Sexual dimorphisms in mechanisms of pressure natriuresis

Abstract

Aim:

The renal pressure-natriuresis response defines the blood pressure set point and maintains circulating volume. In male rats, the mechanisms involve trafficking and inactivation of renal proximal sodium transporters. Mechanisms may differ in females due to sex differences in transporters along the nephron: lower proximal and higher distal nephron salt reabsorption. We aimed to determine impact of sex on pressure- natriuresis mechanisms.

Methods:

In anesthetized 8–10 week old male and female Sprague Dawley rats, hypertension was induced by vasoconstriction (25 min) and mean arterial pressure, urine excretion, glomerular filtration rate, renal transporters’ abundance (immunoblot), and subcellular location (IHC) were determined. A subset received 1-aminobenzotriazole (ABT) to inhibit cytochrome P450-derived arachidonic acid metabolites.

Results.

At baseline, females (vs. males) exhibited lower baseline MAP and, during hypertension, greater sodium excretion at any given pressure, and 1.7-fold higher sodium clearance (P=0.01). With hypertension, immunofluorescence revealed sex- and transporter-specific redistribution of NHE3 and NaPi2 out of microvilli, and, by immunoblot, greater abundance of cortical NHE3p in both sexes (indicating less PT reabsorption), less medullary NHE3 and NKCC2 in females, and less distal NCC in males. ABT provoked leftward-shift in renal function curve in both sexes, and 2-fold higher natriuresis in males.

Conclusion:

Female Sprague Dawley rats exhibit a more robust acute pressure natriuretic response than males, facilitated by baseline sex differences in renal transporter patterns and reduced medullary NHE3 and NKCC2. These female-specific responses augment the pressure natriuresis response which may contribute to their lower baseline blood pressure.

News and Noteworthy:

Three discoveries contribute to understanding sex differences in blood pressure setpoint: 1) Females exhibit a greater natriuresis to acute hypertension (aHTN) than males; 2) aHTN provokes sex-specific molecular adaptations along the nephron: proximal NHE3 and NaPi2 redistribute in both sexes; medullary NHE3 and NKCC2 abundance are reduced in females, distal NCC abundance is reduced in males; 3) Inhibition of arachidonic acid metabolites shifts renal function curves leftward in both sexes, and augments natriuresis in males.

Graphical Abstract

Introduction

Hypertension is a leading global burden of disease risk factor for cardiovascular morbidity and a major contributor to chronic kidney disease (1–3). Blood pressure can increase secondary to elevated effective circulating volume (ECV), e.g., provoked by elevated sodium intake and retention. A rise in blood pressure above its intra-renal set-point is sensed as an error signal that drives renal natriuretic and diuretic responses in order to restore ECV. This important homeostatic response is known as “Pressure Natriuresis.” To maintain ECV over time, pressure natriuresis persists until sodium retention is resolved (with dietary and/or therapeutic approaches). Early investigations of the mechanisms in male rats demonstrated that when arterial pressure was acutely raised experimentally by vasoconstriction (aHTN), the rats exhibited a near-instantaneous increase in salt and water excretion due to reduced reabsorption along the early renal tubule; autoregulation of glomerular filtration rate (GFR) and renal blood flow was maintained after a brief spike at the onset of vasoconstriction (4–6). Subsequent investigations identified renal tubule specific mechanisms underlying the natriuretic response to aHTN including redistribution of sodium transporters out of the proximal tubule (PT) apical microvilli driven by myosin VI: Na/H exchanger (NHE3) to the microvillar base, where activity was inhibited, and endocytosis of Na-phosphate cotransporter (NaPi2) into subapical endosomes, as well as reduced Na,K-ATPase activity (7–10). Together, the responses reduced PT fractional reabsorption increased salt and water delivery to downstream segments (9, 11–14); and provoked Na-Cl cotransporter (NCC) traffic to subapical endosomes (15). Collectively, these acute adaptations increase urinary sodium and volume excretion in response to aHTN. Experimental models of chronic hypertension exhibit analogous persistent transporter responses in male rats (16, 17).

Sex-specific differences in kidney function are widely recognized as important determinants of blood pressure regulation and susceptibility to cardiovascular and kidney disease progression (18–20). We have characterized significant sexual dimorphisms in abundance and covalent modifications of kidney sodium transporters, channels, claudins, and regulatory factors (collectively referred to as “transporters”) along the entire renal tubule (20, 21). Of relevance is the sexual dimorphism in PT NHE3 localization in normotensive rodents: in males, NHE3 is localized to the body of the PT apical microvilli and in females, NHE3 is localized to the base of the microvilli, mimicking the distribution pattern of NHE3 in males during aHTN. Computational modelling that incorporates sex-specific differences in PT dimensions and transporter abundance predicts that males reabsorb 70% of the filtered sodium load along the PT, whereas females reabsorb only 40% with a correspondingly higher fractional reabsorption along the distal nephron (22). This pattern of lower proximal and higher distal reabsorption in females drives a more robust natriuretic response to a saline load challenge (21), suggesting that baseline sex differences along the nephron may “prime” females for a more robust pressure natriuresis response. These observations drive our rationale for investigating whether female Sprague Dawley rats exhibit a more robust pressure natriuretic response than males, and address the mechanisms involved.

Cytochrome P450 (CYP) metabolites of arachidonic acid, notably epoxyeicosatrienoic acids (EET) and 20-hydroxyeicosatetraenoic acid (20-HETE), have been implicated as signaling mediators connecting elevated kidney perfusion pressure to pressure natriuresis, potentially through local release of these metabolites in response to vascular stretch from arterial pressure near target transporters (23–27). However, the role of 20-HETE in blood pressure regulation is complex, because this pathway exerts opposing effects: inhibition of renal tubular sodium reabsorption (natriuretic, anti-hypertensive) and vasoconstriction (pro-hypertensive) (28). Additionally, androgens upregulate 20-HETE production (29, 30), and recent evidence supports a broader role of androgens in contributing to sex-dependent transporter profiles along the nephron, particularly in the proximal tubule (20, 31–33). These findings raise the possibility that CYP-derived eicosanoids contribute differently to pressure natriuresis response in females versus males, and provide a rationale for testing whether inhibition of CYP metabolites differentially affects pressure natriuresis in female and male rats during aHTN.

