Role of the Na⁺/H⁺ exchanger 3 in angiotensin II-induced hypertension

Abstract

The renal mechanisms responsible for angiotensin II (ANG II)-induced hypertension remain incompletely understood. The present study tested the hypothesis that the Na⁺/H⁺ exchanger 3 (NHE3) is required for ANG II-induced hypertension in mice. Five groups of wild-type (Nhe3^+/+) and Nhe3^−/− mice were treated with vehicle or high pressor doses of ANG II (1.5 mg/kg/day ip, via minipump for 2 wk, or 10 pmol/min iv for 30 min). Under basal conditions, Nhe3^−/− mice had significantly lower systolic blood pressure (SBP) and mean intra-arterial pressure (MAP) (P < 0.01), 24 h urine (P < 0.05), urinary Na⁺ (P < 0.01) and urinary K⁺ excretion (P < 0.01). In response to ANG II, SBP and MAP markedly increased in Nhe3^+/+ mice in a time-dependent manner, as expected (P < 0.01). However, these acute and chronic pressor responses to ANG II were significantly attenuated in Nhe3^−/− mice (P < 0.01). Losartan blocked ANG II-induced hypertension in Nhe3^+/+ mice but induced marked mortality in Nhe3^−/− mice. The attenuated pressor responses to ANG II in Nhe3^−/− mice were associated with marked compensatory humoral and renal responses to genetic loss of intestinal and renal NHE3. These include elevated basal plasma ANG II and aldosterone and kidney ANG II levels, salt wasting from the intestines, increased renal AQP1, Na⁺/HCO₃⁻, and Na⁺/K⁺-ATPase expression, and increased PKCα, mitogen-activated protein kinases ERK1/2, and glycogen synthase kinase 3αβ signaling proteins in the proximal tubules (P < 0.01). We concluded that NHE3 in proximal tubules of the kidney, along with NHE3 in intestines, is required for maintaining basal blood pressure as well as the full development of ANG II-induced hypertension.

hypertension is one of the most important risk factors for high morbidity and mortality associated with cardiovascular, stroke, and end-stage kidney diseases. In the United States alone, more than 30% of adults will most likely develop hypertension and require antihypertensive medications in their lifetime. However, only 50% of patients with hypertension have their blood pressure under control with current antihypertensive drugs (10, 32, 37, 46). The factors contributing to the development of hypertension and the reasons underlying the difficulty in controlling hypertension remain incompletely understood (37, 46, 58). Recent genome-wide association studies in humans have identified over 50 common genetic variants or loci associated with blood pressure or hypertension, but the roles of these genetic variant/locus in, and the extent to which these genetic variants would contribute to, blood pressure control remain largely unknown (12, 21, 25, 38, 58). Likewise, while hypertension is well recognized as a multifactorial disease, involving genetics, humoral, dietary and environmental, and neural factors, the development and progression of most, if not all, forms of hypertension appear to converge on a final common pathway, increased salt retention due to abnormal intestinal and renal sodium (Na⁺) handling (16, 17). Thus Na⁺ continues to play a key role in the normal blood pressure regulation as well as the development of hypertension, as diuretics still remain to be the first line of current antihypertensive drugs (10, 46).

In humans, most of daily ingested Na⁺ will be reabsorbed by the gut and the kidney, primarily in small intestines of the digestive system and the proximal tubule of the kidney (61). In the kidney, 70% of the glomerularly filtered Na⁺ load is reabsorbed by the proximal tubule (36, 43, 53, 60). Although various Na⁺ exchangers and/or cotransporters act in concert to contribute to maintaining normal blood pressure homeostasis (9, 41, 52, 60), the sodium and hydrogen exchanger 3, NHE3, is the most important Na⁺ transporter in apical membranes of small intestines and the proximal tubule (33, 35, 49, 51). The key function of NHE3 is to secrete H⁺ from the cells in exchange for luminal Na⁺ entry, therefore contributing to Na⁺ reabsorption in the gut and the proximal tubule and body acid-base balance (1, 4, 9, 43). It is estimated that NHE3 directly contributes to more than 25% of Na⁺ reabsorption (35, 54), but it acts indirectly, after generating a luminal Cl⁻ gradient, to drive passive reabsorption of additional 50% of the filtered Na⁺ load in the proximal tubule (6, 43, 47). Thus, NHE3 is responsible for reabsorbing up to 75% of Na⁺ and 90% of HCO₃⁻ reabsorption in the proximal tubule. Indeed, previous studies have shown that global knockout of the NHE3 gene in mice (Nhe3^−/−) decreases Na⁺ reabsorption in the proximal convoluted tubule by 50%, promotes salt wasting from the gut, and decreases blood pressure (39, 48, 55). This clearly suggests a key role of NHE3 in the normal blood pressure regulation.

