| Objective:Diabetic nephropathy has become the leading cause of end-stage renal disease in the world,and there is currently no effective treatment.Studies have shown that decreased expression of SIRT1 in diabetic environment is one of the causes of diabetes and its complications.A large number of studies have shown that SIRT1 promotes cell survival by promoting neovascularization,inhibiting oxidative stress,anti-apoptotic signaling pathways,and remodeling energy metabolism pathways under stress.At the same time,some studies have found that the decrease of SIRT1 expression in renal tubules rather than glomeruli is associated with diabetic nephropathy.The decrease of SIRT1 expression can directly or indirectly activate the related signaling pathway leading to cell damage and apoptosis,resulting in diabetic complications.However,the upstream signaling pathway leading to impaired SIRT1 function has not been elucidated.microRNA is a 17-25 nucleotide non-coding RNA molecule that negatively regulates the expression of a target gene by degrading the messenger RNA or inhibiting its translation.Studies have shown that microRNAs are involved in the development of a variety of kidney diseases,such as DKD,acute kidney injury,lupus nephritis,polycystic kidney disease.Moreover,studies have found that the expression profile of renal microRNAs changes significantly during diabetes.Therefore,we speculate that the abnormal expression of microRNAs in the kidney during diabetes may be the cause of impaired renal function of SIRT1.For this reason,we have carried out a series of studies.Method: 1.Animal experiment Analysis of the effect of SIRT1 agonist on renal injury in diabetic rats: Spontaneous type 2 diabetic rats(ZDF rats)were used as research subjects and fed with high fat diet.Divided into control group,diabetes group,SIRT1 agonist-resveratrol intervention group.The blood glucose,body weight and glucose tolerance test were recorded.The renal tubular function indexes,liver and kidney function,HE and transmission electron microscope were observed by biochemical detection.The expression of SIRT1 and its downstream protein were detected by immunohistochemistry.To investigate whether SIRT1 agonists can improve the structure and function of kidneys in diabetic rats.2.Clinical study Screening microRNAs involved in the regulation of SIRT1 in the urine of patients with early stage of diabetic nephropathy: analysis of microRNAs expression profiles in patients with early diabetic kidney disease and diabetic control by gene chip technology,screening differentially expressed microRNAs,and performing target protein pathways explore.Biochemical method to detect liver function,renal function,blood lipids,blood sugar;retain morning urine,biochemical detection of glomerular and tubular damage indicators.The dual luciferase reporter assay was used to verify the direct regulation of differential expression of microRNAs on SIRT1.3.Cellular assay To verify the regulation mechanism of differentially expressed microRNAs on SIRT1 and downstream signaling pathway: human renal tubular epithelial cells(HK-2)were studied at normal glucose concentration(5.5 mmol/L)and high glucose(25.5).The culture was carried out in mmol/L).The effects of differentially expressed microRNAs on SIRT1 and its downstream signaling pathway were analyzed,and changes in endogenous fluorescently labeled albumin were observed by laser confocal microscopy.result: 1.Animal experiment results:1)The fasting blood glucose of the diabetic group and the resveratrol group gradually increased with time,while the blood glucose of the control group was stable.There was no difference in the fasting blood glucose between the three groups.The diabetic group and the resveratrol group were significantly higher at 12 weeks of age.In the control group,the difference was statistically significant(P<0.05).The body weight of each group increased steadily with time.The weight of the diabetic group and the resveratrol group was greater than that of the control group,and the difference was statistically significant(P<0.05).2)As the disease progresses,it can be seen that the blood glucose gradually increases at each time point after the glucose load in the resveratrol group and the diabetic group(P<0.05),and the AUC increases.There was no significant difference in glucose tolerance between the groups before drug intervention(P>0.05).Compared with the control group at 12 weeks of age,the resveratrol group and the diabetic group were higher than the control group at each time point(P<0.05).3)There were no significant