Synthesis, secretion, function, metabolism and application of natriuretic peptides in heart failure
心力衰竭中钠利尿肽的合成、分泌、功能、代谢和应用

Synthesis and secretion of BNP
BNP 的合成和分泌

Firstly discovered in porcine brain, BNP is stored in atrial granule with ANP, but not in granule in ventricle [14]. BNP is mainly secreted by normal atrium [15]. However, as a hallmark for maladaptive remodeling of left ventricle (LV), BNP is mainly secreted in ventricle when LV function is insufficient and cardiac wall is stretched due to transmural pressure or volume overload [16]. BNP has more obviously elevated levels than ANP in patients with HF [17]. NPPB gene has the following exons: exon 1 (5’-UTR, a 26-aa signal peptide, and 15 aa of proBNP sequence), exon 2 (most of proBNP sequence) and exon 3 (terminal tyrosine and 3’-UTR) (Fig. ​(Fig.1).1). Various causes, such as tissue hypoxia, transmural pressure or volume overload, induce the transcription of NPPB gene in endoplasmic reticulum to produce 134-aa preproBNP [18]. Pro-inflammatory cell factors, including interleukin-1β, interleukin-6 and tumor necrosis factor-α, induce BNP synthesis in cardiomyocytes, suggesting an active involvement of immune system in the regulation of NP synthesis [19]. Repeated AUUUA in 3’-UTR of BNP mRNA make it easier to be degraded than ANP mRNA. After removing 26-aa signal peptide, 108-aa proBNP is produced and then cleaved upon secretion by furin (or corin) into the active BNP1-32 (or BNP4-32), and the inactive 76-aa N-terminal proBNP (NT-proBNP) [20].
首次在猪脑中发现，BNP 与 ANP 一起储存在心房颗粒中，但不在心室颗粒中[14]。BNP 主要由正常心房分泌[15]。然而，作为左心室（LV）不适应性重塑的标志，当 LV 功能不足且心脏壁由于透壁压力或容积过载而伸展时，BNP 主要在心室中分泌[16]。患有 HF 的患者中，BNP 的水平明显高于 ANP[17]。NPPB 基因具有以下外显子：外显子 1（5'-UTR，26 个氨基酸信号肽和 15 个 proBNP 序列氨基酸），外显子 2（大部分 proBNP 序列）和外显子 3（末端酪氨酸和 3'-UTR）（图​（图 1）。1）。各种原因，如组织缺氧、透壁压力或容积过载，诱导内质网中 NPPB 基因的转录，产生 134 个氨基酸的前蛋白 BNP[18]。 促炎细胞因子，包括白细胞介素-1β，白细胞介素-6 和肿瘤坏死因子-α，在心肌细胞中诱导 BNP 合成，表明免疫系统在 NP 合成调节中的积极参与[19]。BNP mRNA 的 3'-UTR 中重复的 AUUUA 使其比 ANP mRNA 更容易被降解。去除 26-aa 信号肽后，产生 108-aa proBNP，然后通过 furin（或 corin）在分泌时被切割成活性 BNP1-32（或 BNP4-32），以及不活性的 76-aa N-末端 proBNP（NT-proBNP）[20]。

BNP-32 and proBNP-108 BNP-32 和 proBNP-108

Not only proBNP and O-Glycosylated proBNP, but also BNP and NT-proBNP, are present in blood [14]. Current immunoassays for BNP-32 also recognize proBNP-108, and plasma BNP in HF rather includes proBNP-108 and O-Glycosylated proBNP-108 [21]. There is an elevated ProBNP-108/BNP-32 ratio in patients with HF [22]. ProBNP-108/BNP-32 ratio increases in response to ventricular overload rather than atrial overload in patients with HF [23]. Moreover, proBNP-108 has much less ability to induce the synthesis of cyclic guanosine monophosphate (cGMP) than BNP-32 in vascular smooth muscle and endothelial cells [24]. Despite high levels of plasma BNP, there is an attenuation of BNP bioactivity in patients with HF [25]. In mild to moderate HF, plasma cGMP levels increase in proportion to HF severity. However, plasma cGMP levels have an attenuated increase relative to disease state, and no longer correlate with BNP levels in severe HF [26]. Even relatively small increase in proBNP-108/BNP-32 ratio, if it lasts for a long time, can sufficiently reduce total potential of cGMP production and attenuate compensatory benefit of plasma BNP, thereby leading to HF progression.
不仅有 proBNP 和 O-糖基化的 proBNP，还有 BNP 和 NT-proBNP 存在于血液中[14]。目前的 BNP-32 免疫测定也能识别 proBNP-108，并且 HF 患者血浆中的 BNP 主要包括 proBNP-108 和 O-糖基化的 proBNP-108[21]。HF 患者中 ProBNP-108/BNP-32 比值升高[22]。在 HF 患者中，ProBNP-108/BNP-32 比值增加是对室壁过载的反应，而不是对心房过载的反应[23]。此外，与 BNP-32 相比，proBNP-108 在血管平滑肌和内皮细胞中诱导环鸟苷酸单磷酸（cGMP）合成的能力要低得多[24]。尽管血浆中 BNP 水平较高，但在 HF 患者中 BNP 的生物活性有所减弱[25]。在轻度至中度 HF 中，血浆 cGMP 水平与 HF 严重程度成比例增加。然而，在疾病状态下，血浆 cGMP 水平的增加相对较小，并且不再与严重 HF 中的 BNP 水平相关[26]。 即使 proBNP-108/BNP-32 比值略微增加，如果持续时间较长，也足以显著降低 cGMP 产生的总潜力，并减弱血浆 BNP 的代偿益处，从而导致 HF 的进展。

O-Glycosylation and proBNP-108
O-糖基化和 proBNP-108

O-Glycosylated proBNP-108 levels correlate with HF severity [24]. Within Golgi apparatus of ventricular myocytes, proBNP-108 is post-translationally glycosylated to various extent at seven sites of N-terminal region: threonine (Thr)36, serine (Ser)37, Ser44, Thr48, Ser53, Thr58 and Thr71 [27]. Within trans-Golgi network, O-Glycosylated proBNP-108 is cleaved into BNP-32 and NT-proBNP [23]. O-Glycosylation has potential effect on the secretion and processing of proBNP-108. Thr71 is near to cleavage site of proBNP-108, and O-Glycosylation at Thr71 inhibits processing of proBNP-108 in human embryonic kidney (HEK) 293 cells and human leukemia 1 cells [28, 29]. Mechanisms governing cardiac secretion and peripheral metabolism of proBNP-108 remain unclear in patients with severe HF [30]. Venous furin-like enzyme activity has been proposed to correlate with NTproBNP/proBNP-108 ratio, and cleave proBNP-108 into BNP-32 and NT-proBNP in peripheral blood of patients with acute HF [31]. However, it has also been reported that proBNP-108 has similar levels between peripheral vein and artery, and is unlikely to be metabolized in peripheral blood and tissue. These inconsistent results may be caused by the diversity between in vivo and in vitro settings, between glycosylated and non-glycosylated proBNP-108, and between clinical status of patients with HF [32].
O-糖基化的 proBNP-108 水平与 HF 严重程度相关[24]。在室壁肌细胞的高尔基体内，proBNP-108 在 N-末端区域的七个位点（苏氨酸（Thr）36、丝氨酸（Ser）37、Ser44、Thr48、Ser53、Thr58 和 Thr71）后翻译地糖基化到不同程度[27]。在转高尔基网中，O-糖基化的 proBNP-108 被切割成 BNP-32 和 NT-proBNP[23]。O-糖基化可能影响 proBNP-108 的分泌和加工。Thr71 靠近 proBNP-108 的切割位点，Thr71 处的 O-糖基化抑制了 HEK 293 细胞和人类白血病 1 细胞中 proBNP-108 的加工[28，29]。在严重 HF 患者中，控制心脏分泌和外周代谢 proBNP-108 的机制仍不清楚[30]。静脉 furin 样酶活性被认为与 NTproBNP/proBNP-108 比值相关，并在急性 HF 患者外周血中切割 proBNP-108 成 BNP-32 和 NT-proBNP[31]。 然而，也有报道称，proBNP-108 在外周静脉和动脉之间的水平相似，并且不太可能在外周血液和组织中代谢。这些不一致的结果可能是由于体内和体外环境之间的差异，糖基化和非糖基化的 proBNP-108 之间的差异，以及患有 HF 的患者的临床状况之间的差异[32]。