However, the role of NHE3 in the development of angiotensin II (ANG II)-induced hypertension remains virtually unknown. NHE3 expression and activity are also significantly increased in the proximal tubule of spontaneously hypertensive rats (SHRs) (18, 22, 57). ANG II especially plays an important role in upregulating the expression of NHE3 or activity in the proximal tubule (5, 8, 20, 29). In vitro, ANG II increases NHE3 expression in cultured proximal tubule cells (8, 11, 15, 26). In vivo, ANG II has biphasic effects on NHE3 expression, depending on whether an acute and high pressor or a slow and subpressor dose is used to increase blood pressure acutely or chronically (24, 29, 35, 45). Pressor doses of ANG II acutely induce internalization of NHE3 in the proximal tubule and pressure natriuresis (24, 45, 56), whereas long-term subpressor doses of ANG II increase the expression of NHE3 and slowly elevate blood pressure (5, 29, 30, 44). Thus, the current study was designed to test the hypothesis for the first time that NHE3 in the gut and the proximal tubule of the kidney is required for maintaining long-term blood pressure homeostasis and genetic deletion of NHE3 attenuates the development of ANG II-induced hypertension in mice.

METHODS

Animals

Heterozygous breeding pairs of NHE3 mutant mice (Nhe3^+/−) were generously provided by Dr. Gary E. Shull of the University of Cincinnati School of Medicine (48). Homozygous mutant mice with the null mutation of the Nhe3 gene (Nhe3^−/−) were obtained by breeding Nhe3^+/− mice and genotyping them in the Zhuo laboratory using Dr. Shull's protocol (48). In brief, PCR genotyping of wild-type (Nhe3^+/+) and mutant alleles of the Nhe3 gene (Nhe3^−/−) was performed by Southern analysis on DNA samples obtained from the tail biopsy (48). To genotype the Nhe3 gene, the forward primer (5′-CATCTCTATCACAAGTTGCCCACAATCGTG-3′) and reverse primer (5′-GTGACTGCATCGTTGAGCAGAGACTCG-3′), which correspond to sequences near the 5′ and 3′ ends of exon 6 and the 5′ end of the neomycin-resistance gene (5′-GCATGCTCCAGACTGCCTTG-3′), were used as described (23, 48). PCR reactions were performed with denaturing at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s (23). Nhe3^+/+ and Nhe3^−/− mice were maintained on normal rodent chow and had free access to tap water and chow throughout the study. The use of Nhe3^+/+ and Nhe3^−/− mice in the current study was approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.

Experimental Protocols

Blood pressure and renal excretory responses to ANG II in conscious animals.

Three groups of adult male Nhe3^−/− mice and their corresponding wild-type Nhe3^+/+ mice were used in the present study. Mice in group 1 were left untreated to serve as controls. Mice in group 2 were infused with a high pressor dose of ANG II (Bachem, 1.5 mg/kg/day ip) via an osmotic minipump (model 2002) for 2 wk, as we described previously (29, 30, 59). Mice in group 3 were infused with ANG II as in group 2 and concurrently treated with the AT₁ receptor blocker losartan (20 mg/kg/day po).

Basal and weekly systolic blood pressure responses to ANG II or losartan treatment were measured primarily by the tail-cuff technique (29, 30, 59). To determine feces and urinary Na⁺ excretory responses to ANG II infusion, Nhe3^+/+ and Nhe3^−/− mice were placed individually in a mouse metabolic cage to collect feces and urine samples for 24 h weekly before and after ANG II was infused (29, 30, 59). Plasma, urinary, and feces Na⁺ and K⁺ concentrations were determined by NOVA 13 Electrolyte Analyzer (Nova Biomedical).

Blood pressure responses to ANG II in anesthetized animals.

To verify the differences in basal intra-arterial mean blood pressure and its responses to ANG II as identified by the tail-cuff method, two additional groups of adult male Nhe3^+/+ and Nhe3^−/− mice were anesthetized with ketamine (50 mg/kg wt ip) and Inactin (50 mg/kg wt ip), respectively, and placed on a thermally controlled mouse surgical table. A polyethylene tube (PE50) with a fine fabricated tip was inserted into the left carotid artery and jugular vein, respectively, to measure mean arterial pressure (MAP) and to infuse saline containing 2% BSA (10 μl/min iv) or ANG II (10 pmol/min iv), as we described previously (29, 30, 59). In the second group of mice, AT₁ receptor blocker losartan (20 mg/kg/day po) was given 3 days before ANG II was infused to determine the role of the AT₁ receptor.

Morphological and/or absorptive phenotypes of intestines and kidney.

At the end of the experiment, Nhe3^+/+ and Nhe3^−/− mice were killed to collect the entire gut and the kidneys for comparison of their histological and absorptive phenotypes, as described (48). The guts and kidneys were weighed, the fluid contents in the gut, largely in the caecum, were collected from Nhe3^+/+ and Nhe3^−/− mice, and Na⁺ concentrations measured accordingly. Frozen kidney sections (10 μm thick) were stained by hematoxylin and eosin, or Masson Trichrome, respectively, for histological examination (28, 29).

Measurement of plasma and kidney ANG II and aldosterone.

