The Protective Effect Of The Xuan Tong San Jiao Huo Xue Tong Luo Decoction On Renal Tissue Of Rats With Membranous Nephropathy And Impact On The Expression Of CD2AP And Podocin In Kidney

Objective: Membranous nephropathy(MN)is a glomerular disease characterized by diffuse deposition of immune complexes in the glomerular visceral epithelial cells,thickening of the glomerular basement membrane and�spikes� formation.The unknown etiology is called idiopathic membranous nephropathy,taking up about 2/3 of MN.The main clinical manifestation of MN is proteinuria.This study applies the caudal vein injection of cationic bovine serum albumin(C-BSA)method to establish the rat model of MN and applies Xuan tong San jiao Huo xue Tong luo decoction to interventional treatment.According o the observation of the general conditions of rats,24 hours urine total protein(24h UTP),total protein(TP),albumin(ALB),total cholesterol(TC),glycerin three greases(TG),blood urea nitrogen(BUN),serum creatinine(Scr),other biochemical indicators and the expression of CD2 AP and Podocin in renal tissue,exploring the protective effect of the Xuan tong San jiao Huo xue Tong luo decoction on renal tissue of rats with membranous nephropathy and impact on the expression of CD2 AP and Podocin in kidney,providing experimental basis and theoretical basis for prevention and treatment of MN.Methods: 40 clean healthy male SD rats,weighted 160�20g,by feeding normal diet for one week after the urine protein decreasing to negative,10 rats were randomly selected as control group(C group)and 30 rats were randomly selected as marked group.After preimmunization for one week,the marked group began to immunize officially and were injected with C-BSA via caudal vein every other day for four weeks to establish MN rats model.The rats of C group were injected equivalent normal saline.After model,24 hours urine ofthe rats in marked group were preserved to test 24 h UTP in order to confirm the success of modeling.According to body weight were randomly divided into model group(M group),Chinese medicine group(Z group)and tripterygium glycosides combined with prednisone acetate group(L group).Z group and L group were taken the corresponding drugs for gastric lavage respectively,C group and M group were taken the same amount of saline.Each group were taken medicine for four weeks.After the administration,24 hours urine of the rats in each group were preserved to test 24 h UTP.The blood of rats in each group were collected from femoral artery under anesthesia to test TP,ALB,TC,TG,BUN,Scr and other biochemical indexes.The fresh kidney tissues were kept for observing the pathological changes of renal tissue in rats by immunofluorescence,light microscope and electron microscope and detecting expression of CD2 AP and Podocin in the kidney of rats by immunohistochemistry,Western-blot and Real-time PCR.Results:1 General condition of rats The C group rats showed good spirit,normal activities,hair shiny with no shedding,good eating and drinking and normal urine and stool.The rats in marked group showed different degree of listlessness,not actively,less hair luster and began to fall off,eating less.Some rats had scrotal edema.In the period of the experiment,three of marked group rats were dead.The cause of death of rats may be that the purity of C-BSA solution was not enough.After improved filtration,no death occurred in each group.After administration,the rats in Z group and L group had better condition than M group,and there was no change in M group.2 24 hUTP After administration,24 h UTP in marked group were significantly higher than that of C group,the difference was statistically significant(P < 0.05).24 h UTP in Z group and L group were significantly lower than M group,the difference was statistically significant(P < 0.05).There were no significant difference between L group and Z group,the difference was not statistically significant(P > 0.05).3 Results of blood indexes in rats(1)TP,ALB: After the administration,TP and ALB of the marked group were significantly lower than those of C group,the difference was statistically significant(P < 0.05);TP and ALB of Z group and L group were significantly higher than that of M group,the difference was statistically significant(P <0.05);TP and ALB of Z group were significantly higher than L group,the difference was statistically significant(P < 0.05).(2)TC,TG: After the administration,TC and TG of the marked group were significantly higher than those of C group,the difference was statistically significant(P < 0.05);TC and TG of Z group and L group were significantly lower than those of M group,the difference was statistically significant(P <0.05);TC and TG of Z group were significantly lower than L group,the difference was statistically significant(P < 0.05).(3)BUN,Scr: After the administration,BUN and Scr of each group had no significant changes,there was no statistically significant difference(P >0.05).