The Mechanism Of Abp1 And IL-17A Activatign The Axis Of ROS-NLPR3-caspase-1 In Inducing Podocyte Injury

PART ? THE ROLE AND PRELIMINARY MECHANISM OF ABP1 ON REGULATING PODOCYTE FUNCTION SECTION ? THE EXPRESSION AND FUCNTION OF ABP1 IN THE PODOCYTEObjective: To investigate whether Abp1 is expressed in the kidneys and podocytes,further explore the effect of Abp1 on the function of podocyte.Methods: The expression of Abp1 was detected by RT-PCR and immunofluorescence in kidney tissue and podocyte;the spatial relationship of Abp1 and F-actin was detected by immunofluorescence;Abp1 overexpression cell line was constructed by transfected Abp1 lentivirus plasmid,the efficiency of Abp1 was measured by Q-PCR and western blot;the proliferation of podocyte was detected by CCK8 and the apoptosis of podocyte was measured by flow cytometry;crystal violet was stained to detect the adhesion function;the m RNA expression of podocin and nephrin was measured by Q-PCR.Results: Abp1 was expressed in kidney tissue and podocyte.It was distributed in the cytoplasm of foot and it was localized with F-actin.Compared with control group,the m RNA and protein expression of Abp1 were significantly increased in overexpression Abp1 group(P < 0.05);Compared with control group,the proliferation activity of podocyte was decreased while the apoptosis of podocyte was increased in overexpression Abp1 group(P < 0.05);the adhesion function of podocyte was decreased while the structure of F-actin was disrupted in overexpression Abp1 group(P < 0.05);There is no significance in the expression of nephrin and podocin between the groups(P >0.05)Conclusion: Abp1 is expressed in the kidney and podocyte,and overexpression Abp1 has an effect on the proliferation,apoptosis and adhesion function of podocyte.SECTION ? THE MECHANISM OF ABP1 INREGULATING THE FUNCTION OF PODOCYTE Objective: To investigate the potential mechanism of Abp1 regulating cell morphology,proliferation and apoptosis.Methods: Cell cycle was detected by flow cytometry.The protein changes of cell cycle related proteins cyclin D1 and P27 were detected by Q-PCR.The expression of YAP and dendrin were measured by Q-PCR.The expression and activity of Rho A were detected by Western blot and pull down.The spatial relationship of Abp1 with Arp2/3(N-wasp)was detected by Immunofluorescence in the podocyte.The expression of Abp1,Rho A,YAP and dendrin were detected by Q-PCR and western blot in LPS induced podocyte injury.Results: Compared with control group,the cell cycle was arrested in S phase in the overexpression Abp1 group(P < 0.05);the expression of cell cycle protein cyclin D1 was decreased and P27 was increased(P < 0.05).Compared with control group,the expression of YAP was decreased and dendrin was increased(P < 0.05),while the expression and activity Rho A expression were decreased(P < 0.05).Abp1 could co-localize with Arp2/3 and N-wasp in podocyte.Compared with control group,the expression of Abp1 and dendrin was increased(P < 0.05);the expression and activity of Rho A was decreased(P < 0.05),while the expression of YAP was decreased in the LPS group(P < 0.05).Conclusion: Abp1 may inhibit cell proliferation by regulating the expression cyclin D1 and P27 and arresting the cell cycle in S phase;Abp1 may promote cell apoptosis through regulating the signal of Rho A-YAP-dendrin;Abp1 may regulate the F-actin structure by mediating N-wasp/Arp2/3 complex;LPS can promote podocyte apoptosis by upregulating the expression of Abp1 and inhibiting Rho A activity.PART ? IL-17 A INDUCING PODOCYTE BY ACTIVATING ROS-NLRP3-CASPASE-1 AXISObjective: To explore the potential mechanism of IL-17 A inducing podocyte injury by treating podocyte with IL-17 A in vitro.Methods: The cells were divided into 5 groups,control group,IL-17 A group,IL-17 RA group,NAC group and YVAD group.The expression of NLRP3,caspase-1,podocin and desmin was detected by Q-PCR,Western blot and immunofluorescence.Intracellular ROS was detected by flow cytometry and caspase-1 activity was detected with the colorimetric assay.The secretion of IL-1? was detected by ELISA,and F-actin was labelled by phalloidin.Results: Compared with control group,the expression of NLRP3 and caspase-1 were increased,which was time and dosage dependent way in the IL-17 A group(P < 0.05);caspase-1 activity and IL-1? secretion was increased in the IL-17 A group(P < 0.05);the intracellular ROS level and the expression of desmin were increased,while the expression of podocin was decreased in the IL-17 A group(P < 0.05);F-actin structure was disrupted in the IL-17 A group.Compared with IL-17 A group,the intracellular ROS level was decreased,while the expression of NLRP3 and caspase-1 was downregulated in the IL-17 RA group(P < 0.05).Compared with IL-17 A group,the intracellular ROS level was decreased,while caspase-1 activity and IL-1? secretion were downregulated in the NAC group;the expression of desmin expression was decreased and the expression of podocin was increased(P < 0.05).Compared with IL-17 A group,caspase-1 activity and IL-1? secretion were downregulated,while the expression of desmin was decreased and the expression of podocin was increased(P < 0.05).Compared with IL-17 A group,F-actin fibers was restored in YVAD group and NAC group(P < 0.05).Conclusion: IL-17 A could induce podocyte injury by promoting IL-1?secretion via activating the ROS-NLRP3-caspase-1 axis.