IGF-1 vs insulin: respective roles in modulating sodium transport via the PI-3 kinase/Sgk1 pathway in a cortical collecting duct cell line.

Insulin and insulin-like growth factor 1 (IGF-1) may play a role in the regulation of sodium balance by increasing basal and aldosterone-stimulated transepithelial sodium transport in the aldosterone-sensitive distal nephron (ASDN). As insulin and IGF-1 are capable of binding to each other's receptor with a 50- to 100-fold lower affinity than to their cognate receptor, it is not clear which receptor mediates its respective sodium transport response in the ASDN. The aim of the present study was to characterize the IGF-1 regulation of Na(+) transport in the mCCD(cl1) cell line, a highly differentiated cell line which responds to physiological concentrations (K(1/2)=0.3 nM) of aldosterone. IGF-1 increased basal transepithelial Na(+) transport with a K(1/2) of 0.41+/-0.07 nM. Insulin dose-response curve was displaced to the right 50-fold, as compared to that of IGF-1 (K(1/2)=20.0+/-3.0 nM), indicating that it acts through the IGF type 1 receptor (IGF-1R). Co-stimulation with IGF-1 (0.3 nM) (or 30 nM insulin) and aldosterone (0.3 nM), either simultaneously or by pretreating the cells for 5 h with aldosterone, induced an additive response. The phosphatidylinositol-3' kinase (PI3-K) inhibitor LY294002 completely blocked IGF-1 and aldosterone induced and co-induced currents. As assessed by Western blotting, protein levels of the serum-, and glucocorticoid-induced kinase (Sgk1) were directly and proportionally related to the current induced by either or both IGF-1 and aldosterone, effects also blocked by the PI3-K inhibitor LY294002. IGF-1 could play an important physiological role in regulating basal sodium transport via the PI3-K/Sgk1 pathway in ASDN.

The connecting tubule is the main site of the furosemide-induced urinary acidification by the vacuolar H+-ATPase.

Final urinary acidification is achieved by electrogenic vacuolar H(+)-ATPases expressed in acid-secretory intercalated cells (ICs) in the connecting tubule (CNT) and the cortical (CCD) and initial medullary collecting duct (MCD), respectively. Electrogenic Na(+) reabsorption via epithelial Na(+) channels (ENaCs) in the apical membrane of the segment-specific CNT and collecting duct cells may promote H(+)-ATPases-mediated proton secretion by creating a more lumen-negative voltage. The exact localization where this supposed functional interaction takes place is unknown. We used several mouse models performing renal clearance experiments and assessed the furosemide-induced urinary acidification. Increasing Na(+) delivery to the CNT and CCD by blocking Na(+) reabsorption in the thick ascending limb with furosemide enhanced urinary acidification and net acid excretion. This effect of furosemide was abolished with amiloride or benzamil blocking ENaC action. In mice deficient for the IC-specific B1 subunit of the vacuolar H(+)-ATPase, furosemide led to only a small urinary acidification. In contrast, in mice with a kidney-specific inactivation of the alpha subunit of ENaC in the CCD and MCD, but not in the CNT, furosemide alone and in combination with hydrochlorothiazide induced normal urinary acidification. These results suggest that the B1 vacuolar H(+)-ATPase subunit is necessary for the furosemide-induced acute urinary acidification. Loss of ENaC channels in the CCD and MCD does not affect this acidification. Thus, functional expression of ENaC channels in the CNT is sufficient for furosemide-stimulated urinary acidification and identifies the CNT as a major segment in electrogenic urinary acidification.

Inactivation of sodium-transporting proteins in the kidney.

The kidney plays a dominant role in maintaining sodium homeostasis. The control of a nearly constant electrolyte composition and osmotic pressure in the extracellular fluids is achieved by well-regulated vectorial salt and water transport processes. Derangement in function of Na(+) transporting proteins is likely to be responsible for a number of clinical disorders of fluid and electrolyte homeostasis. The identification of the genes implicated in sodium reabsorption in the kidney not only allows a detailed analysis of regulation and function of these proteins in vitro but also the generation of genetically engineered mice that constitute valuable mouse models for human diseases. Our review will focus on recent strategies for generating nephron segment-specific knock-outs for the main apical renal Na(+) transporters and channels.

Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.