(4)Immunofluorescence: In C group,there was no obvious glomerular immune complex deposition;In M group,IgG deposited diffusively,along with the glomerular capillary loops and mesangial region;In Z group and L group showed a small amount of IgG,along with the glomerular capillary loops and mesangial area.(5)light microscope: There was no significant change of glomerular structure in C group;In M group,glomerular capillary basement membran of the rats was thickened diffusively,and �spikes� were formed,and mesangial matrix was increased;In Z group and L group,glomerular capillary basement membran of the rats was thickened slightly,and a small amount of �spikes�were formed,and mesangial matrix was relatively reduced.(6)Electron microscope: In C group,there was no significant change in the structure of glomerular podocytes;In M group,the glomerular basement membrane was thickened,and a large number of electron dense deposits were found in the epithelium,and the foot process diffused or disappeared,and�spikes� were formed;In Z group and L group,the glomerular basement membrane was thickened slightly,and a litttle electron dense deposits were found in the epithelium,and partial foot process diffused.(7)Immunohistochemical method: In C group,a large number of CD2 AP were distributed on the glomerular,a small amount of CD2 AP appeared in renal tubule and collecting duct,showing brown staining;Expression of CD2 AP significantly was reduced in M group;Compared with M group,the expression of CD2 AP in glomeruli of Z group and L group was increased significantly.In C group,Podocin appeared in glomeruli of rats and was not significantly expressed in renal tubules and interstitium.Podocin was linearly distributed along the glomerular basement membrane,showing brown staining;In M group,the expression of Podocin was significantly decreased in the glomeruli of rats;Compared with M group,the expression of Podocin in glomeruli of rats in group Z and group L were significantly increased.(8)Western blot: Compared with the C group,the expression of the CD2 AP in renal tissue of the rats of marked group was significantly reduced,the difference was statistically significant(P < 0.05);Compared with M group,the expression of CD2 AP in renal tissue of Z group and L group was significantly increased,the difference was statistically significant(P < 0.05);There was no significant difference between the expression of CD2 AP in renal tissue of Z group and the expression of CD2 AP in renal tissue of L group(P >0.05).Compared with C group,the expression of Podocin in renal tissue of each marked group was significantly reduced,and the difference was statistically significant(P < 0.05);Compared with M group,the expression of Podocin in renal tissue of Z group and L group was significantly increased,the difference was statistically significant(P < 0.05);There was no significant difference between the expression of Podocin in renal tissue of Z group and the expression of Podocin in renal tissue of L group(P > 0.05).(9)Real-time PCR: Compared with C group,the expression of CD2 AP mRNA in renal tissue of each marked group was significantly reduced,and the difference was statistically significant(P < 0.05);Compared with M group,the expression of CD2 AP mRNA in renal tissue of Z group and L group was significantly increased,the difference was statistically significant(P < 0.05);There was no significant difference between the expression of CD2 AP mRNA in renal tissue of Z group and the expression of CD2 AP mRNA in renal tissue of L group(P > 0.05).Compared with C group,the expression of Podocin mRNA in renal tissue of each marked group was significantly reduced,and the difference was statistically significant(P < 0.05);Compared with M group,the expression of Podocin mRNA in renal tissue of Z group and L group was significantly increased,the difference was statistically significant(P < 0.05);There was no significant difference between the expression of Podocin mRNA in renal tissue of Z group and the expression of Podocin mRNA in renal tissue of L group(P > 0.05).Conclusions:1 Xuan tong San jiao Huo xue Tong luo decoction can effectively improve the MN rats' general condition,significantly reduce the level of the MN rats' 24 h UTP.At the same time,it can regulate lipid metabolism and increase plasma protein.2 Xuan tong San jiao Huo xue Tong luo decoction can effectively inhibit or reduce the deposition of immune complexes in MN rats' glomerular basement membrane and reduce renal damage and pathological changes.3 Xuan tong San jiao Huo xue Tong luo decoction can effectively increase the expression level of CD2 AP protein and mRNA in renal tissue and Podocin protein and mRNA and play a protective role in maintaining the integrity of the podocyte structure and function of SD,may be this is one of the mechanisms that control proteinuria.