Methods

Animal protocols

All animal procedures were approved by the Institutional Animal Care and Use Committee of the Keck School of Medicine of the University of Southern California and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Experiments were performed with 22 male and 33 female Sprague Dawley (SD) rats, 8–10 weeks of age, purchased from Envigo (Indianapolis, IN). Rats were housed on 12-hour light-dark cycles with ad libitum access to water and standard vivarium chow (Rodent diet 5001, LabDiet). Experimental protocols were adapted from previously published methods (13, 15). Rats were anesthetized with intramuscular ketamine (10 mg/kg body weight, VetOne, Boise, ID) followed by intraperitoneal Inactin (thiobutabarbital, 100–125 mg/kg, Sigma Aldrich), and body temperature was maintained at 37°C with a thermostatically controlled heating pad. A polyethylene catheter (PE-50) filled with heparinized saline (20 U/ml) was inserted into the carotid artery and connected to a pressure transducer, signal amplifier, and flatbed recorder for continuous blood pressure monitoring. A second PE-50 catheter was placed in the jugular vein for constant infusion (50–100 ul/min) of 4% bovine serum albumin in 0.9% saline to maintain euvolemia. Urine was collected via bladder cannula directly into pre-weighed microcentrifuge tubes.

Acute hypertension was induced using the method of Roman and Cowley (4). In Set 1, (n=5/group), the protocol of Ivy and Bailey(34) was followed: mean arterial pressure (MAP) was raised by step-wise constriction of the celiac and superior mesenteric arteries with 4–0 braided silk surgical ligatures for 15 min, followed by additional constriction of the abdominal aorta below the renal arteries for 20 min. Sham controls underwent the identical surgical exposure with ligature placement but without constriction. In set 2 (n=2–7/group), MAP was raised by simultaneous constriction of the celiac artery, superior mesenteric artery, and abdominal aorta for 25 min; sham animals underwent the same procedure without constriction. In both sets, arterial blood samples were collected via the carotid artery cannula during pre- and post-constriction periods. In a subset of set 2, rats were pretreated for 5 days with 1-aminobenzotriazole (ABT; 50 mg/kg/day i.p.) or 0.9% saline vehicle, administered under brief isoflurane anesthesia (2% inhaled), in order to inhibit cytochrome P450 metabolism of arachidonic acid as previously described (23, 35).

At the completion of data collection, renal arteries were clamped and kidneys excised. One kidney was decapsulated, dissected into cortex and medulla, and immediately homogenized (Ultra-Turrax T25) in isolation buffer [5% sorbitol, 0.5 mM disodium EDTA, and 5 mM histidine-imidazole buffer, pH = 7.5, with the addition of 0.2 mM phenylmethylsulfonyl fluoride, 9 μg/ml aprotinin, and 5 μl/ml of phosphatase inhibitor cocktail (Sigma, P0044)](36). Homogenates were snap-frozen in liquid nitrogen and stored at −80°C as single-use aliquots. The second kidney was bisected along the coronal plane and fixed by surface immersion in ice-cold periodate-lysine-paraformaldehyde fixative (2% paraformaldehyde, 75 mM lysine, and 10 mM Na-periodate, pH 7.4) for 4 hr, cryoprotected overnight in 30% sucrose-PBS, embedded in Tissue-Tek OCT Compound, and frozen at minus 80°C (37). Note: Kidney homogenization and tissue immunohistochemistry was not conducted in rats used for Glomerular Filtration Rate estimation by FITC sinistrin infusion and clearance. Estrus cycle stage was assessed by vaginal lavage (0.3 ml sterile saline 0.9% saline) and cytological examination under a 10X objective (Nikon TMS, Japan). Consistent with previous reports, no relationship between estrous cycle stage and the physiological measurements in this study was detected (21, 38).

Glomerular Filtration Rate (GFR)

In a subset of rats (n=10 total), GFR was estimated from FITC-sinistrin clearance. FITC-sinistrin (0.1%; MediBeacon, St. Louis, MO) was added to the jugular vein infusate (4% BSA in 0.9% saline). Plasma and urine samples were diluted 1:10 and 1:200, respectively, in 0.5 M HEPES (pH 7.4), and 100 μl aliquots were loaded onto 96-well UV-transparent plates. Fluorescence was measured using SpectraMax iD3 Multi-Mode Microplate Reader (excitation 480 nm, emission 530nm). FITC-sinistrin concentration was calculated from a standard curve as: [FITC-sinistrin]=((mean RFU ± y-intercept)/slope) × dilution factor). GFR (ml/min) was calculated as: GFR = (U_{FITC-sinistrin} × UV)/P_{FITC-sinistrin}. Where P is the plasma sinistrin concentration (mg/ml), V is the urine collected over collection time (ml/min), and U is the urine sinistrin concentration (mg/ml).

Electrolyte and glucose analyses

Urine volumes were determined gravimetrically. Plasma and urine concentrations of sodium, potassium, and lithium were measured by flame photometry (Cole-Parmer, model 02655–10, IL, USA). Endogenous lithium clearance (CLi), an estimate of volume flow leaving the proximal tubule and medullary thick ascending limbs (39–41), was calculated classically as (urinary [Li] × UV)/plasma [Li]). Sodium clearance (CNa), an estimate of overall Na reabsorption along nephron, was calculated as (urinary [Na] × UV)/plasma [Na]. Urinary glucose was assessed at baseline, 5 and 20 min vascular constriction using Aimstrip urine reagent strip rUS-5/US-5B (Germaine Laboratories, San Antonio, TX).

K-Dependent p-nitrophenyl phosphatase (K-pNPPase) enzymatic assay

K-pNPPase activity, a measure of K dependent ATPase activity, which is predominantly Na,K-ATPase in kidney homogenates, was assayed colorimetrically in kidney cortical and medullary homogenates as previously described (9, 27, 42).

