Apoptosis in mesangial cells induced by ionizing radiation and cytotoxic drugs

Abstract

Mesangial proliferation contributes to the pathogenesis of many forms of glomerulonephritis. To evaluate the role of apoptosis on the pharmacologic effects of cytotoxic drugs and ionizing radiation, we studied their effects on cultured rat mesangial cells (MC), whose apoptotic response to these drugs is unknown. Mesangial cells were cultured with or without stimuli to induce apoptosis and were harvested at 24 and 48 hours. MC morphology was examined by light microscopy, in situ end labeling technique using terminal deoxy-transferase (TUNEL) and by electrophoresis of extracted total cellular DNA. MCs exposed to cytotoxic drugs or irradiation demonstrated statistically significant increases in apoptotic cells identified by light microscopy. DNA fragmentation of apoptotic cells was also visualized as characteristic staining by the TUNEL method and statistically significant increases in apoptotic cell number in cells exposed to cytotoxic drugs and irradiation were noted compared to control cultures. In general, the number of TUNEL positive cells was greater than that of morphologically apoptotic cells. DNA extracted from these cells also showed the characteristic ladder pattern of internucleosomal chromatin cleavage of 180 bp fragments on agarose gel electrophoresis. To further analyze whether MC apoptosis induced by these drugs alters the cell cycle, 3H-thymidine incorporation rates were measured in both the cell culture monolayer and in those cells shed into the supernatant when cultured with or without cyclophosphamide (N = 5). 3H-thymidine incorporation corrected for total cellular DNA showed a similar pattern in both control and cyclophosphamide treated groups, suggesting that cyclophosphamide did not alter the mesangial cell cycle. Considering that the dosage of the cytotoxic drugs utilized in these experiments in nearly the therapeutic plasma level in humans, these results suggest that cytotoxic drugs used to treat glomerular disease can induce apoptotic mesangial cell death and may operate in part via this mechanism.