Hypertensive female Sprague-Dawley rats require an intact nitric oxide synthase system for compensatory increases in renal regulatory T cells

Abstract

We have previously shown that hypertensive female rats have more regulatory T cells (Tregs), which contribute more to blood pressure (BP) control in female versus male rats. Based on known protective properties of Tregs, the goal of the present study was to investigate the mechanisms by which female rats maintain Tregs. The present study was designed to 1) compare the impact of three hypertension models on the percentage of renal Tregs and 2) test the hypothesis that nitric oxide synthase (NOS) inhibition prevents increases in renal Tregs and exacerbates renal damage in female Sprague-Dawley rats. Rats (11–14 wk old) were randomized to one of the following four groups: control, norepinephrine (NE) infusion, angiotensin II infusion, or the NOS inhibitor N^ω-nitro-l-arginine methyl ester (l-NAME) in drinking water. BP was measured via tail cuff. After 2 wk of treatment, kidneys were isolated and processed to measure Tregs via flow cytometric analysis and renal injury via urinary albumin excretion, plasma creatinine, and histological analyses. Hypertensive treatments increased BP in all experimental animals. Increases in BP in norepinephrine-and angiotensin II-treated rats were associated with increases in renal Tregs versus control. In contrast, l-NAME treatment decreased Tregs compared with all groups. l-NAME treatment modestly increased albumin excretion. However, plasma creatinine was comparable among the groups, and there was no histological evidence of glomerular or tubular injury. This study provides insights into the mechanisms regulating renal Tregs and supports that an intact NOS system is crucial for female rats to have BP-related increases in renal Tregs.

INTRODUCTION

Clinical (58) and experimental (9, 10, 43, 46, 56) data have shown that young healthy female subjects have a lower incidence of hypertension compared with their age-matched male counterparts. Lower blood pressure (BP) in female versus male animals has been reported in numerous rat models of hypertension, including spontaneously hypertensive rats (SHRs) (12, 46), N^ω-nitro-l-arginine methyl ester (l-NAME)-induced hypertension (9), DOCA-salt-induced hypertension (56), and angiotensin II (ANG II)-induced hypertension (23, 43). Female rats have also been found to have less hypertensive renal injury than age-matched male rats (47). These data suggest a protective effect of the female sex against high BP and hypertensive renal injury. However, the mechanisms conferring cardiovascular and renal protection in female rats are still being investigated.

It is now well established that immune cells contribute to the development of hypertension and end-organ damage in both sexes (54, 55). In particular, T cells are required for the development of hypertension (21, 32, 42). However, not all T cells are prohypertensive. T cell subtypes consist of both prohypertensive T cells and antihypertensive regulatory T cells (Tregs). Adoptive transfer of Tregs limits increases in BP in both male and female subjects (2, 24). Tregs have also been shown to be protective in experimental models of renal injury in male rodents (25, 27). Little is known regarding the role of Tregs in modulating renal injury in female subjects.

There are distinct sex differences in the T cell profile in hypertension. Young adult female SHR and Dahl salt-sensitive rats with established hypertension have more aortic and renal Tregs than male rats (52, 54). Additionally, 13-wk-old female SHRs (53) and female Sprague-Dawley rats treated with ANG II for 2 wk (61) exhibit a compensatory increase in renal Tregs in response to increases in BP that are not seen in male rats. These data suggest that Tregs serve as an important feedback mechanism in female rats that may account for the consistently lower BP relative to male rats. The mechanisms responsible for sex differences in the T cell profile remain unknown.

Evidence in the literature shows that nitric oxide (NO) (35, 60) promotes Treg differentiation and development, and female subjects have greater NO synthase (NOS) expression (34), activity (48), and NO production (48) than male subjects. Although there are compensatory increases in renal Tregs in female SHRs with the development and maintenance of hypertension (53), pharmacological inhibition of NOS with l-NAME did not increase renal Tregs in female SHRs (9). However, l-NAME treatment resulted in pronounced increases in BP and renal injury. These data suggest that an intact NOS system is required for female SHRs to increase Tregs with increases in BP.

