| Objective: Diabetic kidney disease(DKD)is one of the most serious complications of diabetic mellitus(DM),and is the second leading cause of end-stage renal disease.Its pathogenesis is still unclear.Recent studies have found that the injury of podocyte on glomerular filtration barrier is one of the important factors leading to proteinuria.The protein of P-cadherin and transient receptor potential canonical-6(TRPC6)is recently discovered which is closely related to proteinuria.This research is aimed to observe the intervention effect of different combinations of Chinese recipe of stasis-removing-and-collaterals-dredging on the kidney of DKD rats and its effect on the expression of P-cadherin and TRPC6 in the glomerular,and further explore the possible mechanism.Methods:70 male Sprague Dawleg rats,weighing about 100±10g,were randomly divided into control group(C group,n=8),modelling group(n=62).The model of diabetic rats were established with high sugar high fat diet combined with streptozotocin(STZ)by intraperitoneal injection(35 mg/kg).After 72 h,the rats whose three times of random blood glucose(RBG)from tail vein were ≥16.7 mmol/L were considered as successful model and removed if it do not meet the criteria.The 55 rats were randomly divided into model group(M group),stasis-removing-and-collaterals-dredging group(Q group),the Irbesartan group(I group),stasis-removing group(H group)and collaterals-dredging group(T group).The number of each group is 11 and different medicines were intragastrically fed once a day for 16 weeks.The urine volume in 24 hours were collected for the calculation of 24 h urinary total protein(24h UTP),and RBG,food intake,water intake were measured at the third day and the end of 4th,8th,12 th,16th week to observe their dynamic changes.At the end of the 16 th week after anesthesia with chloral hydrate,the kidney of rats were stripped for kidney weight(KW)and hypertrophy index(HI),and the biochemical indexes of blood from abdominal aorta were detected.Then the renal tissues were preserved for light microscope and transmission electron microscope observation,and the expression of mRNA and protein were detected by immunohistochemistry technique(IHC),Real-time PCR and Western Blot,respectively.Results:1 The general status of ratsThe rats in C group were in good condition and flexible activity.Food intake,water intake and urine volume were no abnormal,and weight increased steadily.The rats in M group showed flagging spirit,polydipsia,polyphagia,polyuria and loss of weight gradually.Compared with M group,the treatment groups were relieved to varying degrees.2 Changes of food intake,water intake and urine volume in 24 hours of rats at different time pointsCompared with C group at the same time points,24 hours of food intake,water intake and urine volume were significantly increased in modelling group(P<0.05 or P<0.01).Compared with M group at the same time points,there was no difference in food intake of rats in treatment group at any time(P>0.05).Water intake and urine volume appeared difference in 16 th week,the water intake and urine volume of rats were reduced in Q group and T group(P<0.05 or P<0.01),but I group and H group had no significant change(P>0.05).3 Comparison of body weight,kidney weight and hypertrophy index of ratsThere was no significant difference in kidney weight between the groups(P>0.05).Compared with C group,the body weight was significantly reduced and the hypertrophy index was significantly increased in modelling group(P<0.01).Compared with M group,Q group had higher body weight and lower hypertrophy index(P<0.01),but I group,H group and T group had no significant difference(P>0.05).4 Comparison of 24 h urinary total protein(24h UTP)of ratsOn the third day,there was no difference between the groups of 24 h UTP(P>0.05).At the end of 4th week,compared with C group,the 24 h UTP of the rats in modeling group were increased(P<0.05),and There was no difference between groups(P>0.05).At the end of 8th week,compared with C group,the 24 h UTP of each modelling group were increased(P<0.01).Compared with M group,the 24 h UTP of Q group and I group were decreased(P<0.01),H group and T group had no difference(P>0.05).At the end of 12 th week,compared with C group,the 24 h UTP of the rats in each modelling group were increased(P<0.01).Compared with M group,the 24 h UTP of the rats in each treatment group were decreased(P<0.05 or P<0.01).At the end of 16 th week,compared with C group,the 24 h UTP of the rats in each modelling group were increased(P<0.01).Compared with M group,the 24 h UTP of the rats in each treatment group were decreased(P<0.05 or P <0.01).Compared with I group,Q group were decreased(P<0.05).Compared with Q group,H group and T group were increased(P<0.05).5 Comparison of random blood glucose,blood lipid of ratsThe RBG of rats in C group was roughly stable at different time points.The RBG of each modelling group were significantly increased(P<0.01)and there was no difference between groups(P>0.05).Compared with C group,the total cholesterol(TC),triglyceride(TG),high density