At the end of study or the treatment, all mice were decapitated, and trunk blood samples collected for measurement of plasma ANG II and aldosterone concentrations without the influence of anesthetic administration, as described (29, 30, 59). Plasma and kidney samples were immediately harvested and extracted for measurement of plasma aldosterone, plasma, and whole kidney ANG II with a sensitive aldosterone or ANG II ELISA kit (29, 30, 59). Aldosterone and ANG II concentrations are expressed as fmol/ml or pg/ml.

Western blot analysis of major sodium and water transporters or signaling proteins in the renal cortex.

To determine whether the expression of some key sodium and water transporters as well as major signaling protein responses were upregulated by genetic deletion of the Nhe3 gene, protein samples were extracted from the renal cortex of six representative Nhe3^+/+ and Nhe3^−/− mice for Western blot analysis. The rabbit polyclonal anti-Na⁺/HCO₃ cotransporter (NBC) targeting the NH₂ terminus 338–391 of the rat kidney Na⁺/HCO₃ cotransporter (cat. #AB3212), and the mouse monoclonal anti-Na⁺/K⁺-ATPase recognizing the α1 subunit isoform of Na⁺/K⁺-ATPase (cat. #05-369, lot #DAM1794271) were purchased from Millipore. The rabbit polyclonal anti-protein kinase Cα antibody targeting phospho-S657+Y658 (ab23513) and anti-glycogen synthase kinase 3 α and β antibody (GSKα and β) recognizing phospho-Y216+Y279 (ab4797) were purchased from Abcam. The goat polyclonal anti-aquaporin 1 (AQP1) antibody targeting the COOH terminus of AQP1 of human origin (sc-9878), and the mouse monoclonal anti-MAP kinases ERK1/2 antibody recognizing phospho-Tyr 204 (sc-7383) were obtained from Santa Cruz Biotechnology, respectively. Western blot analyses of these transporter or signaling proteins in the renal cortex of Nhe3^+/+ and Nhe3^−/− mice were carried out as we described previously (27, 29, 30). To ensure equal sample loading, the same membranes were treated with stripping buffer (Pierce) for 20 min, blotted with 5% nonfat dry milk, and reprobed with a mouse anti-β-actin monoclonal antibody at 1:2,000 (Sigma-Aldrich). Western blot signals were detected by enhanced chemiluminescence and analyzed with a Molecular Imager, ChemiDoc XRS⁺ (Bio-Rad Laboratories). The changes in the expression of these proteins were expressed as a ratio to β-actin protein.

Data Analysis and Statistics

All results are presented as means ± SE. The differences between Nhe3^+/+ and Nhe3^−/− mice in systolic blood pressure (SBP), plasma aldosterone, and plasma and kidney ANG II, and other renal excretory responses were analyzed by Student's unpaired t-test. The differences between basal responses and weekly responses to ANG II in the same group of Nhe3^+/+ and Nhe3^−/− mice were analyzed by one-way ANOVA followed by Dunnett's comparisons between experimental treatments. A value of P < 0.05 was considered statistically significant.

RESULTS

Molecular Phenotyping of Nhe3^+/+ and Global Nhe3^−/− Mice

All Nhe3^+/+ and Nhe3^−/− mice used in the present study were genotyped for genetic verification before and after the experiment. Figure 1 shows a representative Southern analysis of tail DNA samples for the presence (Nhe3^+/+ as the wild-type and Nhe3^+/− as the heterozygous) or absence of the Nhe3 gene (Nhe3^−/− as the homozygous) in mice (Fig. 1A). In further experiments, the expression of NHE3 mRNAs in the superficial cortex of the kidney was verified by quantitative RT-PCR (Fig. 1B). Clearly, >90% of NHE3 mRNAs were deleted from the cortex in the kidney of Nhe3^−/− mice.

Fig. 1.

Molecular and morphological phenotypes of wild-type Nhe3^+/+ and global Nhe3^−/− mice. A: representative Southern blot genotyping of wild-type Nhe3^+/+, heterozygous Nhe3^+/−, and global Nhe3^−/− mice. B: RT-PCR analysis of NHE3 mRNA expression in the superficial cortex of the kidney, primarily the proximal tubules (n = 12). C: representative normal structures of the gastrointestinal system in Nhe3^+/+ mice. D: representative abnormal structures of the gastrointestinal system of Nhe3^−/− mice, noting the most strikingly enlarged cecum segment with a very thin wall and marked fluid accumulation inside (indicated by *). E: representative normal light microscopic glomerular and proximal tubule structures of Nhe3^+/+ mice. F: representative abnormal light microscopic glomerular and proximal tubule structures of Nhe3^−/− mice, which show slight to moderate cellular abnormalities of glomerulus especially in the vascular pole and the proximal tubule wall. NHE3, Na⁺/H⁺ exchanger 3; G, glomerulus; PT, proximal tubule.

