Female Spontaneously Hypertensive Rats Research Articles

Sex Differences in the Beneficial Cardiac Effects of Chronic Treatment with Atrial Natriuretic Peptide In Spontaneously Hypertensive Rats

IntroductionThe aim of this study was to investigate both the effects of chronic treatment with atrial natriuretic peptide (ANP) on systolic blood pressure (SBP), cardiac nitric oxide (NO) system, oxidative stress, hypertrophy, fibrosis and apoptosis in spontaneously hypertensive rats (SHR), and sex-related differences in the response to the treatment.Methods10 week-old male and female SHR were infused with ANP (100 ng/hr/rat) or saline (NaCl 0.9%) for 14 days (subcutaneous osmotic pumps). SBP was recorded and nitrites and nitrates excretion (NOx) were determined. After treatment, NO synthase (NOS) activity, eNOS expression, thiobarbituric acid-reactive substances (TBARS) and glutathione concentration were determined in left ventricle, as well as the activity of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). Morphological studies in left ventricle were performed in slices stained with hematoxylin-eosin or Sirius red to identify collagen as a fibrosis indicator; immunohistochemistry was employed for identification of transforming growth factor beta; and apoptosis was evaluated by Tunel assay.ResultsFemale SHR showed lower SBP, higher NO-system activity and less oxidative stress, fibrosis and hypertrophy in left ventricle, as well as higher cardiac NOS activity, eNOS protein content and NOx excretion than male SHR. Although ANP treatment lowered blood pressure and increased NOS activity and eNOS expression in both sexes, cardiac NOS response to ANP was more marked in females. In left ventricle, ANP reduced TBARS and increased glutathione concentration and activity of CAT and SOD enzymes in both sexes, as well as GPx activity in males. ANP decreased fibrosis and apoptosis in hearts from male and female SHR but females showed less end-organ damage in heart. Chronic ANP treatment would ameliorate hypertension and end-organ damage in heart by reducing oxidative stress, increasing NO-system activity, and diminishing fibrosis and hypertrophy.

Female SHR have greater blood pressure sensitivity and renal T cell infiltration following chronic NOS inhibition than males

Nitric oxide is a critical regulator of blood pressure (BP) and inflammation, and female spontaneously hypertensive rats (SHR) have higher renal nitric oxide bioavailability than males. We hypothesize that female SHR will have a greater rise in BP and renal T cell infiltration in response to nitric oxide synthase (NOS) inhibition than males. Both male and female SHR displayed a dose-dependent increase in BP to the nonspecific NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME: 2, 5, and 7 mg·kg(-1)·day(-1) for 4 days each); however, females exhibited a greater increase in BP than males. Treatment of male and female SHR with 7 mg·kg(-1)·day(-1) L-NAME for 2 wk significantly increased BP in both sexes; however, prior exposure to L-NAME only increased BP sensitivity to chronic NOS inhibition in females. L-NAME-induced hypertension increased renal T cell infiltration and indices of renal injury in both sexes, yet female SHR exhibited greater increases in Th17 cells and greater decreases in regulatory T cells than males. Chronic L-NAME was also associated with larger increases in renal cortical adhesion molecule expression in female SHR. The use of triple therapy to block L-NAME-mediated increases in BP attenuated L-NAME-induced increases in renal T cell counts and normalized adhesion molecule expression in SHR, suggesting that L-NAME-induced increases in renal T cells were dependent on both increases in BP and NOS inhibition. Our data suggest that NOS is critical in the ability of SHR, females in particular, to maintain BP and limit a pro-inflammatory renal T cell profile.

Conjugated equine estrogens and estradiol benzoate differentially modulate the natriuretic peptide system in spontaneously hypertensive rats

