Vascular control of kidney epithelial transporters

Abstract

A major pathway in hypertension pathogenesis involves direct activation of ANG II type 1 (AT₁) receptors in the kidney, stimulating Na⁺ reabsorption. AT₁ receptors in tubular epithelia control expression and stimulation of Na⁺ transporters and channels. Recently, we found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT₁ receptors in smooth muscle (SMKO mice). Although impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary Na⁺ excretion in SMKO mice, we considered whether alterations in Na⁺ transporter expression might also play a role; therefore, we carried out proteomic analysis of key Na⁺ transporters and associated proteins. Here, we show that levels of Na⁺-K⁺-2Cl⁻ cotransporter isoform 2 (NKCC2) and Na⁺/H⁺ exchanger isoform 3 (NHE3) are reduced at baseline in SMKO mice, accompanied by attenuated natriuretic and diuretic responses to furosemide. During ANG II hypertension, we found widespread remodeling of transporter expression in wild-type mice with significant increases in the levels of total NaCl cotransporter, phosphorylated NaCl cotransporter (Ser⁷¹), and phosphorylated NKCC2, along with the cleaved, activated forms of the α- and γ-epithelial Na⁺ channel. However, the increases in α- and γ-epithelial Na⁺ channel with ANG II were substantially attenuated in SMKO mice. This was accompanied by a reduced natriuretic response to amiloride. Thus, enhanced urinary Na⁺ excretion observed after cell-specific deletion of AT₁ receptors from smooth muscle cells is associated with altered Na⁺ transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial in the kidney, modulating the expression of Na⁺ transporters and contributing to the regulation of pressure natriuresis.

NEW & NOTEWORTHY The use of drugs to block the renin-angiotensin system to reduce blood pressure is common. However, the precise mechanism for how these medications control blood pressure is incompletely understood. Here, we show that mice lacking angiotensin receptors specifically in smooth muscle cells lead to alternation in tubular transporter amount and function. Thus, demonstrating the importance of vascular-tubular cross talk in the control of blood pressure.

INTRODUCTION

The renin-angiotensin system (RAS) is a key pathway for blood pressure control, and its dysregulation contributes to hypertension pathogenesis (1). The importance of the RAS in human hypertension is highlighted by the broad clinical efficacy of angiotensin receptor blockers (ARBs) in the treatment of essential hypertension (2–5). Angiotensin II (ANG II) is the major biologically active peptide generated by the RAS, and its main effects on blood pressure are mediated by ANG II type 1 (AT₁) receptors (6). AT₁ receptors are expressed in a number of cell lineages important for blood pressure control, including vascular smooth muscle cells (VSMCs), cardiovascular regulatory regions in the brain, kidney epithelia, and zona glomerulosa cells in the adrenal cortex (7, 8). Yet, experiments using kidney cross-transplantation (9) and cell-specific knockout of AT₁ receptors from kidney epithelia (10) have indicated that direct actions of ANG II on AT₁ receptors in the kidney to reduce urinary Na⁺ excretion constitute a major pathway in hypertension pathogenesis (1, 9–11).

Previous studies have clearly documented the actions of AT₁ receptors to regulate abundance and function of a range of critical kidney Na⁺ transporters, including Na⁺/H⁺ exchanger isoform 3 (NHE3), Na⁺-K⁺-2Cl⁻ cotransporter isoform 2 (NKCC2), NaCl cotransporter (NCC), and epithelial Na⁺ channel (ENaC) (12–16). These regulatory actions involve a number of intracellular signaling pathways, including protein kinase C, STE20/SPS1-related proline-alanine-rich kinase, serum- and glucocorticoid-regulated kinase 1, with no lysine-4, and reactive oxygen species (ROS) activation (12, 17, 18). The relevance of these pathways in physiological regulation has also been well documented (12). For example, previous studies by our group and others have shown that elimination of AT₁ receptors from proximal tubule epithelia protects against hypertension, reduces abundance of key Na⁺ exchangers such as NHE3, and promotes natriuresis (13, 19). Taken together, this work strongly indicates that activation of AT₁ receptors in kidney epithelial cells increases abundance and activity of Na⁺ transporters, enhancing tubular Na⁺ reabsorption and promoting hypertension.

AT₁ receptors in smooth muscle cells in the vasculature represent another target for raising blood pressure in hypertension. In previous studies, we confirmed an important direct contribution of AT₁ receptors in VSMCs to blood pressure control and hypertension pathogenesis using mice with cell-specific deletion of AT_1A receptors, the major murine AT₁ receptor isoform (20), from smooth muscle (SMKO mice) (21, 22). These SMKO mice lacking AT_1A receptors in VSMCs have lower baseline blood pressures, preserved renal blood flow (RBF), and attenuated hypertensive responses to chronic ANG II administration (21, 22). Furthermore, they exhibit enhanced natriuresis during chronic ANG II infusion, which we posited was a key mechanism in their resistance to hypertension (21).

ANG II causes kidney vasoconstriction, reducing RBF while preserving glomerular filtration rate (GFR) due to preferential constriction of efferent compared with afferent glomerular arterioles (23, 24). The expected hemodynamic consequences of this ANG II-dependent increase in filtration fraction (GFR/RBF) would be enhanced kidney Na⁺ conservation caused by reduced peritubular capillary and interstitial hydrostatic pressures favoring tubular Na⁺ reabsorption (25–27). Conversely, reversing ANG II-dependent vasoconstriction would promote natriuresis (28). Indeed, we found that RBF was increased in SMKOs at baseline and preserved during ANG II infusion (21). Therefore, we concluded that favorable alterations in kidney hemodynamics might be one mechanism promoting natriuresis and lowering blood pressures in mice lacking AT_1A receptors in VSMCs (21).

