Resveratrol Improves Oxidative Stress and Protects Against Diabetic Nephropathy Through Normalization of Mn-SOD Dysfunction in AMPK/SIRT1-Independent Pathway Open Access

Details of the materials and antibodies used in the current study are available in the Supplementary Data.

Male db/db mice and age-matched db/m mice were purchased from Clea Japan (Tokyo, Japan). At 9 weeks of age, mice were divided into four groups: db/m mice, db/db mice, db/m mice treated with RSV, and db/db mice treated with RSV. RSV was mixed (0.3%) with chow and administered orally. Body weight, blood glucose level, food consumption, and blood pressure were measured every 2 weeks in all animals. The blood pressure of conscious mice was measured at steady state by a programmable tail-cuff sphygmomanometer (BP98-A; Softron, Tokyo, Japan). At 17 weeks of age, individual mice were placed in metabolic cages for 24-h urine collection. The urine samples were stored at −80°C until analysis. Mice were anesthetized by intraperitoneal injection of pentobarbital sodium, and then the right kidneys were removed and stored at –80°C for experiments as described below. After collection of blood samples from the left cardiac ventricle, the left kidney was perfused with ice-cold phosphate-buffered saline (PBS) and 10% neutral buffered formalin and then removed. The Research Center for Animal Life Science of Kanazawa Medical University approved all experiments.

To assess the mesangial expansion, thirty glomeruli, cut at the vascular pole, randomly selected from each mouse were measured the periodic acid/Schiff (PAS)-positive material in the mesangial area and glomerular tuft area by computer-assisted color image analysis (Micro Analyzer; Japan Poladigital, Tokyo, Japan) as previously described (21).

For semiquantitative evaluation of the fibronectin, F4/80 and nitrotyrosine scores, 20 randomly selected glomerulus or tubulointerstitial areas per mouse were graded in a double-blind manner, as reported previously (21–23), with minor modifications.

The mitochondrial DNA (mtDNA) was extracted from the kidney using the mtDNA Extractor CT kit. The 8-OHdG levels in DNase I-digested mtDNA were determined by ELISA using a kit (8-OHdG Check, Institute for the Control of Aging, Shizuoka, Japan) (24). We determined. the deletion mutation, D-17, as reported previously (23). The sequences of primers are listed in Supplementary Table 1.

The mitochondria were isolated from the renal cortex of all groups of mice using the mitochondria isolation kit, according to the manufacturer’s instructions. O²⁻ production from the mitochondria was measured by L-012 chemiluminescence (CL) dye (25). Mitochondrial suspensions were diluted to a final protein concentration of 0.1 mg/mL in 0.2 mL of PBS buffer containing 100 μmol/L L-012. O²⁻ from mitochondria was detected after stimulation with 4 mmol/L succinate and 20 μg/mL antimycin A by L-012. The CL registered at intervals of 30 s over 5 min with a chemiluminometer, and the signal was expressed as counts of CL/min/100 μg protein at 5 min. In ex vivo study, similar experiments were performed in isolated mitochondria from the renal cortex of db/db mice at various concentrations (10⁻³–100 μmol/L) of RSV or 100 U/mL SOD. In addition, O²⁻ from the reaction of hypoxanthine and xanthine oxidase was also measured. L-012 (100 μM) was incubated for 5 min in PBS buffer containing 100 μmol/L diethylenetriamine pentaacetic acid (DTPA) and 1 mmol/L hypoxanthine at room temperature, and then the basal (background) signal was determined in a chemiluminometer at intervals of 30 s for 5 min. The CL was counted after the addition of 10 mU/mL xanthine oxidase at various concentrations of RSV (10⁻³–100 μmol/L) or 100 U/mL SOD at intervals of 30 s over 5 min.

The whole kidney was homogenized in 2 mL of 50 mmol/L Tris HCl buffer containing 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA) at pH 7.0. After centrifugation at 15,000g for 15 min, the supernatant was removed and total protein concentration was measured using a protein assay kit. The Mn-SOD activity was measured by inhibiting extracellular and cytosolic Cu/Zn SOD activity with KCN (1 mmol/L) using an SOD assay kit (WST-1) (26). One unit of SOD activity was defined as the amount causing 50% inhibition of the initial rate of reduction of WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt), a highly water-soluble tetrazolium salt. Mn-SOD activity was calculated in terms of protein content (U/μg) and expressed as a fold increase relative to that found in db/m mice.