Morphological Phenotypes of Intestines and Kidney in Nhe3^−/− Mice

Global deletion of the Nhe3 gene resulted in mild to moderate diarrhea in most adult homozygous Nhe3^−/− mice. One in three newly born homozygous pups with the severe phenotype died during the first week after birth, whereas those having a mild to moderate phenotype were able to survive and grow to adulthood. Figure 1, C–F, compares the morphological phenotypes of the intestines and kidneys between Nhe3^+/+ and Nhe3^−/− mice. All segments of intestines of Nhe3^−/− mice appeared to be significantly enlarged, compared with Nhe3^+/+ mice. However, the most striking morphological abnormality is the extremely enlarged cecum segment with a very thin wall, which was about five to 10 times of the size of wild-type Nhe3^+/+ mice (Fig. 1D). Generally, the kidneys of Nhe3^−/− mice looked no different from those of Nhe3^+/+ mice, with similar kidney weight-to-body weight ratios (Nhe3^+/+: 1.37 ± 0.07% vs. Nhe3^−/−: 1.37 ± 0.05%, not significant). At the microscopic level, however, the vascular pole of the glomeruli of Nhe3^−/− mice showed dense and fibrotic histology in Masson-trichrome staining (Fig. 1F). Furthermore, the wall of proximal tubules appeared to be thinner and slightly disorganized in Nhe3^−/− mice, compared with those of Nhe3^+/+ mice (Fig. 1E).

Absorptive Phenotypes of Intestines in Nhe3^−/− Mice

The overall gut weight of age-matched adult male Nhe3^−/− mice was 2.6-fold heavier than Nhe3^+/+ mice (Nhe3^+/+: 3.19 ± 0.15 g vs. Nhe3^−/−: 7.97 ± 0.31 g; P < 0.01) (Fig. 2A). Accumulated intestinal fluid in the cecum amounted to 1.38 ± 0.3 ml per Nhe3^−/− mouse with an averaged Na⁺ concentration of 94.5 ± 8.4 mmol/l, whereas no fluid accumulation was observed in Nhe3^+/+ mice (Fig. 2B). Similarly, 24 h fecal Na⁺ excretion was about 17-fold higher in Nhe3^−/− mouse than in Nhe3^+/+ counterparts (Nhe3^+/+: 3.02 ± 0.05 μmol/24 h vs. Nhe3^−/−: 50.8 ± 3.6 μmol/24 h, P < 0.01) (Fig. 2C).

Fig. 2.

Comparisons of overall gut weight (A), fluid accumulation in the caecum segment (B), and 24 h fecal Na⁺ excretion (C) in wild-type Nhe3^+/+ (n = 16) and global Nhe3^−/− mice (n = 19), which underlies the functional absorptive abnormality of Nhe3^−/− mice in the absence of NHE3 expression. **P < 0.01 vs. wild-type Nhe3^+/+ mice.

Basal Blood Pressure Phenotype in Nhe3^−/− Mice

In age- and body weight-matched adult male Nhe3^+/+ and Nhe3^−/− mice (Fig. 3A), basal SBP in conscious Nhe3^−/− mice (105 ± 3 mmHg), as determined by the tail-cuff method from five weekly measurements with 20 measurements each, was about 14 mmHg lower than Nhe3^+/+ mice (119 ± 3 mmHg, P < 0.01) (Fig. 3B). Similarly in anesthetized Nhe3^+/+ and Nhe3^−/− mice, mean intra-arterial pressure (MAP) was also 15 mmHg lower in Nhe3^−/− (78 ± 3 mmHg) than in Nhe3^+/+ mice (93 ± 3 mmHg, P < 0.01) (Fig. 3C).

Fig. 3.

Basal blood pressure phenotypes in ∼12 wk old male Nhe3^+/+ and Nhe3^−/− mice. A: similar body weight (n = 15 each). B: systolic blood pressure (SBP) was ∼15 mmHg lower in Nhe3^−/− mice than in wild-type Nhe3^+/+ mice (n = 15 each). C: mean intra-arterial blood pressure (MAP) in anesthetized Nhe3^+/+ (n = 7) and Nhe3^−/− mice (n = 8). **P < 0.01 vs. Nhe3^+/+ mice.

