Genetic and genomic evidence for an important role of the Na⁺/H⁺ exchanger 3 in blood pressure regulation and angiotensin II-induced hypertension

Abstract

The sodium (Na⁺)/hydrogen (H⁺) exchanger 3 (NHE3) and sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase) are two of the most important Na⁺ transporters in the proximal tubules of the kidney. On the apical membrane side, NHE3 primarily mediates the entry of Na⁺ into and the exit of H⁺ from the proximal tubules, directly and indirectly being responsible for reabsorbing ~50% of filtered Na⁺ in the proximal tubules of the kidney. On the basolateral membrane side, Na⁺/K⁺-ATPase serves as a powerful engine driving Na⁺ out of, while pumping K⁺ into the proximal tubules against their concentration gradients. While the roles of NHE3 and Na⁺/K⁺-ATPase in proximal tubular Na⁺ transport under in vitro conditions are well recognized, their respective contributions to the basal blood pressure regulation and angiotensin II (ANG II)-induced hypertension remain poorly understood. Recently, we have been fortunate to be able to use genetically modified mouse models with global, kidney- or proximal tubule-specific deletion of NHE3 to directly determine the cause and effect relationship between NHE3, basal blood pressure homeostasis, and ANG II-induced hypertension at the whole body, kidney and/or proximal tubule levels. The purpose of this article is to review the genetic and genomic evidence for an important role of NHE3 with a focus in the regulation of basal blood pressure and ANG II-induced hypertension, as we learned from studies using global, kidney- or proximal tubule-specific NHE3 knockout mice. We hypothesize that NHE3 in the proximal tubules is necessary for maintaining basal blood pressure homeostasis and the development of ANG II-induced hypertension.

INTRODUCTION

Hypertension, i.e., high blood pressure, is now well recognized to play an important causative role in the pathogenesis of target organ injuries, such as ischemic heart diseases, atherosclerosis, stroke, and renal diseases. The impact and significance of hypertension in public health are best illustrated by the facts that about half of the U.S. adult population will develop hypertension and take antihypertensive drugs in their life time and that the direct and indirect costs of preventing and treating hypertension are estimated at hundreds of billion dollars a year (77, 87). Although hypertension has been extensively studied for decades, the precise mechanisms of hypertension remain incompletely understood. Genetic predisposition, endocrine disorders, increased sympathetic nerve activity, cardiovascular dysfunction, unhealthy dietary styles, and environmental factors all have been shown to contribute independently to the development of hypertension. Indeed, regardless of etiologies, clinical practice has firmly established that diuretics to inhibit sodium (Na⁺) and fluid reabsorption in the kidney and blockers of the renin-angiotensin system (RAS), such as renin inhibitors, angiotensin-converting enzyme inhibitors, angiotensin II (ANG II) type 1 receptor blockers, and aldosterone receptor antagonists, all significantly lower blood pressure in human essential hypertension (3, 87). Furthermore, long-term restriction of dietary Na⁺ intake appears to be clinically beneficial in reducing blood pressure in prehypertensive and hypertensive subjects (36, 51, 102). This strongly suggests that the activation of the RAS, or ANG II specifically, and increased Na⁺ reabsorption by the kidney are two key factors in the maintenance of basal blood pressure and the development of hypertension in humans. However, clinical studies also demonstrated that only half of patients with hypertension have adequate control of their blood pressure with current antihypertensive therapy (77, 87). It is therefore imperative to continue to uncover additional mechanisms of hypertension and devise novel antihypertensive strategies to prevent and treat hypertensive diseases.

The late great renal physiologist Dr. Arthur C. Guyton from the University of Mississippi Medical Center long proposed that the kidney plays a central role in maintaining basal arterial blood pressure and the development of hypertension through a key regulating mechanism, i.e., the pressure-natriuresis response (43, 44). The pressure-natriuresis response is an important feedback mechanism for long-term blood pressure control in which an acute increase in blood pressure will lead to a decrease in Na⁺ reabsorption primarily in the proximal tubule and restore blood pressure to normal (43, 44). In the kidney, the proximal tubules are responsible for reabsorbing ~70% of filtered Na⁺ and H₂O from the glomeruli; thus sustained increases or decreases in Na⁺ reabsorption in the proximal tubules will ultimately alter overall body salt and fluid balance and therefore blood pressure (116, 121, 131). The Na⁺ reabsorption in the proximal tubules is mediated primarily by the Na⁺ and proton (H⁺) exchanger subtype 3 (NHE3) from apical membranes (78, 96, 103, 110, 113), and the Na⁺/K⁺-ATPase from the basolateral membranes (59, 94, 118, 120, 126, 131). NHE3 is the most important apical (AP) membrane Na⁺ transporter in the proximal tubules (6, 14, 78, 103, 115). Indeed, the key function of NHE3 is to release H⁺ into the lumen in exchange for luminal Na⁺ entering the cells; NHE3 is directly and indirectly responsible for reabsorbing more than 50% of filtered Na⁺ and 80% of filtered bicarbonate (${\text{HCO}}_3^{-}$) in the kidney. By contrast, Na⁺/K⁺-ATPase is well recognized as the most important active Na⁺ transporter in the basolateral membranes, playing an indispensable role in driving active Na⁺ transport across the proximal tubules (35). While we do not discount the relatively minor roles of the Na⁺ and glucose cotransporter (sglt2), Na⁺ and bicarbonate cotransporters (Na⁺/${\text{HCO}}_3^{-}$), and aquaporin 1 (AQP1), NHE3, and Na⁺/K⁺-ATPase clearly contribute most to basal blood pressure homeostasis and the development of ANG II-induced hypertension.

