| Backgrounds:Diabetes(diabetes mellitus,DM)incidence increased gradually as the change of lifestyle and aging of population,and the diabetic kidney disease(DKD)patients are also increasing year by year.DKD is the leading cause of end stage renal failure,as well as an important influencing factor of its high morbidity and high mortality rate.Once a large amount of albuminuria occurs,the rapid decline of renal function would usually follow and cause poor prognosis and high mortality.Therefore,it has important clinical significance to find ways of early diagnosis and intervention.At present,DKD early screening is mainly based on microalbuminuria,but recent studies have found that microalbuminuria is not a perfect indicator of early diabetic nephropathy diagnosis and prognosis.It has been confirmed that the immune mediated inflammatory reaction is an indispensable factor during the development of diabetic nephropathy.The changes of cytokines related to inflammation also occur in diabetic patients with normal albuminuria,indicating that microinflammation may be earlier than the occurrence of microalbuminuria.Biomarkers of glomerular or tubular dysfunction associated with microinflammation may have occurred before microalbuminuria.Neutrophil gelatinase-associated lipocalin(NGAL)is a member of the superfamily of lipid carrier proteins.Recent studies have shown that NGAL can serve as an important indicator of renal tubular injury and is an ideal biological marker for the early diagnosis of acute kidney injury.Some studies have found that NGAL may participate in the inflammatory lesion of renal tissue as an inflammatory associated factor in chronic kidney disease.Whether NGAL is involved in the occurrence and development of diabetic nephropathy,and whether it can be used as a marker for the early diagnosis and monitoring of DKD,these are worth exploring.Recent studies have found that metformin can improve albuminuria levels in DKD,although the mechanism is not entirely clear,it is worth noting that it does not depend solely on metformin's hypoglycemic effect.Studies have reported that inflammatory reactions play an important role in DKD and chronic kidney disease,but there are few studies on the changes and roles of inflammatory response in the renal protection of metformin in DKD.Therefore,we hypothesized that metformin could reduce the renal protective effect of proteinuria by inhibiting the inflammatory response in patients with DKD.So the relationship between the expression of NGAL,IL-10,IL-6,TNF-alpha in serum and urine of patients with Type 2 diabetes was validated in the study,followed by observation of changes in the level of proteinuria,IL-10,and NGAL IL-6,TNF-alpha and other inflammatory factors in T2DM patients taking metformin with different doses and after treatment,to explore the role of metformin in the therapeutic value and possible mechanism in DKD,and to provide important theoretical basis for the prevention and application of metformin in DKD.Part 1 The value of blood and urine NGAL protein determination in diabetic nephropathy and its correlation with inflammatory factorsObjectives:To observe the changes of blood,urine,NGAL and some inflammatory factors type 2 diabetic kidney disease,and to investigate whether NGAL is a marker for early diagnosis and monitoring of diabetic kidney disease associated with micro-inflammation.Methods:(1)93 patients with T2DM(WHO,1999)in outpatient and physical examination department of Anhui Provincal Hospital were divided into three groups,31 patients in normal albuminuria group(NA group),31 patients in microalbuminuria group(MA group)and 31 patients in clinical albuminuria group(CA group)according to urine albumin levels.31 cases of normal control group(NC group)are enrolled from people doing health examination in our hospital.Venous blood of patients were collected on an empty stomach,and the levels of blood glucose,lipids,glycosylated hemoglobin,serum creatine,NGAL,IL-10,IL-6 and TNF-a were measured respectively.Clean midstream urine samples were taken to measure urine microalbumin(UAlb),urine creatinine(UCr),urine NGAL and MCP-1.(2)Fasting serum NGAL,IL-10,IL-6,TNF-alpha and urine NGAL and MCP-1 levels were measured by ELISA(enzyme-linked immunosorbent assay);UAlb was measured by turbidimetric immunoassay;SCr and UCr were measured by the method of creatine oxidase;Fasting plasma glucose was measured by glucose oxidase;The blood lipid level was measured by enzyme method;HbA1c was determined by high performance liquid chromatography(HPLC).(3)The differences of the indexes of glycolipid metabolism,inflammatory factors,urinary albumin and serum creatinine in each group were analyzed.The trend of NGAL in vivo and the correlation with the above indexes were analyzed.SPSS 16.0 software was used for statistical analysis.The stepwise regression analysis was used to analyze the influencing factors of the variables.Results:(1)In group T2DM,serum NGAL,IL-6,TNF-alpha and urine MCP-1 levels increased,while blood IL-10 levels decreased,and the difference was statistically significant(P<0.01).Compared with NC group,serum NGAL(84.95±127.30ng/ml vs.30.66± 11.66ng/ml),IL-6(11.72 ±2.54pg/ml vs.7.19±2.19pg/ml)and TNF-alpha(9.87±2.08pg/ml vs.5.84±1.56pg/ml)levels increased significantly in T2DM grouop,the difference was statistically significant(P<0.05).The levels of NGAL(51.24 ± 35.85ng/ml vs.16.15 ± 3.52ng/ml)and MCP-1(98.40 ±28.25pg/ml vs.57.12 ± 14.5pg/ml)in urine were significantly higher,and the difference was statistically significant(P<0.05).Level of serum IL-10(2.31±0.70 pg/ml vs.4.28 ±1.15 pg/ml)decreased significantly,the difference was statistically significant(P<0.05).