Xie Wu

Sex chromosome effects unmasked in angiotensin II-induced hypertension

Sex differences in mean arterial pressure (MAP) are reported in many experimental models of hypertension and are ascribed to gonadal sex based on studies showing that gonadectomy and gonadal hormone replacement affect MAP. The interpretation of these studies, however, has been confounded by differences in the sex chromosome complement (XX versus XY). To investigate the sex chromosome complement independent of gonadal sex, we used the 4 core genotype mouse model in which gonadal sex is separated from the sex chromosome complement enabling comparisons among XX and XY females and XX and XY males. We found that, in the gonadectomized (GDX) 4 core genotype, MAP after 2 weeks of angiotensin II infusion (200 ng/kg per minute) was greater in XX than XY (MAP [in millimeters of mercury]: GDX-XX-female, 148±4.5; GDX-XY-female, 133±4.4; GDX-XX-male, 149±9.4; GDX-XY-male, 138±5.5; P<0.03, XX versus XY; n=8 to 9 per group). In contrast, no sex chromosome effects were found on heart rate, body weight, or plasma angiotensin II 2 weeks after angiotensin II infusion. This study suggests that, in addition to effects of gonadal hormones on blood pressure, X- or Y-linked genes, parental imprinting, or X mosaicism contributes to sex differences in hypertension. Furthermore, the finding that MAP was greater in XX mice compared with XY mice in the GDX state suggests that adverse sex chromosome effects encoded within the XX sex chromosome complement could contribute to hypertension in women with ovarian hormone deficiency, such as postmenopausal women and women with premature ovarian failure.

Role of angiotensin‐converting enzyme 2 and angiotensin(1–7) in 17β‐oestradiol regulation of renal pathology in renal wrap hypertension in rats

17β‐Oestradiol (E2)‐mediated inhibition of angiotensin‐converting enzyme (ACE) protects the E2‐replete kidney from the progression of hypertensive renal disease. Angiotensin‐converting enzyme 2 (ACE2), a homologue of ACE, counters the actions of ACE by catalysing the conversion of angiotensin II (Ang II) to angiotensin(1–7) [Ang(1–7)]. We investigated E2 regulation of ACE2 in the renal wrap (RW) model of hypertension in rats. After 6 weeks on a high‐sodium diet (4% NaCl), the activity of ACE2 was reduced in the renal cortex by 31%, which was mirrored by similar decreases in ACE2 protein (30%) and mRNA expression (36%) in the ovariectomized RW rat (RW‐OVX); E2 replacement prevented these effects. The RW‐OVX rats exhibited greater renal injury, including 1.7‐fold more tubulointerstitial fibrosis and 1.6‐fold more glomerulosclerosis than E2‐replete females (RW‐Intact and RW‐OVX+E2). Angiotensin(1–7) infusion prevented these exacerbating effects of ovariectomy on renal pathology; no differences in indicators of renal injury were observed between RW‐OVX‐Ang(1–7) and RW‐Intact rats. These renal protective effects of Ang(1–7) infusion were not attributable to increased ACE2 activity or to changes in heart rate or body weight, since these parameters were unchanged by Ang(1–7) infusion. Furthermore, Ang(1–7) infusion did not attenuate renal injury by reducing mean arterial pressure (MAP), since infusion of the peptide did not lower MAP but rather caused a slight increase during a 6 week chronic treatment for Ang(1–7). These results suggest that E2‐mediated upregulation of renal ACE2 and the consequent increased Ang(1–7) production contribute to E2‐mediated protection from hypertensive renal disease. These findings have implications for E2‐deficient women with hypertensive renal disease and suggest that therapeutics targeted towards increasing ACE2 activity and Ang(1–7) levels will be renal protective.

Female Protection in Progressive Renal Disease Is Associated with Estradiol Attenuation of Superoxide Production