Semiquantitative immunoblotting

Total protein concentration in each homogenate was determined by bicinchoninic acid (BCA) assay. Samples of equal protein content were denatured in Laemmli buffer for 20 min at 60°C. Uniform loading was confirmed by densitometric analysis of 4–5 bands on Coomassie-stained gels run in parallel; sample sets exhibiting >15% variability across lanes were re-prepared rather than corrected for uneven loading (43). To validate the linearity of the detection system, 1X and ½X protein loads were run on parallel gels and quantitated. Normalized values of 1X and ½X loads were averaged for statistical analysis. Antibody details, loading amounts, dilutions, and incubation conditions are provided in Table S1. Signals were detected and quantified using the Odyssey Infrared Imaging System and the accompanying software (LI-COR, Lincoln, NE). Uncut blots are provided in Supplement.

Immunofluorescence

To assess transporter distribution within the apical microvilli, cryosections (5 μm) were cut from fixed kidneys (see Animal protocols), transferred to charged glass slides, and air-dried. Sections were rehydrated in PBS for 10 min, quenched in 50 mM NH₄Cl in PBS for 10 min, and subjected to antigen retrieval in 1% SDS in PBS for 5 min. After blocking with 5% normal goat serum and 0.3% Triton X-100 in PBS to reduce background, sections were double-labeled for 2 hours with a polyclonal antibody against either NHE3, NaPi2, or SGLT2 together with a monoclonal antibody against villin tagged with Alexa Fluor 594, all diluted in blocking buffer. Sections were then washed and incubated for 1 hr with a mixture of Alexa 488-conjugated goat anti-rabbit secondary antibody (1:500 in blocking buffer), mounted in ProLong Antifade for 1 hour and dried overnight. Sections were examined using the Leica TCS SP8 DIVE confocal/ multiphoton laser scanning microscope system. Leica microsystems, Wetzlar, Germany). Reagent details provided in Table S1.

Computational modeling

The impact of the effects of acute pressure natriuresis-induced changes in transporter abundance on tubular transport was examined by adapting published models of tubule water and electrolyte transport along a superficial nephron in the male and female rat kidneys (22). The models represent the transport of water and 15 solutes along the nephron. Reabsorption and secretion rates are computed based on the membrane abundance and activity of the specific apical and basolateral “transporters” (i.e., channels, exchangers, cotransporters, pumps, and claudins) expressed in each segment. Model parameters consist of filtered loads, tubular and cell dimensions, as well as transporter density and kinetic properties. The model assumes a steady state, and solute concentrations in plasma and interstitium are specified a priori.

Baseline morphologic, hemodynamic, and transport parameters for the male and female models are as published (22). To simulate the effects of acute pressure natriuresis on segmental transport, we incorporate changes in GFR and transporter relative abundance measured 25 min post-vasoconstriction. These changes are summarized in Table 1. A comment about the estimation of ENaC activity is warranted. CNT and CD ENaC is composed of three subunits (α,β,γ); γ ENaC is considered an important determinant of ENaC channel activity as its proteolytic cleavage is associated with activation (44). We estimated ENaC activity by measuring the abundance of both the full length (FL) as well as the proteolytically cleaved (Cl) forms: in females, neither form was changed by aHTN, while in males FL γ increased 1.29 fold and Cl γ did not change. Thus, we estimated that ENaC increased overall 1.15 fold (15%)

Table 1.

Model parameters that describe hemodynamics and transporter activity changes following 25 min vasoconstriction.

Segment	Parameter	Male (HTN-to-Ctrl)	Female (HTN-to-Ctrl)
	GFR	↑20%	↑21%
PCT	NHE3	↓15%	↓20%
	NaPi2	↓50%	↓55%
TAL	NKCC2	↓17%	↓17%
	NHE3	—	↓25%
DCT	NCC	↓16%	—
	ENaC	↑15%	—
CNT, CCD, OMCD	ENaC	↑15%	—

PCT, proximal convoluted tubule; TAL, thick ascending limb; DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct; OMCD, outer medullary collecting duct.

Statistical analysis

Values are expressed as means ± SEM. Statistical analyses were performed using GraphPad Prism 8.4 (San Diego, CA). Group comparisons were made by two-way ANOVA followed by Dunnett’s multiple comparisons post hoc test, or by mixed-effects analysis followed by Sidak’s multiple comparisons post hoc test, as appropriate. Renal function curves were analyzed by Pearson correlation and linear regression; slopes and elevations were compared between groups. Specific P values are provided; P<0.05 was considered statistically significant.

Abbreviations

Standard abbreviations for “transporters” (ion transporters, channels, claudins and regulators): AQP1, aquaporin 1; AQP2, aquaporin 2; AQP2pS256, AQP2 phosphorylated at S256; cldn, claudin; cl, cleaved; FL, full-length; NaPi2, Na-phosphate cotransporter 2A; NCC, Na-Cl cotransporter; NCCpT53, NCC phosphorylated at threonine 53; NCCpS71 NCC phosphorylated at serine 71; NHE3, Na/H exchanger isoform 3; NHE3p, NHE3 phosphorylated at S552; NKA, Na,K-ATPase α1 and β1; NKCC2, Na-K-2Cl cotransporter isoform 2; NKCC2p, NKCC2 phosphorylated at T96 and T101; SGLT2, sodium-glucose cotransporter 2

Results

Effect of vasoconstriction on mean arterial pressure in female versus male rats

To compare acute pressure natriuretic responses in females (F) to males (M), vascular resistance was raised by constriction as previously described (4, 11, 12, 15, 16, 34). In set 1, step-wise constriction of the celiac and mesenteric arteries for 15 min was followed by additional constriction of the abdominal aorta for 20 min (Figure 1A, arrows). At baseline, females exhibited lower MAP than males (91 ± 5 vs 105 ± 3 mmHg, P=0.03). Step-wise vasoconstriction raised MAP from 91 ± 5 to 126 ± 5 mmHg in females and from 105 ± 3 to 130 ± 3 mmHg in males (P<0.001 for both; Figure 1A). The rise in MAP tended to be greater in females than males (ΔMAP: 34 ± 4 vs 25 ± 2 mmHg, respectively, P=0.07). Since constriction of the abdominal aorta did not further increase MAP, subsequent experiments employed simultaneous constriction of all three arterial beds for 25 min (set 2; Figure 1B–D), which raised MAP from 90 ± 2 to 123 ± 3 mmHg in females and from 93 ± 2 to 132 ± 3 mmHg in males (P=0.004 for both). Given the comparable MAP responses within each sex across protocols, data from step-wise (Set 1, n=5/group) and simultaneous (Set 2, n=2–7/group) constriction were pooled for subsequent analyses.

