Dataset Information

Drosophila nephrocyte proteome

ABSTRACT: Podocyte injury is a major cause of chronic kidney disease, but the involved signaling pathways are largely unknown. Here, we used ultrasensitive proteomics to investigate the response of the native renal podocyte to injury and prioritized candidates by aligning proteinuria in individual mice with protein expression data. The analysis revealed large perturbations of signaling pathways known to associate with FSGS, but also showed perturbation in metabolism and proteins responding to mechanical stress. We employed Drosophila nephrocytes for further functional analysis of selected candidates and studied filtration function and endocytosis. Among the candidates identified was the mechanosensor protein Filamin-B which is a central component of a conserved cellular response to mechanical stress in podocytes in vitro. Loss of Cher, the orthologue of Filamin-B, led to an increased endocytosis in nephrocytes. Of note, Filamin-B expression was only elevated in the Doxorubicin-induced proteome of podocytes but not after LPS induced injury despite the regulation of multiple similar pathways on the systems level. The Filamin-B amount in glomeruli of proteinuric PAN rats correlated with the amount of albumin . In conclusion, this study identified conserved mechanisms of podocyte stress response employing state of the art ultrasensitive proteomics in isolated mouse podocytes and Drosophila nephrocytes.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Pericardial Nephrocyte

DISEASE(S): Focal Segmental Glomerulosclerosis

SUBMITTER: Markus Rinschen

LAB HEAD: Markus Rinschen

PROVIDER: PXD012025 | Pride | 2020-07-29

REPOSITORIES: Pride

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Publications

Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis.

Koehler Sybille S Kuczkowski Alexander A Kuehne Lucas L Jüngst Christian C Hoehne Martin M Grahammer Florian F Eddy Sean S Kretzler Matthias M Beck Bodo B BB Höhfeld Jörg J Schermer Bernhard B Benzing Thomas T Brinkkoetter Paul T PT Rinschen Markus M MM

Journal of the American Society of Nephrology : JASN 20200211 3

<h4>Background</h4>Understanding podocyte-specific responses to injury at a systems level is difficult because injury leads to podocyte loss or an increase of extracellular matrix, altering glomerular cellular composition. Finding a window into early podocyte injury might help identify molecular pathways involved in the podocyte stress response.<h4>Methods</h4>We developed an approach to apply proteome analysis to very small samples of purified podocyte fractions. To examine podocytes in early d ...[more]

PMID: 32047005

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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

Dataset Information

Drosophila nephrocyte proteome

Publications

Proteome Analysis of Isolated Podocytes Reveals Stress Responses in Glomerular Sclerosis.

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