Hassan Ali, Tian Ying, Zheng Wei, Ji Hong, Sandberg Kathryn, Verbalis Joseph G
Center for the Study of Sex Differences in Health, Aging and Disease, Georgetown University Medical Center, Washington, District of Columbia 20057, USA.
Physiol Genomics. 2005 May 11;21(3):382-8. doi: 10.1152/physiolgenomics.00147.2004. Epub 2005 Mar 22.
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 V1a and V1b 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 (approximately 50 microg) 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.
精氨酸加压素（AVP）对肾脏的抗利尿作用是由V2型AVP受体（V2R）介导的。为了研究V2R调节在水和钠稳态中的作用，我们开发了一种在体内通过小干扰RNA（siRNA）介导抑制V2R表达的方法。选择了三条21个核苷酸的siRNA序列，它们特异性靶向小鼠V2R，但与任何其他已知的小鼠基因（包括加压素V1a和V1b受体）没有明显的序列同源性。此外，选择了一条与任何已知哺乳动物基因序列均无显著匹配的siRNA序列用作对照。化学合成的siRNA与脂质体转染试剂DOTAP复合。每只小鼠（雄性C57BL/6）通过静脉注射接受3.6 nmol（约50μg）的对照（非沉默）siRNA或一种靶向V2R的siRNA。注射后48小时，从肾脏内髓制备膜，并通过放射性配体结合测定和蛋白质免疫印迹法测量V2R表达。用一种靶向V2R的siRNA（R2）处理后，与对照相比，V2R特异性结合减少了39.7±8.7%（n = 11，P < 0.05），通过蛋白质免疫印迹法测量，V2R蛋白表达减少了37.0±2.3%（n = 4，P < 0.001）。此外，实时PCR显示R2 siRNA处理使V2R mRNA减少了68.8±2.2%。然而，这种siRNA处理在未刺激条件下并未改变动物的基础尿液浓缩能力。在随后的实验中，发现用R2 siRNA处理可显著减弱V2R特异性AVP激动剂1-去氨基-[8-D-精氨酸]加压素（dDAVP）的抗利尿作用。给小鼠输注dDAVP（0.25 ng/h）3天以产生最大抗利尿作用，然后注射R2 siRNA或非沉默对照。处理后第2天，对照动物（n = 12）的尿渗透压从3455±72显著降至R2 siRNA处理动物（n = 12）的3155±129 mosmol/kgH2O（P < 0.05）；同样，在第2天，R2 siRNA处理动物的24小时尿量从0.86±0.07 ml/天显著增加至1.11±0.06 ml/天（P < 0.05）。总之，我们已经证明RNA干扰方法可以在体内成功用于显著降低小鼠肾脏中V2R的功能表达。