However, l-NAME-treated female SHRs have extreme increases in BP (∼200 mmHg mean arterial BP) approaching malignant hypertension, which may be a confounding variable. Moreover, SHRs are a model of essential hypertension (36) and numerous pathways have been shown to contribute to increases in BP in SHRs, many of which are interconnected (14, 16). Therefore, the present approach was designed to limit these potential confounding variables by using female Sprague-Dawley rats infused with a single hypertensive agent. To determine whether an intact NOS system is required for hypertensive female rats to upregulate Tregs, female Sprague-Dawley rats were randomized to receive norepinephrine (NE), ANG II, or l-NAME, and renal Tregs were measured. NE, ANG II, and l-NAME act through distinct pathways to induce hypertension.

The present study sought to 1) compare the compensatory increases in renal Tregs in three distinct models of hypertension and 2) test the hypothesis that NOS inhibition will prevent compensatory increases in renal Tregs and will exacerbate renal damage in female Sprague-Dawley rats. The major finding of the present study was that despite marked increases in BP among all hypertensive groups, l-NAME-treated rats did not show the compensatory increase in Tregs observed in the other experimental groups.

MATERIALS AND METHODS

Animals.

All work with animal subjects was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Augusta University Institutional Animal Care and Use Committee. Female Sprague-Dawley rats were purchased from Envigo (11–14 wk of age) and upon arrival were given 1 wk to acclimate. All rats were group housed on a 12:12-h light-dark cycle with standard chow (Envigo Teklad 2918-041420M) and water available ad libitum. Rats were then randomized to the following groups: 1) untreated control (n = 21), 2) implantation with sterile osmotic minipumps to deliver NE (1.25 µg·kg⁻¹·min⁻¹, n = 11, A7256-5G, Aldrich Chemistry), 3) implantation with sterile osmotic minipumps to deliver ANG II (400 ng·kg⁻¹·min⁻¹, n = 10, no. 002-12, Phoenix Pharmaceuticals), or 4) l-NAME (7 mg·kg⁻¹·day⁻¹, n = 11, N5751-25G, Sigma Life Sciences) in drinking water. For rats that received l-NAME, animal weights and volume of water consumed were measured daily. l-NAME-contining water was made fresh every 3 days to maintain constant dosage. Age-matched female Sprague-Dawley rats maintained on tap water served as controls for each group. After 2 wk of treatment, all rats were anesthetized with ∼2.5% isoflurane. An abdominal incision was made, and a terminal blood sample was collected into a syringe containing 7.5% EDTA (in diH₂O, pH 7.5–8.0). Kidneys were isolated for histological analysis.

Minipump implantation.

The animal was anesthetized with 1.5% isoflurane and placed dorsal side up. Consciousness was measured via toe pinch. The midscapular region was shaved and cleaned with 70% ethanol and betadine. A midscapular incision was made, a small hemostat was inserted, and the jaws opened and then retracted from the incision to spread the subcutaneous tissue. This process was repeated until there was a pocket big enough to hold the osmotic minipump (Alzet). The minipump was inserted with the flow moderator end first. The skin was closed with wound clips, and the area was cleaned with betadine and 70% ethanol. Marcaine was used as a topical anesthetic (0.1 mL/animal). Animals were placed in their home cage under a heat lamp and monitored until conscious.

Metabolic cage experiments.

Rats were placed in metabolic cages at baseline and after 2 wk of treatment. All rats were weighed and placed in individual metabolic cages for an acclimation period of 24 h, during which no data were collected (n = 11–21/group). Following this acclimation period, water bottles, food containers, and urine tubes were emptied and weighed. Metabolic cage data were collected 24 h later (volume of urine excreted, volume of water consumed, and amount of food consumed). Urine samples were centrifuged at 3,000 rpm for 10 min at 4°C, aliquoted, and stored at −80°C.

BP measurement.

Systolic BP (SBP) was measured in the following three separate subsets of rats via tail-cuff plethysmography (IITC Life Sciences BP system) at baseline and after 2 wk of treatment: 1) control and l-NAME treated, 2) control and NE treated, and 3) control and ANG II treated (n = 5/group). All rats were acclimated to the tail-cuff machine by performing several training sessions in the week before the initial data collection and randomization to treatment groups. During each training session, rats were placed in the restraining tube and the chamber was heated to ∼37°C. Tail cuffs were inflated one at a time for a total of 10–20 inflations/rat, a period that totaled ∼15 min. During data collection sessions, at least five BP readings were collected per animal and averaged.

Flow cytometric analysis.

