A new mechanism for the sex differences in angiotensin II-induced hypertension: the role of macula densa NOS1β-mediated tubuloglomerular feedback

Abstract

Females are protected against the development of angiotensin II (ANG II)-induced hypertension compared with males, but the mechanisms have not been completely elucidated. In the present study, we hypothesized that the effect of ANG II on the macula densa nitric oxide (NO) synthase 1β (NOS1β)-mediated tubuloglomerular feedback (TGF) mechanism is different between males and females, thereby contributing to the sexual dimorphism of ANG II-induced hypertension. We used microperfusion, micropuncture, clearance of FITC-inulin, and radio telemetry to examine the sex differences in the changes of macula densa NOS1β expression and activity, TGF response, natriuresis, and blood pressure (BP) after a 2-wk ANG II infusion in wild-type and macula densa-specific NOS1 knockout mice. In wild-type mice, ANG II induced higher expression of macula densa NOS1β, greater NO generation by the macula densa, and a lower TGF response in vitro and in vivo in females than in males; the increases of glomerular filtration rate, urine flow rate, and Na⁺ excretion in response to an acute volume expansion were significantly greater and the BP responses to ANG II were significantly less in females than in males. In contrast, these sex differences in the effects of ANG II on TGF, natriuretic response, and BP were largely diminished in knockout mice. In addition, tissue culture of human kidney biopsies (renal cortex) with ANG II resulted in a greater increase in NOS1β expression in females than in males. In conclusion, macula densa NOS1β-mediated TGF is a novel and important mechanism for the sex differences in ANG II-induced hypertension.

INTRODUCTION

Hypertension, defined by blood pressure (BP) ≥ 130/80 mmHg (64), is a leading risk factor for cardiovascular morbidity and mortality (13, 15), affecting ∼30% of adults in the United States (72). In addition, the prevalence of hypertension is lower in women, before menopause, compared with age-matched men (1, 2, 39, 50). However, the underlying mechanisms for the female sex-related protective benefit on BP have not been fully clarified.

The renin-angiotensin-aldosterone system is one of the most important mechanisms in the control of BP (26, 35, 49, 54). Chronic administration of subpressor doses of angiotensin II (ANG II) is a well-established animal model in various species that mimics the general characteristics of hypertension in clinical patients (10, 11, 34, 44). Moreover, blood pressure responses to ANG II are significantly lessened in female animals compared with males (27, 36, 41, 66). Although the exact mechanisms underlying the sex differences in ANG II-induced hypertension have not been completely elucidated, a large amount of evidence has indicated a potential implication of the neuronal nitric oxide (NO) synthase (NOS1)-derived NO (67–69). A 7-day systemic ANG II infusion has been reported to result in a greater increase in NOS1 protein expression in the subfornical organ and paraventricular nucleus in female than male C57BL/6 mice (67). Furthermore, the central nonselective NOS inhibition with N^G-nitro-l-arginine methyl ester and even the central selective NOS1 inhibition with N⁵-(1-imino-3-butenyl)-l-ornithine were found to significantly potentiate BP responses to ANG II in females but not in males, thereby diminishing the sex differences in ANG II-induced hypertension (69).

Besides the brain, NOS1 is also highly expressed in the kidneys, in particular, at the macula densa (25, 31, 32, 43). NOS1 exists as various isoforms. Alternative 5′-end splicing of NOS1 mRNA results in at least three different NH₂-terminal protein variants, which are NOS1α at 155 kDa, NOS1β at 135 kDa, and NOS1γ at 125 kDa (5, 12, 16, 17). NOS1β has ∼80% of the catalytic activity of NOS1α, whereas NOS1γ has only 2% of the catalytic activity of NOS1α (5, 22). Moreover, we recently found that multiple splice variants of NOS1 are expressed in the macula densa. In addition, NOS1β is the primary isoform of NOS1, and it is responsible for most of the NO production in the macula densa, which is distinct from the pattern of NOS1 splice variants in the brain, where NOS1α is the major isoform (30, 59). NO generated by NOS1β in the macula densa is a key modulator of the tubuloglomerular feedback (TGF), which buffers or attenuates the TGF response via a cGMP-dependent pathway (25, 30, 42, 65). TGF describes an important intrinsic mechanism in the control of renal hemodynamics wherein an increase in NaCl delivery to the macula densa promotes the release and formation of ATP and/or adenosine, which constricts the afferent arteriole and induces an inhibition of single-nephron glomerular filtration rate (GFR) (9, 47, 51, 55).

Recently, several studies from our laboratory have demonstrated the effect of ANG II on macula densa NOS1β-mediated TGF responsiveness as well as the significance of the macula densa NOS1β-mediated TGF mechanism in ANG II-induced hypertension (28, 30, 48). Nevertheless, all of these previous studies were undertaken in male animals without a comparison with female animals. It is unknown whether the effect of ANG II on macula densa NOS1β-mediated TGF responsiveness is different in females, and the significance of the macula densa NOS1β-mediated TGF mechanism in the sexual dimorphism of ANG II-induced hypertension remains to be determined.