The whole kidney was homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer. Solubilized protein (1 mg) was used for immunoprecipitation with rabbit polyclonal anti–Mn-SOD antibody (10 μg/mL) using protein A-Sepharose, and then Western blot analysis was performed with mouse monoclonal antinitrotyrosine antibody (1:1000) or rabbit polyclonal anti–Mn-SOD antibody (1:1000). Samples of protein solutions from the kidney were used for Western blotting with antiphospho-AMPKα (Thr172) antibody, anti-AMPKα (23A3) antibody (1:1000), or anti-SIRT1 antibody (1:1000).

Isolation of total RNA from kidney, and determination of complementary DNA synthesis by reverse transcription and quantitative real-time PCR were performed as described previously (23). PCR primer sets are listed (Supplementary Table 1).

Murine proximal tubular cells (mProx) (derivative, patent WO9927363, Japan, U.S., European Union), kindly provided by CMIC Co., were cultured as described previously (27).

The pSUPERretro and pSUPERretro-SIRT1 RNA interference vectors were kind gifts from Dr. L. Guarente (Massachusetts Institute of Technology, Cambridge, MA). Human embryonic kidney 293T cells were transfected with pSUPERretro or pSUPERretro-SIRT1 RNAi by using Lipofectamine reagent. At 48 h after transfection, the media containing the retroviruses were collected, centrifuged, and transferred to mProx treated by polybrene (1 g/mL). The infected cells were selected by treatment with puromycin (2.5 μg/mL) for several days as described previously (28).

Adenoviruses containing green fluorescent protein (Ad-GFP) or dominant-negative AMPK (DN-AMPK) were added to subconfluent mProx at a concentration of 50 multiplicity of infection for 1 h at 37°C in serum-free Dulbecco’s modified Eagle’s medium (29).

To determine the effect of RSV on oxidative stress in mProx, cells were incubated with RSV (10 μmol/L) for 180 min at 37°C before exposure to H₂O₂ (10 μmol/L). After incubation, the levels of intracellular ROS were measured with the fluoroprobe 2.7-dichlorodihydrofluorescein diacetate. The intracellular ROS was evaluated as the fluorescence intensity (an excitation wavelength 488 nm, an emission wavelength 525 nm) of dichlorofluorescein by Infinite M200 microplate reader (Tecan Japan Co., Kanagawa, Japan). The results of intracellular ROS are expressed in arbitrary units.

Well-conserved nuclear and other mitochondrial genes were selected to quantify mtDNA copy number per nuclear genome. Cytochrome c oxidase subunit 2 was used as a marker for mtDNA and uncoupling protein 2 for nuclear DNA. Renal DNA was extracted from frozen kidney tissues of all animals using the DNeasy tissue kit. Total DNA concentration was determined using a PicoGreen DNA quantitation kit. Specific mouse primer sequences are provided in the Supplementary Table 1. mtDNA per nuclear genome was calculated as the ratio of cytochrome c oxidase subunit 2 DNA to uncoupling protein 2 DNA (30) and expressed as the fold increase relative to that found in db/m mice.

Citrate synthase activity in the kidney was determined using the citrate synthase activity measurement kit, according to the manufacturer’s instructions.

The mitochondria in proximal tubular cells were observed by electron microscopy as previously described (23). Mitochondrial area and number were estimated in 15–18 micrographs, which were taken for renal proximal tubular cells of three animals of each group (20). The mitochondrial area was measured using the ImageJ software, and the number of mitochondria per cell was counted manually by a blind observer.

Data are expressed as means ± SD. The Tukey multiple-comparison test was used to determine the significance of pairwise differences among three or more groups. P < 0.05 was considered significant.

Table 1 details the characteristics of four groups of mice at the end of the experimental period. The whole body and right kidney weights were significantly higher in db/db mice compared with db/m mice. The mean blood pressure (MBP) was significantly higher in db/db mice than in db/m mice. RSV did not affect changes in MBP. db/db mice exhibited markedly elevated blood glucose levels compared with db/m mice throughout the entire experiment. In db/db mice, treatment with RSV induced a partial improvement in blood glucose levels and glycated hemoglobin by the end of the experiments. Serum lipid profiles including total cholesterol, triglyceride, and free fatty acid levels were also significantly elevated in db/db mice compared with db/m mice; the increases in triglycerides and free fatty acids were partially rescued by RSV (Table 1). In addition, an impairment of glucose and insulin tolerance was evident in db/db mice relative to db/m mice (Supplementary Fig. 1), and high levels of fasting glucose and insulin (Table 1) indicating insulin resistance were also observed in db/db mice. Such alteration of insulin resistance in db/db mice was partially improved by treatment with RSV. There were no differences in body weight and food consumption between untreated and RSV-treated db/db mice. The 24-h urine volume was significantly larger in db/db mice compared with db/m mice, and this was partially reduced by treatment with RSV (data not shown).

