Investigating a novel pathway for hypertension and pharmacologic response involving the dicarboxylic acid, hexadecanedioate

Human hypertension is the biggest contributor to the global burden of cardiovascular disease and it is the most prevalent modifiable risk factor for cardiovascular morbidity and mortality. The causation of hypertension is multifactorial and is related to perturbation in the pathways that regulate blood pressure. The known blood pressure regulating pathways include those that regulate intravascular volume, vascular tone and peripheral vascular resistance through renal, the renin-angiotensin-aldosterone and autonomic systems, which are targets of the commonly used antihypertensive drugs. In the largest investigation of blood pressure using a metabolomics approach, a novel pathway for blood pressure regulation was identified, involving a dicarboxylic acid, hexadecanedioate. Hexadecanedioate is a long chain dicarboxylic acid, which is generated during fatty acid omega-oxidation pathway (ω-oxidation). Higher circulating levels of hexadecanedioate were significantly associated with both high blood pressure and mortality. This research project aimed to examine the functional role of hexadecanedioate and investigate its underpinning mechanisms in blood pressure regulation using in-vivo, ex-vivo and metabolomics studies in two animal models; the Wistar Kyoto (WKY) and the Spontaneously Hypertensive Stroke Prone rats (SHRSP). In the first results chapter (chapter 3), we conducted three independent interventional studies to examine the effects of hexadecanedioate on blood pressure regulation. Circulating levels of hexadecanedioate were significantly increased in WKY rats after treatment with oral hexadecanedioic acid leading to blood pressure elevation and impairment of vascular function in WKY rats. However, hexadecanedioic acid treatment was unable to further increase circulating hexadecanedioate levels or blood pressure in SHRSP rats, indicating that hexadecanedioate levels may already be maximally elevated in the hypertensive model. The data also show an increase in adiposity index despite reduced body weight in SHRSP rats after treatment with hexadecanedioic acid. These results may indicate muscle wasting and altered cellular energy homeostasis such as an impairment of the beta-oxidation pathway (β-oxidation). In the second results chapter (chapter 4), a global metabolomics study was carried out in WKY rats to demonstrate the metabolic effect of hexadecanedioic acid treatment in cardiovascular tissues (i.e. heart, kidney, liver, aorta, adipose and brain) in order to identify the metabolic pathways leading to hexadecanedioate-induced blood pressure elevation. Several alterations in metabolic readouts, including changes in metabolites related to lipid and glucose metabolisms, bile acid metabolism, redox homeostasis, and uric acid cycle were observed after hexadecanedioic acid treatment. Hexadecanedioate increased in all tested tissues of hexadecanedioic acid-treated WKY rats except brain, where this metabolite was below the threshold of detection. Our data indicated significant increases in peroxisomal ω-oxidation metabolites (i.e. dicarboxylic fatty acids) along with marked changes in mitochondrial fatty acid β-oxidation metabolites such as phospholipids, lysolipid, sphingolipid, monoacylglycerol and acyl carnitine in all tested tissues. These changes in fatty acid availability indicate that hexadecanedioic acid treatment induced changes in mitochondrial β-oxidation and a shift toward increased use of peroxisomal ω-oxidation. In the third results chapter (chapter 5), the ω- oxidation pathway was investigated by measuring the expression levels of the different enzymes involved in the pathway (i.e. CYP4A, alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH)) in two different rat strains; the normotensive WKY and hypertensive SHRSP rat models. The gene expression results show significant reduction in expression of the ALDH isoform, ALDH1L2, in the liver tissues of SHRSP rats compared to WKY. In addition, the levels of ALDH enzyme were measured in the liver tissues using ELISA assay, which showed a trend towards an increase in SHRSP rats compared to WKY rats. Blood pressure was assessed after modulation of endogenous hexadecanedioate levels by perturbing the ω-oxidation pathway in both WKY and SHRSP by either inhibition or stimulation of enzymes critical to ω-oxidation. Inhibition of the final ω-oxidation pathway enzyme, ALDH by disulfiram resulted in a significant lowering of blood pressure in the hypertensive SHRSP rat. A pilot study was also conducted to examine the effect of HET0016 (CYP4A antagonist) on blood pressure of SHRSP rats, which showed no significant changes. However, stimulation of the ω-oxidation pathway enzyme, CYP4A by fenofibrate caused a significant reduction of blood pressure in WKY rats, along with significant increase in the CYP4A expression in kidney and liver tissues. In the final results chapter (chapter 6), an alternative mechanism was examined that may lead to elevated circulating levels of hexadecanedioate via altered activity of the solute carrier organic anion transporter (SLCO), which encodes the protein organic anion transporting polypeptide (OATP). It has been found that the human SLCO1B1 gene is associated with elevated levels of circulating hexadecanedioate. SLCO1B1 may have a role in hexadecanedioate elimination from the body, and any alteration in this liver transporter may cause increases in circulating hexadecanedioate levels leading to blood pressure elevation. A preliminary investigation was performed to identify the sequence variants in the rat homolog, the Slco1b2 gene. Genotype analysis of the SHRSPGla and WKYGla genome sequence identified an insertion of 9 nucleotides (GTCTATCTA) within an intronic region (intron 3/14) on chromosome 4 at position 240,062,637 bp, band 4q44 forward strand in the WKYGla strain rather than a G nucleotide in the SHRSPGla strain. The deletion of 4 nucleotides (TATC) within the 3’untranslated region (UTR) was also detected on chromosome 4 at position 240,102,755 bp of the WKYGla genome sequence. Total Slco1b2 mRNA expression was measured in liver tissues of WKY and SHRSP, which showed significantly decreased levels in SHRSP compared to WKY. These genetic variants within the Slco1b2 gene may contribute to altered function of the Oatp1b2 transporter, which may in turn contribute to elevated levels of circulating hexadecanedioate in the SHRSP rat model of hypertension. In summary, we have investigated the functional role of hexadecandioate in blood pressure regulation. The findings presented in this thesis demonstrate novel targets associated with hexadecandioate; including the ω-oxidation pathway and the solute carrier organic anion mechanism in regulation of blood pressure.