The purpose of this article is to review and discuss the important role of NHE3 in the regulation of basal blood pressure homeostasis and in the development of ANG II-induced hypertension, as we learned from studies using genetically modified mouse models with global, kidney-, or proximal tubule-specific knockout of NHE3 (68–70). To ensure some relevance to the potential role of Na⁺/K⁺-ATPase in the proximal tubules, we also discuss whether the expression of Na⁺/K⁺-ATPase is upregulated in the proximal tubules of the kidney to compensate for the loss of NHE3 in global, kidney-selective, and proximal tubule-specific NHE3 knockout mice (68–70). Based on the genetic and genomic evidence obtained from studying genetically modified NHE3 mouse models, we hypothesize that NHE3 in the proximal tubules of the kidney may be a novel mechanism and therapeutic target of ANG II-induced hypertension. The new insights to be gained from future studies in this field will help us better understand renal mechanisms of hypertension and develop proximal tubule-targeting diuretics to treat hypertension in the future.

NHE3 IS PREDOMINANTLY EXPRESSED AND LOCALIZED IN THE PROXIMAL TUBULES OF THE KIDNEY

NHE3 is considered to be the most important Na⁺ transporter in the apical membranes of the proximal tubules in the kidney, because its action directly and indirectly contributes to the reabsorption of nearly 50% of filtered Na⁺ and fluid loads (26, 116, 121, 131). NHE3 belongs to the mammalian NHE gene family, which has nine isoforms (26, 127), but NHE3 is most relevant in blood pressure regulation due to its dominant expression in the proximal tubules of the kidney and small intestines and its role in mediating Na⁺ reabsorption (14). Localization of NHE3 on the apical membranes promotes, whereas internalization of NHE3 from the apical membranes to subapical regions inhibits, Na⁺ reabsorption in the proximal tubules (25, 50, 60, 64, 129). Apart from the proximal tubules, the loop of Henle [thick ascending limb (TAL)] also express low levels of NHE3 (26). With respect to the expression and localization of other NHE isoforms in the kidney, NHE1 is primarily found in the basolateral membranes of proximal and distal convoluted tubules, TAL, and collecting duct, where it mediates vectorial Na⁺ transport (15, 62). NHE2 and NHE4 are primarily localized in basolateral membranes of medullary collecting duct (19, 109). It has been shown that NHE3 and NHE5 may traffic between the apical membranes and recycling endosomes under physiological conditions (9, 11, 89, 111). However, NHE 1, 2, and 4 appear to mainly stay on the membranes (26). Like NHE3, NHE2 and NHE8 are also expressed in the apical membranes of the proximal tubules, but their functional roles in mediating Na⁺ transport remain poorly understood. Furthermore, NHE6, 7, 8, and 9 are reportedly localized in other cell types to regulate intraorganellar pH (41, 42, 82, 86, 124). Overall, NHE3 in the proximal tubules of the kidney plays a key role in the regulation of proximal tubule Na⁺ and fluid reabsorption and therefore has an impact on blood pressure homeostasis.

EXPRESSION OF NHE3 IN THE PROXIMAL TUBULES OF THE KIDNEY IS REGULATED BY VASOACTIVE, ANTINATRIURETIC, AND NATRIURETIC FACTORS

