The Mechanism Of Renal Protective Effect Of Metformin On Diabetic Kidney Disease

Diabetic kidney disease is the main complication and cause of death of diabetic patients,and also the main cause of chronic kidney failure in China.The pathogenesis of diabetic kidney disease is not completely clear.In addition to lowering blood glucose,metformin can also reduce the excretion of urinary protein in early diabetic kidney disease,thereby protecting the kidney.The renal protective effect of metformin is closely related to its inhibition of AMPK/mTOR signaling pathway.Klotho is an anti-aging gene,which is expressed in the distal convoluted tubule in the kidney.With the progression of renal disease,the expression of klotho protein decreases in diabetic kidney disease,and the decrease of klotho can activate mTOR signaling pathway to aggravate renal injury.Therefore,it is speculated that metformin regulates the mTOR pathway through klotho and protects renal tubular cells,thereby delaying renal progression in patients with diabetic kidney disease.We will observe the influence of the metformin interventions to klotho protein and the progression of renal injury for diabetic kidney disease patients,analyze the relationship between klotho protein and renal pathological injury in diabetic kidney disease mice,and explore the effect of high glucose and metformin on klotho protein expression and mTOR pathway in Madin-daby canine kidney cells(MDCK).It is preliminaryly discussed the mechanism of renal protection for metformin and klotho in the process of diabetic kidney disease,providing theoretical foundation for the treatment of diabetic kidney disease by metformin.In clinical studies,we found that metformin can improve part of renal outcomes for patients with DKD,considering the clinical data of patients with DKD imbalance and interpretability of medical model,we put forward the unbalanced learning ability and highly interpretability UB-0-TSK-FS model,the system belongs to the artificial intelligence in the field of machine learning,able to build on the classification of patients with DKD prediction model.This model will become an important tool for assisting doctors to monitor the course of DKD,and also provide a solution for nephrologists in intelligent diagnosis.PART ONE Effects of metformin on klotho protein and progression of renal injury in diabetic kidney disease Objective:To observe the effect of metformin intervention on klotho and progression of renal injury in DKD,and to analyze the association between metformin and klotho on progression of renal function in DKD.Methods:80 patients with DKD who visited the department of nephrology and endocrinology of Wuxi People's Hospital affiliated to Nanjing Medical University from January to July 2017 were prospectively collected,with 20 healthy controls.DKD patients were divided into metformin group and non-metformin group according to whether taking metformin.Hematuria samples were collected for detection of klotho protein levels by ELISA,and the differences in clinical indicators and concentrations of klotho between the groups were compared.The progression of renal injury patients was followed up for one year,and the indicators were urinary protein progress and blood creatinine doubling.Multivariate Logister regression was used to analyze the relationship between klotho protein and progression of renal injury in DKD patients after one year.Results:1.One year later,urinary protein progression occurred in 50.0% of DKD patients,and blood creatinine doubled in 37.5%.In the metformin group,40.0% of urine protein progression and 30.0% of serum creatinine doubling occurred.In the non-metformin group,60.0% presented urinary protein progression and 45.0%presented creatinine doubling.There was no significant difference in the probability of endpoint events between the two groups(P protein progression =0.117,P creatinine doubling=0.248).2.Serum and urine klotho levels in DKD patients were significantly lower than those in the healthy control group(489.87±288.64 vs 717.49±296.01pg/ml,703.96 ±649.86 vs 1588.34 ± 1041.07pg/ml·Cr,P<0.05).Serum klotho levels(610.30 ±312.31pg/ml)in the metformin group were significantly higher than those in the non-metformin group(369.43 ± 203.74pg/ml,P<0.001),but there were no difference in urine klotho leves between the two groups(P=0.923).3.Linear correlation analysis showed that serum klotho levels were negatively correlated with the course of diabetes,the quantitative level of urine protein,blood urea and blood creatinine levels(r=-0.308,-0.311,-0.348,-0.355,p<0.05),and positively correlated with blood albumin level and taking metformin(r=0.209,0.460,p<0.05).Urine klotho levels were negatively correlated with urine protein level,blood urea and blood creatinine levels(r=-0.413,-0.215,-0.230,p<0.05),and positively correlated with blood albumin level(r=0.313,p<0.05).4.Multiariable Logistic regression showed that serum klotho as an independent predictor of urinary protein progress and double serum creatinine for diabetic nephropathy patients,after adjusted age,duration of diabetes,glycosylated hemoglobin,mean arterial pressure,and low-density lipoprotein cholesterol,blood albumin,blood urea,serum creatinine,urine protein and urinary klotho(OD urinary protein progress1.010,P urinary protein progress = 0.002,OD double serum creatinine = 1.002,P double serum creatinine= 0.041).Conclusion:1.Metformin can improve the expression of klotho protein in the blood of patients with DKD,but cannot change the levels in the urine.2.The factors affecting the serum klotho protein include the course of diabetes mellitus,urine protein quantification,blood urea,blood creatinine,blood albumin level and history of taking metformin.3.Serum klotho was an independent predictor of urinary protein progression and serum creatinine doubling in patients with DKD,after adjusted age,duration of diabetes,glycosylated hemoglobin,mean arterial pressure,LDL cholesterol,serum albumin,serum urea,serum creatinine,urinary protein quantification and