The organization of Na(+) and Ca(2+) transport pathways along the mouse distal nephron is incompletely known. We revealed by immunohistochemistry a set of Ca(2+) and Na(+) transport proteins along the mouse distal convolution. The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) characterized the distal convoluted tubule (DCT). The amiloride-sensitive epithelial Na(+) channel (ENaC) colocalized with NCC in late DCT (DCT2) and extended to the downstream connecting tubule (CNT) and collecting duct (CD). In early DCT (DCT1), the basolateral Ca(2+)-extruding proteins [Na(+)/Ca(2+) exchanger (NCX), plasma membrane Ca(2+)-ATPase (PCMA)] and the cytoplasmic Ca(2+)-binding protein calbindin D(28K) (CB) were found at very low levels, whereas the cytoplasmic Ca(2+)/Mg(2+)-binding protein parvalbumin was highly abundant. NCX, PMCA, and CB prevailed in DCT2 and CNT, where we located the apical epithelial Ca(2+) channel (ECaC1). Its subcellular localization changed from apical in DCT2 to exclusively cytoplasmic at the end of CNT. NCX and PMCA decreased in parallel with the fading of ECaC1 in the apical membrane. All three of them were undetectable in CD. These findings disclose DCT2 and CNT as major sites for transcellular Ca(2+) transport in the mouse distal nephron. Cellular colocalization of Ca(2+) and Na(+) transport pathways suggests their mutual interactions in transport regulation.

Functional analyses of a N-terminal splice variant of the alpha subunit of the epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is the limiting step for sodium absorption in epithelial cells of the distal nephron, distal colon, airways and excretory ducts of several glands. In vivo and in vitro studies showed that the alpha subunit of ENaC is necessary for the expression of functional channels. Using RT-PCR strategy, a novel N-terminal splice variant has been identified which deletes 49 amino acids in the N-terminal region of the mouse alphaENaC subunit. In oocytes expressing the alphaENaC splice variant, together with beta and gammaENaC subunits, amiloride-sensitive currents were less than 20% of values obtained with the wild type ENaC. The single channel conductance and the ionic selectivity were similar and there was only a minor decrease in the level of expression of the protein at the oocyte surface. These findings indicate that the deleted sequence in the N-terminal part of the mouse and rat alphaENaC subunit might play a role in the regulation of the activity of expressed ENaC channels.

Compensatory up-regulation of angiotensin II subtype 1 receptors in alpha ENaC knockout heterozygous mice.

BACKGROUND: In mice, a partial loss of function of the epithelial sodium channel (ENaC), which regulates sodium excretion in the distal nephron, causes pseudohypoaldosteronism, a salt-wasting syndrome. The purpose of the present experiments was to examine how alpha ENaC knockout heterozygous (+/-) mice, which have only one allele of the gene encoding for the alpha subunit of ENaC, control their blood pressure (BP) and sodium balance. METHODS: BP, urinary electrolyte excretion, plasma renin activity, and urinary adosterone were measured in wild-type (+/+) and heterozygous (+/-) mice on a low, regular, or high sodium diet. In addition, the BP response to angiotensin II (Ang II) and to Ang II receptor blockade, and the number and affinity of Ang II subtype 1 (AT1) receptors in renal tissue were analyzed in both mouse strains on the three diets. RESULTS: In comparison with wild-type mice (+/+), alpha ENaC heterozygous mutant mice (+/-) showed an intact capacity to maintain BP and sodium balance when studied on different sodium diets. However, no change in plasma renin activity was found in response to changes in sodium intake in alpha ENaC +/- mice. On a normal salt diet, heterozygous mice had an increased vascular responsiveness to exogenous Ang II (P < 0.01). Moreover, on a normal and low sodium intake, these mice exhibited an increase in the number of AT1 receptors in renal tissues; their BP lowered markedly during the Ang II receptor blockade (P < 0.01) and there was a clear tendency for an increase in urinary aldosterone excretion. CONCLUSIONS: alpha ENaC heterozygous mice have developed an unusual mechanism of compensation leading to an activation of the renin-angiotensin system, that is, the up-regulation of AT1 receptors. This up-regulation may be due to an increase in aldosterone production.

Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK.