In the present study, we hypothesized that ANG II induces greater increases in macula densa NOS1β expression and activity in females than in males; the greater macula densa NOS1β-derived NO generation in females promotes lower TGF response and higher GFR, thereby facilitating Na⁺ excretion and protecting against ANG II-induced hypertension. A set of sophisticated techniques including microperfusion of juxtaglomerular apparatus (JGA) in vitro, micropuncture of renal tubules in vivo, clearance kinetics of FITC-inulin in conscious animals, and radio telemetry for continuous monitoring of BP were used to examine the sex differences in the effects of ANG II on NOS1β expression and NO generation in the macula densa, TGF responsiveness, natriuresis, and BP in both wild-type and macula densa-specific NOS1 knockout (MD-NOS1KO) mice. In addition, human kidney biopsy samples from cadaveric kidney donors were used to further evaluate the clinical significance of the macula densa NOS1β.

METHODS

Animals.

Animal experiments were conducted in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals with all animal protocols approved by the Institutional Animal Care and Use Committee at the University of South Florida Morsani College of Medicine. C57BL/6 mice (both male and female, 8–10 wk old) were purchased from Jackson Laboratory. MD-NOS1KO (NKCC2^cre/NOS1^flox/flox) mice were generated by crossing NKCC2^cre mice with NOS1^flox/flox mice as we have previously described (30). Mice were housed individually at 23°C on a 12:12-h light-dark cycle while being maintained on water and food (Envigo, Indianapolis, IN) ad libitum.

Implantation of the ANG II-loaded microosmotic pump.

The ANG II-loaded microosmotic pump (model 1002, Alzet, Cupertino, CA) was subcutaneously implanted in mice as we have previously described (30, 73, 75, 78). Briefly, the microosmotic pump was filled with ANG II (600 ng ·kg⁻¹·min⁻¹) and then incubated in sterile saline overnight at 37°C to reach a steady state. The animal was anesthetized with inhaled isoflurane via vaporizer (Vaporizer Sales & Service, Rockmart, GA). Under aseptic conditions, a small incision was made in the midscapular region on the back, and the subcutaneous tissue was spread with a hemostat to create a small pouch. The microosmotic pump was then inserted into the pouch, and the wound was sutured. The microosmotic pump filled with saline was used as the vehicle control.

Measurement of NOS1 expression by Western blot analysis.

As previously described (60–62, 77), protein extracts from renal cortical tissue were separated on 7.5% SDS-PAGE gels (50 μg/lane). After being blocked with 5% skim milk for 1 h at room temperature, membranes were incubated with a COOH-terminal NOS1 antibody (no. 610309, mouse polyclonal IgG, 1:3,000, BD Biosciences, San Jose, CA) overnight at 4°C (30, 77). After being washed three times for 10 min, membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (no. 0300-0108P, goat anti-mouse IgG, 1:300,000, Bio-Rad, Hercules, CA) for 1 h at room temperature. After another three washes for 10 min, the immunoreactive bands were revealed by ChemiDoc System (Bio-Rad) and qualified by ImageLab software (Bio-Rad). For normalization, membranes were stripped with Restore Western Blot Stripping Buffer (Fisher Scientific, Waltham, MA) for 15 min at room temperature and incubated with a β-actin antibody (A5441, mouse monoclonal IgG, 1:5,000, Sigma, St. Louis, MO) (30, 77). The aforementioned COOH-terminal NOS1 antibody has been validated by the KO models (NOS1KO and MD-NOS1KO mice) as well as the expected band size and pattern (molecular weights: NOS1α at 155 kDa and NOS1β at 140 kDa) in our previous studies (30, 59).

Measurement of NO generation at the macula densa in the isolated and perfused JGA.

NO generation at the macula densa was measured in isolated and perfused mouse JGAs with a fluorescent NO probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2 DA) as previously described (30, 61, 74, 78). Briefly, a single afferent arteriole along with its intact glomerulus as well as adherent tubular segments consisting of the thick ascending limb (TAL), macula densa, and early distal tubule were microdissected from fresh mouse kidney slices within 30 min in ice-cold DMEM containing 5% BSA under a stereomicroscope (SMZ1500, Nikon, Yuko, Japan). The isolated JGA was then transferred to a temperature-regulated chamber mounted on an inverted microscope (Axiovert 100TV, Zeiss), in which the bath solution (total volume of 1.5 mL) was exchanged continuously at a rate of 1 mL/min and the temperature was maintained at 37°C throughout the experiment. The afferent arteriole was cannulated with a set of glass pipettes and perfused with DMEM at 60 mmHg. The TAL was cannulated with another set of glass pipettes and perfused with macula densa solution (containing 10 mM HEPES, 1.0 mM CaCO₃, 0.5 mM K₂HPO₄, 4.0 mM KHCO₃, 1.2 mM MgSO₄, 5.5 mM glucose, 0.5 mM Na acetate, 0.5 mM Na lactate, 0.5 mM l-arginine, and 10/80 mM NaCl; pH: 7.4). Following a 30-min equilibration, the macula densa was loaded with DAF-2 DA (10 μM plus 0.1% pluronic acid) through the lumen of TAL for 30 min and then washed for 15 min with macula densa solution. DAF-2 was excited at 490 nm, and the emitted fluorescence was recorded at wavelengths of 510–550 nm. The rate of increase in the fluorescent intensity of DAF-2 was used as the index of NO generation at the macula densa. The imaging system was composed of a microscope (Eclipse Ti, Nikon, Tokyo, Japan), a digital charge-coupled device camera (CoolSnap, Photometrics, Tucson, AZ), a xenon light (LB-LS/30, Sutter Instruments, Novato, CA), and an optical filter changer (Lambda 10-3, Sutter Instrument). Images were displayed and analyzed with NIS-Elements imaging software (Nikon).