Figure 1. Mean arterial pressure (MAP) and GFR during vascular constriction in male (blue circles) and female (red triangles) Sprague Dawley rats.

MAP was measured via carotid artery cannula. A) step-wise constriction of the celiac and mesenteric arteries, at arrow between Pre- and 5 min, followed by additional constriction of abdominal aorta, indicated by arrow at 15 min, in males (n=5) and females (n=5). B) simultaneous constriction of celiac, mesenteric, and abdominal aorta arteries in males (n=4) and females (n=7); C) individual responses over time. D) GFR measured during simultaneous constriction of celiac, mesenteric and abdominal aorta (n=10 combined male and female), and also displayed as comparison at Pre- and 25 min post-constriction. Data are expressed as individual values ± SEM. Statistical comparisons of pre- vs post-constriction conducted by one-way ANOVA followed by Sidaks multiple comparisons post hoc test using GraphPad Prism.

Glomerular filtration rate (GFR) was assessed in a subset of rats (n=10) by FITC-sinistrin clearance via jugular vein infusion. During the first 5 min post-constriction, GFR spiked 16- to 20-fold in both sexes but rapidly autoregulated between 5 and 10 min and remained approximately 2-fold above baseline through 25 min in both sexes (Figure 1D).

Impact of acute hypertension on renal function

At baseline, urinary sodium excretion (UNaV) tended greater in females: (5 ± 1 μmol/min in females vs 2 ± 1 μmol/min in males (P= 0.058; Figure 2A). The pressure natriuretic response was sustained over 25 min of vasoconstriction in both sexes and cumulative UNaV over 25 min did not differ significantly between sexes (Figure 2C,D; P=0.09). Urine volume (UV) was greater in females versus males during aHTN (P=0.04), whereas urinary potassium excretion (UKV) did not differ between sexes. UNaV correlated strongly with MAP in both females (r=0.66; P<0.001) and males (r=0.35; P=0.004; Figure 2B). Linear regression analysis revealed a leftward shift of the renal function curve in females relative to males, reflected by a significant difference in elevation (P<0.001), indicating a greater sodium excretion at any given MAP. A similar leftward shift is also evident for UV versus MAP (Figure 2F). In contrast, UKV peaked during the first 5 min of constriction but returned to pre-constriction levels by 10–15 min (Figure 2G), with no shift in the potassium renal function curve (Figure 2H).

Figure 2. Natriuretic, diuretic and kaliuretic responses to acute hypertension in male (blue circles) and female (red triangles) Sprague Dawley rats.

Urine was collected via bladder cannula directly into pre-weighed microcentrifuge tubes over 5-min intervals in males (n=9) and females (n=12); [Na⁺]and [K⁺] were measured by flame photometry, V by weight. A) urinary sodium excretion post-constriction (UNaV). B) UNaV as a function of MAP. C) accumulated sum-total UNaV over 25 min. D) Mean +/− SEM of sum-total at each collection point. E) UV as a function of constriction time and F) MAP. G) urinary potassium excretion (UKV) as a function of constriction time and of H) MAP. Data expressed as individual measurements or means ± SEM (D). Statistical comparisons via two-way ANOVA followed by Dunnetts multiple comparisons post hoc test; and correlation test followed by linear regression analysis (B, F, H) using GraphPad Prism.

Endogenous lithium clearance (CLi), a measure of volume flow from the proximal tubule and medullary thick ascending limb, as well as sodium clearance, a measure of whole nephron volume flow, were calculated at baseline, 5 min post-constriction (when GFR was elevated >10-fold), and 25 min post-constriction (when GFR had autoregulated to ~2-fold above baseline) (Figure 3A,B). At 25 min, CLi was significantly elevated over baseline in both females (P=0.007) and males (P=0.004), with no significant sex difference. CNa was likewise elevated in both sexes at 25 min (P<0.001 in both); notably, CNa was significantly greater in females than males (P=0.01).

Figure 3. Renal clearance of lithium and sodium during 5 and 25 min of acute hypertension in male (blue circles) and female (red triangles) Sprague Dawley rats.

Urine was collected via bladder cannula directly into pre-weighed microcentrifuge tubes over 5-min intervals in males (n=9) and females (n=12); blood was collected from carotid cannula and spun for plasma. [Na⁺]and [Li⁺] were measured by flame photometry, V by weight. A) Li⁺ clearance and B) Na⁺ Clearance calculated at 5 and 25 min post constriction. Data expressed as individual measurements as well as means ± SEM. Statistical comparisons via two-way ANOVA using mixed effects analysis followed by Tukeys multiple comparison post-hoc test using GraphPad Prism.

Impact of acute hypertension on kidney transporters

Redistribution of PT sodium transporters within the apical microvilli was assessed by immunofluorescence microscopy in kidney cortex from sham (baseline BP) and aHTN (25 min post-constriction) animals (Figure 4). The microvillar body was demarcated by labelling the actin-bundling protein villin with a red-tagged anti-villin, and apical transporters with green-labelled secondary antibodies such that co-localization in microvilli appears yellow and trafficking of transporters out of the body of the microvilli is evident as unmasking of red-tagged villin. At baseline, NHE3 co-localized with villin within the body of the microvilli in males (yellow overlay), whereas in females NHE3 was more concentrated at the base of the microvilli (green), unmasking red-tagged villin in the microvillar body, consistent with previous reports (21). During aHTN, NHE3 in males redistributed toward the microvillar base (green), revealing red-stained villi. In females, NHE3 retracted further to a thin band concentrated at the base, revealing more red-labelled villin (Figure 4A). At baseline, NaPi2 colocalized with villin in both sexes, but there was more sub-apical punctate NaPi2 apparent in males than females (Figure 4B); during aHTN, most of the NaPi2 appeared in green-labelled subapical endocytic vesicles in males, with a more pronounced loss of apical NaPi2 signal in females, revealing pronounced red villin (Figure 4B). In contrast, SGLT2 was similarly evident within the body of the microvilli in both sexes at baseline and during aHTN (Figure 4C).

Figure 4. Distribution of proximal tubule transporters pre- vascular constriction (baseline BP) and post- vascular constriction with elevated blood pressure (aHTN) in relation to apical microvilli.