SBP and MAP Responses to ANG II in Conscious and Anesthetized Nhe3^−/− Mice

There were significant differences in SBP and MAP responses to ANG II stimulation in Nhe3^+/+ and Nhe3^−/− mice (Fig. 4). Under conscious conditions and in response to a high pressor dose of ANG II infusion (1.5 mg/kg/day po), SBP in Nhe3^+/+ mice was increased from its baseline level (117 ± 3 mmHg) to 165 ± 5 mmHg, a net increase of 48 ± 3 mmHg (P < 0.01) (Fig. 4A). By contrast, SBP in Nhe3^−/− mice was increased from its baseline level of 102 ± 4 mmHg to a peak response of 119 ± 5 mmHg (P < 0.01), a net increase of about 17 ± 3 mmHg (Fig. 4A). Concurrent treatment with losartan largely blocked ANG II-induced blood pressure response in Nhe3^+/+ mice (124 ± 3 mmHg, P < 0.01 vs. ANG II infusion alone). By contrast, most of losartan-treated, ANG II-infused Nhe3^−/− mice died during the first week of treatment, so that blood pressure could not be determined in these mice. Under anesthetic conditions, intra-arterial MAP responses to intravenous infusion of a pressor dose of ANG II, 10 pmol/min iv, were also different between Nhe3^+/+ and Nhe3^−/− mice (Fig. 4B). Indeed, MAP in anesthetized Nhe3^+/+ mice was increased by ANG II from a baseline of 93 ± 3 mmHg to a peak response of 137 ± 6 mmHg at 5 min, a net increase of 44 ± 3 mmHg (P < 0.01). By comparison, MAP in anesthetized Nhe3^−/− mice was increased by ANG II from a baseline of 80 ± 5 mmHg to a peak response of 103 ± 5 mmHg at 5 min, a net increase of 23 ± 3 mmHg (P < 0.01). The differences in MAP responses to ANG II between Nhe3^+/+ and Nhe3^−/− mice lasted throughout the duration of ANG II infusion (Fig. 4B). In Nhe3^+/+ mice, ANG II-induced MAP response was again blocked by pretreatment with losartan (20 mg/kg/day po), whereas most of losartan-pretreated Nhe3^−/− mice also died before experiment due to severe hypotension (not shown).

Fig. 4.

Blood pressure responses to ANG II in adult male conscious and anesthetized Nhe3^+/+ and Nhe3^−/− mice. A: comparison of SBP responses to a high pressor dose of ANG II (1.5 mg/kg/day ip, 2 wk) in conscious Nhe3^+/+ (n = 15) and Nhe3^−/− mice (n = 15). B: comparison MAP responses to acute ANG II infusion, 10 pmol/min iv, in anesthetized Nhe3^+/+ (n = 7) and Nhe3^−/− mice (n = 8). In both scenarios, blood pressure responses to ANG II were significantly attenuated in Nhe3^−/− mice, compared with Nhe3^+/+ mice. Concurrent treatment with losartan blocked ANG II-induced hypertension in Nhe3^+/+ mice, but most losartan-treated Nhe3^−/− mice died from severe hypotension during the 1st week of ANG II infusion (A) or during acute ANG II infusion (not shown). *P < 0.05 or **P < 0.01 vs. their basal blood pressure in the same strain of mice. +P < 0.05 or ++P < 0.01 vs. Nhe3^+/+ mice at the same time point. ##P < 0.01 between ANG II-infused Nhe3^+/+ mice treated with or without losartan (n = 8 for each group). ND, animals died before blood pressure measurement was made.

Urinary Na⁺ and K⁺ Excretory Responses to ANG II

Under basal conditions, 24 h urine output (Nhe3^+/+: 1.18 ± 0.10 ml/24 h vs. Nhe3^−/−: 0.82 ± 0.1 ml/24 h, P < 0.05) (Fig. 5A), 24 h urinary Na⁺ excretion (Nhe3^+/+: 193.7 ± 15 μmol/24 h vs. Nhe3^−/−: 55.7 ± 10 μmol/24 h, P < 0.01) (Fig. 5B), and 24 h urinary K⁺ excretion (Nhe3^+/+: 300.8 ± 21.9 μmol/24 h vs. Nhe3^−/−: 238.4 ± 26.4 μmol/24 h, P < 0.05) were significantly lower in Nhe3^−/− than in Nhe3^+/+ mice (Fig. 5C). In response to a pressor dose of ANG II infusion (1.5 mg/kg/day po), Nhe3^+/+ mice showed significant diuretic (Fig. 5A), natriuretic (Fig. 5B), and increased urinary K⁺ excretory responses. By contrast, there were very small or insignificant responses in 24 h urine and urinary Na⁺ and K⁺ excretion in Nhe3^−/− mice (Fig. 5).

Fig. 5.

Renal urine and urinary electrolyte excretory responses to a high pressor dose of ANG II infusion (1.5 mg/kg/day ip, 2 wk) in conscious Nhe3^+/+ (n = 15) and Nhe3^−/− mice (n = 15). A: 24 h urine excretion. B: 24 h urinary sodium excretion (U_NaV). C: 24 h urinary potassium excretion (U_KV). Note that 24 h urine and urinary Na⁺ and K⁺ excretory responses under both basal conditions and during infusion of a high pressor dose of ANG II were significantly attenuated in Nhe3^−/− mice than in wild-type Nhe3^+/+ mice. *P < 0.05 vs. their basal in Nhe3^+/+ or Nhe3^−/− mice. +P < 0.05 or ++P < 0.01 vs. wild-type Nhe3^+/+ mice under basal conditions or during ANG II infusion.

