Expression And Clinical Significance Of Plasm Growth Differentiation Factor-15,Vascular Endothelial Growth Factor And Endostatin In Patients With Type2Diabetic Nephropathy

BackgroundDiabetes mellitus（DM） is a set of clinical syndrome characteristic with disorder of sugar,which is caused by interaction between genetic and environmental factors.With the development of global economy, population aging and the improvement of people’s living standards.the global prevalence of DM is increasing year by year.Global number of DM is280million by2010,and the number will reach430million until2030.DM has become the third serious noninfectious chronic disease,the rates of its prevalence,morbidity and mortality are only behind cancer and cardiac vascular disease. Diabetic nephropathy（DN） is a specific and also one of the most common chronic microvascular complication of DM. According to statistics,about30%to40%patients of type1diabetes mellitus（T1DM） and20%patients of type2diabetes mellitus will occur DN.One of the characteristics of DN is that once renal damage appears, renal function deteriates at a greater rate in those with diabetic renal damage compared to those with non-diabetic renal damage.DN eventually develops into end-stage renal disease（ESRD）,which requires treatment of dialysis or renal transplant. With increasing of prevalence of DM,DN has become the main reason of ERSD in European and American countries and the second reason of ERSD in China.Clinical observation found that the long-term prognosis of patients with DN is worse than that with other kidney disease when patients enter the stage of ERSD even if those patients get therapy by dialysis or renal transplant,and those patients with DN often merge cardiovascular complications,the mortality of those patients is high.Therefore,DN has become a major problem affecting human health.The basic pathological changes of DN is proliferation of mesangial cells and increased extracellular matrix of mesangial area, thickening of glomerular capillary basement membrane and increased capillary permeability,damage and decrease in number of podocyte,hypertrophy of renal tubular,thickening and stratification of renal tubular basement membrane,hyaline degeneration and arteriosclerosis of efferent and afferent glomerular arteriole.With the appearance of proteinuria,they lead to the deterioration of glomerulosclerosis and renal interstitial fibrosis and, eventually leading to ERSD.The pathogenesis of DN is very complex and not yet fully elucidated till now.Current studies consider that DN is the result caused by interaction of multifactors which include change of renal hemodynamics,disorder of glucose and lipid metabolism,oxidative stress,cytokines,insulin resistance and gentic factor.In the process of development and progression of DN, cytokines are involved in many aspects such as glomerular hemodynamics change, cell proliferation and hypertrophy.A wide variety of cytokines function by the way of autocrine,paracrine and endocrine.They influence and restrain each other, constitute the complex network of cytokines,and play an important role in the pathogenesis of DN.Growth differentiation factor-15（GDF-15）,also known as macrophage inhibitory cytokine-1（MIC-1）,is a divergent member of the TGF-β superfamily. GDF-15is a product of macrophage in the condition that a variety of pathological and environmental stress activates macrophage. Activated macrophage up-regulates expression of GDF-15through secreting TNF-α.GDF-15has a effective feedback effect on macrophage by the way of inhibiting TNF-α secretion by activated macrophage,named MIC-1,which is a kind of stress protein and plays a role of a wide range of protective regulation in body. Using gene chip technology,researchers found that GDF-15,as one of the main regulation factors, have obviously up-regulated expression at early stage of renal damage （7days） in animal models of diabetes. Another basic research found that renal damage of GDF-15knockout mice is heavier than that of wild-type mice in both type1and type2models of diabetes.At present,GDF-15has come under increasing scrutiny as a novel cardiovascular protection factor.GDF-15level is closely related to the diagnosis,risk stratification and prognosis of several cardiovascular diseases including hear failure,acute coronary syndrome and atherosclerosis.Recent studies found that circulating GDF-15level of T2DM also increased significantly,which might be associated with internal persistent hyperglycemia environment and systemic inflammation.A small amount of foreign clinical studies found that GDF-15was not only a good predictor of all-cause and cardiovascular mortality in type2diabetic patients with nephropathy,can predict worsening of albuminuria in patients with type2diabetes either. However,clinical study about the expression level and change of plasm GDF-15in different clinical stages of type2diabetic nephropathy is less.Vascular endothelial growth factor （VEGF） is a kind of growth factor acting selectively on endothelial cell,which is mainly secreted by podocytes and tubular epithelial cells in kidney.The biological characteristics of VEGF include increasing vascular permeability,promoting angiogenesis and increasing adhesion molecules,etc.Under physiological conditions,low level of VEGF secreted by podocytes has the function of maintaining endothelial hole, right amount of VEGF can protect and repair glomerular endothelial cells.Hyperglycemia,angiotensin II, TGF-βand advanced glycation end-products can induce overexpression of VEGF.Excessive VEGF is able to decrease the resistance of glomerular endothelial cells and increase permeability of endothelial cells,thus leading to proteinuria,promoting secretion of a variety of fiber factors,inducing deposition of extracellular matrix,promoting thickening of glomerular basement membrane and renal interstitial fibrosis,eventually glomerulosclerosis.Thus,VEGF has an important role in the pathogenesis of DN.In animal models,blocking VEGF could effectively improve DN and alleviate proteinuria.At the same time,abundant clinical studies indicated that plasm VEGF concentration of patients with DN had the varying degree of increasing in different DN stages.Endostatin（ES） is antagonist of VEGF. ES and VEGF are the strongest pair of factors which mediate the angiogenesis,that is,inhibits and promots angiogenesis. In animal model,expression of ES and VEGF in plasm and kidney tissue of diabetic rats increased. Both ES and VEGF were significantly positively associated with severity of DN.Plasm concentration of ES and VEGF are significantly positively related.It was reported that level of plasm ES in patients with chronic kidney or diabetic retinopathy were significantly higher compared to normal controls.However,there is no study which reports the change of ES level in patients with DN.At the same time, clinical study which investigates the expression level of VEGF and ES in type2diabetic patients, the relationship between those two cytokines and DN is less.Objective:1.The fisrt part of this study detected the level and change of serum GDF-15in different stages of DN by means of enzyme-linked immunosorbent assay（ELISA）,analyzed the correlation between GDF-15and clinical indicators refleting the sverity of diabetic renal damage such as microalbuminuria（mAlb） and estimated glomerular filtration rate（eGFR）.The purpose of this study is to investigate the clinical application value of GDF-15in diagnosis and evaluation of severity of type2diabetic nephropathy.2.The second part of this study detected the level and change of serum VEGF and ES in different Mogensen’s stages of DN by means of ELISA,analyzed the correlation between VEGF and ES in different clinical stages.The purpose of this study is to investigate the clinical significance of VEGF and ES in the development and progression of diabetic nephropathy as well as the association of those two cytokines and metabolism index.Two specific contents are as follows:Part1. Expression and clinical significance of plasm growth differentiation factor-15in patients with type2diabetic nephropathyObjective:To investigate the change of plasm growth differentiation factor-15（GDF-15） in type2diabetic nepropathy patients and its clinical significance.Methods:1.Research subjects:T2DM patients with or without diabetic nephropathy （Subjects were recruited from inpatients of department of endocrinology and metabolism,Nanfang Hospital,from June2013to September2013）.Selection criteria:（1）30to75-year-old;（2） All the T2DM patients were confirmed according to the Word Health Organization（WHO）1999diagonstic and classified criteria;（3） All the participants informed consent to participate in the test.Exclusion criteria:（1）Patients with special types of diabetes.