Diclofenac induced in vivo nephrotoxicity may involve oxidative stress-mediated massive genomic DNA fragmentation and apoptotic cell death

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely u sed for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing s pondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this D CLF-induced nephrotoxicity involves apoptotic cell death in addition to nec rosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type g enomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25-45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, 300 mg/Kg, po) and sacrificed 24 h later. Blood was coll ected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdeh yde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitativel y to determine the degree and type of DNA damage, and evaluated histopathol ogically for the presence of apoptotic characteristics in the nucleus of di verse types of kidney cells. Results show that diclofenac is a powerful nep hrotoxicant (at 100, 200, and 300 mg/kg: 4.7-, 4.9, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxida tive stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, a nd 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respective ly). A dose-dependent increase in MDA levels and SOD activity was also obse rved, which indicated a link between oxidative stress and nephrotoxicity. Q ualitative analysis of DNA fragmentation by gel electrophoresis showed a DN A ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across p roximal and distal tubular cell linings. Collectively, these data for the f irst time suggest that DCLF-induced nephrotoxicity may involve production o f reactive oxygen species leading to oxidative stress and massive genomic D NA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF. (C) 2001 Elsevier Science Inc.