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Small interfering RNA-mediated functional silencing of vasopressin V₂ receptors in the mouse kidney

¹ Center for the Study of Sex Differences in Health, Aging and Disease, Georgetown University Medical Center, Washington, District of Columbia
² Department of Medicine, Division of Nephrology and Hypertension, Georgetown University Medical Center, Washington, District of Columbia
³ Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University Medical Center, Washington, District of Columbia

The antidiuretic effects of arginine vasopressin (AVP) on the kidney are mediated by V₂ subtype AVP receptors (V₂R). To investigate the role of regulation of V₂R in water and sodium homeostasis, we have developed a method for small interfering RNA (siRNA)-mediated inhibition of V₂R expression in vivo. Three 21-nt siRNA sequences were chosen that specifically targeted the mouse V₂R but shared no appreciable sequence homology to any other known mouse genes, including the vasopressin V_1a and V_1b receptors. Additionally, an siRNA sequence that shared no significant matches to any known mammalian gene sequences was chosen for use as a control. Chemically synthesized siRNA was complexed with the liposomal transfection reagent DOTAP. Each mouse (male C57BL/6) received 3.6 nmol (50 µg) of either the control (nonsilencing) or one of the V₂R-targeting siRNAs via intravenous injection. Forty-eight hours after injection membranes were prepared from the inner medulla of the kidneys, and V₂R expression was measured by a radioligand binding assay and Western immunoblotting. Treatment with one of the V₂R-targeting siRNAs (R2) caused a 39.7 ± 8.7% reduction in V₂R-specific binding compared with the control (n = 11, P < 0.05) and a 37.0 ± 2.3% reduction in V₂R protein expression as measured by Western immunoblotting (n = 4, P < 0.001). Additionally, real-time PCR revealed that R2 siRNA treatment induced a 68.8 ± 2.2% reduction in V₂R mRNA. However, this siRNA treatment did not alter the animals’ basal urine concentrating capacity under unstimulated conditions. In subsequent experiments, treatment with R2 siRNA was found to significantly attenuate the antidiuretic effects the V₂R-specific AVP agonist 1-desamino-[8-D-arginine]vasopressin (dDAVP). Mice were infused with dDAVP (0.25 ng/h) for 3 days to produce maximal antidiuresis and then were injected with either the R2 siRNA or the nonsilencing control. On day 2 after treatment, urine osmolality was significantly decreased from 3,455 ± 72 in control animals (n = 12) to 3,155 ± 129 mosmol/kgH₂O in R2 siRNA-treated animals (n = 12) (P < 0.05); similarly, on day 2 24-h urine volume was significantly increased from 0.86 ± 0.07 ml/day to 1.11 ± 0.06 ml/day in R2 siRNA-treated animals (P < 0.05). In summary we have demonstrated that RNA interference methodology can be used successfully in vivo to significantly reduce functional expression of the V₂R in the mouse kidney.

inner medulla; in vivo gene silencing; G protein-coupled receptor