Ovariectomy Via 12/15-lipoxygenase Augments Angiotensin II-Induced Hypertension and Its Pathogenesis in Female Mice

Background:

Ang II (angiotensin II) releases arachidonic acid from tissue phospholipids that is metabolized by 12/15-lipoxygenase (ALOX15), generating 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE), which have been implicated in cardiovascular and renal diseases. In this study, we tested the hypothesis that ovariectomy augments Ang II-induced hypertension and renal pathophysiological changes via ALOX15 activation in female mice.

Methods:

Ang II (700 ng/kg/min) was infused subcutaneously by osmotic pumps for 2 weeks in intact and ovariectomized wild-type and Alox15 knockout (ALOX15KO) female mice for evaluation of hypertension and associated pathogenesis.

Results:

Ang II increased blood pressure, impaired autonomic function, and increased renal reactive oxygen species production and plasma 12(S)-HETE level without altering renal function in intact wild-type mice. However, in OVX-wild-type mice with depleted plasma 17β-estradiol, the effects of Ang II on blood pressure, autonomic impairment, renal reactive oxygen species production, and plasma 12(S)- but not 15(S)-HETE was markedly enhanced. In OVX-wild-type mice, Ang II also increased renal alox15 mRNA, urine 12(S)-HETE, water intake, urine output, decreased osmolality, increased urinary excretion of vasopressin prosegment copeptin, protein/creatinine ratio, and caused renal hypertrophy, fibrosis, and inflammation. These effects of Ang II were attenuated in ALOX15KO mice.

Conclusions:

These data suggest that 17β-estradiol protects against Ang II-induced hypertension and associated pathogenesis in female mice, most likely via inhibition of ALOX15-arachidonic acid derived production of 12(S)-HETE. Therefore, the selective inhibitors of ALOX15 or 12(S)-HETE receptor antagonists could be useful for treating hypertension and its pathogenesis in postmenopausal, hypoestrogenic women, or females with ovarian failure.

Novelty and Relevance.

What Is New?

• Evidence that ovariectomy or depletion of 17β-estradiol (E2) contributes to the deleterious effects of Ang II (angiotensin II) including hypertension, impaired autonomic function, proteinuria, renal fibrosis, and renal IL-6 that are fully or in part suppressed in the female mice with endogenous E2 present.

• 12/15-lipoxygenase (ALOX15) is essential for the development of Ang II-induced hypertension and impaired autonomic function and its marked augmentation in female mice depleted of E2.

• ALOX15/12-hydroxyeicosatetraenoic acid is also required for Ang II-induced renal dysfunction, excretion of vasopressin, reactive oxygen species production, renal hypertrophy, and damage in ovariectomized mice lacking E2.

What Is Relevant?

Our study provides novel information on the mechanism of Ang II-induced hypertension and associated pathophysiological changes in females, whereby arachidonic acid metabolism by ALOX15 to eicosanoid 12(S)-hydroxyeicosatetraenoic acid (HETE) with prohypertensive effects is crucial for exaggerated effects of Ang II-induced hypertension in conditions lacking E2.

Clinical/Pathophysiological Implication

Our findings suggest that the development of agents that selectively inhibit ALOX15 activity or 12(S)-HETE receptor-mediated actions could be useful in treating hypertension and its pathogenesis in females specifically postmenopausal, hypoestrogenic women or females with ovarian failure. Moreover, the effect of alox15 gene polymorphism in pre- and postmenopausal females to determine its impact on the contribution of AA-ALOX15 derived 12(S)-HETE in hypertension and its pathogenesis is also warranted.

Hypertension is the most important risk factor for ischemic heart disease, stroke, and renal disease in postmenopausal women.^1,2 Increased cardiovascular and renal incidents in women with premature ovarian failure³ and surgically induced menopause by bilateral oophorectomy^4,5 suggest a protective role of estrogens against cardiovascular and renal disease. Supporting this view, it has been shown that ovariectomy worsened hypertension in SHR⁶ and accelerated in aging Dahl salt–sensitive female rats,⁷ whereas treatment with the major estrogen, 17β-estradiol (E2) in both models minimized hypertension.^6,7 Ovariectomy also enhanced the effect of Ang (angiotensin) II, the main product of the renin-angiotensin system, to increase blood pressure (BP), which was minimized by E2 in mice.^8,9 However, the underlying mechanism of augmentation of Ang II (angiotensin II)-induced hypertension in females with the loss of endogenous estrogens remains undefined, hampering the development of therapeutic agents for treating hypertension in women. Therefore, clinicians need distinct approaches to treat hypertension-related morbidity/mortality in pre- and postmenopausal (natural menopause, hysterectomy, or oophorectomy) females.