Immunofluorescence microscopy of proximal tubule: A, NHE3, B, NaPi2, and C, SGLT2 in males (left column) and females (right column). Apical transporters were detected with transporter specific polyclonal antisera and green-labelled secondary antisera, and counterstained with red-labelled anti-villin antiserum to demarcate location of the proximal tubule microvilli. Scale bar = 20 μm. Protocol details provided in Table S1. Statistical comparisons were not conducted. Results are typical of those from 2–4 samples from different males and females.

The impact of aHTN on transporter abundance was assessed by semiquantitative immunoblotting in cortical and medullary homogenates from sham and aHTN kidneys (25–35 min post-constriction; (Figure 5A, 5B). In the proximal tubule, NHE3 phosphorylated at S552 (NHE3p), a marker for localization to the base of the microvilli (8, 21), tended to increase during aHTN in both males (~20%, P=0.05) and females (22%, P=0.06). In the medulla, transporter abundance decreased during aHTN, reaching significance for medullary NHE3 and NKCC2 (both 20% lower, P=0.04). In the distal nephron, aHTN in males was associated with 16% lower NCC, 29% lower NCCpS71, and 30% greater full-length γENaC (all P<0.05); these changes were not observed in females. Sodium pump activity during 25 min aHTN was assessed with a K⁺-dependent p-nitrophenyl phosphatase (K-pNPPase activity) enzymatic assay performed in kidney cortical and medullary homogenates (Figure 5C). At baseline, females exhibited 18% and 31% greater K-pNPPase activity in the cortex and medulla, respectively, compared to males (P=0.005 and P=0.01). aHTN did not alter K-pNPPase activity in either sex.

Figure 5. Impact of vascular constriction-driven acute hypertension on relative abundance and phosphorylation of renal transporters, channels along the nephron.

Assay and immunoblot details are provided in Table S1. 1X and 1/2X amounts were assayed in parallel to confirm linearity of detection system (one amount shown, see uncut blots in Supplement). A) Immunoblots of proteins from proximal tubule to collecting duct (m prefix denotes medulla). kD indicates apparent molecular weight of the prestained protein ladders (BioRad). Relative abundances (arbitrary density values) were normalized to SHAM = 1 for both male and females; n=5/group is shown but n=7–12/group were assayed and used for statistical comparisons; data are expressed as mean ± SEM; *P< 0.05 within males, ^P< 0.05 within females. B) Impact of acute hypertension (aHTN) vs. sham treatment, defined as 1, on relative abundance of transporters in males (blue) compared to females (red); bars indicate means ± SEM and *P< 0.05 in aHTN vs. shams within sexes. C. K⁺⁻dependent p-nitrophenyl phosphatase (K-pNPPase) activity, a measure of maximal sodium pump activity, assessed in kidney cortical and medullary homogenates in males (blue) and females (red), details in Methods. Statistical comparisons were conducted by unpaired t-test within sexes using GraphPad Prism. Abbreviations: ENaC: epithelial Na⁺ channel γ full length (Fl), cleaved (Cl); NaPi2: Na⁺-phosphate cotransporter 2A; NCC: Na⁺-Cl⁻ cotransporter; NCCp -S71 –T53: NCC phosphorylated at S71 and T53; NHE3: Na⁺/H⁺ exchanger isoform 3; NHE3p: NHE3 phosphorylated at S552; NKA: Na,K-ATPase; NKCC2: Na⁺-K⁺−2Cl⁻ cotransporter isoform 2; NKCC2p -T39T101: NKCC2 phosphorylated at T39, T101;

Predicted impact of acute pressure natriuresis on segmental transport

The impact of acute pressure natriuresis-induced changes in transporter abundance on transport along the renal tubule was examined by adapting published models of tubule water and electrolyte transport along a superficial nephron in the male and female rat kidneys (45). Model parameters that describe hemodynamics and transporter activity changes following vasoconstriction are provided in Table 1 (see Methods for details). Following 25 min vasoconstriction, GFR increases whereas the activities of NHE3 and NaPi2 along the proximal tubule decrease due to transporter retraction from microvilli as well as rise in NHE3p (which is associated with lower activity). Together these changes increase both Na⁺ delivery to the thick ascending limbs (Figures 6A1 and 6A2) and fractional Na⁺ reabsorption along the proximal tubule (Figures 7A1 and 7A2). Therefore, despite the downregulation of NKCC2 in both sexes and NHE3 in females, Na⁺ reabsorption along the TAL segment increases. Nonetheless, downstream Na⁺ flow remains elevated above baseline. In females, distal Na⁺ transporters are assumed unchanged from baseline; in males, NCC is assumed to be downregulated. The abundance of the cleaved form of γENaC, which is a determinant of ENaC activity, is unchanged whereas the abundance of the full length (pre-processed) γENaC increases. The higher luminal flows substantially increase Na⁺ reabsorption along the connecting tubule (Figures 6A1 and 6A2). These adaptations yield Na⁺ excretion that is 13.3 and 7.8 folds higher than baseline in female and male, respectively (Figures 6A1 and 6A2, “urine” bars). The corresponding female-to-male Na⁺ excretion increase is 1.7, consistent with figure 2D. Similar trends are predicted for segmental water transport, with urine output predicted to increase by 7.6 and 5.0 folds in female and male, respectively. The female-to-male urine output increase is 1.5, consistent with Figures 2E,F.

Figure 6. Predicted delivery of Na⁺ (A), K⁺ (B) and volume (C) to the beginning of individual nephron segments in male and female rats at baseline and during aHTN.

PT, proximal tubule; DL, descending limb; mTAL, medullary thick ascending limb; DCT, distal convoluted tubule; CNT, connecting tubule; CCD, cortical collecting duct

Figure 7. Predicted net transport of Na⁺ (A), K⁺ (B) and volume (C) along individual nephron segments of male and female rats at baseline and during aHTN.

Transport is taken positive out of a nephron segment. PT, proximal tubule; DL, descending limb; TAL, thick ascending limb; DCT, distal convoluted tubule; CNT, connecting tubule; CD, collecting duct.

Impact of inhibition of cytochrome P450-derived arachidonic acid metabolites on pressure natriuresis responses in males and females.