Circulating and Kidney ANG II and Plasma Aldosterone Responses

Basal circulating or plasma ANG II (Nhe3^+/+: 228.9 ± 22.1 pg/ml vs. Nhe3^−/−: 322.7 ± 32.1 pg/ml, P < 0.01) and plasma aldosterone levels (Nhe3^+/+: 505.6 ± 58.6 pg/ml vs. Nhe3^−/−: 692.4 ± 28.7 pg/ml, P < 0.01) were significantly higher in Nhe3^−/− than in Nhe3^+/+ mice (Fig. 6, A and B). Basal kidney ANG II was also significantly elevated in Nhe3^−/− mice (Nhe3^+/+: 284.1 ± 14.9 pg/g kidney weight vs. Nhe3^−/−: 399.9 ± 26.9 pg/g kidney weight, P < 0.01) (Fig. 6C). Chronic infusion of ANG II via osmotic minipump for 2 wk elevated plasma and kidney ANG II to similar extents (Fig. 6, A and C), but plasma aldosterone level was not altered by ANG II infusion in Nhe3^−/− mice (Fig. 6B).

Fig. 6.

Plasma ANG II and aldosterone and kidney ANG II levels under basal conditions and their responses to a high pressor dose of ANG II infusion (1.5 mg/kg/day ip, 2 wk) in conscious Nhe3^+/+ (n = 9) and Nhe3^−/− mice (n = 8). *P < 0.05 or **P < 0.01 vs. their basal levels in Nhe3^+/+ or Nhe3^−/− mice. ++P < 0.01 vs. wild-type Nhe3^+/+ mice under basal conditions or during ANG II infusion.

Proximal Tubule Transporter and Signaling Protein Responses in Nhe3^−/− Mice

To help explain the attenuated blood pressure, 24 h urine, and urinary Na⁺ and K⁺ excretory responses to ANG II stimulation, three major representative transporter and three major signaling proteins were measured in the proximal tubules of the superficial renal cortex. Under basal conditions, the expression of the major water transport protein aquaporin 1 (AQP1) increased nearly threefold in Nhe3^−/− mice (Nhe3^+/+: 0.08 ± 0.05 vs. Nhe3^−/−: 0.23 ± 0.04 AQP1/actin ratio, P < 0.01). Similarly, the expression of Na⁺/HCO₃⁻ (Nhe3^+/+: 0.13 ± 0.03 vs. Nhe3^−/−: 0.46 ± 0.08 Na⁺/HCO₃⁻/actin ratio, P < 0.01) and Na⁺/K⁺-ATPase (Nhe3^+/+: 0.32 ± 0.05 vs. Nhe3^−/−: 0.60 ± 0.06 Na⁺/K⁺-ATPase/actin ratio, P < 0.01) were also significantly upregulated in the superficial cortex of the kidney, primarily in the proximal tubules, in Nhe3^−/− mice (Fig. 7). With respect to signaling mechanism responses, phosphorylated protein kinase Cα (PKCα) (Nhe3^+/+: 0.10 ± 0.03 vs. Nhe3^−/−: 0.23 ± 0.05 p-PKCα/actin ratio, P < 0.01), phosphorylated MAP kinases ERK1/2 (Nhe3^+/+: 0.06 ± 0.03 vs. Nhe3^−/−: 0.26 ± 0.05 p-ERK1/2/actin ratio, P < 0.01), and phosphorylated glycogen synthase kinase 3α/β (Nhe3^+/+: 0.38 ± 0.06 vs. Nhe3^−/−: 0.58 ± 0.05 p-GSK3α/β/actin ratio, P < 0.01) were all significantly activated and upregulated in the proximal tubules of Nhe3^−/− mice (Fig. 8).

Fig. 7.

The expression of major water and sodium transporter proteins in the superficial cortex of the kidney, primarily the proximal tubules, in Nhe3^+/+ and Nhe3^−/− mice (n = 6 for each strain). AQP1, aquaporin 1; Na⁺/HCO₃⁻, sodium and bicarbonate cotransporter; Na⁺/K⁺-ATPase α1, sodium and potassium ATPase α1 isoform. **P < 0.01 vs. Nhe3^+/+ mice.

Fig. 8.

The expression of major signaling proteins, including phosphorylated protein kinase C α isoform (p-PKCα), MAP kinases ERK1/2 (p-ERK1/2), and glycogen synthase kinase 3α/β (p-GSK3α/β), in the superficial cortex of the kidney, primarily the proximal tubules in Nhe3^+/+ and Nhe3^−/− mice (n = 6 for each strain). **P < 0.01 vs. Nhe3^+/+ mice.

