| PurposeWater metabolism balance disorder is one of the most common clinical Symptoms of chronic renal failure,often occur at an early stage of CRF.As one of important risk factors,it can cause rapid deterioration of renal function.To rectify timely water metabolism balance disorder,can improve renal function and delay progression of CRF.It has great significance to improve quality of life in patients with CRF.At present,the basic pathogenesis of CRF according to Traditional Chinese Medicine theories was considered to be kidney-yang deficiency.At Early and medium-term of CRF,warm and tonify kidney-yang therapy acquired a better effect in CRF treatment.In this study,we set up the rats model of chronic renal failure with kidney-yang deficiency,to observe renal AQP2 expression changes, changes of angiotensinⅡand cyclic adenosine monophosphate contents in rat renal tissue, and expression changes of angiotensinⅡtype 1 receptor,cyclooxygenase-2 and protein kinase A,by using biology and morphology technique.Order to explore the Pharmacological treatment mechanism of yougui pill in disorder of water metabolism of CRF,and to provide experimental evidence for pathophysiological mechanisms of "obstruction of qi activities" of Kidney-Yang Deficiency.Methods1.Setting up Model and AdministrationThirty Healthy male wistar rats were randomized into normal group,model group,yougui pill group and telmisartan group.From frist day of experiment,rats of model group,yougui pill group and telmisartan group were given by 200mg/Kg/d adenine ig daily morning,the normal control group rats were intragastrically administrated with distilled water of corresponding volume,totally for 21 days.After two weeks adenine ig,rats of yougui pill group and telmisartan group were given with yougui pill(at 3.6g/kg/d) and telmisartan(at 5.4mg/Kg/d) Separately by gastric irrigation daily afternoon,rats of normal control group and model group were intragastrically administrated with distilled water of corresponding volume,,totally for 21 days.2.Indexes were detected.:In experimental process,to observe changes of behavior,average weight,Urine volumes and urine specific gravity,blood Scr and BUN content,renal tissue pathological of rats;Expression of renal tissue AQP2 was detected by immtmohistochemistry method and Western blot analysis;Blood AVP content,renal tissue angiotensinⅡand cyclic adenosine monophosphate content were determined by radioimmunoassay;Renal tissue Protein kinase A expression was detected by immunohistochemistry method;Renal tissue angiotensinⅡtype 1 receptor and cyclooxygenase-2 were determined by Western blot analysis.3.Statistical data analysisThe data were expressed as(?)±SD.Completely randomized design one-way ANOVA was used to deal with data by SPSS10.0 statistical software,P＜0.05 for significantly difference.Results1.Impact of Yougui Pill on renal function of chronic renal failure rats with kidney-yang deficiency.The experimental results show that model group rats appeared intolerance to cold,cold four limbs,loss of appetite,weight loss,less activity.Compared with the normal group,model group rats show a marked increase in urine volume,decreased urine specific gravity,increase of blood Scr and BUN content significantly(P＜0.05) and Renal tissue by light microscopy observation shows glomerular expand cysts,interstitial fibrosis and a large amount of inflammatory cell infiltration,tubular compensatory enlargement.Compared with model group,Yougui Pill group rats show better feeding,stronger activity,more shiny hair,pretty sencitive reaction,body weight increasing,urine volume decreased,urine specific gravity increased,Scr and BUN content in blood decreased(P＜0.05),.Renal tissue pathological injury relieved2.Influence of yougui pill on the expression of renal tissue AQP2 of rats of chronic renal failure with kidney-yang deficiency.In immunohistochemical detection,Compared with the normal group,AQP2 expression of model group rats in principal cell membrane and cytoplasm of kidney connecting duct decreased.It manifested as brown particles reducing significantly and slight loss of color in principal cell membrane and cytoplasm.Imaging analysis showed integrated optical density(IOD) of AQP2 immunoreactivity positive in renal collecting tubular epithelial cells was significantly lower than that in normal group rats(P＜0.05);Compared with model group, AQP2 expression of yougui pill group rats in principal cell membrane and cytoplasm of kidney connecting duct increased.It presented as brown particles increasing significantly and darker in color in principal cell membrane and cytoplasm.Imaging analysis showed integrated optical density(IOD) of AQP2 immunoreactivity positive in renal collecting tubular epithelial cells was significantly higher than that in model group rats(P＜0.05 ).Western blot proved that AQP2 expression of renal tissue decreased obviously in model group rats.Gray scales was determined with image analysis system.Compared with normal group,gray scales of renal tissue AQP2 expression decreased in model group rats(P＜0.05).Compared with model group,AQP2 expression of renal tissue increased obviously in yougui pill group rats with western blot,gray scales of renal tissue AQP2 expression was higher in yougui pill group rats than that in model group(P＜0.05).3.Influence of yougui pill on Contents of blood AVP,angiotensinⅡand cyclic adenosine monophosphate in renal tissue,expression of renal tissue angiotensinⅡtype 1 receptor etc of chronic renal failure rats with kidney-yang deficiency.The results show that blood AVP content of three groups rats was no significant difference(P＞0.05).Compared with the normal group,content of angiotensinⅡ,the expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were elevated significantly.cyclic adenosine monophosphate content and expression of protein kinase A of renal tissue were significantly decreased in model group rats(P＜0.05);Compared with the model group,Content of angiotensinⅡ,the expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were decreased significantly,cyclic adenosine monophosphate content and expression of protein kinase A of renal tissue were significantly increased in yougui pill group rats(P＜0.05).The results showed that one of the mechanisms of water metabolism balance disorder of chronic renal failure is probably reduced AQP2 expression of renal collecting duct.The theory of Chinese medical considered kidney goveming water..It mainly manifested in maintaining the balance of the body water metabolism.During kidney yang deficiency,it will occur obstruction of qi activities and result in disturbance of water metabolism.The experimental results indicate that one of the essence of obstruction of qi activities is AQP2 expression decreased.The possible mechanism of AQP2 expression decreased in renal tissue is due to that increased angiotensinⅡcontent in renal tissue,increased angiotensinⅡtype 1 receptor and cyclooxygenase-2 expression,This results in renal tissue content of cyclic adenosine monophosphate and protein kinase A expression decreased,making the expression of AQP2 in the kidney decreased.After Warmly Nourishing Kidney-yang treatment with Yougui Pill,angiotensinⅡcontent in renal tissue was decreased,expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue was decreased and cyclic adenosine monophosphate content and expression of protein kinase A increased.It results in increased renal expression of AQP2,which corrects water metabolic disorder in chronic renal failure, improves renal function,delay the progress of chronic renal failure..Conclusion:1.Chronic renal failure rats with kidney-yang deficiency have a marked increase in urine volume,decreased specific gravity,increase of blood Scr and BUN content and renal tissue pathological injury significantly,treatment with Yougui Pill can improve above facts apparently.2.AQP2 expression of renal tissue of Chronic renal failure rats with kidney-yang deficiency decreased.After treatment with Yougui Pill,AQP2 expression of renal tissue increased.3.Yougui pill have no apparent effects on blood AVP content.of Chronic renal failure rats with kidney-yang deficiency4.Chronic renal failure rats with kidney-yang deficiency show that AngiotensinⅡcontent and expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were increased,cyclic adenosine monophosphate content and expression of protein kinase A decreased.5.After treatment with Yougui Pill,AngiotensinⅡcontent and expression of angiotensinⅡtype 1 receptor and cyclooxygenase-2 in renal tissue were decreased,cyclic adenosine monophosphate content and expression of protein kinase A increased.
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