Resveratrol as a therapeutic agent for renal fibrosis induced by unilateral ureteral obstruction

Abstract

Aims: Renal fibrosis is a common outcome of chronic kidney disease. This study was designed to examine the protective effects of resveratrol (RSV) against renal fibrosis induced by unilateral ureteral obstruction (UUO). We also attempted to elucidate the potential mechanism involved. Methods: Mice were randomly divided into three groups: sham-operated, UUO, and UUO/RSV (20 mg·kg⁻¹·day⁻¹). Histological changes were examined using periodic acid-Schiff and Masson’s trichrome staining after 14 days. Superoxide dismutase (SOD), malondialdehyde (MDA), and 8-OHdG levels were determined using a commercially available kit. ICAM-1, TNF-α, and TGF-β levels were measured using real-time PCR. Fibronectin levels were measured by western blot, and the Smad3 acetylation and Sirt1 were examined by immunoprecipitation and western blot. Results: Our study showed that RSV treatment significantly attenuated renal injury including extracellular matrix deposition and tubulointerstitium damage. Renal cortical mRNA levels of ICAM-1, TNF-α, and TGF-β, protein expression of fibronectin and Smad3 acetylation were significantly upregulated in the UUO group. However, treatment with RSV significantly decreased the expression of these proteins. Furthermore, RSV also decreased the levels of reactive oxygen species (ROS) including MDA and 8-OHdG, and increased the level of SOD, which protects cells against ROS damage. Conclusion: Our findings suggest that RSV treatment inhibits oxidative stress, Smad3 acetylation, and renal interstitial fibrosis. Therefore, RSV may have potential as a therapeutic target for the treatment of chronic kidney disease.

• Acetylation
• oxidative stress
• renal fibrosis
• resveratrol
• unilateral ureteral obstruction

Introduction

Renal fibrosis is considered as the final stage in the course of chronic kidney disease that eventually leads to end-stage renal disease.1 Renal fibrosis is manifested as hypoxia and ischemia among other indicators. The pathogenesis of renal fibrosis is characterized by monocyte infiltration, fibroblast activation or proliferation, and accumulation of extracellular matrix (ECM).2,3 Reactive oxygen species (ROS) are also believed to play a key role in this condition. ROS, which are known to function as secondary messengers, play a key role in active solute transport and reabsorption; however, increased ROS accumulation leads to renal fibrosis and is involved in various kinds of kidney disease.4,5 Therefore, reducing the production of ROS could be a therapeutic strategy for renal fibrosis. Among the many stressors that regulate renal fibrosis, transforming growth factor-β (TGF-β) plays an important role. Both type I and type II receptors and signal transduction pathways mediate the effects of TGF-β, which eventually lead to renal tubulointerstitial fibrosis.6 Thus, inhibition of the TGF-β/Smad pathway is considered as a therapeutic method for treating renal fibrosis.7

Resveratrol (RSV) is a well-known natural polyphenolic compound that is found in plants such as grapes, peanuts, berries, and pines.8 A growing body of evidence suggests the role of RSV as an anti-inflammatory, antioxidant, anticancer, and antiproliferative agent.9 Besides, RSV was reported to possess cardioprotective benefits.10 In particular, Zhao and co-workers have demonstrated that RSV could attenuate As₂O₃-induced cardiotoxicity.11 RSV treatment also could improve ischemia/reperfusion induced renal injury.12,13 In addition, Kitada and co-workers showed that RSV could improve renal function by enhancing mitochondrial biogenesis and decreasing ROS in db/db mice.14 Based on these findings, it is possible that RSV can be used to treat various diseases in the animal model, including kidney diseases. However, there is not much known about the role of RSV in the development of renal fibrosis. Therefore, the present study aimed to investigate the protective effects of RSV in the progression of renal fibrosis. We also sought to explore the possible mechanism of action of RSV, including its effects on ROS production and inflammatory response.

We used the unilateral ureteral obstruction (UUO) model of renal injury. According to a previous study, UUO is characterized by decreased renal blood flow and increased cortical ROS generation; these events eventually lead to tubulointerstitial inflammation, apoptosis, and fibrosis.15,16 Chronic UUO results in renal functional injury including marked renal hemodynamic and metabolic changes, as well as histological changes including atrophy and dilation, leukocyte infiltration, and inflammation.2,17 We therefore felt that this model would be apt for studying the effects of RSV on renal fibrosis.