DISCUSSION

To the best of our knowledge, this is the first study to determine the specific role of the Nhe3 gene and NHE3 protein in the development of ANG II-induced hypertension and representative signaling mechanisms involved in global Nhe3^−/− mice. We demonstrated that global deletion of the Nhe3 gene in all tissues significantly attenuates SBP and MAP responses to ANG II in both conscious and anesthetized Nhe3^−/− mice. The attenuated blood pressure responses to ANG II were directly related to the loss of NHE3 proteins and its salt-retaining function, since all experimental conditions and approaches, including the sex, age, body weight, and doses of ANG II, were kept identical in both Nhe3^+/+ and Nhe3^−/− mice. These attenuated responses to ANG II might also be due to the fact that, as a result of salt wasting from the gut, the renin-angiotensin-aldosterone system (RAAS) has already been maximally stimulated to conserve Na⁺ from the proximal tubule of the kidney and to maintain overall body salt and fluid homeostasis, and therefore basal blood pressure in Nhe3^−/− mice. Indeed, plasma and kidney ANG II levels were significantly elevated in Nhe3^−/− mice, as were plasma aldosterone levels (Fig. 6). This is also supported by the findings in the present study that blockade of AT₁ receptors by losartan alone, or concurrent treatment with ANG II and losartan, led to high mortality in Nhe3^−/− mice, apparently due to severe hypotension. Furthermore, despite several key water and sodium transporters or cotransporters, such as AQP1, Na⁺/HCO₃⁻, and Na⁺/K⁺-ATPase, or key signaling proteins, such as PKCα, MAP kinases ERK1/2, and GSK3 α/β, were all upregulated in the renal cortex of Nhe3^−/− mice, their compensatory responses were still not enough to maintain normal blood pressure in these mutant mice. Thus our study shows that in the absence of NHE3 in all tissues, blood pressure was maintained at a significantly lower level primarily by the activated RAAS, providing evidence that NHE3 plays a critical role in maintaining normal basal blood pressure and the development of ANG II-induced hypertension.

The role of NHE3 in the regulation of basal blood pressure in health and ANG II-dependent hypertension remains incompletely understood. NHE3 is primarily expressed in the small intestine of the digestive system and the proximal tubule of the kidney (3, 7, 60), where it is the most important Na⁺ transporter in apical membranes of the small intestine and the proximal tubule (33, 49, 51). NHE3 acts to secrete H⁺ from the cells in exchange for luminal Na⁺ entry, therefore contributing to Na⁺ reabsorption in the gut and the proximal tubule and body salt and fluid and acid-base balance (4, 9, 43). Although NHE3 directly contributes to about 25% of Na⁺ reabsorption (35, 54), it acts indirectly, after generating a luminal Cl⁻ gradient, to drive passive reabsorption of additional 50% of the filtered Na⁺ load in the proximal tubule (6, 43, 47). Direct evidence for an important role of NHE3 in blood pressure control was first provided by Schultheis et al. (48). These investigators generated mice lacking the Nhe3 gene (Slc9a3^−/− or Nhe3^−/−) and reported for the first time that Nhe3^−/− mice showed lower basal blood pressure despite marked upregulation of circulating aldosterone and kidney renin expression (48). Nhe3^−/− mice also developed structural and absorptive abnormalities in the intestines with moderate diarrhea, and their fluid and HCO₃⁻ reabsorption were reduced by half in the proximal tubule of the kidney. In the present study, we confirmed similar structural and absorptive abnormalities in the intestines and basal blood pressure phenotype in Nhe3^−/− mice, as previously reported by Schultheis et al. (48). Specifically and strikingly, the overall gut weight, including the stomach, in Nhe3^−/− mice more than doubled that of Nhe3^+/+ mice (Figs. 1D and 2A), and intestinal fluid accumulation in the abnormal cecum segment (Fig. 2B) and overall 24 h fecal Na⁺ excretion in Nhe3^−/− mice (Fig. 2C) were more than 15 times of Nhe3^+/+ mice. These abnormal phenotypes in Nhe3^−/− mice strongly support an important role of intestinal NHE3 in maintaining basal body salt and fluid balance and blood pressure homeostasis. However, the relative contribution of intestinal vs. proximal tubule NHE3 in the kidney has not been studied previously. Since basal blood pressure remains significantly lower in global NHE3-deficient mice with transgenic rescue of the Nhe3 gene in the small intestine, an equally important role of proximal tubule NHE3 is strongly suggested (39).

