Molecular dissection of reactive oxygen species-mediated oncotic cell death

Reactive oxygen species (ROS) are associated with a variety of human diseases and toxicities mediated by redox-active chemicals and/or their metabolites. One such redox active chemical, hydroquinone (HQ), is both a nephrotoxicant and a nephrocarcinogen. Sequential oxidation of HQ and addition of glutathione (GSH) leads to the formation of 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ). TGHQ maintains HQ’s ability to redox-cycle and generate ROS, and to covalently bind to cellular molecules. Thus, TGHQ induces ROS-dependent DNA damage and cell death in renal proximal tubule epithelial cells (LLC-PK1). It is not clear how ROS generated by redoxactive chemicals induce renal cell death. The molecular mechanisms of quinone-mediated nephrotoxicity therefore invite further investigation. We used TGHQ to study ROSmediated renal cell death, and have found that TGHQ induces oncotic rather than apoptotic cell death in LLC-PK1 cells. MAPKs are quickly and robustly activated by TGHQ in a ROS-dependent manner. Pharmacological inhibition of either extracelluar signal regulated kinase (ERK) or p38 MAPK pathways attenuates TGHQ-induced cell death in LLC-PK1 cells. TGHQ also induces ROS-dependent histone H3 phosphorylation in LLC-PK1 cells, which leads to premature chromatin condensation (PCC) and mitotic catastrophe. Our studies show that TGHQ induces epidermal growth factor receptor (EGFR) phosphorylation at multiple tyrosine residues, leading to the EGFR-dependent activation of the ERK cascade. TGHQ also induces EGFR-independent activation of the p38 MAPK cascade. Both ERK and p38 MAPK inhibition attenuates TGHQ-induced histone H3 phosphorylation in LLC-PK1 cells, indicating both ERK and p38 MAPK are associated with ROS-mediated histone H3 phosphorylation. Additionally, proteomics analysis revealed changes in the overall expression and post-translational modification of several proteins in TGHQ-treated LLC-PK1 cells. The identified proteins can be grouped according to function, as follows; antioxidants and cytoskeleton stabilizing proteins (peroxiredoxin II, peroxiredoxin III, Hsp27), membrane fusion (annexin I), nuclear shuttling (nucleophosmin/B23), and calcium binding and chaperoning (calreticulin, Hsp27, peroxiredoxins). The changes are largely due to post-translational modifications, such as phosphorylation (Hsp27) and oxidation (peroxiredoxin). These modifications likely determine cell fate. Finally, we found that in Eker rats, TGHQ induces a timedependent increase in ERK1/2 and histone H3 phosphorylation within the outer stripe of outer medulla (OSOM) in the kidneys, confirming our findings in LLC-PK1 cells. The high constitutive in vivo phosphorylation of p38 MAPK obscured any changes induced by TGHQ within the OSOM. These early molecular changes likely lead to nephrotoxicity followed by excessive proliferation and amplification of genomic instability in Eker rats, and contribute to subsequent nephrocarcinogenicity. This dissertation contributes to the understanding of the molecular mechanisms of TGHQ/ROS-induced renal cell death/nephrotoxicity. I am also of the view that this work will contribute to the future development of therapeutic strategies designed to alleviate acute renal diseases associated with ROS generation.