Complement (C5b-9) induces glomerular epithelial cell DNA synthesis but not proliferation in vitro

Abstract

The C5b-9 membrane attack complex of complement is the principal mediator of injury induced experimentally by antibodies directed at glomerular cell membranes. In experimental membranous nephropathy, C5b-9 induced injury to the glomerular visceral epithelial cell (VEC) is associated with DNA synthesis, but not cytokinesis. In the current study we determined if C5b-9 increases DNA synthesis in VEC in vitro, and defined the mechanisms involved. Rat VEC in vitro were divided into three groups: (1) sensitized with anti-VEC antibody and exposed to sublytic concentrations of C +/PVG serum (normal complement components); (2) anti-VEC antibody and control C-/PVG serum (C6 deficient); (3) no anti-VEC antibody. DNA synthesis (BrdU staining), mitosis (mitotic figures) and cytokinesis (cell counts) were measured at 24 and 48 hours. To examine the expression of specific S-phase and M-phase cell cycle regulatory proteins and their inhibitors, immunostaining and Western blot analysis was performed for cyclin A, CDK2, p21 and p27, cyclin B and cdc2. In the absence of growth factors, sublytic C5b-9 attack did not increase proliferation. In contrast, sublytic C5b-9 attack (group 1) augmented growth factor induced DNA synthesis by 50% compared to controls (groups 2 and 3; P < 0.001), and was accompanied by increased levels of cyclin A and CDK2, and a decrease in the cyclin kinase inhibitor p27 (but not p21). Sublytic C5b-9 attack reduced the expression of the M phase cell cycle proteins, cyclin B and cdc2, accompanied by reduced mitosis (mitotic figures) and cytokinesis (cell number). Our results show that the C5b-9 augmented growth factor entry into the S phase in VEC is regulated by changes in specific cell cycle regulatory proteins. However, antibody and complement decreased the M phase cell cycle proteins, and prevented VEC mitosis and cytokinesis, suggesting a delay or arrest at the G2/M phase.