Ang II, via activation of group IV cytosolic phospholipase A₂α, releases arachidonic acid (AA), which is metabolized by various enzyme systems, including 12/15 lipoxygenase (ALOX 15), into pro- and antihypertensive eicosanoids.^10–15 ALOX15 is expressed in several tissues, including peritoneal and alveolar macrophages, dendritic cells, eosinophils, airway epithelial, renal, and vascular cells, and it has been implicated in atherosclerosis, diabetes, cancer, and hypertension along with its 2 AA-derived metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE) and 15(S)-HETE.^16–20 12(S)-HETE contributes to the effect of Ang II to increase BP and constrict femoral arterial rings of rat,²¹ the human placenta,²² and the rat afferent arterioles,²³ whereas 15(S)-HETE has been reported to mediate hypoxia-induced pulmonary vasoconstriction and hypertension.^18,19 Ang II-induced steroidogenesis is also dependent on the ALOX15 pathway, as indicated by reports that ALOX15 inhibition blocks the aldosterone response to Ang II, and that this effect can be overcome by 12(S)-HETE.^15,24 The basal levels of 12(S)-HETE is higher in the urine of patients with essential hypertension and the plasma of SHR.^25,26 Inhibition of ALOX15 reduced BP in renovascular hypertensive and WKY and SHR male rats.^27,28 Moreover, Alox15 gene disruption minimized Ang II-induced hypertension in male mice.^29,30 However, there is a significant gap in our knowledge of the contribution of ALOX15 and AA-ALOX15-derived eicosanoids 12(S)- and 15(S)-HETE in hypertension and its pathogenesis in females. The present study was conducted to test the hypothesis that ovariectomy augments angiotensin II-induced hypertension and associated renal pathophysiological changes through ALOX15 activation and generation of AA-derived metabolites 12(S)- and 15(S)-HETE in female mice.

Methods

Data Availability

The authors declare that a detailed Methods Section and all supporting data are available within the article and in the Supplemental Material. Other details of analytical methods, study materials, and data will be made available from the corresponding authors upon reasonable request.

Animal Experiments

All experiments were performed in female mice according to protocols approved by the University of Tennessee Health Science center Institutional Animal Care and Use Committee according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Wild-type (WT, Alox15^+/+) and Alox15 gene knockout (ALOX15KO, Alox15^−/−) female mice on the C57BL/6J background were used in this study. WT and ALOX15KO mice were bred and genotyped by PCR. For PCR analysis, genomic DNA was obtained from tail snips using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI) according to the manufacturer’s instructions. All mice were housed under temperature (22°C±2°C)-controlled conditions and a 12-hour(light/dark cycle with free access to drinking water and food. To test our hypothesis, we randomly divided the mice into the following groups: (1) Intact WT; (2) OVX-WT; (3) intact ALOX15KO; and (4) OVX-ALOX15KO with Ang II (Bachem, Torrance, CA) or its (vehicle saline) infusion for 2 weeks. The groups of animals are shown in Flowchart 1 and the experimental protocol with a timeline is depicted in Flowchart 2 in the Supplemental Material. Two separate cohorts were used for the experiments, one for measurement of BP by radiotelemetry and another for metabolic cage study for renal function analysis and collection of blood and tissue samples (kidney and uterus). A third cohort of mice was also used for the measurement of BP by tail-cuff. These mice were placed in the metabolic cage at the end of the experiment to collect metabolic data and urine samples. Ovariectomy was performed when mice were 6 to 7 weeks old and allowed to recover for 3 to 5 days. After that, 1 cohort was trained for tail-cuff for 7 to 10 days. Another cohort underwent surgery for radiotelemetry implantation and 7 to 10 days of recovery before measurement of basal BP (ie, day 0) and implantation of osmotic pump for Ang II/saline infusion. Therefore, Ang II infusion started after about 14 days of ovariectomy surgery.

