EV inhibits mTORC1 and mTORC2.

<p>(A) Western blot analysis to measure the activation level of Akt (representative example from 3 independent experiments). GAPDH = loading control, Akt = total Akt protein levels, pAkt = active, phosphorylated Akt protein. (B) For quantification, total Akt protein was normalized with respect to GAPDH. (C) To quantify the amount of active Akt protein, phosphorylated Akt was normalized with respect to total Akt. (D) Western blot analysis of p70S6K protein (representative example from 3 independent experiments). GAPDH = loading control, p70S6K = total p70S6K protein levels, p-p70S6K = active, phosphorylated p70S6K protein. (E) For quantification, total p70S6K was normalized to GAPDH. (F) Phosphorylated p70S6K was normalized to total p70S6K.</p

Aberrant distribution and size of focal adhesions is recovered by EV in human podocytes.

<p>(A) Actin (phalloidin-TRITC) and paxillin (antibody staining) images are presented in gray scale for maximum contrast. The merge image depicts paxillin in green and actin in red. DAPI was used to visualize nuclei (blue). White arrows depict focal adhesion localization. Scale bar = 25 µm. (B) Quantification of focal adhesion size (n = 3 experiments, ≥10 images per condition). (C) Quantification of the distance of focal adhesions from the cell periphery (n = 3 experiments, ≥10 images per condition. MeOH = solvent for EV. FAs = focal adhesions. Data are means ± SD.</p

Cytoskeletal organization in differentiated human podocytes.

<p>(A) Confocal imaging of the actin cytoskeleton and focal adhesions. Actin was visualized by phalloidin-TRITC and focal adhesions by paxillin antibody staining. Actin and paxillin images are presented in gray scale to preserve maximum contrast. Merge image shows actin in red and paxillin in green. Yellow arrow in the paxillin image: Weak paxillin staining along stress fibers. White arrow in the enlarged image: Strong accumulation of paxillin in focal adhesions at the ends of stress fibers. Scale bar = 20 µm. (B) Confocal imaging of the actin cytoskeleton (phalloidin-TRITC, grey) revealed distinct cellular structures reminiscent of dynamic actin rich protrusions (yellow arrows). Scale bar = 25 µm. (C) Phase contrast movies of control podocytes confirmed several dynamic protrusions generated in more central regions in addition to the peripheral extensions. Upper image depicts the first frame of a representative phase contrast movie (Movie S1). Lover panel shows the image sequence of the enlarged region (white box) with two dynamic protrusions (yellow stars).</p

EV prevents disruption of the actin cytoskeleton in human podocytes.

<p>(A) Actin (phalloidin-TRITC, grey) and nuclear staining (DAPI, blue). Scale bar = 100 µm. (B) Quantification of cell size (n = 5 experiments, ≥25 images per condition). (C) Number of central actin stress fibers within a distinct area (50 µm²) (n = 3 experiments, 20 cells per condition). (D) Quantification of cell numbers (n = 5 experiments, ≥25 images per condition). (E) Hoechst nuclear staining for the detection of apoptosis (n = 3 experiments, ≥50 images per condition). Apoptotic cells were defined as percentage of fragmented nuclei. MeOH = solvent for EV. Data are means ± SD.</p

EV targets RhoA signaling pathway in human podocytes.

<p>(A) Biochemical assay to measure the activation level of the GTPase. The Rho-binding domain (RBD) of the RhoA effector rhotekin was used to affinity-precipitate the active fraction of endogenous RhoA (GTP-RhoA) from cell lysates (representative example from 3 independent experiments). Tubulin was used as loading control. (B) Quantification of total RhoA protein (n = 3 experiments). For quantification, total RhoA protein was normalized with respect to tubulin from whole cell lysates. (C) To quantify the amount of active RhoA protein, GTP-bound RhoA was normalized with respect to total RhoA (n = 3 experiments). (D) Western-blot analysis of MLC protein (representative example from 4 independent experiments). GAPDH = loading control, MLC = total MLC protein levels, pMLC = active, phosphorylated MLC protein. (E) Quantification of total MLC protein (n = 4 experiments). For quantification, total MLC was normalized to GAPDH from whole cell lysates. (F) Quantification of phosphorylated MLC protein (n = 4 experiments). Phosphorylated MLC was normalized to total MLC from whole cell lysates. (G) Western blot analysis of MLC protein after treatment with the ROCK inhibitor Y-27632 (10 µM for 1 h; n = 2 independent experiments). (H) Actin cytoskeleton (phalloidin-TRITC, grey) after treatment with Y-27632. DAPI was used for nuclear staining (blue). Scale bar = 100 µm. MeOH = solvent for EV. Data are means ± SD.</p

EV inhibits migration in human podocytes.

<p>(A) Representative phase contrast images after 0 h and 12 h of migration. Scale bar = 500 µm. (B) Quantification of migration efficiency by measurement of cell-free area after 12 h of migration (n = 3 experiments, ≥5 images per condition). (C) Phase contrast time-lapse studies of living cells. Migration was tracked following the nuclei in the phase contrast movie. Lower panel: Tracks were depicted on white background for better contrast. Scale bar = 500 µm. MeOH = solvent for EV. Data are means ± SD.</p

Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression

<div><p>Background</p><p>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.</p><p>Methods</p><p>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 <i>in vitro</i> model of podocyte injury.</p><p>Results</p><p>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 <i>tubulin beta 2B class IIb</i> (<i>TUBB2B)</i> and <i>doublecortin domain containing 2</i> (<i>DCDC2)</i>, 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, <i>Tubb2b</i>^{<i>brdp/brdp</i>} mice revealed a delay in glomerular podocyte development as showed by podocyte-specific markers Wilm’s tumour 1, Podocin, Nephrin and Synaptopodin.</p><p>Conclusions</p><p>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.</p></div

Transmission electron micrographs of wild type and <i>Tubb2b</i>^brdp/brdp mouse kidneys (E18.5).

<p>(A-F) With TEM, a delay in glomerular endothelial (E) and podocyte (P) development could be observed in <i>Tubb2b</i>^brdp/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 <i>Tubb2b</i> mice had open glomerular capillaries with fenestrated endothelium, differentiated podocyte foot processes linked by SDs (black arrow) and a normal glomerular basement membrane.</p

Western-blot analysis of human podocytes: Verification of selected microarray results.

<p>Quantification of total TUBB2B (A), DCDC2 (B), THBS1 (C), COL4A3 (D), SYNPO2 (E) and PCDH9 (F) protein. GAPDH = loading control. For quantification, total protein was normalized with respect to GAPDH (representative example from 3 independent experiments). PAN = puromycin aminonucleoside. EV = everolimus. MeOH = methanol, solvent for EV. p = p-value. Data are means ± SD. ns = not significant.</p

TUBB2B and DCDC2 are expressed in human kidneys.

<p>(A) Western-blot analysis to measure the existence of TUBB2B and DCDC2 in human kidneys. GAPDH = loading control. CC = Podocyte cell culture. WT = Wild type kidney. (B+C) Immunohistochemical staining of TUBB2B and DCDC2 in healthy human kidneys. 3,3'-diaminobenzidine (DAB) was used as chromogen (brown staining) and nuclei were stained with hematoxylin (blue). The black arrows mark a nuclear as well as cytoplasmic staining of TUBB2B and a cytoplasmic staining of DCDC2 in podocytes. A negative control was performed without the primary antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137043#pone.0137043.s001" target="_blank">S1 Fig</a>). T = tubule. EPC = parietal epithelial cell. Scale bar = 50 μm.</p