YAP Signaling Regulates Podocytes Re-entry Into Cell Cycle And Dedifferentiation In Adriamycin-induced Nephropathy

Background Podocytes,also known as glomerular visceral epithelial cells,cover the glomerular basement membrane and its adjacent foot processes stagger each other,forming a key component of the glomerular filtration membrane barrier.Podocytes also have the function of synthetising basement membrane composition,maintaining glomerular morphology,regulating glomerular blood flow and so on.Podocytes injury results in cellular dysfunction,podocyte loss and proteinuria.Severe damage leads to a reduction in the number of podocyte,resulting in focal and segmental glomerulosclerosis and renal dysfunction.Podocytes are terminally differentiated cells with little or no proliferative capacity.The high expression of cell cycle inhibitory proteins,including p21,p27 and p57,is the main reason for maintaining the low proliferation of mature podocytes.Recent studies found that in some glomerular diseases such as focal and segmental glomerulosclerosis,collapsed glomerular disease and human immunodeficiency virus(HIV)-related nephropathy,podocytes would re-enter into the cell cycle and increased susceptibility to pathogenic compounds.Until now,little is known about the specific molecular biological mechanism of podocyte re-entry into the cell cycle in the pathological state.The Hippo pathway is a kinase chain composed of a series of protein kinases and transcription factors.Activated Hippo signaling negatively regulates the transcriptional activity of its downstream effector,Yes associated protein(YAP),through phosphorylation to limit tissue overgrowth and is an important pathway to maintain cell proliferation and homeostasis.Some studies found that YAP signaling pathway played an important role in the podocyte apoptosis.In the present study,we intended to explore the role of YAP signaling in adriamycin-induced podocyte re-entry into cell cycle and dedifferentiation in vitro and in vivo.Objective To investigate the role of YAP in adriamycin-induced nephropathy podocyte entering into cell cycle and cell dediffierentiation.Methods In vivo experiments,adriamycin nephropathy mouse model was used.The urinary albumin was detected by co-omassie brilliant blue method and the urine creatinine was detected by sarcosine oxidase method.Immunohistochemical staining was used to detect the expression of Proliferating Cell Nuclear Antigen(PCNA),Cyclin-dependent kinase4(CDK4),CyclinD1,YAP and Desmin in mouse kidney.Immunofluorescence staining was used to detect the co-localization of Podocalyxin,Desmin and Snail2.In this study,the conditional immortalized mouse podocyte line5P12,passages between 15-25 were used for vitro studies.The cell cycle was detected by flow cytometry.The expression of PCNA,P-YAP,CDK4,Snail2 and Desmin were detected by immunofluorescence staining.Overexpression of plasmid transfection up-regulated podocyte YAP expression.Real-time PCR was used to detect the expression of cell cycle-related genes and podocyte marker genes and loss-of-differentiation related genes.Protein expression was examed by western blotting.Results Urine albumin creatinine ratio began to rise in adriamycin-induced nephropathy mice after tail vein infusion of doxorubicin(10 mg/kg)for 8 days.Urinary albumin creatinine ratio increased significantly on day 16(56.6±16.03 mg/mmol),P<0.05 as compared to 4.05±0.44 mg/mmol in the control group.(PCNA)positive podocytes were found in adriamycin-induced nephropathy mice.In vitro cultured podocytes,12 h and 24 h after adriamycin administration,podocytes re-entered into the cell cycle,the percentage of cells entering S phase was 39.94±0.84%and41.18±1.46%,respectively,p<0.05 compared with 26.52±0.65%in control group.Immunofluorescence staining showed that after 4h,8h and 12h of adriamycin stimulation,PCNA expression was up-regulated.Adriamycin nephropathy mice expressed cell cycle associated proteins CDK4 and CyclinD1 in podocytes from 16days.In vitro studies,incubation of adriamycin had no significant effect on the expression of cell cycle related genes in podocytes.Immunoblotting showed that CDK4and CyclinD1 were significantly up-regulated after incubation of adriamycin for 4h and8h,but P27 and P21,which are cell cycle-dependent protein kinase inhibitors,remained unchanged.Further experiments showed that YAP signaling may play a key role in the regulation of podocyte re-entry into the cell cycle.Immunohistochemical staining showed that the expression of YAP in podocyte nucli of adriamycin nephropathy mice increased significantly.In vitro experiments,phosphorylated YAP levels podocytes were significantly reduced after 4h and 8h of adriamycin incubation,and 12h restored to baseline,cell immunofluorescence also confirmed these results.Overexpression of YAP in podocytes promoted cells entry into the cell cycle and upregulate CyclinD1expression.Pretreating podocytes with verteporfin,an inhibitor for combination of YAP/TEAD,decreased the adriamycin-induced overexpression of CyclinD1 and reduced the ratio of S phase podocytes.Incubation podocytes with adriamycin showed up-regulation of mesenchymal-related genes?-SMA,PAX2,snail,and desmin with down-regualtion of podocyte marker proteins,including WT1 and ZO-1.Western blot showed that after adriamycin incubation,protein expression of Snail2 and Desmin in podocyte was up-regulated,while the expression of tight junction protein ZO-1 was significantly decreased.Immunofluorescence staining showed that Podocalyxin in adriamycin mice co-localized with Desmin and Snail2,respectively.Immunohistochemical staining showed that adriamycin-incubated podocytes had co-localization of CDK4 and Desmin.In vitro stimulation of mature podocytes by fibroblast growth factor bFGF allows podocytes to re-enter the cell cycle and up-regulate the expression of cell cycle-associated proteins CDK4 and CyclinD1.Overexpression of YAP in podocytes upregulated the expression of Desmin and Snail2.Immunofluorescence showed that bFGF stimulated mature podocytes could increase the proportion of CDK4~+/snail2~+and CDK4~+/desmin~+cells,respectively,and upregulate the expression of genes involved in the dedifferentiation.Immunoblotting results showed that after bFGF stimulation of mature podocytes for 72h,the expressions of podocyte-related marker proteins WT1,podocalyxin and Nephrin were significantly down-regulated and the expressions of Desmin and Snail2 proteins were increased.In addition,Nephrin protein can be found in the cell supernatant.Conclusion Adriamycin can induce the expression of CDK4 and Cyclin D1 in podocytes to re-enter the cell cycle,which is at least partly mediated by YAP signaling.Re-entry into cell cycle induced the marker of matural podocyte to fall off into the culture fluid,while high expression of mesenchymal markers,Desmin and Snail2 leads to cell dedifferentiation.