Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2

Abstract

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na⁺ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na⁺ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na⁺ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

hypertension affects more than 25% of the adult population in most Westernized countries and is a major cause of coronary artery disease and cerebrovascular disease (9, 10). Despite its prevalence, the etiology of more than 90% of hypertension cases is unknown. Numerous studies revealed that interactions between genetic and environmental factors, especially the generous intake of dietary salt, play a critical role in its pathogenesis and a large body of evidence implicates the inappropriate retention of Na(Cl) by the kidney in the pathophysiology of hypertension (25). One of the most important molecular determinants of Na⁺ excretion is the activity of the epithelial Na⁺ channel (ENaC), a highly regulated, three-subunit ion channel complex that is active in the distal nephron (15, 29). The activity of ENaC is regulated by many first and second messengers, the most widely studied of which is aldosterone (13, 22). The importance of ENaC in Na⁺ homeostasis and hypertension is underscored by the discovery that patients with Liddle syndrome, an autosomal dominant form of hypertension, have gain-of-function mutations in ENaC (28).

The mutations in the β- and γ-ENaC subunits leading to Liddle syndrome have focused attention on the specific regions of the COOH termini whose modifications result in increased channel activity. There is now general agreement that ENaC overactivity is caused by the replacement or absence of key amino acid residues that constitute a PY motif. This motif can interact with WW domain proteins and lead to removal of the ENaC complexes from the cell surface. The specific WW domain protein most likely to interact with the PY motifs of ENaC is Nedd4-2, an E3 ubiquitin ligase (30–32). The Nedd4-2 association with ENaC probably leads to the ubiquitination of ENaC and, ultimately, to its removal from the plasma membrane (1). The data from biochemical and heterologous expression experiments thus implicate Nedd4-2 as a central regulator of ENaC activity (16, 17). So far, however, there is no direct evidence demonstrating Nedd4-2's role in ENaC activity, Na⁺ balance, or blood pressure (BP) control.

To examine the in vivo role of Nedd4-2, we generated Nedd4-2 knockout (KO) mice. In the study presented here, we demonstrate that these KO mice have high BP and an impaired ability to downregulate ENaC activity. We also show that a high-salt (HS) diet further increased the BP of Nedd4-2 KO mice. Moreover, persistently elevated BP induced by chronic salt loading in the Nedd4-2 KO mice resulted in cardiac hypertrophy and reduced cardiac function. Overall, our results suggest that variation in the NEDD4-2 gene may contribute to the etiology of salt-sensitive hypertension in humans.

METHODS

Generation of Nedd4-2 KO mice.

Mice expressing a floxed Nedd4-2 gene were created using the methods outlined in the supplemental material (the online version of this article contains supplemental data). These mice were crossed with a strain expressing cre recombinase (EIIa-Cre) universally to yield Nedd4-2 KO mice (20). Mice heterozygous for the deletion (F₁ hybrids between 129/SvJ and C57BL/6) were backcrossed twice to C57BL/6 mice before being intercrossed to generate homozygotes. Unless otherwise stated, mice of both genders at 8–10 wk of age were used for the experiments.

Care of the mice used in the experiments met or exceeded the standards set forth by the National Institutes of Health in their Guide for the Care and Use of Experimental Animals. All procedures were approved by the University Animal Care and Use Committee at the University of Iowa.

Immunoblotting.

Cell lysates from M1 cells and mouse kidneys of all three genotypes were used. SDS-PAGE was performed in 8% gels with DTT, and protein bands were transferred to nitrocellulose membranes and blocked with milk-Tween-PBS. The blots were first incubated with a 1:5,000 dilution of antibody against Nedd4-2 overnight (18). Secondary goat-anti-rabbit antibody was used at a 1:50,000 dilution. Blots were developed using Pierce Super Signal Femto as previously described (2). An antibody against the WW2 domain of Nedd4 (Upstate, Charlottesville, VA) was used at a 1:20,000 dilution to reprobe the blot. Similar methods were used for semiquantitative immunoblotting for the Na⁺ transport proteins. Antibodies against the α-, β-, and γ-ENaC subunits were obtained from Dr. L. Palmer, antibodies against the Na/Cl cotransporter were obtained from Dr. D. Ellison, antibodies against the Na-K-2Cl cotransporter were obtained from Dr. M. Kwon, and antibodies against NHE3 were obtained from Chemicon.

