The Role And Mechanisms Of Zyxin In Podocyte Injury In Hypertensive Nephropathy

Objective: Podocyte injury and effacement are early cellular events in hypertensive nephropathy,but the mechanisms by which they occur are unknown.Zyxin is a mechanotransductor located on focal adhesions,which regulates stress fiber remodeling and has a wide range of biological functions.However,the role of zyxin in podocytes is still unclear.In this study,we constructed hypertensive environment model in immortalized human podocytes in vitro and hypertensive nephropathy model in podocyte-specific zyxin knockout mice in vivo to explore the role and mechanisms of zyxin in podocytes in the hypertensive environment,expecting to provide new ideas for delaying the progression of hypertensive nephropathy in clinical.Methods:1.The model of hypertensive nephropathy was established by implanting DOCA pellet and Ang II osmotic pump subcutaneously after uninephrectomy.The blood,urine,and kidney samples were collected after 5 weeks.2.Construction of podocyte-specific Zyxin knockout mice: podocyte-specific zyxin knockout(Nphs2-Cre+/Zyxinflox/flox,abbreviated Cre+/Zyxinfl/fl)mice were constructed using the Cre-Lox P recombination system.3.Immunohistochemistry was used to observe the expression and distribution of zyxin,nephrin,and WT1 in the glomerulus.4.Periodic Acid-Schiff(PAS)staining was used to evaluate the degree of glomerular damage in each group by the glomerular morphology.5.Transmission electron microscopy(TEM)was used to observe the ultrastructure of the podocyte foot processes and the thickness of the glomerular basement membrane.6.Immunofluorescence was used to detect the expression and co-localization of zyxin and nephrin in the glomerulus,and to detect the expression and localization of α-actinin-4 and focal adhesions(vinculin and paxillin)in the glomerulus.7.In vitro,the cultured human podocytes stimulated by mechanical stretch and Ang II were used to simulate the hypertensive nephropathy environment.Western-blot was used to detect the expression of zyxin.8.Podocytes were transfected with zyxin-sh RNA.The expression levels of zyxin,vinculin,paxillin,and α-actinin-4 were detected by Western blot and q RT-PCR.9.Cell scratch assay and cell adhesion assay were used to evaluate the changes of podocyte migration and adhesion.10.Phalloidin staining and cell spreading assay were used to assess actin cytoskeleton rearrangement in podocytes.11.Co-immunoprecipitation(CO-IP)was used to verify the interaction between Zyxin and Sirt1.Results:1.The expression of zyxin was decreased in the hypertensive nephropathy model.Immunofluorescence staining showed that the expression of zyxin in the glomeruli was decreased in hypertensive nephropathy mice,and the co-localization of zyxin with nephrin was decreased.The expression of zyxin in podocytes cultured in vitro was down-regulated and redistributed during mechanical stretch,while the expression of zyxin remained unchanged but was redistributed during Ang II stimulation.2.Zyxin protected the kidney from hypertension.Compared with the control group,renal functions(urinary protein/creatinine ratio,serum creatinine,and blood urea nitrogen)were significantly increased in hypertensive mice,and podocyte-specific zyxin knockout aggravated renal function damage,glomerulosclerosis,the glomerular basement membrane thickening,foot process effacement,and podocyte detachment induced by hypertension.These results suggested that zyxin has protective effect on kidney and podocytes in hypertensive mice.3.Zyxin regulated podocyte actin cytoskeleton remodeling and the dynamic changes of migration and adhesion.In vitro,actin stress fiber remodeling was observed under mechanical stretch and AngII stimulation in the cultured podocytes.Inhibiting nuclear-cytoplasmic transport of zyxin,podocyte actin cytoskeleton remodeling disordered.After knockdown of the zyxin,stress fiber remodeling was impaired.The expression of focal adhesions(vinculin and paxillin)and integrin in podocytes changed,thus podocyte migration force was increased,while the podocyte adhesion force was decreased.The expression of focal adhesions(vinculin and paxillin)and α-actinin-4 was down-regulated in podocyte-specific zyxin knockout mice,resulting in foot process fusion,podocyte shedding,and glomerulosclerosis.4.Zyxin deacetylation in podocytes was regulated by Sirt1,maintaining actin cytoskeleton integrity.Immunofluorescence showed that zyxin and sirt1 were co-localized in the nucleus,and sirt1 could deacetylate zyxin.Inhibiting the activity of sirt1 resulted in the impaired actin cytoskeleton remodeling and the decreased podocyte migration.Conclusions:Zyxin plays an important protective role in podocyte injury in hypertensive nephropathy.Zyxin regulates podocyte cytoskeleton rearrangement and dynamic changes of adhesion and migration by shuttling between the cytosol and the nucleus,maintaining actin cytoskeleton integrity to protect podocytes and prevent glomerular injury,which may be mediated by sirt1 through deacetylation.