Aldosterone controls sodium reabsorption and potassium secretion in the aldosterone-sensitive distal nephron (ASDN). Although clearance measurements have shown that aldosterone induces these transports within 30--60 min, no early effects have been demonstrated in vivo at the level of the apical epithelial sodium channel (ENaC), the main effector of this regulation. Here we show by real-time RT-PCR and immunofluorescence that an aldosterone injection in adrenalectomized rats induces alpha-ENaC subunit expression along the entire ASDN within 2 h, whereas beta- and gamma-ENaC are constitutively expressed. In the proximal ASDN portions only, ENaC is shifted toward the apical cellular pole and the apical plasma membrane within 2 and 4 h, respectively. To address the question of whether the early aldosterone-induced serum and glucocorticoid-regulated kinase (SGK) might mediate this apical shift of ENaC, we analyzed SGK induction in vivo. Two hours after aldosterone, SGK was highly induced in all segment-specific cells of the ASDN, and its level decreased thereafter. In Xenopus laevis oocytes, SGK induced ENaC activation and surface expression by a kinase activity-dependent mechanism. In conclusion, the rapid in vivo accumulation of SGK and alpha-ENaC after aldosterone injection takes place along the entire ASDN, whereas the translocation of alpha,beta,gamma-ENaC to the apical plasma membrane is restricted to its proximal portions. Results from oocyte experiments suggest the hypothesis that a localized activation of SGK may play a role in the mediation of ENaC translocation.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets.

Previous electrophysiological experiments on renal cortical collecting ducts indicated that dietary sodium intake and variations in aldosterone plasma levels regulate the abundance of functional epithelial Na channels (ENaC) in the apical plasma membrane. In mouse kidney we investigated by immunohistochemistry whether feeding for 3 wk a diet with high (3% Na) and low (0.05% Na) Na content influences the distribution pattern of ENaC. In mice of all experimental groups, ENaC was apparent in cells from the late portion of the distal convoluted tubule (DCT2) down to the medullary collecting duct (CD). In mice on a high-Na diet (plasma aldosterone: 40.8 +/- 2.0 ng/dl), the alpha-subunit was undetectable, and the beta- and gamma-ENaC were detected in the cytoplasm, but not in the apical plasma membrane of the cells. In contrast, in mice on a low-Na diet (plasma aldosterone: 93.6 +/- 9.3 ng/dl) all three ENaC subunits were displayed in the subapical cytoplasm and in the apical membrane of DCT2, connecting tubule (CNT), and, although less prominent, in cortical CD cells. Apical plasma membrane immunostaining progressively decreased along the cortical CD, simultaneously with increasing cytoplasmic staining for beta- and gamma-ENaC. Thus our data on mice adapted to moderately low and high Na intake suggest that regulation of ENaC function in vivo involves shifts of beta- and gamma-subunits from the cytoplasm to the apical plasma membrane and vice versa, respectively. The insertion of these subunits into the apical plasma membrane coincides with upregulation of the alpha-subunit and its insertion into the apical plasma membrane.

Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex.

The amiloride-sensitive epithelial sodium channel (ENaC) and the vasopressin-dependent water channel aquaporin-2 (AQP2) mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption, respectively. Distributions of ENaC and AQP2 have been shown by immunohistochemistry in rats. Functional data from rabbits suggest a different distribution pattern of these channels than in rats. We studied, by immunohistochemistry in the rabbit kidney cortex, the distributions of ENaC and AQP2, in conjunction with marker proteins for distal segments. In rabbit cortex ENaC is restricted to the connecting tubule (CNT) cells and cortical collecting duct (CCD) cells. The intracellular distribution of ENaC shifts from the apical membrane in the most upstream CNT cells to a cytoplasmic location further downstream in the CNT and in the CCD cells. AQP2 is detected in the CCD cells exclusively. The anatomic subdivisions in the rabbit distal nephron coincide exactly with distributions of apical transport systems. The differences between rabbits and rats in the distribution patterns of ENaC and AQP2 may explain functional differences in renal salt and water handling between these species.

Salt- and angiotensin II-dependent variations in amiloride-sensitive rectal potential difference in mice.

