Rapamycin Improved Renal Injury Induced By Iodixanol In Diabetic Rats Through The MTOR/p70S6K Signaling Pathway

Contrast induced nephropathy(CIN)is the application of iodine contrast agent and appear after acute renal failure,has become the iatrogenic etiology of acute renal failure after renal hypoperfusion and use the third reason of renal toxicity drugs,studies have found that high sugar environment are more likely to induce the occurrence of CIN,seriously affect the patients quality of life,although the related research is numerous,but there is no effective treatment.A number of studies have found that the contrast agent,acute kidney injury,increased dephosphorylation and inactivation of mTOR and P70S6K in renal tubular epithelial cells,and that rapamycin is an mTOR inhibitor.In recent years,studies have found that mTOR is closely related to rapamycin and kidney disease,and is involved in the occurrence and development of a variety of acute and chronic kidney diseases[1].Studies have shown that rapamycin can increase autophagy level by inhibiting mTOR and relieve renal tissue ischemia-reperfusion injury in rats.However,there are few studies on whether it can regulate autophagy through mTOR/p70S6K pathway to treat diabetic CIN in rats.Based on the above,this study established a CIN rat model by femoral artery injection of iodixanol,observed the effect of rapamycin on renal function of diabetic CIN rats,and explored the regulatory mechanism of mTOR/p70S6K signaling pathway,providing new ideas for the prevention and treatment of clinical CIN.Part 1 The role of iodixanol with different viscosity in renal injury of diabetic rats and the mediating effect of autophagyObjective:To observe the effect of iodixanol with different viscosity on renal function in diabetic rats,and to explore the mechanism of its action.Materials and Methods:90 SPF SD rats were randomly divided into 6 groups:Control group(Sham group),270 grams of iodixanol group(270 ’IO group),320 grams of iodixanol group(320’ IO group),diabetic mellitus group(DM group),diabetic mellitus+270 grams of iodixanol group(DM+270 ’IO group),diabetic mellitus+320 grams of iodixanol group(DM+320’ IO group),uses the abdominal cavity injection chain urea with cephalosporins(STZ)combined high-fat feed diabetes model was established,by using femoral artery give medicine to intervene,The contents of renal injury molecules-1(KIM-1)and Neutrophils gelatinase-associated lipid delivery proteins(NGAL)in urine of rats in each group were not observed.Changes in serum concentrations of creatinine(Scr),blood urea nitrogen(BUN),superoxide dismutase(SOD),malondialdehyde(MDA)and glutathione peroxidase(GPX);Pathological changes of kidney were observed by HE staining.Apoptosis of renal epithelial cells was detected by TUNEL,and the expression of autophagy related proteins Beclinl and LC3 was detected by immunohistochemistry and Western blot.Results:Compared with the Sham group,each group of rats,Scr,BUN,KIM-1,NGAL and MDA increased significantly,SOD and GPX expression significantly reduced,and appeared different degree of pathological injury and apoptosis,compared with DM group,270 ’IO and 320’ IO group rats Scr,BUN,NGAL,KIM-1 and the expression of MDA was increased,SOD and GPX expression has decreased,but no statistically significant difference(P>0.05).Pathology results suggest DM group,270 IO group and 320’ IO rat renal tubular epithelial cell damage is not apparent,cell arrangement basic rules,only a small number of renal tubule cells cavitation degeneration and apoptosis,and the DM group,270 ’IO group and 320’ IO group compared with DM+320 ’IO group and DM+270’ IO group rats Scr,BUN,KIM-1,the expression of NGAL and MDA increased significantly,SOD and GPX expression reduced significantly(P<0.05),Pathology results show that the DM+270 ’IO group and DM+320’ IO rat renal tubular epithelial cell damage is serious,visible renal tubular vacuoles degeneration,cell falls off to small lumen,renal tubular structure is destroyed,renal tubular epithelial cells apoptosis,moreover under the environment of high sugar,iodine,alcohol significantly lower LC3,Beclin 1 protein expression(P<0.05),compared with the DM group,270 ’IO group and 320’ IO group,significant difference,but compared with DM group+320 ’IO,DM+270’ IO group on diabetic rats damage is lighter.Conclusion:iodixanol can cause severe renal function damage in diabetic rats,and the damage of high viscosity iodixanol is more obvious.The mechanism may be related to the inhibition of renal autophagy in diabetic rats,the initiation of oxidative stress,and the induction of apoptosis of renal tubular epithelial cells.Part 2 Rapamycin reduced the regulation effect of iodixanol on renal injury and autophagy in diabetic ratsObjective:To observe the effect of rapamycin on renal function and the regulation of autophagy related proteins in diabetic rats with iodixanol-induced renal injury,and to explore the mechanism of its