Study On Renal Afferent Arteriolar Responses To Endothelin-1 In Diabetic Mice And Related Mechanisms

Vascular lesion is the most common reason of mortality and morbidity in diabetes, which exerts a series of influence on vascular function. Endothelial dysfunction occurs early in the vascular complications associated with diabetes and has traditionally been ascribed to the imbalanced production of vasodilatory and vasoconstrictor factors. Endothelin-1(ET-1), as the strongest vasoconstrictor factor, released by the endothelial cells causes vascular contraction and has also been shown to play an important role in pathological mechanism of vascular complications in diabetes. The renal afferent arteriolar responses to ET-1 remain unclear although microvesseles are more useful for exploring the mechanisms of occurrence of diabetes and its vascular complications.Researches combined clinical with animal experiments have demonstrated that there is an increased oxidative stress driven by increased production of superoxide radical(O2-) and hydrogen peroxide (H2O2), both of which have been implicated as modulators of smooth muscle contraction in the vasculature. Basal levels of both O2-and H2O2 are increased in diabetic condition, and ET-1 induced more O2- release. Incubation of pulmonary arterial endothelial cells with ET-1 increased catalase activity and decreased cellular H2O2 levels. Therefore, these studies strongly suggest that oxidative stress is closely related with vascular responses to ET-1.It is believed that the abnormal expressions of Wnt pathway, for example, decreased protein expression of Wnt5a/β-catenin, is closely related with the pathogenisis of diabetes. Dickkopfl (Dkkl) is a kind of secretory Wnt inhibitors, previous research showed that, transfection with Dkkl significantly increased the levels of reactive oxygen species. Nucleoredoxin (NRX), a member of thioredoxin family, negatively regulates the Wnt signaling pathway through dishevelled (Dvl). Treatment with H2O2 increased glycogensynthase kinase-3β (GSK-3β) depHospHorylation, and activated T cell factor (TCF) temporarily. Interestingly, many studies showed that H2O2 inhibited the β-catenin/TCF transcriptional activity, however, over expression of Dvl could improved the inhibitory effect of H2O2. These studies strongly suggest that there is a correlation between oxidative stress and the Wnt signaling pathway.However, whether renal afferent arteriolar responses to ET-1 are enhanced in diabetic models or not? What is the relationship between oxidative stress and ET-1 induced vascular responses? whether the canonical Wnt/β-catenin occurs in diabetic models or not? What is the relationship between the canonical Wnt/β-catenin signaling pathway and oxidative stress? If the canonical Wnt/β-catenin signaling pathway does occur in diabetic models, may sulindac, a small molecule inhibitor of the canonical Wnt/β-catenin pathway impaired the abnormal afferent arteriolar responses to ET-1? These problems still remain unknown. In this study, we focused on the relationship between the canonical Wnt/β-catenin pathway and oxidative stress in the diabetic models by using the world’s leading perfused technique and primary cultured human umbilical vein endothelial cells, exploring the possible pathomechanisms of diabetic microvascular complication. Part one:Roles of reactive oxygen species in afferent arteriolarresponses to ET-1 in diabetic miceAims:In this present study, we established diabetic mice models to investigate the afferent arteriolar responses to ET-1 and the roles of ROS in these effects.Methods:1.Diabetic mice were induced by streptozotocin (70 mg/kg/d) through intraperitoneal injection for four consecutive days, C57 BL/6 mice as control.2.By using the world’s leading and first domestic technique to microperfuse afferent arterioles from both diabetic group and control group, ET-1 induced vascular effects were measured and the roles of ET receptors were identified.3.After microperfusion, the roles of ROS in ET-1 induced vascular effects were measured.4.After microperfusion, changes in luminal diameter treated with 10 μM H2O2 were measured at different time