(2)Compared with group NC,blood NGAL(43.26 ± 10.70ng/ml vs.30.66± 11.66ng/ml)and urine NGAL(20.39 ± 4.32ng/ml vs.16.15 ± 3.52ng/ml)levels were significantly higher in group NA,and the difference was statistically significant(P<0.05).(3)Comparison of T2DM groups:the three groups of urine MCP-1(122.13±24.16pg/ml vs.92.37±28.31pg/ml8 vs.0.69±10.91pg/ml),blood TNF-alpha(11.26±1.88pg/ml vs.110.27±1.74pg/ml vs.8.07±1.10pg/ml),the difference was statistically significant(P<0.05),and gradually decreased with the decrease of urinary protein level.The difference between the HBA1C and MA group of CA group(6.96±1.17mmol/l vs.7.25±1.02mmol/l)and NA group(6.96±1.17mmol/l vs.6.19±0.53mmol/l)was statistically significant(P<0.05).The differences between the three groups of urine NGAL(97.33±20.66ng/ml3vs.6.00±8.78ng/ml vs.20.39±4.32ng/ml)were statistically significant(P<0.05).and decreased with the decrease of urinary protein level.The blood NGAL of the CA group was significantly higher than that in the MA group(143.50±208.82ng/ml vs 68.08±18.55ng/ml)and NA group(143.50±208.82ng/ml vs 43.26±10.70ng/ml),which was statistically significant(P<0.05).(3)Stepwise linear regression showed that urine ACR(t=445.034,P<0.001)and blood TNF-alpha(t=13.822,P<0.001)were the influencing factors of urine NGAL in patients with T2DM,and positively correlated with urine NGAL.Glycosylated hemoglobin(t=3.266,P=0.002)and urine ACR(t=2.065,P=0.011)were the blood NGAL influencing factors,and positively correlated with blood NGAL.Conclusions:There are changes of inflammatory cytokine in T2DM,and inflammation may play an important role in pathogenesis of DKD and progression of disease.NGAL has been significantly increased in T2DM normal albuminuria period and is positively correlated with UACR.Blood and urine NGAL are sensitive indicators for early diagnosis and monitoring of DKD.NGAL may be involved in the development of DKD as an important link in chronic inflammation and glucose metabolism disorders.Part 2 Protective effect of metformin on type 2 diabetes mellitus and its relationship with NGAL and inflammatory factorsObjectives:Observe the influence of different dosages and duration of metformin on urinary albumin,NGAL and inflammatory cytokines in vivo in patients with T2DM,and explore the role of metformin in therapeutic value and possible mechanism in DKD,to provide an important theoretical basis for prevention and application of metformin in DKD.Methods:(1)281 patients were divided into the metformin group(N=112)and the non-metformin group(N= 169)according to the accepted antidiabetic regimen.Patients in metformin group were divided into group lg/d(n=91)and group 1.5g/d(n=21)according to the dose.According to the course of taking metformin,patients were divided into less than 1 year group(n=71)and more than 1 year group(n=41).Venous blood of patients were collected on an empty stomach,and the levels of blood glucose,lipids,glycosylated hemoglobin,serum creatine,NGAL,IL-10,IL-6 and TNF-? were measured respectively.Clean midstream urine samples were taken to measure urine microalbumin(UAlb),urine creatinine(UCr),urine NGAL and MCP-1.(2)Fasting serum NGAL,IL-10,IL-6,TNF-alpha and urine NGAL and MCP-1 levels were measured by ELISA(enzyme-linked immunosorbent assay);UAlb was measured by turbidimetric immunoassay;SCr and UCr were measured by the method of creatine oxidase;Fasting plasma glucose was measured by glucose oxidase;The blood lipid level was measured by enzyme method;HbAlc was determined by high performance liquid chromatography(HPLC).The differences of glucose and lipid metabolism indexes,NGAL,inflammatory factors,urinary microalbumin and serum creatinine in each group were analyzed.Statistical analysis was carried out by Graphpad Prism software.Results:(1)Compared with the non-metformin group,UACR[21.27(17.72,25.57)mg/g vs.26.71(23.52,29.13)mg/g]and blood NGAL(48.997 ± 20.830ng/ml vs.64.263 ± 22.148ng/ml),urinary MCP-1(113.85 ± 18.31pg/ml vs.122.46 ±25.82pg/ml)in metformin group were significantly decreased,the difference was statistically significant(P<0.05).The level of serum IL-6(11.52±2.57pg/ml vs.13.35± 3.15 pg/ml)in metformin group was significantly decreased,the difference was statistically significant(P<0.05).(2)Compared with metformin(1g/d)group,urinary MCP-1(97.46 ± 18.62pg/ml vs.17.64 ± 16.10 pg/ml)and urinary ACR[15.32(12.52,20.16)mg/g vs.22.13(19.45,26.32)of mg/g]in metformin(1.5g/d)group were significantly decreased,the difference was statistically significant(P<0.05).(3)Compared with the less than I year group,the blood IL-10(2.42 ± 0.67pg/ml vs.2.71± 0.59pg/ml)levels increased significantly in the more than 1 year group,and the difference was statistically significant(P<0.05),while Urine NGAL(24.51 ±14.47ng/ml vs.35.57 ± 32.47ng/ml)levels decreased significantly,the difference was statistically significant(P<0.05).Conclusions:Metformin can reduce urinary albumin in T2DM patients and have definite renal protection.Metformin can reduce the level of proinflammatory cytokines and NGAL in T2DM patients,and its protective effect on renal function may be related to the regulation of NGAL and inflammatory factors in vivo.The anti-inflammatory and protective effects of metformin may be time and dose dependent.Full text Conclusions:1.There are changes of inflammatory cytokine in T2DM,and inflammation may play an important role in pathogenesis of DKD and progression of disease.2.NGAL may be involved in the development of DKD as an inflammatory associated factor.?Blood and urine NGAL are sensitive indicators for early diagnosis and monitoring of DKD.3.Metformin has a renal protective effect in T2DM and may be related to its regulation of NGAL and inflammatory factors in vivo.The anti-inflammatory and protective effects of metformin may be time and dose dependent. |
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