核受体FXR在肾髓高渗环境建立及髓质细胞功能维持中的作用

The kidney represents a key organ in the maintenance of water and sodium homeostasis. It regulates urine output by a countercurrent mechanism. Urine volume depends on the function of many aquaporin water channels (AQPs) and solute transporters located in the apical and basolateral membrane of epithelial cells along renal tubules. The farnesoid X receptor (FXR) is a bile acid-activated transcription factor belonging to the nuclear receptor superfamily. FXR is mainly expressed in the liver and small intestine, where it plays an important role in bile acid, lipid and glucose metabolism. The kidney also has aboundant FXR expression with its physiological function largely unknown. We have previously demonstrated that inner medullary collecting ducts (IMCDs) and renal medullary interstitial cells (RMICs) has constitutive FXR expression. FXR plays a critical role in the regulation of urine volume, and its activation increases urinary concentrating capacity mainly via up-regulating its target gene AQP2 transcription. In the meanwhile, we also demonstrated a significant increase in FXR expression in the renal medulla following water deprivation in wild-type mice. TonEBP is a member of the nuclear factors of the activated T cells family of transcription factors, which is also the only known tonicity-regulated transcription factor in mammals. Hypertonic stress stimulates TonEBP expression and nulear translocation and controls the transcription of many osmo-protective genes preventing the cell from apoptosis. We found that both TonEBP mRNA and protein levels were significantly induced after water deprivation in FXR +/+ mice, but not in FXR-/- mice, suggesting that FXR may be involved in the establishment and maintenance of countercurrent mechanism in renal medulla. The present proposal aimed to characterize the role of FXR in the regulation of the renal medullary hyperosmolarity and cell viability. We will use the tissue-specific gene targeting strategy to further clarify whether FXR increases medullary osmolarity and promotes the survival of IMCDs and RMICs under hypertonic stress in mice with water restriction. In vitro study will focus on identifying the role of FXR in hypertonicity-induced apoptosis in cultured IMCDs and RMICs. Physical interaction between FXR and TonEBP and synergistic impact of these two transcription factors on cell viability of IMCDs and RMICs after hypertonic exposure will also be investigated. The completion of the present study will not only help understand the cellular and molecular mechanisms involved in the regulation of urinary concentrating process, but also help uncover the pathogenesis of many disorders including hepatorenal syndrome, a state with increased circulating bile acid levels and impaired renal water and sodium excretion.

尿量的多少取决于肾小管水的转运功能及髓质高渗状态的建立。我们前期研究表明核受体FXR在肾髓集合管（IMCDs）和髓质间质细胞(RMICs)有组成性表达，并通过上调AQP2的表达减少尿量；禁水增加野生型小鼠肾髓FXR表达水平；FXR基因缺陷小鼠在禁水后没有野生型小鼠肾髓中出现的TonEBP表达增加，提示FXR可能与肾髓高渗状态的建立和维持有关。本课题将探讨IMCD和RMIC细胞FXR对肾髓高渗状态建立及细胞存活的影响，内容1)使用组织特异性敲除技术明确IMCD和RMIC细胞FXR对髓质高渗状态建立的影响及机制；2)使用离体培养细胞研究FXR在维持高渗环境下IMCD和RMIC细胞存活的作用及机理；3)探讨FXR与TonEBP交互作用、协同促进肾髓细胞在高渗状态下生存的机制。本课题将为阐明肾脏髓质高渗状态建立和维持的机制提供重要实验依据，也将为尿崩症和肝肾综合征等水盐代谢紊乱疾病的治疗提供思路。

尿量的多少取决于肾小管对水的转运能力及髓质高渗状态的建立。我们前期研究表明核受体FXR在肾髓集合管（IMCDs）和髓质间质细胞（RMICs）有组成性表达，集合管FXR通过上调AQP的表达减少尿量；禁水增加野生型小鼠肾髓FXR表达水平；野生型小鼠肾髓中渗透压反应增强子结合蛋白（TonEBP）的表达在禁水后增加，而FXR基因缺陷小鼠TonEBP的表达不受禁水的影响，提示FXR可能与肾髓高渗状态的建立和维持有关。本课题旨在研究核受体FXR在肾脏髓质高渗环境建立及髓质细胞功能维持中的作用。研究结果：1) 通过Cre-LoxP技术获得集合管特异性FXR敲除小鼠FXRflox/flox-AQP2Cre（CD-FXR-/-）和肾髓间质细胞特异性FXR基因敲除小鼠（RMIC-FXR-/-）；2) 通过比较正常饮食、禁水和水负荷情况下尿量及相关指标，发现肾脏内髓集合管特异性FXR敲除对高渗环境下细胞存活及肾髓的高渗梯度建立和维持起到重要作用；3) 利用原代培养细胞技术从离体实验证明FXR在肾髓集合管上皮细胞和间质细胞中参与了高渗所致的细胞凋亡；4) FXR与TonEBP在转录及蛋白质水平的相互促进作用维持髓质高渗环境中细胞存活；结论：1）证明FXR通过与TonEBP在转录及蛋白质水平的交互调节作用，影响高渗环境下肾髓集合管上皮细胞和间质细胞的存活，建立及维持肾髓的高渗梯度，上述部分研究结果已发表在已发表在美国科学院院报（PNAS 2017，PNAS 2018）、美国生理杂志内分泌与代谢病学（AJP-EM 2017）、美国生理杂志肾脏生理学（AJP-RP 2017）等国际高水平期刊上；2）本课题阐明了肾脏髓质高渗状态建立和维持的具体机制,为尿崩症和肝肾综合征等水盐代谢紊乱疾病的治疗提供思路。