The Mechanism Of Glucose Fluctuation Aggravates Renal Injury In Diabetic Rats

Part1 Effect of glucose fluctuation on renal injury in diabetic rats and its related mechanismObjective:1.To investigate the implication of blood glucose fluctuation in type 2 diabetic rats through detecting the changes of urine protein,renal function and tissues.2.To detecte the relevant mechanism of blood glucose fluctuation aggravates renal damage through testing oxidative stress and AKT signal pathway in diabetic rats.Methods:54 male SD rats were randomly divided into 3 groups: Control group(Con,n=18),diabetes group(DM,n=18)and blood glucose fluctuation group(FDM,n=18).The Con group were fed with standard diet,the DM and FDM groups were fed with high sugar and high fat diet for four weeks,then diabetic model were induced by injecting with Streptozotoein(35mg/kg).After 72 h,rats with blood glucose steadily more than 16.7 mmol/L were considered as successful models.FDM group were intraperitoneal injected by short-acting insulin(20U/kg,06:00,12:00,18:00)and perfused with glucose(3g/kg,9:00,15:00,21:00)at different time points every day to induce blood glucose widely varied in one day.While the Con and DM groups received normal saline in same.During the experiment,the body weight was measured every week,the blood glucose variation coefficient(CV),MBG,LAGE and SDBG were calculated by measuring blood glucose(6:30,9:30,12:30,15:30,18:30,21:30)in two days in a week.In 16 th weeks,Hb Al C were detected and 24 h urine was collected to measure 24 h Urinary albumin,BUN,Scr,TC,TG,LDL and HDL-C were tested by collecting blood.SOD activity and MDA content were tested and the kidney tissue morphology was observed by electron microscope.The expression of AKT,phosphorylated AKT(p-AKT,Ser473),GSK-3?,phosphorylated GSK-3?(p-GSK-3?,Ser9)and Cleaved Caspase-3 and the activity of NF-?B were detected by Western blot.Results:1.Body weight in DM and FDM groups were higher than Con group in the 4th week(P<0.05).However,the body weight were lower in DM and FDM groups than Con group in the 12 th and 16 th week(P<0.05).2.Data shown that the Hb A1 c levels in DM and FDM groups were higher than in Con group(P<0.05),and there were no significant different when compared the DM and the FDM group(P>0.05).CV,MBG,SDBG and LAGE in DM and FDM groups were higher than in Con group(P<0.05).Meanwhile,these data in the FDM groups were higher than theses in the DM group(P<0.05).Furthermore,levels of 24 h Urinary albumin,BUN,Scr,TC,TG and LDL were higher in DM and FDM groups(P<0.05),and the levels of 24 h Urinary albumin also were significant higher in FDM group when compared with DM groups(P<0.05).HDL-C levels were higher in the CON group than in DM and FDM groups,and there was no significant different when compared the DM group to the FDM groups3.The GBM thickness and the width of foot processes in three groups was observed by transmission electron microscopy.The FDM and DM rats displayed significant thickening of the GBM compared with the Con animals.There was,however,a significant increase in the thickness of the GBM in the FDM group in comparison with the DM group.The width of foot processes was significantly higher in the DM group and FDM group compared with the Con group.These phenomena in the FDM group was aggravated when compared with the DM group.4.The activity of T-SOD were lower in DM and FDM groups,and this decrease were significant in the FDM group(P<0.05).The content of MDA were increased in diabetic rats,but the FDM group had higher MDA levels than DM group(P<0.05).5.The expressions of p-AKT were lower in DM and FDM group than in Con group(P<0.05),when compared FDM group with DM group,there was significant decrease(P<0.05).Furthermore,p-GSK-3? and Cleaved Caspase-3 levels and NF-?B activity were significant increased in DM and FDM groups,and the FDM group had higher levels than the DM group(P<0.05).Conclusion:1.Blood glucose fluctuation accelerate renal injury in diabetic rats by increasing oxidative stress.2.Blood glucose fluctuation promote the progress and development of diabetic kidney disease is,at least partly,by inhibiting the AKT signal pathway.Part1 Effect of glucose fluctuation on glomerular mesangial cells and its related mechanismsObjective:1.To investigate the effects of blood glucose fluctuation