Sodium pump inhibitors in blood pressure regulation and in profibrotic signaling in salt sensitive hypertension and aging

为了验证这些假设，我们进行了以下实验： 我们用年轻(3个月)和老年(24个月)雄性SD大鼠的培养的VSMC以及PKG-1基因沉默的年轻的VSMC用1nmol/L ANP、1nmol/L MBG或ANP和MBG的组合处理。Western blotting分析胶原-1、Fli-1和PKG-1的表达。 我们已经证明，与年轻大鼠相比，老年大鼠血管中PKG1和FLI1的水平降低。ANP可阻断MBG对年轻VSMC血管NKA的抑制作用，但对老年VSMC则无明显影响。在幼年大鼠的VSMC中，MBG诱导Fli-1表达下调，胶原-1水平升高，而ANP可阻断这一作用。在年轻的VSMC中，PKG1基因沉默导致PKG1和FLI1水平降低。此外，MBG还降低了FLI1，增加了胶原-1的水平，ANP不能对抗这些MBG效应，类似于老年大鼠的VSMC，PKG1的年龄相关性降低。 根据我们的研究结果，我们得出结论，随着年龄的增长，血管PKG1的减少以及由此导致的cGMP信号的下降导致ANP失去对抗MBG诱导的NKA抑制和纤维化发展的能力。PKG1的沉默模仿了衰老的这些影响。 综上所述，在幼年和老年大鼠的VSMC中，MBG预孵育与胶原-1水平的升高有关，而且这种作用在老年VSMC中表现得更强，即在给予MBG后，老年VSMC的胶原水平高于年轻的VSMC。观察到MBG的年龄相关效应是ANP/PKG依赖的，因为在年轻的VSMC中PKG1的沉默与MBG作用的增强有关。ANP不能拮抗PKG1沉默后MBG致VSMC纤维化的作用。因此，ANP/PKG依赖的增强MBG效应的机制很可能促进了血压盐敏感性的发展，并伴随着MBG诱导的心血管纤维化的发展。因此，与3月龄大鼠相比，24月龄大鼠在急性氯化钠负荷后表现出更大的升压反应和更小的利钠反应。我们目前的数据与最近的一项发现是一致的，即位于PKG1基因的一组单核苷酸多态与高血压患者的血压盐敏感性有关。我们先前的研究表明，MBG通过NKA依赖的信号作用，刺激心脏、肾脏和主动脉组织中胶原的合成，并且MBG的免疫中和作用逆转了肾功能衰竭大鼠的心脏纤维化。由于纤维化是心血管衰老的标志，PKG激动剂和利钠肽具有抗纤维化作用，cGMP/PKG信号的增龄性降低对MBG促纤维化作用的影响值得进一步研究。

To test the hypotheses, we have performed the following experiments: We used cultured VSMC from the young (3-mo) and old (24-mo) male Sprague-Dawley rats, and young VSMC with silenced PKG-1 gene to treat them with 1 nmol/L ANP, or with 1 nmol/L MBG, or with a combination of ANP and MBG. Collagen-1, Fli1 and PKG-1 levels were assessed by Western blotting analyses. We have demonstrated, that vascular PKG1 and Fli1 levels in the old rats were reduced versus young counterparts. ANP prevented inhibition of vascular NKA by MBG in young VSMC, but not in old VSMC. In VSMC from the young rats, MBG induced down-regulation of Fli1 and an increase in collagen-1 level, whereas ANP blocked this effect. Silencing of PKG1 gene in young VSMC resulted in a reduction in the levels of PKG1 and Fli1. MBG additionally reduced Fli1 and increased collagen-1 level, and ANP failed to oppose these MBG effects, similar to VSMC from the old rats with the age-associated reduction in PKG1. Based on the results of our study we concluded, that age-associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose MBG-induced inhibition of NKA and fibrosis development. Silencing of PKG1 mimicked these effects of aging. In summary, in VSMC from young and old rats, preincubation with MBG was associated with an increased level of collagen-1, and this effect of MBG became stronger in advanced age, i.e., the older VSMC exhibited higher collagen level after MBG administration than did young VSMC. The observed age-associated effect of MBG was ANP/PKG-dependent, because silencing of PKG1 in young VSMC was associated with augmentation of the MBG effect of collagen-1 level. ANP failed to protect VSMC against the profibrotic effect of MBG following silencing of PKG1. Thus, the ANP/PKG-dependent mechanism of augmentation of the effects of MBG very likely contributed to the development of salt-sensitivity of blood pressure, accompanied by the development of MBG-induced cardiovascular fibrosis. Accordingly, following acute NaCl loading, 24-month-old rats exhibited a greater pressor response and a lesser natriuretic response to NaCl loading as compared to 3-month-old rats. Our present data are in agreement with a recent finding indicating that a group of single nucleotide polymorphisms located in the PKG1 gene is associated with the salt sensitivity of blood pressure in hypertensive human subjects. Our prior studies indicate that MBG, acting via NKA-dependent signaling, stimulates the synthesis of collagen in cardiac, renal, and aortic tissue, and that immunoneutralization of MBG reverses cardiac fibrosis in rats with renal failure. Because fibrosis is a hallmark of cardiovascular aging, the PKG agonists and natriuretic peptides exhibit anti-fibrotic effects, the impact of the age-associated reduction in cGMP/PKG signaling on the pro-fibrotic effects of MBG merits further investigation.