BACKGROUND Several types of renal disease progress at a faster rate in men compared with women, but the reasons for this sex difference are not well understood. Chronic renal disease is associated with elevated levels of toxic reactive oxygen species (ROS). Superoxide, the major ROS in the kidney, is generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. OBJECTIVE To determine if female protection from renal disease progression is consistent with 17beta-estradiol (E2) attenuation of superoxide production, this study was conducted to assess superoxide production in the renal cortex of male and female control and renal wrap (RW) rats, as well as in ovariectomized rats treated with vehicle or E2. METHODS Sprague-Dawley rats were divided into 2 sham operation male (Sham-M) and female (Sham-F) control groups, and 4 RW hypertensive groups: RW-M; RW-F; RW ovariectomized females treated with vehicle (RW-OVX); and RW ovariectomized females treated with E2, supplied as a 0.24 mg/60-day release pellet (RW-OVX+E2). All groups were maintained on a high-sodium (4% NaCl) diet for 6 weeks. RESULTS Mean (SEM) markers of renal injury and oxidative stress, including urinary protein (mg/24 h: RW-M, 298 [31] vs RW-F, 169 [22]; P < 0.001), microalbuminuria (RW/Sham arbitrary units [AU]/24 h: M, 8.78 [0.58] vs F, 4.31 [1.0]; P < 0.005), and malondialdehyde (nmol/24 h: RW-M, 167 [23] vs RW-F, 117 [8.5]; P < 0.05) levels, as well as mean glomerular volume (microm3 x 10(6): RW-M, 2.25 [0.16] vs RW-F, 1.25 [0.04]; P < 0.001) and the glomerulosclerotic index (AU: RW-M, 2.64 [0.19] vs RW-F, 1.10 [0.09]; P < 0.001) were greater in both control and RW males compared with females in the same treatment groups. Though RW surgery increased mean arterial pressure in both male and female rats, no sex difference was observed. Under these conditions, mean (SEM) renal cortical NADPH oxidase activity was 1.3-fold higher in RW males compared with RW females (relative light units [RLU]/180 sec: RW-M, 4080 [240] vs RW-F, 3200 [260]; P < 0.05). Ovariectomy increased NADPH oxidase activity by 1.4-fold (RLU/180 sec: RW-OVX, 4520 [184]; P < 0.01) under conditions in which the mean glomerular volume and glomerulosclerotic index were both increased by 1.5-fold, whereas E2 replacement (RLU/180 sec: RW-OVX+E2, 2745 [440]) prevented these effects. Furthermore, the effects on NADPH oxidase activity were mirrored by changes in the protein abundance of NADPH oxidase subunit p22P(phox). CONCLUSION These results suggest that E2 protects the female kidney in part by attenuating injury-induced increases in renal superoxide production.

Sex differences in vasopressin V2 receptor expression and vasopressin-induced antidiuresis

The renal vasopressin V(2) receptor (V(2)R) plays a critical role in physiological and pathophysiological processes associated with arginine vasopressin (AVP)-induced antidiuresis. Because clinical data suggests that females may be more prone to hyponatremia from AVP-mediated antidiuresis, we investigated whether there are sex differences in the expression and function of the renal V(2)R. In normal Sprague-Dawley rat kidneys, V(2)R mRNA and protein expression was 2.6- and 1.7-fold higher, respectively, in females compared with males. To investigate the potential physiological implications of this sex difference, we studied changes in urine osmolality induced by the AVP V(2)R agonist desmopressin. In response to different doses of desmopressin, there was a graded increase in urine osmolality and decrease in urine volume during a 24-h infusion. Females showed greater mean increases in urine osmolality and greater mean decreases in urine volume at 0.5 and 5.0 ng/h infusion rates. We also studied renal escape from antidiuresis produced by water loading in rats infused with desmopressin (5.0 ng/h). After 5 days of water loading, urine osmolality of both female and male rats escaped to the same degree physiologically, but V(2)R mRNA and protein in female kidneys was reduced to a greater degree (-63% and -73%, respectively) than in males (-32% and -48%, respectively). By the end of the 5-day escape period, renal V(2)R mRNA and protein expression were reduced to the same relative levels in males and females, thereby abolishing the sex differences in V(2)R expression seen in the basal state. Our results demonstrate that female rats express significantly more V(2)R mRNA and protein in kidneys than males, and that this results physiologically in a greater sensitivity to V(2)R agonist administration. The potential pathophysiological implications of these results are that females may be more susceptible to the development of dilutional hyponatremia because of a greater sensitivity to endogenously secreted AVP.

