| Backgrounds:With the development of China’s economy and the improvement of people’s living standard,the incidence of diabetes mellitus(DM)is increasing year by year.Diabetic kidney disease(DKD)has become the second leading cause of End-stage renal disease(ESRD)in recent years,accounting for about 25%of the total ESRD patients,and the DKD patient morbidity is continuously increasing.Microalbuminuria has been accepted as the earliest marker for development of DKD,however,it has been reported that a large proportion of renal impairment occurs even before appearance of microalbuminuria.Once urinary albumin excretion is increased,renal damage will aggravate progressively,so early diagnosis and early treatment of DKD are essential.Podocytes,also called glomerular visceral epithelial cells,are the outermost layer of glomerular filtration membrane,which constitute the last molecular charge barrier of filtration membrane.There are podocyte injury in the early stage of DKD.But the pathophysiology of DKD is complex and not yet fully elucidated.Studies have confirmed that the occurrence and development of DKD are closely related to the inflammation and oxidative stress,but the exact mechanism remains unclear.Recent studies have demonstrated that urinary podocyte marker proteins were significantly elevated in normoalbuminuric type 2 diabetes mellitus(T2DM)patients compared with non-diabetic control subjects and could be used as markers for earlier,specific and accurate prediction of DKD.Metformin is a first-line drug for the treatment of T2DM,many studies have confirmed that metformin has a protective effect on kidney through anti-inflammatory,anti-oxidative stress,which is independently of its hypoglycemic effect.So metformin can prevent and delay the occurrence and development of DKD,the mechanism remains to be exploded.But there are few studies on the relationship between inflammation,oxidative stress and their interaction with podocyte injury and the intervention of metformin.Therefore,we hypothesized that metformin could play the renal protective effect by inhibiting the inflammatory response and oxidative stress in patients with DKD.In this study,we first explored the urinary inflammatory factors such as tumor necrosis factor-alpha(TNF-a),monocyte chemoattractant protein-1(MCP-1),macrophage migration inhibitory factor(MIF),urinary oxidative damage markers 8-hydroxy-deoxyguanosine(8-OhdG)in relation to urinary albumin,urinary podocyte protein marker protein(podocalyxin,PCX)and nephrin excretion in T2DM patients,and then further compared the excretion level of urinary albumin,urinary inflammatory factors,urinary 8-OhdG,urinary PCX and nephrin between the combined metformin treatment group and non combined metformin treatment group with similar blood glucose control.Then the protective effect of metformin on podocyte and its relationship with inhibition of inflammation and antioxidation are discussed in order to provide a theoretical basis for the renal protection of metformin.Part 1 Relationship between inflammatory factors,oxidative stress and glomerular podocyte marker protein excretion in diabetic patientsObjective:To observe the changes of urinary inflammatory factors such as TNF-a,MCP-1,MIF,urinary oxidative damage markers 8-OhdG and glomerular podocyte marker protein(PCX),nephrin excretion and the relationship between urinary inflammatory factors,oxidative damage markers and urinary podocyte marker protein excretion in type 2 diabetic patients with different levels of albuminuria.Methods:(1)180 patients