MES and HS synergistically activate PI3K-Akt signaling, induce Hsp72 expression and regulate permeability activity in podocytes.

<p>(<i>a</i>) Mouse podocytes were treated with MES for 10 min using various pulse durations as indicated. Whole cell proteins were isolated 30 min after MES treatment. Phosphorylated Akt (p-Akt) and total Akt proteins were analyzed by immunoblotting. Actin was used as loading control. (<i>b</i>) Relative amount of p-Akt in cells treated with MES (0.1 ms pulse duration) was quantified and normalized to total Akt. (<i>c</i>) Cells were treated with 10 µM LY294002 for 1 hr, and stimulated with MES for 10 min. Whole cell protein lysates were isolated after 30 min. (<i>d</i>) Whole cell proteins were isolated 5 hr after HS treatment. Hsp72 protein expression was analyzed by Western blotting. (<i>e</i>) Relative amount of Hsp72 was quantified with Image Gauge Software and normalized to HSC70. (<i>f</i>) Cells were treated with HS for 10 min. Total RNA was isolated after 1 hr and Hsp72 mRNA expression was analyzed by Q-PCR. (<i>g</i> & <i>h</i>) Cells were co-treated with MES and HS for 10 min. Thirty minutes or 5 hr after the treatment, proteins were isolated and analyzed with p-Akt and total Akt (<i>g</i>) or with Hsp72 (<i>h</i>) antibodies. (<i>i</i> & <i>j</i>) Cells were plated onto Transwell chamber and cultured for 8 days. Cells were pre-treated with LY294002 (10 µM) or quercetin (100 µM) for 1 hr and treated with MES+HS for 10 min. (<i>i</i>) After treatment, 40 mg/ml BSA in RPMI-1640 solution was added to the bottom chamber. Permeability activity of podocytes was analyzed by measuring the protein concentration in the upper chamber 5 hr after adding the BSA solution. (<i>j</i>) Thirty minutes or 5 hr after the treatment, proteins were isolated and immunoblotted respectively with p-Akt and total Akt (<i>left</i>) or with Hsp72 (<i>right</i>) antibodies. For (<i>b</i>, <i>e</i>, <i>f</i> & <i>i</i>), values are the mean ± S.E. (n = 3). *<i>P</i><0.05, *** <i>P</i><0.001 assessed by unpaired <i>t</i>-test. n.s.; not significant.