Establishing utility of in vivo siRNA to identify therapeutic targets for treatment of hypertension and renal disease
In this proposal we aim to develop strategies to optimize siRNA silencing of gene expression in the rat kidney in vivo. Rats are chosen for this study because of the well studied hypertensive strains (SHR and Dahl salt sensitive) that mimic human hypertension. We propose to target three proteins that are excellent theoretical candidates for decreasing sodium transport and lowering blood pressure. The siRNAs will be designed, constructed, and administered in collaboration with Dr. Rama Natarajan at the City of Hope who is one of the few investigators who has succeeded in using siRNA to depress target gene expression in kidneys in vivo. This success can, at least in part, be attributed to the use of chemically derivatized siRNAs developed and/or implemented at City of Hope to optimize tissue delivery and uptake in vivo.
Aim 1. Develop strategies that can decrease renal gene expression Using different dose and time course protocols, quantitate siRNA delivery and uptake using fluorescently labeled siRNA by in vivo two photon microscopy (Peti-Peterdi core lab, USC). Quantitate silencing biochemically by measuring target RNA and protein levels.
Aim 2. Assess the physiologic impact of target gene silencing by measuring:
- blood pressure reduction in hypertensive rat strains
- efficacy of blocking effects of angiotensin II signaling on renal Na+ transporter distribution and phosphorylation
Developing reproducible strategies to reduce target gene expression in rats will provide a clear cut method to define specific protein-protein interactions that are important in regulatory cascades involved in blood pressure regulation. This strategy will not only specifically identify proteins involved in hypertension and renal disease in whole animals, but will also identify these proteins as good targets for therapeutic intervention to retard or prevent hypertension and renal disease.