On the other hand, previous studies addressing the impact of peritubular capillary flow, peritubular colloid oncotic pressure, and interstitial Starling forces on Na⁺ transport have typically used acute experimental systems with instrumented animals where vascular and epithelial effects of ANG II cannot be distinguished (25, 29). Furthermore, potential contributions of altered abundance of Na⁺ transporters in this process have not been considered (28, 30). The SMKO model, where AT_1A receptors are eliminated from VSMCs but preserved in all kidney epithelial lineages, is a unique experimental system allowing clear delineation of the potential impact of vascular AT₁ receptors on tubular epithelial responses. Using this system, we demonstrate a novel pathway for vascular-epithelial whereby stimulation of AT_1A receptors in VSMCs affects Na⁺ transporter abundance, potentially impacting natriuretic responses.

METHODS

Generation of Mice

To examine the changes in Na⁺ transporter (cotransporter and channel) abundance in mice lacking vascular AT_1A receptors, we used mice with specific deletion of AT_1A receptors from smooth muscle cell lineages, SMKO (KISm22α-Cre^pos;Agtr1a^flox/flox) and control (KISm22α-Cre^neg;Agtr1a^flox/flox), as previously described (21, 22, 31). All mice used in these experiments were male and age-matched littermates (age: 10–20 wk) on an inbred C57BL/6 background.

Details of Reporter Mice

Membrane-targeted tdTomato (mT)/membrane-targeted enhanced green fluorescent protein (EGFP) (mG) mice with loxP sites flanking the membrane-targeted tdTomato cassette followed by an NH₂-terminal membrane-tagged version of EGFP were purchased from The Jackson Laboratory [Strain name: B6.129(Cg)-Gt(ROSA)26Sort m4(ACTB-tdTomato,-EGFP)Luo/J, Stock No. 007676] and crossed with constitutively expressed KIsm22α-Cre. All animal experiments were approved by the Duke University and Durham Veterans’ Affairs Health Care System Institutional Animal Care and Use Committees and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals had free access to standard rodent chow and water unless specified.

ANG II Infusion Model of Hypertension

To induce hypertension, osmotic minipumps (model 2004, Alzet, Durect, CA) filled with ANG II (1,000 ng/kg/min) were implanted in mice. After 5 days of infusion, mice were euthanized to assess the abundance of various Na⁺ transporters. To confirm hypertension, heart-to-body weight ratios were determined immediately after euthanization. The weight of the Alzet minimpump was subtracted from body weight before the ratio was determined.

Quantitative Immunoblot Analysis

Proteomic analysis of transporters and other related proteins in kidneys were determined using quantitative immunoblot analysis as previously described (32). The kidneys were flash frozen after mice were euthanized. The kidney cortex and medulla were dissected on ice and homogenized, respectively, in 5 mL and 3 mL of isolation buffer [5% sorbitol, 0.5 mM disodium EDTA, and 5 mM histidine-imidazole buffer (pH 7.5) with 0.2 mM phenylmethylsulfonyl fluoride, 9 µg/mL aprotinin, and 5 µL/mL of a phosphatase inhibitor cocktail (P2850, Sigma)]. Samples were homogenized for 5 min at low setting with an Ultra-Turrax T25 (IKA Laboratory) and centrifuged at 2,000 g for 10 min. The pellets from the cortex samples were homogenized again in 5 mL isolation buffer, centrifuged, and pooled with the first supernatant. Aliquots were directly frozen in liquid nitrogen and stored at −80°C. Protein quantification was performed using the bicinchoninic acid (BCA) assay (Pierce Thermo Scientific, Rockford, IL). All kidney cortex and medulla homogenates were denatured in SDS-PAGE sample buffer for 20 min at 60°C. To ensure equal protein concentrations across all samples, 12 µg of protein from each sample were resolved by SDS-PAGE stained with Coomassie blue, and random bands were quantified to determine equivalent protein content across all samples (Supplemental Fig. S1, all Supplemental Material is available at https://doi.org/10.6084/m9.figshare.14473539.v1). For each loading sample on the blots, a duplicate was performed with half the volume to ensure the linearity of the detection system (Supplemental Fig. S1). Signals were measured using the Odyssey Infrared Imaging System (LI-COR), and quantification of the signals was performed using the corresponding Image Studio Light software (LI-COR, Lincoln, NE). The antibodies used for protein identification and quantitation are provided in Supplemental Table S1. Arbitrary density units were normalized to the mean intensity of the control group, which was defined as 1.0. The results of the duplicates (1 and ½) were normalized and averaged. Statistical analysis was carried out using two-way ANOVA and a Sidak posttest using Prism GraphPad (San Diego, CA).

Determination of Urinary Aldosterone and Creatinine Content

Aldosterone content was quantified from urine by an enzyme immunoassay (Aldosterone ELISA, No. 501090, Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. Creatinine content in urine was determined using the creatinine companion kit (Mouse Exocell, Philadelphia, PA).

GFR Methods

FITC-inulin clearance was measured in conscious mice as previously described (33, 34). FITC-inulin (5%) was injected into the penile vein of mice that were briefly anesthetized with isoflurane. At 3, 7, 10, 15, 35, 55, and 75 min after the FITC-inulin injection, blood samples were obtained from the lateral saphenous vein (35). Fluorescence intensity was measured at 485-nm excitation/520-nm emission using a FLUOstar Omega plate reader (BMG Labtech). Plasma fluorescence was fitted to a two-phase exponential decay using nonlinear regression (GraphPad Prism). GFR was calculated using standard formulas (36) and is reported as microliters per minute per gram body weight (37).

Natriuretic Response to Furosemide

As previously described (38, 39), furosemide (Sigma-Aldrich, St. Louis, MO) was first dissolved in methanol followed by sterile saline and brought to a pH of ∼7.7 with NaOH and warmed to 38°C to further solubilize. A dose of 25 mg/kg furosemide in a volume of 500 µL was administered by intraperitoneal injection to control (n = 9) and SMKO (n = 9) mice. For all of the diuretic experiments, mice were individually housed overnight in metabolic cages to collect urine and were provided a gel diet containing all nutrients and water (S5769, BioServ Nutragel). Urine was collected for 20 h before and 4 h after furosemide injection. Urine was centrifuged for 5 min at 6,500 rpm to remove particulates, and the resulting supernatant was stored at −80°C for later analysis. Na⁺ concentrations in the urine samples were determined using a flame photometer (model 02655-10, Cole-Parmer).

