Role Of C-Ab1 In Nephrin-mediated Cytoskeleton Remodeling Of Podocytes

Background and objective:Podocyte is the most important component of glomerular filtration barrier,and the slit diaphragm(SD)formed between adjacent foot processes is regarded as the key structure to maintain normal glomerular filtration function.SD destruction leads to podocyte cytoskeleton remodeling,podocyte loss and progressive proteinuria.As a transmembrane protein,nehprin is specifically expressed in the SD region.Its extracellular domain,which contains type Ⅲ fibronectin structure and IgG region,forms the foundation of SD structure by connection with other nephrin molecules and nephrin homologous molecules through homophilic or heterophilic binding ability.The intracellular domain of nephrin contains multiple tyrosine residues with a potential ability to be activated and mediate podocyte cytoskeleton remodeling and apoptosis.Angiotensin Ⅱ(AngⅡ),as a key effector of renin-angiotensin system(RAS),can regulate nephrin phosphorylation and podocyte injury,but the specific mechanism is unclear.c-Abl,a non-receptor protein tyrosine kinase,is widely expressed in terminal differentiated cells.Through its SH2 and SH3 domains,c-Abl can be activated by various molecules.Following activation,c-Abl participates in diverse cellular events,including cytoskeleton remodeling,apoptosis and infection.Previous studies demonstrated that nephrin recruits molecules contained SH2 and SH3 domains,such as Nck、podocin and CD2AP,but whether nephrin interacts with c-Abl is not clarified.In this study,biopsy specimens from podocyte disease patients,AngⅡ-induced model and cultured podocytes were used to evaluate the expression and distribution characteristics of nephrin and c-Abl.By transfection of nephrin specific phosphorylated construct and c-Abl construct,we further explore the role of c-Abl in nephrin-mediated cytoskeleton remodeling of podocytes.Methods:Part 1:Colocalized expression of nephrin and c-Abl was evaluated in glomeruli of patients with podocyte diseases by double immunolabeling analysis.Sixteen male Wistar rats were randomly assigned to receive either Ang Ⅱ(400 ng·kg-1·min-1)as the experimental group or saline as control group using osmotic mini-pumps.24-hour urinary protein were measured at 7d and 14d of the experiment.Rats were sacrificed at 14d and renal cortex tissues were collected.Transmission electron microscopy was used to observe glomerular podocyte ultrastructure changes.Immunofluorescence was performed to evaluate the expression and distribution of c-Abl and nephrin.Part2:In vitro,cultured murine podocytes were devided into AngⅡ group(10-7M AngⅡ)and control group.CD 16 antibody induced nephrin specific phosphorylation in myc-CD16/7-nephrin construct transfected cell lines.c-Abl expression and activation were altered by siRNA and imatinib(STI571).Western blotting was performed to evaluate the expression and phosphorylation of nephrin and c-Abl.Co-localization of nephrin and c-Abl was determined by immunofluorescence and co-immunoprecipitation analysis.Cell migration and spreading assays were used to evaluate the function of cytoskeleton remodeling.Cytoskeleton configuration was evaluated by FITC-phalloidin staining.Part 3:c-Abl full-length plasmids or mutated constructs were co-transfected with myc-CD16-CD7-nephrin in COS7 cells.Cytochalasin D(Cyt D)was performed to disorganize cytoskeleton of COS7 cells.Western blotting was performed to evaluate the expression and phosphorylation of nephrin and c-Abl.Co-localization of nephrin and c-Abl was determined by immunofluorescence and co-immunoprecipitation analysis.Cytoskeleton configuration was evaluated by FITC-phalloidin staining.Results:Part1:(1)The glomerular staining of nephrin and c-Abl indicated that the co-localization in patients with podocyte diseases was obviously decreased compared with that in control patients;(2)Ang II-infused animals developed proteinuria and fusion of foot process;(3)Ang II induced reduction in co-localization of nephrin and c-Abl.Part 2:(1)Ang Ⅱ exposure induced dephosphorylation of nephrin and diminished interaction between nephrin and c-Abl;(2)Cell migration and F-actin disruption were aggravated by exposure of Ang Ⅱ;(3)Phosphotylated nephrin recruited c-Abl,and prevented disorganized cytoskeleton stimulated by AngⅡ.Part 3:(1)Co-transfection with dephosphorylated nephrin and c-Abl full-length constructs failed to restore disorganized cytoskeleton stimulated by Cyt D in COS7 cells;(2)Nephrin specific phosphorylation induced by CD 16 antibody promoted nephrin-c-Abl interaction,and prevented cytoskeleton disorganization of COS7 cells stimulated by Cyt D;(3)SH2/SH3-defective c-Abl was unable to interacted with nephrin,which had no ability to restore disorganized cytoskeleton of COS7 cells.(4)Phosphorylated nephrin interacted with FABD-defective c-Abl,but the interaction failed to prevented COS7 cells cytoskeleton disorganization.Conclusion:The present study suggests that phosphorylated nephrin is able to recruit c-Abl in a SH2/SH3-dependent manner,which contributes to cytoskeleton remodeling of podocytes.