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

实验1.我们发现，与未经处理的细胞或非靶向siRNA转染的细胞相比，在人滋养层细胞和大鼠肾上腺皮质细胞中，转录后沉默的细胞色素P27A1基因的表达减少了70%，总胆汁酸减少了2倍，MBG水平降低了67%。在体内，在高盐摄入实验中，雄性和雌性Dahl-S大鼠在高盐饮食4周后出现高血压，其血浆MBG水平增加一倍，肾上腺皮质细胞色素P27A1基因和蛋白表达增加，雄性增加1.6倍，雌性增加2.0倍。 我们的结果首次证明，哺乳动物类固醇MBG是一种内源性的二烯内酯类钠泵配体，来源于胆汁酸，其生物合成是由CYP27A1酶启动的。这一观察是在大鼠肾上腺皮质和人类胎盘细胞中进行的，这两种细胞被选为胆汁酸途径研究的模型，因为这些组织已知会产生MBG。 结论1.哺乳动物肝外组织胎盘和肾上腺皮质通过酸性胆汁酸途径合成内源性类固醇Na/K-ATPase抑制剂MBG。这些发现将有助于理解MBG在高度流行的人类心血管疾病中的作用。 实验2：有趣的是，CYP27A1 KO小鼠在肾上腺皮质CYP27A1 mRNA(减少了95%；qPCR)和CYP27A1蛋白(通过Western blotting)减少的情况下，MBG的产生没有减少。值得注意的是，在这些小鼠中，肾上腺皮质组织中CYP27A1酶的下调伴随着肝酶的2倍上调，该酶参与了经典的胆汁酸途径AKR1D1(醛酮还原酶家族1，成员D1)。我们用富含全反式维甲酸(atRA；维生素A代谢物)的饮食治疗CYP27A1 KO小鼠，众所周知，全反式维甲酸可以抑制AKR1D1(Yanab等人)。Biochem Pharmacol 2014)(10天)。 我们发现，全反式维甲酸处理后，野生型和CYP27A1 KO小鼠的AKR1D1mRNA和蛋白表达均下降，但全反式维甲酸处理后，只有CYP27A1 KO小鼠的MBG产量下降了75%，而野生型的MBG产量没有下降，野生型的细胞色素P27A1酶正常表达。 结论2：细胞色素P27A1是参与MBG生物合成的主要酶。在小鼠模型中，当CYP27A1被敲除时，参与经典胆汁酸途径的另一种酶AKR1D1上调，并弥补了CYP27A1的缺失。在哺乳动物中，MBG很可能是由酸性和经典胆汁酸途径的产物和中间体合成的。肝脏经典胆汁酸途径的中间产物可到达肝外组织，即肾上腺，并可被酸性胆汁酸途径的酶进一步转化为MBG和其他生物活性类固醇。 我们目前的发现，促高血压和促纤维化类固醇MBG的生物合成是由CYP27A1和AKR1D1酶控制的，这为未来的研究提供了新的方向，可能会出现新的治疗策略来阻断MBG的产生，以减少其对涉及MBG水平升高的高度流行的人类疾病的影响。

Experiment 1. Here we show that post-transcriptional silencing of the CYP27A1 gene in human trophoblast and rat adrenocortical cells reduced the expression of CYP27A1 mRNA by 70%, reduced total bile acids 2-fold, and MBG levels by 67%, compared to non-treated cells or cells transfected with non-targeting siRNA. In vivo, in a high salt administration experiment, male and female Dahl-S rats became hypertensive after 4 weeks on a high NaCl diet, their plasma MBG levels doubled, and adrenocortical CYP27A1 mRNA and protein increased 1.6-fold in males and 2.0-fold in females. Our results demonstrate, for the first time that the mammalian steroid MBG, an endogenous sodium pump ligand of bufadienolide nature, is derived from bile acids, and its biosynthesis is initiated by CYP27A1 enzyme. This observation was made in rat adrenocortical and human placental cells, which were chosen as models for bile acid pathway studies, because these tissues are known to produce MBG. Conclusion 1: Therefore, the endogenous steroidal Na/K-ATPase inhibitor, MBG, is synthesized in mammalian extra-hepatic tissues placenta and adrenal cortex from cholesterol through the novel acidic bile acid pathway. These findings will help to understand the role of MBG in highly prevalent human cardiovascular diseases. Experiment 2: Interestingly, that CYP27A1 KO mice showed no decrease in MBG production in the presence of decreased adrenocortical CYP27A1 mRNA (by 95%; qPCR) and CYP27A1 protein (by Western blotting). Notably, that in these mice, downregulation of CYP27A1 enzyme in adrenocortical tissue was accompanied by a 2-fold upregulation of liver enzyme, which participates in classical bile acid pathway, AKR1D1 (aldo-keto reductase family 1, member D1). We treated CYP27A1 KO mice with a diet enriched in all-trans retinoic acid (atRA; Vitamin A metabolite), which is known to suppress AKR1D1 (Yangab et al. Biochem Pharmacol 2014) (10 days). We have demonstrated that AtRA treatment resulted in decrease in AKR1D1 mRNA and protein in both wild type and CYP27A1 KO mice; however, MBG production decreased by 75% after atRA diet in CYP27A1 KO mice only, and not in wild type, in which CYP27A1 enzyme was normally expressed. Conclusion 2: We conclude that CYP27A1 is a predominant enzyme that participates in MBG biosynthesis. When CYP27A1 is knock-out in mouse model, another enzyme, which participates in classical bile acid pathway, AKR1D1, is upregulated and compensates the lack of CYP27A1. It is very likely that MBG is synthetized in the mammals from the products and intermediates of acidic and classical bile acids pathways. The intermediates from the hepatic classical bile acid pathway can reach the extra-hepatic tissues, i.e. adrenals, and can be further transformed by enzymes from acidic bile acids pathways into MBG and other bioactive steroids. Our present finding, that biosynthesis of pro-hypertensive and pro-fibrotic steroid MBG is controlled by CYP27A1 and AKR1D1 enzymes, gives a new direction for future studies that will likely enable the emergence of novel therapeutic strategies to block MBG production in order to reduce its impact on highly prevalent human diseases involving heightened levels of MBG.