As the major function of NHE3 in the proximal tubules of the kidney involves stimulation of Na⁺ reabsorption, which alters blood pressure, its expression in the proximal tubules is regulated by a complex of vasoactive antinatriuretic and natriuretic factors (17, 48, 54, 72, 80, 83). One of these factors is the Na⁺/H⁺ exchanger regulatory factor-1, NHERF-1 (113, 117). NHERF-1 has been shown to bind to NHE3 and the Na⁺-dependent phosphate transporter 2a (Npt2a) and induce cAMP-mediated phosphorylation and inhibition of NHE3 activity in the apical membranes of the proximal tubules (85, 111, 117). cAMP (EPAC)- and protein kinase A (PKA)-dependent mechanisms may be involved in the role of NHERF-1 in the regulation of NHE3 activity in the proximal tubules, as suggested in NHERF1^−/− mice (85). However, other factors also play important roles in the regulation of NHE3 expression and activity in the kidney. Dopamine, a natriuretic peptide hormone, plays a significant inhibitory role in NHE3 expression and activity in the kidney (10, 18, 30, 50, 116). Conversely, some antinatriuretic peptide hormones or humoral factors, such as ANG II (16, 29, 72, 99), glucocorticoid (92, 114), glucagon, insulin (37), and oxidative stress, upregulate the NHE3 expression and activity and promote Na⁺ and fluid reabsorption in proximal tubules of the kidney. ANG II, via activation of AT₁ receptor-mediated protein kinase Cα (PKCα) and inositol 1,4,5-triphosphate, and Ca²⁺/calmodulin-dependent protein kinase II (48), induces NHE3 expression and activity in the proximal tubules (21, 28, 49, 55, 57, 58, 72, 75). Inositol 1,4,5-triphosphate receptor-binding protein interacted with inositol 1,4,5-triphosphate, which is expressed in the proximal tubules, appears to mediate ANG II-induced activation of NHE3 in a Ca²⁺/calmodulin-dependent protein kinase II-dependent pathway (48). Finally, the circadian clock genes and the circadian clock protein period 1 (Per1) have been shown to play an important role in regulating NHE3 in the kidney (102a, 110). Rohman et al. (102a) demonstrated that NHE3 mRNA and protein expression in the kidney display significant circadian patterns with a decrease in expression when the lights are on and an increase in expression when lights are off. Furthermore, the expression of NHE3 and Per2 appeared to be colocalized in the proximal tubules of the kidney. In contrast, Solocinski et al. (110) showed that pharmacological blockade of nuclear Per1 entry or siRNA knockdown of Per1 expression markedly decreased NHE3 expression in the renal cortex or human proximal tubule cells, suggesting that Per1 rather than Per2 regulates NHE3 in the proximal tubules of the kidney. Whether the circadian clock genes directly and independently regulate NHE3 expression in the proximal tubules of the kidney remains poorly understood (102a, 110). Indeed, the expression and activity of nearly all neural, endocrine, paracrine, vasoactive hormones or factors are theoretically under the regulation of the circadian clock genes. Thus, the expression and activity of NHE3 in the proximal tubules of the kidney may directly respond to the circadian rhythms of the expression and activity of various vasoactive antinatriuretic, neural, and natriuretic hormones or factors.

NHE3 IS NECESSARY FOR MAINTAINING NORMAL BLOOD PRESSURE HOMEOSTASIS, AS REVEALED IN GLOBAL NHE3^−/−, KIDNEY-SELECTIVE, AND PROXIMAL TUBULE-SPECIFIC NHE3 KNOCKOUT MICE

In the proximal tubules of the kidney, the key function of NHE3 is to secret H⁺ ions into the tubular lumen in exchange for luminal Na⁺, thereby mediating proximal tubular Na⁺ reabsorption and contributing to body salt, fluid, and acid-base balance and basal blood pressure homeostasis (5, 7, 8, 20, 94, 112). Previous studies in cultured proximal tubule cells or isolated perfused proximal tubules with NHE3 inhibitors have firmly established that NHE3 plays a key role in proximal tubule Na⁺ and ${\text{HCO}}_3^{-}$ transport. However, these studies were unable to determine directly the role of NHE3 in the blood pressure regulation. Schultheis et al. (78, 103, 115) were the first to generate genetically modified NHE3-deficient mice (Nhe3^−/−) to determine the physiological relevance of NHE3 in body salt and fluid balance and blood pressure. These authors showed that deletion of NHE3 globally caused slight to moderate salt wasting from the digestive system with diarrhea and mild acidosis, but most of the Nhe3^−/− mice survived to adulthood (103). In the kidney, ${\text{HCO}}_3^{-}$ and fluid absorption were reduced by ~50% in proximal convoluted tubules. Although renal renin, aldosterone in the plasma, the AE1 (Slc4a1) Cl⁻/${\text{HCO}}_3^{-}$ exchanger mRNA expression in the kidney, and epithelial sodium channel (ENaC) and H⁺, K⁺-ATPase mRNA expression in the colon were all significantly upregulated, basal blood pressure was still significantly reduced in Nhe3^−/− mice. These landmark studies confirm for the first time that NHE3 is the major absorptive Na⁺/H⁺ exchanger in the kidney and small intestines and that lack of NHE3 impairs body acid and base and Na⁺ and fluid balance and basal blood pressure homeostasis.

Our group has also recently studied the roles of NHE3 in the regulation of body salt and fluid balance and blood pressure using Nhe3^−/− mice with or without transgenic rescue of the NHE3 gene in small intestines of the digestive system (68, 69). Nhe3^−/− mice represent the global NHE3 knockout mouse model (68, 103), whereas tgNhe3^−/− mice with transgenic rescue of the NHE3 gene in small intestines of the digestive system may be considered to be a “kidney-selective” NHE3 knockout model (69, 88). We also found that Nhe3^−/− and tgNhe3^−/− mice grew normally but showed significantly abnormal structural and absorptive phenotypes of the small intestines, as demonstrated by the markedly enlarged cecum segment of the small intestines with accumulation of a large volume of fluid content inside and a marked increase in 24 h fecal Na⁺ excretion (Fig. 1) (68–70). In our study, however, we found that the transgenic rescue of the NHE3 gene in small intestines in tgNhe3^−/− mice was inadequate to fully rescue the abnormal structural and absorptive phenotypes of Nhe3^−/− mice (69, 70). The moderate to severe salt wasting from the digestive system of Nhe3^−/− and tgNhe3^−/− mice caused marked compensatory Na⁺-retaining responses in the kidney with significantly decreases in 24 h urine output and urinary Na⁺ excretion. Although the RAS was markedly activated as demonstrated by increased basal plasma ANG II and aldosterone levels, basal systolic, diastolic, and mean arterial blood pressures were still ~12–15 mmHg lower in Nhe3^−/− and tgNhe3^−/− mice, respectively (Fig. 2) (P < 0.01). Recently, basal arterial blood pressure was also reportedly lower in a new whole kidney nephron segment, panepithelial cell-specific NHE3 knockout mice using the Pax8-Cre/NHE3^loxlox approach (34). Our data are consistent with the results of Schultheis et al. (103), Noonan et al. (88), and Fenton et al. (34) and strongly support the scientific premise that NHE3 plays a critical role in maintaining basal blood pressure homeostasis by mediating Na⁺ reabsorption in the digestive system and the kidney.

