The Na/K-ATPase Oxidant Amplification Loop Regulates Aging

Abstract

As aging involves oxidant injury, we examined the role of the recently described Na/K-ATPase oxidant amplification loop (NKAL). First, C57Bl6 old mice were given a western diet to stimulate oxidant injury or pNaKtide to antagonize the NKAL. The western diet accelerated functional and morphological evidence for aging whereas pNaKtide attenuated these changes. Next, human dermal fibroblasts (HDFs) were exposed to different types of oxidant stress in vitro each of which increased expression of senescence markers, cell-injury, and apoptosis as well as stimulated the NKAL. Further stimulation of the NKAL with ouabain augmented cellular senescence whereas treatment with pNaKtide attenuated it. Although N-Acetyl Cysteine and Vitamin E also ameliorated overall oxidant stress to a similar degree as pNaKtide, the pNaKtide produced protection against senescence that was substantially greater than that seen with either antioxidant. In particular, pNaKtide appeared to specifically ameliorate nuclear oxidant stress to a greater degree. These data demonstrate that the NKAL is intimately involved in the aging process and may serve as a target for anti-aging interventions.

Figure 1

Effects of pNaKtide on adipocyte phenotype, senescence, and apoptosis in C57Bl6 aging mice. (A) H&E staining in visceral adipose tissue. Images taken with 40X objective lens; scale bar represents 100 µm. Arrows mark “crown like structures” indicative of inflammation. (B) Quantitative analysis of adipocytes area in visceral adipose tissue. (C) Representative images of TUNEL assay with quantification in C57B16 aging mice. Images taken with 40X objective lens; scale bar represents 25 µm. (D–F) qRT-PCR analysis of ApoJ, p21 and PPARγ in C57Bl6 aging mice with GAPDH as a loading control. Y, young; Y + P, young + pNaKtide; OB, old baseline; O, old; O + P, old + pNaKtide; O + WD, old + western diet; O + WD + P, old + western diet + pNaKtide. N = 8/group, ^*p < 0.05, ^**p < 0.01 vs Y, ^#p < 0.05, ^##p < 0.01 vs O, ^&p < 0.05, ^&&p < 0.01 vs O + WD.

Figure 2

Effect of pNaKtide on fibrosis, senescence markers, and apoptosis in heart tissue of C57Bl6 aging mice. (A and B) Sirius red staining for detection of cardiac fibrosis with quantitative analysis. Images taken with 20X objective lens; scale bar represents 100 µm. (C–E) qRT-PCR analysis of Fibronectin, p21 and ApoJ in C57B16 aging mice. GAPDH was used as a loading control. (F,G) Representative images of TUNEL assay with quantification in C57B16 aging mice. Images taken with 40X objective lens; scale bar represents 25 µm. N = 8/group, ^*p < 0.05, ^**p < 0.01 vs Y, ^#p < 0.05, ^##p < 0.01 vs O, ^&p < 0.05, ^&&p < 0.01 vs O + WD.

Figure 3

Effects of pNaKtide on H₂O₂-induced senescence markers, apoptosis, and Na/K-ATPase signaling in human dermal fibroblasts (HDFs). (A) Qualitative and quantitative analysis of ß-galactosidase levels. (B) Representative images and quantification of γ-H2AX levels. Images taken with 40X objective lens; scale bar represents 100 µm. (C) Representative images and quantification of the TUNEL assay (D) Quantitative analysis of Caspase-9 activity in cell lysates. (E) pATM, (F) pCHK2, and (G) p53 immunoblot analysis. Data shown are the mean band density normalized to ATM, CHK2, and actin, respectively. (H) Protein carbonylation levels with Coomassie staining as a loading control. (I) pSrc immunoblot analysis with data shown as mean band density normalized to Src. Data are displayed as “scatter plots” showing data points and “box plots” showing the distribution of a continuous variable as described in the Methods section. All gels were cropped above and below the band. N = 6/group, *p < 0.05, **p < 0.01 vs. control, ^#p < 0.05, ^##p < 0.01 vs. H₂O₂.

Figure 4

Effect of anti-oxidants on H₂O₂ induced cellular senescence in HDFs. (A) Representative images of HDFs exposed to H₂O₂ and treated with varying concentrations of NAC and Vitamin E respectively. Images taken with 20X objective lens; scale bar represents 100 µm. (B) Quantitative analysis of TBARS. (C) Representative images and quantification of γ-H2AX levels. Images taken with 40X objective lens; scale bar represents 100 µm. qRT-PCR analysis of (D) p21 and (E) MMP9 in HDFs exposed to H₂O₂. GAPDH was used as a loading control. N = 6/group. ^**p < 0.05 vs. CTR, ^##p < 0.01 vs. H₂O₂, ^&&p < 0.01 vs. H₂O₂ + pNaKtide.

Figure 5

Effects of glucose oxidase (GO) and pNaKtide on oxidative stress, cell proliferation, and apoptosis in HDFs. Concentrations of GO ranging from 0 to 10 mU/ml were administered to HDFs. (A) Quantitative analysis of TBARS (B) Quantitative analysis of the MTT assay (C) Cell count by CyQUANT assay (D) Caspase-9 activity assay. N = 16/group, *p < 0.05, **p < 0.01 vs. corresponding GO concentration groups without pNaKtide.

Figure 6

Effects of pNaKtide and GO on protein carbonylation in HDFs. (A) Representative immunofluorescence images of carbonylated protein. HDFs exposed to various concentrations of GO (1 mU, 3 mU, 5 mU, and 10 mU) with or without pNaKtide treatment. DNP fluorescence intensity was visualized using an EVOS portable microscope with an excitation/emission wavelength of 470/525 nm. (B) Quantification was done by Image J software based on the mean optical density transmitted under fluorescent microscopy. Pictures were taken at 40X with a linear scale bar representation of 50 µm. N = 8/group, **p < 0.01 vs. corresponding GO concentration groups without pNaKtide. (C) Representative immunoblot analysis with Coomassie staining as a loading control and the quantification of protein carbonylation levels in HDFs treated with 10 mU/ml GO and pNaKtide. N = 8/group, **p < 0.01 vs. control, ^#p < 0.01 vs. GO. (D) Confocal images of protein carbonylation and γ-H2AX phosphorylation showing co-occurence of these markers within the nucleus. Nuclei were identified using DAPI staining. DNP and γ-H2A.X double immuno-fluorescence staining were visualized with a Leica SP5 TCSII with Coherent Chameleon multiphoton (MP) Vision II (IR) laser confocal microscope. Pictures were taken with the 100 × objective lens under oil immersion; scale bar representation of 5 µm. N = 8/group. (E) Quantification of DNP intensity and γ-H2AX foci shown as a scatterplot with linear regression demonstrating a strong correlation (R² = 0.847). Gray band around regression line represent 95% confidence interval.