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.

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.

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.

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.

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.

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.

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.

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.

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.