20-HETE-mediated protection against ischemia and reperfusion injury in renal epithelial cells

Acute renal failure is defined as rapid loss of renal function and has been associated with a 50% mortality rate. Ischemia and reperfusion (I/R) injury of the kidney is the most prominent cause of intrinsic acute renal failure. As the pathophysiology of the I/R injury remains to be fully elucidated, mediators which activate pro-survival signaling mechanisms have been shown to confer protection against the ischemic insult. Recently, 20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite, has been reported to be renoprotective against I/R injury, but the intracellular signaling mechanisms activated by 20-HETE in the renal epithelial cells remain to be determined. In this dissertation, we hypothesized that 20-HETE activates pro-survival ERK1/2 and PI3-K-Akt pathways to protect renal epithelial cells against I/R injury. To investigate the validity of this hypothesis, we designed the following specific aims to determine the role of 20-HETE-mediated protection: (1) To examine the effects of 20-HETE and 20-HETE analogs on the activation of pro-survival and pro-apoptotic kinases in renal epithelial cells in vitro; (2) To determine the upstream signaling cascades by which 20-HETE could mediate the activation of pro-survival and pro-apoptotic pathways; and (3) To determine whether 20-HETE could mediate protective effects in vitro and in vivo through interactions with the pro-survival and pro-apoptotic pathways.;Our results indicate that 20-HETE, as well as its analogs activate pro-survival ERK1/2 and Akt pathways in renal epithelial cells. The activation of both pathways was rapid. On the contrary, 20-HETE-mediated activation of pro-apoptotic SAPK/JNK and p38 MAPK pathways were slow and only detectable after prolonged exposure to certain 20-HETE analogs. 20-HETE-mediated activation of the ERK1/2 pathway was mediated through upstream MEK1/2 and Raf-1. Interestingly, with 20-HETE-treatment, phosphorylation of Epidermal Growth Factor Receptor (EGFR) was increased significantly. Blockade of EGFR completely abolished 20-HETE-mediated activation of Raf/MEK/ERK and Akt pathways while SAPK/JNK activity was also reduced, but not totally eliminated. Similarly, inhibition of c-Src also blocked 20-HETE-mediated activation of Raf/MEK/ERK and Akt pathways; and decreased SAPK/JNK activity noticeably. Finally, our studies have shown that 20-HETE and 20-HETE analogs confer significant protection against in vitro I/R injury and this protection was eliminated with inhibition of ERK1/2 and Akt pathways. Moreover, we found that stable 20-HETE analogs also mediate in vivo activation of ERK1/2 and Akt pathways in renal medullas of Sprague Dawley rats. Taken together, our results indicate that, in vitro protection of renal epithelial cells by 20-HETE is mediated by pro-survival ERK1/2 and Akt pathways, which could also partially account for the protection shown in in vivo models.;In conclusion, this dissertation provides new insight into the interaction of 20-HETE with the intracellular signal transduction pathways in the kidney and presents proof for 20-HETE-mediated activation of EGFR. Due to recent findings of the role of 20-HETE in the pathogenesis of several proliferative diseases including cancer and polycystic kidney disease, our findings are of great importance in pre-clinical research for the generation and use of 20-HETE synthesis inhibitors and 20-HETE antagonists. Moreover our studies will lead to the continued progress in the understanding of the mechanisms by which 20-HETE can provide therapeutic protection in the context of I/R injury in the kidney.