To investigate the contribution of CYP-derived arachidonic acid metabolites on pressure natriuresis, a subset of rats was pretreated with 1-aminobenzotriazole (ABT) for 5 days to block formation of EETs and 20-HETE prior to undergoing the identical vasoconstriction protocol. A separate group received saline vehicle or no pretreatment. Since no differences were detected between vehicle-treated and untreated rats (data not shown), these groups were combined and designated as untreated controls. Physiological responses are detailed in Table 2 and Figure 8. At baseline, ABT pretreatment did not affect UV or UNaV in either sex. ABT lowered basal MAP by approximately 10% in females and less than 10% in males (Figure 8A). The magnitude of the MAP rise in response to vasoconstriction was not affected by ABT in either sex. During aHTN, ABT-treated males exhibited greater cumulative UNaV over 25 min compared to untreated males (Figure 8B). In females, cumulative UNaV was not altered by ABT (Figure 8C). Renal function curves relating UNaV to MAP were shifted leftward by ABT in both males (slope P=0.002; Figure 8B) and females (elevation P<0.001; Figure 8C). ABT had no effect on UKV in either sex (not shown).

Table 2.

Impact of ABT on physiological parameters in males and female SD rats at baseline and with acute hypertension

Male Parameters	Untreated M (n=9)		M ABT (n=4)
	Basal Pre-aHTN	20–25 min aHTN	Basal Pre-aHTN	20–25 min aHTN
Mean Arterial Pressure (MAP; mmHg)	100 ± 3	121 ± 4*	91 ± 6	119 ± 1^
Urine Volume (μl/min)	12 ± 3	71 ± 16*	11 ± 4	140 ± 40
Sodium Clearance (μl/min)	15 ± 7	135 ± 22*	25 ± 14	281 ± 64
Potassium Clearance (μl/min)	0.8 ± 0.2	1.4 ± 0.2	0.5 ± 0.2	5 ± 1^

Female Parameters	Untreated F (n=12)		F ABT (n=8)
	Basal Pre-aHTN	20–25 min aHTN	Basal Pre-aHTN	20–25 min aHTN
Mean Arterial Pressure (MAP; mmHg)	90 ± 3	117 ± 4*	80 ± 5*	100 ± 7
Urine Volume (μl/min)	21 ± 4	102 ± 12*	14 ± 5	60 ± 7^
Sodium Clearance (μl/min)	38 ± 8	239 ± 20*	26 ± 6	161 ± 28^
Potassium Clearance (μl/min)	0.8 ± 0.1	1.32 ± 0.09	1.2 ± 0.3	2.2 ± 0.05

Data expressed as mean ± SEM. Statistical comparisons were performed using GraphPad Prism (9.2.0) mixed-effects analysis followed by Tukey’s multiple comparisons post hoc test. P<0.05 considered significant

^*versus untreated basal value,

^{^}versus ABT basal value. M=male. F=female

Figure 8. Impact of ABT pre-treatment on MAP, sodium and volume excretion in response to vascular constriction.

A) Males (M and MABT; n=4–9/group) and Females (F and FABT; n=8–12/group) were treated with ABT (50 mg/kg/day) for 5 days or untreated (vehicle or saline or no injection) prior to acute hypertension (aHTN) protocol of simultaneous constriction of celiac, mesenteric and abdominal aorta. Urinary sodium excretion post-constriction (UNaV), and UNaV plotted as a function of MAP in M and MABT (B), and in F and FABT (C); furine, collected every 5 min, was measured via bladder cannulation directly into pre-weighed microcentrifuge tubes and Na⁺ was measured by flame photometry. Data are expressed as individual measurements or mean ± SEM. Statistical comparisons were performed using Two way ANOVA or Correlation test and linear regression analysis, or mixed effects analysis followed by Sidaks multiple comparisons post hoc test , all with GraphPad Prism (9.2.0)

The impact of ABT pretreatment on PT transporter redistribution during aHTN was assessed by immunofluorescence (Figure S1). The patterns observed were similar to those in un-pretreated rats of both sexes (Figure 4): NHE3 partially redistributed toward the microvillar base (evident as orange overlay with villin), NaPi2 was displaced from apical microvilli into subapical compartments, and SGLT2 distribution was unchanged by aHTN. Confirming these immunohistochemistry findings, the impact of ABT pretreatment on transporter relative abundance during aHTN, assessed by immunoblot, was similar to that observed without ABT pretreatment in both males (Figure S2) and females (Figure S3).

Discussion

Three principal novel findings emerged from the results of this investigation: First, females exhibit a more robust pressure natriuretic response to vascular constriction provoked acute hypertension, shown as a leftward shift in renal function curves, i.e. greater sodium excretion capacity at any given arterial pressure. Second, acute hypertension provokes sex-specific molecular adaptations along the nephron: both sexes exhibit proximal tubule transporter redistribution. Females exhibit reduction in medullary transporter abundance and males show decreased distal NCC phosphorylation. Third, inhibition of cytochrome P450-derived arachidonic acid metabolites with ABT shifts renal function curves leftward in both sexes and augments natriuresis in males, not females, suggesting that the vascular pro-hypertensive actions of 20-HETE predominate over the tubular natriuretic effects in this acute model.

The central observation that females excrete more sodium than males for any given MAP is reflected in the leftward-shifted renal function curves (Figures 2B,F) and extends prior work demonstrating sex differences in pressure-natriuresis relationships: Hilliard et al (46). reported a similar leftward shift in female FVB/N mice subjected to similar vascular constriction surgery protocols, and Reckelhoff et al. (47) demonstrated that testosterone exacerbates hypertension and shifts the pressure-natriuresis curve rightward in male spontaneously hypertensive rats.

