Inhibition of vasopressin (AVP) V2 receptor expression by RNA interference (RNAi) in adult mice

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 antidiuretic effects of administration of the V2R-specific AVP analogue dDAVP. C57B6 mice were infused with dDAVP (0.25 ng/h) for 3 d to produce maximal antidiuresis, and then were injected with either the R2 siRNA or the NS control. On day 2 after treatment, 24 h urine volume was significantly increased (1.07 ± 0.07 vs. 0.69 ± 0.08 ml, P<0.01) and urine osmolality was significantly decreased (3310 ± 94 vs. 4133 ± 313 mOsm/kg H2O, P<0.01) in R2-treated mice (n=10) compared to NS-treated controls (n=7).   In summary we have demonstrated that RNAi can be used successfully to markedly reduce functional expression of V2R in vivo.

Presented at the Georgetown University Student Research Days, Washington, DC, 2004.