points.5.After microperfusion, fluorescent signals of H2O2 and O2- on basal and ET-1 stimulated conditions were measured.6.Western blot was used to measure protein levels of ET receptors.Results:1.ET-1 induced vascular contractions were enhanced in diabetes, and these effects were mainly mediated by ETB receptor.2.Both ETA and ETB receptors, were increased in pre-glomerular arterioles of diabetes.3.Both H2O2 and O2- driven by diabetes were involved in the enhanced ET-1 contractions.4.H2O2 promoted vascular contractions in diabetes in a time-dependent manner.5.Basal fluorescent signals of H2O2 and O2- were increased, ET-1 induced more O2-fluorescent signal rather than H2O2.Conclusions:Both H2O2 and O2- contributed to ETB receptor mediated ET-1 contractions in diabetes, application of ET-1 induced more O2- fluorescent signals rather than H2O2Part two:The canonical Wnt/p-catenin pathway impaired the ET-1 contractions by downregulation of oxidative stress in diabetic miceAims:In this present study, we explored the relationship between the canonical Wnt/β-catenin pathway and oxidative stress, and further determined the influences of the canonical Wnt/β-catenin pathway on the afferent arteriolar responses to ET-1 in diabetic mice models.Methods:1.Four groups including Control group, Control+sulindac group (Control+S group), DM group, Diabetic+sulindac group (DM+S group) were involved in this experiment.2.The levels of mRNA and protein including β-catenin, GSK-3β, catalase, SOD1 and SOD2 in pre-glomerular arterioles were measured.3.The contents of H2O2 and O2-, enzymatic activities of catalase and SOD were measured.4.After microperfusion of afferent arterioles from four groups, ET-1 induced vascular responses were measured.5.After microperfusion, fluorescent signals of H2O2 and O2- on basal and ET-1 stimulated conditions were measured.Results:1.The canonical Wnt/β-catenin pathway was activated in diabetes. Sulindac, an inhibitor of the canonical Wnt/β-catenin pathway, decreased the levels of H2O2 and O2-, and increased enzymatic activities of catalase and SOD.2.Sulindac impaired the enhanced ET-1 vascular contractions in diabetes.Conclusions:Sulindac impaired the enhanced ET-1 vascular contractions by downregulation of oxidative stress in diabetes.Part three:The canonical Wnt/β-catenin pathway promotes high glucose induced oxidative stressAims:In this present study, incubations with different concentrations of glucose in HUVECs were used to investigate the relationship between the canonical Wnt/β-catenin pathway and oxidative stress.Methods:1.HUVECs were incubated with glucose at 5.5,25,50,75,100 mM for 24 or 48 h, or sulindac at 0.15,0.25 and 0.5 mM for 24 or 48 h, or co-incubation with sulindac (0.15,0.25,0.5 mM) in glucose (50 mM) for 24 or 48 h to identify ideal concentrations of glucose and sulindac.2.HUVECs were co-incubated with sulindac (0.5 mM) and glucose (50 mM) for 24 h or incubated with glucose at 50 mM for 24 h before incubation with tempol (1 mM) for 1 h, then the levels of mRNA and protein including β-catenin, GSK-3β, catalase, SOD1 and SOD2 mRNA and protein levels were measured. The mannitol at 26.5 mM for 24 h was used for eliminating the implication of increased osmotic pressure.3.HUVECs were co-incubated with sulindac (0.5 mM) in glucose (50 mM) for 24 h, then fluorescent signals for H2O2 and O2- were measured.Results:1.High glucose decreased cell viabilities in a concentration-dependent manner, which would be eliminated by sulindac, an inhibitor of the canonical Wnt/p-catenin pathway.2.The canonical Wnt/β-catenin pathway was activated after incubation of HUVECs with glucose (50 mM) in an osmotic pressure-independent manner, sulindac decreased the contents of H2O2 and O2-,and increased mRNA and protein levels of catalase and SOD2.3.Co-incubation of glucose (50 mM) and tempol (1 mM) increased the protein levels of SOD2, whereas had no effect on the canonical Wnt/β-catenin pathway.Conclusions:Incubation of HUVECs with high glucose induced the activation of the canonical Wnt/β-catenin pathway by increasing oxidative stress. There was no interaction between the canonical Wnt/β-catenin pathway and oxidative stress.