The Protective Effects And Mechanism Of Metformin On Diabetic Kidney Disease

Objective To investigate the protective effects and mechanism of metformin on diabetic kidney disease(DKD),urinary albumin excretion(UAE), urinary malondialdehyde(MDA) and advanced oxidative protein products(AOPPs) content, superoxide dismutase(SOD) and catalase(CAT) activity in patients with early DKD were testd, which were observed again after treatment by metformin. The relationship between the changes of urinary MDA, AOPPs content, SOD, CAT activity and the change of urinary albumin excretion was also observed.Methods Choose 54 cases of healthy adult in our hospital medical center and 120 cases of outpatient and inpatient of type 2 diabetes mellitus with early nephropathy(diagnostic criteria of UAER 20-200ug/min) from January in 2012 to December in 2013. Randomly grouping as follows: ①Normal control group: healthy volunteers ②Metformin-treatment group:metformin ③Acarbose-treatment group:acarbose With health education on diabetes, reasonable diet and exercise intervention, we collect urine and blood of patients before treatment and in the first, 3, 6 months after treatment respectively. Before and after treatment with metformin or acarbose,body mass index(BMI) changes were observed.Systolic blood pressure(SBP) and diastolic blood pressure(DBP) were tested by mercury sphygmomanometer.Fasting plasma glucose(FPG), 2h-postprandial plasma glucose(2h PG),low density lipoprotein cholesterol(LDL-C) and triglyceride(TG) were detected with automatic biochemical analyzer. The urinary MDA(UMDA) and AOPPs(UAOPPs) content, urinary SOD(USOD) and CAT(UCAT) activity were detected by enzyme-linked immunosorbent assay(ELISA).Glycosylated hemoglobin1c(Hb A1c) was detected by high performance liquid chromatography,UAE was detected by immunoturbidimetry, urinary creatinine(UCr) was detected by sarcosine oxidase method,fasting blood insulin(FINS) were determined by chemiluminescent immunoassay. Insulin resistance index(IRI) was calculated by glucose stability mathematical model evaluation method.Results 1.Comparison of BMI, FPG, 2h PG, Hb A1 c, FINS, IRI between different groups Compared with the normal control group, FPG, 2h PG and Hb A1 c were significantly higher in metformin-treatment and acarbose-treatment group before treatment(P<0.05).Compared with pretherapy,FPG and 2h PG significantly decreased in the first months after treatment with metformin or acarbose(P <0.05),but the reduction of Hb A1 c was not obvious(P>0.05).FPG,2h PG and Hb A1 c were significantly decreased in the 3rd, 6th months after treatment with metformin or acarbose(P <0.05),but the difference was not significant between the two groups(P>0.05). Compared with pretherapy, BMI, FINS, IRI decreased significantly in the first,3rd, 6th months after treatment with metformin(P<0.05),meanwhile IRI decreased significantly(P<0.05),but there are no obvious difference in BMI, FINS after treatment with acarbose.The difference was significant between the two groups(P<0.05) 2.Comparison of TG,LDL-C,SBP,DBP between different groups Compared with the normal control group, TG,LDL-C,SBP and DBP were significantly higher in metformin-treatment and acarbose-treatment group before treatment(P<0.05).Compared with pretherapy,there are no obvious difference in TG,LDL-C,SBP and DBP after treatment with metformin or acarbose(P>0.05). 3.Comparison of UMDA/UCr and UAOPPs/UCr,USOD/UCr and UCAT/UCr between different groups Compared with the normal control group, UMDA/UCr and UAOPPs/UCr increased,UCAT/UCr and USOD/UCr were significantly lower in metformin-treatment and acarbose-treatment group before treatment(P<0.05).Compared withpretherapy,UMDA/UCr and UAOPPs/UCr decreased, UCAT/UCr and USOD/UCr increased significantly in the first, 3, 6 months after treatment with metformin(P<0.05).But there are no obvious difference after treatment with acarbose(P>0.05).The difference was significant between the two groups(P <0.05) 4.Comparison of UACR between different groups Compared with the normal control group,UACR increased in metformin-treatment and acarbose-treatment group before treatment(P<0.05).Compared with pretherapy,UACR decreased significantly in the first, 3, 6 months after treatment with metformin(P<0.05).But there are no obvious difference after treatment with acarbose(P>0.05). The difference was significant between the two groups(P <0.05) 5.Analysis of correlation between UMDA/UCr and UACR The UMDA/UCr was positively correlated with UACR(r=0.834,P<0.05) 6.Analysis of correlation between UAOPPs/UCr and UACR The UAOPPs/UCr was positively correlated with UACR(r=0.821,P<0.05) 7.Analysis of correlation between USOD/UCr and UACR The USOD/UCr was negatively correlated