Natriuretic Response to Amiloride During Chronic ANG II Infusion

ANG II was infused chronically by osmotic mini-pump (1,000 ng/kg/min) in control (n = 5) and SMKO (n = 8) mice as described above. On day 4 of ANG II infusion, sterile saline was administered by intraperitoneal injection (100 µL/25 g body wt) before mice were placed in the metabolic cages and urine was collected for 24 h. A dose of 10 mg/kg amiloride (No. 1019701, Sigma-Aldrich), first dissolved in sterile saline and warmed in a 55°C water bath to solubilize, was then administered by intraperitoneal injection (100 µL/25 g body wt). Urine was collected for 24 h postinjection. Urine samples were processed and Na⁺ concentrations determined as described above.

Statistical Analysis and Experimental Design

All experiments were performed in a blinded fashion. For the physiological study, all genotyping information was excluded from the cage cards and no genotype assignment was entered thereafter. The surgeon and/or technician randomly selected mice from cages (all mice were eventually used), and the physiological study (e.g., GFR and furosemide challenge) was performed. Data were analyzed only after the entire experiment was completed (with a predetermined number of mice based on a power calculation) by an individual with no knowledge of genotype. A genotype key was used only after all data for that particular experiment were complete and analyzed. To ensure correct genotype, each animal’s ear was clipped before and after experiments were performed, and the clipped tissue was sent for regenotyping. All statistical analyses were determined a priori. Values for each parameter within a group are means ± SE. For analyses with multiple treatments (uninfused vs. ANG II) and genotypes (control vs. SMKO), two-way ANOVA with a Sidak posttest for multiple comparisons was used. For furosemide and amiloride challenge, repeated-measures two-way ANOVA with a Sidak posttest for multiple comparisons was used. For analyses with only one variable (control vs. SMKO), an unpaired t test was used (GFR and NKCC2). Heatmaps were plotted using pheatmap (1.0.12), and hierarchical clustering was performed across the columns (proteins) using the Euclidean method. The R version used was 3.6.3.

RESULTS

Absence of AT_1A Receptors in VSMCs Alters the Kidney Epithelial Transporter Profile at Baseline

In three published studies, we have reported that basal blood pressures are reduced by ≈7–8 mmHg in mice with cell-specific deletion of AT_1A receptors from VSMCs (SMKO mice) compared with control mice (21, 22, 40). Although this alteration in blood pressure might be explained by kidney hemodynamic changes (21), the magnitude of these effects is sufficiently large and durable such that we considered whether altered abundance and activity of key Na⁺ transporters might also be contributing. Note that SMKO mice lack AT_1A receptors in VSMCs but have the full complement of receptors in other cell lineages in the kidney, including tubular epithelial cells. Figure 1 shows specific expression of KISm22α-Cre in vascular structures but not tubules of the double-reporter mTmG mouse kidney. To test this hypothesis, we carried out a systematic proteomic analysis of key kidney Na⁺ transporters, cotransporters, and channels in control and SMKO mice (Fig. 2).

Figure 1.

Expression of Cre recombinase in KIsm22 Cre mice using the mTmG double reporter. A: ×40 view of the kidney medulla showing green fluorescence in the perivascular spaces and not tubular structures where red fluorescence is located. Scale bar = 100 μm. B: ×10 view of the kidney cortex showing intense green fluorescence in vascular structures and not in tubular structures. Intense red fluorescence can be seen in glomeruli. Scale bar = 100 μm. C: stitched view of the entire kidney of a KIsm22 Cre+ and mTmG+ mouse. Note the predominance of vascular structures positive (green). Scale bar = 500 μm.

Figure 2.

Na⁺ transporter protein abundance in control mice and mice with cell-specific deletion of angiotensin II (ANG II) type 1 receptors in smooth muscle (SMKO mice). A: proteomic analysis of transporters and channels in kidneys was determined using quantitative immunoblotting. Signals were measured and quantified, and arbitrary density units were normalized to the mean intensity of the control group, which was defined as 1.0. B: mean levels for abundance of all individual Na⁺ transporters in the kidney cortex were normalized to control levels, which were arbitrarily expressed as a value of 1.0. mNHE3 and mNKCC2 refer to the medullary forms of Na⁺/H⁺ exchanger isoform 3 (NHE3) and Na⁺-K⁺-2Cl⁻ cotransporter isoform 2 (NKCC2), respectively. Data were plotted as a heatmap with the color scale as depicted for levels of expression from 0.5 to 2.5. ANG II was infused for 5 days. ENaC-Cl, cleaved epithelial Na⁺ channel (ENaC); ENaC-Fl, full-length EnaC; NaPi2, Na⁺-P_i cotransporter isoform 2; NCC, NaCl cotransporter; NCCpS71, phosphorylated NCC (Ser⁷¹).

Indeed, we found significant differences in transporter/exchanger abundance between SMKO and control mice (Fig. 2). As shown in Fig. 3A, the levels of total NHE3 in the medulla of SMKO mice were reduced by ≈40% compared with controls. Similarly, baseline levels of total NKCC2 were significantly reduced by ≈40% in the medulla and cortical NKCC2 was ≈25% lower in SMKO mice compared with controls (Fig. 4). Phosphorylated NKCC2 was not analyzed in view of the recent discovery of a mutation in the Nkcc2 gene in C57BL/6 mice obscuring epitopes recognized by most widely used anti-pNKCC2 antibodies (41). Notably, several key transporters were not altered at baseline in SMKO mice, including Na⁺-P_i cotransporter isoform 2, NCC, phosphorylated NCC, and ENaC subunits (Fig. 2). In summary, the abundance of key Na⁺ transporters/exchangers [NHE3 in the proximal tubule and thick ascending limb (TAL) as well as NKCC2] are selectively diminished in SMKO mice at baseline compared with controls. These differences would be expected to promote natriuresis, likely contributing to the lower blood pressures seen at baseline in SMKO animals.

Figure 3.

