The present study demonstrates the major new finding that hypertension induced by a high salt diet in mice with a homozygous knockout of the Nedd4-2 gene is largely prevented by icv infusion of benzamil. The −/− mice develop increased CSF [Na] on high salt diet and show markedly enhanced BP and HR responses to sodium in the CSF, which can be blocked by icv benzamil. Altogether, these findings strongly suggest that the salt-induced hypertension in this mouse model of Liddle's syndrome depends critically on increased expression of ENaC in the brain.
In rat models of salt-induced hypertension, an increase in CSF [Na] occurs on high salt diet and appears to precede the increase in BP10
. In rats, chronic elevations in CSF [Na] by icv infusion of Na-rich aCSF increase BP via activation of the same systems in the brain (including ENaC) that produce the hypertensive response to high salt diet in salt-sensitive strains7,8,16
. Taken together, these findings suggest a role for increased CSF [Na] in the etiology of the salt-induced hypertension. The mechanisms responsible for the increase in CSF [Na] in Dahl S and SHR on high salt have not yet been elucidated14
. In rats, ENaC is present on both the basolateral and apical membranes of choroid plexus (CP) cells6,14
, whereas the Na+
-ATPase is located only on the apical (CSF facing) side. In normotensive rats, apical ENaC expression predominates and icv benzamil increases CSF [Na]6
. In the present study, the −/− mice show increased expression of all 3 ENaC subunits on both the basolateral and apical membranes of CP cells. In −/− mice after 8-10 days on high salt diet both plasma [Na+
and CSF [Na] show clear increases. It appears that in −/− mice on high salt diet the increase in plasma [Na]4
and the overexpression of ENaC on the basolateral membrane of CP cells lead to increased Na entry into the cells and elevated [Na]i
in the CP, which presumably causes apical Na+
-ATPase to transport the excess sodium into the CSF. Increased apical ENaC expression may enhance Na+
re-absorption from the CSF, which may contribute to the normal CSF [Na] in −/− mice on regular salt and may attenuate the increase in CSF [Na] on high salt diet. Further studies are needed to assess whether or not increases in both plasma and CSF [Na] precede the increases in BP on high salt diet.
In rats, ENaC not only plays a role in regulation of CSF [Na], but also mediates the sympathoexcitatory and pressor responses to a chronic increase in CSF [Na]8
. The actual cellular, presumably neuronal, location of these channels still needs to be determined. In the −/− genotype, neuronal ENaC staining is significantly increased but – in contrast to the CP and cortex – only for the β subunit in the SON and PVN and not in the SFO. These findings suggest cell-type specific regulation of ENaC ubiquitination by Nedd 4-2. A 90 minutes icv infusion of Na-rich aCSF provokes a ~3-fold greater pressor response in −/− than W/T mice, whereas icv mannitol does not change BP. The enhancement in the BP response to Na+
-rich aCSF is abolished by icv benzamil, indicating that it is due to enhanced ENaC in the brain. Similar to Dahl S rats, the Nedd4-2 −/− mice show both an increase in CSF [Na] on high salt and hypersensitivity to sodium in the CSF. Whether this hypersensitivity persists during a chronic increase in CSF[Na+
], as is the case in Dahl S rats11
, still needs to be assessed.
The current telemetry data show that on high salt diet BP of the −/− mice starts to increase after 3–4 days, and after 7–10 days shows marked increases by 30–35 mm Hg, similar to the increase previously reported4
. HR increases in parallel, consistent with sympathetic hyperactivity. Central infusion of benzamil prevents most of the increases in BP and HR, indicating that ENaC in the brain is critical for the salt-induced hypertension in this model. Peripheral infusion of benzamil at the icv rate does not affect the initial rise in BP or the further increase in daytime BP. It does attenuate the further rise in night BP, but this reduction is substantially less than achieved by icv benzamil (ie 30% for sc versus 85% for icv). This delayed and partial response to sc benzamil may reflect a slowly developing central blockade. Night (ie active phase) BPs may be more sensitive to such blockade, but peripheral effects cannot be excluded. Although brain ENaC appears to mediate (most of) the salt-induced hypertension, it cannot be deduced from the present data whether the brain effects of high salt in −/− mice are secondary to, enhanced by, or independent of renal sodium retention. In balance studies, no evidence for renal sodium retention was found when −/− mice were placed on high salt diet4
. However, even if present, renal effects per se appear not sufficient to cause the hypertension, i.e., they appear to depend on increased brain ENaC.