Submitted on June 30, 2004
Accepted on March 10, 2005
Small interfering RNA-mediated functional silencing of vasopressin V2 receptors in the mouse kidney
Ali Hassan1*, Ying Tian2, Wei Zheng1, Hong Ji1, Kathryn Sandberg1, and Joseph G Verbalis3
1 Center for the Study of Sex Differences in Health, Aging and Disease, Georgetown University Medical Center, Washington, DC, USA; Department of Medicine, Division of Nephrology and Hypertension, Georgetown University Medical Center, Washington, DC, USA
2 Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University Medical Center, Washington, DC, USA
3 Center for the Study of Sex Differences in Health, Aging and Disease, Georgetown University Medical Center, Washington, DC, USA; Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University Medical Center, Washington, DC, USA
* To whom correspondence should be addressed. E-mail: amh56{at}georgetown.edu.
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 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 V1a and V1b receptors. Additionally, a siRNA sequence which 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 C57B6) received 3.6 nmol (~50 µg) of either the control (NS) 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 V2 receptor 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 V2 receptor 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 d to produce maximal antidiuresis, and then were injected with either the R2 siRNA or the non-silencing control. On day 2 after treatment, urine osmolality was significantly decreased from 3455 ± 72 in control animals (n=12) to 3155 ± 129 mOsm/kg H2O 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/d to 1.11 ± 0.06 ml/d in R2 siRNA treated animals (P<0.05). In summary we have demonstrated that RNAi methodology can be used successfully in vivo to significantly reduce functional expression of the V2R in the mouse kidney.
