Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression

• Published in: PloS one Vol. 10; no. 9; p. e0137043
• Main Authors: Jeruschke, Stefanie, Jeruschke, Kay, DiStasio, Andrew, Karaterzi, Sinem, Büscher, Anja K, Nalbant, Perihan, Klein-Hitpass, Ludger, Hoyer, Peter F, Weiss, Jürgen, Stottmann, Rolf W, Weber, Stefanie
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 02-09-2015; Public Library of Science (PLoS)
• Subjects: Actin; Animal euthanasia; Animals; Biochemistry; Brain architecture; Cell Adhesion; Cell Line, Transformed; Cell migration; Children & youth; Cluster analysis; Complications and side effects; Cytoskeleton; Data processing; Developmental biology; Diabetes; DNA microarrays; Dosage and administration; Doublecortin protein; Dyslexia; Everolimus; Everolimus - pharmacology; Extracellular matrix; Gene clusters; Gene expression; Genes; Genetic aspects; Humans; Immunohistochemistry; Immunosuppressive agents; Inhibitor drugs; Inhibitors; Injury analysis; Kidney - metabolism; Kidneys; Laboratory animals; Mice; Mice, Mutant Strains; Microtubule-Associated Proteins - genetics; Microtubules; Microtubules - drug effects; Microtubules - metabolism; Morphogenesis; Muscle proteins; Mutation; Nephrology; Neurons; Pediatrics; Physiological aspects; Podocytes - drug effects; Podocytes - metabolism; Polymerization; Proteins; Puromycin; Rodents; Signal transduction; Target recognition; TOR protein; Transcription; Transcriptome; Tubulin; Tubulin - genetics; Tumors; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0137043

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 actin cytoskeleton. In contrast, a potential stabilization of microtubules by everolimus has not been studied so far.
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 involved in cytoskeletal reorganization, cell adhesion, migration and extracellular matrix composition to be affected. Everolimus was capable of protecting podocytes from injury, both on transcriptional and protein level. Rescued genes included tubulin beta 2B class IIb (TUBB2B) and doublecortin domain containing 2 (DCDC2), both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Validating gene expression data, Western-blot analysis in cultured podocytes demonstrated an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Interestingly, Tubb2bbrdp/brdp mice revealed a delay in glomerular podocyte development as showed by podocyte-specific markers Wilm's tumour 1, Podocin, Nephrin and Synaptopodin.
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.