Materials and methods

Materials

RSV was purchased from Sigma (St. Louis, MO). Goat polyclonal anti-fibronectin antibody, mouse monoclonal anti-Sirt1 and anti-Smad3 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit monoclonal anti-acetylated lysine and anti-Smad3 antibody were purchased from Cell Signaling Technology (Beverly, MA). Superoxide dismutase (SOD) and malondialdehyde (MDA) were purchased from Jiancheng Bioengineering Research Institute (Nanjing, China). The mouse 8-OHdG ELISA kit was purchased from BMAssay (Beijing, China).

Experimental animals

Male C57BL/6J mice that weighed 20–25 g were purchased from Slac Laboratory Animal (Shanghai, China). A UUO was created by ligation of the left ureter with 4-0 silk through a left flank incision; during the procedure, the animals were under anesthesia induced with sodium pentobarbital. Sham mice underwent the same procedure, except that the ureter was not ligated. Mice were randomly divided into three groups: sham-operated vehicle (control mice without UUO, n = 8), UUO + vehicle (UUO model mice that were treated with vehicle, n = 8), and UUO + RSV (UUO model mice that were treated with RSV, 20 mg·kg⁻¹·d⁻¹, by gavage, n = 8).18 On the 14th day after UUO surgery, the mice were sacrificed. The kidney was fixed in 4% buffered paraformaldehyde and embedded in paraffin for histological evaluation; the remaining kidney tissues were frozen at −70 °C for further study.

Histological analysis

The left kidney sample was fixed in 4% paraformaldehyde buffer for subsequent light microscopic examination. Then, 3-µm sections of the formalin-fixed, paraffin-embedded renal tissue samples were dewaxed and stained with periodic acid-Schiff (PAS) and Masson’s trichrome for histological examination. Briefly, glomerular proliferative lesions were classified into five grades as follows: 0, absent; 1, minor; 2, mild; 3, moderate; 4, severe.19 About 20 glomeruli were examined in each PAS-stained specimen and an average score was obtained. The severity of tubulointerstitial fibrosis was assessed by the degree of interstitial collagen deposition using Masson’s trichrome staining as follows: 0, no staining; 1, ≤25% staining; 2, 25%–50% staining; 3, 51%–75% staining; 4, 76%–100% staining.20 A minimum of 20 fields were examined in each Masson-stained specimen, and the above histological measurements were performed in a blind manner by a single observer.

Real-time PCR for detecting the mRNA levels of ICAM-1, TGF-β, and TNF-α

Total RNA was extracted from the mouse renal cortex using an RNA isolation kit (Invitrogen, Carlsbad, CA) and was reverse-transcribed to cDNA according to the manufacturer’s instructions (Fermentas, Glen Burnie, MD). Gene expression was performed using the SYBR Green master mix (Applied Biostems, Carlsbad, CA) and 7500 real-time PCR detection system. The primers used in this study were ICAM-1 (forward: 5′-CCTGCCTCTGAAGCTCGGATA-3′, reverse: 5′-CTAAAGGCATGGCACACGTATGTA-3′), TNF-α (forward: 5′-GCCTCTTCTCATTCCTGCTTG-3′, reverse: 5′-CTGATGAGAGGGAGGCCATT-3′), and TGF-β (forward: 5′-CAACAATTCCTGGCGTTACCTTGG-3′, reverse: 5′-GAAAGCCCTGTATTCCGTCTCCTT-3′). All oligonucleotides were designed using the Primer5 software (Premier Biosoft International, Palo Alto, CA) and synthesized by Invitrogen. The relative amount of mRNA was calculated using the comparative Ct (ΔCt) method with GAPDH for comparison.

Immunoprecipitation and Western blot

Kidney tissues were sonicated and resuspended using modified RIPA buffer (50 mM Tris-HCl, 1% Triton X-100, 0.2% sodium deoxycholate, and 0.2% sodium dodecyl sulfate [SDS]). Detection of acetylated Smad3 was measured by immunoprecipitation with mouse anti-Smad3 antibody followed by Western blot with rabbit monoclonal anti-Smad3 and anti-acetylated lysine antibody.21 Goat anti-fibronectin antibody, mouse anti-Sirt1 were used in Western blot. The blots were incubated with goat anti-fibronectin and mouse anti-Sirt1 antibody at a dilution of 1:500 for 2 h. After washing, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody. Blot intensities were quantified by densitometric analysis, and the results were normalized to β-actin expression.

