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Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.

Jeruschke S, Jeruschke K, DiStasio A, Karaterzi S, Büscher AK, Nalbant P, Klein-Hitpass L, Hoyer PF, Weiss J, Stottmann RW, Weber S - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Transmission electron micrographs of wild type and Tubb2bbrdp/brdp mouse kidneys (E18.5).(A-F) With TEM, a delay in glomerular endothelial (E) and podocyte (P) development could be observed in Tubb2bbrdp/brdp mice. (A+B) Glomerulus with immature, cuboidal podocytes (black arrow). (C+D) Incompletely differentiated podocyte foot processes (FPs); some are extremely wide and linked by occludens junctions (OJs). (D) The glomerular basement membrane (asterisk) appears to be normal. (A+E) Glomeruli with no or small visible capillary lumen (CL) and multiple endothelial cells within the capillary loops (black arrows). (F) Swollen and vacuolated glomerular endothelial cells with decreased fenestrations. (G-I) Glomeruli of wild type Tubb2b mice had open glomerular capillaries with fenestrated endothelium, differentiated podocyte foot processes linked by SDs (black arrow) and a normal glomerular basement membrane.
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pone.0137043.g007: Transmission electron micrographs of wild type and Tubb2bbrdp/brdp mouse kidneys (E18.5).(A-F) With TEM, a delay in glomerular endothelial (E) and podocyte (P) development could be observed in Tubb2bbrdp/brdp mice. (A+B) Glomerulus with immature, cuboidal podocytes (black arrow). (C+D) Incompletely differentiated podocyte foot processes (FPs); some are extremely wide and linked by occludens junctions (OJs). (D) The glomerular basement membrane (asterisk) appears to be normal. (A+E) Glomeruli with no or small visible capillary lumen (CL) and multiple endothelial cells within the capillary loops (black arrows). (F) Swollen and vacuolated glomerular endothelial cells with decreased fenestrations. (G-I) Glomeruli of wild type Tubb2b mice had open glomerular capillaries with fenestrated endothelium, differentiated podocyte foot processes linked by SDs (black arrow) and a normal glomerular basement membrane.

Mentions: On examination with TEM, significant morphologic changes of foot process and glomerular endothelium development were observed in Tubb2bbrdp/brdp glomeruli (Fig 7). Some glomeruli from Tubb2bbrdp/brdp mice had immature, cuboidal podocytes (Fig 7A+7B). In some areas, podocyte foot processes appeared incompletely differentiated. They were extremely wide and linked by occludens junctions (OJs) rather than SDs (Fig 7C+7D). Often, there was no visible capillary lumen in Tubb2bbrdp/brdp glomeruli, even in most mature glomeruli, and multiple endothelial cells were visualized within the capillary loops (Fig 7A+7E). In addition, glomerular endothelial cells were swollen and vacuolated, with decreased fenestrations (Fig 7F). The glomerular basement membrane in Tubb2bbrdp/brdp mice appeared morphologically similar to that seen in controls (Fig 7D). Glomeruli of wild type Tubb2b+/+ mice had, according to age, open glomerular capillaries with fenestrated endothelium, differentiated podocyte foot processes linked by SDs and a normal glomerular basement membrane (Fig 7G–7I).


Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.

Jeruschke S, Jeruschke K, DiStasio A, Karaterzi S, Büscher AK, Nalbant P, Klein-Hitpass L, Hoyer PF, Weiss J, Stottmann RW, Weber S - PLoS ONE (2015)

Transmission electron micrographs of wild type and Tubb2bbrdp/brdp mouse kidneys (E18.5).(A-F) With TEM, a delay in glomerular endothelial (E) and podocyte (P) development could be observed in Tubb2bbrdp/brdp mice. (A+B) Glomerulus with immature, cuboidal podocytes (black arrow). (C+D) Incompletely differentiated podocyte foot processes (FPs); some are extremely wide and linked by occludens junctions (OJs). (D) The glomerular basement membrane (asterisk) appears to be normal. (A+E) Glomeruli with no or small visible capillary lumen (CL) and multiple endothelial cells within the capillary loops (black arrows). (F) Swollen and vacuolated glomerular endothelial cells with decreased fenestrations. (G-I) Glomeruli of wild type Tubb2b mice had open glomerular capillaries with fenestrated endothelium, differentiated podocyte foot processes linked by SDs (black arrow) and a normal glomerular basement membrane.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557973&req=5

pone.0137043.g007: Transmission electron micrographs of wild type and Tubb2bbrdp/brdp mouse kidneys (E18.5).(A-F) With TEM, a delay in glomerular endothelial (E) and podocyte (P) development could be observed in Tubb2bbrdp/brdp mice. (A+B) Glomerulus with immature, cuboidal podocytes (black arrow). (C+D) Incompletely differentiated podocyte foot processes (FPs); some are extremely wide and linked by occludens junctions (OJs). (D) The glomerular basement membrane (asterisk) appears to be normal. (A+E) Glomeruli with no or small visible capillary lumen (CL) and multiple endothelial cells within the capillary loops (black arrows). (F) Swollen and vacuolated glomerular endothelial cells with decreased fenestrations. (G-I) Glomeruli of wild type Tubb2b mice had open glomerular capillaries with fenestrated endothelium, differentiated podocyte foot processes linked by SDs (black arrow) and a normal glomerular basement membrane.
Mentions: On examination with TEM, significant morphologic changes of foot process and glomerular endothelium development were observed in Tubb2bbrdp/brdp glomeruli (Fig 7). Some glomeruli from Tubb2bbrdp/brdp mice had immature, cuboidal podocytes (Fig 7A+7B). In some areas, podocyte foot processes appeared incompletely differentiated. They were extremely wide and linked by occludens junctions (OJs) rather than SDs (Fig 7C+7D). Often, there was no visible capillary lumen in Tubb2bbrdp/brdp glomeruli, even in most mature glomeruli, and multiple endothelial cells were visualized within the capillary loops (Fig 7A+7E). In addition, glomerular endothelial cells were swollen and vacuolated, with decreased fenestrations (Fig 7F). The glomerular basement membrane in Tubb2bbrdp/brdp mice appeared morphologically similar to that seen in controls (Fig 7D). Glomeruli of wild type Tubb2b+/+ mice had, according to age, open glomerular capillaries with fenestrated endothelium, differentiated podocyte foot processes linked by SDs and a normal glomerular basement membrane (Fig 7G–7I).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Pediatric Nephrology, Pediatrics II, University Hospital Essen, Essen, Germany.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus