The Effect Of Yi Qi Yang Yin Fang Intervention On Glomerular Filtration Barrier In Adrimacycin-induced Nephropathy Rats

BackgroundPrimary nephrotic syndrome(PNS) is a common disease during childhood. About 80 percentage of PNS in children is minimal change nephrotic syndrome (MCNS).Although most of the patients were sensitive to steroid,some patients were prone to frequent-relapse or to be resistant to steroid treatment,even developing into renal insufficiency.In view of this,we have been seeking to new drugs and new methods which can improve effectiveness and reduce side effects of glucocorticoid and immuno-suppressive agents.Recently,traditional Chinese medicine has made great progress in treating kidney diseases.However,the suppressive effect and mechanism of TCM on MCNS have not well known yet in spite of many studies on the suppressive effect of TCM with MCNS,in particular proteinuria.In addition to the profound involvement of podocytes in the pathogenesis of glomerular diseases the sophisticated function of podocytes and the molecules associated with podocytes are highly attractive in the research of the mechanisms of proteinuria.Permselectivity of serum protein dertermined by the integrity of glomerular filtration barrier.Heparan sulfate(HS) in the glomerular basement membrane(GBM) is important in maintaining the charge-dependent permeability.It is proposed that The beta-D-endoglyeosidase heparanase should be important in the pathogenesis of proteinuria by degrading the negatively charged HS side chains with in the GBM selectivity Similarly,nephrin is one of important component of podocyte slit diaphragm,precious studies showed that it was involved in several diseases such as nephritic syndrome,and all of these diseases have close correlation with proteinuria.Therefore,in this study we used adrimacycin-induced nephropathy rats to observe the heparanase and nephrin expression and their changes in kidney in the protein and the gene level respectively,and to investigate the protective effect and mechanism of Yi Qi Yang Yin Fang on nephrotic syndrome induced by adriamycin(ARD). PARTⅠEffect of the Yi Qi Yang Yin Fang intervention on adrimacycin-induced nephropathy ratsObjective To observe the 24hr urinary protein,routine biochemical indicators of expression and the pathological changes in kidney morphology within normal and adriamycin -induced nephrotic rats,and in order to investigate the suppressive effect of Yi Qi Yang Yin Fang on nephrotic syndrome induced by Adriamycin(ADR).Methods Adriamycin(ADR)-induced nephrotic rat models were established by a single injection of 5mg/kg adriamycin via the tail vein.30 Sprague-Dawley rats were divided randomly into five groups:ADR group injected with adriamycin and the control group injected with normal saline.The 24hr urinary volume were collected at 7days,14days,21 days and 28days after adriamycin injection respectively,and all of rats were sacrificed at 28days.24hr urinary protein,serum cholesterol,serum triglyceride and renal function of rats were measured by routine biochemical method. The ANOVA test was used to analyse the results.Differences were regarded as significant when P＜0.05.All values are expressed as mean±SEM unless otherwise stated.Results(1) In ADR rats,the level of urinary protein and triglyceride and cholest -erol in serum increased significantly at 14th day(P＜0.01,0.01,0.05,respectively),at same time,albumin in serum decreased significantly(P＜0.01).The changes of these indices reached the peak level at 28th day.Under electron microscope,at 28 days after adriamycin injection,extensive fusion of foot processes was observed in ADR rats.(2) In Yi Qi Yang Yin Fang group(Z),Glucocorticoid group(J) and Yi Qi Yang Yin Fang plus Glucocorticoid group(ZJ),urinary protein and the level of triglyceride and choles -terol in serum decreased,albumin in serum increased at 28th day compared with Model,especially ZJ group significantly(P＜0.01).Under electron microscope,inter -ventions groups dramatically reversed the loss of foot processes back to normal mor -phology,especially ZJ group significantly.Conclusion Yi Qi Yang Yin Fang attenuates foot process injury of ADR-induced kidney,especially ZJ group dramatically.PartⅡEffect of Yi Qi Yang Yin Fang intervention on haparanase and nephrin in adrimacycin-induced nephropathy ratsObjective To detect the HPA and nephrin level in kidney of every group rats,and to discuss mechanism of Yi Qi Yang Yin Fang on adrimacycin-induced nephropathy rats.Methods Real time quantitative RT-PCR was used to determine the expression of HPAmRNA and nephrin mRNA respectively in renal cortex of every group rats at 28 days after adriamycin injection.The level of HPA in urine of every group rats were detected by ELISA.The expression and distribution of nephrin in kidney were detected by immunohistochemical staining(IHC),and were quantitatively analyzed with Motie image 3.2 analysis system.Results(1) The mRNA of HPA in total cortex of kidney in model rat(4.55±0.38) increased at 28th day(P＜0.05) compared to control(1.5±0.79).In invention groups, the level of HPA mRNA in total cortex of kidney decreased respectively at 28th day compared with Model,especially Z(3.566±0.18) and ZJ(3.125±0.807) group significantly(P＜0.05).The expression of HPA in total cortex of kidney correlated significantly to the proteinuria level(r=0.708,P＜0.005).(2) No significant difference of HPA in urine was found between inventions groups and model group at 28day, while model rats had a higher HPA level in urine than control(P＜0.05).(3) The mRNA of nephrin in total cortex of kidney in rat model increased at 28th day compared to the control(P＜0.05).In invention groups,the level of nephrin mRNA in total cortex of kidney decreased respectively at 28th day compared with the Models, especially ZJ(0.91±0.01) group significantly(P=0.031＜0.05).(4) However,The expression of nephrin protein in kidney in model rats(15.1±1.42%) increased at 28th day compared to the control(23.77±4.5%,P＜0.05).In other groups,the level of nephrin in kidneys decreased respectively compared with the Models,especially in ZJ (19.9±3.3) group significantly(P=0.016＜0.05).(5) The expression of nephrin in kidney correlated negatively to the proteinuria level(r=-0.66,P=0.001)Conclusions Yi Qi Yang Yin Fang group(Z),Glucocorticoid group(J) and Yi Qi Yang Yin Fang plus Glucocorticoid group(ZJ) can attenuate the expression of HPA in total cortex of kidney.It is suggested that these inventions groups improve proteinuria through regulation of haparanase on GBM.Proteinuria are correlated with the expression of nephrin in kidney.Yiqiyangyin herbals and/or steroid are capable of regulating the expression of nephrin in kidney.It is suggested that these inventions may decrease the proteinuria through the regulation of nephrin on SD.Conclusions1.Yi Qi Yang Yin Fang attenuates foot process injury of ADR-induced kidney, especially combined with the steroid.2.Yi Qi Yang Yin Fang attenuates the development of proteinuria in ADR-induced rats possibly through inhibiting HPA in kidney3.Yi Qi Yang Yin Fang can regulate the expression of nephrin on SD.The study illustrated that the treatment with Yang Yin Fang plus Glucocorticoid.was better than other singular intervention,and i its pharmacological mechanism was explored by biochemieal and molecular biological analysis to provide the basis for clinical application.