Measurement of TGF in vivo with micropuncture.

The TGF response in vivo was measured in anesthetized mice with micropuncture as previously described (61, 63, 77, 78). Briefly, mice were anesthetized with inactin (70 mg/kg ip) plus ketamine (50 mg/kg im) and placed on a temperature-controlled table kept at 37°C throughout the experiment. A tracheostoma was cannulated to facilitate respiration. The right femoral artery was catheterized to monitor BP with a pressure transducer (ADInstruments, Sydney, NSW, Australia), and the left femoral vein was catheterized to infuse saline solution with 2% BSA at a rate of 1 mL/h per 100 g body wt throughout the experiment. Following an abdominal incision, the left kidney was exposed and immobilized in a kidney holder cup. With a clear visualization of the superficial tubules under a microscope (SZX16, Olympus, Tokyo, Japan), a selected proximal tubule with multiple visible loops was obstructed with a grease block. The stop-flow pressure (P_sf) in the proximal tubule upstream of the grease block was measured by the servo-nulling method with a pressure micropipette (model 900A, World Precision Instruments, Sarasota, FL). The segment of the proximal tubule distal to the grease block was perfused with artificial tubular fluid (ATF; containing 4 mM NaHCO₃, 5 mM KCl, 2 mM CaCl₂, 7 mM urea, 2 mM MgCl₂, 128 mM NaCl, and 1% fast green; pH 7.4) via a perfusion micropipette. P_sf was continuously recorded while the tubular perfusion rate was switched from 0 to 40 nL/min. The change in P_sf (ΔP_sf) was used as the index of TGF response in vivo.

Measurement of the natriuretic response following acute volume expansion.

The natriuretic response to acute volume expansion was measured in anesthetized mice as previously described (30, 78). Briefly, mice were anesthetized with inactin (70 mg/kg ip) plus ketamine (50 mg/kg im) and placed on a temperature-controlled table kept at 37°C throughout the experiment. A tracheostoma was cannulated to facilitate respiration. The left femoral vein was catheterized to infuse saline solution with 2% BSA and 2 mg/mL FITC-inulin at a rate of 1 mL/h per 100 g body wt. The right femoral artery was catheterized to monitor BP with a pressure transducer (ADInstruments) and to collect plasma samples. The left ureter was catheterized to collect urine samples. After a 30-min equilibration, the urine and plasma samples were collected during a 30-min control period. The animal then received an intravenous infusion of isotonic saline (3% of body wt) over a 5-min period. The urine and plasma samples were collected during a 0- to 60-min period and a 60- to 90-min period afterward, respectively. At the end of the experiment, the left kidney was removed and weighed. Na⁺ concentration in the urine samples was measured by using a Flame Photometer (BWB Technologies, Newbury, UK). FITC-inulin concentration in the urine and plasma samples was measured using a plate reader (Cytation3, BioTek).

Measurement of mean arterial pressure with the radio telemetry system.

Mean arterial pressure (MAP) was measured with the radio telemetry system (PA-C10, Data Sciences International) in conscious mice as previously described (73, 75, 76, 78). Briefly, mice were anesthetized with inhaled isoflurane via vaporizer (Vaporizer Sales & Service). Under aseptic conditions, a small incision was made in the middle of the neck to expose the left carotid artery. The pressure catheter of transmitter was carefully inserted into the left carotid artery and further advanced down to the aortic arch. The body of the transmitter was then placed subcutaneously in the right ventral flank. Mice were allowed 7 days of recovery before data were collected. MAP was recorded for 10 s every 1 min for 4 h each day from 1 PM to 5 PM.

Human kidney tissue culture.

Human kidney tissue was cultured with the hanging drop technique as previously described (58, 77). The human kidney biopsy samples (renal cortex) were collected from cadaveric kidney donors in the Transplant Institute of Tampa General Hospital under the approval of the Institution Review Board from Tampa General Hospital and Lifelink Foundation. Table 1 shows demographic information of the kidney donors.

Table 1.

Demographic information of the kidney donors

Donor ID	Sex	Age, yr	Ethnicity	Cause of Death
Donor 1	Male	46	Caucasian	Head trauma
Donor 2	Male	47	Caucasian	Head trauma
Donor 3	Male	30	Caucasian	Head trauma
Donor 4	Female	41	Caucasian	Head trauma
Donor 5	Female	30	Caucasian	Stroke
Donor 6	Female	28	Caucasian	Anoxia

The culture medium was DMEM containing 0.1 mM MEM nonessential amino acids, 2 mM sodium pyruvate, 2 nM l-glutamine, 0.01 mg/mL insulin, 5.5 µg/mL transferrin, 5 µg/mL selenium, 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% FBS. Briefly, a drop of the culture medium (∼30 µL) was prepared on the lid of a petri dish (no. 351008, Corning, Corning, NY). A piece of human kidney biopsy tissue (∼1 mm³) was placed into the drop, and the lid was then carefully inverted to keep the drop intact with the tissue suspended. PBS was added to the bottom of the dish to prevent dehydration. The tissue pieces were incubated at 37°C in a humidified atmosphere of 5% CO₂ and 95% air (model 3530, Fisher Scientific, Hampton, NH).