A separate set of control, l-NAME-, NE-, and ANG II-treated female Sprague-Dawley rats (n = 6/group) was used to measure renal T cells via flow cytometric analysis. The protocol has been previously described (54) and was used with slight modifications. All four groups were processed and included in flow cytometric analyses together to allow for direct comparisons of T cells. After 2 wk of treatment, animals were anesthetized under 1.5% isoflurane and exsanguinated, and hearts were removed. Half of the left kidney was isolated via a transverse cut and placed in RPMI media supplemented with 10% FBS. Single cell suspensions of kidneys were achieved using a 100 μM cell strainer (BD Biosciences, San Diego, CA) followed by centrifugation at 1,400 rpm for 5 min; the pellet was retained. The pellet was incubated for 3 min in 3 mL of ammonium-chloride-potassium lysis buffer and then washed with 1× PBS (in diH₂O, no. 2810305, MP Biomedicals) and centrifuged (1,400 rpm for 5 min). The pellet was brought to a final volume of 5 mL with RPMI media supplemented with 10% FBS. Cells (200 µL of the resulting cell suspension) were then incubated for 20 min on ice in the dark with 2 µL of antibody to the T cell surface markers CD3 (catalog no. 46-0030-82, RRID: AB_2573666, ThermoFisher Scientific) and CD4 (catalog no. 554837, RRID: AB_395547, BD Biosciences). Samples were washed with PBS, 500 µL Fix/Perm (eBioScience) was added to each tube, and cells were incubated on ice for 15 min. Cells were washed with 1× PBS before incubation on ice for 15 min in the dark with 2 µL of antibody for intracellular staining of FoxP3 to identify Tregs (catalog no. 17-5773-82, RRID: AB_469457, ThermoFisher Scientific). Cells were again washed, fixed in 500 µL of 4% paraformaldehyde, and run through a four-color flow cytometer (FACS Calibur, BD Biosciences). Data were collected (CytExpert software version 2.0), and total T cells and Tregs were quantified (FlowJo, version 10). Total cells were gated to include lymphocytes; gating excluded dead cells and debris using forward and side scatterplots (Fig. 1A). To measure total T cells, lymphocytes were gated for expression of CD3⁺ cells (Fig. 1B). To measure total Tregs, CD3⁺ cells were further gated for expression of CD4⁺ and FoxP3⁺ cells (Fig. 1C). Proper compensation was set to ensure that the median fluorescence intensities of negative and positive cells were identical and was then used to gate the population. To confirm the specificity of primary antibody binding and rule out nonspecific Fc receptor binding to cells or other cellular protein interactions, all appropriate single-color controls were included in all experiments.

Fig. 1.

Gating strategy for flow cytometry. Total cells were gated for lymphocytes (A), which were further gated for total CD3⁺cells (B). Finally, CD3⁺cells were gated to include CD4⁺ and FoxP3⁺ cells to identify regulatory T cells (Tregs; C). Q1−Q4, quadrants 1−4, respectively.

Assessment of renal injury.

All renal injury measurements were collected in a subset of animals (control group: 11, l-NAME-treated group: 8, NE-treated group: 5, and ANG II-treated group: 6). Renal structure and fibrosis were assessed in formalin-fixed tissue slices. Briefly, the right kidney was isolated, the poles were removed, and the remainder of the kidney was placed in 10% formalin for ∼24 h. Formalin was replaced with 70% ethanol, and kidneys were then paraffin embedded and sectioned at a thickness of 4 or 15 µm onto Superfrost plus slides. All experimental groups and the control group were represented in each batch of staining to assess injury, and all scoring was performed in a blinded manner. Glomerular injury was assessed via Gomori’s trichrome staining. Five-micrometer-thick kidney sections were stained according to the manufacturer’s instructions (catalog no. 87020, Thermo Scientific Richard-Allan Scientific, Chromaview). Each sample was assigned an injury percentage from 0% to 100% based on the amount of collagen deposition graded by visual inspection. Tubular injury was assessed via hematoxylin and eosin staining of 4-μm-thick kidney slices according to the manufacturer’s instructions (Leica Biosystems, Buffalo Grove, IL). Assessment of tubular epithelial necrosis, brush-border loss, protein cast formation, and apoptotic bodies was performed, and each sample was assigned an injury percentage from 0% to 100% graded by visual inspection. Each sample was then assigned a score of 0–5 according to the following scoring system: 0 = 0–4%, 1 = 5–19%, 2 = 20–39%, 3 = 40–59%, 4 = 60–79%, and 5 = 80–100%. A score of 0 indicates no injury; a score of 5 indicates maximal injury. For both glomerular and tubular injury, a total of 8−10 images/kidney were taken, and injury scores were averaged. Renal sections were imaged (Olympus DP12, Olympus America, Melville, NY) at ×20 magnification (Olympus BX40 microscope, Olympus America) using a bright-field setting. Fibrosis was also measured in 15-μm-thick kidney slices via a Sirius Red/Fast Green Collagen Staining Kit according to the manufacturer’s instructions (catalog no. 9046, Chondrex). Twenty-four-hour urine samples were used to measure urinary albumin excretion via ELISA (Nephrat II Rat Albumin ELISA, Ethos Biosciences). Creatinine concentrations were quantified by a QuantiChrom creatinine assay kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s instructions.

