Proximal tubular-targeted overexpression of the Cyp4a12-20-HETE synthase promotes salt-sensitive hypertension in male mice.

20-Hydroxyeicosatetraenoic acid (20-HETE) has been linked to blood pressure (BP) regulation via actions on the renal microvasculature and tubules. We assessed tubular 20-HETE contribution to hypertension by generating transgenic mice overexpressing the CYP4A12-20-HETE synthase (PT-4a12 mice) under the control of the proximal tubule (PT)-specific promoter, phosphoenolpyruvate carboxykinase (PEPCK). 20-HETE levels in the kidney cortex of male (967±210 vs. 249±69 pg/mg protein), but not female (121±15 vs. 92±11 pg/mg protein) PT-4a12 mice, showed a 2.5-fold increase compared to WT. Renal cortical Cyp4a12 mRNA and CYP4A12 protein in male, but not female PT-4a12 mice increased by 2-3-fold compared to WT. Male PT-4a12 mice displayed elevated BP (142±1 vs. 111±4 mmHg, p<0.0001), whereas BP in females PT-4a12 mice was not significantly different from WT (118±2 vs. 117±2 mmHg; p=0.98). In male PT-4a12 mice, BP decreased when transitioned from a control salt (0.4%) to a low-salt diet (0.075%) from 135±4 to 120±6 mmHg (p<0.01) and increased to 153±5 mmHg (p<0.05) when placed on a high-salt diet (4%). Female PT-4a12 mice did not show changes in BP on either low- or high-salt diet. In conclusion, the expression of Cyp4a12 driven by the PEPCK promoter is sex-specific probably due to its X-linkage. The salt-sensitive hypertension seen in PT-4a12 male mice suggests a potential anti-natriuretic activity of 20-HETE that needs to be further explored.

20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension.

RATIONALE: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction, and vascular diseases. OBJECTIVE: To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. METHODS AND RESULTS: Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified G-protein receptor 75 (GPR75), currently an orphan G-protein-coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Gαq/11 protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src-mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE-mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gαq/11- and GPCR-kinase interacting protein-1-mediated protein kinase C-stimulated phosphorylation of MaxiKß, linking GPR75 activation to 20-HETE-mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension prevented blood pressure elevation and 20-HETE-mediated increases in angiotensin-converting enzyme expression, endothelial dysfunction, smooth muscle contractility, and vascular remodeling. CONCLUSIONS: This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.

High-fat diet-induced obesity and insulin resistance in CYP4a14-/- mice is mediated by 20-HETE.

20-Hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia, and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin resistance. Cyp4a14-/- male mice, a model of 20-HETE overproduction, were fed a regular or high-fat diet (HFD) for 15 wk. 20-SOLA [2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyeicosa-6( Z),15( Z)-dienoate], a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed mice gained significant weight (16.7 ± 3.2 vs. 3.8 ± 0.35 g, P < 0.05) and developed hyperglycemia (157 ± 3 vs. 121 ± 7 mg/dl, P < 0.05) and hyperinsulinemia (2.3 ± 0.4 vs. 0.5 ± 0.1 ng/ml, P < 0.05) compared with regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4 ± 1 vs. 16.7 ± 3 g, P < 0.05) and normalized the hyperglycemia (157 ± 7 vs. 102 ± 5 mg/dl, P < 0.05) and hyperinsulinemia (1.1 ± 0.1 vs. 2.3 ± 0.4 ng/ml, P < 0.05). The impaired glucose homeostasis and insulin resistance in HFD-fed mice evidenced by reduced insulin and glucose tolerance were also ameliorated by 20-SOLA. Circulatory and adipose tissue 20-HETE levels significantly increased in HFD-fed mice correlating with impaired insulin signaling, including reduction in insulin receptor tyrosine (Y972) phosphorylation and increased serine (S307) phosphorylation of the insulin receptor substrate-1 (IRS-1). 20-SOLA treatments prevented changes in insulin signaling. These findings indicate that 20-HETE contributes to HFD-induced obesity, insulin resistance, and impaired insulin signaling.

The Blood Pressure-Lowering Effect of 20-HETE Blockade in Cyp4a14(-/-) Mice Is Associated with Natriuresis.

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) has been linked to pro-hypertensive and anti-hypertensive actions through its ability to promote vasoconstriction and inhibit Na transport in the ascending limb of the loop of Henle, respectively. In this study, we assessed the effects of 20-HETE blockade on blood pressure, renal hemodynamics, and urinary sodium excretion in Cyp4a14(-/-) male mice, which display androgen-driven 20-HETE-dependent hypertension. Administration of 2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyicosa-6(Z),15(Z)-dienoate (20-SOLA), a water-soluble 20-HETE antagonist, in the drinking water normalized the blood pressure of male Cyp4a14(-/-) hypertensive mice (±124 vs. ±153 mmHg) while having no effect on age-matched normotensive wild-type (WT) male mice. Hypertension in Cyp4a14(-/-) male mice was accompanied by decreased renal perfusion and reduced glomerular filtration rates, which were corrected by treatment with 20-SOLA. Interestingly, Cyp4a14(-/-) male mice treated with 20-SOLA displayed increased urinary sodium excretion that was paralleled by the reduction of blood pressure suggestive of an antinatriuretic activity of endogenous 20-HETE in the hypertensive mice. This interpretation is in line with the observation that the natriuretic response to acute isotonic saline loading in hypertensive Cyp4a14(-/-) male mice was significantly impaired relative to that in WT mice; this impairment was corrected by 20-SOLA treatment. Hence, endogenous 20-HETE appears to promote sodium conservation in hypertensive Cyp4a14(-/-) male mice, presumably, as a result of associated changes in renal hemodynamics and/or direct stimulatory action on tubular sodium reabsorption.

