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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: Kidney Int. 2017 Jan 4;91(5):1126–1145. doi: 10.1016/j.kint.2016.10.036

Figure 2. Expression of the reactive oxygen species-generating enzyme, Nox4, is reduced in kidneys of mice with advanced age treated with SS-31.

Figure 2

(A–G) The increase in Nox4 expression in aged kidneys is reduced by SS-31. Nox4 staining is detected in the nucleus and cytoplasm of glomeruli and tubules throughout the kidney but is most readily detectable in the outer cortex. (A–F) Representative immunohistochemistry images of Nox4 staining with a primary antibody (from Abcam, brown) counterstained with hematoxylin (blue, nuclear) in the outer cortex and juxta-medulla in 24m-old aged baseline mice (A, D), 26m-old mice given 8 weeks of saline (B, E), and SS-31-treated 26m-old mouse, which shows reduced cytoplasmic stain relative to other groups. (C, F). Scale bar = 25 μm. (B’, C’) Insets from B and C. Examples of images used to quantify relative levels of cytoplasmic Nox4 expression in the outer cortex by measuring areas of dark brown staining containing color threshold (solid dark red) selection using imageJ software. (G) Quantification of Nox4 staining in the outer cortex shows a significant decrease with SS-31 treatment. 26m-old mice given 8w of SS-31 had less cortical Nox4 staining when compared to aged-matched saline treated mice and baseline 24m-old mice. Error bars show the mean ± standard deviation. (H) Negative control: Omitting the primary Nox4 antibody in 26m-old Saline treated animals showed no dark brown staining.

(I–K) Nox4 expression is reduced in parietal epithelial cells of aged mice treated with SS-31. A second primary antibody (Novus) to Nox4 confirmed the staining pattern in D-I in that Nox4 positive cells (brown) are seen in all treatment groups, including parietal epithelial cells (PECs), podocytes and mesangial cells, based on location within the tuft. (I, J) PECs positive for Nox4 are seen in all mice (brown stain, thin arrows) but this expression is reduced in SS-31 treated animals (blue stain, wide arrow heads). Scale bar = 10μm.

(K) Quantification of Nox4 positive cells shows that SS-31 significantly reduces Nox4 staining of PECs in 26m-old mice in the outer cortex. These results show that Nox4 expression is reduced both in the cytoplasm and nucleus by SS-31 treatment.