To evaluate the effects of RSV on functional abnormalities in db/db mice, we measured the urinary albumin excretion. Values were markedly higher in db/db mice and RSV treatment significantly reduced urinary albumin excretion (Table 1), indicating that RSV ameliorates the functional abnormality of diabetic nephropathy in db/db mice.

Figure 1A a–d shows representative photomicrographs of mesangial matrix accumulation in the PAS-stained kidneys of the four groups. The mesangial matrix was more extensive in the glomeruli of db/db mice than in db/m mice, and treatment with RSV reduced such expansion. Figure 1B shows the results of quantitative analysis of mesangial matrix expansion in all groups. Although the ratio of mesangial matrix/glomerular area was markedly larger in db/db mice than in db/m mice, treatment with RSV significantly reduced this expansion.

Immunohistochemistry for fibronectin (Fig. 1A e–h and i–l) also showed a significantly higher score for renal glomerular and tubulointerstitial expression in db/db mice than in db/m mice (Fig. 1C and D). Treatment with RSV reduced the score for fibronectin in db/db mice but had no effect on db/m (Fig. 1C and D).

The number of cells positive for F4/80 (a macrophage marker) in the renal interstitial lesion was significantly higher in db/db mice than in db/m mice (Fig. 1A m–p), but this pattern was not found in the glomeruli (data not shown). Treatment with RSV reduced the number of F4/80-positive cells in the renal interstitial lesions of db/db mice (Fig. 1E). These results indicate that RSV might improve glomerular and interstitial histological abnormalities including mesangial expansion, fibronectin accumulation and increased interstitial macrophage infiltration in the kidney of db/db mice.

Urinary 8-OHdG excretion was markedly higher in db/db mice compared with db/m mice but diminished after treatment with RSV (Fig. 2A). The 8-OHdG content (Fig. 2B) and subsequent deletion mutation (Fig. 2C) in the mtDNA isolated from the kidneys of db/db mice were significantly higher than in db/m mice. These mitochondrial alterations in mtDNA were not observed in the kidney of db/db mice treated with RSV. In addition, O²⁻ production from the isolated renal mitochondria after stimulation with succinate and antimycin A was significantly increased in db/db mice, but treatment with RSV restored it to normal in db/db mice. In ex vivo experiments, the addition of RSV exogenously attenuated the levels of O²⁻ from the renal mitochondria of db/db mice in a dose-dependent manner (Fig. 2D). Moreover, RSV could also scavenge O²⁻ production from reaction of hypoxanthine and xanthine oxidase in a dose-dependent manner. These results suggest that the enhancement of both systemic oxidative damage and renal mitochondrial oxidative damage was observed in db/db mice and that RSV has antioxidative activity.

Figure 3A a–h show representative immunohistochemical staining for nitrotyrosine in the kidney. The semiquantitative scores for nitrotyrosine in both the glomerular and the tubulointerstitial lesions of the renal cortex were increased in db/db mice compared with those of db/m mice (Fig. 3B and C). Treatment with RSV reduced the scores in the glomeruli and tubulointerstitium of db/db mice but had no effect on the lesions of db/m mice (Fig. 3B and C). These results indicate that tyrosine nitration of proteins is enhanced in the kidney of db/db mice and that RSV seems to attenuate the effect.

The mRNA and protein expression of Mn-SOD was higher in db/db mice than in db/m mice (Fig. 4A–C). The Mn-SOD activity was significantly lower in the kidney of db/db mice compared with that of db/m mice (Fig. 4D). Nitrotyrosine immunoreactivity was significantly higher in Mn-SOD immunoprecipitates from the kidney of db/db mice than in that from db/m mice, and the increase in immunoreactivity was attenuated by treatment with RSV in db/db but not db/m mice (Fig. 4E and F). These findings suggest that Mn-SOD activity could be reduced by modifying tyrosine nitration in the kidneys of db/db mice.

