| Objective:Diabetic kidney disease(DKD),as one of the most common microvascular complications of type 2 diabetes mellitus(T2DM),is the primary cause of the end-stage renal disease(ESRD)and death in patients with diabetes.The pathogenesis of DKD has not been systematically elucidated.The changes of hemodynamics,the disorder of glucose and lipids metabolism,oxidative stress,inflammatory reaction,and the inhibition of autophagy are closely related to the occurrence and development of DKD.Oxidative stress plays an important role in the DKD pathogenesis and can promote inflammatory response,which accelerates the kidney injury.8-hydroxy-2-deoxygnanosine(8-OHdG)is a sensitive indicator of oxidative DNA damage and oxidative stress status.It has been found that the excessive activation of mammalian target of rapamycin(mTOR)pathway involves in the pathogenesis of chronic kidney disease by promoting the hypertrophy of renal cells,increasing the synthesis of protein,participating in oxidative stress,increasing inflammatory reaction and inhibiting the autophagy of renal cells.Adenosine 5’-monophosphate-activated protein kinase(AMPK)is an upper reaches signal molecule of mTOR and an inhibitor of mTOR signaling pathway.Lipoic acid,a potent antioxidant,can scavenge reactive oxygen species.Lipoic acid is widely used and is effective for diabetic peripheral neuropathy.However,the effect of lipoic acid for DKD is not clear.In this study,the rat models of type 2 diabetes were prepared.The expression of AMPK,p-AMPK,mTOR and p-mTOR in the kidney of rats and the level of8-OHdG were detected.This study was to explore the relationship between mTOR signaling pathway and DKD and the intervation of lipoic acid.Methods: Twenty-two Sprague-Dawley(SD)male rats with six weeks of age,weighing 160-200 g,were housed in a clean and temperature-controlledenvironment.The rats were randomly divided into three groups: normal control group(group NC,n=6),diabetic mellitus group(group DM,n=8)and lipoic acid group(group LA,n=8).The rats in group NC were fed with ordinary alex,and others rats were fed with high-sugar,high-fat diet(20%sugar,10% lard,2.5% cholesterol,1% bile acid salt and 66.5% basic alex).Four weeks later,rats in group DM and group LA were given an intraperitoneal injection of 30 mg/kg streptozotocin(dissolved in citric acid buffer,PH 4.2~4.5).At the same time,group NC was injected with the same volume of citric acid buffer.Two weeks after injection,the rats with fasting blood glucose(FBG)higher than 7.8 mmol/L were regarded as the type 2diabetes models.Then,the rats in group LA were given lipoic acid at a dosage of 100 mg/kg by daily intraperitoneal injection and the ones in group NC and group DM received equal volume saline.The intervention lasted for 8 weeks.24-hour urine was collected to exam 24-hour urine protein and a blood sample was collected to inspect serum creatinine,blood urea nitrogen and 8-OHdG level and so on.The left kidneys were weight and fixed by4% polyphosphate formaldehyde.HE staining,PAS staining and Masson staining were used to observe the changes of kidney morphology.The expression of AMPK,p-AMPK,mTOR and p-mTOR in the kidney of rats were detected by immunohistochemistry.Results:1 Body weight and index of kidney hypertrophy in different groupsThe body weight of group NC rats was(455.17±22.46)g and the index of kidney hypertrophy was(3.65±0.24)mg/g.The body weight of group DM rats was(250.67±26.56)g and the index of kidney hypertrophy was(7.12±0.28)mg/g.The body weight of group LA rats was(259.20±15.48)g and the index of kidney hypertrophy was(6.10±0.45)mg/g.Compared with the group NC,the group DM or group LA had lower body weight(P<0.01)and higher index of kidney hypertrophy(P<0.01).Rats in group LA had lower index of kidney hypertrophy than that in group DM(P<0.01),while there was no significant difference between the weightof the two groups(P>0.05).2 Biochemical indexes in different groupsThe blood glucose in group NC was(5.71±0.71)mmol/L