Coordinated Control of ENaC and Na⁺,K⁺-ATPase in Renal Collecting Duct

Functional properties of the Na⁺,K⁺-ATPase

The Na⁺,K⁺-ATPase, also referred to as the Na⁺-pump, is a P-type ATPase (also called E1,E2-ATPases) characterized by the transient phosphorylation of an asparagine residue during its catalytic cycle. Na⁺,K⁺-ATPase activity is stimulated by Na⁺ and K⁺ from the cytoplasmic and extracellular side, respectively. Under conditions of low K⁺ concentration (5–10 mM), the apparent Na⁺ mean affinity constant of Na⁺,K⁺-ATPase (K_0.5Na) is between 5 and 15 mM, while V_max is attained at Na⁺ concentrations of between 60 and 100 mM. Because K⁺ and Na⁺ compete at the cytoplasmic site, K_0.5Na is lower in intact cells where K⁺ concentration is high.³ Therefore, in intact cells where Na⁺ concentration is low (10–20 mM), Na⁺,K⁺-ATPase works largely below its V_max.^4,5 Consequently, an acute increase in intracellular Na⁺ stimulates Na⁺,K⁺-ATPase, leading to a rapid return of intracellular Na⁺ concentration to basal levels. Na⁺ activation of the Na⁺,K⁺-ATPase is strongly cooperative and is close to the enzyme's K_0.5Na implying that small variations in intracellular Na⁺ concentration produce large variations in Na⁺,K⁺-ATPase activity. The apparent mean affinity constant of Na⁺,K⁺-ATPase for extracellular K⁺ (K_0.5K) is between 0.5 and 1.5 mM. Therefore, under physiologic conditions intracellular Na⁺, but not extracellular K⁺, is rate limiting for Na⁺,K⁺-ATPase activity. In intact cells where intracellular ATP concentration is well above the K_0.5 of Na⁺,K⁺-ATPase, ATP is not physiologically rate limiting for Na⁺,K⁺-ATPase activity. ATP may become rate limiting when its synthesis is decreased under ischemic or anoxic conditions.²

Structure of the Na⁺,K⁺-ATPase

The Na⁺,K⁺-ATPase is constituted of two subunits (α and β) associated in a 1:1 ratio and facultative accessory small transmembrane proteins of the FXYD family.

The α subunit contains about 1000 amino acids and its apparent molecular mass is close to 110 kDa. Four Na⁺,K⁺-ATPase α subunit isoforms (α-1 to α-4) have been identified^6,7 but kidney tubule epithelial cells express only the ubiquitous α-1 isoform. The α subunit of Na⁺,K⁺-ATPase displays all the functional properties of the enzyme. It is an integral membrane protein with ten membrane-spanning domains (M1 to M10) and both NH₂- and COOH-terminal domains are intracellular.⁸ The docking site for ATP is located in a large intracellular loop between M4 and M5. This loop also contains the aspartic acid transiently phosphorylated during the catalytic cycle.

The β subunit containing about 300 amino acids exists as a single membrane-spanning domain with a large extracellular end that interacts with the extracellular loop of the α subunit located between transmembrane domains M7 and M8.^9,10 The association of the β subunit with the α subunit is necessary for the adequate folding of newly synthesized subunits.¹¹

Seven FXYD proteins have been identified and four (FXYD-1, -2, -4 and -7) can be associated with Na⁺,K⁺-ATPase.¹² In the kidney, FXYD-2 decreases while FXYD-4 increases the apparent Na⁺ affinity of the Na⁺,K⁺-ATPase.¹³ FXYD-2 is mostly expressed in the PT and the thick ascending loop of Henle (TAL) where Na⁺,K⁺-ATPase displays a low apparent Na⁺ affinity,^3,14,15 while FXYD-4 is specifically expressed in the CD where the apparent affinity of Na⁺,K⁺-ATPase is high.^3,16,17

