A decline in podocyte number correlates with progression to glomerulosclerosis. A mechanism underlying reduced podocyte number is the podocyte's relative inability to proliferate in response to injury. Injury by the podocyte toxin
puromycin aminonucleoside (PA) is mediated via
reactive oxygen species (ROS). The precise role of ROS in the pathogenesis of PA-induced glomerulosclerosis remains to be determined. We sought to examine whether PA-induced ROS caused podocyte DNA damage, possibly accounting for the podocyte's inability to proliferate in response to PA. In vitro, podocytes were exposed to PA, with or without the radical scavenger
1,3-dimethyl-2-thiourea (
DMTU). In vivo, male Sprague-Dawley rats were divided into experimental groups (n = 6/group/time point): PA, PA with
DMTU, and control, killed at days 1.5, 3, or 7. DNA damage was measured by
DNA precipitation, apurinic/apyrimidinic site, Comet, and
8-hydroxydeoxyguanosine assays. Cell cycle checkpoint
protein upregulation (by immunostaining and Western blotting), histopathology, and biochemical parameters were examined. DNA damage was increased in cultured podocytes that received PA, but not PA with
DMTU. PA exposure activated specific cell cycle checkpoint
proteins, with attenuation by
DMTU.
DNA repair enzymes were activated, providing evidence for attempted DNA repair. The PA-treated animals developed worse
proteinuria and histopathologic disease and exhibited more DNA damage than the
DMTU pretreated group. No significant apoptosis was detected by
terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. A mechanism underlying the lack of podocyte proliferation following PA-induced injury in vitro and in vivo may be ROS-mediated DNA damage, with upregulation of specific cell cycle checkpoints leading to cell cycle arrest.