The purpose of this study was to compare the effects of conjugated equine estrogens (CEE) and estradiol benzoate on the blood pressure and body weight of spontaneously hypertensive rats (SHRs) and the associated changes in several components of the natriuretic peptide system. The blood pressure of randomly distributed female SHRs and Wistar rats was determined by tail plethysmography. The rats were ovariectomized and, after 3 weeks, injected daily for 4 days with estradiol benzoate (5 μg/100 g/d), CEE (50 μg/100 g/d), or vehicle (corn oil 0.1 mL/100 g/d). One day after the last injection, the rats were decapitated, and their blood was collected to measure atrial natriuretic peptide (ANP) and estradiol. The atria were removed to measure ANP levels using radioimmunoassay and to quantify ANP messenger RNA expression using real-time polymerase chain reaction. The kidneys and adipose tissue were removed to analyze the expression of natriuretic peptide clearance receptor messenger RNA. A reduction in blood pressure was observed in estradiol-treated SHRs, but CEE treatment had no effect. Estradiol decreased the body weight and parametrial adipose tissue mass of SHRs. Estradiol-induced alterations in SHRs were accompanied by increased synthesis and release of ANP. CEE had no effect on body weight but increased the mesenteric adipose tissue mass of SHRs. These results indicate that estradiol and CEE have different effects on the reduction in body weight and blood pressure. These results are correlated with changes in plasma ANP levels.

The Impact of High Mobility Group Box 1 Protein (HMGB1) on Renal Ischemia‐Reperfusion Injury in Male and Female Spontaneously Hypertensive Rats (SHR)

HMGB1 is an endogenous molecule released by dying cells which contributes to renal ischemia‐reperfusion injury (IRI). We recently published that male SHR have more pro‐inflammatory immune cells in the kidney. The goal of this study was to test the hypothesis that female SHR have less HMGB1 than males resulting in a lower degree of renal IRI. HMGB1 levels were measured via ELISA in kidneys of 12 week old male and female SHR (N=5). Additional age matched rats were pre‐treated with control or HMGB1 neutralizing antibody (300 μg/rat via IP; N=4) 1 hour before occluding renal arteries for 1 hour. Renal blood flow velocity (RBV) was measured via ultrasound before and 24 hours after reperfusion. Kidneys were isolated for mRNA extraction and histology. HMGB1 levels were greater in the renal cortex of female vs. male SHR (0.33±0.05 vs. 0.16±0.02 ng/mg; p=0.01). Medullary HMGB1 levels were comparable between sexes. Pre‐treatment with anti‐HMGB1 did not alter RBV compared to controls (% decrease of male SHR: 34±2 to 35±5, NS; female SHR: 35±6 to 22±6, NS) or expression of renal VCAM1 or ICAM1 in either sex. Using histological analysis we determined that anti‐HMGB1 decreased CD3+ T cell infiltration in both sexes, verifying a role for HMGB1 in inflammation following IRI. In conclusion, despite greater renal HMGB1 levels in female SHR, there were no sex differences in the functional role of HMGB1 inhibition on renal IRI or inflammation.

Neither Hypertension nor Sexual Maturation is Responsible for Elevated Mesenteric Arterial Expression of TGF‐β in Female Spontaneously Hypertensive Rats (SHR)

TGF‐β has been implicated in vascular damage and hypertension. We have demonstrated that adult female SHR express more vascular TGF‐β than males. Moreover, TGF‐β expression increases as female SHR, but not males, age from 5 to 13 wks. This study tested the hypotheses that 1) increased TGF‐β expression in female SHR is due to hypertension not sexual maturation and 2) greater TGF‐β in females does not equate with greater fibrosis. To test our hypotheses 5 wk old female SHR were randomized to the following groups (N=4): vehicle vs hydralazine‐treated (50 mg/kg/day) or gonad‐intact vs ovariectomized (OVX). Mesenteric arteries were isolated at 13 wks and TGF‐β was measured by immunoblotting. TGF‐β expression was similar in vehicle and hydralazine treated SHR (densitometric units (DU): 27±1 vs 33±5, p=0.3) as well as between intact and OVX (DU: 17±2 vs 14±2, p=0.2). These data suggest that increased TGF‐β with age in female SHR is not dependent on hypertension or sexual maturation. Additional studies measured collagen expression by histological staining in 13 wk male and female SHR. Mesenteric arterial collagen deposition was similar in males and females (area μm2: 6632±898 vs 5507±522, p=0.3; N=3), suggesting that greater TGF‐β in females does not result in greater vascular injury. We propose that the increase in TGF‐β in females might instead be compensatory to offer vascular protection.