New forms of BNP BNP 的新形式

Many new plasma forms of BNP with low molecular mass are present, but BNP-32 is nearly absent in patients with HF [33]. DPP-4 cleaves N-terminal serine-proline dipeptide from BNP1-32 in blood to produce BNP3-32 and BNP5-32, which are more rapidly degraded and have much less abilities to induce beneficial responses than BNP1-32 [34]. DPP-4 also cleaves N-terminal histidine-proline dipeptide from proBNP1-108 to produce proBNP3-108. Current immunoassays can not distinguish these forms in blood [35]. Patients using DPP-4 inhibitors have exhibited an increased risk of HF hospitalization in Saxagliptin Assessment of Vascular Outcomes Recorded in patients with diabetes mellitus-Thrombolysis in Myocardial Infarction (SAVOR-TIMI) trial [36]. However, other studies have not confirmed an association between DPP-4 inhibitors and HF hospitalization and shown adverse prognosis in patients with HF using DPP-4 inhibitors [37–41]. Previous meta-analyses can not rule out the concern that DPP-4 inhibitors have cardioprotective roles by affecting BNP1-32/BNP3-32 ratio [42–45].
许多低分子量的新 BNP 等离子形式存在，但在 HF 患者中 BNP-32 几乎不存在[33]。DPP-4 在血液中从 BNP1-32 中切除 N-末端丝氨酸-脯氨酸二肽，产生 BNP3-32 和 BNP5-32，这些形式降解更快，且比 BNP1-32 具有更少的诱导有益反应的能力[34]。DPP-4 还从 proBNP1-108 中切除 N-末端组氨酸-脯氨酸二肽，产生 proBNP3-108。目前的免疫测定无法区分这些形式在血液中[35]。使用 DPP-4 抑制剂的患者在糖尿病患者血管结果评估-心肌梗死溶栓治疗（SAVOR-TIMI）试验中表现出 HF 住院风险增加[36]。然而，其他研究未证实 DPP-4 抑制剂与 HF 住院之间的关联，并显示使用 DPP-4 抑制剂的 HF 患者预后不良[37]-[41]。先前的荟萃分析无法排除 DPP-4 抑制剂通过影响 BNP1-32/BNP3-32 比例具有心脏保护作用的担忧[42]-[45]。

Function of ANP ANP 的功能

ANP boosts natriuresis and diuresis. Na⁺ reabsorption in inner medullary collecting ducts depends mainly on the apical amiloride-sensitive Na⁺ channel (cyclic nucleotidegated ion channel) and basolateral Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-ATPase). Apical amiloride-sensitive Na⁺ channel allows passive Na⁺ entry from renal tubular lumen. Basolateral Na⁺-K⁺-adenosine triphosphatase helps Na⁺-K⁺-2Cl^- cotransporter actively pump Na⁺ out of epithelial cell into peritubular space and eventually bloodstream. ANP inhibits apical Na⁺ channel and basolateral Na⁺-K⁺-ATPase activity, resulting in decreased Na⁺ reabsorption from inner medullary collecting ducts and increased Na⁺ excretion from urine known as natriuresis [53]. ANP induces cGMP to inhibit Na⁺ channel by a mechanism of activating PKG independent of phosphorylation [2]. ANP inhibits basolateral Na⁺-K⁺-ATPase through PKG-induced phosphorylation in a cGMP-dependent manner [53]. ANP inhibits RAAS with reduced angiotensin II-induced sodium and water transport in renal proximal tubules [60]. ANP suppresses renin secretion from juxtaglomerular (granular) cells in a cGMP-dependent manner without changing intracellular Ca²⁺. ANP also suppresses aldosterone synthesis in adrenal glomerulosa (adrenocorticotropic hormone-induced, angiotensin-II-induced and basic aldosterone), which enhances natriuretic effect and reduces extracellular volume [61]. ANP/NPR-A activity induces natriuresis and diuresis, but appears to be downregulated in HF with RAAS activation [62]. Although conflicting studies exist, ANP may suppress angiotensin-II-induced secretion of vasopressin from posterior pituitary without blood-brain barrier, and inhibit V2 receptor-mediated effect of vasopressin on water reabsorption in collecting ducts [63–65]. ANP increases glomerular filtration rate (GFR) through its direct vasodilative effect on afferent arterioles. ANP also reverses norepinephrine-induced vasoconstriction of afferent arterioles [66]. Vasodilative effect of ANP on vascular smooth muscle may involve following mechanisms: reducing Ca²⁺ influx, enhancing Ca²⁺ extrusion, and inhibiting Ca²⁺ release from sarcoplasmic reticulum [67]. ANP directly relaxes contractile intraglomerular mesangial cells and expands glomerular capillary surface area available for filtration. ANP also inhibits angiotensin-II-induced constriction of mesangial cells [67]. There have been controversies on vasoconstrictive effect of ANP on efferent arterioles: some have observed no change in diameter of efferent arterioles, whereas others have reported the dilatation of afferent arterioles and constriction of efferent arterioles [68].
ANP 增加尿钠和利尿。内髓集合管中的 Na⁺重吸收主要取决于顶端的敏感于米诺地尔的 Na⁺通道（环核苷酸门控离子通道）和基底的 Na⁺-K⁺-腺苷三磷酸酶（Na⁺-K⁺-ATP 酶）。顶端的敏感于米诺地尔的 Na⁺通道允许来自肾小管腔的被动 Na⁺进入。基底的 Na⁺-K⁺-腺苷三磷酸酶帮助 Na⁺-K⁺-2Cl^-共转运蛋白将 Na⁺主动泵出上皮细胞进入周围管腔空间，最终进入血液循环。ANP 抑制顶端 Na⁺通道和基底 Na⁺-K⁺-ATP 酶活性，导致内髓集合管中 Na⁺重吸收减少，尿液中 Na⁺排泄增加，即所谓的尿钠作用[53]。ANP 诱导 cGMP 通过激活 PKA 独立于磷酸化的机制抑制 Na⁺通道[2]。 ANP 通过 PKG 诱导的 cGMP 依赖性磷酸化抑制基底侧 Na⁺-K⁺-ATP 酶[53]。ANP 通过减少肾近曲小管中血管紧张素 II 诱导的钠和水转运来抑制 RAAS[60]。ANP 以 cGMP 依赖的方式抑制球旁（颗粒）细胞中的肾素分泌，而不改变细胞内 Ca²⁺。ANP 还抑制肾上腺球状区（促肾上腺皮质激素诱导、血管紧张素 II 诱导和基础醛固酮）的醛固酮合成，增强利尿作用并减少细胞外容积[61]。ANP/NPR-A 活性诱导利尿和利尿，但在 RAAS 激活的 HF 中似乎被下调[62]。尽管存在矛盾的研究，但 ANP 可能抑制后垂体没有血脑屏障的血管紧张素 II 诱导的抗利尿素分泌，并抑制 V2 受体介导的抗利尿素对集合管中水重吸收的作用[63–65]。ANP 通过对肾小球入球小动脉的直接扩张作用增加肾小球滤过率（GFR）。 ANP 还可以逆转去甲肾上腺素诱导的肾小球入球小动脉收缩[66]。ANP 对血管平滑肌的扩张作用可能涉及以下机制：减少 Ca²⁺的流入，增强 Ca²⁺的外排，以及抑制来自肌浆网的 Ca²⁺释放[67]。ANP 直接放松具有收缩性的肾小球系膜细胞，并扩展可供过滤的肾小球毛细血管表面积。ANP 还抑制了血管紧张素 II 诱导的系膜细胞收缩[67]。关于 ANP 对出球小动脉的收缩作用存在争议：有些人观察到出球小动脉直径无变化，而其他人报告了入球小动脉扩张和出球小动脉收缩[68]。

Lowering blood pressure 降低血压

ANP induces hypovolemia and decreases blood pressure (BP). ANP lowers BP with increased permeability of capillaries and fluid efflux from blood [69]. ANP decreases cardiac load by shifting intravascular fluid into interstitial space. ANP stimulates Ca²⁺/calmodulin-dependent endothelial nitric oxide (NO) synthase in aorta, ventricle and kidney to produce more NO for relaxing vascular smooth muscle cells by binding to either NPR-A or NPR-C, causing a decrease in BP levels [70]. ANP decreases vascular resistance also by inhibiting RAAS [71]. Thus, ANP/NPR-A activity reduces basic BP levels through its combined effects on vascular relaxation and intravascular volume. Moreover, ANP relaxes air passages and blood vessels in lung [72]. ANP and BNP have elevated levels due to wall stretch of right ventricle, and inhibit pulmonary hypertension caused by chronic hypoxia [73].
ANP 诱导低血容量并降低血压（BP）。ANP 通过增加毛细血管通透性和血液从血液中流出来降低 BP[69]。ANP 通过将血管内液体转移到间质空间来降低心脏负荷。ANP 刺激主动脉、心室和肾脏中的 Ca²⁺/钙调蛋白依赖性内皮一氧化氮（NO）合酶产生更多 NO，通过结合到 NPR-A 或 NPR-C 来使血管平滑肌细胞松弛，从而导致 BP 水平降低[70]。ANP 还通过抑制 RAAS 来降低血管阻力[71]。因此，ANP/NPR-A 活性通过对血管松弛和血管内容量的综合影响降低基本 BP 水平。此外，ANP 放松肺部气道和血管[72]。由于右心室壁伸展，ANP 和 BNP 的水平升高，并抑制由慢性缺氧引起的肺动脉高压[73]。