Sex-Specific T-Cell Regulation of Angiotensin II–Dependent Hypertension

Studies suggest T cells modulate arterial pressure. Because robust sex differences exist in the immune system and in hypertension, we investigated sex differences in T-cell modulation of angiotensin II–induced increases in mean arterial pressure in male (M) and female (F) wild-type and recombination-activating-gene-1–deficient (Rag1−/−) mice. Sex differences in peak mean arterial pressure in wild-type were lost in Rag1−/− mice (mm Hg: wild-type-F, 136±4.9 versus wild-type-M, 153±1.7; P<0.02; Rag1−/−-F, 135±2.1 versus Rag1−/−-M, 141±3.8). Peak mean arterial pressure was 13 mm Hg higher after adoptive transfer of male (CD3M→Rag1−/−-M) versus female (CD3F→Rag1−/−-M) T cells. CD3M→Rag1−/−-M mice exhibited higher splenic frequencies of proinflammatory interleukin-17A (2.4-fold) and tumor necrosis factor-&agr; (2.2-fold)–producing T cells and lower plasma levels (13-fold) and renal mRNA expression (2.4-fold) of interleukin-10, whereas CD3F→Rag1−/−-M mice displayed a higher activation state in general and T-helper-1–biased renal inflammation. Greater T-cell infiltration into perivascular adipose tissue and kidney associated with increased pressor responses to angiotensin II if the T cell donor was male but not female and these sex differences in T-cell subset expansion and tissue infiltration were maintained for 7 to 8 weeks within the male host. Thus, the adaptive immune response and role of pro- and anti-inflammatory cytokine signaling in hypertension are distinct between the sexes and need to be understood to improve therapeutics for hypertension-associated disease in both men and women.

Sex differences in renal angiotensin converting enzyme 2 (ACE2) activity are 17β-oestradiol-dependent and sex chromosome-independent

BackgroundAngotensin converting enzyme 2 (ACE2) is a newly discovered monocarboxypeptidase that counteracts the vasoconstrictor effects of angiotensin II (Ang II) by converting Ang II to Ang-(1-7) in the kidney and other tissues.MethodsACE2 activity from renal homogenates was investigated by using the fluorogenic peptide substrate Mca-YVADAPK(Dnp)-OH, where Mca is (7-methoxycoumarin-4-yl)-acetyl and Dnp is 2,4-dinitrophenyl.ResultsWe found that ACE2 activity expressed in relative fluorescence units (RFU) in the MF1 mouse is higher in the male (M) compared to the female (F) kidney [ACE2 (RFU/min/μg protein): M 18.1 ± 1.0 versus F 11.1 ± 0.39; P < 0.0001; n = 6]. Substrate concentration curves revealed that the higher ACE2 activity in the male was due to increased ACE2 enzyme velocity (Vmax) rather than increased substrate affinity (Km). We used the four core genotypes mouse model in which gonadal sex (ovaries versus testes) is separated from the sex chromosome complement enabling comparisons among XX and XY gonadal females and XX and XY gonadal males. Renal ACE2 activity was greater in the male than the female kidney, regardless of the sex chromosome complement [ACE2 (RFU/min/μg protein): intact-XX-F, 7.59 ± 0.37; intact-XY-F, 7.43 ± 0.53; intact-XX-M, 12.1 ± 0.62; intact-XY-M, 12.7 ± 1.5; n = 4-6/group; P < 0.0001, F versus M, by two-way ANOVA]. Enzyme activity was increased in gonadectomized (GDX) female mice regardless of the sex chromosome complement whereas no effect of gonadectomy was observed in the males [ACE2 (RFU/min/μg protein): GDX-XX-F, 12.4 ± 1.2; GDX-XY-F, 11.1 ± 0.76; GDX-XX-M, 13.2 ± 0.97; GDX-XY-M, 11.6 ± 0.81; n = 6/group]. 17β-oestradiol (E2) treatment of GDX mice resulted in ACE2 activity that was only 40% of the activity found in the GDX mice, regardless of their being male or female, and was independent of the sex chromosome complement [ACE2 (RFU/min/μg protein): GDX+E2-XX-F, 5.56 ± 1.0; GDX+E2-XY-F, 4.60 ± 0.52; GDX+E2-XX-M, 5.35 ± 0.70; GDX+E2-XY-M, 5.12 ± 0.47; n = 6/group].ConclusionsOur findings suggest sex differences in renal ACE2 activity in intact mice are due, at least in part, to the presence of E2 in the ovarian hormone milieu and not to the testicular milieu or to differences in sex chromosome dosage (2X versus 1X; 0Y versus 1Y). E2 regulation of renal ACE2 has particular implications for women across their life span since this hormone changes radically during puberty, pregnancy and menopause.