Statistical Analysis

The data were expressed as the mean±SEM with P<0.05 considered statistically significant (GraphPad Prism, version 8.4.2; GraphPad Software, Inc, San Diego, CA). For the BP data that were separate for intact and OVX mice, a comparison between the groups was performed using 2-way ANOVA with repeated measures. Multiple groups with normally distributed variables were compared by 3-way ANOVA and Tukey test for multiple comparisons. Univariate linear associations were assessed by calculation of Pearson coefficient of correlation. Power analysis was done using tools from the Institute for Digital Research and Education, University of California, Los Angeles, CA (G*Power software version 3.0.10.). In most of the experiments, the primary outcomes and main comparisons exceeded a power of 80% with the number of animals used.

Results

Ovariectomy Augmented the Ang II-Induced Increase in Blood Pressure Response in WT but Not in ALOX15KO Mice

Consistent with our previous reports,³¹ Ang II enhanced the increase in systolic BP (SBP) measured by tail-cuff (Figure 1A), and mean arterial pressure (MAP; Figure 1B), SBP (Figure S1A), and diastolic BP (Figure S1B) measured by radiotelemetry in OVX compared with intact WT mice. In this study, we determined whether this enhanced pressor effect of Ang II in OVX-WT mice is mediated via ALOX15 activation leading to increased generation of AA metabolite 12(S)-HETE. The increase in SBP (Figure 1A; Figures S1A and S2A), DBP (Figure S1B), and MAP (Figure 1B; Figure S2B) caused by Ang II in WT mice and its augmentation by ovariectomy were minimized in ALOX15KO mice. There were no differences in basal BP amongst these groups of mice and saline infusion also did not alter BP in any of these groups. Also, Ang II did not alter pulse pressure (Figure S1C), locomotor activity (Figure S1D), or heart rate (Figure S1E) in intact or OVX-WT or ALOX15KO mice.

Figure 1.

Ang II (Angiotensin II) increased the systolic blood pressure (SBP, mmHg) measured by tail-cuff and mean arterial pressure (MAP, mmHg) measured by radiotelemetry in intact wild-type (WT) mice, which was exacerbated in ovariectomized (OVX) WT mice and minimized in intact and OVX Alox15 knockout (ALOX15KO) mice. SBP (A) measured by tail-cuff, and MAP measured by radiotelemetry (B) in WT and ALOX15KO mice. Ang II-induced increase in MAP in intact WT mice that was exaggerated in OVX WT mice and blunted in ALOX15KO mice toward the end of the experiment (day 12) corroborated with the decreased MAP in response to ganglionic blocker hexamethonium measured on day 14 (C), bradycardia in response to β-blocker propranolol (D) and tachycardia in response to muscarinic cholinergic antagonist atropine (E), both measured on day 13 in Ang II-infused mice. Saline was used as a vehicle for Ang II. Data are mean±SEM (n=4–6/group). A 2-way repeated measures ANOVA followed by Tukey multiple comparisons in A and B. A 3-way ANOVA followed by Tukey multiple comparisons in C through E; Δ indicates change; bpm=beats per minute. *P<0.05 vs Day 0 value (the day before implantation of the osmotic pump) within the group, #P<0.05 vs WT+Saline in the corresponding groups; $P<0.05 vs WT+Ang II.

Ovariectomy Augmented the Ang II-Induced Impairment of Autonomic Function in WT but Not in ALOX15KO Mice

Systemic Ang II infusion in mice is associated with impairment of autonomic function.^31–34 Therefore, we assessed whether ovariectomy and/or lack of E2 promotes and Alox15 gene disruption protects from this effect of Ang II. Consistent with the Ang II-induced increase in MAP at the end of the experiment (Day 12; Figure S2B) in intact WT mice, Ang II failed to increase the vascular (decreased MAP to ganglionic blocker Hexamethonium, 30 mg/kg, intraperitoneal; Figure 1C) but not cardiac (bradycardia to β-blocker Propranolol, 4 mg/kg, intraperitoneal; Figure 1D) sympathetic activity, and reduced vagal tone (tachycardia to muscarinic antagonist Atropine, 1 mg/kg, intraperitoneal; Figure 1E). Moreover, this Ang II-induced sympatho-parasympathetic imbalance was further exaggerated in OVX-WT mice (Figure 1C through 1E), an effect that was blunted in ALOX15KO mice (Figure 1C through 1E). These changes in OVX-WT or ALOX15KO mice that were measured toward the end of Ang II infusion corroborated with the increase in MAP (Figure S2B).

Uterine Weights and Plasma E2 Levels Were Reduced in OVX Mice

The uterus/body weight ratio (Figure S3) was reduced in the OVX-WT and OVX-ALOX15KO mice as compared to their intact (ovaries intact) counterparts. Ang II infusion for 2 weeks did not increase the uterus/body weight ratio.

