Effects Of Resveratrol On Inhibiting Renal Tubulointerstitial Fibrosis In Diabetic Nephropathy

Diabetic nephropathy is a major complication of diabetes that leads to chronic renal failure. Although the glomerulus has been the focus of intense investigation in diabetes,it was found that residual renal function and long-term survival correlate better with the extent of tubulointerstitial damage than with the degree of glomerular damage, suggesting that tubulointerstitial fibrosis is an important predictor of renal dysfunction independent of glomerular lesions. Thus, development of therapeutic approaches against progression of renal interstitial fibrosis may help us target diabetic nephropathy.To date, the mechanisms of tubulointerstitial fibrosis remain incompletely understood. However, this pathological phenomenon occurs due to the accumulation of interstitial extracellular matrix(ECM) derived from myofibroblasts in the kidney. Myofibroblasts are defined as cells whose phenotype features between fibroblasts and smooth muscle cells. In previous studies, Inflammation, hypoxia, high glucose were all shown to be able to induce fibroblasts proliferation and differentiation into myofibroblasts. In addition, epithelial to mesenchymal transition(EMT) in tubuloepithelial cells is another crucial source of myofibrolasts. Therefore, inhibiting fibroblasts proliferation and EMT of tubuloepithelial cells is an important way to reduce the number of myofibroblasts and ECM synthesis, and the studies on the underlying mechanisms can provide a novel strategy and target for prevention and treatment of renal fibrosis.Reactive oxygen species(ROS) played an important part in renal fibrosis. It was reported that fibrogenic factors as transforming growth factor-β(TGF-β), angiotensin II can stimulate proliferation of fibroblast and tubuloepithelial cells EMT by increasing level of intracelluar ROS. High level of glucose, a unique biochemical characteristic of diabetes, also has been shown to be able to promote ROS generation. Additionally, the intracellular ROS in kidney were mainly derived from nicotinamide adenine dinucleotide phosphate(NADPH) oxidase, which were up-regulated in diabetic nephropathy. Hence, the inhibition of NADPH oxidase-derived ROS will be an important breakthrough for a novel treatment of renal fibrosis in diabetic nephropathy.AMP-activated protein kinase(AMPK) acts as a key cellular energy sensor, and it exerts diverse biological effects, including regulation of both glucose and lipid metabolism, reduction inflammation and inhibition of cell proliferation. However, its activity in renal cells can be down-regulation by high glucose. There is now evidence that AMPK functions as a negative modulator of NADPH oxidase activation. A recent report showed that AMPK inhibited Nox4-dependent activation of p53 and epithelial cell apoptosis in diabetes. Thus, it would be reasonable to expect that AMPK inhibition could likewise induce oxidative stress in diabetic nephropathy. Resveratrol(RSV), a natural antioxidant polyphenols, has been reported to be able to active AMPK signaling pathway and exert renoprotective activity against diabetic and its complications. Nevertheless, the mechanism reglating this process is not fully elucidated. On this basis, we speculate that RSV could decrease high glucose-induced NADPH oxidase and ROS production by activating AMPK signal, thereby inhibting proliferation and activation of fibroblasts and EMT of tubuloepithelial cells, finally ameliorating renal fibrosis.In this study, we used renal interstitium fibroblasts(NRK-49F) and tubuloepithelial cells(HK-2) in vitro experiments, and employed C57BL/Ksj/db/db-(db/db) mice to construct the animal model of type 2 diabetes to study the effects and mechanism of RSV in attenuating renal interstitium fibrosis from three aspects:(1) Firstly we explored the underling mechanism of high glucose on the induction of proliferation and activation in fibroblasts and EMT in tubuloepithelial cells;(2) and evaluated whether RSV can inhibit proliferation and activation of fibroblasts and EMT of tubuloepithelial cells through AMPK/NADPH signaling pathway;(3) and RSV administered orally to a mouse model of type 2 diabetes to study the effects of RSV on FGF2/FGFR and renal fibrosis in vivo.The results and conclusions are listed as follows:1. High glucose induced proliferation and activation of renal fibroblasts by increasing NOX activity and ROS production.As revealed by western blot analysis, high glucose(30 mmol/L) increased the expression of α-smooth muscle actin(α-SMA), an important marker of myofibroblat, and fibronectin, a major extracellular matrix protein in NRK cells, indicating that the cells were undergoing a transition from a interstitial fibroblast phenotype to a myofibroblast phenotype, and increased in secretion and synthesis of ECM. Meanwhile, CCK-8 analysis revealed that high glucose evidently increased proliferation of NRK cells.Intracellular ROS generation is a tightly regulated process which plays an important role induces renal fibrosis. We therefore measured ROS levels of NRK cells after 48 h incubation with high glucose using DCFH-DA analysis. A significant increase in ROS production was observed in NRK cells after exposure to high glucose, whereas DPI(10 μmol/L) inhibited high gluocose-induced ROS production, indicating that high glucose promotes ROS generation derived from NADPH oxidases in NRK cells. We then assessed the expression changes of NADPH oxidase subunits NOX1, NOX2 and NOX4 by western blot analysis. It was found that the expression levels of NOX4, but not NOX1 and NOX2, were up-regulated in the NRK cells. To further investigate the role of NOX4 in high glucose-induced proliferation and activation of NRK cells, the cells were exposed to high glucose after DPI pretreatment. As revealed by western