Physiological and Transcriptomic Changes in the Hypothalamic-Neurohypophysial System after 24 H of Furosemide-Induced Sodium Depletion

Background/Aims: Furosemide is a loop diuretic widely used in clinical practice for the treatment of oedema and hypertension. The aim of this study was to determine physiological and molecular changes in the hypothalamic-neurohypophysial system as a consequence of furosemide-induced sodium depletion. Methods: Male rats were sodium depleted by acute furosemide injection (10 and 30 mg/kg) followed by access to low sodium diet and distilled water for 24 h. The renal and behavioural consequences were evaluated, while blood and brains were collected to evaluate the neuroendocrine and gene expression responses. Results: Furosemide treatment acutely increases urinary sodium and water excretion. After 24 h, water and food intake were reduced, while plasma angiotensin II and corticosterone were increased. After hypertonic saline presentation, sodium-depleted rats showed higher preference for salt. Interrogation using RNA sequencing revealed the expression of 94 genes significantly altered in the hypothalamic paraventricular nucleus (PVN) of sodium-depleted rats (31 upregulated and 63 downregulated). Out of 9 genes chosen, 5 were validated by quantitative PCR in the PVN (upregulated: Ephx2, Ndnf and Vwf; downregulated: Caprin2 and Opn3). The same genes were also assessed in the supraoptic nucleus (SON, upregulated: Tnnt1, Mis18a, Nr1d1 and Dbp; downregulated: Caprin2 and Opn3). As a result of these plastic transcriptome changes, vasopressin expression was decreased in PVN and SON, whilst vasopressin and oxytocin levels were reduced in plasma. Conclusions: We thus have identified novel genes that might regulate vasopressin gene expression in the hypothalamus controlling the magnocellular neurons secretory response to body sodium depletion and consequently hypotonic stress. Overall design: Male rats were sodium depleted by acute furosemide injection (10mg/kg) followed by access to low sodium diet and distilled water for 24 h. The PVN region of the brain was collected from 9 animals per group, and 3 animals were pooled per sequencing replicate to give a final n=3 for assessing gene expression responses via RNAseq technology.

背景与目的：呋塞米（furosemide）是临床广泛用于水肿与高血压治疗的袢利尿剂（loop diuretic）。本研究旨在探究呋塞米诱导钠耗竭后，下丘脑-神经垂体系统（hypothalamic-neurohypophysial system）的生理与分子变化。
方法：通过急性注射呋塞米（剂量10与30mg/kg）构建雄性大鼠钠耗竭模型，随后给予低钠饮食与蒸馏水，持续24小时。评估大鼠的肾脏与行为学变化，同时采集血液与脑组织以检测神经内分泌与基因表达应答。
结果：呋塞米治疗可急性升高尿钠与水排泄量。造模24小时后，大鼠饮水与进食量均降低，血浆血管紧张素II（angiotensin II）与皮质酮（corticosterone）水平升高。给予高渗盐水后，钠耗竭大鼠表现出更高的盐偏好。通过RNA测序（RNA sequencing）分析发现，钠耗竭大鼠的下丘脑室旁核（hypothalamic paraventricular nucleus, PVN）中共有94个基因表达发生显著改变（31个上调、63个下调）。在筛选出的9个基因中，有5个在下丘脑室旁核中通过定量PCR（quantitative PCR）得到验证（上调基因：Ephx2、Ndnf与Vwf；下调基因：Caprin2与Opn3）。同时在视上核（supraoptic nucleus, SON）中对上述基因进行了检测（上调基因：Tnnt1、Mis18a、Nr1d1与Dbp；下调基因：Caprin2与Opn3）。受这些可塑性转录组变化影响，下丘脑室旁核与视上核中的血管加压素（vasopressin）表达量降低，血浆中血管加压素与催产素（oxytocin）水平也出现下降。
结论：本研究鉴定出潜在调控下丘脑内血管加压素基因表达的全新基因，这些基因可调控大细胞神经元对机体钠耗竭及后续低渗应激的分泌应答。
整体实验设计：雄性大鼠经急性呋塞米注射（10mg/kg）构建钠耗竭模型，随后给予低钠饮食与蒸馏水，持续24小时。每组采集9只大鼠的下丘脑室旁核组织，每个测序重复混合3只动物的组织，最终每组样本量n=3，以通过RNA测序（RNAseq）技术评估基因表达应答。