BP measurements using tail-cuff method.

Age- and gender-matched mice were kept on a normal diet for the first 2 wk during the first round of BP measurements. BP was measured via tail cuff, by detection of the return tail pulsations (IITC Life Science, Woodland Hills, CA) (34). Mice were trained for 1 wk before the measurement and were then measured for 1 wk. All testing was done between 1 and 3 PM in a dark and quiet room. All mice were switched to the HS diet at the beginning of the third week, and tail-cuff training started at the beginning of the fourth week. BP measurement started at the beginning of the fifth week, after 2 wk on the HS diet.

BP measurements using radiotelemetry method.

Mice were anesthetized using pentobarbital sodium (50 mg/kg). The catheter (Data Sciences International, Minneapolis, MN) was placed into the left common carotid artery, and the transmitter was placed subcutaneously along the left flank (3). Mice were given 7 days to recover, after which time heart rate (HR) and arterial pressure were continuously recorded (sampling every 5 min for 20-s intervals). Data were collected and stored using Dataquest ART. Basal BP was collected for 7 days while on a normal diet, after which the mice were put on the HS diet for 2 wk. After the mice had been on this diet for 7 days, data were collected for another 7 days while the HS diet was administered. For amiloride treatment, all mice were given a daily intraperitoneal injection of amiloride (1 mg/kg body wt) for 12 days, and BP data were collected during the last 7 days that the mice were on the HS diet and receiving a daily amiloride injection. Mean arterial pressure (MAP) was calculated as the average of each individual sampling segment over a 24-h period.

DOCA treatment.

BP was measured by the tail-cuff method for 2 consecutive days, in age- and gender-matched mice on the normal diet. At this point, a DOCA pellet (25 mg each, 21-day release; Innovative Research of America, Sarasota, FL) was implanted on the back, slightly posterior to the scapulas, and drinking water was switched to 1% NaCl with 0.2% KCl. BP was measured every other day for 9 days.

Measurement of rectal potential difference.

The rectal potential difference (PD) in mice on the normal diet was measured in the absence and presence of amiloride, as previously described (2). One group of mice was switched to the HS diet for 1 wk and PD was measured again. A second group was switched to the low-salt (LS) diet for 1 wk, and then placed on the HS diet for 2 wk. PD was measured at the end of each week. All measurements were conducted by an investigator who was blinded to the mouse genotype.

Plasma and urine Na⁺/K⁺ concentrations.

Blood samples were collected as previously described (2). The plasma and urine concentrations of Na⁺ and K⁺ were measured using a VT250 Chemical Analyzer (Johnson & Johnson Clinical Diagnostics, Rochester, NY) at the Animal Clinical Laboratory Core Facility at the University of North Carolina at Chapel Hill.

Metabolic cages and diet.

Mice were placed in metabolic cages (Nalgene, Rochester, NY) for 3 days for acclimation. After an additional 2 days on a normal diet (0.3% Na⁺), the diet was switched to one containing HS (3.2% Na⁺; Harlan) as indicated. Urine volume, body weight, and food/water consumption data were recorded on a daily basis. Throughout the experiment, the mice had free access to tap water and the indicated diets. Blood samples were collected on day 2 while on the normal diet and on days 5 and 8 on the HS diet.

Plasma aldosterone level and glomerular filtration rate.

During dietary manipulations (3 days on normal diet, 10 days on HS diet, and 6 days on LS diet), urine was collected every day. In addition, blood was collected on day 2 of being on the normal diet, days 5 and 8 of being on the on HS diet, and day 5 of being on the LS diet. Blood was obtained via retro-orbital bleeding. Urine and plasma creatinine concentrations were determined using a creatinine assay kit (Cayman Chemical, Ann Arbor, MI). The glomerular filtration rate (GFR) was estimated based on creatinine clearance, using the standard equation for this calculation. Aldosterone levels were measured in unextracted plasma samples, using the Coat-A-Count Aldosterone radioimmunoassay kit (Diagnostics Products, Los Angeles, CA) as previously described (2).

Echocardiography and image analysis.