To evaluate mitochondrial biogenesis, we assessed peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, nuclear respiratory factor (NRF)-1, and cytochrome c oxidoreductase mRNA expression levels and mtDNA contents in the kidney. The citrate synthase activity was also measured in the kidney. All these were significantly higher in the kidneys of db/db mice than in that of db/m mice (Fig. 5A–E). These changes improved following treatment with RSV, consistent with the observed normalization of Mn-SOD activity and oxidative stress.

To confirm the beneficial effects of RSV on mitochondrial biogenesis, we examined the renal morphology in more detail by electron microscopy. The number and area of the mitochondria were significantly larger in renal proximal tubular cells of db/db mice compared with db/m mice, but treatment with RSV rescued these differences in db/db mice (Fig. 6A–C).

We assessed whether RSV activates AMPK in the kidney using immunoblotting for phosphorylation of AMPK. Activation of AMPK was significantly reduced in the kidney of db/db mice compared with db/m mice, and RSV did not alter AMPK activation in the kidney (Fig. 7A and B). In addition, SIRT1 expression was no different among the groups (Fig. 7A and C).

RSV attenuated intracellular ROS in both the SIRT1-knocked-down and DN-AMPK–overexpressing renal proximal tubular cells as well as in control cells. These results indicate that RSV exerts antioxidative effects independent of the AMPK/SIRT1 pathway (Fig. 7D and E).

In this study, we showed the potential benefits of RSV in ameliorating the renal injury and the enhanced mitochondrial biogenesis with Mn-SOD dysfunction observed in the diabetic kidney. We also demonstrated that RSV seems to exert these effects by improving the oxidative stress status in the kidney via the scavenging of ROS, normalization of Mn-SOD dysfunction, and partial rescue of glucose-lipid metabolism. Also, such antioxidative effects of RSV could be exerted in an AMPK/SIRT1-independent mechanism.

First, we showed that RSV improved renal functional and histological abnormalities such as albuminuria, mesangial expansion, glomerular and interstitial fibronectin accumulation, and interstitial macrophage infiltration in the kidney of db/db mice, a type 2 diabetes animal model. Oxidative stress has been implicated in the pathogenesis of diabetic nephropathy, and high glucose-induced ROS in renal mesangial or tubular cells contribute to overproduction of extracellular matrix proteins and inflammation (31,32). Because it has been reported that RSV is a robust scavenger of ROS (6,7), we assessed whether RSV has beneficial effects against diabetes-induced systemic and renal oxidative stress by measuring urinary 8-OHdG excretion and renal immunostaining for nitrotyrosine. Moreover, not only are mitochondria an important source of ROS, but also they themselves can be damaged by ROS. Therefore, we also assessed renal mitochondrial oxidative stress by measuring accumulation of mitochondrial 8-OHdG and D-17 deletion of mtDNA, which have been observed in diabetic nephropathy (33) and aging (24). In the current study, renal nitrotyrosine expression and mitochondrial oxidative damage indicated systemic, renal and particularly mitochondrial enhanced oxidative stress in the kidney of db/db mice, and there was clear attenuation of these markers following treatment with RSV. In addition, we showed that isolated mitochondrial O²⁻ production by stimulation with succinate and antimycin A was increased in db/db mice, and the RSV could scavenge ROS from the mitochondria.

The mitochondria are an important source of ROS in diabetes (9), and ROS metabolism in the mitochondria is mainly regulated by Mn-SOD, which is an important antioxidative enzyme in the mitochondria. In the diabetic state, high glucose- or FFA-induced overproduction of ROS from the mitochondria is inhibited by overexpression of Mn-SOD in vascular cells and tissues (9,34–37). Moreover, heterozygous Mn-SOD deficient mouse, which only have 50% of the Mn-SOD activity seen in wild-type mice, showed O²⁻-induced mitochondrial oxidative damage (38), together with massive glomerulosclerosis, tubulointerstitial damage and inflammation including macrophage infiltration in the kidney (39). Therefore, regulation of ROS metabolism in the mitochondria via Mn-SOD function is an important protective factor in managing diabetic vascular complications including nephropathy. It is also known that tyrosine nitration at the active site of Mn-SOD is associated with reduction in enzymatic activity in the kidneys of several disease models (26,40–42). In the current study, we showed reduced Mn-SOD activity even when the expression of Mn-SOD was increased, and the former was probably caused by increased tyrosine nitration in the kidney of db/db mice. RSV might reduce tyrosine nitration of Mn-SOD and rescue the activation, resulting in improvement of mitochondrial oxidative stress and renal injury in diabetic kidney. Thus, RSV might ameliorate systemic, renal, and particularly mitochondrial oxidative stress in diabetes via the direct scavenging of ROS and normalization of the Mn-SOD function.