To determine the effect of ANG II on the expression of NOS1 in the macula densa, we cultured the human renal cortex tissue in the hanging drop medium with or without ANG II (10⁻⁶ M) for 60 min and then measured the protein level of NOS1 by Western blot analysis.

Immunofluorescence staining of NOS1.

The immunofluorescence staining of NOS1 in human kidney slices was performed as previously described (77). Briefly, 2-µm slices were incubated with NOS1 antibody (BML-SA227-0100, rabbit polyclonal IgG, 1:500, Enzo Biochem, Farmingdale, NY) overnight followed by fluorescent secondary antibody (ab150080, Alexa Fluor 594 goat anti-rabbit IgG, 1:1,000, Abcam, Cambridge, UK). Negative controls included sections incubated without primary and/or secondary antibodies. All slices were mounted with VECTASHIELD antifade medium with DAPI (H-1500, Vector Laboratories, Burlingame, CA). All images were captured with Nikon Eclipse E600FN Confocal Microscope equipped with a Cascade 131 512F digital camera (Photometrics, Tucson, AZ).

Statistical analysis.

Statistical analysis was performed using Prism 8 (GraphPad Software, San Diego, CA). The effects of interest were tested using two-way ANOVA or repeated-measures two-way ANOVA followed by Sidak’s multiple-comparisons test when appropriate. Data are presented as means ± SD, and P values of <0.05 were considered statistically significant.

RESULTS

ANG II induces a greater increase in macula densa NOS1 expression in female mice than in male mice.

To determine the sex differences in the effect of ANG II on the expression of NOS1 in the macula densa, we measured the protein level of NOS1 in the renal cortex, where most of the NOS1 comes from the macula densa cells, in male and female C57BL/6 mice with a 2-wk administration of ANG II or vehicle (Fig. 1A). Renal cortical NOS1 expression was not significantly different between males and females with infusion of vehicle. Compared with vehicle, infusion of ANG II increased the renal cortical NOS1 expression by 112.6 ± 25.9% in males and by 306.1 ± 65.9% in females (Fig. 1B). The ANG II-induced increase in renal cortical NOS1 expression was significantly greater in female mice than in male mice.

Fig. 1.

Angiotensin II (ANG II) infusion induces greater increases of macula densa nitric oxide (NO) synthase (NOS1) expression and activity in female mice than in male mice. A: immunoblots of NOS1 and the loading control of β-actin. B: renal cortical expression levels of NOS1 in male and female C57BL/6 mice with ANG II or vehicle infusion. n = 3. *P < 0.01 vs. vehicle; #P < 0.01 vs. male. C: tubuloglomerular feedback-induced NO generation in the macula densa was measured in the isolated perfused juxtaglomerular apparatus (JGA) with 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2 DA). The bright-field image exhibited the anatomic structure of the perfused JGA. The fluorescent image of the DAF-2 DA-loaded JGA showed NO generation in the macula densa. D–H: NaCl-induced NO generation in male and female C57BL/6 mice with ANG II or vehicle infusion. n = 15. *P < 0.05 vs. vehicle; #P < 0.01 vs. male.

ANG II induces a greater increase in NO generation at the macula densa in female mice than in male mice.

To determine sex differences in the effect of ANG II on the NO generation in the macula densa, we measured acute NaCl perfusion-induced macula densa NO generation in vitro in isolated JGAs of male and female C57BL/6 mice with a 2-wk administration of ANG II or vehicle (Fig. 1, C–H). Acute NaCl perfusion-induced NO generation in the macula densa, as indicated by the change in the fluorescent intensity of DAF-2 when the NaCl concentration of tubular perfusate was increased from 10 to 80 mM, was not significantly different between males and females with infusion of vehicle. Compared with vehicle, infusion of ANG II increased NO generation by 37.7 ± 12.7% (from 42.7 ± 8.1 to 58.2 ± 8.6 U/min) in males and by 148.8 ± 44.4% (from 39.9 ± 9.2 to 95.5 ± 10.8 U/min) in females. The ANG II-induced increase in NO generation by the macula densa was significantly greater in female mice than in male mice.

ANG II induces a less augmentation in TGF responsiveness in female mice than in male mice.

To determine sex differences in the effect of ANG II on the TGF responsiveness, we measured the TGF response in vivo by micropuncture in male and female C57BL/6 mice with a 2-wk administration of ANG II or vehicle. The TGF response in vivo, as indicated by ΔP_sf when the tubular perfusion rate of ATF was increased from 0 to 40 nL/min, was not significantly different between males and females with infusion of vehicle (Fig. 2, A and B). Compared with vehicle, infusion of ANG II augmented the TGF response in vivo by 49.3 ± 10.2% (from 4.9 ± 0.6 to 7.3 ± 0.9 mmHg) in males and by 20.8 ± 6.2% (from 5.0 ± 0.7 to 6.1 ± 0.9 mmHg) in females (Fig. 2, C and D). The ANG II-induced augmentation in TGF response in vivo was significantly less in female mice than in male mice (Fig. 2E).

Fig. 2.

Angiotensin II (ANG II) infusion induces more inhibition in the tubuloglomerular feedback (TGF) response in vivo in female mice than in male mice. A–E: TGF responses in vivo, as indicated by the change of stop-flow pressure when the tubular perfusion rate was increased from 0 to 40 nL/min, was measured and compared in male and female C57BL/6 mice with ANG II or vehicle infusion. n = 15–17 tubules/4–5 mice. *P < 0.01 vs. vehicle; #P < 0.01 vs. male.