Statistical analysis.

Data were analyzed with Graphpad Prism software (version 7.03). All data are shown as means ± SE. Between-group comparisons were made via one-way ANOVA followed by Tukey post hoc tests. Within-group comparisons of metabolic cage data were made via a dependent-samples t test. Within-group comparisons of BP were analyzed via repeated-measures (RM) ANOVA with a Tukey post hoc test. Significance for all analyses was set at P ≤ 0.05.

RESULTS

Weight and metabolic cage data.

To determine the effect of l-NAME, NE, and ANG II treatments on food intake, water intake, and urine excretion, 24-h metabolic cage data were collected at baseline and after 2 wk of treatment. At baseline (Table 1), all groups consumed similar amounts of water (P_ANOVA = 0.79) and food (P_ANOVA = 0.33), excreted similar amounts of urine (P_ANOVA = 0.68), and had comparable body weights (P_ANOVA = 0.54).

Table 1.

Metabolic cage data at baseline and after 2 wk of hypertensive treatment in young female Sprague-Dawley rats

	Control (n = 21)	l-NAME (n = 11)	NE (n = 11)	ANG II (n = 10)	P Value
Baseline
Water intake, mL	22.9 ± 1.6	24.7 ± 1.1	24.8 ± 1.0	24.0 ± 2.1	0.7855
Food intake, g	16.5 ± 1.1	18.4 ± 0.7	18.4 ± 0.6	18.2 ± 1.3	0.3318
Urine excretion, mL	9.8 ± 1.1	11.5 ± 1.2	11.9 ± 1.0	10.9 ± 2.0	0.6753
Weight, g	218 ± 2	220 ± 2	221 ± 3	216 ± 2	0.5355
Week 2
Water intake, mL	20.6 ± 1.9	28.7 ± 3.4	30.9 ± 2.7*†	26.9 ± 2.2	0.027
Food intake, g	21.9 ± 3.0	12.7 ± 1.3†	21.1 ± 3.4	13.8 ± 1.3	0.0561
Urine excretion, mL	10.3 ± 1.1*	20.3 ± 3.2*†	19.3 ± 1.9*†	18.0 ± 2.1*†	0.0004
Weight, g	242 ± 3†	232 ± 2†‡	246 ± 3†	241 ± 3†	0.0407

Values are means ± SE. l-NAME, N^ω-nitro-l-arginine methyl ester; NE, norepinephrine; ANG II, angiotensin II. Metabolic cage data were collected via 24-h metabolic cage experiments at baseline and following 2 wk in untreated female rats and in rats randomized to receive l-NAME, NE, or ANG II. Between-group comparisons at each time point were made via one-way ANOVA followed by a Tukey post hoc test; P values for the ANOVA are as shown. Within-group comparisons were made via a dependent-samples t tests.

^*P < 0.05 vs. control;

^†P < 0.05 vs. baseline;

^‡P < 0.05 vs. NE.

