Effect Of STAMP2 On Autophagy In Diabetic Nephropathy

Part Ⅰ:Effect of STAMP2 on autophagy in diabetic nephropathyBackgroundDiabetic nephropathy(DN)is a serious microvascular complication of diabetes.About 35%～45%of diabetic patients suffer from diabetic nephropathy,which is the most common cause of end-stage renal disease(ESRD).The significantly reduced level of renal autophagy in diabetes mellitus plays an important role in the occurrence and development of DN.Six transmembrane protein of prostate2(STAMP2)plays an important role in inflammation and metabolic regulation.Our previous studies have shown that STAMP2 has a protective effect on diabetic atherosclerotic model mice,improving insulin resistance and reducing atherosclerotic plaque formation.However,the relationship between STAMP2 expression and autophagy in type 2 DN is unclear.Therefore,in this study,we established a rat model of type 2 diabetic nephropathy to investigate whether overexpression of STAMP2 can reduce renal injury in type 2 DN and activate autophagy through autophagy-related nutrient-sensing signaling pathway,in order to provide a new target for the treatment of DN.ObjectivesTo investigate the effects of STAMP2 overexpression on metabolism,renal function,renal fibrosis,autophagy and nutrient-sensing signaling pathways in type 2 diabetic nephropathy rats.MethodsThe rat model of type 2 diabetic nephropathy was established by high-fat feeding combined with intraperitoneal low-dose STZ injection.STAMP2 is overexpressed by jugular injection of STAMP2 adeno-associated virus.Metabolism,renal function,histological changes and autophagy related molecular pathways were detected.Results1.Body weight and blood pressure resultsAfter the overexpression of STAMP2,the kidney weight to body weight ratio was significantly reduced(P<0.05).No significant differences in the blood pressure parameters including systolic pressure,diastolic pressure,and mean arterial pressure.2.IPGTT and IPITTAt the end of experiment,rats in STAMP2 overexpression group showed improved glucose and insulin tolerance,and improved insulin resistance.3.Determination of biochemical parametersFasting glucose levels,total cholesterol,triglycerides and urinary albumin-to-creatinine ratio(UACR)in STAMP2 overexpression group were significantly decreased,while ISI was increased.During the experiment,serum creatinine and urea nitrogen showed no statistical difference at each time point in the experimental group.4.Histopathological changes of renal tissueCompared with diabetic rats,STAMP2 overexpression group had less pathological changes such as glomerular hypertrophy,increased mesangial matrix,mild proliferation of mesangial cells,vacuolar degeneration of renal tubular epithelial cells,widened renal interstitium,and increased collagen fiber deposition in mesangial area and renal interstitium.Transmission electron microscopy showed that the ultrastructural changes of STAMP2 overexpressed diabetic kidney,such as widened foot process,thickened glomerular basement membrane,fusion of foot process and significant reduction of autophagy lysosome,were alleviated.5.Immunohistochemistry and Western blot resultsIn DN group,the expression level of STAMP2 was significantly decreased and the positive expressions of Collagen Ⅰ and Ⅳ were significantly increased(P<0.001).Compared with DN+Vehicle group,the expression level of STAMP2 in renal tissues of rats in DN+STAMP2 overexpression group was significantly increased,while the positive expressions of Collagen Ⅰ and Ⅳ were significantly decreased.Overexpression of STAMP2 activated autophagy,significantly decreased the levels of p62,p-mTOR/mTOR ratio and p-ULKl,and markedly increased the expressions of LC3Ⅱ/Ⅰ and Beclinl,p-AMPK/AMPK ratio and SIRT1.Conclusions1.The combination of high-fat diet and low-dose STZ intraperitoneal injection successfully established type 2 diabetic nephropathy rat model.2.The expression of STAMP2 in kidney of type 2 DN rats was decreased,and the transfection of STAMP2 adeno-associated virus could effectively promoteSTAMP2 protein expressing.3.Overexpression of STAMP2 can reduce blood glucose and lipids,improve metabolic disorders and insulin resistance in type 2 DN rats.4.Overexpression of STAMP2 can significantly reduce UACR,improve renal histopathological changes,reduce the expression of Collagen Ⅰ and Ⅳ in kidney,and alleviate renal fibrosis in type 2 DN rats.5.Overexpression of STAMP2 can activate renal autophagy in type 2 DN rats,resulting in increased Beclinl and LC3 Ⅱ/Ⅰ protein expression and decreased p62 protein expression.6.Overexpression of STAMP2 may activate renal autophagy in DN by activating AMPK/SIRT1 pathway and down-regulating mTOR pathway.Part Ⅱ:CIDEC gene silencing alleviates type 2 diabetes related vasculitisBackgroundIn the first part of the study,we found that STAMP2 overexpression can significantly reduce diabetic microangiopathy--diabetic nephropathy,and diabetic macrovascular disease is an important pathological mechanism leading to cardiovascular and cerebrovascular events in diabetic patients.Chronic vascular inflammation is considered to be the common pathological basis of vascular remodeling and atherosclerosis.However,the mechanism of insulin resistance and vascular inflammation remains unclear.To this end,we further explored the underlying molecular mechanism of vascular inflammation in diabetes.It has been reported that cell death-inducing DFF45-like effector C(CIDEC)is significantly upregulated in adipose tissue of type 2 diabetes mellitus,and CIDEC silencing can significantly reduce visceral fat,reduce lipid oxidation,and significantly improve insulin sensitivity and lower blood glucose levels.The down-regulation of CIDEC expression in liver and kidney can inhibit the expression of inflammatory indicators in tissues,exert anti-inflammatory and anti-apoptotic effects,and significantly improve insulin resistance.No studies have shown whether CIDEC is involved in the process of vascular inflammation in diabetes mellitus.To explore the above issues,we used type 2 diabetic rats as a model to study the role of CIDEC in the occurrence and development of diabetic vascular inflammation.ObjectivesTo study the effect and mechanism of CIDEC gene silencing on vascular inflammation in type 2 diabetes.MethodsA rat model of type 2 diabetes was established.CIDEC-shRNA adenovirus was injected into the jugular vein to silence the CIDEC gene.The metabolism of rats,the content of collagen and the expression of inflammatory factors,CIDEC and CTRP3 were detected.ResultsCompared with the control group,the aorta media was thickened,collagen synthesis was increased,the expression of CTRP3 was down-regulated,and the expression of CIDEC,IL-6,IL-1β and TGF-β1 was increased in diabetic group.CIDEC gene silencing significantly reduced blood glucose and fasting insulin levels,and improved glucose and insulin tolerance.The media thickness of aorta and the content of collagen Ⅰ and Ⅲ were significantly decreased in the DM+CIDEC-shRNA group compared with the DM+vehicle group.With CIDEC-shRNA treatment,the expression of inflammatory cytokines(TGF-β1,IL-6 and IL-1β)and CIDEC in aorta were significantly decreased,and the expression of CTRP3 protelin was significantly increased in the DM+CIDEC-shRNA group compared with the DM+vehicle group.Conclusions1.Aortic vascular remodeling was observed in rats in the DM group,accompanied by increased expression of CIDEC in the aortic tissue,suggesting that CIDEC may be involved in the vascular progression of type 2 diabetes;2.CIDEC expression was up-regulated,CTRP3 expression was down-regulated,and inflammatory and fibrosis factors were increased,which may be an important molecular mechanism of diabetic macrovascular disease;3.CIDEC silencing increased the expression of CTRP3 and inhibited the expression of IL-6,IL-1β and TGF-β1;4.CIDEC silencing can improve aortic remodeling in diabetes mellitus.