Specificity of the Na⁺,K⁺-ATPase in kidney tubule epithelial cells

Kidney tubule epithelial cells are functionally polarized and express different transporters and receptors along their apical and basolateral membrane domains delineated by tight junctions.¹⁸ Na⁺,K⁺-ATPase is located along the basolateral membrane¹⁹ where it extrudes intracellular Na⁺ to maintain the Na⁺ gradient that would be otherwise dissipated by apical Na⁺ entry.²⁰ There is a close relationship between the expression levels and activity of Na⁺,K⁺-ATPase and the Na⁺ reabsorption capacity of kidney tubule segments.²¹ It is high in TAL and the distal convoluted tubule, intermediate in PT, low in CD, and extremely low in the thin segments of the loop of Henle.²² The distribution profile of Na⁺,K⁺-ATPase was similar as estimated by ³H-ouabain binding²³ or by Western blotting.²⁴

Physiologic properties of the CD

Both intercalated cells and principal cells are found in connecting tubules and CD. Intercalated cells are key players in acid/base and potassium control and will not be discussed further.^25,26 Principal cells perform H₂O and Na⁺ reabsorption as well as K⁺ secretion (Figure 1). Their detailed function and regulation has been recently reviewed.^27–29 These cells mostly express the epithelial sodium channel (ENaC), the renal outer medullary potassium channel (ROMK), as well as calcium-activated K⁺ channels and the water channel aquaporin-2 (AQP2) in their apical membranes. Both apical and basolateral Cl⁻ channels have been described in CD principal cells.^30,31 Na⁺,K⁺-ATPase is located along the basolateral membrane including deep, basal invaginations surrounding the mitochondrial network. Na⁺,K⁺-ATPase along with basolateral K⁺ channels of the Kir family participate in the generation of the negative intracellular potential.³² Active Na⁺ transport generates a lumen-negative transepithelial voltage (0 to −60 mV), which is highest in the cortical collecting duct (CCD) (−10 to −60 mV) and then decreases along the medullary CD. This feature results from decreased Na⁺ reabsorption by principal cells combined with increased electrogenic H⁺ secretion by intercalated cells. Water flows through the apical membrane via AQP2 and leaves the cell through basolateral aquaporin-3 and -4 water channels. It is worth noting that Na⁺ reabsorption by principal cells provides the driving force for K⁺ secretion. Basolateral transport of K⁺ into the cell by Na⁺,K⁺-ATPase supplies apical ROMK for K⁺ secretion that is enhanced by the lumen-negative membrane potential generated by Na⁺ reabsorption.³³

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Figure 1.

Ion and water transporters in CD principal cells. Sodium reabsorption occurs sequentially through apical ENaC and basolateral Na⁺,K⁺-ATPase. This vectorial Na⁺ transport generates a lumen-negative transepithelial potential that drives potassium secretion via luminal K⁺-channels, i.e., ROMK and Big K⁺-channels (BK). The Na⁺,K⁺-ATPase generates the driving forces for both Na⁺ entry and K⁺ exit at the apical side of the cell. Basolateral K⁺ influx through Na⁺,K⁺-ATPase is also equilibrated by K⁺ efflux through basolateral K⁺-channels (Kir) to generate the negative resting transmembrane potential. Water permeability is mediated by sequential flow through apical AQP2 water channels and basolateral aquaporin-3 and -4 water channels.

In CD principal cells Na⁺ reabsorption is tightly controlled by hormonal, neural and local factors as well as chemical (Na⁺ concentration) or physical (luminal flow, membrane potential) factors. These regulatory mechanisms have been recently reviewed.^28,29 In this review we will focus on the coordinated effects of aldosterone and vasopressin on apical ENaC and basolateral Na⁺,K⁺-ATPase. We will also analyze the role of apical Na⁺ influx and/or intracellular Na⁺ concentration that may play a major role in the coordination of transepithelial Na⁺ transport.