with T2DM in endocrinology clinic of Anhui Provincal Hospital were divided into two groups according to urinary albumin levels,whose glycated haemoglobinA1c(HbA1c)were<9%,82 patients in normal albuminuria group(UACR<30mg/gCr,NA group),98 patients in microalbuminuria group(30mg/gCr<UACR<300mg/gCr,MA group).60 cases of normal control group(NC group)are enrolled from people doing health examination in our hospital.Venous blood of patients were collected and the levels of fasting blood glucose(FBG),postprandial blood glucose(PBG),HbAlc,serum creatinine(SCr)were measured respectively.Urinary albumin,TNF-a,MCP-1,MIF,8-OhdG,PCX and nephrin were measured and expressed as a ratio of urinary creatinine(abbreviated as UACR,UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR).(2)FBG,PBG were measured by glucose oxidase;HbAlc was determined by high performance liquid chromatography(HPLC);Urinary albumin was measured by turbidimetric immunoassay;SCr and UCr were measured by the method of creatine oxidase;Urine TNF-a、MCP-1、MIF、8-ohdG、PCX and nephrin levels were measured by enzyme-linked immunosorbent assay(ELISA).(3)The differences of the indexes of blood glucose,SCr,UACR,UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR in each group were analyzed.SPSS22.0 software was used for statistical analysis.Independent sample t test was used to compare the mean of two samples,variance analysis SNK method was used to compare the mean of multiple samples,and Kruskal Wallis method was used to test the data which did not conform to normal distribution.Correlation factors of UACR,UPCR,UNCR in group T2DM were analyzed by Pearson correlation analysis and multiple linear stepwise regression analysis.P-values<0.05 were considered to be statistically significant.Results:(1)Compared with NC group,UACR(3 3.19±16.3 5mg/gCr vs.10.38±1.42mg/gCr),UTCR(112.51±29.16pg/ngCr vs.63.17±8.79pg/ngCr),UMCR(96.70±20.53pg/ngCr vs.58.47±6.13pg/ngCr),UMIFCR(71.04±19.35ng/mgCr vs.29.25±4.40ng/mgCr),U8CR(24.90±8.68ng/mgCr vs.12.83±2.33ng/mgCr),UPCR(60.26±14.09ng/mgCr vs.34.37±4.88ng/mgCr)and UNCR(69.78±17.16ng/mgCr vs.22.46±5.15ng/mgCr)increased significantly in DM group,the difference was statistically significant(P<0.05).(2)The NC,NA and MA three groups of UTCR(63.17±8.79pg/ngCr vs.89.36±21.55pg/ngCr vs.131.88±18.70pg/ngCr),UMCR(58.47±6.13pg/ngCr vs.82.18±15.56pg/ngCr vs.108.85±15.75pg/ngCr),UMIF CR(29.25±4.40ng/mgCr vs.59.20±16.40ng/mgCr vs.80.95±15.77ng/mgCr),U8CR(12.83±2.33ng/mgCr vs.19.89±7.47ng/mgCr vs.29.08±7.32ng/mgCr),UPCR(34.37±4.88ng/mgCr vs.50.91±12.00ng/mgCr vs.68.09±10.50ng/mgC),UNCR(22.46±5.15ng/mgCr vs.58.65±13.11ng/mgCr vs.79.09±14.41ng/mgCr),the difference was statistically significant(P<0.05),and gradually increased with the increase of urinary albumin level.(3)UACR had positive correlation with UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR in DM group(r=0.85,P<0.01;r=0.75,P<0.01;r=0.68,P<0.01;r=0.66,P<0.01;r=0.74,P<0.01;r=0.68,P<0.01).UPCR had positive correlation with UTCR,UMCR,UMIFCR and U8CR in DM group(r=0.71 P<0.01;r=0.73,P<0.01;r=0.68,P<0.01;r=0.57,P<0.01);UNCR had positive correlation with UTCR,UMCR,UMIFCR and U8CR(r=0.71 P<0.01;r=0.76,P<0.01;r=0.68,P<0.01;r=0.61,P<0.01).Conclusions:There is increased microinflammation and oxidative stress in patients with T2DM,which is closely related to the podocyte injury.Increased excretion of urinary podocyte marker protein is a sensitive indicator of early DKD podocyte injury.Part 2 Protective effect of metformin on glomerular podocyte in type 2 diabetic patients