Ali Hassan

Ali Hassan, Ying Tian, Wei Zheng, Kathryn Sandberg & Joseph G. Verbalis

Department of Medicine, Georgetown University, 4000 Reservoir Rd, NW, Washington, DC 20057.

The antidiuretic effects of AVP on the kidney are mediated by V2 subtype AVP receptors (V2R). To investigate the role of regulation of V2R in water and salt balance we have developed a method for small interfering RNA (siRNA)-mediated inhibition of V2R expression in vivo. Three 21 nt siRNA sequences were selected that specifically targeted the mouse V2R but shared no appreciable sequence homology to any other known mouse genes, including the V1a and V1b vasopressin receptors. Additionally, an siRNA sequence which shared no significant matches to any known mammalian gene sequences was selected for use as a control. Chemically synthesized siRNA was complexed with the liposomal transfection reagent DOTAP. Each mouse (male C57B6) received 3.6 nmol (˜50 μg) of either the control (NS) or one of the V2R-targeting siRNAs via intravenous injection. 48 h after injection membranes were prepared from kidneys and V2R-specific binding was measured. Treatment with one of the V2R-targeting siRNAs (R2) caused a 52 ± 13% (n=10, P<0.05) reduction in binding compared with NS-treated controls. In subsequent experiments, treatment with R2 siRNA was found to significantly attenuate the ntidiuretic effects of administration of the V2R-specific AVP analogue dDAVP (Tian, et al EB2004 Abstracts). In summary we have demonstrated that RNAi can be used successfully to markedly reduce functional expression of V2R in vivo.

Experimental Biology 2004, Washington, DC, Abstract 6580, 2004.