Counteracting sympathetic activity
对抗交感神经活动

ANP not only modulates baroreflex mechanism, but also inhibits sympathetic activity and enhances vagal afferent. SNS is inhibited in peripheral vessels, perhaps by lowering activation threshold of baroreceptors, by decreasing catecholamine release from nerve endings, and by reducing sympathetic outflow [74]. It lowers activation threshold of vagal afferent, reflex tachycardia and vascular constriction, and causes a sustained decrease in mean arterial pressure [75]. ANP decreases sympathetic outflow by modulating ganglionic neurotransmission rather than increasing discharge from cardiac mechanoreceptors with inhibitory vagal afferent [76].
ANP 不仅调节压力感受机制，还抑制交感神经活动并增强迷走神经传入。SNS 在外周血管中受到抑制，可能是通过降低压力感受器的激活阈值，减少神经末梢的儿茶酚胺释放，以及减少交感神经输出。它降低迷走神经传入的激活阈值，反射性心动过速和血管收缩，并导致平均动脉压持续下降。ANP 通过调节节后神经传递而不是增加心脏机械感受抑制性迷走神经传入来降低交感神经输出。

Inhibiting cardiac hypertrophy
抑制心肌肥大

Both prolonged exposure to systemic hypertension and lacking the inhibition of heart growth lead to cardiac hypertrophy [77, 78]. Moreover, ANP has direct effects on heart, and inhibits cardiac hypertrophy and fibrosis [79]. ANP induces cardiomyocyte apoptosis and inhibits fibroblast growth [3]. ANP suppresses fibroblast migration and proliferation by counteracting angiotensin-II, aldosterone and endothelin-1, as well as inflammatory reaction including pro-inflammatory factor and macrophage infiltration [80, 81]. ANP inhibits stress-activated protein kinase and extracellular-regulated kinase-2 induced by platelet-derived growth factor [82]. ANP exhibits antimitogenic and antineoplastic properties by reducing cell adhesion and inflammatory reaction through p38 mitogen-activated protein kinase [83]. ANP attenuates the growth of cardiomyocytes and fibroblasts by inhibiting norepinephrine-induced Ca²⁺ influx in a cGMP-mediated manner [84]. Meanwhile, ANP and BNP reduce systemic and pulmonary BP, and inhibit cardiac hypertrophy in HF [85]. ANP and CNP exert direct effects on vessels by reducing adhesion molecules of endotheliocytes (P-selectin and monocyte chemotactic protein-1) and inflammatory infiltration on atheromatous plaques [86].
长期暴露于全身高血压和缺乏心脏生长抑制会导致心肌肥厚[77, 78]。此外，ANP 对心脏有直接影响，抑制心肌肥厚和纤维化[79]。ANP 诱导心肌细胞凋亡并抑制成纤维细胞生长[3]。ANP 通过抵消血管紧张素-II、醛固酮和内皮素-1，以及包括促炎因子和巨噬细胞浸润在内的炎症反应，抑制成纤维细胞迁移和增殖[80, 81]。ANP 通过抑制血小板源性生长因子诱导的应激激活蛋白激酶和细胞外调节激酶-2 来抑制细胞增殖[82]。ANP 通过减少细胞粘附和通过 p38 丝裂原活化蛋白激酶抑制炎症反应来表现抗有丝分裂和抗肿瘤特性[83]。ANP 通过抑制去甲肾上腺素诱导的 Ca²⁺通量来减弱心肌细胞和成纤维细胞的生长，这是一种 cGMP 介导的方式[84]。同时，ANP 和 BNP 降低全身和肺动脉血压，并在 HF 中抑制心肌肥厚[85]。 ANP 和 CNP 通过降低内皮细胞的粘附分子（P-选择素和单核细胞趋化蛋白-1）以及动脉粥样硬化斑块上的炎症浸润，对血管产生直接影响[86]。

Regulating energy homeostasis
调节能量平衡

ANP stimulates exercise-induced lipolysis through cGMP and PKG in primates with an increased NPR-A/NPR-C ratio [87]. ANP affects the conversion from white to brown fat through mitochondrial uncoupling protein-1 and p38 mitogen-activated protein kinase [88]. BNP may also have hypoglycemic effect and regulate energy homeostasis [89]. ANP and BNP stimulate oxidative ability of skeletal muscle and lipolytic action in subcutaneous adipose [90]. ANP and BNP induce hormone-sensitive lipase of adipocytes in a cGMP-mediated manner [91].
ANP 通过 cGMP 和 PKG 在具有增加的 NPR-A/NPR-C 比例的灵长类动物中刺激运动诱导的脂解作用[87]。ANP 通过线粒体解耦联蛋白-1 和 p38 丝裂原活化蛋白激酶影响白色脂肪向棕色脂肪的转化[88]。BNP 可能还具有降糖作用并调节能量稳态[89]。ANP 和 BNP 刺激骨骼肌的氧化能力和皮下脂肪的脂解作用[90]。ANP 和 BNP 以 cGMP 介导的方式诱导脂肪细胞的激素敏感性脂解酶[91]。

Function of BNP BNP 的功能

In addition to the well documented natriuresis, diuresis and vasodilation, BNP also has direct effects on heart [14]. BNP may provide compensatory protection, such as inhibiting myocardial apoptosis and necrosis and reducing cardiac hypertrophy and fibrosis [92, 93]. BNP may also modulate immune and inflammatory reaction to cardiac injury. BNP depletes monocytes, B lymphocytes and natural killer cells in peripheral blood [94]. BNP regulates the chemotaxis of monocytes and production of inflammatory molecules by macrophages [95]. BNP may promote cardiac neutrophil infiltration and metalloprotease-9 expression after myocardial infarction (MI), and also have direct effects on matrix remodeling and wound healing [96].
除了已经有记录的利尿、利尿和血管舒张作用外，BNP 还对心脏有直接影响[14]。BNP 可能提供补偿保护，如抑制心肌凋亡和坏死，减少心肌肥大和纤维化[92，93]。BNP 还可能调节免疫和炎症反应以应对心脏损伤。BNP 耗尽外周血液中的单核细胞、B 淋巴细胞和自然杀伤细胞[94]。BNP 调节单核细胞的趋化作用和巨噬细胞产生炎症分子[95]。BNP 可能促进心脏中性粒细胞浸润和心肌梗死（MI）后金属蛋白酶-9 表达，并对基质重塑和伤口愈合产生直接影响[96]。

Affecting cardiac embryogenesis
影响心脏胚胎发育

BNP plays significant roles in cardiac embryogenesis. There are high BNP levels in embryonic heart during the midgestation, and peaks of BNP secretion correlate with cardiac development [97]. Plasma BNP levels in humans are high at birth, progressively declining thereafter, to stabilize at around ten years of age to the levels found in adults [98]. ANP and BNP may regulate cardiomyocyte differentiation and proliferation in the developing embryo [99]. Embryonic stem cells express high levels of BNP, which are crucial for their proliferation and differentiation [100]. BNP may also be involved in the process of angiogenesis following skeletal muscle ischemia [101]. BNP secretion by vascular satellite cells has been found to activate the regeneration of adjacent endothelium in a paracrine manner. Moreover, BNP has been addressed in cardiac regeneration by evaluating the relationships between cardiac precursor cells (CPCs) and BNP in neonatal and adult mice. Firstly, all forms of proBNP are more abundant in neonatal heart than in adult heart [102]. Secondly, CPCs express NPR-A and NPR-B, supporting that CPCs can respond to BNP [102]. NPR-A contributes to self-renewal and maintenance of CPC pluripotency, whereas NPR-B is involved in CPC proliferation [103]. ANP, BNP and CNP stimulate CPC proliferation and differentiation into new cardiomyocytes by NPR-B binding, cGMP increase and PKG activation [104]. Thirdly, exogenous BNP has increased proliferating CPCs and new cardiomyocytes, which are associated with improved cardiac function and remodeling after MI [102]. Finally, CPCs stain positive for BNP, suggesting that CPCs can also synthesize and secrete BNP in an autocrine manner to regulate their proliferation and differentiation into new cardiomyocytes. Thus, BNP and CPCs may be useful therapies for HF and MI [102].
BNP 在心脏胚胎发育中发挥重要作用。在中期妊娠期间，胚胎心脏中的 BNP 水平很高，BNP 分泌的高峰与心脏发育相关[97]。人类血浆中的 BNP 水平在出生时很高，之后逐渐下降，稳定在约十岁时达到成人水平[98]。ANP 和 BNP 可能调节发育中胚胎心肌细胞的分化和增殖[99]。胚胎干细胞表达高水平的 BNP，这对它们的增殖和分化至关重要[100]。BNP 也可能参与骨骼肌缺血后血管生成的过程[101]。已发现血管卫星细胞分泌的 BNP 以旁分泌方式激活相邻内皮的再生。此外，通过评估新生儿和成年小鼠心脏前体细胞（CPCs）与 BNP 之间的关系，已经探讨了 BNP 在心脏再生中的作用。首先，所有形式的 proBNP 在新生儿心脏中比成年心脏中更丰富[102]。 其次，CPCs 表达 NPR-A 和 NPR-B，支持 CPCs 可以对 BNP 做出反应[102]。NPR-A 有助于 CPC 的自我更新和多能性维持，而 NPR-B 参与 CPC 的增殖[103]。ANP、BNP 和 CNP 通过 NPR-B 结合、cGMP 增加和 PKG 激活刺激 CPC 的增殖和分化为新的心肌细胞[104]。第三，外源性 BNP 增加了增殖的 CPC 和新的心肌细胞，这与 MI 后心功能和重塑的改善有关[102]。最后，CPC 对 BNP 呈阳性染色，表明 CPC 也可以自分泌和分泌 BNP，以调节其增殖和分化为新的心肌细胞。因此，BNP 和 CPC 可能是 HF 和 MI 的有用疗法[102]。

