Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.
Stefanie
S
Jeruschke
Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Duesseldorf, Germany.
Kay
K
Jeruschke
Divisions of Human Genetics and Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
Andrew
A
DiStasio
Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.
Sinem
S
Karaterzi
Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.
Anja K
AK
Büscher
Center for Medical Biotechnology, Molecular Cell Biology, University of Duisburg-Essen, Essen, Germany.
Perihan
P
Nalbant
Institute of Cell Biology, University of Duisburg‑Essen, Essen, Germany.
Ludger
L
Klein-Hitpass
Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.
Peter F
PF
Hoyer
Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Duesseldorf, Germany.
Jürgen
J
Weiss
Divisions of Human Genetics and Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.
Rolf W
RW
Stottmann
Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.
Stefanie
S
Weber
D000818:Animals; D002448:Cell Adhesion; D002461:Cell Line, Transformed; D000068338:Everolimus / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D050199:Podocytes / Q000187:drug effects* / Q000378:metabolism; D059467:Transcriptome; D014404:Tubulin / Q000235:genetics*
C510451:DCDC2 protein, human; D008869:Microtubule-Associated Proteins; C576987:TUBB2B protein, human; D014404:Tubulin; D000068338:Everolimus