Fig. 1.

Structural and absorptive phenotypes of small intestines of the digestive system in mutant mice with global (Nhe3^−/−), kidney- (tgNhe3^−/−), and proximal tubule-specific deletion of NHE3 (PT-Nhe3^−/−). A: a representative normal cecum segment between small and large intestines in a wild-type mouse (*). B: a representative cecum segment between small and large intestines in a global Nhe3^−/− mouse, showing the extremely enlarged cecum segment accumulated with a large volume of fluid content inside (*). C: a representative cecum segment between small and large intestines in a PT-Nhe3^−/− mouse, showing the lack of enlarged cecum segment and no accumulation of a large volume of fluid content in the cecum segment (*). Please note that the overall gut weight more than double in global Nhe3^−/− mice than wild-type (WT) and PT-Nhe3^−/− mice (D), whereas 24 h fecal Na⁺ excretion (E) and accumulation of fluid content (F) are ~10 times higher in global Nhe3^−/− mice than WT and PT-Nhe3^−/− mice. These results strongly suggest that there is no off-side NHE3 deletion in small intestines in PT-Nhe3^−/− mice. Reproduced from reference (70) with permission.

Fig. 2.

The basal blood pressure phenotype in genetically modified mice with global (Nhe3^−/−), kidney- (tgNhe3^−/−), and proximal tubule-specific deletion of NHE3 (PT-Nhe3^−/−). Basal systolic and intra-arterial blood pressure is significantly lower in adult male Nhe3^−/−, tgNhe3^−/− and PT-Nhe3^−/−mice. Blood pressure was measured continuously for 7 days by the direct implanted telemetry technique. Modified from references (68–70) with permission.

To further determine the role of NHE3 in the proximal tubules of the kidney in body salt and fluid balance and blood pressure regulation, Soleimani at the University of Cincinnati and our group at the University of Mississippi Medical Center generated a novel mouse model with deletion of NHE3 selectively in the proximal tubules of the kidney, PT-Nhe3^−/− mice, using the sglt2-Cre/LoxP approach (65, 70). The effectiveness of this approach to genetically delete NHE3 only in the proximal tubules is based on the scientific premise that sglt2 is primarily expressed in the early S1 and S2 segments of the proximal tubules, and thus the sglt2 promoter is expected to drive the Cre expression exclusively in the S1 and S2 segments (65, 70, 101). Using PT-Nhe3^−/− mice, we directly tested the hypothesis that deletion of NHE3 selectively in the proximal tubules of the kidney lowers basal blood pressure by increasing the pressure-natriuresis response in mice. To test the hypothesis, adult male and female, age-matched wild-type and PT-Nhe3^−/− mice were studied for basal body electrolytes and pH, blood pressure, and kidney function, the pressure-natriuresis response, and the natriuretic responses to acute saline expansion (0.9% NaCl at 10% body wt ip) or to 2 wk of 2% NaCl diet. Under physiological conditions, PT-Nhe3^−/− mice showed significantly lower systolic, diastolic, and mean arterial blood pressure than wild-type mice (Fig. 2) (P < 0.01), at the levels basically similar to those of global Nhe3^−/− and tgNhe3^−/− mice (70). However, unlike Nhe3^−/− and tgNhe3^−/− mice, which show significant compensatory Na⁺-retaining responses (68, 69), PT-Nhe3^−/− mice showed significantly greater diuretic and natriuretic responses than wild-type mice (P < 0.01). Twenty-four hour fecal Na⁺ excretion, plasma pH, Na⁺, and bicarbonate levels were not significantly different from wild-type mice. Furthermore, the pressure-natriuresis response (Fig. 3) and natriuretic responses to acute saline expansion or to 2 wk 2% NaCl diet were all significantly augmented compared with wild-type mice (70).

Fig. 3.

The pressure-natriuresis response in male wild-type and PT-Nhe3^−/− mice. In response to an increase of ~30 mmHg in renal perfusion pressure, the pressure-natriuresis response increased ~5-fold in male wild-type mice, whereas the response increased ~8-fold in male PT-Nhe3^−/− mice (**P < 0.01). Both net urinary Na⁺ excretion and as a response to 10 mmHg blood pressure increase were significantly higher in male PT-Nhe3^−/− mice (P < 0.01; n = 10) than in wild-type mice (P < 0.01; n = 8). Reproduced from reference (70) with permission.