Selective deletion of NOS1 from the macula densa eliminates the sex differences in the effect of ANG II on TGF responsiveness in mice.

To determine the significance of macula densa NOS1 in the sex differences in the effect of ANG II on TGF responsiveness, we measured the TGF response in vivo by micropuncture in male and female MD-NOS1KO mice as well as NOS1^flox/flox mice with a 2-wk administration of ANG II or vehicle.

In NOS1^flox/flox mice, the TGF response in vivo was not significantly different between males and females with infusion of vehicle. Compared with vehicle, infusion of ANG II enhanced the TGF response in vivo by 47.9 ± 10.6% (from 4.9 ± 0.8 to 7.2 ± 0.9 mmHg) in males and by 19. ± 10.6% (from 5.1 ± 0.7 to 6.0 ± 0.7 mmHg) in females. The ANG II-induced enhancement in TGF response in vivo was significantly less in female NOS1^flox/flox mice than in male NOS1^flox/flox mice (Fig. 3, A–D).

Fig. 3.

Sex differences in the effect of angiotensin II (ANG II) infusion on the tubuloglomerular feedback (TGF) response are mediated by macula densa nitric oxide synthase (NOS1). A–I: the TGF response in vivo was measured and compared in male and female NOS1^flox/flox mice as well as macula densa-specific NOS1 knockout mice with ANG II or vehicle infusion. n = 15 tubules/4–5 mice. *P < 0.0 vs. vehicle; #P < 0.01 vs. male.

In MD-NOS1KO mice, the TGF response in vivo was significantly enhanced compared with that in NOS1^flox/flox mice but not significantly different between males and females with infusion of vehicle. Compared with vehicle, infusion of ANG II augmented the TGF response in vivo by 47.4 ± 9.4% (from 8.0 ± 0.9 to 11.8 ± 1.0 mmHg) in males and by 44.7 ± 11.3% (from 7.8 ± 0.8 to 11.3 ± 1.1 mmHg) in females. The ANG II-induced augmentation in TGF response in vivo was not significantly different between male and female MD-NOS1KO mice (Fig. 3, E–H).

These results demonstrated that the selective deletion of NOS1 from the macula densa eliminates sex differences in ANG II-induced enhancement in the TGF response in vivo, which indicates that macula densa NOS1 mediates sex differences in the effect of ANG II on TGF responsiveness in mice (Fig. 3I).

Selective deletion of NOS1 from the macula densa reduces sex differences in the effect of ANG II on natriuresis in mice.

To determine the significance of macula densa NOS1 in sex differences in the effect of ANG II on natriuresis, we measured the changes in GFR, urine flow rate, and Na⁺ excretion after an acute volume expansion in male and female MD-NOS1KO mice as well as NOS1^flox/flox mice with a 2-wk administration of ANG II.

In NOS1^flox/flox mice, basal levels of GFR, urine flow rate, and Na⁺ excretion were not significantly different between males and females. In male NOS1^flox/flox mice, an acute volume expansion increased GFR by 30.7 ± 2.5% (from 630.8 ± 21.1 to 824.8 ± 33.5 μL·min⁻¹·g kidney wt⁻¹), urine flow rate by 92.2 ± 12.8% (from 4.4 ± 0.6 to 8.4 ± 0.8 μL·min⁻¹·g kidney wt⁻¹), and Na⁺ excretion by 266.2 ± 21.4% (from 0.86 ± 0.07 to 3.16 ± 0.10 μeq·min⁻¹·g kidney wt⁻¹) from baselines. In female NOS1^flox/flox mice, an acute volume expansion increased GFR by 56.2 ± 6.5% (from 621.9 ± 37.3 to 970.6 ± 52.3 μL·min⁻¹·g kidney wt⁻¹), urine flow rate by 133.8 ± 18.1% (from 4.4 ± 0.7 to 10.3 ± 1.1 μL·min⁻¹·g kidney wt⁻¹), and Na⁺ excretion by 362.8 ± 24.1% (from 0.85 ± 0.06 to 3.95 ± 0.18 μeq·min⁻¹·g kidney wt⁻¹) from baselines. The acute volume expansion-induced increases in GFR, urine flow rate, and Na⁺ excretion were significantly greater in female than male NOS1^flox/flox mice by 154.7 ± 34.7 μL·min⁻¹·g kidney wt⁻¹, 1.8 ± 0.3 μL·min⁻¹·g kidney wt⁻¹, and 0.79 ± 0.14 μeq·min⁻¹·g kidney wt⁻¹, respectively (Fig. 4, A–C).

Fig. 4.

Sex differences in the effect of angiotensin II (ANG II) on natriuretic responses to acute volume expansion are dependent on macula densa nitric oxide synthase (NOS1). A−C: changes in glomerular filtration rate (GFR; A), urine flow rate (B), and Na⁺ excretion (C) during the 0- to 60-min period and 60- to 90-min period after acute volume expansion in male and female NOS1^flox/flox mice as well as macula densa-specific NOS1 knockout mice with ANG II infusion. n = 8. *P < 0.01 vs. baseline; #P < 0.05 versus male. KW, kidney weight.

