The role of red cell eNOS in the regulation of vascular tone

高血压是心血管疾病最重要的危险因素。肾脏主要通过调节钠和水的体内平衡来控制血压。在肾脏中，由内皮NO合成酶（eNOS）产生的一氧化氮（NO）被证明可以调节肾血流量，从而调节尿钠、利尿，进而调节血压。然而，生物活性NO的细胞来源尚不清楚。有趣的是，红细胞（rbc）被认为通过释放NO生物活性和诱导缺氧血管舒张来调节血流。值得注意的是，缺氧条件普遍存在于肾髓质，因此红细胞介导的NO生物活性释放可能在髓质血流调节中发挥核心作用。在之前的资助期内，我们发现红细胞特异性eNOS KO小鼠的循环亚硝酸盐/硝酸盐水平和高血压水平降低。反之，红细胞中eNOS的再激活可使eNOS KO小鼠免于高血压。支持红细胞enos介导的血压控制及其在肾脏中的作用的分子机制尚不清楚。我们假设RBC eNOS信号控制肾脏中NO生物活性的输出，这反过来有助于调节肾脏血流、钠排泄和血压控制。从机制上讲，红细胞eNOS可以调节髓质中NO代谢产物的合成（在常氧条件下）和输出（在缺氧条件下），从而诱导血管舒张和血流调节。本研究旨在分析红细胞eNOS/sGC信号在肾血流和血压调节中的作用。具体来说，我们将分析红细胞eNOS/sGC信号在一氧化氮生物活性输出中的作用(1)，以及在体外缺氧条件下抵抗性动脉血管反应的调节；(2)在稳态条件下和血管紧张素II （AngII）治疗后，对红细胞eNOS KO和KI小鼠以及红细胞sGC KO小鼠体内和体外肾血流的调节；(3)高血压患者eNOS/sGC信号、HbNO形成及NO生物活性释放的变化。我们预计红细胞eNOS/sGC信号将在红细胞NO生物活性释放和缺氧血管扩张作用中发挥重要作用。缺乏红细胞eNOS/sGC信号会通过减少红细胞eNOS依赖性NO生物活性的输出，加剧血管诱导的髓质血流减少，从而增加高血压。与正常对照相比，高血压患者的红细胞在缺氧条件下会表现出血管舒张功能下降，这可能通过激活eNOS/sGC信号来恢复。这些研究将揭示红细胞eNOS/sGC信号、NO生物活性释放和肾脏局部血流控制之间的联系。此外，我们的研究可能有助于发现新的临床诊断标志物和新的治疗高血压的药物靶点。

Hypertension is the most important risk factor for cardiovascular disease. The kidney contributes to blood pressure control, mainly via the regulation of sodium and water homeostasis. In the kidney nitric oxide (NO) produced by the endothelial NO synthase (eNOS) was shown to regulate renal blood flow and thereby natriuresis, diuresis, and consequently blood pressure. However, the cellular source of the bioactive NO is still unknown. Interestingly, red blood cells (RBCs) were proposed to regulate blood flow by releasing NO bioactivity and inducing hypoxic vasodilation. Notably, hypoxic conditions prevail in the renal medulla, and thus RBC-mediated release of NO bioactivity may play a central role in medullary blood flow regulation. In the previous funding period, we found that RBC-specific eNOS KO mice had decreased circulating nitrite/nitrate levels and hypertension. Vice versa, reactivation of eNOS in the RBCs rescues the global eNOS KO mice from hypertension. The molecular mechanisms underpinning RBC eNOS-mediated blood pressure control and its role in the kidney are unknown. We hypothesize that RBC eNOS signaling controls the export of NO bioactivity in the kidney, which in turn contributes to the regulation of renal blood flow, sodium excretion, and blood pressure control. Mechanistically, RBC eNOS may regulate synthesis (under normoxic conditions) and export of NO metabolites (under hypoxic conditions) in the medulla, which then may induce vasodilation and regulation of blood flow. This study aims to analyze the role of red cell eNOS/sGC signaling in the regulation of renal blood flow and blood pressure. Specifically, we will analyze the role of red cell eNOS/sGC signaling (1) in the export of NO bioactivity, and regulation of vascular responses in resistive arteries in hypoxic conditions ex vivo; (2) in the regulation of renal blood flow ex vivo and in vivo in RBC eNOS KO and KI mice, as well as RBC sGC KO mice under homeostatic conditions and after treatment with angiotensin II (AngII); and (3) changes in eNOS/sGC signaling, HbNO formation and release of NO bioactivity in patients with hypertension. We anticipate that red cell eNOS/sGC signaling in the RBCs will play a significant role in the release of NO bioactivity, and hypoxic vasodilatatory effects of RBCs. Lack of RBC eNOS/sGC signaling will exacerbate AngII-induced decrease in medullary blood flow via a reduced export of eNOS-dependent NO bioactivity from the RBCs, thereby increasing hypertension. Compared to normotensive control, RBCs from hypertensive patients will show decreased vasodilatory function under hypoxia, which may be recovered by activating the eNOS/sGC signaling. These studies will reveal the link between red cell eNOS/sGC signaling, the release of NO bioactivity, and control of local blood flow in the kidney. Moreover, our studies may help identify novel clinical diagnostic markers and new druggable targets for treating hypertension.