NEP and IDE NEP 和 IDE

NEP is a dominant enzyme for NP degradation. In heart, NEP is expressed on membrane of cardiomyocytes, fibroblasts, vascular smooth muscle and endothelial cells [123]. Although expressed in many epithelial tissues, NEP levels are particularly high at the luminal side of renal proximal tubules [124]. Initial attack of breaking ring and inactivating peptide occurs between cysteine and phenylalanine (Fig. ​(Fig.1)1) [125]. Cleavage sites in ring structure are crucial for degradation [48]. ANP and CNP have differences of zero or one aa between species, and are similarly degraded by NEP. BNP differs obviously between species, and is species-specifically degraded by NEP [126]. BNP is a worse substrate than ANP and CNP, and human NEP cleaves BNP at three sites [127]. Most BNP is degraded by NEP in rat renal membrane, but NEP dose not degrade all BNP in human renal membrane [128]. IDE, a zinc-dependent protease with broad substrates, degrades not only insulin, but also ANP [129, 130].
NEP 是降解 NP 的主要酶。在心脏中，NEP 表达在心肌细胞、成纤维细胞、血管平滑肌和内皮细胞的膜上[123]。尽管在许多上皮组织中表达，但 NEP 水平在肾近曲小管腔侧尤为高[124]。环和肽的失活的初始攻击发生在半胱氨酸和苯丙氨酸之间（图​（图 1）1）[125]。环结构中的裂解位点对降解至关重要[48]。ANP 和 CNP 在物种之间差异为零或一个氨基酸，被 NEP 类似地降解。BNP 在物种之间明显不同，并且被 NEP 物种特异性地降解[126]。BNP 是比 ANP 和 CNP 更差的底物，人类 NEP 在三个位点裂解 BNP[127]。大部分 BNP 在大鼠肾膜中被 NEP 降解，但 NEP 在人类肾膜中并不降解所有 BNP[128]。IDE 是一种具有广泛底物的锌依赖性蛋白酶，不仅降解胰岛素，还降解 ANP[129, 130]。

Genetic manipulation 基因操纵

In order to determine the function of NPs and medication of NPRs, genetic manipulation has been widely applied through the knockout of NPRs in animal experiments. For example, ANP-dependent natriuresis and diuresis are mediated exclusively by NPR-A in mice because these effects are completely lost after NPR-A knockout [62]. Mice lacking ANP or NPR-A have an enlarged heart, whereas mice over-expressing ANP have a smaller heart [79]. Mice over-expressing ANP are resistant to hypoxia-induced hypertension, whereas mice lacking ANP exhibit increased pulmonary hypertension in response to chronic hypoxia [73]. Moreover, designer NPs have been engineered through genetic alteration of native NPs. Compared with native NPs, designer NPs have improved efficacy and safety [7].
为了确定 NPs 的功能和 NPRs 的药物作用，遗传操纵已广泛应用于动物实验中的 NPRs 敲除。例如，在小鼠中，ANP 依赖性的利尿和利尿作用仅由 NPR-A 介导，因为在 NPR-A 敲除后这些效应完全丧失[62]。缺乏 ANP 或 NPR-A 的小鼠有扩大的心脏，而过度表达 ANP 的小鼠有较小的心脏[79]。过度表达 ANP 的小鼠对缺氧诱导的高血压具有抗性，而缺乏 ANP 的小鼠在慢性缺氧情况下表现出增加的肺动脉高压[73]。此外，通过对原生 NPs 进行基因改变，设计师 NPs 已被工程化。与原生 NPs 相比，设计师 NPs 具有改进的功效和安全性[7]。

Roles of micro-RNA 微小 RNA 的作用

MiR-100 has been demonstrated to inhibit NPR-C expression in rat MI tissues and human LV cells, and increased miR-100 in HF may reflect a compensatory mechanism to prolong half-life of NPs [149]. MiR-425 inhibits ANP synthesis in human heart by interacting with 3’-UTR of NPPA gene, and antagonists of miR-425 may be a potential therapy for HF [144]. MiR-21 interacts with ANP in vascular smooth muscle cells through modulating downstream cGMP signaling [150]. MiR-30 is expressed in healthy heart, but suppressed in failing heart. MiR-30 inhibits GalNAc-transferase (GALNT) 1 and 2-mediated glycosylation of proBNP-108. MiR-30-GALNT pathway may be a novel therapeutic target for HF, and more researches are necessary in humans [32]. Although many miRs targeting NPRs have been suggested in silico analyses to modulate NP signaling pathways in HF, it is necessary to confirm their actual interactions in vivo experiments [151].
MiR-100 已被证明能抑制大鼠 MI 组织和人类 LV 细胞中的 NPR-C 表达，并且 HF 中 miR-100 的增加可能反映了一种延长 NPs 半衰期的代偿机制[149]。MiR-425 通过与 NPPA 基因的 3'-UTR 相互作用，在人类心脏中抑制 ANP 的合成，miR-425 的拮抗剂可能是 HF 的潜在治疗方法[144]。MiR-21 通过调节下游 cGMP 信号与血管平滑肌细胞中的 ANP 相互作用[150]。MiR-30 在健康心脏中表达，但在衰竭心脏中受抑制。MiR-30 抑制 GalNAc 转移酶（GALNT）1 和 2 介导的 proBNP-108 的糖基化。MiR-30-GALNT 途径可能是 HF 的一种新的治疗靶点，需要在人类中进行更多的研究[32]。尽管许多通过计算分析提出的 miRs 被认为在 HF 中调节 NP 信号通路，但有必要通过体内实验来确认它们的实际相互作用[151]。

Confounding factors 混杂因素

Although applying BNP and NT-proBNP as diagnostic biomarkers of HF has brought significant improvement in treating HF, several confounding factors, such as aging, obesity, anemia, sepsis, hypertension, MI, cardiac hypertrophy, pulmonary hypertension, atrial fibrillation, diabetes mellitus, renal failure, liver cirrhosis, severe burn and cancer chemotherapy, limit their accuracy [165]. Plasma NP levels have been inversely related to body mass index in epidemiological investigations [166, 167]. Plasma NPs have lower levels in patients with diabetes mellitus, insulin resistance or metabolic syndrome, perhaps contributing to HF risk [168, 169]. Plasma BNP has higher levels in patients with hypertension or LV hypertrophy than in those without them [170, 171]. Plasma BNP has higher levels in patients with LV concentric hypertrophy than in those with LV eccentric hypertrophy or in those with normal LV structure and hypertension [172]. BP-lowering therapy reduces BNP levels and LV mass [173, 174].
尽管将 BNP 和 NT-proBNP 应用作 HF 的诊断生物标志物在治疗 HF 方面取得了显著进展，但多种混杂因素，如衰老、肥胖、贫血、败血症、高血压、心肌梗死、心肌肥厚、肺动脉高压、心房颤动、糖尿病、肾功能衰竭、肝硬化、严重烧伤和癌症化疗，限制了它们的准确性[165]。流行病学调查显示，血浆 NP 水平与体重指数呈负相关[166, 167]。患有糖尿病、胰岛素抵抗或代谢综合征的患者血浆 NPs 水平较低，可能增加 HF 风险[168, 169]。患有高血压或左心室肥厚的患者血浆 BNP 水平较高，而没有这些情况的患者血浆 BNP 水平较低[170, 171]。左心室浓厚肥厚患者的血浆 BNP 水平较左心室偏心肥厚或正常左心室结构和高血压患者的水平更高[172]。降压治疗可降低 BNP 水平和左心室质量[173, 174]。

Coronary artery disease 冠状动脉疾病

Plasma NPs have higher levels in patients with acute coronary syndrome or exercise-induced myocardial ischemia but without ventricular dilation [175]. Plasma ANP levels rise until admission and then decline in patients with acute MI. Plasma BNP levels rise until 12-24 hours after acute MI, then decline and peak once more after 5-7 days [176]. Height of the second peak is an useful indicator of LV remodeling [177]. Although there is a gradual decrease, plasma BNP levels are still increased in chronic phase, showing LV damage and remodeling [178]. However, plasma BNP has almost normal levels if early coronary reperfusion successfully prevents LV remodeling.
Plasma NPs 在急性冠状动脉综合征或运动诱发的心肌缺血患者中水平较高，但没有室壁扩张[175]。急性心肌梗死患者的血浆 ANP 水平在入院前升高，然后下降。急性心肌梗死后 12-24 小时，血浆 BNP 水平上升，然后下降，并在 5-7 天后再次达到峰值[176]。第二次峰值的高度是 LV 重塑的一个有用指标[177]。尽管有逐渐减少的趋势，血浆 BNP 水平在慢性阶段仍然增加，显示 LV 损伤和重塑[178]。然而，如果早期冠状动脉再灌注成功预防 LV 重塑，血浆 BNP 几乎达到正常水平。