Echocardiography was performed on mice that had received normal or HS diets for 8 to 10 mo using a previously described protocol (14). The investigator who performed the echocardiography (R. M. Weiss) was unaware of genotype.

Statistics and data analysis.

Data analysis between different groups of animals was performed by unpaired Student's t-test. Two-way ANOVA was used for analysis with repeated measurements (see Fig. 2B). We considered P values 0.05 statistically significant. All values are expressed as means ± SE.

Fig. 2.

Nedd4-2 KO mice have increased blood pressure (BP). Both radiotelemetry (A) and tail-cuff (B) methods were used. Age (8 to 12 wk old)- and gender-matched mice were used for this study. A: effect of high-salt (HS) diet: starting 7 days after implantation of telemetry transmitter, heart rate and arterial pressure were continuously recorded for 7 days while mice were maintained on normal salt diet. All mice were then placed on HS diet for 2 wk in the absence of recording. Then, heart rate and arterial pressure were continuously recorded for 7 days while mice were maintained on the HS diet. Systolic BP, red symbols; diastolic BP, blue symbols; mean BP, green symbols. B: effect of HS and DOCA: all mice were kept on normal diet for the first 2 wk during the first round of BP measurement. BP was measured via tail cuff. Numbers shown are averages of readings from 4 days. All mice started DOCA/HS treatment at the beginning of the third week, and tail-cuff BP measurement was carried out for 9 days. DOCA-HS treatment increased BP in both groups (P < 0.01 by 2-way ANOVA with repeated measures). The difference in BP observed on a normal diet (P < 0.05) was eliminated by DOCA-HS treatment. The number of mice in each group is in parentheses.

RESULTS

Generation of Nedd4-2 KO mice.

We used conditional gene targeting in mouse ES cells to generate mice in which the Nedd4-2 gene is inactivated (Fig. S. 1). Mice in which the gene was globally deleted from both chromosomes are viable and are born at the predicted Mendelian ratio. Immunoblotting (Fig. 1A) showed that the 130-kDa Nedd4-2 protein was completely absent from the Nedd4-2 KO kidney in these animals and that it was present in the Nedd4-2 +/− kidney at a level about half that present in the Nedd4-2 +/+ kidney. An antibody directed against the WW2 domain of the related E3 ligase Nedd4 cross-reacts with Nedd4-2 (18). Using it, we found that Nedd4 expression levels are not altered in Nedd4-2 −/− mice (Fig. 1B). Also, the Nedd4-2 −/− mice appeared to be normal, both males and females were fertile, and there was no increased mortality when the mice were maintained on a normal diet. Histologically, the kidney and heart structures were normal when the animals were 6 mo of age (data not shown).

Fig. 1.

Nedd4-2 protein is expressed in both cultured mouse kidney cells and normal mouse kidney, but not in Nedd4-2 knockout (KO) kidneys. Cell lysates prepared from M1 cells (a mouse line derived from collecting duct cells) and from kidneys of the 3 tested mouse genotypes were used in Western blot analyses. The amount of protein loaded in each lane is indicated. A: probed with antibody against Nedd4-2. Top arrow (∼130 kDa) indicates Nedd4-2. Bottom MW bands could represent WW domain proteins that react nonspecifically. B: probed with antibody against Nedd4. The blot shown in A was stripped, exposed to film to ensure that the Nedd4-2 signal was removed, and reprobed with a Nedd4 antibody (obtained from Upstate) raised against the WW2 domain of Nedd4. The arrowhead indicates a band that is not recognized by the antibody used in A.

Hypertension in Nedd4-2 KO mice.

On a normal diet (0.3% Na⁺), with free access to drinking water, Nedd4-2 KO mice had a higher BP than did their wild-type (WT) littermates (Fig. 2A, left). A HS diet (3.2% Na⁺) for 2 wk had no effect on BP in the WT mice. In contrast, a HS diet increased the BP even further in the Nedd4-2 KO mice (Fig. 2A, right). The changes in systolic BP were the same whether measured by tail cuff (data not shown) or radiotelemetry (Fig. 2A). These results demonstrate salt-sensitive hypertension in the Nedd4-2 KO mice. In addition, we saw that the normal diurnal variation in BP disappeared in Nedd4-2 KO mice that were maintained on the HS diet.