The mechanistic basis for the leftward shift likely resides, at least in part, in the sexually dimorphic patterns of renal sodium transporters at baseline. As characterized by our group and others, the female rodent nephron, compared to the male nephron, exhibits a “downstream shift” in fractional sodium and volume reabsorption due to lower overall abundance and/or activity of proximal tubule NHE3, NaPi2, AQP1, and cldn2, compensated for by greater activity and abundance of NKCC2, NCC, and ENaC along the distal nephron (20–22, 45). Supporting this concept, female rats excrete an acute sodium load more rapidly than male rats. (21) Computational modeling incorporating these sex-specific transporter profiles predicts that females reabsorb only ~40% of the filtered sodium load along the proximal tubule versus ~70% in males, (22, 45) resulting in greater fractional delivery to downstream segments. Here we show that acute hypertension inhibits proximal reabsorption (via NHE3 retraction and NaPi2 internalization), which further amplifies the baseline differences contributing to enhanced natriuretic responses in females due to their lower proximal reabsorptive set-point. Said another way, the female nephron is constitutively poised with a pattern that resembles the male pressure-natriuretic state, providing what we term a “head-start” for sodium excretion when challenged with acute hypertension.

Notably, baseline MAP was lower in females than males (91 ± 5 vs 105 ± 3 mmHg), consistent with both experimental(48) and clinical(49) observations of lower blood pressure in premenopausal females. Although vasoconstriction raised MAP to a comparable level in both sexes in this study, we recognize that the slightly greater rise in BP in females vs. males (ΔMAP 34 ± 4 vs 25 ± 2 mmHg) may contribute to the enhanced natriuretic response. However, the leftward shift in renal function curves persists after accounting for MAP (Figure 2B), indicating that intrinsic renal mechanisms, rather than hemodynamic differences alone, are key determinants of the sex difference.

A key mechanistic finding is that both sexes exhibit acute redistribution of proximal tubule NHE3 and NaPi2 during aHTN. Initial reports in males demonstrated, using subcellular fractionation, that these transporters rapidly and reversibly redistributed from low density plasma membranes to higher density intracellular vesicles (9, 16, 50), and subsequent studies using confocal microscopy established that NHE3 (in lipid rafts), redistributed to the base of the microvilli while NaPi2, in non-rafts were internalized to sub apical endosomes (11–13). In females, where NHE3 resides predominantly at the microvillar base at baseline (21), aHTN provoked further retraction to a thin band at the base (but not into sub-apical vesicles). This observation suggests that the machinery governing NHE3 redistribution, likely involving myosin VI-mediated transport along the actin cytoskeleton (10), coupled to local pH microdomain-dependent inhibition of transport (7), is conserved in both sexes albeit at different baseline starting points. NaPi2, which resides in non-rafts, is internalized into subapical endosomes during aHTN in both sexes, with a visually more pronounced loss of apical signal in females.

The finding that SGLT2 distribution was unaltered by aHTN in either sex (Figure 4) is noteworthy in light of the literature describing NHE3-MAP17-SGLT2 structural interactions that play a role NHE3-SGLT2i driven natriuresis (51–53). While NHE3 and NaPi2 are well-established effectors of pressure natriuresis, SGLT2 has not been previously examined in this context. The stability of SGLT2 localization during acute hypertension may reflect distinct membrane domains and/or trafficking machinery governing SGLT2 versus NHE3 and shows that not all proximal tubule sodium transport pathways participate in the acute pressure natriuretic response. In support of this idea, we did not detect any change in urine glucose from baseline during the aHTN protocol (not shown).

We did not predict that the pool sizes of sodium transporters along the nephron would change rapidly during acute (25 min) hypertension. NHE3 total abundance (by immunoblot) was, indeed, not altered by 25 min aHTN, consistent with the paradigm that pressure natriuresis operates primarily through NHE3 redistribution within microvilli (14, 54). Covalent modification of NHE3 by phosphorylation at S552 (NHE3p), a marker for localization to the microvillar base where activity is reduced (8), tended to increase during aHTN in both males (P=0.05) and females (p=0.06); combining data of sexes reduced P value to 0.01, consistent with the redistribution results (Figure 4). In the medullary compartment during aHTN, abundance of NHE3 and NKCC2 decreased in both sexes, supporting a role for medullary thick ascending limb participation in pressure natriuresis as reported previously (55, 56). This rapid reduction in antibody detection of total NHE3 and NKCC2 in the medulla during 25 min aHTN has not been previously reported, to our knowledge, and the mechanism underlying the reduction remains to be explored. A plausible link is suggested by recent reports of cGMP-dependent ubiquitination and proteasomal degradation of NKCC2 in the thick ascending limb (57, 58), coupled to earlier reports that acute elevations in renal perfusion pressure increase medullary nitric oxide production in a pressure-dependent manner (59, 60), which activates soluble guanylyl cyclase to generate cGMP (61). However, this link requires validation and cannot exclude alternative explanations.

Computational modeling analysis that incorporated measured changes in GFR and previously reported transporter abundance into sex-specific nephron transport models(22) predicted that female Na⁺ excretion would increase 13.3-fold over baseline versus 7.8-fold in males during aHTN, yielding a female-to-male excretion ratio of 1.7. This prediction closely approximates the experimentally observed ratio, providing independent validation that the measured transporter changes are sufficient to account for the observed sex difference in pressure natriuresis. The models also predict that the proximal tubule is the dominant site of transport inhibition in both sexes, with increased downstream delivery overwhelming the reabsorptive capacity of distal segments.

We previously reported that NCC traffics to subapical vesicles during aHTN in male rats(15) and in this study report significant reductions in total abundance of NCC and NCCpS71 (a phosphorylation marker of apical NCC localization(62)) in males, but not females; both responses would reduce NCC in apical membrane and amplify pressure natriuresis in a male specific manner. Simultaneously with NCC suppression, the rise in full-length, unprocessed γENaC in males, but not females, suggests an accumulation of near silent channels (44). Importantly, the decrease in transport activity of NCC during aHTN in males would also amplify pressure natriuresis by counteracting flow dependent stimulation of sodium reabsorption in response to delivery from the proximal nephron in males (63). The lack of a similar response in females could be attributed to the lower fractional sodium reabsorption along the proximal nephron at baseline and during aHTN.

Endogenous lithium clearance, an established marker of volume flow from the proximal tubule and medullary thick ascending limb (39, 41), spiked approximately 6-fold at 5 min aHTN ,before settling to ~3.5-fold above baseline at 25 min in both sexes (Figure 3A). The spike paralleled the 7-fold transient increase in GFR, which settled to a 2-fold increase at 25 min. The results indicate equivalent reductions in proximal and loop reabsorption of sodium (not potassium) reabsorption, consistent with the comparable NHE3 redistribution and medullary NKCC2 reductions observed in both sexes. Sodium clearance, which reflects whole-nephron sodium handling, was significantly greater in females than males at 25 min (Figure3B, P=0.01) consistent with the greater UNaV versus MAP evident in Figure 2B. Although males exhibited decreased NCC phosphorylation during acute hypertension, which would likely reduce sodium reabsorption in the distal convoluted tubule and favor natriuresis, this adaptation was not sufficient to close the gap in sodium excretion between sexes.

