The Roles Of Nephrin In Angiotensin Ⅱ-induced Phenotypic Alteration Of Podocytes

Background and Aim The slit diaphragm(SD), an unique structure of podocyte, is the key component of glomerular filtration barrier to prevent leakage of serum protein. Nephrin, the first identified component of SD, has been demonstrated to play a crucial role in the development of proteinuric glomerular disease. In addition to its structural role, accumulating data has implicated that nephrin also acts as a signaling protein involved in complex signal reactions via its intracellular domain. AngiotensinⅡ(AngⅡ), a key factor of renin-angiotensin system(RAS), has been demonstrated as a crucial mediator in the progression of chronic kidney diseases. In our recently published work, AngiotensinⅡinduces down-regulation of nephrin expression and apoptosis of podocytes via mini-osmotic pumps in vivo and the AngⅡ-infused rats develop proteinuria and foot process effacement. Moreover, the podocyte apoptosis and effacement of foot process are the characteristic phenotypes of podocyte injury in several glomerular diseases. However, the direct role of nephrin in AngⅡ-induced podocyte injury is not fully understood. In the present study, we evaluated the effects of AngⅡon phenotypes of apoptosis and F-actin cytoskeleton remodeling and expression of nephrin in conditionally immortalized mouse podocytes. In addition, the PI3K/Akt signaling pathway was assessed in the process of podocyte apoptosis and cytoskeleton remodeling.Methods PartⅠ. Differentiated mouse podocytes were exposed to AngⅡat different concentrations(10-12M,10-10M,10-8M,10-6M) for 18h or at 10-8M for variable incubation time(3h,6h,12h,18h,24h). In addition, the pretreated cells with angiotensinⅡtype 1 receptor antagonist (losartan, at 10-6M) for 1h were co-incubated with AngⅡ(10-8M) for 18h and medium in 1% FCS treated cells were used as control. F-actin was stained with FITC-conjugated phalloidin and semi-quantitative system with cortical F-actin score(CFS) was introduced to analyze the degree of actin cytoskeleton arrangement. Apoptosis was evaluated by Hoechst-33342 staining and flow cytometry respectively. The expression of nephrin was assessed by quantitative real-time PCR and immunofluorescence. PartⅡ. The pcDNA3.1-mNPHS 1 vector (gift of Dr. Lawrence. Holzman, Michigan University, Ann Arbor, USA) was cut with HindⅢand EcoR I and sequenced to verify whether the full-length ORF of mouse nephrin was incorporated in pcDNA3.1. Undifferentiated mouse podocytes were transfected using Lipofectamine 2000 with the pcDNA3.1-mNPHS1 plasmid and stably transfected cell lines was generated with G418 selection. Levels of mRNA and protein expression of nephrin were measured by RT-PCR and Western-blotting. PartⅢ. In separated experiments, untransfected mouse podocytes(MPC) and stably transfected podocytes with pcDNA3.1-neo and pcDNA3.1-mNPHS1 were exposed to AngⅡ(10-8M) or LY2940027 (a selective Akt inhibitor,50μM) for indicated times. The phosphorylation level of Akt was determined by Western-blotting. The actin cytoskeleton remodeling and apoptosis were assessed with the same methods indicated in Part I.Results PartⅠ:AngⅡpromoted podocyte apoptosis in a dose- and time -dependent manner. AngⅡ-stimulated cells revealed a morphologic shift with lose of filopodium and formation of lamellipodia, which was associated with cytoskeletal rearrangement including cortical F-actin ring formation and stress fiber attenuation. Pretreatment with losartan significantly prevented AngⅡ-induced apoptosis and actin cytoskeleton reorganization. Nephrin mRNA and protein were obviously decreased in podocytes exposed to AngⅡfor a least 12h than in vehicle-treated cells. PartⅡ:The full-length ORF of mouse nephrin was ligated to pcDNA3.1-mNPHS1 vector. Levels of nephrin mRNA and protein in cells stably transfected with pcDNA3.1-mNPHS 1 were about 2 times higher than those in untransfected and pcDNA3.1-neo transfected podocytes. PartⅢ:AngⅡexposure for more than 15min inhibited the phosphorylation of AKT in MPC, which were dramatically reversed by pcDNA3.1-mNPHS1 transfection, but not by pcDNA3.1-neo transfection. Podocyte apoptosis and cytoskeletal rearrangement were also promoted by LY2940027. Conversely, AngⅡ-induced podocyte apoptosis was significantly alleviated by pcDNA3.1-mNPHS1 transfection. However, pcDNA3.1-mNPHS1 transfection did not completely prevent AngⅡ-induced F-actin remodeling, but still induced filopodium formation under AngⅡ-stimulated condition.Conclusion These studies demonstrate that AngⅡinduces mouse podocyte apoptosis and actin cytoskeleton rearrangement and suppresses the expression of nephrin through PI3K/Akt signaling pathway.