Treatment with the mineralocorticoid DOCA in combination with HS diet reliably produces hypertension in normal animals. We asked whether Nedd4-2 KO mice would respond differently to this treatment. As shown in Fig. 2B, DOCA treatment led to an increase in BP in both groups using tail-cuff measurement. Nedd4-2 KO mice had 12 mmHg higher BP on a normal diet than WT mice before treatment. After 9 days of DOCA/HS treatment, both groups had an increase in BP but the difference between groups disappeared (Nedd4-2 KO mice, from 129 to 140 mmHg; WT mice, from 117 to 141 mmHg). The lack of additive effects of genotype and DOCA treatment is consistent with convergent pathways for increasing BP.

Defective ENaC regulation in Nedd4-2 KO mice.

After establishing that Nedd4-2 KO mice have salt-sensitive hypertension, we asked whether the effect could be caused by defective ENaC regulation. We used the amiloride-sensitive rectal transepithelial PD as an assessment of Na⁺ absorption and ENaC function. As shown in Fig. 3, WT and Nedd4-2 KO mice have similar, low rectal PD values when maintained on a normal diet. Switching the diet to HS suppressed PD in WT, but not in Nedd4-2 KO, mice (Fig. 3A). We further tested ENaC regulation by interposing a 1-wk period of a LS diet (0.01% Na⁺) before initiating the HS diet. The LS diet produced a substantial increase in PD in both groups of mice (Fig. 3B). As expected, following this stimulus to ENaC activity, the HS diet suppressed rectal PD to values that were less negative than those produced in WT animals on a normal diet. In contrast, Nedd4-2 KO mice showed a markedly slower rate of suppression of the negative PD values. At each stage after the LS diet was imposed, the PD of Nedd4-2 KO mice was more negative than that of the WT controls. These results suggest that ENaC activity is inappropriately high in Nedd4-2 KO mice.

Fig. 3.

Nedd4-2 KO mice have elevated amiloride-sensitive rectal potential difference (PD). Age- and gender-matched mice (8 to 12 wk of age) were used for this experiment. Initially, PD of mice on normal diet was measured in the absence and presence of amiloride. A: these groups of mice were switched directly to a HS diet for 1 wk, at the end of which PD was again measured, or these groups of mice were switched to low-salt diet (0.01% Na⁺) for 1 wk (B), before being placed on an HS diet for 2 wk. PD was measured at the end of each week. The amiloride-sensitive PD was determined and plotted. The number of mice in each group is shown in parentheses.

The loss of Nedd4-2 would be predicted to decrease ENaC internalization and degradation. To test whether the absence of Nedd4-2 caused an increase of ENaC protein in the kidney, we conducted semiquantitative immunoblotting of each of the three ENaC subunits. As shown in Fig. 4, A and B, all three subunits were expressed at significantly higher levels in the kidneys of Nedd4-2 KO mice (ranging from 30 to 50% increases) than in those of their WT counterparts, whereas other Na⁺ transporters were not affected (Fig. 4C).

Fig. 4.

Nedd4-2 KO mice have higher ENaC levels. Protein levels of ENaC subunits in kidney. A: immunoblots of whole kidney tissue from 12 mice [6 wild-type (WT) and 6 KO] fed on normal-NaCl diet. Kidneys were isolated from age-matched (10–12 wk) mice, and total tissue lysates were prepared as previously described (2). B: quantitative assessment of the amount of α-, β-, and γ-ENaC protein in Nedd4-2 +/+ and KO mice. All ENaC bands are expressed relative to WT values and normalized to the amount of actin. C: quantitative assessment of the amounts of other epithelial Na⁺ transporters in Nedd4-2 +/+ and KO mice. NHE3, Na⁺/H⁺ exchanger type 3; NCCT, Na/Cl cotransporter; NKCC2, Na-K-2Cl cotransporter type 2. All bands are expressed relative to WT values and normalized to the amount of actin. *P < 0.05. **P < 0.01. Open bars, +/+ mice; filled bars, KO mice.

