Nuclear translocation of IQGAP1 protein upon exposure to puromycin aminonucleoside in cultured human podocytes: ERK pathway involvement

IQGAP1, a protein that links the actin cytoskeleton to slit diaphragm proteins, is involved in podocyte motility and permeability. Its regulation in glomerular disease is not known. We have exposed human podocytes to puromycin aminonucleoside (PAN), an inducer of nephrotic syndrome in rats, and studied the effects on IQGAP1 biology and function. In human podocytes exposed to PAN, a nuclear translocation of IQGAP1 was observed by immunocytolocalization and confirmed by Western blot after selective nuclear/cytoplasmic extraction. In contrast to IQGAP1, IQGAP2 expression remained cytoplasmic. IQGAP1 nuclear translocation was associated with a significant decrease in its interaction with nephrin and podocalyxin. Activation of the ERK pathway was observed in PAN treated podocytes with a preponderant nuclear localization of the phosphorylated form of ERK (P-ERK). The interaction between IQGAP1 and P-ERK increased upon podocyte exposure to PAN. Inhibitors of ERK pathway activation blocked IQGAP1 nuclear translocation (p<0.02). Chromatin interaction protein assays demonstrated an interaction of IQGAP1 with chromatin and with Histone H3, which increased in response to PAN. In summary, PAN induces the ERK dependent translocation of IQGAP1 into the nuclei in human podocytes which leads to the interaction of IQGAP1 with chromatin and Histone H3, and decreased interactions between IQGAP1 and slit-diaphragm proteins. Therefore, IQGAP1 may have a role in podocyte gene regulation in glomerular disease

Protective effects of the mTOR inhibitor everolimus on cytoskeletal injury in human podocytes are mediated by RhoA signaling

Podocytes are highly differentiated kidney cells playing an important role in maintaining the glomerular filtration barrier. Particularly, the integrity of the actin cytoskeleton is crucial as cytoskeletal damage associated with foot process effacement and loss of slit diaphragms constitutes a major aspect of proteinuria. Previously, the mammalian target of rapamycin (mTOR) was linked to actin regulation and aberrant activity of the kinase was associated with renal disease. In this study, actin-related effects of mTOR inhibition by the immunosuppressant everolimus (EV) were investigated in human podocytes using an in vitro model of puromycin aminonucleoside (PAN) induced proteinuria. EV substantially recovered aberrant podocyte behavior by re-establishing a stationary phenotype with decreased migration efficiency, enhanced cell adhesion and recovery of actin stress fibers. Biochemical studies revealed substantial increase in the activity of RhoA and the effector pathway Rho-associated protein kinase (ROCK) and myosin light chain (MLC) by EV, all known regulators of stress fiber generation. Taken together, we show for the first time cytoskeleton stabilizing effects of the mTOR inhibitor EV and establish RhoA signaling as a key mediator in this process

The cytoprotective role of autophagy in puromycin aminonucleoside treated human podocytes

Autophagy is a ubiquitous catabolic process involving degradation of damaged organelles and protein aggregates. It shows cytoprotective effects in many cell types and helps to maintain cell homeostasis. In many glomerular diseases, podocyte damage leads to the disruption of the renal filtration barrier and subsequent proteinuria. Puromycin aminonucleoside (PAN) which induces podocyte apoptosis in vitro and in vivo is widely used for studying the pathophysiology of glomerular diseases. It has been shown that PAN induces autophagy in podocytes. However, the relationship between autophagy and apoptosis in PAN treated human podocytes is not known and the role of PAN-induced autophagy in podocyte survival remains unclear. Here we demonstrate that PAN induced autophagy in human podocytes prior to apoptosis which was featured with the activation of mTOR complex 1 (mTORC1). When the PAN-induced autophagy was inhibited by 3-methyladenine (3-MA) or chloroquine (CQ), podocyte apoptosis increased significantly along with the elevation of active caspase-3. Under such circumstance, the podocyte cytoskeleton was also disrupted. Collectively, our results suggested that the induced autophagy may be an early adaptive cytoprotective mechanism for podocyte survival after PAN treatment.Department of Health Technology and Informatic

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

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 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

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

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

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