Departments of Medicine, University of Florida, Gainesville, Florida 32610; Departments of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610.
Department of Medicine, University of Chicago, Chicago, Illinois 60637.
J Biol Chem. 2014 Apr 25;289(17):11791-11806. doi: 10.1074/jbc.M113.531095. Epub 2014 Mar 7.
It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent (22)Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.
已经证实，血压和肾功能会发生昼夜波动。我们已经证明，昼夜节律蛋白 Per1 调节肾脏集合管中参与钠转运的多个基因。然而，Per1 在肾单位的其他部分的作用尚未被研究。远曲小管（DCT）在肾脏钠重吸收中起着关键作用。在这个节段中，钠通过 NaCl 共转运体（NCC）的作用被重吸收，NCC 受无赖氨酸激酶（WNK）调节。本研究的目的是测试 Per1 是否通过调节 NCC 和 WNK 激酶 WNK1 和 WNK4 来调节 DCT 中的钠转运。核 Per1 进入的药理学阻断导致小鼠肾皮质中 NCC 和 WNK1 的 mRNA 表达减少，但 WNK4 的表达增加。这些发现通过使用 Per1 siRNA 和 Per1 核进入的药理学阻断在 mDCT15 细胞（小鼠远曲小管的模型）中得到了证实。转录调控通过短暂异质核 RNA 的变化得到证实。染色质免疫沉淀实验表明 Per1 和 CLOCK 与 NCC、WNK1 和 WNK4 的启动子相互作用。这种相互作用受到 Per1 核进入的阻断的调节。重要的是，NCC 蛋白表达和 NCC 活性，如噻嗪敏感、氯依赖性（22）Na 摄取测量所示，在 Per1 核进入的药理学抑制下降低。总之，这些数据表明 Per1 在 NCC、WNK1 和 WNK4 的转录调节中起作用。
