Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury

Abstract

Background: Two important issues in the aging kidney are mitochondrial dysfunction and oxidative stress. An Nrf2 activator, resveratrol, is known to have various effects. Resveratrol may prevent inflammation and oxidative stress by activating Nrf2 and SIRT1 signaling. We examined whether resveratrol could potentially ameliorate the cellular condition, such as renal injury due to cellular oxidative stress and mitochondrial dysfunction caused by aging.

Methods: Male 18-month-old C57BL/6 mice were used. Resveratrol (40 mg/kg) was administered to aged mice for 6 months. We compared histological changes, oxidative stress, and aging-related protein expression in the kidney between the resveratrol-treated group (RSV) and the control group (cont). We performed experiments using small-interfering RNAs (siRNAs) for Nrf2 and SIRT1 in cultured HK2 cells.

Results: Resveratrol improved renal function, proteinuria, histological changes and inflammation in aging mice. Also, expression of Nrf2-HO-1-NOQ-1 signaling and SIRT1-AMPK-PGC-1α signaling was increased in the RSV group. Transfection with Nrf2 and SIRT1 siRNA prevented resveratrol-induced anti-oxidative effect in HK2 cells in media treated with H₂O₂.

Conclusions: Activation of the Nrf2 and SIRT1 signaling pathways ameliorated oxidative stress and mitochondrial dysfunction. Pharmacological targeting of Nrf2 signaling molecules may reduce the pathologic changes of aging in the kidney.

Keywords: SIRT1-Nrf2; aging; kidney; oxidative stress; resveratrol.

Figure 1

Effects of resveratrol on aging-related histological renal injury. Less expansion of the mesangial area (A; periodic acid–Schiff (PAS), original magnification ×400) and significantly less tubulointerstitial fibrosis (B; Masson’s trichrome, original magnification ×200) were found in the RSV group compared to that in the control (Cont) group. Quantitative assessment of the areas of extracellular matrix in the glomerulus (C) and tubulointerstitial fibrosis (D) in the control and RSV groups, respectively. (*P<0.001).

Figure 2

Effects of resveratrol on the renal phenotypes of collagen IV (Col IV), TGF-β1 and F4/80. Immunohistochemical staining with Col IV, TGF-β1, and F4/80 in RSV and control (Cont) groups. Representative images of Col IV and TGF-β1 in aging kidney glomerulus (A, B; original magnification ×400) are shown. In addition, the F4/80-positive cells in glomeruli and F4/80-positive areas in the tubules (C, D; original magnification ×400) are shown. Expression of Col IV and TGF-β1 was decreased in the RSV group (E, F). F4/80-positive cells in glomeruli and positive areas in the tubules were observed as significantly smaller in the RSV group (G, H) (*P<0.001).

Figure 3

Effects of resveratrol on the expression of BCL2 and BAX proteins. Representative western blot analysis of BCL-2 and BAX expression (A). BCL-2 protein levels were increased in the RSV group, while BAX protein levels were not different between the two groups (B). Quantitative analysis of the results is shown (*P<0.05).

Figure 4

Effects of resveratrol on the expression of Nrf2-related proteins. Representative western blot analysis of Nrf2 expression in total and nuclear proteins, and Keap1 expression in total protein (A). The results showed that the total and nuclear Nrf2 protein levels were decreased in the RSV group compared to that in the control (Cont) group (B, C). There were no differences in Keap1 protein expression between both groups (D). Quantitative analysis of the results is shown (*P<0.05, †P<0.01).

Figure 5

Effects of resveratrol on the HO-1 and NQO-1 protein expressions. Representative western blots of HO-1 and NQO-1 protein levels. (A) The protein levels of HO-1 and NQO-1 were higher in the RSV group than in the control (Cont) group (B, C). Quantitative analysis of the results is shown (*P<0.05, †P<0.01).

Figure 6

Effects of resveratrol on the expression of SIRT1-related proteins. Representative western blots of SIRT1, phospho-Thr¹⁷² AMPK, PPARα, PGC-1α, and ERR-1α protein levels (A). Compared with the control (Cont) group, expression of SIRT1 was significantly increased in the RSV group (B). The phospho-Thr¹⁷² AMPK/total AMPK ratio was also increased in the RSV group (C). PPARα, PGC-1α and ERR-1α protein levels were higher in RSV than in Cont (D-F). Quantitative analysis of the results is shown (*P<0.05, †P<0.01).

Figure 7

Effects of resveratrol on the expression of SOD1 and SOD2. Representative western blots of SOD1 and SOD2 protein levels. (A). Expression of SOD1 and SOD2 was significantly increased in the RSV group compared to that in the control (Cont) group (B, C). Quantitative analysis of the results is shown (*P<0.05).

Figure 8

Effects of resveratrol on the expression of cytochrome c oxidases. Representative western blots of cytochrome c oxidase I and IV protein levels (A). The cytochrome c oxidase I/cytochrome c oxidase IV expression ratio was increased in the RSV group compared to that in the control (Cont) group (B). Quantitative analysis of the results is shown (*P<0.05).

Figure 9

Effects of resveratrol on renal oxidative stress. The 24-h urinary 8-epi-prostaglandin F2α (isoprostane) levels were decreased in the RSV group (A). Moreover, the 24-h concentration of urinary 8-hydroxy-deoxyguanosine was also decreased in the RSV group compared to that in the control (Cont) group (B). Significantly, the positive area expression of 8-OH-dG in renal tissue was decreased in the RSV group (C, D) (*P<0.05, †P<0.01, ‡P<0.0001).

Figure 10

Effects of resveratrol in HK2 cells. Representative western blots of Nrf2, HO-1 and NQO-1 protein levels (A) and SIRT1, SOD1 and SOD2 protein levels (E). The Nrf2 was increased in the RSV group compared to H₂O₂ group (B). HO-1 as well as NQO-1 were significantly increased in the RSV group (C, D). Also, the expression of SIRT1 was significantly increased in the RSV group (F). Both SOD1 and SOD2 were higher in the RSV group than the H₂O₂ group (G, H). Quantitative analysis of the results is shown (*P<0.05, †P<0.001).

Figure 11

Small interfering RNA in HK2 cells. Representative western blots of Nrf2 and SIRT1 protein levels by transfected siRNA for Nrf2 and SIRT1 (A). Protein expression of Nrf2 and SIRT1 by transfection with siNrf2 and siSIRT1 were suppressed than the control group (B, C). Representative western blots of Nrf2, HO-1 and NQO-1 protein levels (D) and SIRT1, SOD1 and SOD2 protein levels by transfection with Nrf2 and SIRT1 (E). In groups with siNrf2 and siSIRT1 treated with resveratrol in exposure to H₂O₂ media, Nrf2, HO-1 and NQO-1 protein expressions were lower compared to the resveratrol group exposed H₂O₂ media (F-H). Similarly, the resveratrol-induced increase in protein levels of SIRT1, SOD1 and SOD2 were inhibited by siNrf2 and siSIRT1 (J-K). Quantitative analysis of the results is shown (*P<0.05, †P<0.01, ‡P<0.0001).