action,so as to provide theoretical basis for the prevention and treatment of clinical contrastive nephropathy.Materials and Methods:45 SPF SD rats,the chain of intraperitoneal injection of urea with cephalosporins(STZ)combined high-fat feed diabetes model was established,after the success of the model,were randomly divided into 3 groups:diabetic mellitus group(DM group),diabetic mellitus+320 grams of iodixanol group(CM),diabetic mellitus+320 grams of iodixanol+rapamycin group(RAPA),the femoral artery injection of iodine,alcohol method based contrast medium kidney disease animal model,RAPA group by intraperitoneal injection of rapamycin,the other groups give equal amounts by intraperitoneal injection of 25%DMSO,observation group rats blood express,Scr,BUN,Expressions of urine injury markers KIM-1,NGAL and oxidative stress markers SOD,MDA and GPX were detected by ELISA,pathological changes were observed by HE staining and TUNEL staining,and the expressions of autophagy related proteins Beclin-1 and LC3 were observed by immunohistochemistry and Western blot.Results:Compared with DM group,CM group rats Scr,BUN,KIM-1,the expression of NGAL and MDA increased significantly,SOD and GPX expression significantly decreased(P<0.05),renal tubular epithelial cell damage is serious,visible renal tubular vacuoles degeneration,cell falls off to small lumen,renal tubular structure is destroyed,renal tubular epithelial cells apoptosis,about 32%of renal tubular epithelial cells(P<0.05),after rapamycin intervention,Scr,BUN,RAPA group rats,NGAL,KIM-1 the expression of MDA decreased obviously,SOD and GPX expression increased significantly(P<0.05),pathology of rat renal tubular epithelial cells swelling,loss reduction,glomerular atrophy,reduce and inflammatory cell infiltration relieve edema,congestion,compared with CM group,RAPA group rat renal tubular epithelial cells apoptosis,significantly reduce about 18%of renal tubular epithelial cells(P<0.05),in addition,rapamycin can significantly raise the expression of Beclin1and LC3.Conclusion:rapamycin can significantly improve renal function damage induced by iodixanol in rats under high glucose environment,and its mechanism may be achieved by increasing the level of autophagy in rats’ kidneys,inhibiting oxidative stress and apoptosis.Part 3 Rapamycin activates autophagy via mTOR/P70S6K to inhibit renal injury induced by iodixanol in diabetic ratsObjective:To observe the regulatory effect of rapamycin on renal mTOR/P70S6K pathway in diabetic rats with iodixacol-induced renal injury,and to preliminarily explore the regulatory mechanism of rapamycin on autophagy in CIN rats,so as to provide theoretical basis for clinical prevention and treatment of contrastive nephropathy.Materials and Methods:45 SPF grade SD rats were given streptozotocin(STZ)intra－abdominal injection combined with high-fat diet to establish a diabetes model.After the model was successful,they were randomly divided into 3 groups:diabetic mellitus+320 grams of iodixanol group(CM),diabetic mellitus+320 grams of iodixanol+rapamycin group(RAPA),diabetic mellitus+320 grams of iodixanol+rapamycin+autophagy inhibitor 3-MA group(3-MA),using femoral artery injection of iodixanol,method based contrast medium kidney disease animal model,RAPA group by intraperitoneal injection of rapamycin,3-MA in RAPA group by intraperitoneal injection of 2 hours prior to the modeling based on autophagy inhibitor,other groups was given more than amount of intraperitoneal injection of 25%DMSO group,observation group rats blood express,Scr,BUN,The expressions of urine injury markers KIM-1,NGAL and oxidative stress markers SOD,MDA and GPX in each group were detected by ELISA,pathological changes were observed by HE staining and TUNEL staining,and the expressions of autophagy related proteins Beclinl and LC3,and the regulation of mTOR/P70S6K pathway protein were observed by immunohistochemistry and Western blot.Results:Compared with the CM group,group RAPA Scr,BUN,KIM-1,the expression of NGAL and MDA significantly reduced,SOD and GPX expression increased significantly(P>0.05),pathology of rat renal tubular epithelial cells swelling,loss reduction,glomerular atrophy,reduce and inflammatory cell infiltration relieve edema,congestion,compared with CM group,RAPA group rat renal tubular epithelial cells apoptosis,significantly reduce about 21%of renal tubular epithelial cells(P<0.05).In addition,rapamycin can significantly up-regulate the expression of LC3,and Beclinl(P<0.05),and reduce p-mtor and p-p70S6k protein(P<0.05).However,with the addition of autophagy inhibitor,the improvement and regulation mechanism of RAPA were significantly inhibited.Conclusion:Rapamycin can significantly improve renal function damage in rats caused by iodixanol-induced hyperglycemia,and its mechanism may be related to the regulation of mTOR/P70S6K signaling pathway,the increase of rat renal autophagy level,the inhibition of oxidative stress and apoptosis.