All animals gained weight over the 2-wk treatment period compared with baseline body weights (P_{t test} < 0.05). NE-treated rats exhibited an increase in water intake following 2 wk of treatment (P_{t test} = 0.029); water intake was comparable with baseline in the other hypertensive groups (l-NAME: P_{t test} = 0.32 and ANG II: P_{t test} = 0.39). As a result, water intake was greater in NE-treated rats versus control rats after 2 wk of treatment (P_ANOVA = 0.03). Food intake decreased in l-NAME-treated rats (P_{t test} = 0.005) but was maintained in NE-treated rats (P_{t test} = 0.43) and ANG II-treated rats (P_{t test} = 0.10). As a result, increases in body weight were attenuated in l-NAME-treated rats, and, following 2 wk of treatment, l-NAME rats had a lower body weight versus NE-treated rats (P_ANOVA = 0.04 and P_{post hoc} = 0.03). All hypertensive rats had an increase in urine excretion versus baseline (l-NAME-treated rats: P_{t test} = 0.02, NE-treated rats: P_{t test} = 0.006, and ANG II-treated rats: P_{t test} = 0.03), and urine excretion was greater in all hypertensive groups versus the control group (P_ANOVA = 0.0004 and P_{post hoc} < 0.05).

l-NAME-, NE-, and ANG II-induced hypertension in female Sprague-Dawley rats.

To confirm that treatments increased BP, SBP was measured via tail-cuff plethysmography in three separate groups of female rats at baseline and following 1 and 2 wk of treatment with l-NAME, NE, or ANG II (n = 5); each group included control female Sprague-Dawley rats (n = 5; Fig. 2A). There were significant differences in baseline SBP between groups (control: 136 ± 2 mmHg, ANG II: 128 ± 2 mmHg, NE: 144 ± 1 mmHg, and l-NAME: 132 ± 1 mmHg, P_ANOVA = 0.005). Prior to treatment, control rats had higher SBP than ANG II-treated rats (P_{post hoc} = 0.04), and SBP was higher in rats treated with NE compared with control rats and rats treated with l-NAME or ANG II (P_{post hoc} = 0.04, 0.005, and 0.0004, respectively). BP was significantly increased by all treatments versus control rats following both 1 wk (P_{RM ANOVA} < 0.0001) and 2 wk of treatment (P_{RM ANOVA} < 0.0001). There were no differences in SBP between treated groups at either week 1 or week 2. Because of significant differences in baseline BP, the percent increase in SBP from baseline values was also calculated (Fig. 2B). All treatments resulted in increases in SBP versus control rats (P_ANOVA < 0.0001). However, greater increases in BP were observed in ANG II-treated animals compared with animals treated with NE or l-NAME (P_{post hoc} < 0.05).

Fig. 2.

N^ω-nitro-l-arginine methyl ester (l-NAME), norepinephrine (NE), and angiotensin II (ANG II) induce hypertension in young female Sprague-Dawley rats. A: systolic blood pressure (BP) was measured via tail-cuff plethysmography in rats randomized to receive l-NAME (n = 5), NE (n = 5), ANG II (n = 5), or control (n = 15) treatment at baseline and after 1 wk and 2 wk of treatment. B: due to differences in baseline systolic BP (SBP) measurements, data are also presented as percent increases in SBP from baseline. Between-group comparisons at each time point were made via one-way ANOVA followed by a Tukey post hoc test; *P < 0.05 vs. control. Within-group comparisons were made via repeated-measures ANOVA; #P < 0.05 vs. baseline. Values are means ± SE.

Compensatory increase in renal Tregs was absent in l-NAME-treated rats.

To determine the effect of the hypertensive treatments on renal T cells, percentages of total T cells and Tregs were measured via flow cytometric analysis in kidneys from control, l-NAME, NE, and ANG II-treated female Sprague-Dawley rats. Total CD3⁺ T cells were comparable among all groups (P_ANOVA = 0.1; Fig. 3A). In contrast, the percentage of renal Tregs was altered among treatment groups (P_ANOVA< 0.0001; Fig. 3B). Tregs increased relative to control in rats treated with both NE (P_{post hoc} = 0.0005) and ANG II (P_{post hoc} = 0.0001). In contrast, l-NAME treatment decreased renal Tregs versus control (P_{post hoc} = 0.02), and l-NAME-treated animals had fewer Tregs than NE- and ANG II-treated animals (P_{post hoc} < 0.0001).

Fig. 3.

