The Antioxidant Effect Of HGF On Rat Mesangial Cells Exposed To High Glucose Conditions And Its Mechanism

Diabetic nephropathy, the severest and commonest long-term microvascular complications in diabetes mellitus, is the leading cause of end-stage renal failure and mortality in diabetic patients. Evolution of these will depend on elucidating the molecular mechanisms driving development and progression of diabetic nephropathy, which attracts attention of basic research of nephrology all along. The unifying mechanism for the pathogenesis of diabetic complications theory presented by Brownlee in 2001 provided a new breakthrough in understanding of pathogenesis of diabetic nephropathy. Many factors and pathways related to pathogenesis of diabetic nephropathy such as genetic susceptibility, metabolic and haemodynamatic disturbances, and upregulation of various cytokine,vasoactive hormone and chemokine are all involved in a crucial common pathway--oxidative stress. Reactive oxygen species (ROS) mediate hyperglycaemia-induced renal damage through activating the polyol way, promoting advanced end product formation, increasing activity of protein kinase C (PKC) and hexosamine pathway.Hepatocyte growth factor originally identified and cloned as a potent mitogen for primary hepatocytes exhibits multiple biological activities including mitogenic, morphogenic, motogenic, angiogenic, anti-apoptotic activities in a wide variety of cells. Since kidney is one of the most highly expressed c-Met organs, growing researches have focused on clarifying the role of HGF in renal physiology and pathology during the past decade. Though considerable in vivo animal studies have shown that HGF has the therapeutic potential to retard development and progression of diabetic nephropathy, it remains unknown that suppressing high glucose-mediated oxidative stress is the underlying mechanism for the protective effect of HGF in diabetic nephropathy. Explaining the renoprotective actions of HGF from antioxidant point of view may help us get a better knowledge of the beneficial role of HGF in various renal diseases including diabetic nephropathy.As intrinsic glomerular cells, mesangial cells play a central regulatory role in mesangial functions both in physiological and pathological conditions. There is increasing evidence that ROS act as intracellular signaling molecules activate the PKC and mitogen-activated protein kinase (MAPK) pathways, which lead to the activation of transcription factor including nuclear faetor-kB (NF-kB) and activator protein-1(AP-1) . These enhance the transactivation of genes coding for extracellular matrix (ECM) components as well as cytokines,chemokine,and growth factors such as transforming growth factor beta1(TGF-β1),connective tissue growth factor (CTGF),monocyte chemoattractant protein-1(MCP-1) and plasminogen activator inhibitor-1(PAI-1) that mediate and/or amplify excessive ECM deposition and lastly glomerular sclerogenesis.In vitro, we performed many molecular biologic technologies, including RT-PCR, Western blot, isoenzyme electrophoresis, flowcytometer detection and EMSA, to examine the influence of HGF on c-Met expression and high glucose (HG)-induced oxidative stress in rat mesangial cells and explore the relevant mechanisms. The purpose of our research was to determine the antioxidant role of HGF in diabetic nephropathy and elucidate the involved mechanisms, thus bringing new insights into the mechanisms implicated in HGF ameliorating diabetic nephropathy and opening new avenues toward the therapeutic potential of HGF for prevention and treatment renal disease.PartⅠThe effect of HG on transcription and expression of c-Metin cultured rat MsCObjective To observe the effect of HG on transcription and expression of c-Met in cultured rat MsC and explore its mechanism.Methods Primary cultured rat MsC were treated with HG (30mM D-glucose) for different time (0, 12, 24, 48, 96h). The transcription and expression of c-Met were examined by semiquantitative RT-PCR and Western blot analysis, respectively. Mithramycin A was used to block Sp1 binding DNA. The activity of Sp1 binding to c-Met promoter was detected by electrophoretic mobility shift assay (EMSA).Results Upregulation of c-Met transcription and expression were observed in rat mesangial cells exposed to HG conditions at 12, 24, and 48h, then downregulation was shown at 96h. The difference was statistically significant. In comparison