized open-source quartz crystal microbalance coupled with graphene biointerface sensors (G-QCM), to quantify antibodies in undiluted patient serum. We demonstrate its efficacy for a specific antibody against the phospholipase A2 receptor (anti-PLA2R), which is a biomarker in primary membranous nephropathy. A novel graphene-protein biointerface was constructed by adsorbing a low concentration of denatured bovine serum albumin (dBSA) on the reduced graphene oxide (rGO) sensor surface. The dBSA film prevents the denaturation of the protein receptor on the rGO surface and serves as the cross-linker for immobilization of the receptor for anti-PLA2R antibodies on the surface. The detection limit and selectivity of this G-QCM biosensor was compared with a commercial QCM system. The G-QCM immunoassay exhibited good specificity and high sensitivity toward the target, with an order of magnitude better detection limit (of 100 ng/mL) compared to the commercial system, at a fraction of the cost and with considerable time saving. The results obtained from patient sera compared favorably with those from enzyme-linked immunosorbent assay, validating the feasibility of use in clinical applications. The multifunctional dBSA-rGO platform provides a promising biofunctionalization method for universal immunoassay and biosensors. With the advantages of inexpensive, rapid, and sensitive detection, the G-QCM sensor and instrument form an effective autoimmune disease screening tool.

The anti-PLA2R antibody in membranous nephropathy: what we know and what remains a decade after its discovery.

The discovery in 2009 of the M-type phospholipase A2 receptor (PLA2R) as the primary target in membranous nephropathy (MN) greatly advanced basic and clinical research. Primary MN is now considered a renal-limited autoimmune disease, with antibodies against PLA2R (aPLA2Rab) identified in 70-80 % of patients of various ethnic groups. Although the use of aPLA2Rab as a diagnostic and prognostic biomarker is now widely accepted, many questions related to the development of the auto-immune response, the role of IgG subclasses and antigenic epitopes, and the pathways to podocyte injury remain unresolved. PLA2R-associated MN most likely develops governed by factors such as genetic susceptibility, loss of tolerance, alterations in antigen expression with a role for environmental factors like air pollution, smoking, and infections. More detailed knowledge of genetic factors, the relevant B- and T-cell epitopes, and the mechanisms of podocyte injury is needed to identify patients at risk for disease progression and to develop optimized, targeted treatment strategies. In this review we highlight unresolved issues, addressing initiation of antibody formation, the timeline of antibody production, the role of IgG subclass, and the pathogenicity of the antibodies in concert with complement to produce glomerular pathology and proteinuria.

PLA2R binds to the annexin A2-S100A10 complex in human podocytes.

Phospholipase A2 receptor (PLA2R) is a member of the mannose receptor family found in podocytes in human kidney. PLA2R is the target of the autoimmune disease, membranous nephropathy, characterised by production of anti-PLA2R autoantibodies which bind to the podocyte. However the function of PLA2R in health and in disease remains unclear. To gain insight into the molecular mechanisms of PLA2R function, we searched for its endogenous binding partners. Proteomic analysis identified annexinA2 as a potential interactor with the extracellular domains of PLA2R. We confirmed that PLA2R binds to annexinA2-S100A10 (A2t) complex with specific high affinity to the S100A10 component. The binding occured within the PLA2R NC3 fragment and was increased in acidic pH. Furthermore Ca2+ promoted the association of the PLA2R-A2t complex with phospholipid membranes in vitro. Within the podocyte, all three proteins were enriched in the plasma membrane and organelle membrane compartments. PLA2R co-localised with S100A10 at the cell surface and in extracellular vesicles. This novel interaction between PLA2R and the A2t complex offers insights into the role of PLA2R in podocytes and how autoantibodies might disrupt PLA2R function. The ability of podocytes to secrete vesicles containing PLA2R provides a route for engagement of PLA2R with the immune system.

Glucocorticoid therapy regulates podocyte motility by inhibition of Rac1.

Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes excessively permeable leading to massive proteinuria. In childhood NS, immune system dysregulation has been implicated and increasing evidence points to the central role of podocytes in the pathogenesis. Children with NS are typically treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids that are activating ligands for the glucocorticoid receptor (GR) transcription factor. Although Gc-therapy has been the cornerstone of NS management for decades, the mechanism of action, and target cell, remain poorly understood. We tested the hypothesis that Gc acts directly on the podocyte to produce clinically useful effects without involvement of the immune system. In human podocytes, we demonstrated that the basic GR-signalling mechanism is intact and that Gc induced an increase in podocyte barrier function. Defining the GR-cistrome identified Gc regulation of motility genes. These findings were functionally validated with live-cell imaging. We demonstrated that treatment with Gc reduced the activity of the pro-migratory small GTPase regulator Rac1. Furthermore, Rac1 inhibition had a direct, protective effect on podocyte barrier function. Our studies reveal a new mechanism for Gc action directly on the podocyte, with translational relevance to designing new selective synthetic Gc molecules.

Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy.

Phospholipase A2 receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.

Armet/Manf and Creld2 are components of a specialized ER stress response provoked by inappropriate formation of disulphide bonds: implications for genetic skeletal diseases.

Mutant matrilin-3 (V194D) forms non-native disulphide bonded aggregates in the rER of chondrocytes from cell and mouse models of multiple epiphyseal dysplasia (MED). Intracellular retention of mutant matrilin-3 causes endoplasmic reticulum (ER) stress and induces an unfolded protein response (UPR) including the upregulation of two genes recently implicated in ER stress: Armet and Creld2. Nothing is known about the role of Armet and Creld2 in human genetic diseases. In this study, we used a variety of cell and mouse models of chondrodysplasia to determine the genotype-specific expression profiles of Armet and Creld2. We also studied their interactions with various mutant proteins and investigated their potential roles as protein disulphide isomerases (PDIs). Armet and Creld2 were up-regulated in cell and/or mouse models of chondrodysplasias caused by mutations in Matn3 and Col10a1, but not Comp. Intriguingly, both Armet and Creld2 were also secreted into the ECM of these disease models following ER stress. Armet and Creld2 interacted with mutant matrilin-3, but not with COMP, thereby validating the genotype-specific expression. Substrate-trapping experiments confirmed Creld2 processed PDI-like activity, thus identifying a putative functional role. Finally, alanine substitution of the two terminal cysteine residues from the A-domain of V194D matrilin-3 prevented aggregation, promoted mutant protein secretion and reduced the levels of Armet and Creld2 in a cell culture model. We demonstrate that Armet and Creld2 are genotype-specific ER stress response proteins with substrate specificities, and that aggregation of mutant matrilin-3 is a key disease trigger in MED that could be exploited as a potential therapeutic target.

Loss of matrilin 1 does not exacerbate the skeletal phenotype in a mouse model of multiple epiphyseal dysplasia caused by a Matn3 V194D mutation.

OBJECTIVE: Mutations in matrilin 3 can result in multiple epiphyseal dysplasia (MED), a disease characterized by delayed and irregular bone growth and early-onset osteoarthritis. Although intracellular retention of the majority of mutant matrilin 3 was previously observed in a murine model of MED caused by a Matn3 V194D mutation, some mutant protein was secreted into the extracellular matrix. Thus, it was proposed that secretion of mutant matrilin 3 may be dependent on the formation of hetero-oligomers with matrilin 1. The aim of this study was to investigate the hypothesis that deletion of matrilin 1 would abolish the formation of matrilin 1/matrilin 3 hetero-oligomers, eliminate the secretion of mutant matrilin 3, and influence disease severity. METHODS: Mice with a Matn3 V194D mutation were crossed with Matn1-null mice, generating mice that were homozygous for V194D and null for matrilin 1. This novel mouse was used for in-depth phenotyping, while cartilage and chondrocytes were studied both histochemically and biochemically. RESULTS: Endochondral ossification was not disrupted any further in mice with a double V194D mutation compared with mice with a single mutation. A similar proportion of mutant matrilin 3 was present in the extracellular matrix, and the amount of retained mutant matrilin 3 was not noticeably increased. Retained mutant matrilin 3 formed disulfide-bonded aggregates and caused the co-retention of matrilin 1. CONCLUSION: We showed that secretion of matrilin 3 V194D mutant protein is not dependent on hetero-oligomerization with matrilin 1, and that the total ablation of matrilin 1 expression has no impact on disease severity in mice with MED. Mutant matrilin 3 oligomers form non-native disulfide-bonded aggregates through the misfolded A domain.

Structural and functional investigations of Matrilin-1 A-domains reveal insights into their role in cartilage ECM assembly.