1. In the rectum and distal nephron, sodium reabsorption is mediated by the amiloride-sensitive epithelial sodium channel (ENaC). The ENaC-mediated sodium transport is electrogenic and creates an amiloride-sensitive transepithelial potential difference (PD). 2. We have evaluated the salt- and angiotensin (Ang)II-dependent variations in amiloride-sensitive rectal PD in mice and assessed their relationship with renal sodium handling. 3. Rectal PD was measured in vivo in mice maintained on a medium-, low- or high-sodium diet. On a medium-salt diet, the mean (+/- SEM) amiloride-sensitive PD was larger in the afternoon than in the morning (-26.1 +/- 0.9 and -11.2 +/- 0.7 mV, respectively; P = 0.001), indicating a circadian cyclicity. Rectal PD increased on a low-sodium diet and decreased on a high-sodium diet. 4. Amiloride-sensitive rectal PD correlated significantly with the urinary Na+/K+ ratio (P < 0.001) and with sodium reabsorption in the distal nephron as measured by the lithium clearance technique (P < 0.001). 5. In mice treated with an AngII AT1 receptor antagonist, amiloride-sensitive rectal PD was increased in the afternoon compared with controls (-32.8 +/- 2.0 vs -24.4 +/- 0.9, respectively; P < 0.001). 6. At high doses, AngII decreased the amiloride-sensitive rectal PD and this effect was blunted by an AT1 receptor antagonist. 7. These results show the presence of a salt-dependent daily cyclicity of sodium transport in the mouse rectum that follows circadian changes in sodium handling in the distal nephron. Angiotensin II appears to modulate this diurnal pattern of rectal amiloride-sensitive sodium transport.

A mouse model for Liddle's syndrome.

Liddle's syndrome (or pseudoaldosteronism) is an autosomal dominant form of salt-sensitive hypertension, due to abnormal sodium transport by the renal tubule. To study the pathophysiology of salt sensitivity, a mouse model for Liddle's syndrome has been generated by Cre/loxP-mediated recombination. Under normal salt diet, mice heterozygous (L/+) and homozygous (L/L) for Liddle mutation (L) develop normally during the first 3 mo of life. In these mice, BP is not different from wild type despite evidence for increased sodium reabsorption in distal colon and low plasma aldosterone, suggesting chronic hypervolemia. Under high salt intake, the Liddle mice develop high BP, metabolic alkalosis, and hypokalemia accompanied by cardiac and renal hypertrophy. This animal model reproduces to a large extent a human form of salt-sensitive hypertension and establishes a causal relationship between dietary salt, a gene expressed in kidney and hypertension.

Identification of a highly conserved sequence at the N-terminus of the epithelial Na+ channel alpha subunit involved in gating.

The epithelial Na+ channel (ENaC) is responsible for Na+ reabsorption in aldosterone target tissues such as distal nephron and colon. ENaC is a heterotetramer composed of three homologous subunits, alpha, beta, and gammaENaC. Mutations leading to loss of function or reduced channel activity have been identified in all three subunits in patients with pseudohypoaldosteronism type-1. One missense mutation substituting a glycine (G95S) which is completely conserved throughout the gene family reduced ENaC open probability, Po. In this study we have performed systematic alanine substitutions of 28 residues of alphaENaC encompassing the glycine (G95). This screen identified a stretch of ten consecutive amino acids (alphaT92-alphaC101) which, when mutated, lead to a decrease in Na+ current (I(Na)) expressed with no significant changes in channel surface expression. This inhibitory effect was strongest for G95 and for two additional highly conserved amino acids--H94 and R98. The R98A mutant led to an important reduction in channel Po with no change in single-channel conductance, indicating that the segment encompassing H94, G95 and R98 is involved in modulation of channel gating kinetics.

Functional expression of a pseudohypoaldosteronism type I mutated epithelial Na+ channel lacking the pore-forming region of its alpha subunit.

The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na+ channel (ENaC). A PHA-I stop mutation (alpha(R508stop)) of the ENaC alpha subunit is predicted to lack the second transmembrane domain and the intracellular COOH-terminus, regions of the protein involved in pore function. Nonetheless, we observed a measurable Na+ current in Xenopus laevis oocytes that coexpress the beta and gamma subunits with the truncated alpha subunit. The mutant alpha was coassembled with beta and gamma subunits and was present at the cell surface at a lower density, consistent with the lower Na+ current seen in oocytes with the truncated alpha subunit. The single-channel Na+ conductance for the mutant channel was only slightly decreased, and the appearance of the macroscopic currents was delayed by 48 hours with respect to wild-type. Our data suggest novel roles for the alpha subunit in the assembly and targeting of an active channel to the cell surface, and suggest that channel pores consisting of only the beta and gamma subunits can provide significant residual activity. This activity may be sufficient to explain the absence of a severe pulmonary phenotype in patients with PHA-I.

Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.

The final control of sodium balance takes place in the cortical collecting duct (CCD) of the nephron, where corticosteroid hormones regulate sodium reabsorption by acting through mineralocorticoid (MR) and/or glucocorticoid (GR) receptors. A clone of principal CCD cells (mpkCCDc14) has been established that is derived from a transgenic mouse (SV40 large T antigen under the control of the SV40 enhancer/L-type pyruvate kinase promoter). Cells grown on filters form polarized monolayers with high electrical transepithelial resistance (R(T) approximately 4700 ohm x cm2) and potential difference (P(D) approximately -50 mV) and have an amiloride-sensitive electrogenic sodium transport, as assessed by the short-circuit current method (Isc approximately 11 microA/cm2). Reverse transcription-PCR experiments using rat MR primers, [3H]aldosterone, and [3H]dexamethasone binding and competition studies indicated that the mpkCCDc14 cells exhibit specific MR and GR. Aldosterone increased Isc in a dose- (10(-10) to 10(-6) M) and time-dependent (2 to 72 h) manner, whereas corticosterone only transiently increased Isc (2 to 6 h). Consistent with the expression of 11beta-hydroxysteroid dehydrogenase type 2, which metabolizes glucocorticoids to inactive 11-dehydroderivates, carbenoxolone potentiated the corticosterone-stimulated Isc. Aldosterone (5x10(-7) M)-induced Isc (fourfold) was associated with a three- to fivefold increase in alpha-ENaC mRNA (but not in those for beta- or gamma-ENaC) and three- to 10-fold increases in alpha-ENaC protein synthesis. In conclusion, this new immortalized mammalian CCD clonal cell line has retained a high level of epithelial differentiation and sodium transport stimulated by aldosterone and therefore represents a useful mammalian cell system for identifying the genes controlled by aldosterone.

Developmental expression of sodium entry pathways in rat nephron.

During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11beta-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins.

Epithelial Na+ channel subunits in rat taste cells: localization and regulation by aldosterone.

Amiloride-sensitive Na+ channels play an important role in transducing Na+ salt taste. Previous studies revealed that in rodent taste cells, the channel shares electrophysiological and pharmacological properties with the epithelial Na+ channel, ENaC. Using subunit-specific antibodies directed against alpha, beta, and gamma subunits of rat ENaC (rENaC), we observed cytoplasmic immunoreactivity for all three subunits in nearly all taste cells of fungiform papillae, and in about half of the taste cells in foliate and vallate papillae. The intensity of labeling in cells of vallate papillae was significantly lower than that of fungiform papillae, especially for beta and gamma subunits. Dual localization experiments showed that immunoreactivity for the taste cell-specific G protein, gustducin, occurs in a subset ofrENaC positive taste cells. Aldosterone is known to increase the amiloride sensitivity of the NaCl taste response. In our study, increases in blood aldosterone levels enhanced the intensity of apical immunoreactivity for beta and gamma rENaC in taste cells of all papillae. In addition, whole cell recordings from isolated taste cells showed that in fungiform papillae, aldosterone increased the number of amiloride-sensitive taste cells and enhanced the current amplitude. In vallate taste cells, which are normally unresponsive to amiloride, aldosterone treatment induced an amiloride sensitive current in about half of the cells. Immunoreactivity for rENaC subunits also was present in nonsensory epithelial cells, especially in the anterior portion of the tongue. In addition, immunoreactivity for all subunits, but especially beta and gamma, was associated with some nerve fibers innervating taste papillae. These extragustatory sites of rENaC expression may indicate a role for this channel in paracellular transduction of sodium ions.

Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta-subunit of the amiloride-sensitive epithelial sodium channel.

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (alpha-, beta-, and gamma-subunits). To study the role of the beta-subunit in vivo, we analyzed mice in which the betaENaC gene locus was disrupted. These mice showed low levels of betaENaC mRNA expression in kidney (approximately 1%), lung (approximately 1%), and colon (approximately 4%). In homozygous mutant betaENaC mice, no betaENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na+ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult betaENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as betaENaC m/m mice showed no weight loss, normal plasma Na+ and K+ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, betaENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that betaENaC is required for Na+ conservation during salt deprivation.