| Objectives: Diabetic nephropathy (DN)is one of the most common complications of diabetes mellitus and it is single largest cause of end-stage renal disease (ESRD) in western countries. The initial stages of diabetic nephropathy are characterized pathologically by glomerular and tubular cell hypertrophy with a thickening of basement membranes, and clinically by the development of hyperfiltration and microalbuminuria. Diabetic nephropathy progresses to glomerulosclerosis with an accumulation of extracellular matrix proteins in the glomerular mesangium. As the glomerular filtration rate declines, progressive tubulo- interstitial fibrosis leads to end-stage renal disease. Recent studies have showed that hyperglycemia induced increase of reactive oxygen species(ROS),oxidative stress and activation of the four pathways including polyol pathway flux,advanced glycosylation end product formation, protein kinase C pathway and increased hexosamine pathway flux which caused increase of ROS ulteriorly .As a result oxidative stress was considered to be the physiopathological basis of diabetic nephropathy. At the same time hyperglycemia could activate the RAS systems and lead to oxidative stress.Excessive ROS further induced activation of RAS system and the beneficial effects of RAS inhibitors on diabetic nephropathy also suggested activation of RAS could play a role in the pathogenesis of diabetic nephropathy. As more and more attention was paid on the roles of activation of RAS and oxidative stress in the pathogenesis of diabetic nephropathy,the prophylactic and therapeutic effects on diabetic nephrpathy of inhibition of RAS and antioxidant therapy are the focus of recent studies.Melatonin and alpha-Lipoic acid,two strong antioxidants,could decrease production of free oxygen radicals and protect antioxidant enzyme and TongLuo could promote blood flow and stabilize endothelial function.This study aims to demonstrate the protective effects of these medicines on kidney and explore their effects on RAS and the mechanisms of protective effects in diabetic nephropathy.Methods: 1,Diabetes mellitus was induced in SD rats by streptozotocin (STZ 60 mg/ kg) abdominal injection.Blood glucose levels were measured three days after the injection to ensure a diabetic state(>16.7 mmol/L).Then diabetes rats were assigned to following groups:group A(DM,n=9)intragastic administration of 2% ethanol; group B(TL1,n=7):treatment with low dose of Tongluo (0.5g/ kg) administrated by gavages;group C(TL2,n=8):treatment with high dose of Tongluo (1.0g/ kg) administrated by gavages;group D(MT1,n=7):treatment with low dose of melatonin (0.5mg/ kg) administrated by gavages;group E ( MT2,n= 7):treatment with high dose of melatonin (10mg/ kg) administrated by gavages;group F(LA,n=7):treatment with alpha-Lipoic acid(100mg/kg); group G(TL1+ MT1,n=8):treatment with low dose of Tongluo (0.5g/ kg) and melatonin (0.5mg/kg) administrated by gavages;and NC group ( NC ,n=7):nondiabetic controls(or normal control),intragastic administration of 2% ethanol.Blood samples were collected from the vena caudalis and were measured twice a week to assure the blood glucose below the preestablished limit.If the blood glucose beyond the limit 2-4U Novolin N was injected subcutaneously.Vena caudalis glucose and body weight were measured twice a week and once a week respectively in all rats.Four rats were dead in the study,two of group A,each of group C and G.2,All rats were treated for 10 weeks then were killed. In each rat 4ml artery blood was collected and anticoagulated with EDTA,and 2ml blood was anticoagulated with the specific reagents (0.3M EDAT, 40ml+0.32M dimercaptopropanol, 20ul +Hydroxyquinoline Sulfate 40ul).All the blood samples were centrifugated in low temperature and plasma were stored -20℃. Two kidneys of each rat were also excised and weighed.The left kidney was incised coronally. Cortex and outer medulla of the half one were stored in -80℃. 3,plasma triglyceride (TG),cholesterol(TC), high density lipoprotein (HDL) and low density lipoprotein(LDL) levels were measured as clinical and biochemimial parameters.4,Kidney weight/body weight ratio was evaluated histological damage index.5,Radioimmunoassay was used to measure plasma and nephritic tissue homogenate angiotensin I and angiotensin II (AI,A II)levels.6,Real-time PCR was used to measure the levels of AT1aR mRNA and AT2R mRNA in kidney cortex.Results: 1,Blood glucose was increased and body weights were declined in all DM rats compared with normal control rats(P<0.001). However any medicine had no effects on blood glucose(P>0.05).Except the weights of the rats in group E(P<0.05),which increased lightly under the effect of high dose of melatonin,the weights of all rats in other groups did not effected by the medicine(P>0.05).There was no significant difference between normal controls and group A with respect to HDL,LDL,TG(P>0.05). Medicines had no effect on blood lipids(P>0.01). 2,kidney weight/body weight ratio were significantly increased in group A(P <0.001),but those were improved in all treated rats to different extent(P<0.01),most significantly in group E .3,By radioimmunoassay both plasma and kidney tissue homogenate A I and A II in group A were significant higher than those in normal controls(P<0.05). Medicines could decrease the A I and A II to different extent especially significant in group E and G(P<0.001).4,By Real-time PCR the levels of AT1a mRNA,AT2R mRNA and AT1R/AT2R ratio in group A were upregulated(P<0.001), especially for AT1a mRNA(39 times of AT1aR mRNA,4 times of AT2R mRNA respectively).Except that alpha-Lipoic acid had no effect on up-regulated AT2R mRNA(P>0.05),all other medicines could reverse the upregulation of the levels of AT1aR mRNA and AT2R mRNA in DM rats (P<0.001)and the effect on AT1aR mRNA was stronger than AT2aR mRNA .AT1aR mRNA levels decreased most in group G,however, the AT2R mRNA levels decreased most in group C. Medicines could decrease AT1R/AT2R ratio(P<0.01).Conclusion:1,Hyperglycemia could activate the RAS systems in kidney,activation of RAS could impair the kidney by increase of local angiotensin and up-regulated AT1aR or by imbalance of AT1aR and AT2R. This effect did not dependent on circulation RAS.2,The study indicated Tongluo , melatonin and alpha- Lipoic acid all had protective effects on diabetic nephropathy, which were independent of blood glucose and plasma lipid .3,Tongluo,Melatonin and alpha-Lipoic acid could inhibit activation of the RAS in diabetic nephropathy to different extent, which maybe one of the mechanisms on protecting diabetic nephropathy.4,"Luo bing"is the base of diabetic nephropathy.Oxidative stress induced activation of RAS in kidney maybe one of the important pathophysiology mechanisms of development of diabetic nephropathy.Tongluo and antioxidants could effectively inhibit activation of RAS and have broad practical prospect.
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