Matrilin-1 is expressed predominantly in cartilage and co-localizes with matrilin-3 with which it can form hetero-oligomers. We recently described novel structural and functional features of the matrilin-3 A-domain (M3A) and demonstrated that it bound with high affinity to type II and IX collagens. Interactions preferentially occurred in the presence of Zn(2+) suggesting that matrilin-3 has acquired a requirement for specific metal ions for activation and/or molecular associations. To understand the interdependence of matrilin-1/-3 hetero-oligomers in extracellular matrix (ECM) interactions, we have extended these studies to include the two matrilin-1 A-domains (i.e. M1A1 and M1A2 respectively). In this study we have identified new characteristics of the matrilin-1 A-domains by describing their glycosylation state and the effect of N-glycan chains on their structure, thermal stability, and protein-protein interactions. Initial characterization revealed that N-glycosylation did not affect secretion of these two proteins, nor did it alter their folding characteristics. However, removal of the glycosylation decreased their thermal stability. We then compared the effect of different cations on binding between both M1A domains and type II and IX collagens and showed that Zn(2+) also supports their interactions. Finally, we have demonstrated that both M1A1 domains and biglycan are essential for the association of the type II·VI collagen complex. We predict that a potential role of the matrilin-1/-3 hetero-oligomer might be to increase multivalency, and therefore the ability to connect various ECM components. Differing affinities could act to regulate the integrated network, thus coordinating the organization of the macromolecular structures in the cartilage ECM.

A recessive skeletal dysplasia, SEMD aggrecan type, results from a missense mutation affecting the C-type lectin domain of aggrecan.

Analysis of a nuclear family with three affected offspring identified an autosomal-recessive form of spondyloepimetaphyseal dysplasia characterized by severe short stature and a unique constellation of radiographic findings. Homozygosity for a haplotype that was identical by descent between two of the affected individuals identified a locus for the disease gene within a 17.4 Mb interval on chromosome 15, a region containing 296 genes. These genes were assessed and ranked by cartilage selectivity with whole-genome microarray data, revealing only two genes, encoding aggrecan and chondroitin sulfate proteoglycan 4, that were selectively expressed in cartilage. Sequence analysis of aggrecan complementary DNA from an affected individual revealed homozygosity for a missense mutation (c.6799G --> A) that predicts a p.D2267N amino acid substitution in the C-type lectin domain within the G3 domain of aggrecan. The D2267 residue is predicted to coordinate binding of a calcium ion, which influences the conformational binding loops of the C-type lectin domain that mediate interactions with tenascins and other extracellular-matrix proteins. Expression of the normal and mutant G3 domains in mammalian cells showed that the mutation created a functional N-glycosylation site but did not adversely affect protein trafficking and secretion. Surface-plasmon-resonance studies showed that the mutation influenced the binding and kinetics of the interactions between the aggrecan G3 domain and tenascin-C. These findings identify an autosomal-recessive skeletal dysplasia and a significant role for the aggrecan C-type lectin domain in regulating endochondral ossification and, thereby, height.

Novel mutations in exon 2 of MATN3 affect residues within the alpha-helices of the A-domain and can result in the intracellular retention of mutant matrilin-3.

Multiple epiphyseal dysplasia (MED) is a clinically variable and genetically heterogeneous chondrodysplasia characterized by mild to moderate short stature and early onset osteoarthritis. Some forms of MED result from mutations in the gene encoding the cartilage structural protein matrilin-3 (MATN3). The majority of MATN3 mutations affect conserved residues within the beta-sheet of the single A-domain of matrilin-3. These mutations cause the protein to misfold and prevent its secretion from the rER, both in vitro and in vivo. More recently a single mutation (p.Phe105Ser) has been identified within the alpha1-helix of the A-domain, but its affect on the structure and/or function of matrilin-3 is unknown. In this paper we describe the characterization of two additional alpha-helical mutations (p.Ala173Asp and p.Lys231Asn) and show that both p.Phe105Ser and pAla173Asp prevent the secretion of A-domain in vitro. In contrast, p.Lys231Asn does not prevent the secretion of matrilin-3 A-domain, nor does it disrupt the structure of this domain or inhibit its binding to type II or type IX collagen. Therefore, despite extensive biochemical analysis the disease mechanism of p.Lys231Asn remains unresolved and care should be taken in counseling for these types of mutation in MATN3.

Structural and functional characterization of recombinant matrilin-3 A-domain and implications for human genetic bone diseases.

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the beta-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn(2+). In the presence of Zn(2+) the A-domain adopts a more stable "tighter" conformation. However, after the removal of Zn(2+) a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more "relaxed" conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn(2+)-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.