N^ω-nitro-l-arginine methyl ester (l-NAME) decreased renal regulatory T cells (Tregs) in young female Sprague-Dawley rats. Female Sprague-Dawley rats were randomized to receive norepinephrine (NE; n = 6), angiotensin II (ANG II; n = 6), the nitric oxide synthase inhibitor l-NAME (n = 6), or untreated control (n = 6). After 2 wk of treatment, kidneys were processed via flow cytometric analysis of total CD3⁺ cells (A) and CD3⁺CD4⁺FoxP3⁺ Tregs (B). Data in each graph were analyzed via one-way ANOVA and a Tukey post hoc test. *P < 0.05 vs. control; P_ANOVA < 0.0001 for Tregs. All data are shown as means ± SE.

Hypertensive female Sprague-Dawley rats were resistant to renal damage.

Following 2 wk of treatment, only l-NAME treatment increased urinary albumin excretion compared with control animals (P_ANOVA = 0.007; Fig. 4A). Plasma creatinine was similar among groups at the end of the 2-wk treatment period (P_ANOVA = 0.38; Fig. 4B).

Fig. 4.

Female Sprague-Dawley rats treated with N^ω-nitro-l-arginine methyl ester (l-NAME) had the highest albumin excretion. Female SD rats were randomized to receive norepinephrine (NE), angiotensin II (ANG II), the nitric oxide synthase inhibitor l-NAME, or untreated control. A: a 24-h urine sample was collected to measure urinary albumin excretion via ELISA after 2 wk of treatment. B: a terminal blood sample was collected to measure plasma creatinine via the QuantiChrom creatinine assay. Data were analyzed via one-way ANOVA. All data are shown as means ± SE.

Glomerular and tubular injury and renal fibrosis were assessed in a blinded fashion. There was no histological evidence of glomerular injury in any of the treatment groups (P_ANOVA = 0.53; Fig. 5, A and B). Fibrosis was measured using the Sirius Red/Fast Green Collagen Staining Kit, and consistent with the lack of glomerular injury, all groups had similar amounts of fibrosis measured as the ratio of collagenous to noncollagenous tissue (control: 1.7 ± 0.1, l-NAME: 2.2 ± 0.2, NE: 1.7 ± 0.2, and ANG II: 1.7 ± 0.5; P_ANOVA = 0.30). Similarly, hypertensive treatments did not induce tubular injury (P_ANOVA = 0.09; Fig. 6, A and B).

Fig. 5.

There was no glomerular injury in hypertensive female Sprague-Dawley rats. Female Sprague-Dawley rats were randomized to control (n = 5), norepinephrine (NE; n = 4), angiotensin II (ANG II; n = 5), or the nitric oxide synthase inhibitor N^ω-nitro-l-arginine methyl ester (l-NAME; n = 5) treatment. After 2 wk of treatment, kidneys were isolated and glomerular structure was assessed via Gomori’s trichrome staining and scored blindly. A: quantification of glomerular injury. B: representative trichrome-stained kidney samples. Data are presented as means ± SE. Data were analyzed via one-way ANOVA.

Fig. 6.

There was no tubular injury in female Sprague-Dawley rats. Two-week treatments were administered via osmotic minipumps for norepinephrine (NE; n = 5) and angiotensin II (ANG II; n = 6) and drinking water for the nitric oxide synthase inhibitor N^ω-nitro-l-arginine methyl ester (l-NAME; n = 8) or control (n = 11). A: slides were stained with hematoxylin and eosin staining and blindly scored for tubular injury. B: representative images stained with hematoxylin and eosin. Data in A were analyzed via one-way ANOVA. All data are shown as means ± SE.

DISCUSSION

The goals of the present study were to 1) compare the increases in renal Tregs in three distinct models of hypertension in female Sprague-Dawley rats and 2) determine whether NOS inhibition prevents increases in renal Tregs and exacerbates renal damage. The major finding of the present study was that despite having comparable increases in BP with the other treatment groups, hypertensive female rats treated with l-NAME showed decreased renal Tregs. These data support the hypothesis that an intact NOS system is required for female rats to upregulate Tregs. In addition, female Sprague-Dawley rats were relatively resistant to hypertensive renal injury.

ANG II, NE, and l-NAME treatments were selected due to their potential to induce an inflammatory response and/or their contribution to sex differences in BP control in hypertension. ANG II (4, 50) and l-NAME (9, 22) treatments are well known to promote inflammation. There are also sex differences in BP responses to these treatments. Male subjects have greater BP sensitivity to ANG II infusion than female subjects (15, 19, 40, 59), whereas female subjects are more dependent on NOS for BP control versus male subjects (10, 37, 44). We are unaware of other studies that have examined the impact of 2 wk of NE infusion on T cells in either sex or on BP responses between the sexes. Additionally, these treatments were of interest to this study based on potential differential impacts on NO; ANG II stimulates NO production (29, 39), and NE stimulates NOS activity (51). A similar high dose of l-NAME administered in this study has been previously shown to inhibit NOS (41). In contrast, lower doses have been shown to stimulate NO production (5, 30).