In MD-NOS1KO mice, the basal levels of GFR, urine flow rate, and Na⁺ excretion were neither significantly different between males and females nor significantly different compared with those in NOS1^flox/flox mice. In male MD-NOS1KO mice, an acute volume expansion increased GFR by 14.6 ± 4.6% (from 606.4 ± 36.7 to 694.1 ± 35.2 μL·min⁻¹·g kidney wt⁻¹), urine flow rate by 35.4 ± 10.3% (from 4.1 ± 0.5 to 5.5 ± 0.5 μL·min⁻¹·g kidney wt⁻¹), and Na⁺ excretion by 114.4 ± 8.5% (from 0.85 ± 0.06 to 1.83 ± 0.10 μeq·min⁻¹·g kidney wt⁻¹) from baselines. In female MD-NOS1KO mice, an acute volume expansion increased GFR by 21.4 ± 4.6% (from 616.1 ± 31.9 to 747.4 ± 39.7 μL·min⁻¹·g kidney wt⁻¹), urine flow rate by 52.5 ± 10.0% (from 4.2 ± 0.6 to 6.4 ± 0.5 μL·min⁻¹·g kidney wt⁻¹), and Na⁺ excretion by 130.1 ± 18.5% (from 0.89 ± 0.08 to 2.03 ± 0.13 μeq·min⁻¹·g kidney wt⁻¹) from baselines. The acute volume expansion-induced increases in GFR, urine flow rate, and Na⁺ excretion were significantly greater in female than male MD-NOS1KO mice by 43.6 ± 9.8 μL·min⁻¹·g kidney wt⁻¹, 0.7 ± 0.2 μL·min⁻¹·g kidney wt⁻¹, and 0.17 ± 0.06 μeq·min⁻¹·g kidney wt⁻¹, respectively (Fig. 4, A–C).

These results demonstrated that the selective deletion of NOS1 from the macula densa significantly reduced sex differences in the effect of ANG II on the natriuretic response to acute volume expansion, which indicates that macula densa NOS1 contributes to sex differences in the effect of ANG II on natriuresis in mice.

Selective deletion of NOS1 from the macula densa diminishes sex differences in ANG II-induced hypertension in mice.

To determine the significance of macula densa NOS1 in the sex differences in ANG II-induced hypertension, we measured BP responses to ANG II infusion in male and female MD-NOS1KO mice as well as NOS1^flox/flox mice.

In NOS1^flox/flox mice, the basal level of MAP was not significantly different between males and females. Following a 2-wk administration of ANG II, MAP raised by 33.7 ± 6.4% (from 95.9 ± 2.2 to 128.1 ± 5.7 mmHg) in males and by 12.5 ± 5.7% (from 96.2 ± 5.5 to 108.1 ± 5.0 mmHg) in females from baselines. The ANG II-induced rise in MAP was significantly less in female than male NOS1^flox/flox mice by 20.4 ± 4.7 mmHg (Fig. 5, A–C).

Fig. 5.

The sex differences in angiotensin II (ANG II)-induced hypertension are dependent on macula densa nitric oxide synthase (NOS1). A–C: mean arterial pressure responses to ANG II infusion in male and female NOS1^flox/flox mice as well as macula densa-specific NOS1 knockout mice. n = 12. *P < 0.01 vs. male.

In MD-NOS1KO mice, the basal level of MAP was neither significantly different between males and females nor significantly different compared with that in NOS1^flox/flox mice. Following a 2-wk administration of ANG II, MAP raised by 46.5 ± 4.9% (from 96.5 ± 2.1 to 141.5 ± 4.4 mmHg) in males and by 35.8 ± 6.6% (from 96.7 ± 3.6 to 131.2 ± 4.4 mmHg) in females from baselines. The ANG II-induced rise in MAP was significantly less in female than male MD-NOS1KO mice by 10.5 ± 3.6 mmHg (Fig. 5, A–C).

These results demonstrated that selective deletion of NOS1 from the macula densa significantly diminished sex differences in the effect of ANG II on BP, which indicates that macula densa NOS1 plays a significant role in sex differences in ANG II-induced hypertension in mice.

ANG II induces a greater increase in macula densa NOS1 expression in female humans than in male humans.

To determine the clinical significance of macula densa NOS1 in sex differences in ANG II-induced hypertension, we examined sex differences in the effect of ANG II on macula densa NOS1 expression in humans by measuring the protein level of NOS1 in male and female human renal cortex tissue cultured with ANG II or vehicle. NOS1 expression was not significantly different between male and female human renal cortex tissue cultured with vehicle. Compared with vehicle, ANG II increased NOS1 expression by 43.8 ± 12.6% in male human renal cortex tissue and by 122.0 ± 27.6% in female human renal cortex tissue. The ANG II-induced increase in renal cortical NOS1 expression was significantly greater in female than male humans, which indicates a strong clinical relevance of macula densa NOS1 in sex differences in ANG II-induced hypertension (Fig. 6, A and B). In addition, as shown in Fig. 6C, NOS1 was primarily located at the macula densa with minor expression at the other segments of nephron, such as the collecting duct, within human kidney biopsies.

Fig. 6.

