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

National Institute On Aging

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Abstract

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.