Taken together, the direct evidence for an important role of NHE3 in maintaining normal basal blood pressure has been confirmed in global Nhe3^−/−, kidney-selective tgNhe3^−/− or Pax8-Cre/NHE3^loxlox, and proximal tubule-specific Nhe3^−/− mice (34, 68–70, 88, 103). However, it should be pointed out that the levels of basal blood pressure are not remarkably different in global, kidney-, and proximal tubule-specific NHE3 knockout models. Since the hypotensive phenotype is accompanied by marked absorptive defects in small intestines of Nhe3^−/− and tgNhe3^−/−, but not in those of PT-Nhe3^−/− mice, our data support the physiological importance of NHE3 and the hypothesis that NHE3 in the proximal tubules of the kidney plays an essential role in maintaining basal blood pressure homeostasis by regulating proximal tubule Na⁺ reabsorption and the pressure and natriuretic response (70).

NHE3 IS NECESSARY FOR THE FULL DEVELOPMENT OF ANG II-INDUCED HYPERTENSION, AS REVEALED IN GLOBAL NHE3^−/− AND KIDNEY-SELECTIVE NHE3 KNOCKOUT MICE

We have previously shown that ANG II, at nonpressor concentrations, acts on the AT₁ (AT_1a) receptor to increase NHE3 expression and activity in rabbit and mouse proximal tubule cells in vitro (66, 67, 71). In vivo, however, the effects of ANG II on the expression or activity of NHE3 in the proximal tubules of the kidney appear to be dose and blood pressure dependent (64, 72, 80, 97). Acute infusion of highly pressor doses of ANG II induces rapid internalization of NHE3 in the proximal tubules and pressure natriuresis, whereas long-term infusion of subpressor doses of ANG II increases the expression and activity of proximal tubule NHE3 and slowly elevates blood pressure (72, 73). Indeed, we have recently demonstrated that NHE3 proteins were significantly increased in the apical membranes of the proximal tubules after 2 wk infusion of a subpressor dose of ANG II (0.5 mg/kg/day ip) but are decreased by 2 wk infusion of a highly pressor dose of ANG II (1.5 mg/kg/day ip) (72). These results suggest that NHE3 may play an important role in the regulation of blood pressure by ANG II through its actions on proximal tubule Na⁺ transport.

We have recently tested the hypothesis that NHE3 is necessary for the full development of ANG II-induced hypertension in global Nhe3^−/− and kidney-selective tgNhe3^−/− mouse models (68, 69). Our argument is that NHE3, especially in the proximal tubules of the kidney, may play an important role in the renal mechanisms of ANG II-induced hypertension. To test the hypothesis, age- and body weight-matched adult male wild-type, Nhe3^−/− and tgNhe3^−/− mice were infused with or without ANG II (1.5 mg/kg/day ip, via osmotic minipump), or ANG II plus treatment with the AT₁ receptor blocker losartan (20 mg/kg/day po) for 2 wk. In response to ANG II infusion for 2 wk, blood pressure markedly increased in wild-type mice in a time-dependent manner (P < 0.01). However, the hypertensive response to ANG II was attenuated by more than 50% in Nhe3^−/− and tgNhe3^−/− mice although plasma ANG II and aldosterone levels were significantly elevated during ANG II infusion (Fig. 4) (68, 69). Similar attenuation of the pressor response to ANG II was observed in anesthetized Nhe3^−/− and tgNhe3^−/− mice (68, 69). In further preliminary studies, we infused the same dose of ANG II in PT-Nhe3^−/− mice and confirmed that ANG II-induced hypertension was also attenuated in PT-Nhe3^−/− mice to a similar magnitude of Nhe3^−/− and tgNhe3^−/− mice (68, 69). Our studies therefore provide unequivocal evidence to support the hypothesis that NHE3 in the proximal tubules of the kidney, along with NHE3 in small intestines, is necessary for the development of ANG II-induced hypertension.

Fig. 4.

The systolic blood pressure responses to ANG II are attenuated in adult male conscious global Nhe3^−/− and “kidney-selective” tgNhe3^−/−mice, compared with wild-type mice (A, **P < 0.01 vs. the same groups' respective control or baseline levels; ⁺⁺P < 0.01 vs. the corresponding wild-type groups with the same treatment), and the compensatory upregulation of basal water (aquaporin 1, AQP1) and major Na⁺ cotransporter proteins, including Na⁺/${\text{HCO}}_3^{-}$ and Na⁺/K⁺-ATPase α1, in the proximal tubules of global Nhe3^−/− mice (B, ⁺⁺P < 0.01 vs. wild type). Reproduced from references (68, 69).

