Splinter Patrick L, Masyuk Anatoliy I, LaRusso Nicholas F
Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic, and Foundation, Rochester, Minnesota 55905, USA.
J Biol Chem. 2003 Feb 21;278(8):6268-74. doi: 10.1074/jbc.M212079200. Epub 2002 Dec 4.
Cholangiocytes express water channels (i.e. aquaporins (AQPs)), proteins that are increasingly recognized as important in water transport by biliary epithelia. However, direct functional studies demonstrating AQP-mediated water transport in cholangiocytes are limited, in part because of the lack of specific AQP inhibitors. To address this issue, we designed, synthesized, and utilized small interfering RNAs (siRNAs) selective for AQP1 and investigated their effectiveness in altering AQP1-mediated water transport in intrahepatic bile duct units (IBDUs) isolated from rat liver. Twenty-four hours after transfection of IBDUs with siRNAs targeting two different regions of the AQP1 transcript, both AQP1 mRNA and protein expression were inhibited by 76.6-92.0 and 57.9-79.4%, respectively. siRNAs containing the same percent of base pairs as the AQP1-siRNAs but in random sequence (i.e. scrambled siRNAs) had no effect. Suppression of AQP1 expression in cholangiocytes resulted in a decrease in water transport by IBDUs in response to both an inward osmotic gradient (200 mosm) or a secretory agonist (forskolin), the osmotic water permeability coefficient (P(f)) decreasing up to 58.8% and net water secretion (J(v)) decreasing up to 87%. A strong correlation between AQP1 protein expression and water transport in IBDUs transfected with AQP1-siRNAs was consistent with the decrease in water transport by IBDUs resulting from AQP1 gene silencing by AQP1-siRNAs. This study is the first to demonstrate the feasibility of utilizing siRNAs to specifically reduce the expression of AQPs in epithelial cells and provides direct evidence of the contribution of AQP1 to water transport by biliary epithelia.
胆管细胞表达水通道（即水孔蛋白（AQP）），这类蛋白在胆管上皮细胞的水转运中所起的重要作用日益受到认可。然而，直接证明AQP介导胆管细胞水转运的功能研究有限，部分原因是缺乏特异性AQP抑制剂。为解决这一问题，我们设计、合成并利用了对AQP1具有选择性的小干扰RNA（siRNA），并研究了其改变大鼠肝脏分离的肝内胆管单位（IBDU）中AQP1介导的水转运的有效性。用靶向AQP1转录本两个不同区域的siRNA转染IBDU 24小时后，AQP1 mRNA和蛋白表达分别被抑制了76.6 - 92.0%和57.9 - 79.4%。含有与AQP1 - siRNA相同碱基对百分比但序列随机的siRNA（即乱序siRNA）则没有效果。胆管细胞中AQP1表达的抑制导致IBDU对内向渗透梯度（200 mosm）或分泌激动剂（福斯高林）的水转运减少，渗透水通透系数（P(f)）降低高达58.8%，净水分泌（J(v)）降低高达87%。在转染了AQP1 - siRNA的IBDU中，AQP1蛋白表达与水转运之间的强相关性与AQP1 - siRNA使AQP1基因沉默导致IBDU水转运减少一致。本研究首次证明了利用siRNA特异性降低上皮细胞中AQP表达的可行性，并提供了AQP1对胆管上皮细胞水转运作用的直接证据。