Endothelial Dysfunction and Enhanced Contractility in Microvessels From Ovariectomized Rats: Roles of Oxidative Stress and Perivascular Adipose Tissue

Ovarian hormone loss increases reactive oxidative species, endothelial dysfunction, and cardiovascular disease. Because perivascular adipose tissue (PVAT) regulates endothelial function, we hypothesized that reactive oxidative species in PVAT mediate adverse microvascular effects of ovarian hormone deficiency. Rats were ovariectomized or sham operated and given vehicle or tempol for 6 weeks. Mesenteric resistance arterioles from ovariectomized compared with sham-operated rats had dysfunctional responses to acetylcholine (ACh) including decreased ACh-induced endothelium-dependent relaxation (50±6% versus 72±2%) and endothelium-dependent relaxation factor (17±4% versus 37±2%) and increased endothelium-dependent contracting factor (27±5% versus 9±3%). OVX rat mesenteric arterioles had increased contractions to the thromboxane/prostanoid receptor agonist U-46 619 (58±3% versus 40±5%) and increased reactive oxidative species (tempo-9-AC fluorescence) with U-46 619 (0.65±0.17 versus 0.14±0.06 Δ unit) or ACh (0.49±0.09 versus 0.09±0.05 Δ unit) and increased p22phox protein expression (0.89±0.05 versus 0.18±0.04 Δ unit), whereas nitric oxide activity (DAF-FM [4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate] fluorescence) with ACh was reduced (0.39±0.1 versus 0.70±0.10 Δ unit). No differences were found in endothelium-dependent hyperpolarizing factor or contractile responses to phenylephrine. PVAT enhanced ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide only in sham-operated rats. Tempol prevented ovariectomy-induced endothelial dysfunction and restored the enhancing effects of PVAT on ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide in ovariectomized rat vessels, but both tempol and PVAT were required to normalize the enhanced U-46 619 contractions after ovariectomy. In conclusion, ovariectomy redirects endothelial responses from relaxation to contraction by reducing vascular nitric oxide, augmenting thromboxane/prostanoid receptor signaling, and attenuating the vasodilatory effects of PVAT, all of which were dependent on reactive oxidative species. # Novelty and Significance {#article-title-37}Ovarian hormone loss increases reactive oxidative species, endothelial dysfunction, and cardiovascular disease. Because perivascular adipose tissue (PVAT) regulates endothelial function, we hypothesized that reactive oxidative species in PVAT mediate adverse microvascular effects of ovarian hormone deficiency. Rats were ovariectomized or sham operated and given vehicle or tempol for 6 weeks. Mesenteric resistance arterioles from ovariectomized compared with sham-operated rats had dysfunctional responses to acetylcholine (ACh) including decreased ACh-induced endothelium-dependent relaxation (50±6% versus 72±2%) and endothelium-dependent relaxation factor (17±4% versus 37±2%) and increased endothelium-dependent contracting factor (27±5% versus 9±3%). OVX rat mesenteric arterioles had increased contractions to the thromboxane/prostanoid receptor agonist U-46 619 (58±3% versus 40±5%) and increased reactive oxidative species (tempo-9-AC fluorescence) with U-46 619 (0.65±0.17 versus 0.14±0.06 &Dgr; unit) or ACh (0.49±0.09 versus 0.09±0.05 &Dgr; unit) and increased p22phox protein expression (0.89±0.05 versus 0.18±0.04 &Dgr; unit), whereas nitric oxide activity (DAF-FM [4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate] fluorescence) with ACh was reduced (0.39±0.1 versus 0.70±0.10 &Dgr; unit). No differences were found in endothelium-dependent hyperpolarizing factor or contractile responses to phenylephrine. PVAT enhanced ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide only in sham-operated rats. Tempol prevented ovariectomy-induced endothelial dysfunction and restored the enhancing effects of PVAT on ACh-induced relaxation, endothelium-dependent relaxation factor, and nitric oxide in ovariectomized rat vessels, but both tempol and PVAT were required to normalize the enhanced U-46 619 contractions after ovariectomy. In conclusion, ovariectomy redirects endothelial responses from relaxation to contraction by reducing vascular nitric oxide, augmenting thromboxane/prostanoid receptor signaling, and attenuating the vasodilatory effects of PVAT, all of which were dependent on reactive oxidative species.

Effect of dietary sodium on estrogen regulation of blood pressure in Dahl salt-sensitive rats.