EXPRESSION, LOCALIZATION, AND ACTION OF Na⁺/K⁺-ATPase IN THE PROXIMAL TUBULES

In the basolateral membranes of the proximal tubules of the kidney, Na⁺/K⁺-ATPase, sodium-potassium adenosine triphosphatase, plays a critical role in driving active Na⁺ transport across the proximal tubular epithelium against a sodium concentration gradient (35, 119–121). Na⁺/K⁺-ATPase was discovered in 1957 by the Danish scientist Jens Christian Skou (108), who isolated an enzyme from the cell membranes of nerves that was activated by Mg²⁺, Na⁺, and K⁺ and mediated the active transport of Na⁺ and K⁺ across the cell membrane. Skou received the Nobel Prize in Physiology and Medicine in 1997 for discovering Na⁺/K⁺-ATPase (24). Na⁺/K⁺-ATPase was cloned in the 1980s (104–106) and was found to belong to the P-type family of ATPase with two major subunits, i.e., the catalytic α- and glycosylated β-subunits (27, 35, 56). The α-subunit consists of four isoforms, α₁–α₄, with a molecular weight of 110 kDa (27, 35), 10 transmembrane domains (M₁–M₁₀), and binding sites for ouabain and K⁺ at the extracellular domain (27, 35). By comparison, the β-subunit includes three isoforms, β₁–β₃, with a single transmembrane domain and a large extracellular domain (27, 35, 56). The α-subunit plays a key role in the Na⁺ transport activity of the Na⁺/K⁺-ATPase, while the β-subunit provides a supportive role. Additionally, a γ-subunit was cloned with 53 amino acids, but it does not have an effect on the formation and expression of functional Na⁺/K⁺-ATPase α/β-subunit complexes (13, 35, 81).

In the kidney, the Na⁺/K⁺-ATPase is predominantly localized on the basolateral plasma membranes of the proximal convoluted and proximal straight tubules (33, 59, 100). However, the basolateral membranes of the loop of Henle, distal tubules, and collecting duct also express Na⁺/K⁺-ATPase. The role of Na⁺/K⁺-ATPase in mediating active Na⁺ transport in the proximal tubules of the kidney has been extensively investigated since the 1970s. Ouabain, a natural inhibitor of Na⁺/K⁺-ATPase, has been widely used in in vitro epithelial Na⁺ transport studies to determine the role of this enzyme (35). In vitro microperfusion and in vivo micropuncture studies have shown that inhibition of Na⁺/K⁺-ATPase by ouabain markedly decreased proximal tubular Na⁺ reabsorption (38, 40, 45). These early studies have established that Na⁺/K⁺-ATPase drives active Na⁺ extrusion from basolateral plasma membranes and helps promote Na⁺ entry into proximal tubular cells via the actions of NHE3 and sodium and glucose cotransporters (sglt2), sodium bicarbonate transporter etc. from apical membranes (35, 38, 40, 45).

In addition to mediating active Na⁺ transport, Na⁺/K⁺-ATPase may act as a signal transducer in the proximal tubules of the kidney (1). Vasoactive peptide hormones, such as ANG II, atrial natriuretic peptide, dopamine, and endothelin 1, high-salt diet, increased oxidative stress, regulate proximal tubular Na⁺ transport in part by regulating the expression or activity of Na⁺/K⁺-ATPase, or the endocytosis of Na⁺/K⁺-ATPase into proximal tubule cells (23, 35, 61, 76). Indeed, as the most relevant to the subject of this review article, ANG II was shown to directly stimulate the activity and/or the phosphorylation of Na⁺/K⁺-ATPase in the proximal tubules of the rat kidney in vitro or in vivo (31, 39, 122, 123, 125, 126). The stimulatory effect of ANG II on Na⁺/K⁺-ATPase activity is primarily mediated by AT₁ receptors (46). By contrast, ANG II and/or ANG III, via activation of AT₂ receptors (90, 129) and nitric oxide-cGMP-dependent signaling (107), may induce the internalization of apical membrane NHE3 and basolateral Na⁺/K⁺-ATPase, leading to the inhibition of proximal tubular Na⁺ reabsorption and natriuresis, and consequently lower blood pressure in rats (60, 91).

LACK OF GLOBAL, KIDNEY-, OR PROXIMAL TUBULE-SPECIFIC OVEREXPRESSION OR DELETION OF Na⁺/K⁺-ATPase ANIMAL MODELS

In contrast to NHE3 in the proximal tubules of the kidney, the role of Na⁺/K⁺-ATPase or its α- or β-subunits in the regulation of active Na⁺ transport or reabsorption in the proximal tubules and basal blood pressure homeostasis has not been studied in genetically modified animal models. To date no transgenic mice with either global or tissue-specific deletion or overexpression of Na⁺/K⁺-ATPase in the proximal tubules of the kidney have been reported. The difficulty in generating and breeding these mice for further studies is due to the fact that mice with global deletion of the α₁-subunit gene die during embryogenesis, and mice with global deletion of α₂-subunit gene also die, immediately after birth (12, 27, 52, 53, 84). It would be interesting to determine the roles of Na⁺/K⁺-ATPase in the proximal tubules or other tubular segments of the kidney using mutant mouse models with proximal tubule-specific or other nephron segment- or cell-specific deletion or overexpression of α- or β-subunits of Na⁺/K⁺-ATPase.