Total Na⁺/H⁺ exchanger isoform 3 (NHE3) protein levels in control mice and mice with cell-specific deletion of angiotensin II (ANG II) type 1A receptors in smooth muscle (SMKO mice) at baseline and during ANG II infusion (5 days). A: total NHE3 protein levels in the kidney cortex. At baseline, levels of NHE3 in the kidney cortex tended to be lower in SMKO mice (open circles) compared with control mice (closed circles) (0.78 ± 0.09 vs. 1 ± 0.11 arbitrary units, P = 0.3). In control mice during ANG II infusion, levels of NHE3 in the cortex (closed squares) were lower than uninfused (unif) control mice (0.6 ± 0.06 vs. 1 ± 0.11 arbitrary units, P = 0.004). ANG II infusion had no significant effect on cortical NHE3 levels in SMKO mice (open squares) compared with uninfused SMKO mice (0.9 ± 0.04 vs. 0.78 ± 0.09 arbitrary units, P = 0.9). Two-way ANOVA for treatment: F(1,11) = 4.7, P = 0.05, and for genotype: F(1,12) = 0.17, P = 0.7. B: total NHE3 protein levels in the kidney medulla. At baseline, NHE3 levels in the kidney medulla were significantly lower in SMKO mice compared with control mice (0.60 ± 0.07 vs. 1 ± 0.07 arbitrary units, **P < 0.0001). In control mice during ANG II infusion, levels of NHE3 in the medulla (closed squares) were lower than in uninfused control mice (0.64 ± 0.07 vs. 1 ± 0.07 arbitrary units, *P = 0.002). ANG II infusion had no significant effect on medullary NHE3 levels in SMKO mice (open squares) compared with uninfused SMKO mice (0.47 ± 0.01 vs. 0.60 ± 0.07 arbitrary units, P = 0.4). However, medullary NHE3 levels were lower in ANG II-infused SMKO mice compared with uninfused control mice (0.47 ± 0.01 vs. 1 ± 0.07 arbitrary units, ***P < 0.0001). Two-way ANOVA for treatment: F(1,11) = 26.9, P < 0.0005, and for genotype: F(1,12) = 22.5, P < 0.0005. Dot plots were derived from densitometry data from immunoblots shown in Fig. 2. For both the cortex and medulla, n = 7 for ANG II-infused and uninfused SMKO mice, n = 7 for uninfused control mice, and n = 6 for ANG II-infused mice.

Figure 4.

Reduced levels of Na⁺-K⁺-2Cl⁻ cotransporter isoform 2 (NKCC2) at baseline in mice lacking vascular angiotensin II (ANG II) type 1 A (AT_1A) receptors (SMKO mice). A: total NKCC2 protein levels in the medullary thick ascending limb. At baseline, NKCC2 levels in the kidney medulla were significantly lower in SMKO mice compared with control mice (0.60 ± 0.02 vs. 1 ± 0.05 arbitrary units, **P < 0.0001). In control mice during ANG II infusion (5 days), levels of NKCC2 in the medulla (closed squares) were reduced compared with uninfused (unif) control mice (0.71 ± 0.04 vs. 1 ± 0.05 arbitrary units, *P < 0.0001). ANG II infusion had no significant effect on medullary NKCC2 levels in SMKO mice (open squares) compared with uninfused SMKO mice (0.56 ± 0.03 vs. 0.59 ± 0.02 arbitrary units, P = 0.99). However, medullary NKCC2 levels were reduced in ANG II-infused SMKO mice compared with uninfused control mice (0.56 ± 0.03 vs. 1 ± 0.05 arbitrary units, ***P < 0.0001). Two-way ANOVA for treatment: F(1,23) = 19.6, P = 0.0002, and for genotype: F(1,23) = 56.8, P < 0.0001. B: total NKCC2 protein levels in the cortical thick ascending limb. At baseline, levels of NKCC2 in the kidney cortex were lower in SMKO mice (open circles) compared with control mice (closed circles) (0.8 ± 0.03 vs. 1 ± 0.03 arbitrary units, ***P = 0.007). In control mice during ANG II infusion, levels of NKCC2 in the cortex (closed squares) were similar to uninfused control mice (0.9 ± 0.05 vs. 1 ± 0.03 arbitrary units, P = 0.82). ANG II infusion had no significant effect on cortical NKCC2 levels in SMKO mice (open squares) compared with uninfused SMKO mice (0.7 ± 0.07 vs. 0.8 ± 0.03 arbitrary units, P = 0.79). However, cortical NKCC2 levels were reduced in ANG II-infused SMKO mice compared with uninfused control mice (0.67 ± 0.07 vs. 1 ± 0.03 arbitrary units, *P = 0.0003), and cortical NKCC2 levels in ANG II-infused SMKO mice were reduced versus ANG II-infused control mice (0.67 ± 0.07 vs. 0.9 ± 0.05 arbitrary units, **P = 0.01). Two-way ANOVA for treatment: F(1,23) = 2.9, P = 0.1, and for genotype: F(1,23) = 26.8, P < 0.0001. Dot plots were derived from densitometry data from Fig. 2. For both the cortex and medulla, n = 7 for ANG II-infused and uninfused SMKO mice, n = 7 for uninfused control mice, and n = 6 for ANG II-infused mice.

Impaired Acute Response to Furosemide in SMKO Mice

To determine whether the reduced abundance of NKCC2 protein levels along the medullary and cortical TAL observed at baseline in SMKO mice has functional consequences, we compared responses to acute administration of the specific NKCC2 blocker furosemide in SMKO and control mice. As shown in Fig. 5, urine volumes and Na⁺ excretion were similar in SMKO and control mice after vehicle administration. In control mice, administration of furosemide significantly increased urine volume by ≈100% (P < 0.0001) and Na⁺ excretion by ≈220% (P < 0.00001). Furosemide also significantly increased urine volume and Na⁺ excretion in SMKO mice, by ≈66% and ≈116%, respectively (P = 0.01). However, the magnitudes of both diuretic (1.5 ± 0.07 vs. 1.8 ± 0.2 mL/4 h, P < 0.05) and natriuretic (33.6 ± 1.7 vs. 48.6 ± 1.9 μmol/h, P < 0.0001) responses to furosemide were significantly less in SMKO compared with control mice (Fig. 5). Thus, the lower abundance of NKCC2 protein in SMKO animals was associated with less responsiveness to the diuretic furosemide.

