Upregulation of NADH/NADPH oxidase 4 by angiotensin II induces podocyte apoptosis

Figures

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  Figure 1.. Ang II-induced generation of superoxide anions in cultured podocytes. Ang II significantly increases the generation of superoxide anions from 6 hours (A: n = 3, *p < 0.05 and **p < 0.01) and significantly suppresses SOD activity after 12 hours (B: n = 3, *p < 0.05 and #p < 0.05) in a time-dependent manner. In contrast, probucol improves Ang II-induced superoxide production and suppresses SOD activity (A, B: n = 3, #p < 0.05). Ang, angiotensin; SOD, superoxide dismutase.

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  Figure 2.. Ang II-induced generation of mitochondrial oxidative stress in cultured podocytes. (A) Ang II increases the intracellular ROS levels in both dose- and time-dependent manners (n = 3); (B) these levels also improve with probucol application (n = 3). Magnification, ×200. MitoSOX Red and MitoTracker Green fluorescence (Invitrogen) is localized in cytoplasm (C), and 10–6 M of Ang II increases the MitoSOX index of MitoSOX intensity per MitoTracker Green-positive area, which is shown as scatter/whisker-box plots (D: n = 6). Magnification, ×720; bar = 20 μm. The 8-oxo-dG fluorescence is also localized in cytoplasm (E) and increased by 10–6 M of Ang II, shown as scatter/whisker-box plots (F: n = 6). Nuclei colored blue by DAPI. Magnification, ×720; bar = 20 μm. *p < 0.05 and **p < 0.01 vs. control and #p < 0.05, and ##p < 0.01 vs. each Ang II-treated condition. Control (100%); compared to conditions without Ang II. All data are presented as mean ± standard deviation. Ang, angiotensin; DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; ROS, reactive oxygen species.

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  Figure 3.. Ang II-induced Nox4 expression. (A) Nox4 (green) and ZO-1 (red) co-localized in cytoplasm, and 10–6 M of Ang II concentrates both and increases the intensity of Nox4. Nuclei colored blue by DAPI. Magnification, ×400; bar = 10 μm. Ang II significantly increases Nox4 protein level at 24 hours of incubation in western blotting analysis (B: n = 4) and Nox4 expression in a time-dependent manner on quantitative polymerase chain reaction analysis (C: n = 4) in Ang II-treated podocytes. (B) Probucol reduces the Nox4 protein level significantly in control and Ang II-treated podocytes. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01 vs. control and ##p < 0.01 vs. Ang II-treated condition. Control (100%); compared to conditions without Ang II. Ang, angiotensin; DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride; Nox4, NADH/NADPH oxidase 4; ZO-1, zonula occludens-1.

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  Figure 4.. Nox4 inhibition by siRNA mitigates Ang II-induced oxidative stress. (A) Nox4 inhibition by siRNA reduces Nox4 protein as expected, and 50 nM of Nox4 siRNA is a sufficient transfection dose. Nox4 siRNA significantly reduces Nox4 protein level (B: n = 3) and the oxidative stress (C: n = 4) increased by Ang II. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01 vs. control and #p < 0.05, and ##p < 0.01 vs. each Ang II-treated condition. Control (100%); compared to conditions without Ang II. Magnification, ×200. Ang, angiotensin; Nox4, NADH/NADPH oxidase 4; siRNA, small interfering RNA.

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  Figure 5.. Probucol and Nox4 siRNA reduce Ang II-induced podocyte apoptosis. Ang II induces podocyte apoptosis as determined using fluorescence-activated cell sorting assay, and that is improved with probucol (A) and Nox4 siRNA but not by scrambled siRNA (B). Data are expressed as mean ± standard deviation (n = 3). **p < 0.01 vs. control and #p < 0.05 vs. Ang II-treated condition. Control (100%); compared to conditions without Ang II. Ang, angiotensin; Nox4, NADH/NADPH oxidase 4; siRNA, small interfering RNA.

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  Figure 6.. AT1R siRNA inhibits Ang II-induced oxidative stress and subsequent podocyte apoptosis. (A) AT1R inhibition by siRNA reduces AT1R protein, and 400 nM of AT1R siRNA is a sufficient transfection dose. (B) AT1R siRNA significantly reduces AT1R protein upregulated by Ang II (n = 3). Ang II-induced oxidative stress and podocyte apoptosis are improved by AT1R siRNA (C and D, respectively; n = 3). Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01 vs. control and #p < 0.05, and ##p < 0.01 vs. each Ang II-treated condition. Control (100%); compared to conditions without Ang II. Magnification, ×200. Ang, angiotensin; AT1R, angiotensin II type 1 receptor; siRNA, small interfering RNA.

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  Figure 7.. Schematic view of oxidative stress and apoptosis induced by Ang II in a podocyte. Ang II induces podocyte oxidative stress and subsequent apoptosis with AT1R and via upregulation of Nox4. Ang, angiotensin; AT1R, angiotensin II type 1 receptor; Nox4, NADH/NADPH oxidase 4.

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