Na⁺/K⁺-ATPase IN THE PROXIMAL TUBULES OF THE KIDNEY IS UPREGULATED IN GLOBAL, KIDNEY-SELECTIVE, AND PROXIMAL TUBULE-SPECIFIC NHE3-KNOCKOUT MICE

In the proximal tubules of the kidney, Na⁺ and fluid are reabsorbed in proportion to the glomerular filtered load through a unique mechanism, i.e., glomerulotubular balance through the regulation of physical and humoral factors (29, 30, 130, 131). This physiological response involves the actions of NHE3, the sglt2, Na⁺ and amino acid cotransporter, Na⁺ and phosphate cotransporter 2, and AQP1 on the apical membranes, and Na⁺/K⁺-ATPase and Na⁺ and bicarbonate cotransporter on the basolateral membranes. However, NHE3 and Na⁺/K⁺-ATPase play the most important or a predominant role in mediating Na⁺ reabsorption in the proximal tubules of the kidney (131). We have recently bred global (Nhe3^−/−) (68), kidney-selective (tgNhe3^−/−) (69), and proximal tubule-specific NHE3-knockout mice (PT-Nhe3^−/−) (70) to determine the roles of NHE3 in the regulation of overall body Na⁺ and fluid balance, proximal tubule Na⁺ reabsorption, and basal blood pressure homeostasis. We next determined whether Na⁺/K⁺-ATPase and other Na⁺ cotransporters in the proximal tubules of the kidney are upregulated to compensate for the loss of global, kidney, and proximal tubule NHE3 in global, kidney-selective, and proximal tubule-specific NHE3-knockout mice. Finally, we investigated whether ANG II-induced hypertension is attenuated in global, kidney-selective, and proximal tubule-specific NHE3-knockout mice (68, 69).

We focused on three key Na⁺ transporter and three key signaling proteins in the proximal tubules of the superficial renal cortex of the kidney in global Nhe3^−/− (68), kidney-selective tgNhe3^−/− (69), and proximal tubule-specific PT-Nhe3^−/− mice (70). In the absence of apical membrane NHE3 in these three different strains of NHE3 genetically modified mice, the expression of sglt2 and the water channel protein AQP 1 in apical membranes and the α1-subunit of Na⁺/K⁺-ATPase, and Na⁺ bicarbonate cotransporter (Na⁺/${\text{HCO}}_3^{-}$) proteins in basolateral membranes was significantly increased in the superficial cortex of the kidney (Fig. 4, P < 0.01) (68–70). Furthermore, the expression of ENaC and AQP2 proteins in the renal medulla was also significantly upregulated (70). The upregulation of sglt2, Na⁺/K⁺-ATPase, Na⁺/${\text{HCO}}_3^{-}$^-, ENaC, AQP1, and AQP2 in the kidney was associated with significant increases in signaling responses, including phosphorylated PKCα, phosphorylated MAP kinases ERK1/2, and phosphorylated glycogen synthase kinase-3α/β (GSK-3α/β) in the proximal tubules of Nhe3^−/− or tgNhe3^−/− mice (68–70). In wild-type mice, ANG II infusion for 2 wk significantly increased the expression of the α1-subunit of Na⁺/K⁺-ATPase, Na⁺/${\text{HCO}}_3^{-}$, and AQP1 in the proximal tubules of the kidney, but this response was not observed in Nhe3^−/− or tgNhe3^−/− mice (68, 69). These data suggest that the deletion of NHE3 globally, in the whole kidney, or in the proximal tubules of the kidney leads to the compensatory upregulation of the expression of sglt2, Na⁺/K⁺-ATPase, Na⁺/${\text{HCO}}_3^{-}$, ENaC, AQP1, and AQP2 or activation of PKCα, MAP kinases ERK1/2, and GSK-3α/β in the proximal tubules of the kidney. This in turn increases Na⁺ and fluid reabsorption through the actions of these Na⁺ cotransporters or AQP1 and AQP2 proteins in the proximal tubules or distal nephron segments of the kidney to minimize salt wasting due to the loss of NHE3 in these tissues. However, it should be recognized that the upregulation of the expression of sglt2, Na⁺/K⁺-ATPase, Na⁺/${\text{HCO}}_3^{-}$, ENaC, AQP1, and AQP2 in Nhe3^−/−, tgNhe3^−/−, and PT-Nhe3^−/− mice is inadequate to compensate for the loss of NHE3 in small intestines or the proximal tubules of the kidney, with the consequence of lower systolic, diastolic, and mean arterial blood pressure in these animals (68–70). Taken together, NHE3 in the small intestines of the digestive system and the proximal tubules of the kidney plays a key role in maintaining basal blood pressure homeostasis and may be considered to be a potential therapeutic target of hypertension.

CONCLUSIONS AND FUTURE PERSPECTIVES

In summary, we and others have used global, kidney-, and proximal tubule-specific NHE3 knockout mouse models to determine the roles of NHE3 in maintaining body salt and fluid balance, basal blood pressure homeostasis, and the development of ANG II-induced hypertension (34, 65, 68–70, 103, 115). The data derived from these studies using these global and tissue-specific, genetically modified NHE3 knockout mice provide unequivocal evidence to support the scientific hypothesis that NHE3 is necessary, or required, for the maintenance of body salt and fluid balance, basal blood pressure homeostasis, and the development of ANG II-induced hypertension (Fig. 5). Other Na⁺ and glucose cotransporters, Na⁺/K⁺-ATPase, Na⁺ and bicarbonate cotransporters, Na⁺ and amino acid cotransporters, ENaC, and AQP1 and 2 in the kidney may also contribute to the blood pressure regulation, but their roles likely pale in comparison to that of NHE3. This conclusion is supported by our observations that most, if not all, of these Na⁺ cotransporters are markedly upregulated in the proximal tubules, or the medulla, of the kidney in global, kidney-, and proximal tubule-specific Nhe3^−/− mice. However, activation or upregulation of these transporters is still inadequate to compensate for the loss of the action of NHE3 and restore blood pressure to normal. These findings have significant and potentially translational or clinical implications for hypertension in humans.

