Effect of astragalus polysaccharin on expression of nephrin and podocin in podocytes of early diabetic nephropathy rats

AIM: To observe the effects of astragalus polysaccharin (APS) on the expression of nephrin and podocin in podocytes of diabetic nephropathy (DN) rats.METHODS: The rat model of diabetes was induced by intraperitoneal injection of streptozotocin (STZ).The diabetic rats were randomly divided into 2 groups by the treatment without or with APS: STZ group (n=8) and STZ+APS group (n=8).In addition, 8 non-treated rats served as control.All the rats were treated with APS or normal saline orally by gavage for 8 weeks.The concentration of blood glucose was monitored on week 2, 5 and 8 after treatment.Eight weeks later, the body weight and renal index were measured.Total urine protein in 24 h, blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by biochemical methods.The pathological changes of the kidneys were also observed under light microscope.The protein levels of nephrin and podocin in the kidney tissues were also determined by Western blotting.RESULTS: After APS intervention, the levels of renal index, blood glucose concentration, 24-hour total urine protein, BUN and SCr were significantly lower and body weight was higher than those in STZ group (P<0.05).The renal pathological status in APS group was significantly improved and the expression levels of nephrin and podocin also markedly increased (P<0.05).CONCLUSION: APS might protect kidney against STZ-induced injury via increasing the expression of nephrin and podocin in podocytes.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

Role of nephrin in preventing angiotensinⅡ-induced cytoskeletal rearrangement in glomerular podocytes

AIM: To investigate the role of nephrin, a slit diaphragm-associated protein, in angiotensinⅡ (AngⅡ)-induced cytoskeleton rearrangement in podocytes. METHODS: Immortalized mouse podocytes were exposed to AngⅡ (10^-8 mol/L) with or without AngⅡ receptor antagonist lorsatan and Akt inhibitor LY294002. FITC-conjugated phalloidin was used to stain F-actin, and semi-quantitative system with cortical F-actin score (CFS) was introduced to analyze the degree of actin cytoskeleton arrangement. The expression of nephrin was assessed by quantitative real-time RT-PCR,RT-PCR and Western blotting. Undifferentiated podocytes were transfected with pcDNA3.1-mNPHS1 plasmid containing the full length of nephrin. The stably transfected cell line was generated by G418 selection. Phosphorylation level of Akt was assessed by Western blotting, and F-actin distribution was further evaluated in transfected cells exposed to AngⅡ or not. RESULTS: Cytoskeletal rearrangements including cortical F-actin ring formation and stress fiber attenuation were observed in Ang II-and LY294002-stimulated podocytes. Pretreatment with losartan significantly prevented Ang II-induced actin cytoskeleton reorganization. The mRNA and protein levels of nephrin and phosphorylation of Akt were obviously decreased in the podocytes exposed to Ang II, which were dramatically reversed by pcDNA3.1-mNPHS1 transfection. Transfection of pcDNA3.1- mNPHS1 induced the formation of short filopodia and partially prevented AngⅡ-induced F-actin remodeling. CONCLUSION: PI3K/Akt signaling is a common downstream pathway of nephrin and Ang Ⅱ. Nephrin is able to stabilize AngⅡ-induced cytoskeletal rearrangement via PI3K/Akt signaling pathway.     

Effects of Zhenwu decoction on rat kidney injury induced by adriamycin

AIM: To observe the effects of Zhenwu decoction on the expression of podocin and nephrin in podocytes of adriamycin nephropathy (AN) rats, and to clarify the mechanism of Zhenwu decoction in decreasing adriamycin-induced proteinuria in rats. METHODS:Biochemical assay and pathological observation (HE staining, Masson trichrome staining and transmission electron microscopy) were used to evaluate the effects of Zhenwu decoction on renal function, pathological morphology and hydroxyproline (Hyp) content in AN rats with renal fibrosis. Western blotting was used to observe the effect of Zhenwu decoction on the expression of podocin and nephrin which are marker proteins in podocytes. RESULTS:In model group, the levels of urinary total protein (TP), blood urea nitrogen (BUN), serum creatinine (SCr) and renal Hyp were significantly increased, and the clearance of creatinine (CCr) level was decreased (P<0.05). The expression of podocin and nephrin was significantly decreased (P<0.05). Atrophic renal tubules, thickened basement membranes, fusion of foot processes, concentrating renal glomerules, expansion of some renal tubules, degeneration of renal tubular epithelial cells, protein casts, and proliferation of fibroblasts and infiltration of inflammatory cells in renal interstitium were also observed. After treatment with drugs (Zhenwu decoction and valsartan), the above-mentioned parameters were significantly changed. TP, BUN, SCr and Hyp were down-regulated to different levels, and CCr was significantly up-regulated (P<0.05). The expression of podocin and nephrin was up-regulated by treatment with Zhenwu decoction, and renal histological changes were alleviated compared with model group. CONCLUSION: Zhenwu decoction can reduce Hyp content in kidney tissue, alleviate kidney histological changes, and improve renal function in AN rats. It might protect kidney against adriamycin-induced proteinuria via increasing the expression of podocin and nephrin in podocytes.