The present study is significantly different from previous studies in that we determined whether global deletion of the Nhe3 gene, and therefore completely removal of NHE3 function, in the proximal tubules of the kidney as well as in small intestines would attenuate blood pressure responses to ANG II in Nhe3^−/− mice. We and others have previously showed that in vitro in cultured proximal tubule cells or in the proximal tubule of the kidney (5, 29, 30, 44), ANG II significantly increases NHE3 expression and activity, which promotes Na⁺ reabsorption by the proximal tubules and contributes to normal blood pressure control. Hypertension induced by nonpressor or slow-pressor doses (not by acute high pressor doses) of ANG II was mediated in part by stimulating NHE3 expression and activity in the proximal tubules of the kidney and consequent Na⁺ retention (5, 29, 30, 44). The hypothesis to be tested was that if intestinal and proximal tubule NHE3 and its function is genetically removed, the Na⁺ reabsorption-dependent blood pressure response to ANG II would be significantly attenuated in Nhe3^−/− mice. Indeed, our results clearly confirmed this hypothesis by showing that both systolic blood pressure responses to ANG II in conscious Nhe3^−/− mice and intra-arterial MAP responses to ANG II in anesthetized Nhe3^−/− mice were markedly attenuated (Fig. 3). These attenuated blood pressure responses to ANG II clearly implicate an important role of NHE3 not only in intestines but also in the proximal tubules of the kidney. We found that the glomerular and proximal tubular structures in Nhe3^−/− mice also appear to be abnormal, compared with those of wild-type animals (Fig. 1F). It was especially noted that the changes in the vascular pole of the glomerulus and the proximal tubules are consistent with the reported increases in the expression of kidney renin (48), most likely in the juxtaglomerular apparatus (48), and kidney ANG II levels (Fig. 6C), as well as significantly reduced glomerular filtration rate (23). Significantly attenuated 24 h urine excretion (Fig. 5A) and 24 h urinary Na⁺ excretion (Fig. 5B) before and after ANG II infusion in Nhe3^−/− mice strongly suggest that in the absence of NHE3 primarily in the proximal tubules and small intestines and consequent salt wasting from the gut, the primary role of the kidney, the proximal tubules specifically, is to conserve body Na⁺ and fluid in these mutant mice to maintain blood pressure. Our results further show that three key water and Na⁺ transporters or cotransporters, AQP1, Na⁺/HCO₃⁻, and Na⁺/K⁺-ATPase, and three key signaling proteins, p-PKCα, p-ERK1/2, and p-GSK3 α/β were markedly upregulated in the superficial cortex of the kidney (Figs. 7 and 8). Nevertheless, these renal compensatory responses under basal conditions were still not sufficient to maintain basal blood pressure and its response to ANG II stimulation at the levels of Nhe3^+/+ mice. Overall, the present study demonstrates that NHE3 in the proximal tubule of the kidney (as well as small intestines) is required to maintain basal blood pressure physiologically and for the development of ANG II-induced hypertension.

However, it should be emphasized that one significant limitation of the present study is that only global Nhe3^−/− mice were used to test our hypothesis. This approach is still unable to dissect the relative contribution of intestinal vs. proximal tubule NHE3 in blood pressure responses to ANG II. In future studies, either global Nhe3^−/− mice with transgenic rescue of the Nhe3 gene selectively in small intestines (tgNhe3^−/−) or mice with conditional deletion of the Nhe3 gene selectively in the proximal tubule of the kidney are required to further determine the respective role of intestinal and proximal tubule NHE3 in basal blood pressure control and in the development of ANG II-dependent hypertension.

Perspective

The present study demonstrates two key findings with important and potentially clinical implications. First, NHE3 is absolutely required for maintaining basal blood pressure homeostasis due to its actions on Na⁺ absorption from the small intestines and Na⁺ reabsorption from the proximal tubules of the kidney. Second, NHE3 is also necessary for the full development of ANG II-dependent hypertension. Furthermore, all known prohypertensive factors, including ANG II (5, 8, 19, 29), glucocorticoid (40, 50), hyperinsulinemia (14), hyperglucagonemia (2), increased oxidative stress (5), and renal nerve stimulation (42), induce NHE3 expression and activity and promote Na⁺ reabsorption in the proximal tubules. NHE3 expression and activity are also significantly increased in the proximal tubules of SHRs (18, 22, 57). Thus, NHE3 in the proximal tubules of the kidney may represent a therapeutic target for hypertension treatment, and NHE3 inhibitors may serve as novel proximal tubule-selective diuretics and antihypertensive drugs especially for those with resistant hypertension. Currently, three types of diuretics are used as anti-hypertensive drugs: thiazides, which inhibit the thiazide-sensitive Na⁺/Cl⁻ symporter; loop diuretics, which inhibit the Na⁺-K⁺-2Cl⁻ symporter in the thick ascending limb; and K⁺-sparing diuretics, which block epithelial Na⁺ channels (13, 34). However, use of these distal nephron diuretics is associated with the significant compensatory activation of the RAAS in the kidney, which augments NHE3 expression and activity and increases Na⁺ reabsorption in the proximal tubules (13, 34). A recent proof of the concept study has shown that a nonabsorbable NHE3 inhibitor SAR218034, which only inhibits intestinal NHE3, significantly decreased blood pressure in old SHRs-lean rats by inhibiting intestinal Na⁺ reabsorption (31). Further preclinical studies are required to determine whether global or tissue-specific deletion or inhibitors of NHE3 will have significant antihypertensive effects in different animal models of human hypertension, and if they do, whether the clinical benefits will outweigh the side effects.

GRANTS

This work was supported in part by National Institutes of Health Grants 2R01DK-067299-06A2 and R01DK-102429-01 (to J. L. Zhuo). Dr. Zhuo is also supported by a Robert Hearin Foundation Medical Research Scholar Award.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: X.C.L., G.E.S., and J.L.Z. conception and design of research; X.C.L., E.M.-Q., and J.L.Z. performed experiments; X.C.L., E.M.-Q., and J.L.Z. analyzed data; X.C.L. and J.L.Z. interpreted results of experiments; X.C.L. and J.L.Z. prepared figures; X.C.L. and J.L.Z. drafted manuscript; X.C.L. and J.L.Z. edited and revised manuscript; X.C.L., G.E.S., E.M.-Q., and J.L.Z. approved final version of manuscript.