lipid cholesterol(HDL-C),low density lipid cholesterol(LDL-C),very low density lipid cholesterol(VLDL-C)of each modelling group were increased(P<0.05 or P<0.01).Compared with M group,the TG,LDL and VLDL of the rats in each treatment group were decreased(P<0.05 or P<0.01).In HDL,Q group and T group increased(P<0.01),the difference between I group and H group was not statistically significant(P>0.05).6 Comparison of serum protein and renal function of ratsThere was no significant difference in total protein(TP)and albumin(ALB)among the groups(P>0.05).Compared with C group,rat blood urea nitrogen(BUN)and serum creatinine(Scr)and uric acid(UA)were increased in M group(P<0.01).Compared with M group,rats of Q group,I group and T group were decreased in BUN(P<0.05 or P<0.01),no difference in H group(P>0.05).Scr and UA of rats in each treatment group were decreased(P<0.05 or P<0.01).7 Morphological observation of kidney7.1 The observations by light microscopeUnder the light microscope rats in C group had clear glomerular structure.M group rats’ kidney showed glomerular hypertrophy,part of the renal capsule narrowed as slit-like,with the accumulation of mesangial matrix,thickening of basement membrane and disorder of renal tubular.Compared with M group,rats in the treatment group were relieved to varying degrees.7.2 The observations by transmission electron microscopeUnder the transmission electron microscope,the structure of glomerular structure in C group was relatively normal.In M group the basement membrane were diffusely thickened,and the foot process appeared wide,flat,and even disappeared.Compared with M group,the pathological changes of each treatment group were slightly reduced.8 Detection of the expression of P-cadherin and TRPC6 in renal tissue by immunohistochemistryIn C group,P-cadherin can be observed as the linear distribution along the glomerular basement membrane and protein expression was more by the light microscope.Compared with C group,expression of M group was significantly less.Compared with M group,Q group and I group was significantly increased,H group and T group had no significant difference.The expression of TRPC6 in C group was decreased.Compared with C group,the expression of TRPC6 was increased in M group,and there was no significant difference between the treatment group.9 Detection of the expression of P-cadherin and TRPC6 mRNA in renal cortex by Real-time PCRCompared with C group,the expression of P-cadherin mRNA in M group was significantly lower(P<0.01).Compared with M group,the expression of P-cadherin mRNA in Q group and I group were significantly increased(P<0.01),H group and T group were not significantly up-regulated(P>0.05).Compared with Q group and I group,H group and Tgroup were relatively decreased(P<0.01 or P<0.05).Compared with C group,the expression of TRPC6 mRNA in M group was increased(P<0.05).Compared with M group,the expression of TRPC6 mRNA in each treatment group was significantly lower(P<0.05),and there was no significant difference between the groups(P>0.05)10 Detection of the protein expression of P-cadherin and TRPC6 in renal cortex by Western BlotCompared with C group,P-cadherin expression in M group significantly decreased(P<0.01).Compared with M group,Q group and I group were significantly increased(P<0.01 or P<0.05),and there was no significant difference between this two groups(P>0.05),H group and T group was no significantly increased(P>0.05).Compared with Q group and I group,H group and Tgroup were relatively decreased(P<0.01 or P<0.05).Compared with C group,the protein expression of TRPC6 in M group were significantly increased(P < 0.01).Compared with M group,the expression of TRPC6 in each treatment group was significantly lower(P<0.05),and there was no significant difference between the groups(P>0.05).Conclusions:1 The DKD model with high sugar high fat diet combined with low dose STZ intraperitoneal injection was basically successful.There were polydipsia,polyphagia,polyuria and gradual proteinuria,corresponding to the pathological manifestations.2 Stasis-removing-and-collaterals-dredging medicine can improve the general status of DKD rats and the disorder of blood lipid,reduce urinary protein,protect renal function,relieve renal pathological damage,and play a protective role in the kidney.3 Stasis-removing-and-collaterals-dredging medicine may maintain the stability of podocytes,decrease the urine protein and slow down the progression of DKD possibly through up-regulating the expression of P-cadherin and down-regulating the expression of TRPC6.4 The combination of Chinese recipe of stasis-removing-and-collateralsdredging is reasonable,and its the effect on reducing urinary protein is superior to stasis-removing medicine and collaterals-dredging medicine respectively.And stasis-removing medicine and collaterals-dredging medicine may have synergistic effect.The effect of collaterals-dredging medicine on reducing blood urea nitrogen and protecting renal function is better than that of stasis-removing medicine. |
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