The spike in GFR at 5 min was coincident with a transient spike in potassium excretion, likely reflecting increased filtered potassium delivery during the autoregulatory transition. However, unlike sodium, UKV returned to baseline by 10–15 min aHTN indicating that the pressure natriuretic response is sodium-selective and that potassium-handling mechanisms that could provoke natriuresis, such as inhibition of potassium secretion by distal ROMK or flow-dependent BK channels, are not engaged by aHTN. Neither Na,K-ATPase subunit abundance nor K-pNPPase activity were altered over 25 min aHTN, consistent with our prior report that Na,K-ATPase activity decreases during the first min of acute hypertension but recovers by 15–20 min (9), a time frame that encompasses the end point of our enzymatic assay.

Can sex differences in pressure natriuresis be related to sex differences in salt sensitivity of blood pressure, which is more prevalent in females than males (64)? Most women are not salt sensitive: while 42% of middle-aged women are hypertensive, only half (~20%) develop salt sensitivity with age. The Sprague Dawley strain, used herein, is salt resistant, as it does not exhibit hypertension when provided with 4% NaCl lab chow (65). Additionally, in this study the dietary potassium to sodium ratio is ~ 2 which augments natriuresis (66). An increased incidence of salt-sensitivity with age in either sex is defined by a reduced ability to excrete dietary salt: we recently reported, in normal female SD rats injected with a bolus of normal saline, that 12 mo old rats required much longer to excrete the sodium bolus than 4.5 mo old rats (67).

Our hypothesis that CYP450 inhibition would blunt pressure natriuresis in both sexes was based on earlier reports in males alone that showed that ABT or cobalt chloride pre-treatment contribute to the pressure natriuresis response to vascular constriction by blunting: natriuresis, rise in lithium clearance, redistribution of proximal NHE3, and the rise in interstitial hydrostatic pressurez. However, in our current study, ABT pre-treatment followed by aHTN versus aHTN without pre-treatment with ABT shifted renal function curves leftward in both sexes, and augmented cumulative sodium excretion in males (not females). Aligning with the immunohistochemistry findings, the impact of ABT pretreatment on transporter relative abundance during aHTN, assessed by immunoblot, was similar to that observed without ABT pretreatment in both males (Figure S2) and females (Figure S3). In males, ABT pretreatment did lower phosphorylation of NCC (pT53 and pS71), but this did not provide insight into the mechanism(s) responsible for the enhanced pressure natriuresis as these changes would not contribute to a leftward shift in renal function curves. There were no significant changes in transporter abundance with ABT pretreatment in females.

The discrepancy between the current findings and earlier reports of attenuated natriuresis with CYP inhibition may reflect the well characterized opposing impact of ABT on the vasculature and the renal tubule epithelia, as well as methodological differences: the present study employed systemic ABT pretreatment (50 mg/kg/day for 5 days), which inhibits both vascular and tubular CYP activity, whereas prior studies used acute intrarenal administration. When both vascular and tubular 20-HETE production are inhibited simultaneously, the reduction in renal vascular resistance (lowering the blood pressure set-point) may outweigh the loss of tubular 20-HETE-mediated natriuresis, resulting in a net leftward shift. Consistent with this interpretation, ABT lowered baseline MAP in both sexes, reaching statistical significance only in females, suggesting that CYP-derived metabolites contribute to basal vascular tone. The greater natriuretic augmentation observed in ABT-treated males may reflect the higher androgen-driven CYP4A/20-HETE production in males, (24, 30) such that removal of this vascular pro-hypertensive signal has a larger impact on the male pressure-natriuresis set-point. The current findings align more closely with recent work demonstrating that the pro-hypertensive vascular actions of 20-HETE can predominate over its anti-hypertensive tubular effects: The Schwartzman group showed that proximal tubule-targeted overexpression of the CYP4A12–20-HETE synthase promotes salt-sensitive hypertension and a rightward shift in renal function curves in male mice, and also demonstrated that 20-HETE receptor blockade lowers blood pressure in mice with vascular smooth muscle-specific CYP4A12 overexpression (68, 69).

Several limitations of the current study warrant consideration. First, the use of Inactin anesthesia, while standard for acute renal physiological studies, increases systemic adrenergic activity (70) and may attenuate sex differences in sympathetic reflex responses (71), potentially masking the full magnitude of sex-specific transporter trafficking responses observed in the conscious state. Second, the immunofluorescence analysis of transporter trafficking provides spatial, not quantitative information about transporter redistribution, and was based on a subset of rats. Third, interpretation of the ABT findings is constrained by both smaller sample sizes in the male ABT-treated group (n=4) and the nature of ABT which blocks all CYP450-dependent arachidonic acid metabolism, simultaneously eliminating 20-HETE (pro-hypertensive in the vasculature but natriuretic in the tubule) and EETs (vasodilatory). Because these metabolites exert opposing vascular effects, the net hemodynamic and tubular consequences of ABT is a function inhibiting both eicosanoid pathways.

In summary, this study establishes that female Sprague Dawley rats exhibit a more robust acute pressure natriuretic response than males, facilitated by baseline sex differences in nephron transporter patterns and sex-specific acute molecular adaptations to hypertension. The female nephron, with its baseline pattern of lower proximal and higher distal transport capacity, is inherently positioned for efficient sodium excretion when perfusion pressure rises. Males partially compensate with lower distal NCC activity. Taken together, females excrete more sodium than males at any given perfusion pressure and achieve comparable cumulative sodium excretion through distinct, sex-specific molecular strategies.

Supplementary Material

Supplemental Figs. S1-S4: DOI:10.6084/m9.figshare.33136472.

Supplemental Table S1: DOI:10.6084/m9.figshare.33136472.

Data Availability Statement

The data that supports the findings of this study are available in the manuscript as well as Supporting information of this article.