Ang (1–7) Has a Greater Contribution to the Blood Pressure Lowering Effects of AT1 Receptor Blockade in Female Spontaneously Hypertensive Rats (SHR) Compared to Males

Ang (1–7) contributes to the blood pressure (BP) lowering effects of renin angiotensin system inhibitors in male SHR. We have previously published that female SHR have greater Ang (1–7) levels than males, however, the functional implications of this with regards to BP control remains largely unknown. Therefore, the goal of this study was to test the hypothesis that female SHR have a larger Ang (1–7) contribution to the BP lowering effects of the angiotensin receptor blocker (ARB) candesartan than male SHR. BP was measured via telemetry in 12 week old male and female SHR (n=4–8). Animals were pre‐treated with either the Ang (1–7) antagonist A‐779 (4 days; 48 μg/kg/hr via osmotic minipump) or vehicle. Candesartan was then co‐administered (0.5 mg/kg/day via drinking water) for a week. A‐779 alone did not alter basal BP in either sex. In male SHR, candesartan‐mediated decreases in BP were comparable in vehicle (15% decrease: 143±2 to 122±3 mmHg; p<0.05) and A‐779 treated (12% decrease: 147±3 to 129±4 mmHg; p<0.05). In contrast, A‐779 attenuated candesartan‐mediated decreases in BP (5% decrease: 127±1 to 121±2 mmHg; NS) in female SHR compared to vehicle treatment (10% decrease: 128±1 to 115±1 mmHg; p<0.05). In conclusion, female SHR have a greater Ang (1–7) contribution to ARB‐mediated decreases in BP than males. This study was funded by National Institutes of Health grant 1R01 HL093271–01A1 to J.C.S.

Sex does not impact asymmetric dimethylarginine (ADMA) or L‐arginine (L‐arg) levels in spontaneously hypertensive rats (SHR).

ADMA and L‐arg are endogenous amino acids that impact nitric oxide synthase (NOS) activity. ADMA has been implicated in nitric oxide (NO) deficiency and hypertension. We have reported that female SHR have significantly greater NO bioavailability and NOS enzymatic activity in the renal inner medulla compared to age‐matched males, yet the mechanism responsible is unknown. We hypothesized that male SHR have greater ADMA levels and less L‐arg compared to females. ADMA and L‐arg levels in plasma (N=6–7) and renal inner medullas (N=10) were measured via HPLC from 12–13 week old male and female SHR. ADMA (Male: 20±6; Female: 31±8 nmols/ml, p=0.7), L‐arg (Male: 152±7; Female: 141±11 nmols/ml, p=0.7) levels and the L‐arg/ADMA ratio (p=0.2) in plasma were comparable between sexes. Renal inner medullary ADMA (Male: 172±55 Female: 192±58 grams wet weight (gww), p=0.8), L‐arg (Male: 507±107; Female: 552±127 gww, p=0.8) levels and L‐arg/ADMA ratio (p=0.8) were also comparable between groups. We conclude that while ADMA and L‐arg may be important in maintaining renal NOS activity in SHR, they do not contribute to sex differences in renal inner medullary NOS activity. Funded by the American Heart Association.

Aortic depressor nerve morphometry in male and female adult spontaneously hypertensive rats.

Previous studies form our laboratory [1,2] in spontaneously hypertensive rats (SHR) have demonstrated morphological alterations on the aortic depressor nerve (ADN), known to contain mainly barorreceptor fibers. Nevertheless, those studies used male and female rats indistinctly. We are investigating if there are morphological differences related to gender in the ADN of adult rats. Male and female SHR aged 20‐week were anesthetized for arterial pressure (AP) and heart rate (HR) recordings, together with the ADN spontaneous activity. After the recordings, the left ADN were prepared for light microscopy and morphometry. Comparisons were made between proximal and distal segments in the same gender and between genders. Males are significantly heavier than females but no differences in AP and HR between genders were found. There was a tendency towards larger fascicle values on males compared to females. Interestingly, this tendency was inverted on the myelinated fibers and respective axons sizes. Nevertheless, no significant differences were observed between genders on the morphometric parameters. We conclude that adult males and females can be used indistinctly for morphometric studies of the ADN if physiological parameters are similar between genders.