Dexamethasone promotes hypertension by allele-specific regulation of the human angiotensinogen gene.

The human angiotensinogen (hAGT) gene has polymorphisms in its 2.5-kb promoter that form two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, and -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A is associated with human hypertension, whereas Hap -6G/-217G reduces cardiovascular risk. Hap -6A/-217A has increased promoter activity with enhanced transcription factor binding, including to the glucocorticoid receptor (GR). Glucocorticoid therapy frequently causes hypertension, the mechanisms for which are incompletely understood. We have engineered double transgenic (TG) mice containing the human renin gene with either Hap of the hAGT gene and examined the physiological significance of glucocorticoid-mediated allele-specific regulation of the hAGT gene. We have also studied the consequential effects on the renin angiotensin system and blood pressure. TG mice with Hap -6A and -6G were treated with and without a low dose of a GR agonist, dexamethasone (2.5 µg/ml), for 72 h. We found greater chromatin-GR binding with increased GR agonist-induced hAGT expression in liver and renal tissues of Hap -6A mice. Additionally, dexamethasone treatment increased circulating hAGT and angiotensin II levels in Hap -6A mice, as compared with -6G mice. Importantly, GR agonist significantly increased blood pressure and redox markers in TG mice with Hap-6A of the hAGT gene. Taken together, our results show, for the first time, that glucocorticoids affect hAGT expression in a haplotype-dependent fashion with SNPs in Hap -6A favoring agonist-induced GR binding. This leads to increased expression of the hAGT, up-regulation of the renin angiotensin system, and increased blood pressure and oxidative stress in Hap -6A mice.

A haplotype of angiotensin receptor type 1 associated with human hypertension increases blood pressure in transgenic mice.

The renin-angiotensin system plays an important role in the regulation of blood pressure via angiotensin II and the angiotensin II receptor type 1 (AT1R). Human AT1R gene promoter has four SNPs: T/A at -777, T/G at -680, A/C at -214, and A/G at -119, that are in linkage disequilibrium. Variants -777T, -680T, -214A, and -119A almost always occur together (named haplotype I), and variants -777A, -680G, -214C, and -119G almost always occur together (named haplotype II) in Caucasian subjects. Genomic DNA analyses, from 388 normotensive and 374 hypertensive subjects, link haplotype I of the human AT1R (hAT1R) gene with hypertension in Caucasians (p = 0.004, χ(2) = 8.46). Our results show increased basal promoter activity of the hAT1R gene in cells (H295R and A7r5) transfected with reporter construct containing haplotype I. We also show increased binding of the transcription factor, USF2, to oligonucleotide containing nucleoside -214A as opposed to -214C. Recombineering of a 166-kb bacterial artificial chromosome containing 68 kb of the 5'-flanking region, 45 kb of the coding sequence, and 53 kb of the 3'-flanking region of the hAT1R gene was employed to generate transgenic mice with either haplotype. We show that (a) hAT1R mRNA level is increased in the kidney and heart of transgenic mice containing haplotype I as compared with haplotype II; (b) USF2 binds more strongly to the chromatin obtained from the kidney of transgenic mice containing haplotype I as compared with haplotype II; and (c) blood pressure and oxidative stress are increased in transgenic mice containing haplotype I as compared with haplotype II.

Transgenic mice with -6A haplotype of the human angiotensinogen gene have increased blood pressure compared with -6G haplotype.

Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at -6, and the -6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with -6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with -6G. We have found that the hAGT gene has three additional SNPs (A/G at -1670, C/G at -1562, and T/G at -1561). Variants -1670A, -1562C, and -1561T almost always occur with -6A, and variants -1670G, -1562G, and -1561G almost always occur with -6G. Therefore, the hAGT gene may be subdivided into either -6A or -6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with -6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with -6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the -6A or -6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing -6A haplotype. Our studies also show that transgenic mice containing -6A haplotype have increased plasma AGT level and increased blood pressure as compared with -6G haplotype. Our studies explain the molecular mechanism involved in association of the -6A allele of the hAGT gene with hypertension.

Regulation of angiotensin II type 2 receptor gene expression in the adrenal medulla by acute and repeated immobilization stress.

While the renin-angiotensin system is important for adrenomedullary responses to stress, the involvement of specific angiotensin II (Ang II) receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT(1A)) and type 2 (AT(2)) receptors in rat adrenal medulla in response to immobilization stress (IMO). AT(2) receptor mRNA levels decreased immediately after a single 2-h IMO. Repeated IMO also decreased AT(2) receptor mRNA levels, but the decline was more transient. AT(1A) receptor mRNA levels were unaltered with either single or repeated IMO, although binding was increased following repeated IMO. These effects of stress on Ang II receptor expression may alter catecholamine biosynthesis, as tyrosine hydroxylase and dopamine ß-hydroxylase mRNA levels in PC12 cells are decreased with Ang II treatment in the presence of ZD7155 (AT(1) receptor antagonist) or with CGP42112 (AT(2) receptor agonist) treatment. Involvement of stress-triggered activation of the hypothalamic-pituitary-adrenocortical or sympathoadrenal axis in AT(2) receptor downregulation was examined. Cultured cells treated with the synthetic glucocorticoid dexamethasone displayed a transcriptionally mediated decrease in AT(2) receptor mRNA levels. However, glucocorticoids are not required for the immediate stress-triggered decrease in AT(2) receptor gene expression, as demonstrated in corticotropin-releasing hormone knockout (Crh KO) mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase-activating polypeptide also reduced AT(2) receptor gene expression and may mediate this response. Overall, the effects of stress on adrenomedullary AT(1A) and AT(2) receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis.