胆汁酸受体FXR在肾脏水钠代谢调节中的作用及机制

The kidney is the central organ in the regulation of water and salt homeostatis. Antidiuretic hormone (ADH) and aldosterone control water and salt reabsorption by activation of V2 receptor and mineralocorticoid receptor localized in the epithelial cell of the collecting ducts, controlling the expression of aquaporin 2 (AQP2) and sodium channel ENaC. Bile acid receptor (FXR), highly expressed in the liver and small intestine, is a vital regulatory factor in the synthesis and transport of bile acids. Our previously study showed that FXR abundantly expressed in the kidney, especially in the collecting duct. In addition, FXR knockout mice exhibited more 24-hour urine volume than wide type mice after treated with a high salt diet. Recently, several groups have reported that chenodeoxycholic acid (CDCA), an endogenous activator of FXR, can increase the reabsorption of water and salt by inhibiting the activity of 11beta-hydroxysteroid dehydrogenase (11β-HSD2). These findings suggest that FXR may play an important role in water and salt reabsorption in the collecting ducts. The present study is designed to clarify the role of FXR in renal water and salt metabolism. We will first study the role of FXR in the water and salt reabsorption by using FXR gene knockout mice. Secondly, we will define the molecular mechanisms by which FXR regulates sodium and fluid reabsorption in cultured epithelial cells of the collecting ducts. The completion of this project will improve our understanding of renal water and salt homeostasis regulation. It may also help identify novel therapeutic target for the treatment of the disorders of water and salt dysregulation such as hepatorenal syndrome.

肾脏在水钠代谢稳态中发挥中心的调节作用。抗利尿激素和醛固酮分别通过结合集合管上皮细胞V2受体及盐皮质激素受体，控制水通道-2及钠通道表达，从而调控水、钠重吸收。胆汁酸受体（FXR）是胆汁酸合成及转运的重要调节因子，在肝脏和小肠高表达。我们的前期研究表明FXR在肾脏特别是集合管高表达；与野生型相比，FXR基因敲除小鼠在高盐饮食时24小时尿量显著增加。最近的报道也显示FXR内源激动剂鹅去氧胆酸可通过抑制11β-羟基类固醇脱氢酶2活性，增加水钠重吸收，以上研究均提示FXR可能在集合管水钠转运调节中有重要作用。本课题将通过体内、外实验探讨其对水钠代谢的影响。内容一将使用FXR基因缺失小鼠探讨其在肾脏水钠代谢调节中的作用；内容二将在培养的集合管上皮细胞研究FXR调控水钠转运的分子机制。本课题的开展不仅为阐明肾脏水钠稳态调控的机制提供实验依据，也可能为肝肾综合征等常见水钠代谢紊乱疾病的治疗提供新思路。

肾脏在水钠代谢稳态中发挥中心调节作用。传统观点认为抗利尿激素和醛固酮分别通过结合集合管上皮细胞V2受体及盐皮质激素受体，控制水通道-2及钠通道表达，是调控水、钠重吸收的重要激素。胆汁酸受体（FXR）是胆汁酸合成及转运的重要调节因子，在肝脏和小肠高表达。我们前期研究表明FXR在肾脏特别是集合管上皮细胞也高水平表达；与野生型相比FXR基因敲除小鼠高盐饮食24h尿量显著增加。最近报道也显示FXR内源激动剂CDCA可通过抑制11β-HSD2的活性增加水钠重吸收。以上研究均提示FXR可能在肾脏水钠转运调节中发挥重要作用。本课题旨在通过体内、外实验深入探讨其对水钠代谢稳态的影响。研究结果：1)FXR在小鼠肾脏的表达分布。FXR表达水平从高到低依次为皮质、外髓和内髓。其中FXR在近端小管、远端小管、髓袢升支粗段和集合管上皮细胞均有较高的表达，而在肾小球中表达相对较低；2）FXR激活或阻断对尿量及尿渗透压的影响。用代谢笼收集小鼠24h尿，测定尿量及尿渗透压。结果表明FXR激动剂可显著降低24h尿量，增加尿渗透压；而FXR基因敲除小鼠尿量增加，尿渗透压降低； 3）肾脏FXR靶基因研究。基因芯片结果发现CDCA显著增加肾脏水通道蛋白AQP2 mRNA的表达；4）FXR调节AQP2基因转录。在内髓集合管细胞(IMCDs)，FXR激动剂GW4064时间依赖性增加AQP2的mRNA和蛋白水平。GW4064明显上调FXR+/+ IMCDs的AQP2表达，但对FXR-/- IMCDs的AQP2表达没有影响。在AQP2基因的启动子-328bp至-316bp之间发现FXRE位点。萤光素酶报告基因和ChIP assay实验表明FXR可直接与AQP2启动子的FXRE结合，并可被GW4064显著增强，但FXRE突变后AQP2启动子活性不再被GW4064上调；5）FXR拮抗高渗对IMCDs的损伤。野生型小鼠禁水24h后FXR表达增加；高渗处理IMCDs引起FXR转录和表达增加。禁水24h后正常小鼠IMCDs发生凋亡， FXR基因敲除后凋亡显著加重。结论:1）在IMCDs中 FXR直接与AQP2启动子的FXRE结合并增加其基因转录，从而促进肾脏水的重吸收。上述部分研究结果已发表在美国科学院院报（PNAS 2014）；2）FXR拮抗高渗对IMCDs的损伤，可能是肾脏内髓细胞在高渗环境下维持生存的重要机制。