Figure 5.

Furosemide resistance in mice with cell-specific deletion of angiotensin II (ANG II) type 1 receptors in smooth muscle (SMKO mice). A: diuretic responses to furosemide. Urine volumes were similar in control mice (closed circles) and SMKO mice (open circles) after vehicle injection. After injection of 25 mg/kg furosemide, urine volumes increased in both groups, but post-furosemide urine volume (mL/h) was significantly lower in SMKO mice compared with control mice. Two-way repeated-measures ANOVA for treatment: F(1,16) = 37.7, P < 0.0001. Sidak post tests: 0.46 ± 0.04 mL/h for furosemide-treated control mice vs. 0.37 ± 0.02 mL/h for furosemide-treated SMKO mice (*P < 0.05), 0.24 ± 0.02 mL/h for vehicle-treated SMKO mice vs. 0.37 ± 0.02 mL/h for furosemide-treated mice (**P < 0.01), and 0.24 ± 0.02 mL/h for vehicle-treated control mice vs. 0.46 ± 0.04 mL/h for furosemide-treated control mice (***P < 0.0001). B: natriuretic responses to furosemide. Furosemide administration increased Na⁺ excretion in both groups. However, the natriuretic response in SMKO mice was blunted compared with control mice. Two-way repeated-measures ANOVA for treatment: F(1,16) = 93.7, P < 0.0001. Sidak post tests: 48.6 ± 2 µmol Na⁺/h for furosemide-treated control mice vs. 33.7 ± 2 µmol Na⁺/h for furosemide-treated SMKO mice (*P < 0.0001), 15.5 ± 3 µmol Na⁺/h for vehicle-treated SMKO mice vs. 33.7 ± 2 µmol Na⁺/h for furosemide-treated SMKO mice (**P > 0.0005), and 15.3 ± 3 µmol Na⁺/h for vehicle-treated control mice vs. 48.6 ± 2 µmol Na⁺/h for furosemide-treated control mice (***P < 0.00001). n = 9 per group.

Altered Profile of Kidney Epithelial Transporters in Control Mice During ANG II Hypertension

As previous studies have documented substantial alterations in kidney transporter abundance contributing to blood pressure elevation in ANG II hypertension (15, 42, 43), we first examined the effects of 5 days of ANG II infusion on transporter profiles in control mice compared with noninfused control mice and found a number of changes. After 5 days of ANG II infusion, control mice developed significant cardiac hypertrophy (6.7 ± 0.5 mg/g) compared with noninfused control mice (4.5 ± 0.1 mg/g, P < 0.0001), consistent with the presence of hypertension (Fig. 6). In previous studies, we found that SMKO mice had attenuated hypertension, exaggerated natriuresis, and diminished cardiac hypertrophy in response to chronic ANG II infusion (21, 22, 40). Furthermore, we have reported in this model (40) and others (9) that the magnitude of cardiac hypertrophy is directly correlated with the extent of blood pressure elevation in ANG II hypertension. As has been previously observed (13), we found significant reductions of cortical NHE3, both total protein and phosphorylated NHE3, by 41% and 40%, respectively, with ANG II infusion (Figs. 2 and 3). Levels of NKCC2 in the medulla were similarly reduced (Fig. 4). In contrast, ANG II significantly increased the abundance of NCC by ≈62% and phosphorylated NCC (Ser⁷¹) by ≈206%. Similarly, cleaved (activated) forms of the α- and γ subunits of ENaC increased by 258% and 66%, respectively, with a coincident reduction in the full-length form of γ-ENaC by 35% (P < 0.0001; Figs. 2 and 7). Thus, in control mice, chronic ANG II infusion causes significant increases of key distal Na⁺ transporters known to promote hypertension, including NCC, phosphorylated NCC (Ser⁷¹), and activated ENaC subunits.

Figure 6.

Attenuation of cardiac hypertrophy during angiotensin II (ANG II)-induced hypertension in mice with cell-specific deletion of ANG II type 1 receptors in smooth muscle (SMKO mice) and no change in glomerular filtration rate (GFR) at baseline. A: cardiac hypertrophy was attenuated in SMKO mice. No difference was seen in heart weights between control (n = 7) and SMKO (n = 7) groups for uninfused (unif) mice (4.5 ± 0.1 vs. 4.9 ± 0.1 mg/g body wt, P = 0.89). After 5 days of ANG II infusion, control mice (n = 7) developed cardiac hypertrophy (control: 4.5 ± 0.1 mg/g body wt vs. ANG II-infused control: 6.7 ± 0.5 mg/g body wt, ***P < 0.0001), whereas SMKO mice (n = 7) did not (SMKO: 4.9 ± 0.1 mg/g body wt vs. ANG II-infused SMKO: 5.5 ± 0.2 mg/g body wt, P = 0.53). Furthermore, SMKO mice had less cardiac hypertrophy compared with control mice (5.5 ± 0.2 vs. 6.7 ± 0.5 mg/g body wt, *P = 0.02). Two-way ANOVA for treatment: F(1,24) = 27.7, P < 0.0001, and for genotype: F(1,24) = 2.2, P = 0.14. B: no change in GFR in SMKO mice. GFR was measured in conscious mice using the FITC-inulin method. Control mice had a GFR of 22.4 ± 2.4 μL/min/g body wt versus 22.7 ± 1.9 μL/min/g body wt in SMKO mice (P = 0.9, unpaired t test).

Figure 7.

