We have shown that retinoid signaling is engaged during 11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) mediatedcytoprotection against reactive oxygen species (ROS) induced necrotic/oncotic cell death. Proteomics analysesrevealed that cytoprotection is associated with the increased synthesis of a select number of proteins, includingretinol binding protein (RBP), actin, and glucose-regulated protein 78 (Grp78). We subsequently confirmed thatall-trans-retinoic acid (aTRA) replicates DDM-PGE2-mediated cytoprotection in vitro, and more importantly, asingle dose of aTRA (1 mg/kg, 6h pretreatment) completely protects mice from renal ischemia/reperfusion (I/R)injury. Furthermore, at this therapeutic dose, aTRA induces Nrf2-responsive antioxidant HO-1 and NQO1 genes,as well as nuclear retinoic acid receptors RAR?, RAR?2, RAR?2, and retinoid X receptors RXR? in the kidney.The revised application is designed to determine the molecular mechanisms by which aTRA affordscytoprotection in vitro, and the extent to which this mechanism(s) of cytoprotection is recapitulated in vivo. Ourcentral hypothesis is that aTRA-induced cytoprotection is mediated by mechanisms similar to ischemicpreconditioning.
In Specific Aim 1 we propose to determine the ability of aTRA to offer cytoprotection in an invitro model (human renal epithelial HK-2 cells) of hypoxia/reoxygenation injury, and to optimize protocols foraTRA-mediated cytoprotection in an in vivo ischemia/reperfuson model (IR). We will also ascertain whether theprotective effects are mediated, at least in part, via the upregulation of anti-oxidant enzymes. The biologicaleffects of retinoids are typically mediated via interaction with their cognate nuclear receptors, namely, retinoicacid receptors (RAR) and retinoid X receptors (RXR). The extent to which RAR and/or RXR participate in aTRA-mediated cytoprotection is not known, and Specific Aim 2 will determine, in both the in vitro and in vivo modelsof I/R, whether aTRA-mediated cytoprotection requires interaction with RAR and/or RXR.
Specific Aims 1 and 2are therefore designed to establish the recruitment of retinoid signaling as a potential therapeutic intervention inconditions where ROS play an important role in the pathology of the disease, such as those involving ischemiareperfusion injury (Specific Aim 1), and to initially characterize the pharmacological basis of this effect (SpecificAim 2). The third and final Specific Aim is designed to identify the molecular mechanisms by which aTRAaccomplishes cytoprotection, with each sub- aim focusing on a target that has already been identified inpreliminary studies as playing an important role in the cytoprotective response. Specifically, those mediators areNrf2, Grp78, and p38 MAPK, each of which is also a key mediator of ischemia preconditioning.
Specific Aim 3 will therefore determine whether (i) aTRA- mediated induction of the anti-oxidant stress response is dependentupon Nrf2; (ii) aTRA-mediated cytoprotection requires the recruitment of the ER (Grp78) mediated stressresponse pathway; (iii) the recruitment of Nrf2 by aTRA is dependent on p38 MAPK-Grp78 interactions; and (iv)the mechanism(s) of cytoprotection identified in the in vitro model are recapitulated in the in vivo model by testingaTRA induced renoprotection in Nrf2-/- mice. The significance of the current studies resides in their potential toenhance our understanding of retinoid mediated cytoprotection at the molecular and cellular level, which cansubsequently provide insights into novel therapeutic strategies effective for clinical interventions during chemicalinduced tissue injury or hypoxia/ischemia-reperfusion injury.
Oxidative stress has been shown to cause or contribute to many diseases including different forms of renaldisease such as acute renal failure; rhabdomyolysis; obstructive nephropathy; hyperlipidemia; glomerulardamage; chronic renal failure; and ischemia/reperfusion. An understanding of the factors that regulate the cellularresponse to oxidative stress and of the molecular mechanisms; by which they interact with cellular constituents;and the consequences of such interactions; remain important fundamental goals of biomedical research. All-trans-retinoic acid (aTRA) may offer an alternative therapeutic strategy offering renoprotection throughmechanisms similar to ischemia preconditioning; and therefore studies on the mechanism by which aTRAconfers cytoprotection could have important therapeutic implications. Assessment of the beneficial effects ofretinoids in animal model and human renal disease warrants further investigation.