（2）Patients have had acute compliactions of diabetes such as diabetic ketoacidosis and hyperosmotic coma during the last one month.（3）Patients have had cerebral infarction or TIA during the last one month.（4）Patients with coronary heart disease（according to ECG,clinical manifestations and hitory）,acute or chronic heart failure.（5）Patients with ALT or AST two times or more than normal upper limit.（6）Patients with fever,acute infection,operation or other stress;（7）Patients with history of cancer;（8）Patients with urinary tract infection or other kidney diseases,or without diabetic nephropathy.（9）Patients have started renal replacement treatment or dialysis.2. Research methods:2.1Accoding to Mogensen criteria,all patients were divided into three groups:（1）Normoalbuminuria（Group A）:30patients with microalbuminuria<30mg/24h（2）Microalbuminuria group（Group B）:20patients with microalbuminuria≥30mg/24h and <300mg/24h.（3）Macroalbuminuria group（Group C）:30patients with microalbuminuria≥300mg/24h.There were no differences in age,sex and body-mass index（BMI） among three groups.2.2Based on age,sex and creatinine,glomerular filtration rate was calculated with Chronic Kidney Disease Epidemiology Collaboration（CKD-EPI） formula,named estimated glomerular filtration rate（eGFR）.According to eGFR, all patients were divided into three groups.31patients in group D were with normal renal function （eGFR≥90ml/min/1.73m2）,26patients in group E were with mild renal dysfunction（60≤eGFR<90ml/min/1.73m2）,23patients in group E were with chronic renal failure（eGFR<60ml/min/1.73m2）. There were no differences in age,sex and BMI among three groups.2.3Concentrations of GDF-15in plasm in all patients were measured by ELISA.And the clinical and laboratory data of all patients were collected.3.Statistical analysis:Statistical analysis were performed using the SPSS19.0software Package.Variables were expressed as mean±standard deviation（SD） if fitting the normal distribution,and were expressed as median（interquartile range） if not. Comparison of rate were analyzed by Chi-square test.The pairwise comparison were analyzed by Independent-Samples T test or Mann-Whitney U test.Statistical significance of differences among groups was evaluated by one-way ANOVA or Kruskal-Wallis ANOVA.Correlation between GDF-15and common clinical index were analyzed by the partial correlation analysis.Receiver operating characteristic（ROC） curve analysis was used to examine the validity of mAlb and eGFR respectively for diagnosing renal dysfunction（eGFR<90ml/min/1.73m2）.Finally,we performed multiple linear regression analysis with mAlb as dependengt variable and GDF-15as well as other clinical index as independent variables.P<0.05was considered as significant.Results:1.The GDF-15of normoalbuminuria and microalbuminuria group was respectively704.5（548.8-975.8） pg/ml and864.0（636.1-994.3） pg/ml.The level of GDF-15in normoalbuminuria and microalbuminuria group were lower than that of macroalbuminuria group[1773.9（1099.1-2357.4）pg/ml] respectively（P<0.01）.The level of GDF-15in normoalbuminuria was lower than that of microalbuminuria group（P>0.05）.2. The GDF-15of normal renal function group and mild renal dysfuntion group was respectively752.2±340.9pg/ml、1399.5±1145.1pg/ml.The level of GDF-15in normal renal function group and mild renal dysfuntion group were lower than chronic renal failure group（2184.1±1673.8pg/ml）（P<0.01, P<0.05）.The level of GDF-15in normal renal function group was lower than that in mild renal dysfuntion group（P<0.01）.3.Partial correlation showed that the level of plasm GDF-15had significantly positive correlation with diabetes duration,mAlb,blood urea nitrogen（BUN） and serum creatinine（sCr）（r=0.246,0.493,0.390,0.471,P<0.05）,had significantly negative correlation with eGFR and plasm albumin（r=-0.438,-0.397,P<0.01）.4.The regression equation obtained from multiple linear regression analysis was mAlb=-2675.46+19.18SBP+0.49GDF₁5,which showed that GDF-15has an independent correlatin to the level of albuminuria.5.In the diagnosing of decline of renal function（eGFR<90ml/min/1.73m2）,the areas under ROC of GDF-15and mAlb was0.801and0.717respectively.The area under ROC of GDF-15was