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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Prostaglandins Other Lipid Mediat. 2014 Nov 4;0:88–98. doi: 10.1016/j.prostaglandins.2014.10.006

FIGURE 3. Ethanol increases superoxide generation in podocytes.

FIGURE 3

A. Immortalized podocytes were incubated with ethanol 0.2 or 0.4 mmol/mL. Dihydroethidium (15 μM) was used to detect superoxide. Significant increase in fluorescence due to ethanol (0.2 or 0.4 mmol/mL) was detectable at 1, 4 and 24 hr compared to control. Fluorescence caused by 0.4 mmol/mL was significantly greater than that by 0.2 mmol/mL ethanol only at 1 hr (P<0.05, n=20 cells in two separate experiments). Ethanol at 0.2 and 0.4 mmol/mL caused significant increase in superoxide production at 1, 4 and 24 hr compared to the their respective control groups (*, P<0.05)

B. Pre-treatment of cells with 100 nM 20-HETE blocked the effect of 0.2 mmol/mL ethanol on superoxide production at 4 hr (P<0.001 vs. ethanol treated group).

C. Pre-treatment of cells with 100 nM 20-HETE blocked the effect of 0.4 mmol/mL ethanol on superoxide production at 4 hr (P<0.001 vs. ethanol treated group).

D. Total RNA was prepared from untreated control and ethanol-treated podocytes (0.2 or 0.4 mmol/mL, 4 hr) and analyzed using RT-qPCR. CYP2e1 gene expression was significantly increased in podocytes treated with 0.4 mmol/mL (P<0.001 vs control). (n=4/group, each value represents average of 3 determinations).