Chronic kidney disease 慢性肾病

Renal function has systematical effects on NP system. In patients with chronic kidney disease (CKD), plasma NP levels are elevated as compensatory protection of renal function. CKD is always related to cardiovascular abnormalities. However, due to unclear mechanisms, there are elevated BNP levels in patients with CKD but without cardiovascular abnormalities [179]. Plasma NP levels may be regulated both by synthetic/secretory rate from heart and by extraction rate from blood. Key mechanism may not be an increase in extraction rate from kidney. Other mechanisms include the decreases in functional renal mass, second messenger synthesis and clearance receptor degradation in kidney [180, 181]. Elevated NP levels in CKD are correlated with a counter-regulatory response directed from heart to kidney, suggesting NPs as potential biomarkers of LV remodeling in patients with CKD [181]. Plasma NP levels in CKD reflect the stress on cardiac wall caused by LV hypertrophy or dysfunction [182].
肾功能对 NP 系统有系统性影响。在慢性肾病（CKD）患者中，血浆 NP 水平升高，作为肾功能的代偿性保护。CKD 通常与心血管异常相关。然而，由于机制不清楚，患有 CKD 但没有心血管异常的患者血浆 BNP 水平升高。血浆 NP 水平可能受心脏合成/分泌速率和血液中提取速率的调节。关键机制可能不是来自肾脏的提取速率增加。其他机制包括功能性肾脏质量减少、第二信使合成和肾脏中清除受体降解的降低。CKD 中升高的 NP 水平与从心脏到肾脏的反调节反应相关，表明 NPs 可能是 CKD 患者 LV 重塑的潜在生物标志物。CKD 中的血浆 NP 水平反映了由 LV 肥厚或功能障碍引起的心脏壁的应激。

End-stage renal disease 晚期肾脏疾病

Plasma BNP and NT-proBNP have higher levels in patients with end-stage renal disease. There is a decrease of about 20–40% in plasma BNP levels after hemodialysis (HD). Peritoneal dialysis (PD) may not alter plasma BNP levels [183]. HD promotes fluid clearance and alleviates volume overload, leading to reduced wall stress and NP release [184]. Plasma BNP has lower levels in patients with PD than in those with HD, supporting that PD may lead to slower ultrafiltration rate, higher urine output, better hemodynamic condition, less cardiac load and lower BP levels than HD [185]. However, it remains inconclusive and needs more researches [186]. Meanwhile, due to reduced ultrafiltration rate, continuous volume overload and more LV hypertrophy, patients with automated PD (APD) may have higher BNP levels than those with continuous ambulatory PD (CAPD) [187]. Moreover, plasma BNP levels have a significant potential to identify LV hypertrophy or dysfunction in patients with different dialysis and renal transplant [188–190].
Plasma BNP 和 NT-proBNP 在晚期肾病患者中水平较高。血液透析（HD）后，血浆 BNP 水平下降约 20-40％。腹膜透析（PD）可能不会改变血浆 BNP 水平[183]。HD 促进液体清除，减轻容量过载，导致减少壁应力和 NP 释放[184]。PD 患者的血浆 BNP 水平低于 HD 患者，支持 PD 可能导致较慢的超滤速率，更高的尿量，更好的血流动力学状况，较少的心脏负荷和较低的血压水平[185]。然而，这仍然没有定论，需要更多的研究[186]。同时，由于超滤速率降低，持续容量过载和更多的 LV 肥厚，自动腹膜透析（APD）患者可能比持续间歇腹膜透析（CAPD）患者具有更高的 BNP 水平[187]。此外，血浆 BNP 水平在不同透析和肾移植患者中有显著潜力用于识别 LV 肥厚或功能障碍[188]-[190]。

Practical application in CKD
慢性肾病中的实际应用

Renal function limits current use of NPs in patients with CKD [191]. Plasma NP levels in patients with CKD are related to CKD severity, and cutoff levels are increased as CKD stages advance. Plasma BNP levels rise to almost 200 pg/ml in patients with CKD but without HF. Compared with plasma BNP levels, plasma NT-proBNP levels may be more strongly correlated with GFR and affected by age-related decrease in GFR, suggesting careful use of NT-proBNP in elderly with CKD [192]. In patients with CKD, plasma NT-proBNP levels > 1200 pg/ml suggest chronic HF in patients < 50 years of age and > 4502 pg/ml in patients between 50 and 75 years old [180]. It remains unclear if elevated NP levels in CKD effectively reflect the activation of NP system and effects on target organ. Elevated NP levels may have reduced ability to activate NP system and affect target organ in CKD. NP resistance in CKD may be caused by downregulated NPR-A expression in renal medulla and upregulated NPR-C expression in renal cortex [193]. NP resistance in CKD results in the invalidity of NP infusion in protecting renal function and treating cardiorenal syndrome in patients with HF [194].
肾功能限制了目前在慢性肾病患者中使用 NPs [191]。慢性肾病患者的血浆 NP 水平与慢性肾病的严重程度相关，随着慢性肾病阶段的进展，截断水平也会增加。慢性肾病患者的血浆 BNP 水平在没有心力衰竭的患者中升高到近 200 pg/ml。与血浆 BNP 水平相比，血浆 NT-proBNP 水平可能与 GFR 更强相关，并受年龄相关 GFR 降低的影响，这表明在患有慢性肾病的老年人中要谨慎使用 NT-proBNP [192]。在慢性肾病患者中，血浆 NT-proBNP 水平> 1200 pg/ml 提示 50 岁以下患者患有慢性心力衰竭，而在 50 至 75 岁之间的患者中> 4502 pg/ml [180]。目前尚不清楚慢性肾病中升高的 NP 水平是否有效反映了 NP 系统的激活和对靶器官的影响。在慢性肾病中，升高的 NP 水平可能降低激活 NP 系统和影响靶器官的能力。慢性肾病中的 NP 抵抗可能是由于肾髓质中 NPR-A 表达下调和肾皮质中 NPR-C 表达上调引起的 [193]。 NP 抗性在 CKD 中导致 NP 输注在保护肾功能和治疗 HF 患者的心肾综合征中无效[194]。

Point-of-care systems 现场系统

Previous assays for BNP are invasive and time-consuming with the discomfort caused by venipuncture. Some ideal point-of-care (POC) systems have been developed to allow rapid and repeated assays for BNP from capillary blood, like measuring blood sugar from fingertip [195]. A system at bedside would not only be useful for repeated assays at home, but also achieve routine monitoring in a remote way. Additionally, POC systems would also be used in the hospitals for BNP-guided therapy of HF and rapid triage of dyspnea. Two POC systems for BNP use either venipuncture ethylenediaminetetraacetic acid (EDTA) plasma (Alere Triage) or EDTA whole blood (Abbott i-STAT) [196–199]. In contrast, Alere Heart Check BNP Test is the first POC system easily used as a rapid assay for BNP from untreated fingertip capillary whole blood. Previous studies have demonstrated the safety and feasibility of Alere Heart Check BNP Test at home [200–202].
以前的 BNP 检测方法是侵入性的和耗时的，由静脉穿刺引起的不适。一些理想的即时检测（POC）系统已经开发出来，允许从毛细血管血液中快速和重复地检测 BNP，就像从指尖测量血糖一样[195]。床边的系统不仅对家庭中的重复检测有用，而且可以实现远程的常规监测。此外，POC 系统还将用于 BNP 引导 HF 治疗和呼吸困难的快速分诊。两种用于 BNP 的 POC 系统分别使用静脉穿刺乙二胺四乙酸（EDTA）血浆（Alere Triage）或 EDTA 全血（Abbott i-STAT）[196–199]。相比之下，Alere Heart Check BNP Test 是第一个易于使用的 POC 系统，可从未经处理的指尖毛细血管全血中快速检测 BNP。以前的研究已经证明了 Alere Heart Check BNP Test 在家中的安全性和可行性[200–202]。