Treatment doses were selected to produce comparable increases in BP between groups, thereby removing BP as a variable that may affect renal T cells. Indeed, BP was not significantly different at the end of treatment among the experimental groups, although when differences in baseline BP values were taken into account, rats treated with ANG II exhibited the greatest overall increase in BP. Therefore, treatment effects with l-NAME on T cells were not mediated by a greater increase in BP. Additional studies are needed to assess the relative contribution of Tregs to BP control within each of these experimental models. We would expect decreasing Tregs to exacerbate ANG II- and NE-induced hypertension with little to no effect in l-NAME-induced hypertension.

Consistent with previously published data, the present study showed increases in renal Tregs in ANG II-treated female Sprague-Dawley rats (61). Novel data showed similar increases in renal Tregs in NE-treated female Sprague-Dawley rats. These data together demonstrate that this outcome is present in distinct models of hypertension. In contrast, l-NAME-treated female Sprague-Dawley rats in the present study exhibited a decrease in renal Tregs consistent with previously published work from Brinson et al. (9) in SHRs, supporting a critical role for an intact NOS system to induce increases in renal Tregs in hypertensive female rats. Brinson et al. (9) further went on to show that l-NAME-induced decreases in renal Tregs were less prominent in male SHRs compared with female SHRs. Because female subjects have greater NOS protein (34), enzymatic activity (48), and NO production (18) and a greater capacity for NO to induce vasodilation (31) compared with male subjects, it is tempting to speculate that males are less dependent on NO to modulate renal Tregs; this will be the topic of future research.

There are many ways that NOS may help maintain Treg populations. In vitro analysis of T cells extracted from the spleen and lymph nodes of BALB/c mice found that treatment with the NO donor NOC-18 caused proliferation of CD4⁺CD25⁻ T cells but not CD4⁺CD25⁺ T cells (35). Although these T cells did not express Foxp3, they still had suppressive actions and were able to suppress proliferation of CD4⁺CD25⁻ effector cells (35). These authors concluded that NO-induced p53 expression resulted in increased production of IL-2 and CD25 (IL-2 receptor α-chain). IL-2 has been shown to induce Treg proliferation (33) and function (13). Because only Foxp3⁺ T cells were quantified in the present study, we do not know whether NO changed the percentage of Foxp3⁻ cells. However, consistent with our results, a study in cell culture has further shown that treatment of human CD4⁺ T cells with a NO donor, NOC 18, resulted in a robust increase in the percentage of CD25⁺Foxp3⁺ Tregs by increasing FoxP3 expression in a soluble guanylyl cyclase-cGMP-dependent manner (60). However, the molecular mechanisms by which cGMP increased Tregs were not determined. Contrary to results from the present study, NO treatment decreased Foxp3 expression in naïve T cells cultured from OT-II mice on a C57BL/6 background (28). Instead, NO allowed transforming growth factor-β to preferentially develop T helper instead of Treg lineages. However, neither BP nor sex was reported in the studies with BALB/c mice or OT-II mice discussed above. Therefore, the role of NO to regulate the T cell profile may be distinct in hypertensive female rats. Additionally, treatment with NO was performed in cell culture in the studies with BALB/c and OT-II mice reported above, as opposed to the use of a whole animal strategy in the Brinson et al. study (9) and present study.

Although female Sprague-Dawley rats treated with l-NAME exhibited a modest yet significant increase in albumin excretion compared with all other groups, there was no histological evidence of glomerular or tubular injury in any of the hypertensive treatment groups. These data suggest that female Sprague-Dawley rats are relatively resistant to renal damage in response to 2-wk treatments with l-NAME, ANG II, and NE. This finding is in line with previous studies in male Wistar rats where treatment for 4 wk with l-NAME resulted in increased urinary albumin compared with controls in the absence of tubular dilation, glomerulosclerosis, or glomerular atrophy as scored by a pathologist (20) or collagen deposition (26). The average glomerular injury of the control animals was higher than zero, although small amounts of collagen deposition have been seen in the kidneys of control animals (1, 11, 57). Male Wistar rats treated with l-NAME for 12 wk showed evidence of glomerular and tubular damage (22), and male Sprague-Dawley rats treated for 3 wk with l-NAME showed glomerular sclerosis (45), suggesting that a longer treatment period may be necessary for l-NAME to cause renal damage in otherwise healthy animals.