with UACR(r=-0.867,P<0.05) 8.Analysis of correlation between UCAT/UCr and UACR The UCAT/UCr was negatively correlated with UACR(r=-0.877,P<0.05)Conclusions The urinary MDA,AOPPs and albumin excretion increased, and the SOD and CAT activity decreased in diabetic patients. Metformin can reduce urinary MDA, AOPPs and albumin excretion, and increase the SOD and CAT activity, which may be its partly mechanism of renal protection.Objective To investigate the protective effect and mechanism of metformin on oxidative stress of rat glomerular mesangial cells,the generation of reactive oxygen species(ROS), expression of P22 phox m RNA and protein, p38 mitogen-activated protein kinase(p38MAPK) protein phosphorylation of rat glomerular mesangial cells cultured in high glucose medium in vitro,and the malondialdehyde(MDA)content, advanced oxidative protein products(AOPPs) content,superoxide dismutase(SOD) activity,catalase(CAT) activity in supernatant fluid were detected,which were observed again after intervention by metformin.Methods The rat glomerular mesangial cells were cultured in the glucose medium of different concentrations and then divided into groups as follows: ①Group NC(the normal control group): Low glucose DMEM(5.6 mmol/L); ②Group HG(the high glucose group):High glucose DMEM(30mmol/L) ③Group MET(the metformin-intervention group):High glucose DMEM(30mmol/L) plus 5mmol/L metformin; ④Group SB(the SB203580-intervention group,SB203580-p38 MAPK inhibitor):High glucose DMEM(30mmol/L) plus 10umol/L SB203580 ⑤Group NAC(the N-acetylcysteine-intervention group, NAC- antioxidant):High glucose DMEM(30mmol/L) plus 100umol/L N-acetylcysteine Rat glomerular mesangial cells and supernatant fluid in every group were collected after 48 hours. Intracellular ROS production of rat glomerular MCs was detected fluorometrically by flow cytometry.SOD activity,CAT activity and MDA content,AOPPs content in supernatant fluid were detected by ELISA respectively. P22 phox m RNA expression ofrat glomerular MCs was determined by real-time quantitative PCR.Expression of p22 phox protein and p38 mitogen- activated protein kinase protein phosphorylation of rat glomerular MCs were determined by Western blot.Results 1. Comparison of ROS production of rat glomerular MCs between different groups 1.1 Change of ROS production of rat glomerular MCs cultured in high glucose medium After rat glomerular MCs have been cultured in high glucose medium for 48 hours, the intracellular ROS level increased significantly to 19.4±2.5.Compared with Group NC(0.7±0.2), differences are significant(P<0.05) 1.2 Effects of metformin,SB203580,N-acetylcysteine on ROS production of rat glomerular MCs cultured in high glucose medium Compared with Group HG(19.4±2.5),the intracellular ROS level decreased to 7.2±1.4 in goupe MET,12.8±1.8 in goupe SB,4.2±0.9 in goupe NAC, respectively. differences are significant(P<0.05) 2.Comparison of P22 phox m RNA expression of rat glomerular MCs between different groups 2.1 Change on of P22 phox m RNA expression of rat glomerular MCs cultured in high glucose medium After rat glomerular MCs have been cultured in high glucose medium for 48 hours, the P22 phox m RNA expression increased significantly to 4.5±0.7.Compared with Group NC(1±0), differences are significant(P<0.05) 2.2 Effects of metformin,SB203580,N-acetylcysteine on P22 phox m RNA expression of rat glomerular MCs cultured in high glucose medium Compared with Group HG(4.5±0.7),the P22 phox m RNA expression decreased to 3.5±0.5 in goupe MET,3.6±0.5 in goupe SB,3.0±0.4 in goupe NAC,respectively. differences are significant(P<0.05) 3.Comparison of P22 phox protein expression of rat glomerular MCs between different groups3.1 Change of P22 phox protein expression of rat glomerular MCs cultured in high glucose medium After rat glomerular MCs have been cultured in high glucose medium for 48 hours,the P22 phox protein expression increased significantly to 0.64±0.2.Compared with Group NC(0.24±0.1), differences are significant(P<0.05) 3.2 Effects of metformin,SB203580,N-acetylcysteine on P22 phox protein expression of rat glomerular MCs cultured in high glucose medium Compared with Group HG(0.64±0.2),the P22 phox protein expression decreased to 0.36±0.1 in goupe MET,0.46±0.1 in goupe SB,0.33±0.1 in goupe NAC,respectively. differences are significant(P<0.05) 4.Comparison of p38 MAPK protein phosphorylation expression of rat glomerular MCs between different groups 4.1 Change of p38 MAPK protein phosphorylation expression of rat glomerular MCs cultured in high glucose medium After rat glomerular MCs have been cultured in high glucose medium for 48 hours,the p38 MAPK protein phosphorylation expression increased significantly to 0.54±0.2.Compared with