Doxorubicin-Induced In Vivo Nephrotoxicity Involves Oxidative Stress- Mediated Multiple Pro- and Anti-Apoptotic Signaling Pathways

Tejas      S. Lahoti; Darshan      Patel; Venkatesh      Thekkemadom; Robert      Beckett; Sidhartha      D. Ray

Abstract

Doxorubicin (DOX), a prominent anticancer agent has enjoyed considerable popularity in the last few decades because of its usefulness in the management of various forms of cancers, but its organotoxic potential (cardio-, hepatoand nephrotoxicity) has constrained on its clinical use. This study investigated whether DOX has the ability to cause nephrotoxicity in vivo and if so, whether it is linked to oxidative stress (OS). Another important goal was to describe whether expression of pro- and anti-apoptotic genes in kidneys was driven by OS. In order to explore DOX’s nephrotoxic potential, male rats (Sprague Dawley; 500–520g; fed ad libitum) were administered i.p. with a single dose of DOX (12 mg/kg) on day one and sacrificed seven days later (day 8). Changes in serum chemistries (i.e., serum urea nitrogen, SUN, and creatinine) were determined immediately upon sacrifice, whereas kidney tissues were subjected to several sensitive biomarkers for OS, such as, lipid peroxidation, Superoxide dismutase (SOD) activity and chromatin fragmentation. The most important goal was to evaluate the select expression of Apaf-1, Caspase-3, Bad, Bax, Bcl-2, Bcl-xL, p53 and Mdm2 genes in order to understand the underlying link between extrinsic and intrinsic pathways of cell death. Data revealed that DOX-exposed animals showed significant nephrotoxicity as reflected in increased SUN (5.6-fold) and creatinine (2.65 fold) levels with considerably decreased body weight. Increases in kidney injury markers reflected parallel elevations in lipid peroxidation (1.7-fold) and genomic DNA fragmentation (2.9 fold) coupled with a proportionate reduction in total SOD activity suggesting DOX-assaulted kidneys encountered massive OS. Western blot showed very striking changes: i) substantial increases in the expression of pro-apoptotic APAF-1, Caspase-3, Bax and Bad proteins; ii) Reduction in the expression of anti-apoptotic Bcl-2 and Bcl-xL genes; iii) considerable increase in the expression of p53 and suppression of its regulator Mdm2. Serum chemistry and tissue biochemistry mirrored histopathology. In conclusion, this study for the first time may have shown a close link between mitochondrial perturbations and cell death regulating genes during DOXinduced nephrotoxicity, and described DOX’s potential to inflict kidney injury in addition to other organs during chemotherapy in clinical setting.

Keywords: APAF-1, apoptosis, Bad, Bax, Bcl-2, Bcl-xL, Caspase-3, doxorubicin, lipid peroxidation, Mdm2, necrosis, nephrotoxicity, oxidative stress, p53, SOD