Effects of Perinatal Lipopolysaccharide (LPS) Exposure on the Developing Rat Brain; Modeling the Effect of Maternal Infection on the Developing Human CNS

In the present study, we examined the effect of perinatal Escherichia coli lipopolysaccharide (LPS) exposure on the developing rat cerebellum and tested the hypothesis that maternal infections impact brain structure and function by mechanisms involving increase in oxidative stress and changes in brain type 2 iodothyronine deiodinase (D2)- and thyroid hormone (TH)-responsive genes. Spontaneously hypertensive rat (SHR) and Sprague-Dawley (SD) rat dams were challenged with LPS (200 μg/kg body weight) exposure during pregnancy (G10-G15) and lactation (P5-P10), the time periods corresponding, respectively, to the first/second and the third trimesters of human pregnancy. LPS exposure resulted in a significantly decreased motor learning in SD male (29.8 %) and in female (55.0 %) pups (p < 0.05); changes in rollover and startle response showed only a trend. The LPS challenge also resulted in a trend (p = 0.09) toward increased cerebellar levels of the oxidative stress marker 3-nitrotyrosine (3-NT) in SD male (16.2 %) and female (21.2 %) neonates, while 3-NT levels were significantly decreased (p < 0.05) in SHR female pups. D2 activity, responsible for local intra-brain conversion of thyroxine (T4) to the active hormone, 3',3,5-triiodothyronine (T3), was significantly (p < 0.05) decreased in LPS-challenged SHR male (40.3 %) and SD female (47.4 %) pups. Several genes were affected by LPS. Notably, D2 (DIO2) and brain-derived neurotrophic factor (BDNF) were significantly elevated in SHR females, while transthyretin (TTR) expression was decreased in both SD males and females (P < 0.05). In vitro chronic exposure of cerebellar cultures to LPS resulted in decreased arborization of Purkinje cells while D2 was only increased transiently. Our data demonstrate that perinatal LPS exposure impacts the developing cerebellum in strain- and sex-dependent manner via complex mechanisms that involve changes in oxidative stress, enzymes involved in maintaining local TH homeostasis, and downstream gene expression.

The chronic blockade of angiotensin I-converting enzyme eliminates the sex differences of serum cytokine levels of spontaneously hypertensive rats

Sex hormones modulate the action of both cytokines and the renin-angiotensin system. However, the effects of angiotensin I-converting enzyme (ACE) on the proinflammatory and anti-inflammatory cytokine levels in male and female spontaneously hypertensive rats (SHR) are unclear. We determined the relationship between ACE activity, cytokine levels and sex differences in SHR. Female (F) and male (M) SHR were divided into 4 experimental groups each (n = 7): sham + vehicle (SV), sham + enalapril (10 mg/kg body weight by gavage), castrated + vehicle, and castrated + enalapril. Treatment began 21 days after castration and continued for 30 days. Serum cytokine levels (ELISA) and ACE activity (fluorimetry) were measured. Male rats exhibited a higher serum ACE activity than female rats. Castration reduced serum ACE in males but did not affect it in females. Enalapril reduced serum ACE in all groups. IL-10 (FSV = 16.4 ± 1.1 pg/mL; MSV = 12.8 ± 1.2 pg/mL), TNF-α (FSV = 16.6 ± 1.2 pg/mL; MSV = 12.8 ± 1 pg/mL) and IL-6 (FSV = 10.3 ± 0.2 pg/mL; MSV = 7.2 ± 0.2 pg/mL) levels were higher in females than in males. Ovariectomy reduced all cytokine levels and orchiectomy reduced IL-6 but increased IL-10 concentrations in males. Castration eliminated the differences in all inflammatory cytokine levels (IL-6 and TNF-α) between males and females. Enalapril increased IL-10 in all groups and reduced IL-6 in SV rats. In conclusion, serum ACE inhibition by enalapril eliminated the sexual dimorphisms of cytokine levels in SV animals, which suggests that enalapril exerts systemic anti-inflammatory and anti-hypertensive effects.

Modulation by 17β-estradiol of anandamide vasorelaxation in normotensive and hypertensive rats: A role for TRPV1 but not fatty acid amide hydrolase