Coordinated control of Na⁺ transport by aldosterone

Aldosterone is the principal hormone that stimulates Na⁺ reabsorption by distal segments of the renal tubule. Aldosterone synthesized in the zona glomerulosa of the adrenal gland is secreted mainly in response to circulating angiotensin II generated in response to renin release by the afferent glomerular artery. High extracellular K⁺ is also an important stimulus for aldosterone secretion.³⁴

The response to aldosterone is biphasic with a short-term (after 30 minutes) and a long-term (after 1 day) effect.^35–37 The short-term increase of Na⁺ reabsorption by the CD in response to aldosterone relies on coordinated stimulation of both ENaC and Na⁺,K⁺-ATPase activities. Stimulation of ENaC activity is in part mediated by the early induction of SGK1 that phosphorylates the ubiquitin-ligase Nedd-4.2. Nedd-4.2 phosphorylation prevents its interaction with ENaC, thereby decreasing ENaC subunits ubiquitination, endocytosis and degradation resulting in an increased number of active ENaC at the apical cell surface.^38,39 Early animal studies have shown that Na⁺,K⁺-ATPase activity decreases slowly (days) after adrenalectomy but returns to basal levels 1–3 hours after administration of aldosterone.^40,41 We have shown that in rat CCD as well as in cultured mpkCCD_c14 principal cells aldosterone stimulates Na⁺,K⁺-ATPase activity via increased expression at the cell surface (Figure 2). This effect is independent of de novo Na⁺,K⁺-ATPase synthesis² and may rely in part on SGK1.⁴² The physiologic role of SGK1 kidney Na⁺ handling has been confirmed in both constitutive and kidney tubule-specific inducible Sgk1-knockout mice.^43,44 These mice display increased levels of urinary Na⁺ excretion and plasma aldosterone in response to a low Na⁺ diet as compared with wild-type mice. After prolonged stimulation (hours), aldosterone also increases the biosynthesis of α-ENaC as well as α-and β-subunits of Na⁺,K⁺-ATPase in a coordinated manner.⁴⁵ Stimulation of α-ENaC transcription by aldosterone involves both direct binding of mineralocorticoid receptors to responsive elements and Sgk1-dependent disruption of Dot1a-Af9 and Dot1a-Sirt1 complexes and/or inhibition of their effects on chromatin.⁴⁶

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Figure 2.

Control of sodium transport by mineralocorticoids in CD principal cells. The steroid hormone aldosterone binds to intracellular mineralocorticoid receptor (MR) that translocates into the nucleus where it activates a transcriptional program inducing expression of modulators of sodium transport such as SGK1. SGK1 phosphorylates and inhibits Nedd-4.2, thereby increasing apical ENaC density via decreased ubiquitination, internalization and degradation of ENaC. SGK1 also increases expression of Na⁺,K⁺-ATPase and ENaC subunits. Sustained aldosterone stimulation increases the transcription of αENaC and Na,K-ATPase.

Coordinated control of Na⁺ transport by vasopressin

In CD principal cells, arginine vasopressin (AVP) binds to basolateral V2 receptors coupled to the cAMP/PKA pathway.²¹ Stimulation of water reabsorption is the major effect of AVP in the CD.⁴⁷ However, AVP also stimulates Na⁺ reabsorption, albeit to a lesser extent. This effect has been verified by in vitro microperfusion studies in rat CCD, where vasopressin directly stimulates Na⁺ reabsorption,⁴⁸ and by the use of V2 receptor antagonists in humans.⁴⁹ An aldosterone-independent control of ENaC expression has been identified in adrenalectomized mice and in cultured CD principal cells.^50,51 This AVP-dependent ENaC expression is more likely aimed at enhancing water reabsorption than maintaining Na⁺ balance. Both AVP and cAMP stimulate Na⁺ reabsorption by coordinated activation of ENaC and Na⁺,K⁺-ATPase (Figure 3). The stimulatory effect of cAMP on ENaC was demonstrated by patch-clamping experiments performed on the apical membrane of principal cells from isolated rat CDD.⁵² In the same preparation as well as in cultured mpkCCD_c14 principal cells, cAMP rapidly stimulates Na⁺,K⁺-ATPase activity and cell surface expression^53,54 independently of phosphorylation of its α−subunit.⁵⁵

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Figure 3.