The effects of high-sodium (HS) and normal-sodium (NS) diets on ovarian hormone modulation of mean arterial pressure (MAP) were examined in Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Ovariectomy increased MAP (OVX-Sham) to a greater extent in DS rats maintained for 2 wk on a HS (22 mmHg) compared with a NS (6 mmHg) diet. Ovariectomy had no effect on MAP in DR rats on NS but did increase MAP in rats on HS (10 mmHg) diets. On HS diets, glomerular filtration rate (GFR) was 36% less in the DS-Sham than DR-Sham animals; ovariectomy increased GFR in both strains by 1.4-1.5-fold; glomerular angiotensin II type 1 receptor (AT(1)R) densities were 1.6-fold higher in the DS-Sham than in the DR-Sham group; ovariectomy increased glomerular AT(1)R densities by 1.3-fold in DR rats but had no effect in DS rats; 17beta-estradiol (E(2)) downregulated adrenal AT(1)R densities in both strains on either diet; ovariectomy reduced estrogen receptor-alpha (ER-alpha) protein expression in the renal cortex by 40-50% although renal ER-alpha expression was 34% lower in DS than in DR rats. These observed effects of gonadectomy were prevented by E(2) treatment, suggesting that E(2) deficiency mediates the effects of ovariectomy on MAP, GFR, AT(1)R densities, and renal ER-alpha protein expression. In conclusion, ovariectomy-induced increases in MAP are augmented by HS diet in both strains, and this effect is not mediated by a reduction in GFR. Aberrant renal AT(1)R regulation and reduced renal ER-alpha expression are potential contributors to the hypertensive effects of E(2) deficiency in DS rats. These findings have implications for women with salt-sensitive hypertension and women who are E(2) deficient, such as postmenopausal women.

Loss of Resistance to Angiotensin II–Induced Hypertension in the Jackson Laboratory Recombination-Activating Gene Null Mouse on the C57BL/6J BackgroundNovelty and Significance

Resistance to angiotensin II (Ang II)–induced hypertension in T-cell–deficient male mice with a targeted mutation in the recombination-activating gene-1 (Rag1) on the C57BL/6J background (B6.Rag1−/−-M), which was reported by 5 independent laboratories including ours before 2015, has been lost. In mice purchased from Jackson Laboratory in 2015 and 2016, the time course and magnitude increase in mean arterial pressure induced by 2 weeks of Ang II infusion at 490 ng/kg per minute was identical between B6.Rag1−/−-M and male wild-type littermates. Moreover, there were no differences in the time course or magnitude increase in mean arterial pressure at the lowest dose of Ang II (200 ng/kg per minute) that increased mean arterial pressure. This loss in Ang II resistance is independent of T cells. Angiotensin type 1-receptor binding was 1.4-fold higher in glomeruli isolated from recently purchased B6.Rag1−/−-M suggesting an increase in renal angiotensin type 1-receptor activity masks the blood pressure protection afforded by the lack of T cells. The phenotypic change in B6.Rag1−/−-M has implications for investigators using this strain to study mechanisms of T-cell modulation of Ang II–dependent blood pressure control. These findings also serve as a reminder that the universal drive for genetic variation occurs in all animals including inbred mouse strains and that spontaneous mutations leading to phenotypic change can compromise experimental reproducibility over time and place. Finally, these observations illustrate the importance of including experimental details about the location and time period over which animals are bred in publications involving animal studies to promote rigor and reproducibility in the scientific literature.

Aging-related impairment of urine-concentrating mechanisms correlates with dysregulation of adrenocortical angiotensin type 1 receptors in male Fischer rats

To investigate age-associated impairments in fluid homeostasis, 4-mo (young) and 32-mo (old) Fischer 344/BN male rats were studied before and after a dietary sodium load. Transferring young rats from a low-sodium (LS) to a high-sodium (HS) diet increased water intake and urine volume by 1.9- and 3.0-fold, respectively, while urine osmolality and plasma aldosterone decreased by 33 and 98%. Concomitantly, adrenocortical angiotensin type 1 receptor (AT1R) density decreased by 35%, and AT1bR mRNA decreased by 39%; no changes were observed in AT1aR mRNA. In contrast, the increase in water intake (1.4-fold) was lower in the old rats, and there was no effect of the HS diet on urine volume or urine osmolality. AT1R densities were 29% less in the old rats before transferring to the HS diet, and AT1R densities were not reduced as rapidly in response to a HS diet compared with the young animals. After 6 days on the HS diet, plasma potassium was lowered by 26% in the old rats, whereas no change was detected in the young rats. Furthermore, while plasma aldosterone was substantially decreased after 2 days on the HS diet in both young and old rats, plasma aldosterone was significantly lower in the old compared with the young animals after 2 wk on the LS diet. These findings suggest that aging attenuates the responsiveness of the adrenocortical AT1R to a sodium load through impaired regulation of AT1bR mRNA, and that this dysregulation contributes to the defects in water and electrolyte homeostasis observed in aging.