Recent studies suggest that endocannabinoid signaling is modulated by 17β-estradiol (17Eβ) however it is unclear if this applies to the cardiovascular actions of anandamide, a major endocannabinoid. This study examined the in vitro effects of 17Eβ on vasorelaxation to anandamide in myograph-mounted small mesenteric arteries obtained from Wistar rats and Spontaneously Hypertensive Rats (SHRs) of both sexes. Treatment with 1μM 17Eβ but not its enantiomer 17Eα significantly enhanced relaxation to anandamide in male Wistar rats. This effect was independent of a functional endothelium but was blocked by the Transient Receptor Potential Vanilloid type 1 (TRPV1) receptor antagonist SB366791 (2μM) or prolonged treatment with the TRPV1 agonist capsaicin (10μM). A TRPV1-dependent potentiation by 17Eβ was also observed in male SHRs, but not in female Wistar rats or female SHRs. Whilst inhibition of anandamide hydrolysis by 1μM URB597 (an inhibitor of fatty acid amide hydrolase; FAAH) similarly augmented anandamide relaxation in male, but not female, Wistar rats and SHRs, URB597 did not affect the 17Eβ-induced potentiation. Female SHRs displayed a larger maximal relaxation to anandamide; however sex difference was not found in Wistar rats. We conclude that pharmacological levels of 17Eβ potentiate mesenteric relaxation to anandamide through mechanisms dependent on TRPV1 receptors but not FAAH-mediated hydrolysis in male Wistar rats and male SHRs. Sexual dimorphism was observed in the modulatory effects of 17Eβ and URB597, which does not necessarily lead to a greater anandamide response in female rats.

Roles played by 20-HETE, angiotensin II and endothelin in mediating the hypertension in aging female spontaneously hypertensive rats

Prevalence of hypertension (HT) increases in women after menopause, and there is evidence that HT is not as well controlled in postmenopausal women as men. The reasons for this are not clear but may be related to the lack of adequate blockade of the systems contributing to HT in women. This study aimed to determine the roles of three of the systems known to contribute to HT in animal studies: angiotensin II (ANG II; enalapril inhibitor), eicosanoids [1-aminobenzotriazole (1-ABT) inhibitor], and endothelin (ET(A) receptor antagonist), on blood pressure (BP) in three groups of female spontaneously hypertensive rats (SHR), aged 18 mos (postmenopausal rat, PMR). After baseline telemetry BP, three drug periods were performed for 5 days each: single blockade (ABT or enalapril), double blockade (ABT+enalapril or enalapril+ABT), and triple blockade (all 3 drugs). Controls received no treatment until the third period when they received ET(A) receptor antagonist alone. Single drug blockade reduced BP in PMR to similar levels. Double blockade reduced mean arterial pressure more in ABT+enalapril rats than in the other group (enalapril+ABT). Triple drug blockade reduced BP to similar levels in both groups, but the BP remained ∼110 mmHg. The data suggest that these three systems, ANG II, eicosanoids, and endothelin, contribute together and independently to BP control in old female SHR. However, other systems also contribute to the HT since the BP was not normalized, supporting the notion that HT in postmenopausal women may require complex multidrug therapy to be better controlled and that may require the development of additional drugs.

Adverse cardiac remodelling in spontaneously hypertensive rats: acceleration by high aerobic exercise intensity

In the present study it was hypothesized that voluntary aerobic exercise favours a pro-fibrotic phenotype and promotes adverse remodelling in hearts from spontaneously hypertensive rats (SHRs) in an angiotensin II-dependent manner. To test this, female SHRs at the age of 1 year were started to perform free running wheel exercise. Captopril was used to inhibit the renin-angiotensin system (RAS). Normotensive rats and SHRs kept in regular cages were used as sedentary controls. Training intensity, expressed as mean running velocity, was positively correlated with the left ventricular mRNA expression of TGF-β(1), collagen-III and biglycan but negatively correlated with the ratio of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)2a to Na(+)-Ca(2+) exchanger (NCX). A pro-fibrotic phenotype was verified by Picrosirius red staining. Sixty-seven per cent of SHRs performing free running wheel exercise died either spontaneously or had to be killed during a 6 month follow-up. In the presence of captopril, aerobic exercise did not show a similar positive correlation between training intensity and the expression of fibrotic markers. Moreover, in SHRs receiving captopril and performing free running wheel exercise, a training intensity-dependent reverse remodelling of the SERCA2a-to-NCX ratio was observed. None of these rats died spontaneously or had to be killed. In captopril-treated SHRs performing exercise, expression of mRNA for decorin, a natural inhibitor of TGF-β(1), was up-regulated. Despite these differences between SHR-training groups with and without captopril, positive training effects (lower resting heart rate and no progression of hypertension) were found in both groups. In conclusion, high aerobic exercise induces an angiotensin II-dependent adverse remodelling in chronic pressure overloaded hearts. However, high physical activity can potentially induce reverse remodelling in the presence of RAS inhibition.