Detection of ROS

SOD activity and MDA levels were detected using commercial kits, and the level of immunoreactive functionally active mouse 8-OHdG was also determined using a commercially available kit according to the manufacturer’s instructions.

Statistical analysis

All values were expressed as mean ± standard error of the mean (SEM). Statistical data were analyzed using one way-ANOVA and the Student’s t-test, and p < 0.05 was considered to be significant.

Results

RSV attenuated renal fibrosis in mice with UUO

Glomerular histological findings with PAS staining are shown in Figure 1(A–D). The sham group showed normal glomeruli and tubulointerstitium (Figure 1A). However, mice with UUO exhibited cell proliferation, increased ECM deposition, and sclerosis (Figure 1B). Treatment with RSV significantly attenuated glomerular injury in the UUO + RSV group (Figure 1C, D). In addition, we also evaluated UUO-induced renal fibrosis by Masson’s trichrome staining. Figure 2(A) shows that there was no interstitial collagen deposition in the sham group (blue staining). However, compared with the sham group, the UUO group showed much more collagen deposition and more prominent renal fibrosis at day 14 (Figure 2B). Treatment with RSV largely inhibited renal interstitial collagen accumulation (Figure 2C, D).

Figure 1. Photomicrographs of glomeruli (A–C). Glomerular injury scores (D) were evaluated as described in “Materials and methods”. Compared with the sham group (A), the UUO group showed cell proliferation and sclerosis (B). Treatment with RSV markedly attenuated UUO-induced glomerular injury (C, D). *p < 0.01 versus the sham group; #p < 0.01, UUO versus UUO/RSV group. Magnification, ×400; PAS staining.

Figure 2. Collagen deposition in mouse kidney subjected to UUO (A–C). The interstitial fibrosis index was determined as described in “Materials and methods” (D). The sham group showed no interstitial collagen deposition (A). The UUO group demonstrated much more collagen deposition and more prominent renal fibrosis at day 14 (B). However, RSV could improve renal fibrosis induced by UUO (C, D). *p < 0.01 versus the sham group; #p < 0.01, UUO versus the UUO/RSV group. Magnification, ×400; Masson’s trichrome staining.

RSV inhibited increased ROS production in UUO mice

We assessed whether ROS production was triggered in UUO-induced renal fibrosis. As shown in Figure 3(A), the content of SOD in the UUO group was significantly lower than that in the sham group at day 14. However, treatment with RSV could inhibit the downregulation of SOD. Similarly, compared with the sham group, the levels of MDA and 8-OHdG were markedly higher in the UUO group, while RSV decreased the expression of MDA and 8-OHdG in the UUO + RSV group (Figure 3B, C).

Figure 3. Reactive oxygen species production in the UUO model was evaluated by measuring the SOD (A), MDA (B), and 8-OHdG levels (C). Data are presented as mean ± SEM; n = 8 for each group; *p < 0.01 versus the sham group; #p < 0.01, UUO versus the UUO/RSV group.

RSV attenuated inflammation in UUO mice

ICAM-1 and TNF-α mRNA expression is shown in Figure 4(A, B). Compared with mice in the sham group, mice that underwent UUO treatment demonstrated increased ICAM-1 mRNA levels; however, treatment with RSV markedly decreased ICAM-1 mRNA levels. TNF-α mRNA was significantly elevated in the UUO group relative to the sham group, but treatment with RSV markedly decreased the mRNA expression of this protein.

Figure 4. Effect of RSV on the renal mRNA levels of ICAM-1 (A), TNF-α (B), and TGF-β (C). The data are expressed as the relative difference after normalization to the expression of GAPDH. All values are expressed as mean ± SEM, *p < 0.01 versus the sham group; #p < 0.01, UUO versus the UUO/RSV group.

RSV decreased TGF-β and fibronectin expression in UUO mice

As shown in Figure 4(C), compared with the sham group mice, the UUO group showed significantly elevated TGF-β mRNA expression. However, the TGF-β levels were normalized in the mice treated with RSV. Western blot analysis revealed that the expression of fibronectin was higher in the UUO group than in the sham group. However, this altered protein expression was significantly normalized in RSV-treated mice (Figure 5).

Figure 5. Effect of RSV on the protein level of fibronectin in the kidney samples of UUO mice. The data are expressed as the relative difference after normalization to the expression of β-actin. All values are expressed as mean ± SEM, *p < 0.01 versus the sham group; #p < 0.01, UUO versus the UUO/RSV group.