to non-mithramycin A-treated control, mithramycin A suppressed HG-increased c-Met mRNA and protein expression in a dose-independent manner. EMS A analysis indicated that statistically significant band of Sp1 binding to c-Met promoter was presented in HG-treated group whereas not revealed in NG, MNT, and mithramycin A-treated groups.Conclusions Exposure MsC to HG resulted in an increase in c-Met mRNA and protein expression at first 48h then a following decrease at 96h. Mithramycin A suppressed HG-elevated c-Met expression in a dose-dependent manner. Compared with NG-treated MsC, enhanced Sp1 binding to c-met promoter was shown in HG-treated MsC.PartⅡThe effect of HGF on HG-induced oxidative stressin cultured rat MsCObjective To observe the effect of HGF on oxidative stress in rat MsC cultured in HG conditions.Methods RT-PCR and Western blot were used to analyze the influence of HGF on c-Met mRNA and protein expression, respectively. With fluorescence microscope and flowcytometer, intracellular ROS was examined qualitatively and quantitatively in MsC loaded with the fluoroprobe 2'7'-dichlorofluorescin diacetate. The intracellular levels of glutathione (GSH) and malonaldehyde (MDA) as well as antioxidant enzymes activities were measured by spectrophotometry following instructions of the commercial kit (Nanjing Jiancheng Bioengineering Institute). The mRNA expressions of antioxidant enzymes in rat MsC were assessed by RT-PCR.Results There is no significant difference in upregulated c-Met mRNA and protein expression between HG-treated group and HG plus HGF-treated group. The level of c-Met phosphorylation in response to HGF (20ng/ml) was markedly increased in HG-treated group compared with NG-treated control. Added 6h before HGF supplementation, c-Met specific inhibitor SU11274 can completely block c-Met phosphorylation in response to HGF. HGF obviously suppressed enhanced oxidative stress as evidenced by elevated ROS and MDA levels and decreased GSH level in rat MsC exposed to HG conditions. There were no significant differences for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) in mRNA expression. The activities of these antioxidant enzymes except for GR were significantly reduced in rat MsC cultured in HG. HGF effectively prevent HG-decreased antioxidant enzymes activities. The effects of HGF mentioned above were wholly inhibited by SU11274．Conclusions HGF had no significant effect on c-Met transcription and expression whereas evidently increased c-Met phosphorylation in rat MsC cultured with HG. HGF/c-Met obviously suppressed high glucose-induced oxidative stress and restored intracellular GSH and MDA to normal levels. The impairment of antioxidant enzymes activities induced by HG was also inhibited by HGF.PartⅢThe relevant mechanism of HGF suppressing HG-induced oxidativestress in cultured rat MsCObjective To explore the underlying mechanism of HGF suppressing HG-induced oxidative stress.Methods RT-PCR and Western blot were used to detect the mRNA and protein expression of p22phox, aldose reductase (AR), glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD), respectively. Analysis of EMSA was performed to determine the activity of transcription factor upstream stimulatory factor (USF) binding to negative regulatory region of GCLC. Using isoenzyme electrophoresis, activity of G6PD was assessed.Results RT-PCR and Western blot analysis showed HGF prevented GCLCdownreglation as well as p22phox and AR upregulation in HG-treated rat MsC. EMSA analysis indicated that USF binding ability markedly enhanced in rat MsC cultured in HG compared with those cultured in NG and HGF (20ng/ml) attenuated HG-enhanced USF binding ability. HGF restored not only HG-reduced G6PD mRNA and protein expression but also its impaired activity. In the presence of SU11274 (5μM), all these effects of HGF were entirely blocked.Conclusions HGF evidently inhibited HG-increased p22phox and AR expression. HGF prevented HG-reduced GCLC expression through inhibiting USF binding to negative regulatory region of GCLC promoter. HG-decreased G6PD activity and expression was also rescued by HGF.Conclusions1．HG may activate c-Met expression in MsC through Sp1．2．HGF can protect MsC against HG-mediated oxidative stress. Phosphorylated c-Met and its downstream signaling pathway are indispensable in the antioxidant action of HGF.3．HGF may balance the ROS production and ROS scavenging in MsC through enhancing GSH synthesis.