and urine protein was(15.27±1.45)mg/24 h.Serum creatinine was(37.23± 4.97)μmol/L.Blood urea nitrogen was(6.38±1.18)mmol/L.The blood glucose in group DM was(21.38±1.66)mmol/L and urine protein was(314.00±16.14)mg/24 h.Serum creatinine was(36.70±4.80)μmol/L.Blood urea nitrogen was(7.52±1.21)mmol/L.The blood glucose in group LA was(20.60±0.60)mmol/L and urine protein was(249.88±28.61)mg/24 h.Serum creatinine was(37.80±4.03)μmol/L.Blood urea nitrogen was(7.76±1.24)mmol/L.Compared with rats in group NC,the blood glucose and 24-hour urine protein of group DM rats and group LA rats were significantly higher(P<0.01).The 24-hour urine protein in group LA were lower than that in group DM(P<0.01).There was no significant difference between the blood glucose of the latter two groups(P>0.05).No significant difference was found among the blood urea nitrogen and serum creatinine in these three groups(P>0.05).3 8-OHdG level in different groupsThe 8-OHdG of group NC rats was(1.63±0.62)ng/ml.The 8-OHdG of group DM rats was(5.09±0.89)ng/ml and the 8-OHd G of group LA rats was(2.84±0.64)ng/ml.The 8-OHdG in group DM and group LA were significantly higher than that in group NC(P<0.05).The 8-OHd G level in group LA was significantly lower than that in group DM(P<0.01).4 Rat kidney morphology in different groupsHE staining: In the group NC,the structures of glomerular and tubular were normal and clear.No infiltration of inflammatory cells was observed in the renal interstitium.In group DM,the glomerular capillary opened badly.Mesangial expansion was obvious.Vacuoles degeneration appeared in some renal tubular cells and there was inflammatory cell infiltration in renal interstitium.The above pathological changes were alleviated in group LA.PAS staining: The mesangial matrix index in group NC was(0.1754±0.0149),the mesangial matrix index in group DM was(0.4052±0.0523),the mesangial matrix index in group LA was(0.2259±0.0103).Compared with group NC,the basement membrane thickening and mesangial matrix increased in group DM and group LA(P<0.05).Compared with group DM,group LA had the lower level of mesangial matrix index(P<0.01).Masson staining: Compared with the group NC,group DM and group LA had more blue collagen fiber in glomerular.The level of collagen deposition in group LA was significantly lower than that in group DM.5 The expression of mTOR,p-mTOR,AMPK and p-AMPK in the kidneyThere were no statistical differences in mTOR and AMPK among three groups(P > 0.05).The IOD/Area of p-mTOR in group NC was(0.0230±0.0034).The IOD/Area of p-mTOR in group DM and group LA was(0.0547±0.0089)and(0.0316±0.0039),respectively.The expression of p-mTOR in group DM and group LA was significantly higher than that in group NC(P<0.05).Compared with the group DM,group LA had lower the expression of p-mTOR(P < 0.01).The IOD/Area of p-AMPK in group NC was(0.0317±0.0040).The expression of p-AMPK in group NC was significantly higher than that in group DM,which IOD/Area was(0.0183±0.0024)(P < 0.01).Compared with the group DM,group LA had higher the expression of p-AMPK,which IOD/Area was(0.0273±0.0026)(P<0.01).Conclusion:1 There were typical renal pathological changes in diabetic rats,with the index of kidney hypertrophy,24-hour urine protein,the level of 8-OHdG and the phosphorylation of mTOR higher than that of normal rats.It indicated that the increase oxidative stress and the activation of mTOR signaling pathway were both correlated with DKD.2 Lipoic acid could reduce index of kidney hypertrophy,24-hour urine protein,the level of 8-OHdG and the phosphorylation of mTOR,which indicated that lipoic acid could lower oxidative stress and could delay progressive renal lesions in DM rats.3 The p-mTOR protein was significantly decreased and the p-AMPK protein was significantly increased after using lipoic acid.The results showed that lipoic acid played a protective effect on diabetic kidney by inhibiting the downstream signal of mTOR. |
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