E2 levels measured in the plasma (Figure 2A) collected after the completion of the experiment were also attenuated in OVX mice as compared to their intact counterparts, thus confirming the effectiveness of ovariectomy in these mice. Ang II infusion for 14 days did not alter the plasma E2 levels in these mice.

Figure 2.

Ang (angiotensin) II did not alter plasma 17β-estradiol (E2, pg/mL) level but increased 12(S)-hydroxyeicosatetraenoic acid (HETE, ng/mL) level in intact wild type (WT). Measurement of plasma E2 level (A), plasma 12(S)-HETE level (B), kidney alox15 mRNA expression (C), and urine 12(S)-HETE level (D) in WT and Alox15 knockout (ALOX15KO) mice. Plasma E2 level was minimized in ovariectomized (OVX)-WT and ALOX15KO mice. Ang II infusion exacerbated the increase in plasma 12(S)-HETE level (B) in OVX-WT mice, indicating increased 12/15-lipoxygenase (ALOX15) activity. Ang II increased kidney alox15 mRNA expression in intact WT, which was exacerbated in OVX-WT mice and minimized in ALOX15KO mice (C). Ang II also increased urinary excretion of 12(S)-HETE in OVX-WT mice compared to intact WT mice (D). Both plasma and urine 12(S)-HETE level remained lower or undetected in ALOX15KO mice. Saline was used as vehicle control for Ang II. Data are mean±SEM (n=6–10/group). A 3-way ANOVA followed by Tukey multiple comparisons in A–D. a.u. indicates arbitrary unit.

Alox15 mRNA and ALOX15-AA Generated Metabolite 12(S)-HETE But Not 15(S)-HETE is Increased in OVX-WT Mice in Response to Ang II

Ang II has been shown to produce its hypertensive effect via ALOX15-generated metabolite 12(S)-HETE in male mice.³⁵ ALOX15 in mice is known to metabolize AA and produce 12(S)- and 15(S)-HETE in the ratio 3:1.³⁶ In our study, the plasma level of AA-derived ALOX15 metabolite 12(S)-HETE (Figure 2B) was increased in Ang II infused intact WT mice, which was further exaggerated in plasma of OVX-WT mice. Ang II failed to increase the plasma level of 15(S)-HETE (Figure S4) in intact or OVX-WT mice. Plasma levels of both 12(S)-HETE and 15(S)-HETE were very low or undetected in Ang II or saline-infused intact and OVX-ALOX15KO mice.

We have shown previously that Ang II increased the 12(S)-HETE level in kidneys in WT (C57BL/6 background) male mice.²⁰ In this study we observed that Ang II also increased alox15 mRNA in the kidney (Figure 2C) and the urinary excretion of 12(S)-HETE in OVX-WT mice compared to intact WT mice (Figure 2D). Similar to plasma, the urinary 12(S)-HETE levels also remained low or undetected in intact and OVX-ALOX15KO mice.

Alox15 Gene Disruption Prevented Increased Water Intake and Renal Dysfunction and Increased Urinary Copeptin Levels Associated With Ang II-Induced Hypertension in OVX Mice

Water intake (Figure 3A) and urine output (Figure 3B) were not different between saline-treated intact and OVX-WT and ALOX15KO mice. Infusion of Ang II did not alter 24-hour water consumption or urine output in intact WT mice; however, in OVX-WT mice, infusion of Ang II caused a marked increase in water consumption and urine output (Figure 3A and 3B). Ang II-induced increase in water intake was markedly reduced, and there was no increase in urine output in OVX-ALOX15KO mice.

Figure 3.

Ang II (angiotensin II) increased water intake, urine output, decreased urine osmolality, and increased urine copeptin level in ovariectomized (OVX) wild-type (WT) mice, which were attenuated in intact WT, and intact or OVX-Alox15 knockout (ALOX15KO) mice. Measurement of water intake (A), urine output (B), urine osmolality (C), and urine copeptin level (D) in WT and ALOX15KO mice. Saline was used as vehicle control for Ang II. Data are mean±SEM (n=8/group). A 3-way ANOVA followed by Tukey multiple comparisons in A through D.