on glomerular mesangial cellular damage.2.To investigate the effect of blood glucose fluctuation on oxidative stress and AKT signal pathway,explore the mechanism of blood glucose fluctuation aggravates glomerular mesangial cells apoptosis.Methods:Rat glomerular mesangial cells were purchased from Boster(Wuhan,China)and were cultured in DMEM(5.6mmol/L Glucose)supplemented with 10 % fetal bovine serum(FBS)at 37? in a 5 % CO2 for 24 h.We selected the 5–10th generation of mesangial cells in the present study.Cells were divide into 4 groups: the normal glucose group(NG,5.6 mmol/L glucose),the high glucose group(HG,25 mmol/L glucose,and then normal glucose conditions incubated for the night),the fluctuation glucose(FG,alternated 5.6 mmol/L glucose and 25 mmol/L glucose every 3 h,repeated 3 times,and then normal glucose conditions incubated for the night)and the mannitol group(MG,5.6 m M glucose plus 24.4 m M mannitol as an osmotic pressure control).Cells viability,oxygen species(ROS)level,superoxide Dismutase(SOD)activity and Malonaldehyde(MDA)level were detected for 12,24,48 and 72 h respectively to choose the optimal time point in next study.After deciding the optimal time point(48 h),we added the IGF-1 group(FG+ IGF,25 ng/m L IGF-1 incubated for 30 min,then received same treatment to the FG group)and insulin group(FG+ RI,100nmol/L insulin incubated for 30 min,then received same treatment to the FG group).Cell apoptosis was assayed by flow cytometry,the level of ROS?MDA and SOD activity were analyzed using specific reagents according to the protocols provided by the manufacturer.The expression of AKT,p-AKT(Ser473),GSK-3?,p-GSK-3?(Ser9),and Cleaved Caspase-3 and the activity of NF-?B were detected by Weston Blot.Results:1.Mesangial cell proliferation rate was increased in HG and FG group when compared with NG group at 12 h(P<0.05),no significant difference was found between HG group and FG group.Mesangial cell proliferation rate was increased in HG and FG group when compared with NG group at 24 h(P<0.05),no significant difference was found between HG group and FG group.Mesangial cell proliferation rate at 48 h and 72 h was decreased in HG group and FG group,and mesangial cell proliferation rate was obviously decreased in FG group when compared to HG group(P<0.05).The cells in MG group floated in the culture medium at 72 h.Therefore,in subsequent study,24 h and 48 h was selected as the cultured time.2.The SOD activity in the HG and FG group at 24 h were decreased when compared with the NG group.While comparing with the HG group,the SOD activity was decreased in the FG group at 24 h.The MDA levels at 24 h were increased in the HG and FG group than those of the NG group.Compared with the HG group,the MDA levels were most increased in the FG group at 24 h.However,glucose fluctuation obviously inhibit SOD activity and increased MDA levels in mesangial cell at 48 h when compared with the HG group.Therefore,in subsequent study,48 h was selected as the optimal time.3.Fluctuated glucose increased the level of intracellular ROS compared to both high sugar and nomal condition at 48 h,but the ROS level in FG+IGF group and FG+RI group were lower than that of FG group(P<0.05).4.Compared to NG group and HG group,the numbers of glomerular mesangial apoptosis in the FG group was increased in 48 h(P<0.05).After adding AKT agonist,the glomerular mesangial cell apoptosis in FG+IGF group and FG+RI group were less compared to FG group(P<0.05).5.There was no significantly difference of AKT and GSK-3? expression level in six groups.The level of p-AKT was less in both HG group and FG group compared with the NG group but least in the FG group(P<0.05).High sugar increased the level of p-GSK-3? and Cleaved-caspase-3 and activity of NF-?B in glomerular mesangial cells compared to these in normal sugar condition,and there were obviously increased induced by glucose fluctuation(P<0.05).Compared with FG group,the expression of p-AKT in FG+IGF group and FG+RI group were increased,and the expression of p-GSK-3?,Cleaved-caspase-3 and the activity of NF-?B were significantly decreased(P<0.05).Conclusion:1.Glucose fluctuation could increased mesangial cell apoptosis.2.Glucose fluctuation aggravate mesangial cell apoptosis by increasing oxidative stress and down-regulating AKT signaling pathway.