To further test the involvement of ENaC in the development of the hypertensive phenotype seen in the Nedd4-2 KO mice, we asked whether amiloride, a specific blocker of ENaC activity, could ameliorate the HS diet-induced BP elevation in Nedd4-2 KO mice. To this end, mice were treated with amiloride for 12 days and BP was measured during the last 7 days. After amiloride treatment, there was a significant reduction in systolic, diastolic, and mean BP in Nedd4-2 KO mice, while the same treatment had no effect on WT mice (Fig. 5A). The effect of amiloride on the KO mice is clearly shown in the change in 24-h MAP (Fig. 5B). Amiloride treatment had no significant effect on MAP in WT mice, whereas it caused a significant reduction in MAP in the Nedd4-2 KO mice.

Fig. 5.

Increased BP in Nedd4-2 KO mice produced by the HS diet is sensitive to amiloride. A: radiotelemetry method was used to monitor BP. For amiloride treatment, all mice that had been on the HS diet for at least 2 wk were given a daily intraperitoneal injection of amiloride (1 mg/kg body wt) for 12 days, and BP data were collected during the last 7 days while on the HS diet and receiving daily amiloride injections. Symbols as in Fig. 2. B: effect of amiloride on mean arterial pressure (MAP). MAP was determined by the average of each individual sampling segment over a 24-h period. The MAP difference before and after amiloride treatment was plotted. Amiloride reduced MAP in KO mice but produced no reduction in WT mice.

Metabolic effects of a HS diet.

We conducted metabolic balance studies to determine whether the HS diet produced differences in WT and Nedd4-2 KO mice. We found that on a normal diet, the daily food and water intake, as well as the amounts of feces and urine produced, were the same in both groups of mice. Although the plasma [Na⁺] was also the same for the two genotypes, plasma [K⁺] in the Nedd4-2 KO mice was slightly higher than in WT mice (Fig. 6). Switching to a HS diet produced some differences; although both groups increased their water consumption and urine output, the Nedd4-2 KO mice did so to a greater extent. In addition, the HS diet increased plasma [Na⁺] more in Nedd4-2 KO mice than in WT mice (Fig. 6E). The difference in plasma [K⁺] seen on the normal diet was not present when mice were maintained on the diet. This is because although the HS diet resulted in a small increase in plasma [K⁺] in WT mice, it also led to a decrease in the [K⁺] in the KO mice.

Fig. 6.

WT and Nedd4-2 KO mice show some differences in metabolic parameters. After being placed into metabolic cages for 3 days for adaptation, mice were provided with normal diet (0.3% Na⁺) for an additional 2 days and were then switched to a HS diet (3.2% Na⁺) for 10 days. Mice had free access to tap water and indicated diets. Data on food/water consumption (A and B), as well as on feces amount/urine volume (C and D), were collected and averaged for each dietary condition. Blood samples were collected at day 2 on the normal diet and at days 5 and 8 on the HS diet, and plasma Na⁺ and K⁺ were measured.

With respect to Na⁺ or K⁺ excretion, there was also no difference between WT and Nedd4-2 KO mice, whether on the normal or HS diet, or during the transition (Fig. 7). Also, the weights of the WT and KO mice were similar on both normal and HS diets (Fig S. 2). In the case of plasma aldosterone, however, there were some differences: on the LS diet the Nedd4-2 KO mice had slightly elevated aldosterone levels than their WT counterparts (Fig. 8). The GFR (as estimated by creatinine clearance) was similar in both groups of mice during all dietary challenges (Fig. S. 3). Thus the Nedd4-2 KO mice may have retained Na⁺, as evidenced by higher plasma [Na⁺], greater water intake, and higher BP, but the difference was too small to detect by balance studies of Na⁺ intake and excretion.

Fig. 7.

Effect of HS diet on urine Na⁺ and K⁺ excretion. After equilibration on a normal diet, WT and Nedd4-2 KO mice were fed an HS diet and Na⁺ and K⁺ excretion was measured daily. Each group included 8 mice. The measured levels of excreted ions did not differ significantly between WT and Nedd4-2 KO mice.

Fig. 8.

Plasma aldosterone concentration under different dietary conditions. Plasma aldosterone levels were measured on blood samples collected on day 2 on the normal diet, on days 5 and 8 on the HS diet, and on day 5 on the low-salt diet, using a Coat-A-Count RIA kit (Diagnostic Products, Los Angeles, CA). Average aldosterone levels during each period are reported. There were 20 WT mice and 18 Nedd4-2 KO mice. Open bars, WT mice; gray bars, Nedd4-2 KO mice.