Levels of protease-cleaved forms of α- and γ-subunits of the epithelial Na⁺ channel (ENaC) before and during angiotensin II (ANG II) infusion. Protease cleavage is essential for ENaC activation. Thus, we measured cleaved fragments of the ENaC subunits. A: cleaved α-ENaC in the kidney cortex. At baseline, levels of cleaved α-ENaC in the kidney cortex were similar in control mice (closed circles) and mice with cell-specific deletion of ANG II type 1 receptors in smooth muscle (SMKO mice; open circles). During ANG II infusion (5 days), cleaved α-ENaC levels increased in both control mice (closed squares) and SMKO mice (open squares), but this increase was significantly attenuated in SMKO mice. Two-way ANOVA for treatment: F(1,23) = 72.1, P < 0.0001, and for genotype: F(1,23) = 13.7, P < 0.0005. Sidak posttests: 1 ± 0.05 arbitrary units for uninfused (unif) control mice vs. 2.6 ± 0.17 arbitrary units for ANG II-infused control mice (*P < 0.04), 2.6 ± 0.17 arbitrary units for ANG II-infused control mice vs. 1.6 ± 0.17 arbitrary units for ANG II-infused SMKO mice (**P < 0.0001), and 1.1 ± 0.05 arbitrary units for uninfused SMKO mice vs. 1.6 ± 0.17 arbitrary units for ANG II-infused SMKO mice (***P < 0.0001). B: cleaved γ-ENaC in the kidney cortex. At baseline, there was no difference in cleaved γ-ENaC abundance in the kidney cortex between control mice (closed circles) or SMKO mice (open circles). During ANG II infusion, levels of cortical cleaved γ-ENaC increased significantly in control mice (closed squares) but did not change in SMKO mice (open squares). Two-way ANOVA for treatment: F(1,23) = 37.5, P < 0.0001, and for genotype: F(1,23) = 10.2, P < 0.01. Sidak posttests: 1.7 ± 0.1 arbitrary units for ANG II-infused control mice vs. 1.2 ± 0.07 arbitrary units for ANG II-infused SMKO mice (**P = 0.0001) and 1 ± 0.01 arbitrary units for uninfused control mice vs. 1.7 ± 0.1 arbitrary units for ANG II-infused control mice (***P < 0.0001). Dot plots were derived from densitometry data from immunoblots shown at the top of each figure. For both the cortex and medulla, n = 7 for ANG II-infused and uninfused SMKO mice, n = 7 for uninfused control mice, and n = 6 for ANG II-infused control mice.

Absence of AT_1A Receptors in VSMCs Modifies the Impact of ANG II on Kidney Transporter Abundance

In a previous study, we found that SMKO mice had attenuated hypertension and exaggerated natriuresis in response to chronic ANG II infusion (21). As shown in Fig. 6A, the extent of cardiac hypertrophy with ANG II infusion was significantly diminished in SMKO mice (5.5 ± 0.2 mg/g) compared with control mice (6.7 ± 0.5 mg/g, P = 0.02), consistent with a reduced severity of hypertension. Thus, we compared kidney transporter profiles after 5 days of ANG II infusion in SMKO and control mice to determine whether alterations in the abundance of kidney Na⁺ transporters might contribute to the resistance to hypertension we observed in SMKO mice (21). Indeed, the absence of AT_1A receptors in SMKO mice significantly modified responses to ANG II (Fig. 2). In particular, the significant increases in activated, cleaved α-ENaC and γ-ENaC seen in the cortex of hypertensive control mice were significantly blunted in ANG II-treated SMKO mice (Figs. 2 and 7). Moreover, although the levels of cortical total NHE3 and phosphorylated NHE3 were significantly reduced in control mice infused with ANG II, levels of NHE3 in SMKO mice were not changed by ANG II (Figs. 2 and 3). In contrast, ANG II significantly increased the levels of cortical total NCC and phosphorylated NKCC2 in SMKO mice to levels indistinguishable from ANG II-infused control mice (Fig. 2). Otherwise, the levels of the remaining transporters were not significantly affected by the absence of vascular AT_1A receptors in SMKO mice. Thus, the absence of AT_1A receptors in VSMCs significantly modifies ANG II-dependent changes in epithelial transporter profiles across the nephron. Notably, the expression of activated forms of ENaC was significantly attenuated in SMKO animals.

Impaired Acute Response to Amiloride in SMKO Mice During ANG II

To test the functional consequences of reduced abundance of cleaved α- and γ-ENaC subunit protein levels in SMKO mice during chronic ANG II infusion, we compared responses to acute administration of the specific ENaC blocker amiloride in SMKO and control mice. As shown in Fig. 8, urine volumes and Na⁺ excretion were similar in ANG II-infused SMKO and control mice after vehicle administration. In the control mice, administration of amiloride significantly increased urine volume by ≈25% (P = 0.01) and Na⁺ excretion by ≈75% (P < 0.0001). In contrast, amiloride had no significant effect on urine volume in SMKO mice, but Na⁺ excretion increased by ≈50% (P = 0.01). Moreover, the magnitude of the natriuretic response to amiloride was significantly lower in SMKO mice (11.9 ± 0.9 μmol/h) compared with control mice (16.8 ± 1.9, P = 0.02). Thus, reduced abundance of cleaved α- and γ-ENaC subunit protein in ANG II-infused SMKOs was associated with an impaired response to the diuretic amiloride and perhaps contributes to their exaggerated natiuresis in ANG II hypertension.

Figure 8.

