The Effect Of Angiopoietin-like3and Its Different Domains On Actin Filament Regulation In Podocyte

BackgroundsSignificant proteinuria is the main character of nephrotic syndrome which is related with diffused podocyte foot process effacement, is an independant risk factor for renal diseases. Foot process is a characteristic structure of glomerular podocytes, which is maintained by actin filaments of podocyte. Diffused foot process effacement and proteinuria is related with the increase of podocyte motility.Actin filament in podocyte is under highly regulation of one than100kinds of moleculi, in which integrin of podocyte basement memebrane area plays an important part. Rho family small GTPases, espacially RhoA, Racl and Cdc42are believed to be the most important moleculi in actin filament regulation for the effect of passing cell signal from membrane molecule to actin filament.Angiopoietin-like3(Angptl3) is a secreted protein weakly expressed in normal kidney, and the expression of which increases greatly in proteinuic diseases. In our previous study, podocyte secreted Angptl3was proved to involve in proteinuria development. And up-regulation of Angptl3increases the motility of podocyte in vitro, the molecular pathway of which is not clear. There are two different domains in Angptl3which is the coiled-coil domain (CCD) and the fibrinogen-like domain (FLD), which is the structural basis for its multiple biological functions. Whether the two domains involve in the effect on podocytes is still not sure. Part I. Study on molecular mechanisms of Angiopoietin-like3on podocytes actin cytoskeleton rearrangement through small GTPasesObjectives To investigate the molecular mechanisms of Angptl3induced podocyte actin filaments rearrangement.Methods1. To identify the cell line by detecting WT-1and podocin expression with immonofluorescence staining.2. To observe the rearrangement of podocytes actin filaments by staining podocyte with FITC-labelled phalloidine following Angptl3treatment.3. To detect the small GTPase activation level in podocyte treated with Angptl3by G-LISATM assay.4. To observe whether blocking the small GTPases could inhibit the effect of Angptl3on actin filament rearrangement.5. To observe whether blocking integrin αvβ3could inhibit the effect of Angptl3on actin filament rearrangement and small GTPases activation.6. To observe whether blocking downstream molecules of integrin αvβ3could inhibit the effect of Angptl3on actin filament rearrangement and small GTPases activation. And to detect key downstream molecule phosphorylation with Western Blot after blocking upstream molecule followed by Angptl3treatment.7. To detect small GTPases expression with Western Blot after Angptl3treatment.Results1. The WT-1and podocin were both expressed in our podocyte cell line, demonatrating the cell line could be used in the following experiment.2. Angptl3treatment could induce podocyte actin filament rearrangement, mainly expressed as lamellipodia and cell spikes formation.3. Angptl3could lead to podocyte small GTPases Racl and RhoA acivation. Among which Racl expressed a stronger and long lasting activation while RhoA only expressed a low level and short term activation.4. Blocking Racl could block the podocyte lamellipodia formation effect induced by Angptl3.5. Blocking integrin αvβ3could block the lamellipodia formation effect and small GTPases activation induced by Angptl3.6. Blocking FAK or PI3K, which are downstream molecules of integrin avP3, could block the lamellipodia formation effect and small GTPases activation induced by Angptl3. Recombinant Angptl3treatment could lead to FAK and PI3K phosphorylation in podocytes. Blocking integrin avP3could block the phosphorylation of both FAK and PI3K induced by Angptl3, while blocking FAK could block the phosphorylation of PI3K induced by Angptl3.7. Recombinant Angptl3treatment could increase the Racl expression in podocyte. Summary1. Angptl3could induce podocyte actin filaments rearrangement, which mainly expressed as lamellipodia formation.2. Small GTPases Racl and RhoA, especially Racl activation were of great important part in Angptl3induced podocyte F-actin rearrangement.3. Angptl3induced podocyte lamellipodia formation through integrin αvβ3-FAK-PI3K-Racl pathway.4. Angptl3could increase Racl expression but not RhoA or Cdc42expression in podocytes. Part Ⅱ. Effects of different domains of Angptl3on podocyte actin filaments rearrangement and podocyte detachmentObjectives1. Study the effect of different domains of Angptl3on podocyte actin filament rearrangement.2. Study the effect of Angptl3and its different domains on podocytes detachment injury induced by Puromycin aminonucleoside (PAN).Methods1. To observe the podocyte actin filaments rearrangement with FITC-labelled phalloidine staining after Angptl3, or its CCD or FLD domain fragments treatment.2. To study whether Angptl3or its different domains have effect on PAN induced podocyte detachment by pretreating podocytes with Angptl3or its CCD or FLD domain fragments followed by PAN treatment.Results1. Angptl3-FLD fragment could induce podocyte F-actin rearrangement, while Angptl3-CCD did not have such effect.2. Angptl3treatment alleviates the PAN induced podocytes detachment, and the anti-detachment effects were time and dose dependent. Angptl3-FLD treatment showed the similar effects as complete Angptl3on PAN induced podocyte detachmen. Angptl3-CCD fragement increased podocyte detachment followed a resistant to PAN induced podocyte detachment.Summary1. FLD fragement was the key domain inducing podocyte F-actin rearrangement.2. Angptl3could resist the PAN induced podocyte detachment. Both FLD domain and CCD domain were involved in this effect but played different part. FLD domain was the most important domain in this process. CCD domain also had its effect of podocyte, the receptor and molecular pathway still needs further study.