It has been reported recently that reduced mitochondrial biogenesis in insulin resistant tissues such as skeletal muscle, liver, or fat is associated with the pathogenesis of type 2 diabetes (11). In addition to its function as a ROS scavenger, several reports have implicated RSV in activating the AMPK/SIRT1 pathway to induce mitochondrial biogenesis (14), leading to life span prolongation and improvement of high-fat diet–induced metabolic impairment such as obesity and insulin resistance (12,13). We therefore examined mitochondrial biogenesis in the kidney based on mtDNA contents, mitochondria area and number, citrate synthase activity and overexpression of PGC-1α, NRF-1, cytochrome c oxidoreductase, and Mn-SOD. Interestingly, these factors were all enhanced in the kidney of db/db mice compared with db/m mice, and were rescued by RSV treatment, consistent with attenuation of systemic and renal mitochondrial oxidative stress including tyrosine nitration of Mn-SOD. Moreover, RSV did not alter AMPK activation or induction of Mn-SOD expression in the kidney. We also showed that RSV attenuated intracellular ROS in both the SIRT1 knocked-down and the DN-AMPK–overexpressing renal proximal tubular cells. These results suggest that systemic and/or renal mitochondrial oxidative stress contributes to the enhanced mitochondrial biogenesis in the kidney of the db/db mice and that RSV might improve mitochondrial biogenesis through the attenuation of oxidative stress, rather than through the activation of the AMPK/SIRT1 pathway. In this regard, Shen et al. (20) demonstrated that oxidative stress–induced mitochondrial biogenesis was increased in the myocardium of OVE26 diabetic mice as a possible compensatory reaction against oxidative stress–induced mitochondrial damage. Moreover, they showed that the myocardium-specific Mn-SOD transgenic mice crossed with OVE26 diabetic mice showed improvement of oxidant-induced mitochondrial biogenesis and dysfunction (37). Therefore, we suggest that the enhanced mitochondrial biogenesis in the kidney of db/db mice might also be a compensatory response to oxidative stress–induced mitochondrial damage; however further study is needed to elucidate the role of enhanced mitochondrial biogenesis associated with Mn-SOD dysfunction.

RSV has also been shown to reduce elevated blood glucose levels and metabolic impairment in diabetes, to be associated with mitochondrial biogenesis through the AMPK/SIRT1 activation pathway, as discussed above (12,13,43). In the current study, the elevated levels of glucose, glycated hemoglobin, triglycerides, and free fatty acids and impairment of glucose/insulin tolerance in db/db mice improved to some extent by treatment with RSV. It is therefore possible that RSV acts as an enhancer of mitochondrial biogenesis through AMPK/SIRT1 activation in liver and skeletal muscle, resulting in demonstrated improvements in abnormal glucose-lipid metabolism in db/db mice. In fact, we found that the phosphorylation of AMPK was reduced in the liver of db/db mice, and RSV could restore its alteration (data not shown). Therefore, RSV is likely exerting some of its effects via improvement of glucose homeostasis, and the systemic improvement of metabolism might at least in part contribute to the amelioration of renal oxidative stress and renal injury.

In conclusion, our study indicated that the enhanced mitochondrial biogenesis with Mn-SOD dysfunction by tyrosine nitration was observed in the diabetic kidney. RSV seems to improve these functional and histological abnormalities and ameliorate the enhanced mitochondrial biogenesis in diabetic kidney, possibly by attenuating the oxidative stress through scavenging of the ROS, by the normalization of the Mn-SOD dysfunction in an AMPK/SIRT1-independent mechanism, and by the partial improvement of the glucose and lipid metabolism.

This work was supported by a Grant-in-Aid for Scientific Research (21591148) to D.K., and the grant from the Uehara Memorial Foundation to D.K.

No potential conflicts of interest relevant to this article were reported.

M.K. collected and analyzed the data and wrote the manuscript. S.K. collected and analyzed the data and contributed to discussion. N.I. collected and analyzed the data. D.K. contributed to discussion and reviewed and edited the manuscript.

The authors thank Yuki Tanaka and Eiko Kobori for the technical support on fibronectin immunohistochemistry and maintenance of animals and collecting their physiological data. The authors also thank Dr. Issa F.G., Word-Medex Pty Ltd. for help with preparing the manuscript.