Amiloride resistance in mice with cell-specific deletion of angiotensin II (ANG II) type 1 receptors in smooth muscle (SMKO mice) during ANG II infusion (5 days). A: diuretic responses to amiloride on day 5 of ANG II infusion. Urine volumes (mL/h) were similar in control mice (closed squares) and SMKO mice (open squares) after vehicle injection. After injection of 10 mg/kg amiloride, urine volumes increased significantly in mice of the control group but had no significant effect on urine volume (mL/h) in the SMKO groups. After amiloride, urine volumes (mL/h) were not significantly different between groups. Two-way repeated-measures ANOVA for treatment: F(1,11) = 13.6, P = 0.004. Sidak posttests: 0.22 ± 0.03 mL/h for amiloride-treated control mice vs. 0.18 ± 0.02 mL/h for amiloride-treated SMKO mice (P = not significant), 0.14 ± 0.02 mL/h for vehicle-treated SMKO mice vs. 0.18 ± 0.02 mL/h for amiloride-treated SMKO mice (P = not significant), and 0.16 ± 0.03 mL/h for vehicle-treated control mice vs. 0.22 ± 0.03 mL/h for amiloride-treated control mice (*P = 0.02). B: natriuretic responses to amiloride on day 5 of ANG II infusion. Amiloride administration significantly increased Na⁺ excretion in both groups. However, the natriuretic response in SMKO mice was blunted compared with control mice. Two-way repeated-measures ANOVA for treatment: F(1,11) = 37.3, P < 0.0001. Sidak posttests: 16.8 ± 1.9 μmol Na⁺/h for amiloride-treated control mice vs. 11.9 ± 2 μmol Na⁺/h for amiloride-treated SMKO mice (*P = 0.04), 7.9 ± 1.4 μmol Na⁺/h for vehicle-treated SMKO mice vs. 11.9 ± 0.9 μmol Na⁺/h for amiloride-treated SMKO mice (**P = 0.01), and 9.5 ± 2.4 μmol Na⁺/h for vehicle-treated control mice vs. 16.8 ± 1.9 μmol Na⁺/h for amiloride-treated control mice (***P < 0.001). n = 5 control mice; n = 8 SMKO mice.

Similar Levels of Urinary Aldosterone Excretion in Control and SMKO Mice

Since aldosterone is a major regulator of ENaC expression and activity, we measured urinary aldosterone levels in control and SMKO mice at baseline and after 5 days of chronic ANG II infusion. There were no differences in urinary aldosterone levels at baseline in the control and SMKO groups (578 ± 76 vs. 663 ± 38 pg/24 h, P = not significant). As expected, ANG II increased urinary aldosterone levels in both groups by ≈60% (control: 944 ± 117 pg/24 h and SMKO: 1,082 ± 110 pg/24 h, P < 0.01). However, there was no significant difference between urinary aldosterone levels in ANG II-infused control and SMKO mice (P = not significant). Thus, the significant attenuation of cleaved ENaC subunits in SMKO mice with ANG II hypertension cannot be explained by differences in aldosterone levels.

GFR Is Similar in SMKO and Control Mice

Differences in the filtered load of Na⁺ might also explain some of the differences in transporter profiles that we observed in SMKO mice. To address this issue, we measured GFR in control and SMKO mice using FITC-inulin in conscious mice (Fig. 6B). We found that GFR was virtually identical between the two groups (control: 22.4 ± 2.4 µL/min/g vs. SMKO: 22.7 ± 1.9 μL/min/g, P = 0.9), suggesting that a systematic difference in filtered solute load is unlikely to explain the differences in transporter profiles.

DISCUSSION

The RAS is a key hormonal system controlling blood pressure (1, 44). Effects of the RAS on blood pressure are primarily mediated through activation of AT₁ receptors by ANG II (6, 45), and blockade of this pathway provides effective therapy for hypertension and its complications (2). There is considerable evidence suggesting that ANG II actions in the kidney play a critical role in the pathogenesis of hypertension by increasing reabsorption of Na⁺ (44). Understanding the key cellular targets of ANG II in the kidney that control blood pressure can therefore provide important insights into the causes of hypertension and molecular mechanisms of action for blood pressure lowering by RAS blockade. In this regard, previous studies have clearly shown that direct actions of AT₁ receptors in specific kidney epithelial lineages control the expression of Na⁺ transporters, impairing natriuresis and contributing to the development of hypertension (1, 9, 11). Similarly, powerful vasoconstrictor effects of AT₁ receptors in VSMCs can directly influence blood pressure by increasing peripheral vascular resistance (22) or by complex effects in the kidney circulation to promote solute reabsorption (1). Here, we demonstrate an unexpected interaction of vascular AT₁ receptors with the kidney tubular epithelium resulting in alterations of transporter abundance, impacting blood pressure.

In previous studies, we directly examined the physiology of vascular AT₁ receptors by generating mice with cell-specific deletion of AT_1A receptors from VSMCs (SMKO mice) (21). These animals have reduced baseline blood pressure and are protected from the full development of ANG II-dependent hypertension. Their resistance to hypertension is associated with exaggerated natriuresis, consistent with the critical role of kidney Na⁺ handling in chronic blood pressure regulation (42). Classical studies by Earley and Friedler (25) and associates documented the capacity for changes in hydrostatic and osmotic pressures in peritubular capillaries to affect tubular reabsorptive processes. Subsequent work showed that various maneuvers reducing or increasing peritubular capillary blood flow cause parallel changes in urinary Na⁺ excretion (29). Indeed, we found that, overall, RBF was increased in SMKO mice and vasoconstrictor responses to ANG II in their kidney vasculature were markedly impaired (21), both of which would be expected to favor a natriuretic state. Our finding that GFR is similar in SMKO and control mice is consistent with this construct. Nonetheless, previous work on the influence of kidney hemodynamics on Na⁺ excretion has largely used acute interventions in instrumented animals (9, 25, 29, 46, 47). Furthermore, because of the magnitude and durable nature of the blood pressure reduction in SMKO animals, we speculated that changes in transporter abundance and activity might also be contributing, and this was the impetus for the current study.

In the basal state, blood pressures are significantly decreased in SMKO animals relative to controls (21). We show here that this reduction in basal blood pressure is associated with lower abundance of two key Na⁺ transporters/exchangers: NHE3 and NKCC2. NHE3 is the major Na⁺ transporter in the proximal tubule (48), and genetic deletion of NHE3 from the proximal tubule causes hypotension and salt wasting (49). NKCC2, expressed in the TAL of the loop of Henle, is the molecular target for furosemide (50), an important diuretic agent used in the treatment of hypertension and heart failure (51–55). Indeed, we showed that diuretic and natriuretic responses to furosemide were significantly attenuated in SMKO animals, consistent with reduced functional activity of NKCC2. These findings suggest that actions of AT₁ receptors in VSMCs, perhaps in the kidney vasculature, support the abundance and function of NHE3 and NKCC2 in the kidney epithelium. Reduced activity of these transporters in SMKO animals would be expected to promote urinary Na⁺ loss, contributing to their lower blood pressures.