differences in TF,IgG,and ?2-MG between the three groups.The expression of RBP,UMA,NAG and NGAL in the diabetic group was significantly higher than that in the control group(P<0.05).There was a statistically significant difference in the expression of UMA,NAG and NGAL between the resveratrol group and the diabetic group(P<0.05).4)There was no significant change in liver function and renal function of rats in each group.There was no significant difference between the two groups after resveratrol intervention(P>0.05).5)HE staining results showed that the mesangial area of the diabetic group was slightly widened,the mesangial cells and mesangial matrix were not increased,the wall of the small arteries was thickened,the capillaries were dilated,and the tubular epithelial cells were slightly swollen and tubed.The cavity becomes smaller.Resveratrol intervention group: glomerular,tubular structure improved compared with diabetic group.There was no obvious increase in the mesangial area,no glomerular glomeruli,and no obvious fibrous hyperplasia and inflammation infiltration in the renal interstitial.6)Electron microscopy results showed that ultrastructural structure showed swelling of small blood vessels in the proximal convoluted tubules and small tubules in the diabetic group,mild mitochondrial swelling in the mesangial cells,ambiguity in the fusion of some sputum and membrane,and mild endoplasmic reticulum in the rough surface.The expansion has particle fusion and degranulation.The above changes in the resveratrol group were significantly improved.7)Immunohistochemistry showed that the expression of SIRT1 was decreased in the kidney tissue of diabetic rats,the expression of Beclin representing autophagy function was decreased,and the expression of p62 was increased;the expression of Cubilin representing renal tubular function was decreased.Resveratrol can improve this phenomenon.2.Population research results:1)TF,IgG,and NAG were elevated in the DM group compared with the NC group(P<0.05).There was a statistically significant difference in the expression of UMA,TF,IgG,RBP and ?2-MG in the DKD group compared with the DM group(P<0.05).2)The miRNAs up-regulated relative to the DKD group of the DM group are: hsa-miR-6785-5p,hsa-miR-494-3p,hsa-miR-7641,hsa-miR-7977,hsa-miR-7975,hsa-miR-601,hsa-miR-7150,hsa-miR-193b-5p.The elevated miRNAs relative to the NC group DM group are: microRNAs including hsa-miR-4484,hsa-miR-6860,hsa-miR-5692 a,hsa-miR-671-5p,hsa-miR-4470,hsa-miR-642a-3p,hsa-miR-7641,hsa-miR-155-5p,hsa-miR-6515-5p,hsa-miR-6824-5p,sa-miR-6125,hsa-miR-7846-3p,hsamiR-6792-5p,hsa-miR-1914-3p,hsa-miR-92a-3p,hsa-miR-6134,hsa-miR-1295 a,hsa-miR-4259,hsa-miR-7977,hsa-miR-7975.3)The regulation of SIRT1 is hsa-miR-601,hsa-miR-7641,hsa-miR-7150,in which hsa-miR-7641 expression is up-regulated.3 cell experiment:The dual luciferase results showed that mir-7641 can directly regulate SIRT1.After high glucose intervention in renal tubular epithelial cells,the expression of mir-7641 was significantly increased(P<0.05),and SIRT1 protein and mRNA levels were decreased(P<0.05).Compared with the control group,the expression of SIRT1 was increased in the mir-7641 inhibitor group,p-AMPK/AMPK and Beclin-1 were increased(P<0.05),and p62 expression was decreased(P<0.05).Compared with the control group,the expression of SIRT1 was further decreased in the mir-7641 mimics group,p-AMPK /AMPK,Beclin-1 were decreased(P<0.05),and p62 expression was increased(P<0.05).conclusion: 1.There is a decrease in SIRT1 expression in renal tubules during diabetes,and structural and functional changes in renal tubules.The application of SIRT1 agonists can improve the structure and function of renal tubules in DM rats.At the same time,we found that SIRT1 agonists can improve the autophagy function of the renal tubules.Therefore,it is speculated that SIRT1 may improve the structure and function of renal tubules by improving autophagy of renal tubules.2.Patients with diabetes have different degrees of renal damage before the appearance of microalbumin,and there is an increase in urinary transferrin,immunoglobulin,and N-acetyl-?-glucosaminidase.At the same time,multiple microRNA changes occur in the urine of diabetic patients.Among them,mir-7641 is significantly elevated,which may be a molecular marker for early diagnosis of diabetic kidney injury.3.In diabetes,mir-7641 may target down regulation of SIRT1/AMPK/autophagy signaling pathway,causing damage to renal tubular epithelial cells and leading to decreased albumin reabsorption. |
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