and its relationship with anti-inflammatory and anti-oxidant effectsObjective:To observe the effects of metformin treatment on urinary cytokines(TNF-a,MCP-1 and MIF),8-OhdG and podocyte marker proteins(PCX and nephrin)excretion in T2DM patients and explore its protective effects on diabetic renal injury.Methods:(1)① The first part 180 cases of T2DM with HbAlc<9%were named diabetes mellitus group A(A-DM group),whose blood pressure,SCr were in normal range and UACR was less than 300 mg/gCr.Then the A-DM group was further divided into insulin group(A-INS group,n=93)and sulfonylurea group(A-SU group,n=87),and then the two subgroups were divided according to whether or not combined with metformin(at least 3 months),grouped as follows:A-INS-Ⅰ group(insulin only,n=47)and A-INS-Ⅱ group(insulin combined with metformin,n=46),A-SU-I group(sulfonylurea only,n=42)and A-SU-Ⅱ group(sulfonylurea combined with metformin,n=45).60 cases of normal control group(NC group)are enrolled from people doing health examination in our hospital.Venous blood of patients were collected and the levels of FBG,PBG,HbAlc,SCr were measured respectively.Urinary albumin,urinary TNF-a,MCP-1,MIF,8-OhdG,PCX and nephrin were measured and expressed as a ratio of urinary creatinine(abbreviated as UACR,UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR).② 60 cases of T2DM patients with poor blood glucose control(9%<HbAlc<13%)In endocrinology clinic or inpatients of Anhui Provincal Hospital were further divided into insulin group(B-INS group,n=60),and then the B-INS group was divided according to whether or not combined with metformin,grouped as follows:B-INS-I group(n=27):Adjust insulin dose according to blood glucose,B-INS-Ⅱ group(n=33):Insulin therapy plus metformin treatment.After 6 months of observation,HbAlc<9%was reviewed.60 cases of normal control group(NC group)are enrolled from people doing health examination in our hospital.Before and after 6 months,venous blood of patients were collected and the levels of FBG,PBG,HbAlc,SCr were measured respectively.Urinary albumin,TNF-a,MCP-1,MIF,8-OhdG,PCX and nephrin were measured and expressed as a ratio of urinary creatinine(abbreviated as UACR,UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR).(2)The differences of the indexes of blood glucose,SCr,urinary albumin,urinary inflammatory factors,urinary 8-OhdG,urinary PCX and nephrin excretion in each group were analyzed.SPSS22.0 software was used for statistical analysis.The values of all parameters between diabetic groups and NC group,the differences between the subgroups were assessed by independent-sample t test,the differences between the pre-observation and post-observation were assessed by student’s paired t test.The data that do not conform to normal distribution are tested by rank sum test with two samples.P-values<0.05 were considered to be statistically significant.Results:(1)Comparison of A-DM groups:Compared with A-INS-Ⅰ,UACR(19.22±7.54mg/gCr vs.44.27±11.27mg/gCr),UTCR(81.43±13.21pg/ngCr vs.134.79±14.23pg/ngCr),UMCR(78.25±9.15pg/ngCr vs.116.86±15.33pg/ngCr),UMIFCR(55.98±14.37ng/mgCr vs.83.42±16.35ng/mgCr),U8CR(19.33±7.21ng/mgCr vs.28.84±7.07ng/mgCr)、UPCR(48.85±10.59ng/mgCr vs.71.14±9.93ng/mgCr)、UNCR(55.52±8.65ng/mgCr vs.83.97±15.99ng/mgCr)decreased significantly in A-INS-Ⅱ group,the difference was statistically significant(P<0.05);Compared with A-SU-I,UACR(21.95±8.80mg/gCr vs.48.15±12.29mg/gCr),UTCR(94.54±13.18pg/ngCr vs.140.87±16.64pg/ngCr),UMCR(83.03±10.33pg/ngCr vs.109.00±12.70pg/ngCr),UMIFCR(62.37±14.82ng/mgCr vs.82.98±14.62ng/mgCr),U8CR(22.35±8.69ng/mgCr vs.29.30±7.44ng/mgCr),UPCR(52.48±9.89ng/mgCr vs.68.91±10.18ng/mgCr),UNCR(60.47Q9.50ng/mgCr vs.79.48±13.02ng/mgCr)decreased significantly in A-SU-II group,the difference was statistically significant(P<0.05).