Abstract 440: Female Spontaneously Hypertensive Rats are More Dependent on Nitric Oxide in Modulating Blood Pressure and Renal Injury Than Males

We recently demonstrated that female spontaneously hypertensive rats (SHR) have higher nitric oxide (NO) bioavailability in the kidney compared to males. Therefore, we hypothesize that female SHR are more dependent on the NO synthase (NOS) system to modulate their blood pressure, and as a result will have a greater rise in blood presure and renal injury in response to chronic NOS inhibition compared to males. Mean arterial pressure in SHR was very sensitive to NOS inhibition, with both sexes exhibiting a significant increase in blood pressure to the non-specific NOS inhibitor N G -nitro-L-arginine methyl ester (L-NAME) at a dose of 2 mg/kg/day. However, there was not a sex difference in the percent increase in mean arterial pressure. Treatment of female and male SHR with 7 mg/kg/day L-NAME for 2 weeks significantly increased mean arterial pressure and indices of renal injury in both females and males; the percent increases in mean arterial pressure and injury were greater in females during the last three days of L-NAME treatment (% increase in mean arterial pressure was 44± 1.3 in females vs. 35± 2.9 in males, P&lt;0.05). L-NAME treatment also increased indices of inflammation and oxidative stress as urinary monocyte chemoattractant protein-1 (MCP-1) and thiobarbituric acid reactive substances (TBARs) excretion levels were increased by L-NAME; however, the increases were greater in females. Renal cortical macrophage and T cell infiltrations and soluble intracellular adhesion molecule-1 (sICAM-1) were also elevated following L-NAME treatment (cortical sICAM-1 levels increased from 4.7± 0.2 to 9± 0.1 ng/mg in males vs. 4.6± 0.1 to 7.9± 0.3 ng/mg in females, P&lt;0.05). Although renal cortical macrophage infiltration was greater in female SHR vs. males following L-NAME treatment, the increase in cortical T cell infiltration and sICAM-1 were greater in males. These data suggest that the NO system plays an important role in modulating blood pressure, renal injury and inflammation in SHR, with females being more dependent on the NOS system than males.

Abstract 155: Female Sex Hormones Blunt Renal Pro-inflammatory T Cell Infiltration Compared to Males

Lymphocytes contribute to hypertension in male spontaneously hypertensive rats (SHR); data in females is lacking, yet young females have a lower blood pressure compared to age-matched males. We hypothesized that 12 week old female SHR have less T cell infiltration and cytokine expression in the kidney compared to males. Kidneys were isolated from male and female SHR and normotensive Wistar Kyoto rats (WKY) and the T cell profile was determined using analytical flow cytometry. In both sexes of SHR, all T cell subtypes and cytokines measured were significantly higher in the kidney compared to the same sex WKY (p&lt;0.05, n=6-8). Male SHR had greater CD4 + T cell infiltration vs. female SHR (p&lt;0.0001), whereas the CD8 + T cell numbers were higher in female SHR (p=0.0114). Analysis of T cell subpopulations showed female SHR has greater expression of regulatory T cells (Foxp3 + CD4 + CD3 + ) than male SHR (p=0.0008), however, male SHR had more infiltrating pro-inflammatory Th17 cells (RORγ + CD4 + CD3 + ; p&lt;0.0001). Male SHR also had greater expression of pro-inflammatory cytokines, IL-17 and IL-6, on cells in the kidney than females, but female SHR expressed more IL-10, an anti-inflammatory cytokine (p&lt;0.0001, all). Gonadectomy of female SHR significantly increased total T cell (CD3 + ; p=0.0009, n=3-6), CD4 + T cell (p=0.0010) and Th17 cell (p=0.0025) infiltration into the kidneys. Infiltration of CD8 + and Foxp3 + cells remained comparable between groups. Together these data suggest that hypertension in both sexes are associated with an increase in renal inflammatory markers; however female SHR have a more anti-inflammatory profile vs. males which may contribute to their lower blood pressure.

