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

Kidney Int. 1999 Aug;56(2):538-48. doi: 10.1046/j.1523-1755.1999.00560.x.

Abstract

Background: 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.

Methods: 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.

Results: 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).

Conclusions: 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies / pharmacology
  • Cell Division / drug effects
  • Cells, Cultured
  • Complement Membrane Attack Complex / metabolism
  • Complement Membrane Attack Complex / pharmacology*
  • Cyclin-Dependent Kinases / antagonists & inhibitors
  • Cyclin-Dependent Kinases / metabolism
  • DNA / biosynthesis
  • Epithelial Cells / cytology*
  • Epithelial Cells / drug effects*
  • Epithelial Cells / enzymology
  • Fibroblast Growth Factor 2 / immunology
  • Fibroblast Growth Factor 2 / pharmacology
  • G2 Phase / drug effects
  • In Vitro Techniques
  • Kidney Glomerulus / cytology*
  • L-Lactate Dehydrogenase / metabolism
  • Mitogens / metabolism
  • Mitosis / drug effects
  • Neutralization Tests
  • Rats
  • S Phase / drug effects
  • Transforming Growth Factor beta / pharmacology

Substances

  • Antibodies
  • Complement Membrane Attack Complex
  • Mitogens
  • Transforming Growth Factor beta
  • Fibroblast Growth Factor 2
  • DNA
  • L-Lactate Dehydrogenase
  • Cyclin-Dependent Kinases