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EPB41L5 controls podocyte extracellular matrix assembly by promoting integrin adhesome reinforcement - CDM dataset

ABSTRACT: The integrity of the kidney filtration barrier essentially relies on the balanced interplay of podocytes, endothelial cells and the glomerular basement membrane. Dysfunction in any of these components results in proteinuria and can progress to chronic kidney disease. While the relevance of cell-matrix interactions for podocytes is well established, it is only incompletely understood whether and how adhesion receptors reciprocally influence the basement membrane and maintain glomerular function. Here, we show by analysis of in vitro and in vivo models that a loss of the podocyte specific FERM-domain protein EPB41L5 results in impaired extracellular matrix integrity and stability. Employing quantitative proteomics analysis of the secretome, matrisome and integrin adhesome revealed that EPB41L5 promotes mechanosensitive properties of podocyte integrin complexes by recruitment of PDLIM5/ACTN4. Consecutively, EPB41L5 knockout podocytes showed insufficient reinforcement of integrin adhesion sites, which translated into impaired ECM assembly. These observations build the framework for a model where EPB41L5 as a cell type specific regulator of the podocyte adhesome controls a localized adaptive module in order to prevent podocyte detachment and ensures GBM integrity.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Oliver Schilling

PROVIDER: MSV000086766 | MassIVE | Tue Jan 26 05:19:00 GMT 2021

SECONDARY ACCESSION(S): PXD023833

REPOSITORIES: MassIVE

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Project description:Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte cytoskeleton. To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury. Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involving cytoskeletal-associated pathways, adhesion, migration and extracellular matrix composition to be affected. Everolimus is capable of protecting podocytes from injury, both on the transcriptome and protein level. Rescued genes included TUBB2B and DCDC2, both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Confirming gene expression data, Western-blot analysis in cultured podocytes showed an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Microtubule-inhibitor experiments led to a maldistribution of TUBB2B and DCDC2 as well as an aberrant reorganization of the actin cytoskeleton. Tubb2bbrdp/brdp mice showed a delay in glomerular podocyte and capillary development. Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development We analyzed the effect of everolimus on gene expression in a puromycin aminonucleoside experimental in vitro model of podocyte injury using microarrays

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EPB41L5 controls podocyte extracellular matrix assembly by promoting integrin adhesome reinforcement - CDM dataset

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