Salt-induced hypertension in a mouse model of Liddle syndrome is mediated by epithelial sodium channels in the brain

Abstract

Neural precursor cell expressed and developmentally downregulated 4-2 protein (Nedd4-2) facilitates the endocytosis of epithelial Na channels (ENaCs). Both mice and humans with a loss of regulation of ENaC by Nedd4-2 have salt-induced hypertension. ENaC is also expressed in the brain, where it is critical for hypertension on a high-salt diet in salt-sensitive rats. In the present studies we assessed whether Nedd4-2 knockout (-/-) mice have the following: (1) increased brain ENaC; (2) elevated cerebrospinal fluid (CSF) sodium on a high-salt diet; and (3) enhanced pressor responses to CSF sodium and hypertension on a high-salt diet, both mediated by brain ENaC. Prominent choroid plexus and neuronal ENaC staining was present in -/- but not in wild-type mice. In chronically instrumented mice, ICV infusion of Na-rich artificial CSF increased mean arterial pressure 3-fold higher in -/- than in wild-type mice. ICV infusion of the ENaC blocker benzamil abolished this enhancement. In telemetered -/- mice on a high-salt diet (8% NaCl), CSF [Na(+)], mean arterial pressure, and heart rate increased significantly, mean arterial pressure by 30 to 35 mmHg. These mean arterial pressure and heart rate responses were largely prevented by ICV benzamil but only to a minor extent by SC benzamil at the ICV rate. We conclude that increased ENaC expression in the brain of Nedd4-2 -/- mice mediates their hypertensive response to a high-salt diet by causing increased sodium levels in the CSF, as well as hyperresponsiveness to CSF sodium. These findings highlight the possible causative contribution of central nervous system ENaC in the etiology of salt-induced hypertension.

Figure 1

Immunoreactivity to α, β and γ ENaC subunits in the choroid plexus of Nedd4-2 −/− mice compared to W/T (+/+) controls. Diffuse and mostly cytoplasmic staining is present in the CP cells of W/T (+/+) controls as compared to clear staining on the plasma membrane in the Nedd4-2 −/− mice. Sections were counterstained with hematoxylin. bar: 100 μm

Figure 2

Immunoreactivity to α, β and γ ENaC subunits in pyramidal neurons of the cingulate cortex and to β ENaC in the PVN in Nedd4-2 −/− mice compared to W/T (+/+) controls. In −/− mice distinct immunoreactivity for all 3 subunits is present in the cell-bodies of cortical neurons as well as for β ENaC in axons. The PVN shows diffuse staining for β ENaC in −/− mice, compared to modest staining in the W/T mice. Sections were counterstained with hematoxylin. bar: 100 μm

Figure 3

Enhanced responses of mean arterial pressure (MAP) and heart rate (HR) to icv infusion of Na-rich aCSF (235 mM Na) at 0.4 μl/min in Nedd4-2 −/− mice versus W/T controls (n=5/group). Blood pressures were averaged over 10-min intervals. Values represent the mean ± SEM of the change from baseline. Baseline MAP and HR preceding the start of aCSF were: 114 ± 3 mmHg, 513 ± 35 b/min for W/T vs. 114 ± 2 mmHg, 516 ± 27 b/min for −/− mice. * p < 0.05 vs W/T

Figure 4

Enhanced responses of mean arterial pressure (MAP) and heart rate (HR) to icv infusion of Na-rich aCSF (235 mM Na) in Nedd4-2 −/− mice are prevented by icv benzamil. Groups of W/T or −/− mice (n=5-6) received icv vehicle in aCSF and Na-rich aCSF, while other groups (n=5-6) received the same two solutions containing benzamil (7.4 ng/min). Values represent the mean ± SEM of the change from baseline. Baseline MAP and HR prior to infusion of aCSF were: 124 ± 5 mmHg, 490 ± 39 b/min and 119 ± 7 mmHg, 507 ± 7 b/min for −/ − and W/T mice receiving icv vehicle vs. 127 ± 6 mmHg, 440 ± 20 b/min and 117 ± 6 mmHg, 480 ± 236 b/min for the respective benzamil groups. * p = 0.0015 vs other 3 groups ^a p < 0.05 for treatment effect (vehicle vs benzamil)

Figure 5

Central infusion of the ENaC blocker, benzamil largely prevents the salt-induced hypertension in Nedd 4-2 −/− mice. Panels show the day (7 am - 6 pm) and night (7 pm - 6 am) BP responses to high salt diet in −/− mice receiving either vehicle (n=4) or benzamil icv (n=5) or sc (n=5). Infusions of vehicle or benzamil (2.64 μg/day) started on day -2 and continued until the end of the study on day 10. High salt diet (8% NaCl) was given on days 0-10. Values represent the means ± SEM of the changes from baseline (average from day -1). The daytime baseline mean MAP and HR (average of day -1, in mm Hg and b/min) were: 115 ± 3 and 544 ± 28, for the icv vehicle group; 115 ± 7 and 509 ± 25, for the icv benzamil group and 108 ± 3 and 509 ± 36 for the sc benzamil group. The nighttime baseline mean MAP and HR (night -1 values, in mm Hg and b/min) were: 115 ± 2 and 543 ± 26, for the icv vehicle group; 125 ± 5 and 515 ± 30, for the icv benzamil group and 114 ± 2 and 534 ± 36 for the sc benzamil group. * p < 0.001 vs icv benzamil group; ^a p < 0.05 vs sc benzamil group