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Cytoskeleton-associated protein 4 (CKAP4) affects podocyte cytoskeleton dynamics in diabetic kidney disease

ABSTRACT: Cytoskeleton-associated protein 4 (CKAP4) stabilizes the endoplasmic reticulum, by regulating its folding and anchoring to the microtubular cytoskeleton. Since podocytes are highly dependent on an intact cytoskeleton to exert their function, the role of CKAP4 was explored during normal conditions and in diabetic kidney disease (DKD). Methods. The differences in CKAP4 gene expression between patients with chronic kidney disease (CKD) and controls were explored in publicly available databases. Expression of CKAP4 in patients with DKD was investigated with in situ hybridization. Localization in human kidney tissue was determined using immunofluorescence, qPCR, and western blot. The CKAP4 homolog was knocked down (KD) in zebrafish. Proteinuria and glomerular morphology were investigated. CKAP4 KD and overexpression in podocytes were investigated using proteomics, immunofluorescence, and western blot. Results. Glomerular CKAP4 gene expression was reduced in DKD patients compared to healthy individuals, but not in other CKDs. The glomerular expression of CKAP4 was most pronounced in podocytes. CKAP4 KD zebrafish became proteinuric and podocyte FPE (foot process effacement) was observed. CKAP4 KD in podocytes in vitro led to disruption of actin stress fibers, microtubular rearrangement and loss of integrin expression. Conclusion. CKAP4 reduction causes podocyte FPE and proteinuria in vivo. Loss of CKAP4 disrupts the podocyte actin cytoskeleton, its microtubular orientation, and integrin-based glomerular basement membrane attachment. This indicates that CKAP4 is a crucial connector between cell body and structural elements in podocytes. The reduction of CKAP4 observed in glomeruli from DKD patients may thus be coupled to impaired podocyte function and proteinuria.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Danio Rerio (ncbitaxon:7955)

SUBMITTER: Roberto Boi

PROVIDER: MSV000093243 | MassIVE | Thu Nov 02 08:46:00 GMT 2023

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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Cytoskeleton-associated protein 4 (CKAP4) affects podocyte cytoskeleton dynamics in diabetic kidney disease

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