Complement-mediated killing of mesangial cells in experimental glomerulonephritis: cell death by a combination of apoptosis and necrosis

Abstract

Immune system mediated, particularly antibody- and complement-mediated, glomerular injury triggers glomerulonephritis (GN). To characterize complement-mediated cytotoxicity in GN, we assessed the process of mesangial cell death induced by C5b-9 attack in Thy-1 GN. Cell injury was recognized morphologically, and nuclear DNA breaks were confirmed by the DNA nick end labeling (TUNEL) method as well as DNA gel electrophoresis. Thy-1 GN was induced in rats with anti-Thy-1.1 antibody injection. Mouse IgG (administered antibody) and rat C3 were detected in all glomeruli within 5 min after antibody injection. Damaged mesangial cells with condensed as well as TUNEL-positive nuclei could be observed at 20 min and became prominent at 40-60 min. Ultrastructurally, damaged mesangial cells contained condensed apoptotic nuclei from 40 to 60 min, whereas the cytoplasm showed necrotic degeneration. This was followed by progressive lysis of both nuclei and cytoplasm. The DNA 'ladder' pattern was observed by gel electrophoresis of extracted DNA between 40 and 60 min and correlated with the increased number of TUNEL-positive damaged mesangial cells. To examine the role of complement in this form of cell death, complement depletion was induced in rats by cobra venom factor. Complement-depleted rats showed no rat C3 deposition, rare TUNEL-positive mesangial cells, rare ultrastructural degenerated mesangial cells with apoptotic nuclei and necrotic cytoplasm, and no DNA 'ladder' pattern on gel electrophoresis at 40 min, although prominent mouse IgG was seen in glomeruli. To analyze milder forms of complement injury, a low dose of the antibody was administered to rats with a normal complement level. A few TUNEL-positive mesangial cells were detected in the glomeruli which contained apoptotic nuclei and necrotic cytoplasm. Our results indicate that an apoptotic death mechanism accompanies cell necrosis in complement-mediated mesangial cell destruction in GN and that this unusual form of cell death may represent a combination of apoptosis-necrosis within the same cell. Complement injury activates a 'death program' which in turn leads to irreversible damage of mesangial cells and which may contribute to initiation and development of GN.