Abstract 644: Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats

Tetrahydrobiopterin Supplementation Increases Renal Inner Medullary Nitric Oxide Synthase Enzymatic Activity in Male, But Not Female Spontaneously Hypertensive Rats Krystal N. Brinson and Jennifer C. Sullivan, Experimental Medicine, Georgia Health Sciences University, Augusta, GA 30912 We previously published that male spontaneously hypertensive rats (SHR) having less nitric oxide synthase (NOS) enzymatic activity in the renal inner medullary (IM) than female SHR, which cannot be explained by protein expression alone. Tetrahydrobiopterin (BH4) is an essential cofactor for NOS activity. BH4 is easily oxidized to BH2, which is not a NOS cofactor, and male SHR have greater levels of oxidative stress than females. Therefore, we hypothesized that 1) male SHR will have greater increases in NOS enzymatic activity following BH4 supplementation than females and 2) antioxidant treatment will result in greater increases in NOS enzymatic activity in male SHR regardless of BH4 supplementation. Studies measured renal IM NOS enzymatic activity in the presence and absence of BH4 from 12-13 week old control (N=5-6) or antioxidant treated (tempol, 30 mg/kg/day in drinking water) male and female SHR (N=4-5). Supplementation with BH4 increased NOS activity in male SHR (177±35 vs. 315±52 pmol/mg; p=0.04), with no effect in females (434±112 vs. 548±84 pmol/mg). Following 2 weeks of antioxidant treatment, NOS enzymatic activity in male SHR was significantly increased compared to control SHR (p=0.04), and supplementation with BH4 no longer altered NOS enzymatic activity in males (588±143 vs. 648±153 pmol/mg). Antioxidant treatment did not significantly alter renal IM NOS enzymatic activity in female SHR compared to untreated controls, and BH4 supplementation did not alter NOS activity in tempol-treated females (473±82 vs. 580±66 pmol/mg). Tempol treatment also abolished the sex difference in renal IM NOS enzymatic activity. In conclusion, our data suggest that greater levels of oxidative stress in male SHR contributes to lower levels of NOS enzymatic activity compared to females. We hypothesize that this results in greater BH4 deficiency in males contributing to decreases in NO and increases in blood pressure.

Abstract 158: Sex Differences in Response to Chronic Administration of Immunosuppressive Agents in Spontaneously Hypertensive Rats

Hypertension in male spontaneously hypertensive rats (SHR) is thought to be mediated in part by enhanced immune function. Tacrolimus (FK-506) and Mycophenolate Mofetil (MMF); potent immunosuppressive agents are widely used for the prevention of graft rejection in organ transplantation. Tacrolimus inhibits proliferation of T cells, whereas MMF inhibits proliferation of T cells, B cells and monocytes. However, the impact of the drug on the cardiovascular system of different sexes is unknown. The present study tested the hypothesis that there are sex differences in response to FK-506 and/or MMF in SHR. Young male (YM, age 3 mos; n= 6) and young female (YF, aged 3 mos; n= 6) SHR received tacrolimus (0.25 mg/kg/day i.p. for 14 d) and (YM, age 3 mos; n= 6) and young female (YF, aged 3 mos; n= 6) SHR received MMF (20 mg/kg/day i.p.) for 14 d. Rats were implanted with radiotelemeters and mean arterial pressure (MAP) was measured chronically. Tacrolimus increased MAP in males (Control: 143 ±3 mmHg vs. Tacrolimus-treated: 163 ±4 mmHg, p=S, p&lt;0.05), but had no effect in females (Control: 132 ±3 mmHg vs. Tacrolimus-treated: 133 ±2 mmHg, p=NS). In contrast, Mycophenolate Mofetil significantly reduced MAP in both males (Control: 153 ±2 mmHg vs. MMF-treated: 140 ± 2 mmHg; p &lt;0.05), and females (Control: 128 ±2 mmHg vs. MMF-treated: 114 ±2 mm Hg, p&lt;0.05). These data suggest that immunosupproessive drugs may have different effects in men and women.

Effect of tempol on myocardial vascular remodeling in female spontaneously hypertensive rats.

