Obestatin对心力衰竭大鼠肾脏水通道蛋白2的短时和长时调控

Enhanced membrane targeting (short-term regulation) and selective increase expression (long-term regulation) of aquaporin 2 (AQP2) in collecting duct cells is a key mechanism for the development of water retention in heart failure. Previous stueies have found that central obestatin can act in brain to inhibit water drinking and also inhibit vasopressin secretion. Whereas circulating and local obestatin may act in kindey to upregulate expression and membrane targeting of AQP2, and may be an important contributor to the physiologic regulation of fluid homeostasis. We have recently found circulating obestatin is increased in patients with heart failure and intense staining for obestatin in cytoplasm of collecting duct cells. These results suggest that obestatin may upregulate the short- and long-term regulation of kidney AQP2 through an endocrine and autocrine/paracrine modes, and play a role in fluid retention of heart failure. So we are intending to study the role and mechanism of obestatin in the short- and long-term regulation of kidney AQP2 in heart failure rat and collecting duct cells. Fisrtly, we will study the role of obestatin in AQP2 trafficking events such as increased intracellular cAMP levels and activation of PKA, AQP2 phosphorylation, increased intracellular Ca2+, depolymerization of cytoskeleton. Secondly, we are going to confirm the role of obestatin in synthesis regulation of AQP2. Thirdly, we are to investigate the role of its receptor GPR39 in short- and long-term regulation of AQP2.

肾脏集合管水通道蛋白2（AQP2）上调导致水重吸收增加是心力衰竭水潴留的重要机制。中枢obestatin通过抑制精氨酸加压素（AVP）减少肾脏水重吸收。集合管细胞丰富表达其受体GPR39，但外周obestatin对肾脏水重吸收的作用未明。我们前期研究发现心衰患者循环obestatin水平明显升高，同时集合管丰富表达obestatin，推测obestatin可能通过内分泌和自分泌/旁分泌方式上调肾脏AQP2，促进心衰水潴留。本项目拟以心衰大鼠和集合管细胞为研究对象，采用组织学、分子生物学等方法观察obestatin对AQP2磷酸化、细胞骨架、钙离子等的作用（短时调控）以及对AQP2 mRNA和蛋白合成的影响（长时调控），并利用RNAi等技术探讨obestatin受体及受体后信号转导通路。研究结果将有助于阐明obestatin在心衰水潴留中的病理生理作用及机制，为心衰的治疗提供理论和实验依据。

肾脏水通道蛋白2（AQP2）表达异常和易位引起全身水代谢失衡，在心力衰竭水潴留的病理生理机制中发挥重要作用。中枢obestatin通过抑制AVP-V2R-AQP2途径减少肾脏水重吸收，心衰患者外周循环obestatin水平升高，对肾脏水重吸收的作用未明。本项目以心衰大鼠和集合管细胞为研究对象，采用组织学和分子生物学方法观察obestatin对AQP2磷酸化、细胞内分布的作用、对AQP2 mRNA和蛋白合成的影响以及水代谢、心衰指标等表型变化，利用高通量基因芯片及RNAi等技术探讨obestatin受体及受体后信号转导通路。研究结果显示，obestatin在心衰大鼠循环及肾脏表达增高。Obestatin作用15min对 AQP2分布及表达未见影响，作用30min即可降低AQP2在集合管细胞膜分布的比例，作用60min后可通过下调AQP2的总体表达来降低其在膜上的表达，obestatin对AQP2的Ser256位点磷酸化水平并无明显作用。尾静脉注射obestatin2周后，心衰大鼠尿量明显增加，血浆BNP下降，但对大鼠心功能、心室重构、血浆电解质、血浆AVP水平无明显影响，肾脏皮质、外髓、内髓集合管的AQP2表达均有所下降，下降幅度以内髓区域最为明显。不同浓度的obestatin对内髓集合管细胞AQP2的调控效果不一样，以10-7mmol/L浓度下调最明显。Obestatin的干预还能同时下调PPARG、V2R表达，对GPR39受体无明显影响。单纯地敲减V2R、PPARG能使AQP2表达水平下降，而敲减GPR39则对AQP2表达无显著影响。但敲减GPR39能部分阻断obestatin对V2R及PPARG的调控。据此我们认为，obestatin对于肾脏AQP2具有短时调控作用，可能通过增加AQP2内在化即促进胞吞作用来下调其在集合管细胞膜上的比例。Obestatin对于肾脏AQP2具有长时调控作用，其分子机制在于AQP2是V2R、PPARG的直接靶向基因，obestatin依赖于GPR39受体相互作用来实现对V2R、PPARG的下调作用，从而降低肾脏集合管AQP2的表达。据此我们推断，心衰时循环中obestatin代偿性上调，并通过对AQP2的短时和长时调控机制发挥利尿作用、减缓心衰且对血电解质无明显影响，将来有望成为新的心衰干预靶点。

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