Effects of Nedd4-2 deletion on cardiac morphology and function.

We determined the long-term cardiac consequence of the HS diet in the absence of Nedd4-2. To that end, we fed WT and Nedd4-2 KO mice either a normal or the HS diet for 8 to 10 mo. As shown in Fig. 9A, the KO mice had greater heart weights on even a normal diet, and the difference was greater when they were fed the HS diet. To assess the changes in cardiac function in live animals, we used echocardiography. On a normal diet, HR, left ventricular (LV) mass, and the ejection fraction (EF) were similar or marginally different between WT and KO mice. However, the HS diet produced marked differences in each parameter in WT and KO mice (for typical echocardiogram images of WT and KO on the HS diet, see supplemental movies): KO mice had a lower HR (Fig. 9B), increased LV mass (Fig. 9C), and severely reduced EF (Fig. 9D).

Fig. 9.

Nedd4-2 KO mice showed cardiac hypertrophy and reduced cardiac function when challenged with HS diet. Age-matched mice (8 wk old) were placed on either a normal or HS diet, and echocardiography was performed on all mice after 8 to 10 mo, and the heart was isolated and weighed. In the case of Nedd4-2 KO mouse, heart weight normalized to body weight was significantly increased regardless of which diet the animals received (A). Analyses of data obtained from echocardiography shows that the HS diet significantly reduced the heart rate (B), increased left ventricular (LV) mass (C), and reduced the ejection fraction (D) for Nedd4-2 KO mice. Open bars, +/+ mice; filled bars, KO mice. Number of mice in each group is shown in parentheses.

DISCUSSION

The results presented here extend our understanding of the molecular regulation of ENaC in vivo, add to the evidence implicating ENaC in BP regulation, and provide a new model of salt-sensitive hypertension that is defined by a single gene defect. The specific physiological characteristics of the Nedd4-2 KO mouse demonstrate that many of the predictions made based on the deduced functions of Nedd4-2 are correct. In addition, however, some characteristics of this mouse reveal unsuspected features of ENaC and BP regulation.

The phenotype of this Nedd4-2 KO mouse suggests that the major function of the Nedd4-2 protein is to regulate Na⁺ balance and BP. The KO mice survive normally and have the predicted Mendelian distribution at birth, but have NaCl-sensitive hypertension. Evidently, Nedd4-2 is not required for development. However, the hypertensive phenotype of the Nedd4-2 KO mouse suggests that this protein is responsible for the normalization of elevated BP and the enhancement of Na⁺ excretion, particularly in the setting of increased NaCl intake. Thus, by extrapolation, genetic abnormalities causing inactivation or ineffective NEDD4-2 function in humans would produce hypertension.

The characterization of the Nedd4-2 KO mice provides mechanistic and in vivo support for the notion that Nedd4-2 regulates ENaC activity through its ubiquitination and possible degradation of this transporter. We provide three lines of evidence. First, we show that, in the absence of Nedd4-2, ENaC activity (as measured by rectal PD), which can be suppressed by increased dietary Na⁺ intake, declines at a much slower rate than in WT mice (Fig. 3). Second, in the absence of Nedd4-2, the amounts of all three ENaC subunits in the kidney are higher in KO mice than in WT mice; this difference is not found for the other major Na⁺ transporters (Fig. 4). Third, the elevated BP in the Nedd4-2 KO mice produced by the HS diet can be ameliorated by the application of amiloride, a specific ENaC inhibitor, whereas amiloride has no such effect in WT mice on a HS diet. In addition, administration of DOCA, a mineralocorticoid hormone that produces salt-sensitive hypertension, did not produce an additive effect in the Nedd4-2 KO mouse (Fig. 2B). Taken together, our data provide strong in vivo evidence that Nedd4-2 influences BP and Na⁺ balance by regulating ENaC activity.

