Trehalose induced autophagy in human podocytes.

<p>(<b>A</b>) The expression of LC3-II increased in a dosage dependent manner. Conditionally immortalized human podocytes were treated with 0, 10, 50 and 100 mM of trehalose (Tre) for 48 h. LC3-II was measured by Western blotting. The data (means ± SEM) was expressed as the relative changes compared with Tre-0 mM group. Representative immunoblot images were shown along with the statistical results. *<i>p</i><0.05 versus Tre-0 mM, n = 5. (<b>B</b>) LC3-II increased in a time dependent manner. Podocytes were treated with 50 mM Trehalose for 0, 12, 24, 36, 48 and 60 h. **<i>p</i><0.01, ***<i>p</i><0.001 versus Tre-0 h, n = 7. (<b>C–D</b>) LC3-II puncta increased after trehalose treatment. LC3 immunostaining in podocytes was performed at 0, 12, 24, 36, 48 and 60 h after trehalose treatment (50 mM). Significant increased green bright puncta (indicated by white arrows) can be observed in cytoplasm after 48 h-trehalose treatment. The representative LC3 immunostaining images were shown along with statistical results from 6 independent experiments. **<i>p</i><0.01, ***<i>p</i><0.001 versus Tre-0 h. Podocyte nuclei were stained with DAPI (blue). (<b>E</b>) The expression of Atg5 was up-regulated in trehalose-treated podocytes (50 mM). Podocytes were treated with 50 mM Trehalose for 0, 12, 24, 36, 48 and 60 h. The expression of Atg5 significantly increased at the time point of 60 h. **<i>p</i><0.01 versus Tre-0 h, n = 5.</p

Trehalose decreased PAN-induced apoptosis in human podocytes.

<p>Podocyte were treated with PAN (30 µg/ml) or/and Trehalose (50 mM) for 48 h. (<b>A</b>) Trehalose induced autophagy in PAN-treated human podocytes. The expression of LC3-II slightly increased after 48 h PAN treatment, while it dramatically up-regulated in Tre and PAN+ Tre groups. Representative immunoblot images were shown along with the statistical results. **<i>p</i><0.01 versus CON, n = 6. (<b>B–C</b>) The findings of (A) were confirmed by LC3 immunostaining. Obvious elevated LC3-II bright green puncta (indicated by white arrows) were visualized in trehalose-treated groups (Tre and PAN+ Tre groups), the representative images and statistical results were shown. Nuclei were stained in blue. **<i>p</i><0.01, ***<i>p</i><0.001 versus CON, n = 6. (<b>D</b>) No significant changes were observed in podocyte necrosis. LDH in culture medium of 4 groups was measured, n = 4. (<b>E</b>) Elevated apoptosis in PAN-treated podocytes was decreased by trehalose. Apoptosis was measured by flow cytometry with YO-PRO-1/PI assay. Podocyte apoptosis was induced by PAN and decreased significantly by trehalose. *<i>p</i><0.05, **<i>p</i><0.01 versus CON, n = 8. (<b>F</b>) The findings of (E) were confirmed by the data of active caspase-3 measurement. The active caspase-3 positive podocytes were measured by flow cytometry. The changes pattern was similar to podocyte apoptosis measured by YO-PRO-1/PI assay. **<i>p</i><0.01 versus CON, n = 7.</p

Inhibition of trehalose-induced autophagy abolished its cytoprotective effects in preventing podocyte apoptosis.

<p>CQ (25 µM) or WT (0.2 µM) was used to inhibit podocyte autophagy which was induced by trehalose (50 mM) or trehalose (50 mM) + PAN (30 µg/ml) for 48 h. (<b>A–C</b>) CQ and WT inhibited trehalose-induced autophagy. The expression of LC3-II drastically increased in Tre+CQ and PAN+ Tre+CQ groups, while it decreased significantly in Tre+WT and PAN+ Tre+WT groups. p62 slightly decreased in PAN+ Tre group, whereas it significantly increased in Tre+CQ and Tre+WT groups. The immunoblot images were shown along with statistical data. *<i>p</i><0.05, **<i>p</i><0.01 versus Tre group, n = 7. (<b>D</b>) Necrosis increased after the inhibition of trehalose-induced autophagy. The LDH in culture medium was measured. *<i>p</i><0.05, **<i>p</i><0.01 versus PAN+Tre group, n = 6. (<b>E</b>) Podocyte apoptosis increased after the inhibition of trehalose-induced autophagy. The percentage of apoptotic podocytes was much higher in PAN+Tre+CQ and PAN+Tre+WT groups than the PAN+ Tre group. *<i>p</i><0.05, **<i>p</i><0.01 versus PAN+ Tre group, n = 7. (<b>F</b>) The percentage of active caspase-3 positive podocytes increased after inhibition of trehalose-induced autophagy. The changes in the percentage of active caspase-3 positive podocytes were similar to the data in (E). *<i>p</i><0.05, **<i>p</i><0.01 versus PAN+ Tre group, n = 8.</p

Trehalose-induced podocyte autophagy was independent of ROS.

<p>Trehalose-induced autophagy was not associated with energy restriction and ROS. (<b>A</b>) Trehalose-treated podocytes (50 mM) were harvested for p-AMPK measurement at the time points of 0, 12, 24, 36, 48 and 60 h. The phosphorylation level of p-AMPK did not change significantly. n = 6. (<b>B</b>) ROS level was recorded every half an hour after trehalose treatment (50 mM), the data representing immunofluorescence intensity within 2.5 h were shown (n = 6). No significant changes were noted.</p

Trehalose diminished cell motility in PAN-treated human podocytes.

<p>Podocyte were treated with PAN (30 µg/ml) or/and Trehalose (50 mM) for 12 h. (<b>A</b>) PAN-increased podocyte motility was suppressed by trehalose. The representative images were taken under inverted microscope (50×). (<b>B</b>) The number of cells migrating into the gap was calculated for evaluating podocyte motility. The migrated podocytes after PAN treatment increased significantly, whereas trehalose suppressed this enhanced motility. **<i>p</i><0.01, ***<i>p</i><0.001 versus CON, n = 6.</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 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 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

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

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