Angiotensin II (ANG II) induces a greater increase in macula densa nitric oxide synthase (NOS1) expression in female humans than in male humans. A: immunoblots of NOS1 and the loading control of β-actin. −, tissue culture with vehicle; +, tissue culture with ANG II. B: expression levels of NOS1 in male and female human renal cortex tissues cultured with ANG II or vehicle. n = 3. *P < 0.05 vs. vehicle; #P < 0.05 vs. male. C: immunofluorescence staining of NOS1 in human kidney slices. Red indicates NOS1; blue indicates the nucleus.

DISCUSSION

The present study demonstrated the significance of the macula densa NOS1β-mediated TGF mechanism in the sexual dimorphism of ANG II-induced hypertension. We found that chronic administration of subpressor ANG II induced higher expression of macula densa NOS1β, greater NO generation at the macula densa, and lower TGF responsiveness in female than male wild-type mice. Moreover, female wild-type mice exhibited a greater natriuretic response and lower MAP to ANG II infusion than males. In contrast, these sex differences in the effects of ANG II on the TGF response, natriuresis, and BP were largely diminished in MD-NOS1KO mice. In addition, tissue culture of the human renal cortex with ANG II resulted in higher expression level of NOS1β in females than in males.

It has been well known that females are protected against the development of ANG II-induced hypertension compared with males in various experimental animals (27, 40, 41, 52). However, the underlying mechanisms for these sex differences have not been fully understood. TGF is a vital mechanism in the regulation of renal hemodynamics, Na⁺ excretion, and BP (3, 30, 51, 55). Acute administration of ANG II was found to enhance the TGF response both in vitro in isolated JGAs (57) and in vivo in anesthetized animals (46). Moreover, it has been recently reported by Brown et al. (6) that both the responsiveness of TGF, as indicated by the maximal change in ΔP_sf, and sensitivity of TGF, as indicated by the turning point in the TGF response curve, were augmented in FVB/N mice with less extension in females than males in response to acute ANG II infusion. Nevertheless, the changes as well as sex differences in the TGF mechanism under the condition of chronic ANG II infusion remain unknown. In the present study, we measured the TGF response in vivo by micropuncture in male and female C57BL/6 mice following a 2-wk administration of a subpressor dose of ANG II. Consistent with the previous findings in FVB/N mice with acute ANG II infusion (6), our results showed that chronic ANG II infusion enhanced the TGF response in C57BL/6 mice and the augmentation was significantly less in females than in males.

It has been demonstrated that the effect of ANG II on the TGF response is primarily mediated by the angiotensin type 1 receptor (AT₁R). Inhibition of AT₁R was found to completely block the ANG II-induced enhancement of the TGF response in vitro in the rabbit kidney (57). Besides its effect of enhancing TGF responsiveness via AT₁R, ANG II has also been shown to stimulate NO production in the macula densa (28), which blunts or buffers the ANG II-induced TGF augmentation, which might be involved with the angiotensin type 2 receptor (AT₂R) (56, 79). Moreover, Brown et al. (6) demonstrated the significance of AT₂R in the sexual dimorphism of TGF in response to ANG II. They found that the renal AT₂R expression level was over threefold higher in female than male wild-type mice, and the sex differences in the ANG II-induced alterations in TGF were completely abolished in global AT₂R knockout mice. In addition, recent studies from our laboratory have demonstrated that NOS1β is the major splice variant of NOS1 as well as the primary isoform of NOS that contributes to most of the NO production in the macula densa, and the NO derived from macula densa NOS1β is a key modulator of the TGF, which inhibits or attenuates the TGF response (30, 59). Furthermore, chronic administration of subpressor ANG II was reported to induce a greater upregulation of NOS1 in the subfornical organ and paraventricular nucleus in female than male C57BL/6 mice (67, 69). Therefore, based on these previous findings, it is postulated that sex differences in the effect of ANG II on TGF response are associated with macula densa NOS1β via AT₂R.

In the present study, we measured the protein expression level of all splice variants of NOS1 with a COOH-terminal antibody in the renal cortex, where most of the NOS1 comes from macula densa cells (24, 30), in male and female C57BL/6 mice following a 2-wk administration of subpressor dose of ANG II. The Western blot results showed that chronic ANG II infusion increased the expression of macula densa NOS1β in C57BL/6 mice and that the upregulation was significantly greater in females than in males. In accordance with the changes in expression, the activity of macula densa NOS1β, as indicated by TGF-induced NO generation, was found to be increased in C57BL/6 mice with a greater extension in females than in males in response to chronic ANG II infusion. Moreover, we further determined the potential clinical relevance by examining sex differences in the effect of ANG II on macula densa NOS1β in human kidney biopsy samples (renal cortex) collected from cadaveric kidney donors. Consistent with the findings in mice, tissue culture with ANG II induced a greater increase in renal cortical NOS1β expression in females than in males. In addition, since the phosphatidylinositol 3-kinase/Akt (20, 29, 45, 53) and cAMP/PKA (4, 21, 71) pathways have been recognized to participate in the regulation of NOS1 expression, we speculate that the sex differences existing in these signaling pathways (7, 8, 33, 37, 38) might contribute to the sexual dimorphism of ANG II-induced changes in macula densa NOS1β expression, which will be examined in our future studies.