Our experiments also demonstrate that the regulation of ENaC activity in vivo does not depend solely on the presence of Nedd4-2. The fact that basal rectal ENaC activity is similar in Nedd4-2 KO and WT mice and that it is greatly enhanced by a LS diet (in which case stimulation is maximal; Fig. 3B) supports the idea that other pathways must also be capable of activating ENaC. In addition, the fact that ENaC is inactivated slowly in a Nedd4-2 loss-of-function background following the shift from the LS to the HS diet suggests that Nedd4-2 is not absolutely required for the eventual inactivation of ENaC. Although the Nedd4-2-independent pathways of ENaC regulation remain unclear, it is possible that some of the genes regulated by aldosterone could act through pathways independent of Nedd4-2. Alternatively, it is possible that another WW domain-containing protein, perhaps Nedd4 or other members of Nedd4 family of HECT ubiquitin ligases, partially substitutes for the actions of Nedd4-2, albeit with less efficiency (18, 24, 30, 31). Inactivation of mouse Nedd4 gene resulted in perinatal lethality (Cao et al., unpublished data) and therefore precluded us from assessing its role in Na⁺ homeostasis and BP regulation.

Another area of ENaC regulation that has recently gained more recognition is the balance between the ubiquitination (by Nedd4-2) and deubiquitination (by Usp2-45) of ENaC (7, 33). The importance of this regulation is highlighted by the fact that both Sgk1, which regulates Nedd4-2 function directly, and Usp2-45 are rapidly and substantially upregulated by aldosterone in kidney and distal colon (33). The role of these processes in regulating ENaC surface expression, cleavage, endocytosis, and degradation is an area for future investigation.

Could loss of function of Nedd4-2 be a cause of hypertension in some patients? Some evidence suggests that it might. First, ∼50% of hypertensive patients demonstrate salt sensitivity (12), and the majority of these have no readily identifiable phenotype. Like such patients, the Nedd4-2 KO mice have only a minimal phenotype (besides NaCl-sensitive hypertension); notably, they have normal plasma [K⁺] and aldosterone levels. Second, Nedd4-2 KO mice on a HS diet lose their normal diurnal variation in BP, a feature that is becoming increasingly recognized in some patients with hypertension (5, 6). Finally, certain genetic data link Nedd4-2 to hypertension in humans. The human NEDD4-2 gene is located on chromosome 18q21 (4), in a region of linkage with essential hypertension (19, 27). A large population study demonstrated that a NEDD4-2 variant (rs4149601) is associated with increasing BP over time and that it may be associated with defective diurnal BP variation (8). In addition, a naturally occurring missense mutation in exon 15 (P355L) can lead to defective ENaC downregulation (11). Thus genetic NEDD4-2 variants that result in loss of transporter function may contribute significantly to the polygenic background of hypertension.

Given the markedly elevated BP in the Nedd4-2 KO mice fed a HS diet, it is perhaps not surprising that these animals develop cardiac hypertrophy and cardiac systolic dysfunction. It is becoming evident that the cardiac remodeling that takes place under conditions of hypertension is not simply the outcome of the raised hemodynamic load, but involves endocrine and autocrine-paracrine factors, such as the increased plasma aldosterone concentration (26). Since Nedd4-2 functions downstream from the interaction between aldosterone and the mineralocorticoid receptor, it is possible that the cardiac remodeling caused by aldosterone and prevented by blockade of the mineralocorticoid receptor is mediated through Nedd4-2 (23). However, this is not supported by the fact that the absence of Nedd4-2 failed to prevent cardiac hypertrophy and heart failure (Fig. 9). Interestingly, we found that Nedd4-2 KO mice did not have reduced aldosterone levels (Fig. 8) and that on the LS diet, they in fact had higher aldosterone levels than the WT mice. We do not have a clear explanation for this unexpected finding, but suggest that Nedd4-2 may play a role in the normal regulation of aldosterone secretion.

Our findings suggest that Nedd4-2 joins the kinase WNK4 as a gene whose normal function is to downregulate Na⁺ transport and to lower BP (21). Such genes may be important in the pathophysiology of the hypertension that develops in the increasing number of people whose diet contains generous amounts of salt.

GRANTS

This work was supported in part by US National Institutes of Health Grants P50-DK-52617 (B. Yang, P. M. Snyder, and J. B. Stokes), DE-16215 (B. Yang), HL-55006 (C. D. Sigmund), and RR-017369 (R. W. Weiss), and a Merit grant from the Department of Veteran's Affairs (J. B. Stokes).

DISCLOSURES

The authors declare that they have no competing financial interests.