larger than that of mAlb（P<0.05）.When733.78pg/ml was set as the cut-off value for the diagnosis of renal dysfunction,the specificity and sensitivity of GDF-15achieved88.1%and58.1%.Conclusions:1.The expression of GDF-15increased as the development of type2diabetic nephropathy.There was significatn correlation between GDF-15and classic clinical index which reflect the damage of kidney.And GDF-15had an independent correlatin to the level of albuminuria, could reflect the severity of kidney disease.Thus,GDF-15is expected to become a new kind of blood markers to evaluate the severity of type2diabetic nephropathy.2.There was a significant increase of the concentration of GDF-15at the stage of mild renal dysfunction（eGFR<90ml/min/1.73m2）,and the concentration of GDF-15increased as the decline of eGFR. In the diagnosing of decline of renal function （eGFR<90ml/min/1.73m2）,the area under ROC of GDF-15was larger than that of mAlb.Thus,GDF-15is expected to become a new kind of blood markers to early found type2diabetic nephropathy. Part2. Study on relationship between vascular endothelial growth factor,endostatin and type2diabetic nephropathyObjective:To investigate the possible association between vascular endothelial growth factor（VEGF）,endostatin（ES） and type2diabetic nephropathy.Methods:1.Research subjects,selection and exclusion criteria were same as the first part.2. Research methods:Accoding to Mogensen criteria,all patients were divided into three groups:（1）normoalbuminuria（Group A）:30patients with microalbuminuria<30mg/24h（2） Microalbuminuria group（Group B）:20patients with microalbuminuria≥30mg/24h and<300mg/24h.（3） Macroalbuminuria group（Group C）:30patients with microalbuminuria≥300mg/24h.There were no differences in age,sex and BMI among three groups. Concentrations of VEGF and ES in plasm in all patients were measured by ELISA.And the clinical and laboratory data of all patients were collected.3.Statistical analysis:Spearman’s rank correlation was computed to assess association between variables,the remainder was same as the first part.Results:1.The plasm VEGF level of microalbuminuria group and macroalbuminuria group was respectively50.93（26.99-72.24）pg/ml and63.89（42.84-93.38）pg/ml.The level of VEGF in microalbuminuria group and macroalbuminuria group were higher than that of normoalbuminuria[45.96（24.94-64.37） pg/ml] respectively（P<0.05, P<0.01）.The level of VEGF in macroalbuminuria group was higher than that of microalbuminuria group（P<0.05）.2.The plasm ES level of microalbuminuria group and macroalbuminuria group was respectively95.64（79.65-125.63） pg/ml and170.13（105.52-195.25）pg/ml.The level of ES in microalbuminuria group and macroalbuminuria group were higher than normoalbuminuria[86.67（76.69-119.02） pg/ml] respectively（P<0.05, P<0.01）.The level of ES in macroalbuminuria group was higher than that of microalbuminuria group（P<0.01）.3.In all patients,the level of VEGF in plasm had significantly positive correlation with SBP （r=0.226,P<0.05）, severity of DR （r=0.221,P<0.05）,mAlb （r=0.226,P<0.01） and HDL （r=0.227,P<0.05）.Meanwhile,VEGF had significantly negative correlation with eGFR （r=-0.237,P<0.05） and plasm albumin （r=-0.233,P<0.05）4.In all patients,the level of ES in plasm had significantly positive correlation with diabetes duration （r=0.252,P<0.05）, severity of DR （r=0.229,P<0.05）, SBP (（r=0.312,P<0.01）, BUN （r=0.459,P<0.01）,sCr （r=0.585,P<0.01）,UA （r=0.320,P<0.01） and mAlb （r=0.491,P<0.01）.Meanwhile,ES had significantly negative correlation with eGFR （r=-0.620,P<0.01） and plasm albumin （r=-0.432,P<0.01）5.In normoalbuminuria and microalbuminuria group,the level of VEGF in plasm had significantly positive correlation with ES （r1=0.633, P1<0.01; r2=0.532, P2<0.05）.However,there was no correlation between VEGF and ES in macroalbuminuria group（r3=0.175, P3>0.05）.Conclusion:1.The expression of VEGF and ES increased as the development of type2diabetic nephropathy.There was significat correlation between GDF-15and mAlb as well as eGFR. The joint detection of VEGF and ES is expected to become a new kind of way to evaluate the severity of DN.2.The correlation between VEGF and ES changed in different stages of type2diabetic nephropathy.We speculate that the imbalance of thos two cytokines may promote the development and progression of type2diabetic nephropathy.