In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na ؉ transport is associated with increased expression of the epithelial Na ؉ channel ␣ subunit (␣ENaC). In H441 lung cells, 100 nM dexamethasone increases amiloride-sensitive shortcircuit current (3.3 A/cm 2 to 7.5 A/cm 2 ), correlating with a 5-fold increase in ␣ENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of ␣ENaC, the human ␣ENaC 5-flanking region was cloned and tested in H441 cells. By deletion analysis, a ϳ150-base pair region 5 to the upstream promoter was identified that, when stimulated with 100 nM dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na ؉ transport, m␣ENaC expression and luciferase expression from ␣ENaC genomic fragments were also increased by 100 nM dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated ␣ENaC gene transcription. We conclude that glucocorticoids stimulate ␣ENaC expression in kidney and lung via activation of a hormone response element in the 5-flanking region of h␣ENaC and this response, in part, is the likely basis for the up-regulation of Na ؉ transport in these sites.
. Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2. Am J Physiol Renal Physiol 295: F462-F470, 2008. First published June 4, 2008 doi:10.1152 doi:10. /ajprenal.90300.2008 has been proposed to play a critical role in regulating epithelial Na ϩ 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 ϩ 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 ϩ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaClsensitive 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. ion channels; kidney; sodium channels 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 ϩ excretion is the activity of the epithelial Na ϩ channel (ENaC), a highly regulated, threesubunit 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 ϩ 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 -and ␥-ENaC subunits leading to Liddle syndrome have focused attention on the specific regions of the COOH termini whose modificati...
H441 cells, a bronchiolar epithelial cell line, develop a glucocorticoid-regulated amiloride-sensitive Na(+) transport pathway on permeable supports (R. Sayegh, S. D. Auerbach, X. Li, R. Loftus, R. Husted, J. B. Stokes, and C. P. Thomas. J Biol Chem 274: 12431-12437, 1999). To understand its molecular basis, we examined the effect of glucocorticoids (GC) on epithelial Na(+) channel (ENaC)-alpha, -beta, and -gamma and sgk1 expression and determined the biophysical properties of Na(+) channels in these cells. GC stimulated the expression of ENac-alpha, -beta, and -gamma and sgk1 mRNA, with the first effect seen by 1 h. These effects were abolished by actinomycin D, but not by cycloheximide, indicating a direct stimulatory effect on ENaC and sgk1 mRNA synthesis. The GC effect on transcription of ENaC-alpha mRNA was accompanied by a significant increase in ENaC-alpha protein levels. GC also stimulated ENaC-alpha, -beta, and -gamma and sgk1 mRNA expression in A549 cells, an alveolar type II cell line. To determine the biophysical properties of the Na(+) channel, single-channel currents were recorded from cell-attached H441 membranes. An Na(+)-selective channel with slow kinetics and a slope conductance of 10.8 pS was noted, properties similar to ENaC-alpha, -beta, and -gamma expressed in Xenopus laevis oocytes. These experiments indicate that amiloride-sensitive Na(+) transport is mediated through classic ENaC channels in human lung epithelia and that GC-regulated Na(+) transport is accompanied by increased transcription of each of the component subunits and sgk1.
The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6(-/-) mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6(-/-) mice that survived were fertile but matings between Trpm6(-/-) mice produced no viable pregnancies. Trpm6(+/-) mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6(+/-) mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.
H441 cells, a bronchiolar epithelial cell line, develop a cAMP-regulated benzamil-sensitive Na+ transport pathway on permeable supports (Itani OA, Auerbach SD, Husted RF, Volk KA, Ageloff S, Knepper MA, Stokes JB, Thomas CP. Am J Physiol Lung Cell Mol Physiol 282: L631-L641, 2002). To understand the molecular basis for the stimulation of Na+ transport, we delineated the role of specific intracellular pathways and examined the effect of cAMP on alphabetagamma-epithelial Na+ channel (ENaC) and sgk1 expression. Na+ transport increases within 5 min of cAMP stimulation and is sustained for >24 h. The sustained effect of cAMP on Na+ transport is abolished by LY-294002, an inhibitor of phosphatidylinositol 3-kinase, by H89, an inhibitor of PKA, or by SB-202190, an inhibitor of p38 MAP kinase. The sustained effect of cAMP was associated with increases in alpha-ENaC mRNA and protein but without a detectable increase in betagamma-ENaC and sgk1. The early effect of cAMP on Na+ transport is brefeldin sensitive and is mediated via PKA. These results are consistent with a model where the early effect of cAMP is to increase trafficking of Na+ channels to the apical cell surface whereas the sustained effect requires the synthesis of alpha-ENaC.
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