Previous studies have shown that chronic RAS activation, which can be mimicked by long-term infusion of ANG II, causes remodeling of transporter expression along the nephron, promoting enhanced tubular Na⁺ reabsorption along the distal nephron and collecting duct, driving the development of a hypertension-provoked depression in the abundance of proximal and TAL NHE3 and NKCC2 to maintain Na⁺ balance (9, 56–58). This remodeling was clearly seen in our experiments of ANG II hypertension in control mice (Fig. 2), where ANG II infusion caused a striking increase in distal NCC and activation (cleavage) of ENaC subunits. The role of these various transporters in hypertension has been well established (15, 59, 60). For NCC, ANG II has been shown to stimulate its expression and activity through direct effects in the epithelium of the cortical TAL and distal tubule, respectively, involving complex signaling pathways (15, 61). Evidence supports direct activation of ENaC by ANG II (62–66) as well as by aldosterone, which is circulating at high levels in this model of ANG II hypertension (67, 68).

The absence of AT₁ receptors in VSMCs was associated with significant modifications in the profile of transporter expression induced by ANG II (Fig. 2). In particular, the increase in the expression of activated ENaC subunits seen in control mice with ANG II infusion was substantially attenuated, as was the natriuretic response to amiloride in SMKO mice. As ENaC is the major Na⁺ channel at the final location of Na⁺ reabsorption in the collecting duct, it plays a critical role in Na⁺ homeostasis (69). Lower activity of ENaC in SMKO animals would be expected to preserve the impact of the lower Na⁺ and volume reabsorbed premacula densa in SMKO animals, promote natriuresis, and shift the kidney function curve to the left, thus protecting against the development of hypertension. As aldosterone levels were appropriately elevated in SMKO animals during ANG II infusion, the regulation of ENaC by vascular AT₁ receptors seems to be independent of mineralocorticoid actions. The ratio of cleaved to full-length ENaC is increased, indicating activating cleavage of ENaC subunits during ANG II infusion (70). Notably, ANG II also caused robust accumulation and phosphorylation of NCC in SMKO animals to levels not different from controls, and enhanced activity of these transporters may contribute to the residual blood pressure elevation seen in SMKO animals given ANG II. On the other hand, the significant attenuation of hypertension in SMKO animals occurred despite enhancement of key Na⁺ transporters, indicating powerful counterbalancing actions of kidney hemodynamic changes and reductions in the activities of TAL NHE3 and NKCC2 and ENaC.

The predominant effect of ANG II in control mice is to increase the abundance of Na⁺ transporters, consistent with its actions to increase avidity of the kidney for Na⁺. The NHE3 response to ANG II infusion is biphasic: increasing at 3 days of infusion (in response to ANG II) and decreasing at 14 days (in response to elevated blood pressure) (71). In agreement with previous studies (13, 19, 65), NHE3 was significantly reduced in ANG II-infused control mice, but, in contrast, chronic ANG II infusion had no effect on NHE3 levels in SMKO animals, likely reflecting a balance between stimulation by ANG II and their modest rise in blood pressure during ANG II infusion to only ≈50% of control levels (21).

The mechanism by which AT₁ receptors in the vasculature can exert powerful regulation of transporters expressed in tubular epithelial cells is not clear from our study. One possibility might be release of paracrine mediators triggered by activation of vascular AT₁ receptors that would affect transporter expression and/or activity in adjacent epithelial populations. As one potential example, AT₁ receptor activation typically generates ROS, which can have wide-ranging biological actions within tissues (72). In the vasculature, ROS can inactivate nitric oxide, which is known to regulate NKCC2 (73). Alternatively, AT₁ receptors in VSMCs may affect transporter abundance indirectly, through effects on mechanical stretch in vessels (74) or kidney baroreceptors (75). Stretch responses have been shown to trigger the release of mediators such as VEGF (76) and 20-HETE (77), and the actions of baroreceptors to control the macula densa in the distal tubule are well established (1, 78, 79).

In summary, our study highlights the potent actions of AT₁ receptors in VSMCs to control the expression of Na⁺ transporters and associated proteins in renal epithelia, providing a mechanism for regulating Na⁺ excretion independent of kidney hemodynamic changes. In this system of vascular-epithelial, signals in VSMCs are transmitted to the renal epithelial cell compartment modulating transporter abundance and thus influencing blood pressure. Moreover, this pathway functions in concert with diverse downstream effects of RAS activation to promote conservation of salt and water.

Recent studies have reported significant species differences in AT₁ receptor expression between mice and humans (80) and suggested that expression of AT₁ receptors in human tubular epithelia tends to be lower than in mice (81, 82). This apparent diminution in the role of the ANG II-AT₁ receptor pathway in direct regulation of epithelial functions across evolution raises the possibility that other indirect mechanisms impacting natriuresis, such as the vascular-epithelial interaction we describe here, may take on added importance in humans.

Significance Statement

Precise regulation of urinary Na⁺ excretion by the kidney plays a critical role in the control of blood pressure, and impaired natriuresis is a final common pathway causing hypertension. Increased abundance and activity of kidney epithelial Na⁺ transporters triggered by circulating mediators such as ANG II constrains natriuresis. Similarly, vasoconstriction of the kidney with consequent fall in RBF and altered Starling forces in the interstitial circulation also promotes Na⁺ reabsorption, contributing to the development of hypertension. Here, we show an unexpected link between vasoconstrictor responses in the kidney and regulation of epithelia transporter abundance, where vascular responses to ANG II are linked to stimulation of key transporters including NKCC2 and ENaC. This study identifies a novel pathway for vascular-epithelial cross talk contributing to the pathogenesis of hypertension.

GRANTS

This work was supported by Career Development Award IK2BX002240 from the Biomedical Laboratory Research and Development Service of the Department of Veterans Affairs Office of Research and Development (to M.A.S.). The research reported in this publication was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01DK105049, R01DK098382, and P30DK096493 (to T.M.C.) and DK083786 (to A.A.M.).

DISCLAIMERS

The views expressed in this article are those of the authors and do not necessarily represent the policy or position of the United States Department of Veterans Affairs or the United States Government.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

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