The Mechanism Of Huangqi Guizhi Wuwu Decoction Protect Podocyte Skeleton In IgA Nephrathy Rat By Regulating AT1R/Nephrin/c-Abl Pathway

Objective: IgAN rat model was established by using BGG.To study the effect of Huangqi Guizhi Wuwu Decoction on AT1R/Nephrin/c-Abl pathway,and explore the mechanism of Huangqi Guizhi Wuwu Decoction in alleviating the damage of podocyte skeleton protein and protecting renal podocyte.Methods: 120 male SD rats were randomly divided into two groups,normal group(20),model group(100).IgAN rat model was established by taking 0.1% BGG acidified water orally for 8 weeks and injecting 1 mg BGG into tail vein for 3 consecutive days.Modeling for 13 weeks,at the 5,7,10,12 and 13 week of the experiment,24-hour urine was collected to detect 24-hour urinary protein,UACR,24-hour Alb and Alb;at the 5 and 12 week,serum BUN,Scr,UA,Alb,ALT,AST,TC and TG also were detected.Two rats in the normal group and nine rats in the model group were randomly selected.The kidneys were removed to detect the fluorescent expression of IgA,Ig G,Ig M,C3 and Clq and the pathological staining of the kidneys.At the end of the 13 th week,90 rats in the model group were randomly divided into 5 groups according to the 24-hour urinary protein level of IgAN rats,18 rats in each group: model group,low-dose group of Huangqi Guizhi Wuwutang,medium-dose group of Huangqi Guizhi Wuwutang,high-dose group of Huangqi Guizhi Wuwutang and valsartan group.After 4 weeks of drug treatment,urine and serum indexes,renal pathology,IgA fluorescence and electron microscopy were measured.Nephrin,Podocin,ACTN4,TNF-alpha,TNFR1,AT1 R,p-Nephrin and c-Abl were detected by immunohistochemistry,Real-Time PCR and Western blot.Immunofluorescence was used to detect the localized expression of Podocin in the kidney.The co-expression of F-actin,TNFR1,AT1 R,c-Abl and Nephrin was detected by double immunofluorescence staining.Results: At the 12 th week of IgAN rats,proteinuria(P<0.05)was observed.Significant deposition of IgA and C3 was observed in glomerular mesangial area(P<0.05),accompanied by a small amount of deposition of Ig G and Ig M(P<0.05),and no deposition of Clq was observed(P>0.05).Significant mesangial cell proliferation and matrix increase were observed in renal pathology.After drug treatment,Huangqi Guizhi Wuwu Decoction improved the general symptoms of mental depression,diet and lassitude in rats.Compared with the model group,proteinuria decreased(P<0.05),Scr,UA,TG decreased(P<0.05),Alb level increased(P<0.05),systolic and diastolic blood pressure ameliorated(P<0.05)in treatment group,and the middle dose group of Huangqi Guizhi Wuwu Decoction had the best effect.Compared with the normal group,the expression of Nephrin,Podocin,p-Nephrin and ACTN4 in the model group decreased significantly(P<0.05),while the expression of TNF-?,TNFR1,AT1 R and c-Abl increased significantly(P<0.05).Compared with model group,Huangqi Guizhi Wuwu Decoction could increase the expression of Nephrin,Podocin,p-Nephrin and ACTN4(P<0.05),and inhibit the expression of TNF-?,TNFR1,AT1 R and c-Abl(P<0.05)at gene and protein levels.The expression of Nephrin,Podocin protein and positive area in the medium dose group of Huangqi Guizhi Wuwu Decoction was better than that in the low and high dose group of Huangqi Guizhi Wuwu Decoction(P<0.05),and the expression of Nephrin and Podocin gene in the medium dose group was better than that in the low dose group of Huangqi Guizhi Wuwu Decoction(P<0.05).The expression of ACTN4 protein in low and medium dose group of Huangqi Guizhi Wuwu Decoction was better than that in high dose group(P<0.05),and the expression of ACTN4 gene and positive area was better than that in low and high dose group(P<0.05).Double immunofluorescence staining showed that the expression of Nephrin in glomeruli decreased(P<0.05),while the expression of AT1 R,TNFR1 and c-Abl increased(P<0.05).Nephrin/TNFR1,Nephrin/AT1 R,Nephrin/c-Abl co-localized expression was observed in glomerular basement membrane.Huangqi Guizhi Wuwu Decoction could increase Nephrin in glomeruli of IgAN rats(P<0.05),and decrease the expression of AT1 R,TNFR1 and c-Abl(P<0.05).Conclusion: 1.IgAN rat model can be successfully established by oral 0.1% BGG acidified water for 8 weeks combined with tail vein injection of 1 mg BGG for 3 consecutive days.2.The middle dose group of Huangqi Guizhi Wuwu Decoction has the best curative effect.3.One of the mechanisms of Huangqi Guizhi Wuwu Decoction in protecting kidney is to reduce the damage of podocyte cytoskeleton protein by regulating AT1R/Nephrin/c-Abl pathway,inhibit the expression of inflammatory factor TNF-? and its receptor TNFR1,and inhibit the " Cross-talk " between mesangial cells and podocytes.4.The second mechanism is that Huangqi Guizhi Wuwu Decoction can repair the damage of Nephrin,Podocin,ACTN4 and F-actin by alleviating the damage of podocyte skeleton protein caused by immune complex deposition.