Infusion of Ang II markedly decreased the urine osmolality (Figure 3C) and increased urinary excretion of Copeptin (Figure 3D), a cleaved peptide from the COOH terminus of pre-pro-arginine vasopressin³⁷ in OVX-WT but not in intact WT mice. Alox15 gene disruption blunted the Ang II-induced decrease in urine osmolality and increase in copeptin excretion in urine in OVX mice (Figure 3C and 3D).

Ang II Caused Renal Injury, Hypertrophy, Fibrosis, and Reactive Oxygen Species Production in OVX but Not in Intact WT and ALOX15KO Mice

Infusion of Ang II increased the urinary protein/creatinine ratio (Figure 4A), an index of renal injury, and increased total kidney/body weight ratio (Figure 4B), a marker for renal hypertrophy in OVX-WT mice but not in intact WT and ALOX15KO, and OVX-ALOX15KO mice.

Figure 4.

Ang II (angiotensin II) increased urinary protein/creatinine ratio, total kidney/body weight (BW) ratio, and renal fibrosis as indicated by renal collagen deposition detected by Masson’s Trichrome staining in ovariectomized (OVX) wild-type (WT) mice, which were minimized in intact WT, and intact or OVX Alox15 knockout (ALOX15KO) mice. Measurement of urinary protein/creatinine ratio (A), total kidney/body weight (BW) ratio (B), and renal collagen deposition (C, representative images and D, quantitation) in WT and ALOX15KO mice. Saline was used as vehicle control for Ang II. Data are mean±SEM (n=6–8/group). A 3-way ANOVA followed by Tukey multiple comparisons in A through C.

Staining of kidney sections with Masson’s trichrome revealed marked renal fibrosis as indicated by increased collagen deposition (Figure 4C and 4D) in the interstitial spaces of kidneys from Ang II-treated OVX-WT mice, but not in intact WT, and OVX-ALOX15KO mice.

Infusion of Ang II increased renal production of superoxide, a reactive oxygen species (ROS), as indicated by increased 2-hydroxyethidium fluorescence intensity in kidney sections (Figure 5A and 5B), specifically in the glomerulus in intact WT mice. The level of Ang II-induced renal 2-hydroxyethidium fluorescence was exacerbated in the OVX-WT mice. Ang II failed to increase 2-hydroxyethidium fluorescence in the kidneys of intact or OVX-ALOX15KO mice.

Figure 5.

Ang II (Angiotensin II) increased the production of reactive oxygen species (ROS) measured by the quantitation of 2-hydroxyethidium (2-OHE) fluorescence in the kidney in intact wild-type (WT) mice, which was exacerbated in ovariectomized (OVX) WT mice and attenuated in intact and OVX Alox15 knockout (ALOX15KO) mice. Effect of Ang II in intact and OVX WT and ALOX15KO mice on renal (A, representative images, and B, quantitation) ROS production. Saline was used as vehicle control for Ang II. Data are mean±SEM (n=4/group). A 3-way ANOVA followed by Tukey multiple comparisons in B.

Ovariectomy Augmented the Ang II-Induced Renal Inflammation in WT but Not in ALOX15KO Mice

The activation of inflammatory cells has been implicated in Ang II effects including increased BP and tissue remodeling.³⁸ To determine the contribution of ALOX15 to inflammatory cell infiltration in the kidney associated with Ang II-induced end-organ damage, we examined the localization of CD68+ monocyte/macrophages and also measured the renal mRNA expression of proinflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 in WT and ALOX15KO mice. Ang II caused infiltration of CD68+ monocyte/macrophage cells (Figure 6A and 6B) in the kidney, mostly in the glomerulus of intact WT mice, which was exacerbated in OVX-WT mice; these effects of Ang II were inhibited in both intact and OVX-ALOX15KO mice (Figure 6A and 6B).

Figure 6.

Ang II (angiotensin II) increased renal infiltration of monocyte/macrophage cells as indicated by increased number of CD68+ cells in ovariectomized (OVX) wild-type (WT) mice, which were minimized in intact WT, and intact or OVX Alox15 knockout (ALOX15KO) mice. Measurement of renal infiltration of CD68+ cells (A, representative images, and B, quantitation), kidney il6 (C) and il10 (D) mRNA expression in WT and ALOX15KO mice. Saline was used as vehicle control for Ang II. Data are mean±SEM (n=6/group). A 3-way ANOVA followed by Tukey multiple comparisons in B through D.