Increasing NP bioactivity
增加 NP 生物活性

Novel therapies are under development based on an augment in cardioprotective effects of NPs to re-balance neuroendocrine dysregulation in HF [216]. Current NP-augmenting strategies include the synthesis of NPs or agonists to increase NP bioactivity and inhibition of NEP to reduce NP breakdown [217, 218]. Nesiritide, a rhBNP, has been shown to induce hemodynamic and clinical improvements in Vasodilatation in the Management of Acute CHF (VMAC) and other trials [219]. Nesiritide is successfully approved by Food and Drug Administration (FDA) and routinely used for both acute and chronic HF. However, nesiritide has been questioned in two subsequent meta-analyses to worsen renal function and increase mortality rate [220]. Other studies, such as Acutely Decompensated Heart Failure Registry (ADHERE) trial, have not confirmed these unfavorable effects of nesiritide [221, 222]. Acute Study of Clinical Effectiveness of Nesiritide in Decompensated Heart Failure (ASCEND-HF) trial has reported that nesiritide has no significant relationship with mortality rate, nor is it related to worsening renal function [223]. Neutral conclusions may be correlated with nesiritide dose. Large dose of nesiritide is strongly vasodilative, causes severe hypotension and neutralizes beneficial roles. Recent studies have reported that small dose of nesiritide, particularly when administered through subcutaneous route, induces hemodynamic and clinical improvements, without increasing nephrotoxicity and mortality, thus reopening the debates about its usefulness in patients with HF [224]. Long-term antiapoptotic, antiremodeling and antihypertrophic actions of nesiritide are beneficial if NPR-A could be activated chronically [225]. Nesiritide administered twice daily for eight weeks through subcutaneous route has improved clinical symptoms and reduced LV mass in patients with HF [226]. Additionally, nesiritide has been suggested to protect LV function in patients with MI [227]. Carperitide, recombinant form of human ANP, has alleviated clinical symptoms and been recommended in Japan for acute decompensated HF [125]. But short half-life limits clinical application of carperitide. Moreover, oral forms of ANP and BNP are too unstable to be routinely used in HF. CNP is unsuitable for treating HF due to relatively short half-life and no renal-enhancing action. In Safety and Efficacy of an Intravenous Placebo-controlled Randomised Infusion of Ularitide (SIRIUS I and II), synthetic urodilatin (ularitide) has induced hemodynamic and clinical improvements, without worsening renal function and obvious BP change, in patients with acute decompensated HF [228, 229]. Another Phase III Trial of Ularitide Efficacy and Safety in Acute Heart Failure (TRUE-AHF) has shown that ularitide reduces systolic BP and cardiac stress as indicated by plasma NT-proBNP levels, but has no effect on clinical composite end point, cardiovascular mortality and myocardial injury as indicated by cardiac troponin T levels [230].
新疗法正在开发中，基于 NPs 的心脏保护效应增强，以重新平衡 HF 中的神经内分泌失调[216]。目前的 NP 增强策略包括合成 NPs 或激动剂以增加 NP 的生物活性，以及抑制 NEP 以减少 NP 的降解[217, 218]。Nesiritide，一种 rhBNP，已被证明在急性 CHF 管理中诱导血流动力学和临床改善（VMAC）和其他试验中的血管舒张作用[219]。Nesiritide 已成功获得食品药品监督管理局（FDA）批准，并常规用于急性和慢性 HF。然而，两项随后的荟萃分析质疑了 Nesiritide 会恶化肾功能并增加死亡率[220]。其他研究，如急性失代偿性心力衰竭登记（ADHERE）试验，未证实 Nesiritide 的这些不利影响[221, 222]。 急性失代偿性心力衰竭（ASCEND-HF）试验报告称，奈西立肽与死亡率无显著关系，也与肾功能恶化无关[223]。中性结论可能与奈西立肽剂量相关。大剂量奈西立肽具有强烈的扩血管作用，会导致严重低血压并中和有益作用。最近的研究报告称，小剂量奈西立肽，特别是经皮下途径给药时，可引起血流动力学和临床改善，而不会增加肾毒性和死亡率，因此重新引发了关于其在 HF 患者中的有用性的争论[224]。奈西立肽的长期抗凋亡、抗重塑和抗肥厚作用在 NPR-A 能够慢性激活时是有益的[225]。经皮下途径每日两次给予奈西立肽治疗八周，改善了 HF 患者的临床症状并减少了左心室质量[226]。此外，奈西立肽被建议用于保护心肌梗死患者的左心室功能[227]。 卡普利肽，人类 ANP 的重组形式，已经缓解了临床症状，并在日本推荐用于急性失代偿性 HF [125]。但卡普利肽的短半衰期限制了其临床应用。此外，ANP 和 BNP 的口服形式过于不稳定，不能常规用于 HF。CNP 不适用于治疗 HF，因为其半衰期相对较短且没有肾脏增强作用。在 Ularitide 的静脉安慰剂对照随机输注的安全性和有效性（SIRIUS I 和 II）中，合成尿利肽（Ularitide）已经在急性失代偿性 HF 患者中引起了血流动力学和临床改善，而且没有恶化肾功能和明显的血压变化 [228, 229]。另一个急性心力衰竭中 Ularitide 疗效和安全性的 III 期试验（TRUE-AHF）显示，Ularitide 降低了收缩压和心脏应激，如血浆 NT-proBNP 水平所示，但对临床复合终点、心血管死亡率和心肌损伤，如心肌肌钙蛋白 T 水平所示，没有影响 [230]。

Designer NPs 设计师 NPs

Severe hypotension and short half-life make recombinant agents, including nesiritide, carperitide and ularitide, not very suitable for clinical use. Designer NPs are developed by altering genetic forms or aa structures of native NPs. These hybrid peptides have normal binding to NPR-A and increased resistance to degradation [7]. DNP is firstly discovered in snake venom, and much about DNP remains unclear in humans. Cenderitide-NP (CD-NP) is not easy to be degraded as a 37-aa hybrid NP designed by fusing native CNP-22 with 15-aa C-terminal of DNP. This first-generation designer NP retains vasodilative, antifibrotic and antihypertrophic roles of CNP, and natriuretic and diuretic roles of DNP [231]. Both NPR-A and NPR-B can be effectively activated by CD-NP to increase more GFR and cause less hypotension than nesiritide, with reduced atrial pressure and improved cardiac-unloading effect [232]. FDA has provided a fast-track designation for CD-NP in Phase II trials [233]. CU-NP has been designed by fusing 17-aa ring structure of native CNP with C- and N-terminal of urodilatin [234]. As an experimental agent in early stage, CU-NP exerts cardiac-unloading, renal-enhancing and RAAS-suppressing effects through activating cGMP. CU-NP has direct antihypertrophic effect through inhibiting sodium-hydrogen exchanger 1 (NHE-1)/calcineurin pathway [235]. Mutant ANP (M-ANP) has been designed as a 40-aa peptide by fusing native ANP with 12-aa extension to C-terminal [236]. M-ANP has exerted beneficial cardiac and renal effects, such as boosting natriuresis and diuresis, regulating BP and GFR, inhibiting RAAS and SNS, and promoting antifibrosis and antiproliferation in experiments [237]. Novel NPs are currently under clinical development programs for further trials [238]. An alternative RNA spliced transcript for BNP (AS-BNP) has a unique 34-aa C-terminal, with remaining structure of native BNP [205]. ANX-042 has been designed as a 42-aa peptide by fusing 16 aa from C-terminal of AS-BNP and 26 aa from native BNP. ANX-042 can activate cGMP to boost natriuresis and diuresis and suppress renin and angiotensin-II, but not activate cGMP to relax blood vessels. As a designer NP in a first-in-human trial, FDA has suggested ANX-042 as an investigated new drug for HF with renal protection and less hypotension [239]. CNP analog (BMN111) is one of the most promising therapy for achondroplasia, and obviously improves skeletal parameters in animal experiments [240].
严重低血压和短半衰期使得重组药物，包括奈西立肽、卡佩立肽和乌拉立肽，不太适合临床使用。设计师 NPs 通过改变基因形式或氨基酸结构来开发。这些混合肽对 NPR-A 具有正常结合能力，并且对降解具有增加的抵抗力[7]。DNP 最初在蛇毒中发现，关于 DNP 在人类中的许多情况仍不清楚。Cenderitide-NP（CD-NP）是一种 37-aa 混合 NP，通过将原生 CNP-22 与 DNP 的 15-aa C-末端融合设计而成，不容易被降解。这种第一代设计师 NP 保留了 CNP 的扩血管、抗纤维化和抗肥厚作用，以及 DNP 的利尿和利尿作用[231]。CD-NP 可以有效激活 NPR-A 和 NPR-B，比奈西立肽引起更多的 GFR 和更少的低血压，降低心房压力并改善心脏卸载效果[232]。FDA 已为 CD-NP 在 II 期试验中提供了快速通道指定[233]。CU-NP 是通过将原生 CNP 的 17-aa 环结构与尿利肽的 C-和 N-末端融合设计而成。 作为早期实验性药物，CU-NP 通过激活 cGMP 发挥心脏卸载、肾脏增强和 RAAS 抑制作用。CU-NP 通过抑制钠-氢交换蛋白 1（NHE-1）/钙调神经磷酸酶途径具有直接的抗肥厚作用。突变 ANP（M-ANP）已被设计为一个由原生 ANP 与 12 个氨基酸延伸至 C 端的 40 个氨基酸肽。M-ANP 已在实验中发挥了有益的心脏和肾脏作用，如促进尿钠和利尿、调节血压和 GFR、抑制 RAAS 和 SNS，以及促进抗纤维化和抗增殖。新型 NPs 目前正在进行临床开发计划，以进行进一步的试验。BNP 的一种替代 RNA 剪接转录本（AS-BNP）具有独特的 34 个氨基酸 C 端，其余结构与原生 BNP 相同。ANX-042 被设计为一个 42 个氨基酸的肽，由 AS-BNP 的 C 端 16 个氨基酸和原生 BNP 的 26 个氨基酸融合而成。ANX-042 可以激活 cGMP 以促进尿钠和利尿，并抑制肾素和血管紧张素 II，但不能激活 cGMP 以放松血管。 作为一名设计师 NP 在第一例人体试验中，FDA 已建议将 ANX-042 作为 HF 肾保护和较少低血压的新药进行研究[239]。CNP 类似物（BMN111）是骨发育不全症最有前途的疗法之一，显然在动物实验中改善了骨骼参数[240]。

Reducing NP degradation 减少 NP 降解

Although NP breakdown can be blocked by affecting NPR-C and inhibiting IDE, the more commonly used approach to reduce NP degradation is NEP inhibition. However, there are plentiful substrates of NEP, such as angiotensin-I, angiotensin-II, bradykinin, substance P, adrenomedullin, endothelin-1, opioid peptide, insulin β-chain, glucagon, oxytocin, chemotactic peptide, neurotensin, enkephalins, gastrin and amyloid-β peptide. NEP inhibition has potential to increase levels of these substrates, leading to conflicting effects on kidney and vessels [7]. Moreover, NEP hydrolyzes angiotensin-I to angiotensin-(1-7), which counteracts angiotensin-II. As the first pure NEP inhibitor, candoxatril is stable when administered orally. However, due to its effects on other systems, candoxatril has no benefit for patients with hypertension or HF [241]. Candoxatril is characterized by both NP augment (elevated NP levels and natriuresis) and RAAS activation (elevated angiotensin-II levels and vasoconstriction), leading to unaltered vascular resistance and unavailable antihypertensive role.
尽管 NP 降解可以通过影响 NPR-C 并抑制 IDE 来阻止，但更常用的减少 NP 降解的方法是 NEP 抑制。然而，NEP 有丰富的底物，如血管紧张素-I、血管紧张素-II、激肽、物质 P、肾上腺髓质素、内皮素-1、阿片肽、胰岛素β链、胰高血糖素、催化肽、神经肽、恩啡肽、胃泌素和淀粉样蛋白-β肽。NEP 抑制有可能增加这些底物的水平，导致对肾脏和血管产生矛盾的影响[7]。此外，NEP 将血管紧张素-I 水解为血管紧张素-(1-7)，与血管紧张素-II 相抗衡。作为第一个纯 NEP 抑制剂，坎多沙曲稳定性口服给药。然而，由于其对其他系统的影响，坎多沙曲对高血压或 HF 患者没有益处[241]。坎多沙曲的特点是 NP 增加（升高 NP 水平和利尿）和 RAAS 激活（升高血管紧张素-II 水平和血管收缩），导致血管阻力不变和无法发挥降压作用。

Dual ACE/NEP inhibitors 双重 ACE/NEP 抑制剂

Pure NEP inhibitors have disappointing clinical effects, which may be improved by combining RAAS blockade. Due to no improvement in clinical symptoms and an increase in aplastic anemia, it is unpractical to choose an addition of NEP inhibitors (ecadotril) to standard therapies including angiotensin-converting enzyme (ACE) inhibitors [242]. Dual ACE/NEP inhibitor (vasopeptidase inhibitor, sampatrilat) has shown beneficial effects, but then been dropped due to short half-life [243]. Omapatrilat (BMS-186716) has almost affinity and inhibition for NEP and ACE. In experimental HF and hypertension models, omapatrilat has not only improved clinical symptoms and survival, but also relieved cardiac dysfunction and hypertension [244]. Moreover, omapatrilat has improved cardiac function and remodeling, and decreased cardiac hypertrophy and fibrosis in mice with MI [245]. Omapatrilat has been evaluated in patients with HF or hypertension in Omapatrilat Cardiovascular Treatment Versus Enalapril (OCTAVE), Inhibition of Metalloprotease by Omapatarilat in a Randomized Exercise and Symptoms Study of Heart Failure (IMPRESS) and Omapatrilat Versus Enalapril Randomized Trial of Utility in Reducing Events (OVERTURE) trials [246–248]. Omapatrilat has more obviously lowered vascular resistance and BP levels than candoxatril. As bradykinin is degraded by both NEP and ACE, simultaneous inhibition of them by omapatrilat increases bradykinin levels that favor the development of angioedema. Compared with enalapril, omapatrilat has increased angioedema and hypotension, and shown no superior benefit in patients with HF or hypertension, precluding its clinical use and final approval of FDA [249].
纯 NEP 抑制剂在临床效果上令人失望，可能通过联合 RAAS 阻断得到改善。由于临床症状没有改善且再生贫血增加，选择将 NEP 抑制剂（ecadotril）添加到包括血管紧张素转换酶（ACE）抑制剂在内的标准治疗方案中是不切实际的[242]。双重 ACE/NEP 抑制剂（血管肽酶抑制剂，sampatrilat）显示出有益效果，但由于半衰期短而被放弃[243]。Omapatrilat（BMS-186716）对 NEP 和 ACE 几乎具有亲和力和抑制作用。在实验性 HF 和高血压模型中，Omapatrilat 不仅改善了临床症状和生存率，还缓解了心脏功能障碍和高血压[244]。此外，Omapatrilat 改善了心脏功能和重塑，并减少了心肌梗死小鼠的心肌肥厚和纤维化[245]。 Omapatrilat 已在 Omapatrilat 心血管治疗与依那普利（OCTAVE）、心力衰竭的随机运动和症状研究中通过 Omapatarilat 抑制金属蛋白酶（IMPRESS）以及 Omapatrilat 与依那普利随机试验中进行评估，用于降低事件（OVERTURE）试验[246–248]。Omapatrilat 明显降低了血管阻力和血压水平，优于卡多沙曲。由于 NEP 和 ACE 均降解激肽，Omapatrilat 同时抑制它们会增加有利于血管水肿发展的激肽水平。与依那普利相比，Omapatrilat 在心力衰竭或高血压患者中引起更多的血管水肿和低血压，并未显示出更好的益处，因此无法在临床上使用，也未获得 FDA 的最终批准[249]。

Triple ACE/ECE/NEP inhibitors
三重 ACE/ECE/NEP 抑制剂

Endothelin-1 is a multifunctional vasoconstrictor and contributes to HF progression [250]. Most endothelin-1 receptor antagonists have no prognostic improvement in patients with acute and chronic HF. In experimental HF model, endothelin-converting enzyme (ECE) inhibition has suppressed endothelin-1 synthesis, improved cardiorenal function and reduced the neurohormones, such as renin, angiotensin-II and aldosterone [251]. NPs may be degraded by ECE. Thus, ECE inhibition may simultaneously augment the NPs and suppress the endothelin [252]. ECE inhibition has induced hemodynamic improvement in patients with HF [253]. However, there is no long-term study about ECE inhibition. Dual ECE/NEP inhibitor SLV-306 (daglutril) has not only lowered LV pressure in patients with HF, but also improved cardiac function and remodeling in rats with LV hypertrophy [254]. Moreover, daglutril has inhibited BP elevation and increased NP levels in healthy humans [255]. Another dual ECE/NEP inhibitor (SLV-338) has improved cardiac fibrosis in experiment [256]. Triple ACE/ECE/NEP inhibitors may inhibit the synthesis of angiotensin-II and endothelin-1, and enhance the effects of NPs and bradykinin. In experimental HF model, triple ACE/ECE/NEP inhibition has been superior to ACE inhibition and dual ECE/NEP inhibition in improving LV structure and function [257]. However, the development of triple ACE/ECE/NEP inhibitors may be obstructed by negative conclusions about endothelin-1 receptor antagonists from large HF trials and practical concerns about the safety with ACE/NEP inhibitors. It needs to be emphasized that endothelin-1 receptor antagonism and ECE inhibition should be distinguished in further human trials.
Endothelin-1 是一种多功能的血管收缩剂，对 HF 的进展起着贡献[250]。大多数内皮素-1 受体拮抗剂对急性和慢性 HF 患者没有预后改善作用。在实验性 HF 模型中，内皮素转化酶（ECE）抑制已经抑制了内皮素-1 的合成，改善了心肾功能并减少了肾素、血管紧张素-II 和醛固酮等神经激素[251]。NPs 可能会被 ECE 降解。因此，ECE 抑制可能同时增加 NPs 并抑制内皮素[252]。ECE 抑制已经在 HF 患者中引起了血流动力学改善[253]。然而，关于 ECE 抑制的长期研究尚无。双重 ECE/NEP 抑制剂 SLV-306（达格鲁曲）不仅降低了 HF 患者的 LV 压力，还改善了 LV 肥厚大鼠的心脏功能和重塑[254]。此外，达格鲁曲已经抑制了健康人体内的血压升高并增加了 NP 水平[255]。另一种双重 ECE/NEP 抑制剂（SLV-338）已经在实验中改善了心脏纤维化[256]。 三重 ACE/ECE/NEP 抑制剂可能抑制血管紧张素-II 和内皮素-1 的合成，并增强 NPs 和激肽酶的作用。在实验性 HF 模型中，三重 ACE/ECE/NEP 抑制优于 ACE 抑制和双重 ECE/NEP 抑制，改善 LV 结构和功能[257]。然而，三重 ACE/ECE/NEP 抑制剂的发展可能会受到大型 HF 试验中对内皮素-1 受体拮抗剂的负面结论以及与 ACE/NEP 抑制剂安全性相关的实际担忧的阻碍。需要强调的是，在进一步的人体试验中应区分内皮素-1 受体拮抗和 ECE 抑制。

Development of LCZ696 LCZ696 的发展

Angiotensin receptor blocker NEP inhibitor (ARNI, LCZ696) is a major advance in the therapies for HF in the last 15 years. Molecular moieties of NEP inhibitor prodrug sacubitril (AHU377), and valsartan, an angiotensin receptor blocker (ARB), are present in this single molecule in 1:1 molar ratio (sacubitril/valsartan). Sacubitril (AHU377) becomes active NEP inhibitor LBQ657 after cleaving ethyl ester. ARNI preserves ACE mechanism for bradykinin degradation [258]. ARNI augments beneficial effects of NPs and inhibits harmful effects of angiotensin-II. As the first-in-class ARNI, LCZ696 has improved cardiac dysfunction, fibrosis, remodeling and hypertrophy in an animal model [245]. Compared with valsartan alone, LCZ696 has more effectively lowered BP levels, without increased angioedema, in patients with hypertension [259]. In patients with HF with reduced ejection fraction (HFrEF), LCZ696 has more effectively reduced all-cause, cardiovascular and sudden death, prevented HF progression and hospitalization, and improved life quality and renal function than enalapril in Prospective comparison of ARNI with ACE inhibitor to Determine Impact on Global Mortality and morbidity in Heart Failure (PARADIGM-HF) trial [260–263]. LCZ696 has recently been approved by FDA for treating HFrEF. But translating the results of this trial into guideline recommendation has raised some concerns [264]. Firstly, this study has been discontinued ahead of schedule due to overwhelming benefit of LCZ696, and there is a doubt about efficacy and safety of LCZ696 used for longer time [265]. Secondly, LCZ696 was administered twice daily at a dose of 200 mg (160 mg of valsartan), and enalapril was administered twice daily at a dose of 10 mg. Both two doses are target doses in most HF guidelines but higher than many patients with HFrEF may tolerate. Thirdly, more symptomatic postural hypotension in LCZ-696 group limited its clinical use, particularly in patients with borderline BP before therapy. It is necessary to observe this agent according to baseline BP in further studies. Fourthly, bradykinin levels have previously been shown to increase with ARB, and there were more patients with angioedema in LCZ-696 group [266]. Fifthly, amyloid-β peptide is a key peptide in Alzheimer disease, and NEP may block its breakdown to induce Alzheimer disease [267]. However, Alzheimer disease and cancer were not increased using LCZ696, and cognitive decline related to vascular diseases may be reduced by LCZ696. Finally, drug denials have already been increased, and applying LCZ696 would be further complicated by cost. Compared with valsartan, LCZ696 has not only reduced NT-proBNP levels and caused GFR elevation, but also improved overall clinical status and decreased left atrial pressure in Prospective comparison of ARNI with ARB on Management Of heart failUre with preserved ejectioN fracTion (PARAMOUNT) trial [268]. Whether it would benefit patients with HF with preserved ejection fraction (HFpEF) is currently being tested in ongoing Efficacy and Safety of LCZ696 Compared to Valsartan on Morbidity and Mortality in Heart Failure Patients With Preserved Ejection Fraction (PARAGON-HF, NCT01920711) trial. ARNI class has sparked considerable excitement in other cardiovascular diseases including hypertension [269]. Prospective comparison of Angiotensin Receptor neprilysin inhibitor with Angiotensin receptor blocker MEasuring arterial sTiffness in the eldERly (PARAMETER) trail is also underway to compare the relationships of LCZ696 and olmesartan with central BP in patients with resistant hypertension [270].
Angiotensin 受体拮抗剂 NEP 抑制剂（ARNI，LCZ696）是过去 15 年心力衰竭治疗中的重大进展。NEP 抑制剂前药沙库比曲（AHU377）和缬沙坦，一种血管紧张素受体拮抗剂（ARB），以 1:1 的摩尔比例（沙库比曲/缬沙坦）存在于这种单一分子中。沙库比曲（AHU377）在裂解乙酸乙酯后成为活性 NEP 抑制剂 LBQ657。ARNI 保留 ACE 机制以降解激肽酶。ARNI 增强 NPs 的有益效果并抑制血管紧张素-II 的有害效果。作为首个 ARNI，LCZ696 在动物模型中改善了心脏功能障碍、纤维化、重塑和肥大[258]。与单独使用缬沙坦相比，LCZ696 在高血压患者中更有效地降低了血压水平，而不增加血管性水肿[259]。 在 HF 伴随降低射血分数（HFrEF）的患者中，LCZ696 在前瞻性比较 ARNI 与 ACE 抑制剂对心力衰竭全球死亡率和发病率的影响（PARADIGM-HF）试验中，比依诺普利更有效地降低了全因、心血管和猝死，预防了 HF 的进展和住院，改善了生活质量和肾功能[260–263]。LCZ696 最近已获 FDA 批准用于治疗 HFrEF。但将该试验结果转化为指南建议引起了一些关注[264]。首先，由于 LCZ696 的巨大益处，这项研究提前终止，对 LCZ696 长期使用的疗效和安全性存在疑虑[265]。其次，LCZ696 每日两次给药，剂量为 200 毫克（缬沙坦 160 毫克），依诺普利每日两次给药，剂量为 10 毫克。这两个剂量是大多数 HF 指南中的目标剂量，但高于许多 HFrEF 患者可能耐受的剂量。 第三，LCZ-696 组中更多的症状性体位性低血压限制了其临床应用，特别是在治疗前边缘血压的患者中。有必要根据基线血压观察这种药物在进一步研究中的作用。第四，已经显示 ARB 会导致激肽酶原水平升高，LCZ-696 组中有更多患者出现血管性水肿。第五，淀粉样蛋白β是阿尔茨海默病的关键蛋白，NEP 可能阻止其降解以诱发阿尔茨海默病。然而，使用 LCZ696 并未增加阿尔茨海默病和癌症的发生，LCZ696 可能减少与血管性疾病相关的认知衰退。最后，药物拒绝已经增加，使用 LCZ696 将进一步受到成本的复杂化影响。与缬沙坦相比，LCZ696 不仅降低了 NT-proBNP 水平并导致 GFR 升高，还改善了整体临床状况并降低了左心房压力在心力衰竭保留射血分数（PARAMOUNT）试验中。 无论是否会使 HF 患者受益，目前正在进行的 PARAGON-HF（NCT01920711）试验中正在测试 HFpEF 患者的效果和安全性与 Valsartan 相比的 LCZ696 对心力衰竭患者的发病率和死亡率。ARNI 类已经在其他心血管疾病中引起了相当大的兴奋，包括高血压[269]。与老年人中的血管硬度测量相关的 Angiotensin Receptor neprilysin inhibitor 与 Angiotensin receptor blocker 的前瞻性比较（PARAMETER）试验也正在进行，以比较 LCZ696 和 olmesartan 与难治性高血压患者中央血压的关系[270]。

Mechanisms of LCZ696 LCZ696 的机制

It remains unclear about the mechanisms responsible for the superiority of LCZ696 over ACE inhibitor. Considering elevated levels of plasma BNP and urinary cGMP, systemic vasodilation and renal natriuresis could be important mechanisms. But it remains uncertain whether its superiority is direct effect on heart or indirect effect secondary to beneficial effects of this agent on vessels (lower BP levels) and kidney (less renal injury) [271]. Lower NT-proANP and troponin levels support that LCZ696 directly reduces myocardial stretch or ischemia. LCZ696 reduces proteinuria, focal segmental glomerulosclerosis and retinopathy, and plays beneficial effects on microvascular and renal complications. Without the evidence in PARADIGM-HF, further studies are required to fully address this issue. Meanwhile, positive effects of BNP on cardiac regeneration may also play an important role, and should be fully addressed in ongoing experimental and clinical studies. Exogenous BNP or NEP inhibition may induce endogenous cardiac regeneration, and achieve the therapies for HF and MI [4]. In experimental hypertension models, either alone or combined with MI or diabetes mellitus, ARNI has improved cardiac hypertrophy and fibrosis in a BP-independent manner. Since sacubitril is largely cleared in kidney, drug accumulation may occur in patients with impaired renal function, and hypotension is a potential adverse effect in patients with CKD.
目前尚不清楚 LCZ696 优于 ACE 抑制剂的机制。考虑到血浆 BNP 和尿 cGMP 水平升高，系统血管舒张和肾脱钠可能是重要的机制。但目前尚不确定其优越性是对心脏的直接影响还是对血管（降低血压水平）和肾脏（减少肾损伤）的有益影响的间接影响[271]。较低的 NT-proANP 和肌钙蛋白水平支持 LCZ696 直接减少心肌伸展或缺血。LCZ696 减少蛋白尿、局灶节段性肾小球硬化和视网膜病变，并对微血管和肾脏并发症产生有益影响。在 PARADIGM-HF 中没有证据，需要进一步研究来全面解决这个问题。同时，BNP 对心脏再生的积极影响也可能起重要作用，并应在正在进行的实验和临床研究中得到充分解决。外源性 BNP 或 NEP 抑制可能诱导内源性心脏再生，并实现 HF 和 MI 的治疗[4]。 在实验性高血压模型中，ARNI 已经改善了心脏肥大和纤维化，无论是单独使用还是与 MI 或糖尿病结合，都是独立于血压的方式。由于沙库比特利主要在肾脏中清除，患有肾功能受损的患者可能会出现药物积累，并且低血压是慢性肾病患者的潜在不良影响。

Consent for publication

Not applicable.

Competing interests

The authors have no competing interest.