The present study evaluated whether the treatment with the superoxide anion dismutase mimetic tempol prevents the worsening in hypertension and in myocardial vascular remodeling induced by ovariectomy in female spontaneously hypertensive rats (SHR). Experiments were performed in ten week old female SHRs randomly assigned to the groups: intact (INT: given vehicle; INT+T: treated with tempol, 90 mg/kg/day), ovariectomized (OVX: vehicle and OVX+T: tempol, respectively) and ovariectomized treated with 17β-estradiol (OVX+E2 and OVX+E2+T). Evolution of systolic blood pressure (SBP) was determined every other week in lightly restrained awake rats using a noninvasive computerized tail-cuff plethysmography system. At 18 weeks of age the heart was excised and structural changes in histopathological sections of coronary vessels were quantified on a computerized imaging system analyzer. SBP was significantly lower in female SHRs treated with tempol compared to the values measured in untreated animals. In the vascular remodeling of myocardial arterioles, OVX+T rats had a lower media cross sectional area and media-to-lumen ratio than those observed in the OVX SHR. Interestingly, treatment with tempol in the presence of estradiol (in female INT and OVX+E2 SHR ) increased media cross sectional area and wall-to-lumen ratio of myocardial arterioles, despite the fact that it lowered arterial pressure in those groups. These results indicate that tempol prevents arterial hypertension and blunts myocardial vascular remodeling in ovariectomized SHR. Paradoxically, when tempol is given in presence of estradiol it has a detrimental effect on myocardial arteriolar remodeling.

Female spontaneously hypertensive rats have greater renal anti-inflammatory T lymphocyte infiltration than males

T cells contribute to hypertension in male experimental models; data in females is lacking even though women are more likely to develop immune disorders. The goal of this study was to determine whether immune cells contribute to hypertension in female spontaneously hypertensive rats (SHR) and define the T cell profile in whole blood and kidneys of male and female SHR. We hypothesized that inflammatory cells contribute to hypertension in female SHR; however, male SHR have a higher blood pressure so we hypothesize they will have a heightened inflammatory profile. The lymphocyte inhibitor mycophenolate mofetil (MMF) was administered in a dose-dependent manner to SHR. At the highest dose (50 mg·kg(-1)·day(-1)), blood pressure was significantly decreased in both sexes, yet the percent decrease in blood pressure was greater in females (female: 12 ± 1%; males: 7 ± 1%, P = 0.01). Circulating and renal T cell profiles were defined using analytical flow cytometry. Female SHR had more circulating CD3(+), CD4(+), and pro-inflammatory CD3(+)CD4(+)RORγ(+) Th17 cells, whereas males had more immune-suppressive CD3(+)CD4(+)Foxp3(+) T regulatory cells. In the kidney, females had greater numbers of CD8(+) and T regulatory cells than males, whereas males had greater CD4(+) and Th17 cell infiltration. MMF decreased circulating and renal T cells in both sexes (P < 0.0001), although the effect of MMF on T cell subtypes was sex specific with females having greater sensitivity to MMF-induced decreases in lymphocytes. In conclusion, there is a lymphocyte contribution to the maintenance of hypertension in the female SHR and sex of the animal impacts the T cell profile.

Female Spontaneously Hypertensive Rats (SHR) have greater increases in NOS in mesenteric arteries than males

Female SHR have greater levels of Ang (1–7) and greater NO production than male SHR. This study tested the hypothesis that infusion of Ang (1–7) increases NO synthase (NOS) activity and expression more effectively in female SHR than in males. 11 week old male and female SHR received minipumps to deliver Ang (1–7) (24 μg/kg/hr; n=3–13). After 2 week, the renal cortex and mesenteric arteries were isolated to assess total NOS activity, NOS1 and NOS3 protein expression. NOS activity and protein expression were similar in the renal cortex of untreated male and female SHR and Ang (1–7) infusion did not alter NOS activity or protein expression in either sex. Similarly, NOS activity and protein expression were comparable in mesenteric arteries from untreated female and male SHR. Ang (1–7) infusion increased NOS activity in mesenteric arteries from female (47±5 to 82±12 pmol/mg, p≤.01) and male SHR (38±3 to 50±6 pmol/mg, p≤.05); however the increase in NOS activity was greater in females. Increased NOS activity was not associated with changes in NOS1 protein expression in either sex, however, Ang (1–7) increased NOS3 (endothelial NOS) protein expression in females (0.6±0.05 to 0.8±0.08 densitometric units, p≤.01), with no effect in male SHR. In conclusion, female SHR more sensitive to tissue specific Ang (1–7) mediated increases in NOS activity and expression, which may contribute to lower blood pressure in female SHR.