Ang II increased renal il6 mRNA expression in OVX-WT compared to intact WT mice, which was minimized in intact and OVX-ALOX15KO mice (Figure 6C). We observed increased il10 mRNA expression in the kidney of intact WT but not in ALOX15KO mice in response to Ang II (Figure 6D). Renal il10 mRNA expression was also higher in OVX-ALOXKO compared to OVX-WT mice infused with Ang II (Figure 6D).

Discussion

The novel findings of this study are that augmentation of Ang II-induced hypertension caused by loss of endogenous estrogen by ovariectomy and associated autonomic and renal dysfunction, renal hypertrophy, fibrosis, ROS production, and inflammation are dependent on ALOX15 activation and generation of 12(S)-HETE but not 15(S)-HETE in female mice. It is well established that after menopause, the incidence of chronic renal disease in women increases suggesting that the loss of sex hormones contributes to the development and progression of kidney disease.³⁹ These observations are concordant with the studies reporting that estrogen/progestin replacement therapy improved renal function.^40–42 In our study, infusion of Ang II caused a small but significant increase in BP and cardiac (higher decrease in heart rate by propranolol) sympathetic tone, and decreased parasympathetic tone (lower heart rate increase by atropine), and increased plasma level of 12(S)-HETE but not 15(S)-HETE in intact WT mice. However, in these mice that have endogenous E2, Ang II failed to increase water intake, urine output, urine osmolality, urinary vasopressin prosegment, copeptin levels,³⁷ and to cause renal injury as assessed by urinary protein: creatinine ratio, renal hypertrophy determined by kidney/body weight ratio, and fibrosis measured by renal interstitial collagen deposition. Our finding that Ang II-induced increase in BP and associated impaired autonomic function in intact WT mice were attenuated in ALOX15KO mice suggests that AA-ALOX15-generated eicosanoid most likely12(S)-HETE contributes to these actions of Ang II. Since Ang II caused a small increase in renal ROS production in the intact WT mice as detected by 2-hydroxyethidium fluorescence, the increase in BP and associated impaired autonomic function in these mice is most likely mediated by the increased ROS production via the effect of 12(S)-HETE. Recently, it was reported that 12(S)-HETE enhanced the vasoconstrictor response of the aorta to Ang II from WT male mice, which were dependent on superoxide production as it was inhibited in aortas from p47^phox-/- male mice.³⁵ Whether p47^phox also contributes to 12(S)-HETE-dependent Ang II-induced increase in ROS production in female mice remains to be explored.

In the present study, Alox15 gene disruption did not alter the basal BP or renal function in intact female mice, suggesting that it is independent of ALOX15 and 12(S)-HETE. Moreover, the small increases in BP and associated impairment of autonomic function produced by Ang II did not cause renal dysfunction, hypertrophy, or fibrosis in intact WT mice. From these observations, it follows that in intact WT mice, E2 by minimizing Ang II-induced increase in ALOX15 activity as indicated by decreased production of 12(S)-HETE protects against the enhanced increase in BP (indicating a positive correlation between SBP and plasma 12(S)-HETE level, Figure S5A and, consequently, kidney dysfunction and damage). Supporting this conclusion were our findings that in OVX-WT mice depleted of E2, Ang II markedly augmented the increase in BP and increased both the vascular and cardiac sympathetic tone and reduced the parasympathetic tone compared to intact WT mice. Moreover, in OVX-WT mice, Ang II increased water intake and urine output, decreased urine osmolality, increased urinary copeptin level, and caused renal injury (increased urinary protein: creatinine ratio), hypertrophy (increased kidney/body weight ratio), fibrosis (renal interstitial collagen deposition), and ROS production (indicating a positive correlation between SBP and renal fibrosis; Figure S5B). Furthermore, Ang II also markedly increased the renal alox15 mRNA expression, plasma 12(S)-HETE without altering 15(S)-HETE levels, and increased 12(S)-HETE in urine in OVX-WT mice compared to intact WT mice, suggesting increased activation of ALOX15 and generation of 12(S)-HETE in the absence of E2 plays a critical role in augmenting the effects of Ang II to increase BP, impair autonomic and renal function, produce renal hypertrophy, fibrosis, and ROS. However, further studies are required to determine if E2 directly or via its cytochrome P450 1B1 (CYP1B1)-generated metabolite 2-methoxyestradiol (2-ME) inhibits ALOX15 activity and/or by reducing cytosolic phospholipase A2 activity⁴³ decreases the release of arachidonic acid for 12(S)-HETE production by ALOX15 in various cell types including infiltrating macrophages in the kidney. 12(S)-HETE is produced in various kidney tissues including glomeruli, cortical and distal convoluted tubules, macrophages, and platelets.^16,29,35,44 Previously, we have measured 12(S)-HETE levels in the kidney of male mice and found that Ang II infusion increased its tissue content almost 3-fold.²⁰ However, to determine the relative contribution of ALOX15-AA derived 12(S)-HETE in the plasma and that generated locally by various cell types of the kidney, macrophages, and other immune cells to renal function in Ang II-induced hypertension and its pathogenesis in intact and OVX mice would require studies in cell-specific ALOX15KO and reconstitution of Alox15 gene in ALOX15KO mice.

