Dual Factors Of SC5B-9and HBx Affect Slit Diaphragm Protein Expression In Podocytes

Objective Glomerular podocytes are highly differentiated cells, the adjacent footprocesses interconnect to form slit diaphragm as glomerular final filtration barrier. A lot ofclinico-pathological studies have shown that patients with hepatitis B virus associatedmembranous nephropathy (HBV-MN) having different degrees of foot process effacement,suggesting that the abnormal of slit diaphragm maybe occurs in the process of HBV-MN.Compared with idiopathic membranous nephropathy, not only the complement membraneattack complex but also HBV proteins can cause podocyte to injury in HBV-MN. Therefore,in this study sublytic C5b-9(sC5b-9) model on podocyte in vitro was constructed andreplication-defective adenovirus carrying the HBx gene were used to transfect podocytes inorder to observe how the sC5b-9and the HBx affect the podocyte’s slit diaphragm proteinson their expression and distribution. Methods The concentrations of antibody andcomplement of the sC5b-9model on podocyte were determined by detecting the LDHactivity in the supernatants of cultured podocytes through ‘checkerboard experiment’.Switzerland-Giemsa staining was used to observe whether sC5b-9affected podocytesmorphology or not. Laser scanning confocal microscope and indirect immunofluorescencestaining were used to observe the distribution of slit diaphragm proteins. Western-blot wasused to analyze the changes of the expression of slit diaphragm proteins. Results The concentrations of antibody and complement were determined both1%in podocytes sC5b-9model by detecting LDH activity in the supernatants of cultured podocytes. Podocytesaffected by sC5b-9were found that cytoplasm had slight vacuolar degeneration, footprocess reduced, nucleus showed granular changes, and boundaries of the nuclear envelopebecame blurred. Immunofluorescence staining showed the distribution of slit diaphragmproteins was more abnormal caused by the dual of sC5b-9and HBx than any one alone:nephrin mainly gathered towards perinuclear and the expression in cytoplasm and footprocesses was drastically reduced; however, podocin and CD2AP distributed on the cellmembrance or foot processes like coarse filamentous or large particles characteristics ratherthan normal evenly distributed in the cytoplasm, granular characteristics on the cellmembrance. Western blot analysis showed that the expression of CD2AP and nephrin bothreduced significantly when podocytes were affacted by the dual injury factors of HBx andsC5b-9compared with the only factor of sC5b-9, but the expression of podocin increaced;the expression of nephrin, podocin and CD2AP all reduced when the dual injury factors ofHBx and sC5b-9affected podocytes together rather than HBx alone. Conclusion Thedual factors of sC5b-9and HBx can aggravate the damage of slit diaphragm proteins tocompare with any of them alone. This reveals complement membrance attack complex andHBx may cause the injury of podocytes together.