urinary klotho.PART TWOStudy on the renal protection mechanism of metformin in mice with diabetic kidney disease Objective:To study the protective effect and underlying mechanism of metformin on the kidney of mice with DKD.Methods:Ten db/db mice were equally divided into the metformin group and the non-metformin group.Five db/m mice were taken as the normal control group.Using radio immunoturbidimetric method to detect 24-hour urine protein quantitative,enzymatic method to detect serum creatinine,ELISA to detect klotho protein,staining to observe the pathological changes of renal tissue in mouse,immunohistochemical and western blot method to detect klotho,mTOR and p-mTOR protein levels in the kidney.Results:1.The urinary protein and serum creatinine levels were significantly increased in mice with DKD(p<0.001),and metformin could reduce the degrees of elevation(p<0.05),improving the renal injury in mice with DKD2.The klotho protein levels in blood and urine of DKD mice decreased(p blood<0.001,purine =0.001),while they significantly increased after the intervention of metformin(p blood <0.001,purine=0.028).3.In DKD mice,glomerular volume and renal tubular epithelial cells increased,basement membrane stiffened,and mesangial cells and matrix increased.All the above renal pathological changes were alleviated to some extent after the treatment with metformin.4.In the renal tissue of DKD mice,the level of klotho protein decreased,while the levels of mTOR and p-mTOR protein increased.After the application of metformin,the level of klotho protein increased,and the mTOR and p-mTOR proteins decreased in the renal tissue of diabetic nephropathy mice.Conclusion:Metformin can inhibit the progress of urinary protein and serum creatinine in DKD mice,increase the klotho protein in serum,urine and kidney tissue,and reduce the expressions of mTOR and p-mTOR protein in kidney tissue,so as to play a protective role in renal function.PART THREE Protective mechanism of metformin against hyperglycemia in MDCK cells Objective:The effects of metformin on klotho protein expression and mTOR pathway in Madin-Darby Canine kidney cells(MDCK)stimulated by high glucoseat were analyzed at the cellular level,and the possible renal protective mechanism of metformin and klotho protein in the process of diabetic nephropathy was preliminarily discussed.Methods:MDCK cells were cultured in vitro and worked by high glucose for different times.Morphological changes of cells were observed and cell activity was detected by MTT and Hoechst 33258 staining.MDCK cells were acted by metformin at different concentrations,and the expressions of mTOR,p-mTOR and klotho proteins in each group were detected by Western blot.SiRNA was used to inhibit klotho protein,and Western blot was used to detect the expression of mTOR and p-mTOR in cells of the hyperglycemic group and metformin group.Results:1.MDCK cells were grown as polygonal adherent cells in cell culture flasks.After 48 h of stimulation with 30mmol/l high-glucose medium,MDCK cells lost their intrinsic morphology and showed a long spindle shape.Hoechst 33258 staining showed that the nuclei of the normal control group were clearly demarcated and the chromatin distribution was uniform light blue,while the apoptotic nuclei of MDCK were strongly stained and the brightness was significantly deepened under the action of high glucose.With the extension of time,the cell viability of MDCK gradually decreased.The damage of MDCK cells was the greatest when 48 h after the hyperglycemic stimulation,and the average cell survival rate was 86.230%(p<0.001).2.Metformin improved the survival rate of MDCK cells under hyperglycemia,with the highest cell activity in the 0.3 g/m L DMBG group(p<0.001).Under Hoechst33258 staining,cell activity was highest in the 0.3 g/m L DMBG group,with clear nuclear boundaries and uniform light blue chromatin distribution,while the rest of the DMBG group showed uneven distribution of chromatin,and some of the nuclei were lobed and fragmented,showing a bright blue color.3.High glucose can down-regulate klotho protein and up-regulate the expression of mTOR and p-mTOR protein.With the prolonged hyperglycemic stimulation,MDCK cells expressed gradually increased levels of mTOR and p-mTOR protein(p<0.001),suggesting that hyperglycemic stimulation could activate the mTOR pathway in MDCK cells.4.After siRNA inhibited the expression of klotho protein,there was no significant difference in mTOR and p-mtor protein levels between the high-glucose group and the metformin intervention group.Conclusion:1.High glucose can down-regulates the expression of klotho,activates the mTOR signaling pathway,and damage MDCK cells,The degree of damage increases with the extension of time.2.Metformin mitigates the damage of high glucose to MDCK,and the optimal concentration is 0.3 g/m L.3.Klotho plays an important role in the process of metformin inhibiting mTOR pathway to protect renal function.PART FOUR Intelligent prediction of renal injury in diabetic kidney disease patients Based on a novel unbalanced Zero-order TSK Fuzzy System Objectives:In the nephrology field,DKD is a common complication of diabetes mellitus.Thus,if we can intelligent predict the renal injury in DKD,it is an important way to help the doctor to monitor the course of this disease.Methods:Since the distribution of the DKD patients' dataset is unbalanced,a novel unbalanced fuzzy learning mechanism based on the Havrda-Charvat entropy is firstly presented,and then a novel unbalanced zero-order Takagi–Sugeno–Kang fuzzy system(UB-0-TSK-FS)and its learning algorithm is presented.Result:Based on the UB-0-TSK-FS model,an intelligent prediction method,which inherits the high interpretability from the classical zero-order TSK fuzzy system,is proposed to help a young doctor to develop his level of expertise.Conclusion:The proposed UB-0-TSK-FS method enhances the generalization performance of the built model and hence is very promising for Intelligent predicting the renal injury in diabetic kidney disease patients.