Affirming that ALOX15/12(S)-HETE mediates Ang II-induced hypertension, renal dysfunction, and associated pathophysiological changes in OVX-WT mice that were partially or completely suppressed in intact WT mice was our demonstration that the effects of Ang II described above were attenuated in OVX-ALOX15KO mice. A corollary of this conclusion is that E2, by inhibiting the effect of Ang II on the ALOX15/12(S)-HETE system maintains the water and possibly electrolyte homeostasis, osmolality by reducing vasopressin release and protects against high BP and renal damage. Since in the intact female mice, Ang II produced only a small increase in BP without causing renal dysfunction, renal injury, or damage, it appears in OVX mice, the renal dysfunction and injury caused by Ang II could also result from enhanced increased BP. In the present study, the basal plasma levels of 15(S)-HETE was lower compared to 12(S)-HETE because mice ALOX15 produces 12(S)- and 15(S)-HETE in the ratio 3:1.³⁶ Since the plasma levels of 15(S)-HETE was not altered by Ang II in the intact and OVX mice, it is unlikely to contribute to Ang II-induced hypertension and its pathogenesis. This is consistent with our previous report that 15(S)-HETE does not contribute to Ang II-induced hypertension in male mice.²⁰

Immune cells are known to contribute to the development of hypertension.^45,46 In the present study, Ang II also caused greater infiltration of macrophages in the kidney, mainly in the glomerulus in OVX-WT than in the intact WT mice, which was markedly reduced in ALOX15KO mice, suggesting that females with E2 present or with reduced ALOX15 activity are protected against Ang II-induced renal inflammation. The infiltration of CD68-positive cells in response to Ang II in the kidney of intact WT mice could be due the presence of M2 type of macrophages because CD68 is a marker for monocyte/macrophage that does not differentiate M1 (proinflammatory/tissue damage) and M2 (anti-inflammatory/tissue repair) subtypes of macrophages. Further studies are required to address this issue. The protection against Ang II-induced renal inflammation in intact WT mice was most likely due to increased anti-inflammatory cytokine IL-10 production, as Ang II increased renal il10 but not il6 mRNA in the intact WT mice. Ang II infusion increased the renal il6 mRNA in OVX-WT, which was minimized in OVX-ALOX15KO mice, suggesting increased proinflammatory cytokine IL-6 contributes to the proinflammatory effect of Ang II in OVX-WT mice. The lower increase in BP in females compared to males by Ang II has been attributed to a higher number of T-regulatory cells in females.⁴⁷ However, whether E2, by inhibiting the activity of ALOX15/12(S)-HETE system and increasing T-regulatory cells and inhibiting cytotoxic T-cells, contributes to Ang II-induced hypertension and its pathogenesis remains to be explored.

The mechanism by which ALOX15-generated 12(S)-HETE contributes to Ang II-induced hypertension in male rats or mice has been attributed to the effects of 12(S)-HETE to stimulate vascular smooth muscle cell hypertrophy and extracellular matrix proteins production,^16,48 vasoconstriction,^21–23 and enhanced vascular contraction to Ang II by macrophage generated 12(S)-HETE and endothelial cell ROS production.³⁵ However, Ang II also regulates BP by maintaining salt and water homeostasis through its actions in the kidney and the brain.^49,50 Peripheral and centrally administered Ang II increases water intake, and vasopressin release, which is inhibited by lesions of the subfornical organ that project to the vasopressin-containing neurons in the supraoptic nucleus and paraventricular-nuclei of the anterior hypothalamus.^51,52 In a double transgenic mouse model (“sRA”) expressing increased brain-specific activity of renin-angiotensin in the supraoptic nucleus, the urine volume, levels of copeptin, and BP were elevated in both male and female mice.⁵³ However, intracerebroventricular administration of Ang II in OVX but not in intact rats increased water and salt intake and plasma levels of vasopressin which was inhibited by subcutaneous E2.⁵⁴ Our demonstration that Ang II in OVX but not in intact mice caused a marked increase in BP that was associated with increased water intake and urinary output of water and copeptin, decreased urinary osmolality, and increased plasma levels of 12 (S)-HETE that were attenuated in intact mice with endogenous E2 present and in ALOX15KO mice raises the possibility that 12(S)-HETE might mediate this effect of Ang II in the brain. Since vasopressin is known to increase sympathetic activity and BP,^53,55 it is possible that E2, by inhibiting 12(S)-HETE-dependent vasopressin release, also contributes to the protection against Ang II-induced hypertension in females. However, further studies need to be conducted on selective Alox15 gene disruption and the effect of 12(S)-HETE and the underlying signaling mechanism including 12(S)-HETE receptors³⁵ in brain regions involved in the actions of Ang II on water and salt homeostasis, sympathetic activity, neuroinflammation, and development of hypertension.