We have previously shown that 2-wk treatment with l-NAME causes pronounced renal injury in male and female SHRs (9). Therefore, a longer treatment period of a different rat strain may be required to assess the role of Tregs in renal injury with l-NAME-induced hypertension. There is evidence in the literature suggesting that the female sex attenuates hypertensive renal injury relative to the male sex, with the most evidence to support this found with ANG II-induced hypertension. Treatment with ANG II for as little as 8 days (17) to 14 days (38) causes pronounced renal injury in male Sprague-Dawley rats. However, the degree of kidney injury is attenuated in female animals during comparable time periods. We have previously published that female Sprague-Dawley rats have minimal increases in urinary protein excretion following 2 wk of ANG II infusion compared with male rats (61), although other measures of renal injury were not assessed. Treatment of male and female SHRs with ANG II for 2 wk resulted in significant increases in renal injury in both sexes, although the degree of injury was much less severe in female SHRs (46). Female C57BL/6 mice infused with ANG II for 2 wk showed no increases in renal collagen deposition compared with controls. The mechanisms protecting female subjects from hypertensive renal injury have not been widely explored. However, renal Tregs are unlikely to explain this relative protection. In the present study, l-NAME-treated rats exhibited a decrease in renal Tregs yet no increases in renal damage compared with the other treatment groups. In addition, we recently reported that decreasing Tregs with anti-CD25 treatment in female DOCA-salt-induced hypertensive rats did not exacerbate renal damage compared with animals with intact Tregs (3).

There are some limitations in the present study. Tail-cuff plethysmography was used to measure SBP as opposed to telemetry. However, the main purpose of measuring SBP was to show an increase in experimental groups versus baseline and versus the control group. Thus, the tail cuff was accurate to measure this difference. Additionally, this method was sensitive enough to detect differences among groups at baseline. Localization of Tregs to regions of the kidney was not done in the present study, and, indeed, very little is known regarding the regional localization of Tregs in the kidney of either male or female rats. However, because female subjects are known to have a better oxygenation of the inner medulla, including higher expression of the angiotensin type II receptor (46), prostaglandins (49), and NOS (48), it is of interest to know whether the increase in renal Tregs in female rats is consistent across all regions of the kidney or found in specific regions. The whole kidney was used in flow cytometry to measure a representation of Tregs in the entire organ. Measurements of NO were not collected in the present study, but many studies have shown the effect of ANG II to increase NO (29, 39) and NE to increase NOS (51). Additionally, l-NAME has been shown to decrease NOS activity in male Wistar rats (6–8). Furthermore, this decreased activity is present in many tissues, including the aorta (6–8), kidneys (8), and brain (8).

In conclusion, novel data in the present study have demonstrated a crucial role for the NOS system to increase renal Tregs in female rats that underwent distinct hypertensive treatments. Because NO promotes differentiation and development of Tregs and female subjects have more NOS expression and availability, this is a possible mechanism through which hypertensive female subjects increase renal Tregs and could contribute to the relative protection from hypertension in young healthy female subjects.

GRANTS

This work was funded by National Institutes of Health Grants R01HL127091 and P01HL134604 (to J. C. Sullivan) and by American Heart Association Grant 17EIA33410565 (to J. C. Sullivan).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.A.R., E.E.G., and J.C.S. conceived and designed research; L.A.R., E.E.G., J.B.M., E.S., and J.C.S. performed experiments; L.A.R., E.E.G., R.M., and A.A.E.-M. analyzed data; L.A.R., R.M., and J.C.S. interpreted results of experiments; L.A.R., R.M., and A.A.E.-M. prepared figures; L.A.R. drafted manuscript; L.A.R., E.E.G., J.B.M., R.M., E.S., A.A.E.-M., and J.C.S. edited and revised manuscript; L.A.R., E.E.G., J.B.M., R.M., E.S., A.A.E.-M., and J.C.S. approved final version of manuscript.