Mechanism Study Of Danggui-shaoyao San On Nitric Oxide And Aquaporin-2 In ADR-induced Nephritic Syndrome Model Rats

Aim: To investigate the treatment effect of Danggui-Shaoyao San(DSS)in ADR-induced nephritic syndrome model rats,biochemical indicators,renal pathomorphology,expression of renal AQP2,i NOS,n NOS were observed,to further explore the possible mechanism of DSS in model rats on NO and AQP2.Methods: The rats were randomly divided into normal control group,adriamycin treated group flow by single dose of 6mg/kg,4mg/kg+3.5mg/kg and4mg/kg+2mg/kg.The 24 h urine protein,levels of serum BUN?Cr?TC?TG?TP?ALB were detected by the automatic biochemistry analyzer and pathological changes were observed.After the establishment of the model method,the rats randomly divided into 5groups: untreated adriamycin nephritic group,adriamycin nephrotic group treated by Danggui-Shaoyao San,adriamycin nephrotic group treated by L-arginine,adriamycin nephrotic group treated by benazepril and normal control group.8 weeks later,metabolic cages were used to collect 24 h urine,and 24 h urine protein content was measured by BCA method.We also detected serum and urine biochemical indicators(BUN?Cr?TC?TG?TP?ALB)by the automatic biochemistry analyzer and pathology changes of renal tissues were observed.The activity of NOS and the urine NO level were detected by spectrophotometry.The level of AQP2 in urine was detected by enzyme-linked immunoabsorbent assay(ELISA).The expressions of AQP2,i NOS and n NOS protein in the kidney of rats were detected by Western Blot and immunohistochemistry.Results:(1)After a single injection of adriamycin 6mg/kg in tail vein,24 urine protein were increased significantly after 4 weeks,then it had a slight decline at 8 weeks.Adriamycin treated group flow by 4mg/kg+3.5mg/kg and 4mg/kg+2mg/kg manifested as a continuous increase in proteinuria,biochemical indicators and renal pathological changes were more obvious.Overall,adriamycin treated group flow by4mg/kg+3.5mg/kg showed obvious nephrotic syndrome,biochemical indicators changes has the significant difference(P<0.01)and the model was stable.(2)Compared with the untreated nephritic syndrome rats,the rats treated by Danggui-Shaoyao San,L-arginine and benazepril could significantly decrease the levels of urinary protein,serum cholesterol,triglyceride,urea nitrogen and creatinine,increase the protein content of rats(P<0.05 or P<0.01),and enhance the activity of NOS and the urine NO Level(P<0.05 or P<0.01),reduced urine AQP2 content(P<0.01).(3)WB results revealed that the expression of AQP2,i NOS and n NOS were reduced(P<0.01).The treatment with DSS could significantly increase the expression of AQP2,i NOS and n NOS(P<0.01),it was consistent with the results of immunohistochemistry.AQP2 is mainly expressed in plasma membrane of renal tubular epithelial cells,i NOS is mainly expressed in glomerular epithelial cells and glomerular mesangial cells,n NOS is expressed in macula densa,glomerular mesangial and medullary thick ascending limb.Conclusion: Danggui-Shaoyao San can reduce the model rats proteinuria,control serum and urine biochemical indicators,enhance the activity of NOS and increase the expression of AQP2,i NOS and n NOS in the kidney.DSS improve the pathological status of Adriamycin nephropathy may be related to the expression of NOS and AQP2 in kidney in rats.