(2)Compared with NC group,UACR(50.23±9.49mg/gCr vs.10.3 8± 1.42mg/gCr),UTCR(144.82±14.93pg/ngCr vs.63.17±8.79pg/ngCr),UMCR(132.32±11.15pg/ngCr vs.58.47±6.13pg/ngCr),UMIFCR(101.98±8.48ng/mgCr vs.29.25±4.40ng/mgCr),U8CR(55.33±5.78ng/mgCr vs.12.83±2.33ng/mgCr),UPCR(87.86±8.58ng/mgCr vs.34.37±4.88ng/mgCr)and UNCR(86.74±10.96ng/mgCr vs.22.46Q5.15ng/mgCr)increased significantly in B-DM group,the difference was statistically significant(P<0.05).(3)Comparison of B-DM groups:After 6 months of the observation,UACR,UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR decreased significantly compared those of pre-observation in B-INS-I group,which were(45.68±9.46mg/gCr vs.50.69±8.30mg/gCr),(135.95±14.78pg/ngCr vs.145.94±13.42pg/ngCr),(117.54±11.11pg/ngCr vs.131.06±12.02pg/ngCr),(86.23±8.70ng/mgCr vs.99.79±9.67ng/mgCr),(41.27±6.68ng/mgCr vs.54.33±15.95ng/mgCr),UPCR(73.38±8.25ng/mgCr vs.89.11±9.26ng/mgCr)and(78.00±8.49ng/mgCr vs.87.98±12.35ng/mgCr),respectively,(P<0.05).After 6 months of the observation,UACR,UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR decreased significantly compared those of pre-observation in B-INS-Ⅱ group,which were(30.28±7.07mg/gCr vs.49.86±10.47mg/gCr),(116.32±12.82pg/ngCr vs.143.91±16.22pg/ngCr),(93.54±9.64pg/ngCr vs.133.34±10.45pg/ngCr),(70.26±8.52ng/mgCr vs.103.76±7.02ng/mgCr),(27.25±4.65ng/mgCr vs.56.15±5.60ng/mgCr),(51.50±8.52ng/mgCr vs.86.85±7.99ng/mgCr)and(59.39±8.00ng/mgCr vs.85.73±9.76ng/mgCr),respectively,(P<0.05).Before observation there was no difference in UACR,UTCR,UMCR,UMIFCR,U8CR,UPCR and UNCR between B-INS-I group and B-INS-II group,which were(50.69±8.30mg/gCr vs.49.86±10.47mg/gCr),(145.94±I3.42pg/ngCr vs.143.91±16.22pg/ngCr),(131.06±12.02pg/ngCr vs,133.34±10.45pg/ngCr),(99.79±9.67ng/mgCr vs.103.76±7.02ng/mgCr),(54.33±5.95ng/mgCr vs.56.15±5.60ng/mgCr),(89.11±9.26ng/mgCr vs.86.85±7.99ng/mgCr)and(87.98±12.35ng/mgCr vs.85.73±9.76ng/mgCr),(P>0.05).Compared with B-INS-Ⅰ group,UACR(30.28±7.07mg/gCr vs.45.68±9.46mg/gCr),UTCR(116.32±12.82pg/ngCr vs.135.95± 14.7 8pg/ngCr),UMCR(93.54±9.64pg/ngCr vs.117.54±11.1 lpg/ngCr),UMIFCR(70.26±8.52ng/mgCr vs.86.23±8.70ng/mgCr),U8CR(27.25±4.65ng/mgCr vs.41.27±6.68ng/mgCr),UPCR(51.50±8.52ng/mgCr vs.73.38±8.25ng/mgCr)and UNCR(59.39±8.00ng/mgCr vs.78.00±8.49ng/mgCr)decreased significantly in B-INS-Ⅱ group after the observation,the difference was statistically significant(P<0.05).Results:Metformin can effectively reduce the urinary excretion of inflammatory factors,oxidative damage markers and podocyte marker proteins,which may be related to the renal protective function.Full text Conclusions:1.There is increased microinflammation and oxidative stress in patients with T2DM,which is closely related to the podocyte injury.2.Increased excretion of urinary podocyte marker protein is a sensitive indicator for early diagnosis of DKD in T2DM patients.3.Metformin can effectively reduce the urinary excretion of podocyte marker proteins,suggesting that it has a protective effect on podocytes and is not entirely dependent on the decrease of blood glucose.4.Metformin can effectively reduce the urinary excretion of inflammatory factors and 8-OhdG,which is partly related to its inhibition on increased inflammation and oxidative stress in Vivo and kidney. |
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