RSV decreased Smad3 acetylation in UUO mice

Smad3 acetylation and Sirt1 expression levels were shown in Figure 6. Immunoprecipitation of Smad3 showed the direct interaction between Sirt1 and Smad3. Compared with mice in the sham group, mice that underwent UUO treatment showed increased acetylation levels of Smad3, but treatment with RSV markedly inhibited Smad3 acetylation levels. Mice that underwent UUO demonstrated decreased Sirt1 activity. However, RSV treatment significantly improved Sirt1 expression.

Figure 6. Effect of RSV on acetylation of Smad3 and Sirt1 levels in UUO model. Immunoprecipitation/Western blot showed increased acetylation levels of Smad3 and decreased Sirt1 activity in UUO model. However, RSV treatment significantly reversed these protein levels. All values are expressed as mean ± SEM, *p < 0.01 versus the sham group; #p < 0.01, UUO versus the UUO/RSV group. Ac-Smad3: acetylation of Smad3; IB: immunoblotting.

Discussion

In the present study, administration of RSV blocked the induction of proinflammatory and fibrogenic cytokines. Interestingly, our results further revealed that RSV markedly reduced ROS generation and Smad3 acetylation. These data suggest that RSV may exert its protective effects against renal fibrosis by inhibiting ROS, inflammatory mediators, and Smad3 pathway.

Recently, ROS have been proposed to be critical factors responsible for the progression of chronic kidney disease and thought to play an important role in renal fibrosis associated with obstructive nephropathy.22 Modi showed that the levels of MDA were significantly higher in rats with ureteral obstruction compared with the sham-treated group.23 In response to ROS, anti-oxidant enzymes including catalase, SOD are thought to protect cellular function. Deficiency of these anti-oxidant enzymes could enhance ROS production and renal tubulointerstitial fibrosis.24 In our study, we found that the activity of SOD was markedly lower in mice with UUO compared with sham-treated mice. The content of MDA and 8-OHdG, which are oxidative damage markers of DNA, was increased in mice with UUO, and this upregulation was correlated with increased expression of TGF-β and fibronectin. However, treatment with RSV could significantly decrease ROS and tubulointerstitial fibrosis. These data suggest that increased ROS production may lead to an increase in fibrogenic cytokines and renal fibrosis. Antioxidants may therefore be a therapeutic option for the treatment of renal fibrosis.

Although the precise mechanism of renal fibrosis is not fully understood, recent studies have shown that TGF-β, a key fibrogenic cytokine, played a determinant role in regulating inflammation and tissue fibrosis.25 TGF-β binds with its type I and type II receptors and activates transmembrane receptor serine/threonine kinases, which initiates a cascade of signaling pathways that lead to renal fibrosis.26 A previous study has shown that TGF-β levels increased from day 3 after UUO surgery.27 Similarly, we also found that TGF-β mRNA expression was significantly elevated in the UUO group. However, treatment with RSV significantly decreased TGF-β expression. A growing body of evidence suggests that Smad3 plays a key role in the pathogenesis of renal fibrosis.7 Li et al. found that acetylation of Smad3 contributed to renal fibrosis, which was inhibited by RSV.21 Our study also demonstrated that mice that underwent UUO treatment showed increased acetylation levels of Smad3, RSV markedly decreased Smad3 acetylation levels via Sirt1 activating. The result is consistent with the research of Hao, who found that Sirt1 activation protected the mouse renal medulla from oxidative injury.28 Inflammatory responses could also initiate collagen deposition and lead to renal fibrosis. Recent research has demonstrated that various cytokines and chemokines including TGF-β, TNF-α, and ICAM-1 are involved in renal fibrosis induced by UUO.27,29,30 Our study also showed that ICAM-1 and TNF-α mRNA expression was upregulated in the UUO group. Furthermore, treatment with RSV markedly decreased these mRNA levels. These data suggest that RSV attenuated tubulointerstitial fibrosis and improved renal function through its anti-inflammatory, antioxidant, and deacetylation properties.

Conclusion

Our findings suggest that RSV significantly attenuated interstitial inflammation and fibrosis in the UUO model. The mechanism of action of RSV may include, at least in part, decreasing ROS levels, anti-inflammatory activity, and deacetylation of Smad3. RSV may therefore be a powerful therapeutic agent for the treatment of fibrotic kidney disease.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgements

The authors thank Dr Junxia Han for his technical help in handling unilateral ureteral obstruction experimental model.