Group NC(0.14±0.04), differences are significant(P<0.05) 4.2 Effects of metformin,SB203580,N-acetylcysteine on p38 MAPK protein phosphorylation expression of rat glomerular MCs cultured in high glucose medium Compared with Group HG(0.54±0.2),the p38 MAPK protein phosphorylation expression decreased to 0.39±0.1 in goupe MET,0.26±0.1 in goupe SB,0.43±0.1 in goupe NAC,respectively.differences are significant(P<0.05) 5.Comparison of MDA and AOPPs content in supernatant fluid between different groups 5.1 Change of MDA and AOPPs content in supernatant fluid of high glucose concentrations After rat glomerular MCs have been cultured in high glucose medium for 48 hours,the MDA content increased significantly to 846.8±87.2.Compared with GroupNC(627.2±62.9), differences are significant(P<0.05).The AOPPs content increased significantly to 851.4±127.3.Compared with Group NC(557.6±94.7), differences are significant(P<0.05). 5.2 Effects of metformin,SB203580,N-acetylcysteine on MDA and AOPPs content in supernatant fluid of high glucose concentrations Compared with Group HG(846.8±87.2),the MDA content decreased to 733.2±71.3 in goupe MET,770.4±76.0 in goupe SB,720.2±69.1 in goupe NAC,respectively. differences are significant(P<0.05). Compared with Group HG(851.4±127.3),the AOPPs content decreased to 649.8±109.8 in goupe MET,695.6±112.9 in goupe SB,598.6±103.4 in goupe NAC,respectively.differences are significant(P<0.05). 6.Comparison of SOD and CAT activity in supernatant fluid between different groups 6.1 Change of SOD and CAT activity in supernatant fluid of high glucose concentrations After rat glomerular MCs have been cultured in high glucose medium for 48 hours,the SOD activity decreased to 130.2±28.4. Compared with Group NC(171.8±42.6), differences are significant(P<0.05).As well,the CAT activity decreased to 14.0±3.5.Compared with Group NC(34.4±8.1), differences are significant(P<0.05) 6.2 Effects of metformin,SB203580,N-acetylcysteine on SOD and CAT activity in supernatant fluid of high glucose concentrations Compared with Group HG(130.2±28.4),the SOD activity increased to 145.4±33.5 in goupe MET,149.4±34.3 in goupe SB,151.6±36.5 in goupe NAC,respectively. differences are significant(P<0.05) Compared with Group HG(14.0±3.5),the CAT activity increased to 25.4±5.8 in goupe MET,21.0±5.1 in goupe SB,26.6±6.3 in goupe NAC,respectively.differences are significant(P<0.05) 7 Analysis of correlation between p38 MAPK protein phosphorylation expression of rat glomerular MCs and intracellular ROS levelThe p38 MAPK protein phosphorylation expression was positively correlated with intracellular ROS level(r=0.770,P<0.05) 8 Analysis of correlation between p38 MAPK protein phosphorylation expression of rat glomerular MCs and MDA content in supernatant fluid The p38 MAPK protein phosphorylation expression was positively correlated with MDA content(r=0.909,P<0.05) 9 Analysis of correlation between p38 MAPK protein phosphorylation expression of rat glomerular MCs and AOPPs content in supernatant fluid The p38 MAPK protein phosphorylation expression was positively correlated with AOPPs content(r=0.941,P<0.05) 10 Analysis of correlation between p38 MAPK protein phosphorylation expression of rat glomerular MCs and SOD activity in supernatant fluid The p38 MAPK protein phosphorylation expression was negatively correlated with SOD activity(r=-0.882,P<0.05) 11 Analysis of correlation between p38 MAPK protein phosphorylation expression of rat glomerular MCs and CAT activity in supernatant fluid The p38 MAPK protein phosphorylation expression was negatively correlated with CAT activity(r=-0.890,P<0.05) 12 Analysis of correlation between p38 MAPK protein phosphorylation expression and P22 phox protein expression of rat glomerular MCs The p38 MAPK protein phosphorylation expression was positively correlated with P22 phox protein expression(r=0.858,P<0.05) 13 Analysis of correlation between p38 MAPK protein phosphorylation expression and P22 phox m RNA expression of rat glomerular MCs The p38 MAPK protein phosphorylation expression was positively correlated with P22 phox m RNA expressionm(r=0.875,P<0.05)Conclusions(1)High concentration of glucose decreased the activity of SOD and CAT in supernatantfluid significantly,and increased the level of MDA and AOPPs in supernatant fluid, intracellular p22 phox m RNA and protein, phosphorylation of p38 MAPK protein expression as well as ROS production of rat glomerular MCs.(2)Metformin can inhibit oxidative stress induced by high concentration of glucose in rat glomerular mesangial cells,and it may be related to the inhibition of over expression of P22 phox m RNA and protein,p38 MAPK protein phosphorylation,which may be one of the mechanisms of its renal protection.