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 used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic 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 collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (SOD) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (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 oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and 300 mg/kg: 3-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and SOD activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA 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.
Hickey
E J
EJ
Molecular Toxicology Program, Division of Pharmacology, Toxicology & Medicinal Chemistry, Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY 11201, USA.
Raje
R R
RR
Reid
V E
VE
Gross
S M
SM
Ray
S D
SD
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004008:Diclofenac / Q000633:toxicity*; D004706:Endodeoxyribonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008315:Malondialdehyde / Q000097:blood; D051379:Mice; D008813:Mice, Inbred ICR; D009336:Necrosis; D018384:Oxidative Stress / Q000187:drug effects; D013482:Superoxide Dismutase / Q000097:blood
D000894:Anti-Inflammatory Agents, Non-Steroidal; D004008:Diclofenac; D008315:Malondialdehyde; D013482:Superoxide Dismutase; D004706:Endodeoxyribonucleases; C029367:calcium magnesium dependent endodeoxyribonuclease