Effects Of Carnosine On AKT/mTOR Pathway And Autophagy In Renal Tissue Of Rats With Diabetic Kidney Disease

ObjectiveTo observe the protective effects of carnosin on renal tissue damage in rats with DKD,and to explore the role of AKT/mTOR signaling pathway and autophagy.MethodsSeventy healthy male SD rats with body weight of 150-160 g were randomly divided into the following two groups: normal control group(Ctrl)and high glucose and high lipid group(HF).After 7 days of adaptive feeding,the Control group rats were fed with maintenance diet,and the HF group rats were fed with high sugar and high fat diet in different groups.At the 35 th day of the experiment,rats in HF group were fasted 12 hours in advance,during which they could not refrain from water.Type 2 diabetes rat model was established by intraperitoneal injection of streptozotocin(STZ)after fasting.On the37 th day of the experiment,the blood glucose of rats in HF group was measured,and the model of type 2 diabetic rats was considered to be successful if the blood glucose was ≥ 16.7 mmol/L.The rats were randomly divided into diabetic kidney disease group(DKD)and carnosine group(CAR).According to different carnosine dosage,CAR group was divided into low(CAR100),medium(CAR300)and high(CAR900)dose groups.The CAR groups were given gavage at 100mg/kg,300 mg/kg and 900 mg/kg,once a day.Control group and DKD group were given the same volume of distilled water;The body weight of each group was recorded every 7 days.The blood glucose of each group was measured by glucose meter regularly.On the 120 th day of the experiment,the urine volume and protein content of the rats were measured for 24 hours.The serum creatinine was measured by protein removal method.The kidney tissues of rats were taken out,weighed,and frozen at-80℃.HE staining was used to stain the kidney sections of rats to observe the morphological and pathological changes of glomeruli and renal tubules of rats.The content of malondialdehyde(MDA)in kidney tissue was determined by thiobarbituric acid(TBA)method.The activity of superoxide dismutase(SOD)in renal tissue of rats was detected by NBT method.The protein expressions of AKT,mTOR,LC3 B and p62 in renal tissues were detected by Western Blot.ResultsCompared with Ctrl group,DKD group and carnosine group showed dull hair color,wet and yellow hair,listless,curled back and so on.But the rats in the Ctrl group were smooth,neat and active.The weight of rats in the DKD group decreased significantly: on day 35,the body weight of rats in the Ctrl group and the DKD group fluctuated in the range of 407.9~452.5 g,with no significant difference.After STZ injection,the body weight of rats in DKD group decreased significantly to 301.7~345.2 g on day 42 of feeding,and the body weight of rats in DKD group did not change significantly after 42 days.On day 120,the body weight of DKD group decreased from 605.5±18.3 g to 322.2±31.4g(P < 0.01).On the 35 th day of the experiment,the blood glucose of the rats in each group was relatively stable,and the blood glucose of the rats in the DKD group increased significantly from 5.6± 0.5mmol /L in the Ctrl group to 24.4± 2.7mmol /L on the 37 th day of the experiment after 72 hours of STZ injection(P < 0.01).Blood glucose was measured on the 65 th,92nd and 120 th day,and the changes in blood glucose were not significant.Compared with Ctrl group,urine protein content in DKD group was increased from 5.3± 0.6 mg to 204.7±31.3mg(P < 0.01).24 h urine volume increased from 11.8±1.2 ml to535.3±38.4 ml(P < 0.01).Serum creatinine increased from51.87±2.62 μ mol /L to 124.29±7.93 μ mol /L(P < 0.01).The mean mass of kidney increased from 2.1±0.2g to 2.5±0.2 g(P < 0.01).In the DKD group,the glomerulus showed swelling and deformation,the mesangial hyperplasia of glomerulus and the basement membrane thickened,accompanied by tubular stenosis and deformation.The activity of SOD in renal tissue of diabetic rats decreased obviously,while the content of MDA increased.p-mTOR/mTOR ratio decreased in renal tissue of diabetic rats.The p-AKT/AKT ratio in renal tissue of diabetic rats was decreased.LC3 B II/I ratio and p62 protein expression were increased in renal tissues of diabetic rats.Compared with DKD group,the fur of CAR group became dry and recovered luster obviously.There were no differences in body weight,blood glucose,serum creatinine and renal mass among CAR groups(P <0.05);The urine protein level of rats in CAR100 group was decreased from 204.7±31.3mg to 131.1±22.5mg(P < 0.01).24-hour urine volume decreased from 535.3±38.4ml to 302.9±23.7ml(P < 0.01).The morphological structure of glomerulus in the CAR group was observed.The swelling degree of glomerulus was significantly reduced,and the thickening degree of basement membrane and the hyperplasia degree of mesangial membrane were restored.The peripheral glomerulus became round and smooth,and the stenosis degree of renal tubules was reduced.SOD activity increased and MDA content decreased in kidney tissue of CAR groups.There was no difference in the total protein contents of AKT and mTOR in kidney tissues of CAR groups,but the content of p-AKT and p-mTOR increased in a dose-dependent manner.The protein contents of LC3 II/LC3 I and p62 in renal tissue of rats in carnosine group were decreased with the increase of carnosine dosage.ConclusionsDKD can be induced in type 2 diabetic rat models prepared by high glucose and fat diet combined with intraperitoneal injection of STZ,and carnosine has a protective effect on it,which is related to the inhibition of oxidative stress by carnosine,activation of AKT/mTOR pathway,and recovery of autophagy function.