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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. Idiopathic pulmonary fibrosis has been associated with aberrant expansion of KRT5-expressing basal cells. Here the authors show how changes in the ECM glycoprotein SPARC restrict the movement of KRT5+ cells, affecting their retention within fibrotic tissue.

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.

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.

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.

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

Alport syndrome is a rare genetic disorder characterized by progressive kidney disease, hearing loss, and eye abnormalities. Gene therapy for Alport syndrome has not yet been realized due to technical challenges, including effective transduction of target cells. In this study, we established a quantitative testing platform using podocytes in culture, ex vivo glomeruli, and a mouse model of Alport syndrome to evaluate the transduction efficacy of adeno-associated Virus-9 (AAV9) as a gene delivery vehicle. We compared transduction levels of AAV9-GFP vectors between healthy and Alport podocytes in culture, revealing that both cell types exhibited similar transduction rates. We then incubated ex vivo glomeruli with AAV9-GFP and found enhanced transduction in Alport compared to wild type podocytes. Finally in mice following a peripheral intravenous injection of AAV9-GFP we found a striking increase in transduction in Alport podocytes suggesting that the pathological environment may facilitate higher penetration of the vector. These findings underscore the potential of AAV9 for effective gene delivery in the context of Alport syndrome, providing a foundation for future therapeutic strategies aimed at correcting the underlying genetic defects. This study highlights the clinical significance of AAV9 as a potential gene delivery vehicle for Alport syndrome treatment. Our findings demonstrate that Alport podocytes exhibit enhanced transduction levels in ex vivo and in vivo environments compared to healthy podocytes. This suggests that AAV9 could effectively target podocytes in Alport syndrome, paving the way for innovative gene therapy strategies that may improve patient outcomes and quality of life.

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.Competing Interest StatementP.L.M. received, unrelated to the submitted work, industry-academic funding from AstraZeneca via his institution and speaker and consultancy fees from Boehringer Ingelheim Trevi and Hoffman-La Roche. A.J.B. received, unrelated to the submitted work, consultancy fees and/or industry/academic funding from Ammax, Devpro, and Ionis pharmaceuticals, via his institution. A.G.N. is or has been a scientific advisor relating to IPF trials for Medical Quantitative Image Analysis, Galapagos, Boehringer Ingelheim and Roche, as well as receiving payment for educational activities relating to interstitial lung disease from Boehringer Ingelheim and UpToDate. T.M.M. received, unrelated to the submitted work, consultancy fees in relation to pulmonary fibrosis from Abbvie, Agomab, Apellis, Astra Zeneca, Bayer, Biogen Idec, Blade Therapeutics, BMS, Boehringer Ingelheim, Bridge Therapeutics, Carthronix, Chieisi, CohBar, CSLBehring, Daewoong, Daiatchi, DevPro, Endeavor, Fibrogen, Galapagos, Galecto, GlaxoSmithKline, Insilico, IQVIA, Kinevent, Pliant, Pfizer, Puretech, Qureight, Redx, Remedy Cell, Respivant Sciences, Roche, Shinogi, Surrozen, Theravance, Three Lakes Partners, Trevi, UCB, United Therapeutics, Veracyte, Vicore.

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 localized to BMs. Network analysis and screening in C. elegans and zebrafish identified new BM regulators, including ADAMTS, ROBO, and TGFβ. More than 100 BM-network genes associate with human phenotypes and by screening 63,039 genomes from families with rare disorders, we discovered loss-of-function variants in LAMA5, MPZL2, and MATN2, and show they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity and role of BMs in human health.