| Background and Objective:Podocyte is the most important cellular component constituting the glomerular filtration barrier with complex and subtle structure. Podocyte is composed of three compartments:cell body, primary process and foot process. A special structure, slit diaphragm (SD), is formed between adjacent foot processes. Podocyte injury and SD destruction are the important mechanisms of pathogenesis of proteinuria and glomerulosclerosis. Nephrin is an important structural and signalling molecule specifically expressed in podocytes. It belongs to the immunoglobulin superfamily members, which is composed of extracellular, intracellular and transmembrane domains. The extracellular domain is cross-linked with the adjacent Nephrin molecules or the homologous nephl through its addicted homophilic or heterophilic binding capacity, which contributes to the formation of SD. The intracellular domain contains three evolutionarily conserved tyrosine residues, with an important signal transduction function. The changes of Nephrin expression, distribution and phosphorylation have been demonstrated in many studies of podocyte injury model. Abnormal activation of the renin-angiotensin-aldosterone system (RAAS) is an important mechanism leading to renal injury, in which angiotensin Ⅱ (Ang Ⅱ) is the main effector. It has been shown that Ang II can induce podocyte apoptosis, F-actin reorganization and the changes of Nephrin expression, distribution and phosphorylation in a dose and time-dependent manner, but its regulatory mechanism is unclear. Caveolae is a special structure of the lipid rafts, with many functions such as endocytosis, cholesterol transport, membrane stability and signal transduction. Caveolin-1is a key molecule constituting the caveolae, which plays an important role in signal transduction or cross-talking between different signaling pathways. Studies have demonstrated that Nephrin and other podocyte-specific molecules co-locate in caveolae. Caveolin-1may play an important role in Ang Ⅱ-induced Nephrin phosphorylation alteration. In the present study, we evaluated the effect of Ang Ⅱ on the expression of Nephrin and Caveolin-1and podocyte injury; By transfection of Caveolin-1full-length plasmid or siRNA in the cultured podocytes, we further explored the mechanisms of Ang Ⅱ-induced podocyte injury and Nephrin phosphorylation alteration. Methods:Part I:Thirty-six Wistar rats were subcutaneously embedded with osmotic minipumps and randomly divided into three groups according to receiving either Ang Ⅱ at a dose of400ng·kg-1-min-1or AngⅡ+telmisartan at a dose of3mg·kg-1·d-1, or normal saline as a control group. The animal models continued for28days. The rat tail artery pressure and24-hour urinary albumin were measured prior to modeling and at7d,14d,21d and28d of the expriment. Rats were sacrificed at14d and28d respectively. Simultaneously, the blood and renal cortex were collected. Serum creatinine (Scr) and creatinine clearance rate (Ccr) were analyzed; The concentrations of Ang Ⅱ both in blood plasma and kidney tissue were detected by radioimmunoassay; Renal pathological and podocyte ultrastructure were observed under light and transmission electron microscopy; Nephrin and Caveolin-1expression and their phosphorylation were analyzed by immunostaining and Western-blot. Part II:In vitro, cultured murine immortalized podocytes were exposed to Ang Ⅱ (10-6M) for variable time periods (3h,6h,12h and24h). or pretreated with losartan (10-5M) for6h. Nephrin and Caveolin-1expression and their phosphorylation were analyzed by Western-blot and immunofluorescence. Caveolae membrane fractions were isolated by sucrose density gradient centrifugation, and the distribution and interactions between Ang Ⅱ type1receptor (AT1R), Nephrin, C-terminal Src kinase (Csk) and Caveolin-1were then evaluated using Western-blot and co-immunoprecipitation. Part Ⅲ:Caveolin-1full-length plasmid (pEGFPC3-cav-1) was constructed and transfected into cultured podocytes using lipofection to up-regulated Caveolin-1expression, in which G418was used to establish stably transfected cell lines. Caveolin-1-specific small interfering RNA (cav-1siRNA) was designed, synthesized and transfected into cultured podocytes to down-regulated Caveolin-1expression. Cells were then divided into six different groups:the normal control group, the Ang Ⅱ-simulated group, the AngⅡ+pEGFPC3-cav-1transfected group, the AngⅡ+empty vector (pEGFPC3) transfected group, the AngⅡ+cav-1siRNA transfected group, and the Ang Ⅱ+scrambled siRNA transfected group. Nephrin expression and its phosphorylation were analyzed by Western-blot. Podocyte apoptosis was evaluated by cell nucleus staining with Hoechst-33342. The distribution of F-actin was presented by FITC-phallodin labeling, and the recombination of F-actin was evaluated by cortical F-actin score index (CFS).Results:Part I:The blood pressure, urinary albumin excretion and Ang Ⅱ concentration in Ang Ⅱ-infused rats were significantly increased more than that in control or telmisartan treatment group at the same time point (p<0.05), but no significant difference in Ccr among three groups(p>0.05); Ang Ⅱ-received rats exhibited significant glomerular pathological changes, such as mesangial area widened, matrix increased, or even focal segmental sclerosis, podocyte foot process fusion obviously or even disappeared. Telmisartan treatment could significantly mitigate the glomerular pathological changes and podocyte injury induced by Ang Ⅱ; In Ang Ⅱ-infused rat glomerular, Nephrin exprenssion and phosphorylation was obviously decreased(p<0.05), and Caveolin-1phosphorylation was increased markedly compared with control or telmisartan treatment group(p<0.05), but with no significant change in total Caveolin-1protein expression(p>0.05). Part II:Podocytes had abundant expression of Caveolin-1but a low level of phosphorylation in nomal conditions. Ang Ⅱ stimulation led to the decrease in Nephrin expression and phosphorylation (p<0.05), and an increase in Caveolin-1phosphorylation (p<0.05), but no change in total Caveolin-1protein expression compared with control or losartan treated group (p>0.05). Lipid raft separation showed that Nephrin and Caveolin-1were co-localized in caveolae fractions; AT1R and Csk were mainly localized in non-caveolae fractions. After Ang Ⅱ stimulation for3h, AT1R and Csk expression were increased in caveolae fractions, and had an interaction with Caveolin-1. Part Ⅲ:pEGFPC3-cav-1transfection increased the expression of Caveolin-1approximately2-fold more than that of control cells or cells transfected with pEGFPC3. After Ang II stimulation, the level of Caveolin-1phosphorylation was significantly increased, the podocyte apoptosis rate and CFS score increased obviously in a short period of time, but Nephrin phosphorylation was diminished markedly compared with the nomal control or pEGFPC3transfected group (p<0.05); cav-1siRNA transfection resulted in a60%down expression of Caveolin-1compared with control or the scrambled siRNA transfected group. Cav-1siRNA significantly inhibited Ang Ⅱ-induced Caveolin-1phosphorylation, Nephrin dephosphorylation, and markedly ameliorated Ang Ⅱ-induced podocyte apoptosis and F-actin recombination.Conclusion:These studies indicate that Nephrin phosphorylation is critical for the podocyte survival and Ang Ⅱ-induced Nephrin dephosphorylation may be an important mechanism for Ang Ⅱ-induced podocyte injury; Ang Ⅱ induces Nephrin dephosphorylation and podocyte injury through a Caveolin-1-dependent mechanism. |
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