Recently, our laboratory developed the MD-NOS1KO mouse model (NKCC2^cre/NOS1^flox/flox) by crossing an NKCC2^cre line with a NOS1^flox/flox line (30). As this NOS1^flox/flox line (18) (kindly provided by Dr. Paul Huang) targets exon 6, a common exon for all splice variants of NOS1 mRNA, the excision of this exon by Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) promoter-driven Cre recombinase results in a complete ablation of all splice variants of NOS1 in the macula densa. Thus, in the present study, this transgenic mouse model was used to examine the significance of macula densa NOS1β in the sex differences in the effects of ANG II on TGF. Our results showed that selective deletion of NOS1 from the macula densa almost abolished sex differences in the ANG II-induced augmentation of theTGF response, indicating that macula densa NOS1β mediates sex differences in the effect of ANG II on TGF.

Several studies from our laboratory have demonstrated the significance of the macula densa NOS1β-mediated TGF mechanism in the control of natriuresis and BP. Mice with selective deletion of NOS1 from the macula densa exhibit an augmented TGF response, impaired natriuresis, and exaggerated ANG II-dependent hypertension (30, 59, 78). However, all of these studies were performed in male mice only. It is unknown whether the sex differences in the macula densa NOS1β-mediated TGF mechanism contribute to the sexual dimorphism of ANG II-induced hypertension. In addition, it has been demonstrated that in the chronic subpressor ANG II infusion model, GFR and Na⁺ excretion are reduced in the initial phase and then gradually compensated to the normal level along with the retention of fluid and rise of BP (19, 23). Thus, in the present study, to determine the significance of the macula densa NOS1β-mediated TGF mechanism in sex differences in the effect of ANG II on natriuresis, we measured the changes in GFR, urine flow rate, and Na⁺ excretion following an acute stimulation with intravenous isotonic saline infusion of 3% body wt in male and female MD-NOS1KO mice as well as NOS1^flox/flox mice under the condition of chronic administration of subpressor ANG II. Our results showed that the increases of GFR, urine flow rate, and Na⁺ excretion in response to the acute volume expansion were significantly greater in female than male NOS1^flox/flox mice, whereas these sex differences were significantly reduced in MD-NOS1KO mice, indicating that sex differences in the effect of ANG II on natriuresis are dependent on the macula densa NOS1β-mediated TGF mechanism. Moreover, to determine the significance of the macula densa NOS1β-mediated TGF mechanism in sex differences in the effect of ANG II on BP, we measured the BP response to a 2-wk subpressor ANG II infusion in male and female MD-NOS1KO mice as well as NOS1^flox/flox mice. Our results showed that selective deletion of NOS1 from the macula densa largely diminished sex differences in ANG II-induced increase in BP, which indicates that the macula densa NOS1β-mediated TGF mechanism plays an essential role in the sexual dimorphism of ANG II-induced hypertension.

Although the macula densa NOS1β-mediated TGF mechanism is the focus of the present study, we are aware that the deletion of macula densa NOS1 does not completely eliminate the differences in the BP response to ANG II between male and female mice, and the other mechanism also contributes to the sexual dimorphism of ANG II-induced hypertension. Several previous studies have demonstrated the significant involvement of sex hormones, vasculatures, the heart, and the brain (66, 68–70). For example, BP responses to ANG II were found to be attenuated by castration and augmented by ovariectomy in C57BL/6 mice (70). Moreover, testosterone was reported to enhance TGF response by stimulating superoxide production in macula densa cells via androgen receptors in Sprague-Dawley rats (14). Nevertheless, whether these mechanisms are parallel to or have interactions with the macula densa NOS1β-mediated TGF pathway still remain to be determined in future studies. In addition, ANG II-induced hypertension is salt sensitive. In a recent study, we have demonstrated that a high-salt diet induces higher levels of macula densa NOS1β expression and activity in female than male mice, which results in a greater inhibition in the TGF response, thereby facilitating Na⁺ excretion and protecting against salt sensitivity in BP (78). We expect that the sex differences in both the effects of ANG II and high salt intake on the macula densa NOS1β-mediated TGF response additively contribute to the sexual dimorphism of salt-sensitive hypertension.

This study demonstrated a novel mechanism for the sex differences in ANG II-induced hypertension, wherein ANG II induces higher levels of macula densa NOS1β expression and activity in females than in males, which promotes a lower TGF response and higher GFR, thereby facilitating natriuresis and protecting against the BP response to ANG II. These findings establish a critical role of the macula densa NOS1β-mediated TGF mechanism in the sexual dimorphism of ANG II-induced hypertension.

GRANTS

This work was supported by American Heart Association Predoctoral Fellowship and American Society of Nephrology Ben J. Lipps Research Fellowship Awards (to J. Zhang), an American Society of Nephrology Ben J. Lipps Research Fellowship Award (to J. Wei), an American Physiological Society STRIDE Summer Research Fellowship Award (to L. Qu), American Heart Association Career Development Award 18CDA34110441 (to L. Wang), and National Institutes of Health Grants DK099276, HL142814, and HL137987 (to R. Liu).

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

J.Z., J.W., and R.L. conceived and designed research; J.Z., L.Q., J.W., and S.J. performed experiments; J.Z., J.W., L.X., F.C., and K.J. analyzed data; J.Z. interpreted results of experiments; J.Z. and S.J. prepared figures; J.Z. and J.W. drafted manuscript; J.Z., L.Q., J.W., S.J., L.X., L.W., F.C., K.J., J.B., and R.L. edited and revised manuscript; J.Z., L.Q., J.W., S.J., L.X., L.W., F.C., K.J., J.B., and R.L. approved final version of manuscript.