blot and CCK-8 analysis, DPI evidently inhibited high glucose-induced proliferation and activation of NRK cells, suggesting that high glucose induced proliferation and activation of NRK cells by increasing NOX4 activity.2. RSV inhibited high glucose-induced proliferation and activation of renal fibroblasts by down-regulating of NOX4-derived ROS production.To further investigate the effects of RSV on high glucose-induced proliferation and activation of NRK cells, the cells were pretreated with RSV(5-10 μmol/L) before exposed to high glucose. It was shown that RSV significantly inhibited high glucose-induced NRK cells proliferation using CCK-8 analysis. RSV also inhibited activation of NRK cells as assessed by western blot analysis. In addition, RSV, similar to DPI, significantly blocked NOX4-derived ROS production, indicating that RSV inhibits high glucose-induced proliferation and activation of NRK cells by down-regulating of NOX4-derived ROS production.3. RSV decreased high glucose-induced NADPH oxidase and ROS production by activating AMPK signal, thereby inhibiting proliferation and activation of fibroblasts.Evidence demonstrates that AMPK functions as a negative modulator of NADPH oxidase activation. As revealed by western blot analysis, high glucose blocked AMPK activity in NRK cells, whereas RSV inhibited the down-regulatation of AMPK activity induced by high glucose. Moreover, Compound C(10 μmol/L), an AMPK inhibitor, abolished the effect of RSV on high glucose-induced proliferation and activation of NRK cells and NOX4 activity, suggesting RSV inhibited proliferation and activation of NRK cells through AMPK/NADPH signaling pathway.4. RSV inhibited high glucose-induced EMT in tubuloepithelial cells by blocking NADPH oxidase-derived ROS production.HK-2 cells treated with high glucose for 48 h lost their cobblestone appearance and obtained a spindle-like fibroblastic shape. Western blotting analysis revealed that high glucose reduced E-cadherin expression, a tubular epithelial marker, and increased α-SMA expression, whereas RSV abrogated the effect of high glucose on phenotypic changes of cells, indicating that RSV has a potent property to repress EMT in HK-2 cells. Moreover, high glucose significantly increase ROS production in dose-dependent manner as assessed by DCFH-DA analysis. Additionally, the intracellular ROS in kidney were mainly derived from NADPH oxidase, we then assessed the expression changes of NADPH oxidase subunits NOX1, NOX2 and NOX4 by western blot analysis. It was found that the expression levels of NOX1 and NOX4, but not NOX2, were increased in time-dependent manner in HK-2 cells. RSV, similar to DPI, significantly blocked ROS production derived from NOX1 and NOX4, indicating that RSV inhibits high glucose-induced EMT in HK-2 cells by down-regulating of ROS production derived from NOX1 and NOX4.5. RSV decreased high glucose-induced NADPH oxidase and ROS production by activating AMPK signal, thereby inhibiting EMT in tubuloepithelial cells.As revealed by western blot analysis, high glucose blocked AMPK activity in HK-2 cells, whereas RSV inhibited the down-regulatation of AMPK activity induced by high glucose. However, Compound C(10 μmol/L) abolished the effect of RSV on high glucose-induced EMT and NOX1 and NOX4 activity, suggesting RSV inhibited EMT in HK-2 cells through AMPK/NADPH signaling pathway.6.RSV treatment significantly mitigated renal fibrosis and inhibited proliferation and activation of renal fibroblasts in db/db mice probably through AMPK/NADPH signaling pathway.To investigate the effects of RSV on the progression of renal fibrosis and AMPK/NADPH signaling pathway, RSV(400 mg/kg/d) was mixed with chow and administered orally to db/db mice. Physical and biochemical characteristics of mice were assessed after 9 weeks experiment. Blood glucose, body weight and levels of urinary albumin were markedly increased in db/db mice compared with db/m mice. Nevertheless, RSV treatment significantly elevated urinary albumin levels in db/db micem,but there were no difference in blood glucose and body weight between untreated and RSV-treated db/db mice, indicating that RSV ameliorate renal fibrosis independ of its effects on blood glucose and body weight. As revealed by H&E and Masson staining, RSV treatment significantly attenuated renal fibrosis in db/db mice. Furthermore, RSV treatment significantly inhibited the expression of FSP-1, a marker of fibroblats, and fibronectin in renal interstitium of db/db mice, and meanwhile increased in the number of interstitial cells stained with α-SMA and PCNA, a proliferation marker, indicating that the renoprotective activity of RSV in renal fibrosis closely associated with its inhibitory action on fibroblasts proliferation and activation. To further investigate the mechanism of RSV in inhibiting progression of renal fibrosis, we then assessed the expression of p-AMPK, NOX1, NOX2, NOX4 in renal interstitium using western blot and immunohistochemistry analysis. It was found that the expression level of p-AMPK significantly decreased, whereas NOX4,but not NOX1 and NOX2, markedly increased in db/db mice compared with db/m mice, suggesting that NOX4-derived ROS is a major culprit of oxidative stress in diabetic nephropathy. The expression level of p-AMPK in renal interstitium in RSV-treated db/db mice was much lower than that of db/m, and the activation of NOX4 was significantly attenuated.In conclusion, our results suggested that high glucose could induce proliferation of fibroblast and EMT of tubuloepithelial cells to promote renal fibrosis, which could be related to p-AMPK inhibition and NADPH-derived ROS activation, and RSV could decrease high glucose-induced proliferation and activation of fibroblasts and EMT of tubuloepithelial cells by regulating AMPK/NADPH signaling pathway, finally ameliorating renal fibrosis.