We have previously shown that in OVX mice, Ang II increased water intake and urine output, decreased urine osmolality, and caused proteinuria, which was minimized by treatment with 2-ME, a CYP1B1-generated metabolite of E2.⁵⁶ Moreover, we have reported that 2-ME produced locally in the brain from E2 by CYP1B1, most likely in the paraventricular-nuclei, protects against Ang II-induced hypertension and neuroinflammation in female mice.³¹ Therefore, these findings, together with the results of the present study, raise the possibility that E2-derived CYP1B1 metabolite 2-ME by inhibiting the production of AA/ALOX15 generated 12(S)-HETE in the brain protects against Ang II-induced hypertension and the associated pathogenesis. Studies to test this hypothesis and the role of ALOX15/12(S)-HETE in immune cells in the brain in the development of hypertension and neuroinflammation are currently under investigation.

In conclusion, depletion of E2 caused by ovariectomy in mice enhanced Ang II-induced hypertension and associated pathophysiological changes, including impaired autonomic and renal function, increased water intake and urinary level of vasopressin, renal hypertrophy, fibrosis, and ROS production. These effects of Ang II were dependent on the increased generation of AA-ALOX15 derived prohypertensive metabolite 12(S)—but not 15(S)-HETE and they were attenuated by Alox15 gene disruption in female mice (Figure S6). Therefore, the development of selective inhibitors of ALOX15 or 12 (S)-HETE receptor antagonists could be useful for treating hypertension and its pathogenesis in postmenopausal, hypoestrogenic women or females with ovarian failure.

Perspectives

This study furthers our knowledge of the mechanism whereby endogenous E2 by inhibiting ALOX15 activity and generation of 12(S)-HETE protects against Ang-II-induced hypertension and associated autonomic and renal dysfunction, increased vasopressin release, renal hypertrophy, ROS production, and damage. Recently, we reported that the protective effect of E2 against Ang II-induced hypertension and associated neuroinflammation is mediated by E2-CYP1B1 generated metabolite 2-ME in the paraventricular-nuclei.³¹ Therefore, further studies are required to elucidate the interaction of CYP1B1 and AA-ALOX15 in brain regions, including subfornical organ, paraventricular-nuclei, and supraoptic nucleus involved in the central actions of Ang II in regulating drinking, sympathetic activity, and vasopressin release and development of hypertension. Furthermore, it has been reported that 2 single-nucleotide polymorphism in the Alox12 gene, rs9904779, and rs434473 (encoding a replacement of asparagine by serine in the protein) was associated with the onset of natural menopause in a small set of White women studied.⁵⁷ Therefore, it would be important to explore ALOX15 gene polymorphism further in a larger population of diverse groups of normal aging and pre- and postmenopausal females to determine its impact on the contribution of AA-ALOX15 derived 12(S)-HETE in hypertension and its pathogenesis.

Article Information

Acknowledgments

We thank Dr Jay H. Fowke, Professor, Department of Preventive Medicine, University of Tennessee Health Science Center for his help in Biostatistics.

Sources of Funding

This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute grants R01HL-19134–47 (K.U. Malik), American Heart Association-Career Development Award No. 940538 (P. Singh), and UTHSC-CGHS Frank W. Dugan Fellowship stipend support to S.R. Dutta. The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Heart, Lung, and Blood Institute.

Disclosures

None.

Supplemental Material

Methods

Flowcharts 1 and 2

Authentication of Antibodies

Figures S1–S6