Fig. 5.

A schematic hypothesis that systemic and tissue ANG II activates AT₁ receptors on the apical and basolateral membranes of the proximal tubules of the kidney and downstream signaling pathways to increase the expression and activity of NHE3 and Na⁺/K⁺-ATPase, which stimulates proximal tubular Na⁺ reabsorption, causes body Na⁺ and fluid retention, and induces ANG II-dependent hypertension. Increased arterial pressure will induce the pressure-natriuresis response to prevent further increase in blood pressure at the very early stage of hypertension. However, the initially increased pressure-natriuresis response is insufficient to restore blood pressure to normal in established ANG II-induced hypertension due to the resetting of the response to higher pressures.

Hypertension is currently treated with loop- or distal tubule-selective diuretics, RAS inhibitors, Ca²⁺ channel blockers, adrenergic receptor antagonists, or aldosterone receptor antagonists (22, 32, 79). Three types of diuretics have been used as antihypertensive drugs: thiazides, which inhibit Na⁺ and Cl⁻ reabsorption from the distal convoluted tubules by blocking the thiazide-sensitive Na⁺-Cl⁻ symporter; loop diuretics, which inhibit the Na⁺-K⁺-2 Cl⁻ symporter (cotransporter) in the TAL of the loop of Henle; and K⁺-sparing diuretics, which either block ENaC or act as aldosterone receptor antagonists (32, 79). However, only 50% of patients with hypertension have their blood pressure under adequate control with these classes of antihypertensive drugs, and some patients still develop resistant hypertension. Against this background, no proximal tubule-selective diuretics, with the exception of carbonic anhydrase inhibitors that are mild diuretics, have been developed for antihypertensive treatment. Proximal tubules are responsible for reabsorbing 70% of filtered Na⁺ load, whereas NHE3 in the proximal tubules directly and indirectly accounts for reabsorbing >50% of filtered Na⁺ and ~80% of filtered ${\text{HCO}}_3^{-}$ in the kidney (4, 7, 83, 103). It is not surprising to hypothesize that the upregulation of the intratubular RAS and NHE3 expression and activity in the proximal tubules will stimulate Na⁺ and ${\text{HCO}}_3^{-}$ reabsorption, induce body salt and fluid retention, and contribute to blood pressure control in health and hypertension. Indeed, increased NHE3 expression and activity have been reported in the proximal tubules of spontaneously hypertensive rats (SHR), an animal model of human essential hypertension (47, 63, 128). Even in 5 wk old SHR before blood pressure begins to rise, NHE3 activity is increased by 48% (2) and remains substantially elevated after hypertension is firmly established (63). Furthermore, all known prohypertensive factors, including ANG II, glucocorticoid, hyperinsulinema, hyperglucagonemia, increased oxidative stress, and renal nerve stimulation (16, 37, 48, 72, 92, 93, 116), induce NHE3 expression and activity and promote Na⁺ reabsorption in the proximal tubules. Thus this raises the possibility to target NHE3 pharmacologically in the proximal tubules of the kidney for additional antihypertensive therapy for those patients with poorly controlled hypertension. A recent proof of concept study by Linz et al. (74) showed that an orally active, nonabsorbable NHE3 inhibitor, SAR218034, significantly inhibited intestinal Na⁺ reabsorption and reduced blood pressure in 78 wk old SHR-lean rats, which supports this hypothesis. Perhaps, the development of orally active, absorbable, and potent NHE3 inhibitors as proximal tubule-selective diuretics to inhibit NHE3 in the proximal tubules of the kidney may have significant translational impact on future antihypertensive treatment in humans.

GRANTS

This work was supported in part by National Institutes of Health Grants 2RO1DK-067299-06A2, 2R01DK067299-10A1, 1R01DK-102429-01, 2R01DK-102429-03A1, and 1R56HL-130988-01 to J. L. Zhuo.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

X.C.L., X.Z., and J.L.Z. conceived and designed research; X.C.L., X.Z., X.C., C.Z., D.Z., J.Z., and J.L.Z. performed experiments; X.C.L., X.Z., X.C., C.Z., D.Z., J.Z., and J.L.Z. analyzed data; X.C.L., X.Z., X.C., C.Z., D.Z., J.Z., and J.L.Z. interpreted results of experiments; X.C.L., X.Z., and J.L.Z. prepared figures; X.C.L., X.Z., and J.L.Z. drafted manuscript; X.C.L., X.Z., and J.L.Z. edited and revised manuscript; X.C.L., X.Z., X.C., C.Z., D.Z., J.Z., and J.L.Z. approved final version of manuscript.