Control of sodium and water transport by vasopressin in CD principal cells. The peptide hormone AVP binds to basolateral V2 receptors coupled to the activation of adenylyl cyclase via protein Gαs (Gαs). Generated cAMP binds to PKA regulatory subunits leading to release and activation of PKA catalytic subunit (PKAc) that phosphorylates a subset of substrates. PKA activation induces both post-transcriptional effects such as activation of ENaC and Na⁺,K⁺-ATPase as well as translocation of AQP2 water channels, and transcriptional effects such as increased AQP2 and ENaC subunits mRNA expression.

Control of Na⁺,K⁺-ATPase by apical Na⁺ entry and/or intracellular concentration

In the CD Na⁺,K⁺-ATPase displays an apparent affinity for Na⁺ that is two-fold higher than in the PT and TAL.^14,16 Consequently, the so called “kinetic reserve” for Na⁺,K⁺-ATPase activation by increased intracellular Na⁺ concentration is much lower in the CD than in the more proximal nephron segments. CD luminal Na⁺ concentration is rather low but it is subjected to wide variations according to dietary Na⁺ intake and fluctuations in GFR. Therefore, CD cells must face large variations in apical Na⁺ entry according to requirements of body fluid homeostasis while maintaining intracellular Na⁺ concentration within a narrow range despite a lower kinetic reserve of the Na⁺,K⁺-ATPase. Indeed, tight control of intracellular Na⁺ concentration is required for many cellular functions including ENaC activity. In the absence of simultaneous stimulation of ENaC and Na⁺,K⁺-ATPase, any increase in Na⁺ entry would raise intracellular Na⁺ concentration and subsequently decrease ENaC activity by feedback inhibition.⁵⁶ Coordinate control of apical and basolateral fluxes would preserve ENaC permeability over a wide range of luminal Na⁺ concentrations.

In the early nineties two independent research groups reported that increasing intracellular Na⁺ concentration in mammalian CCD rapidly and proportionally increased Na⁺,K⁺-ATPase activity and number of functional Na⁺,K⁺-ATPase units.^20,57 This effect was observed after dissipation of the transmembrane Na⁺ gradient by either a Na⁺ ionophore that generates ion-permeable pores across the plasma membrane or by incubating cells in the absence of K⁺ which inhibits Na⁺,K⁺-ATPase activity. The Na⁺-induced increase in Na⁺,K⁺-ATPase activity was independent of de novo protein synthesis. We subsequently showed that increasing intracellular Na⁺ concentration with a Na⁺ ionophore increased both Na⁺,K⁺-ATPase activity and cell surface expression in isolated rat CCDs and in cultured mpkCCD_c14 principal cells.⁵⁸ These results suggest that intracellular Na⁺ concentration may control the number of active Na⁺,K⁺-ATPase units expressed at the cell surface of principal cells and thereby the rate of basolateral Na⁺ extrusion (Figure 4). These early findings suggested that either Na⁺,K⁺-ATPase units stored in an intracellular compartment are recruited to the cell surface or that internalization of resident plasma membrane Na⁺,K⁺-ATPase units is reduced. Activation of silent preexisting Na⁺,K⁺-ATPase units at the plasma membrane is unlikely since variations in intracellular Na⁺ concentration are associated with a parallel increase in Na⁺,K⁺-ATPase protein abundance at the cell surface.⁵⁸

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Figure 4.

Control of sodium transport by intracellular sodium in CD principal cells. The proinflammatory NF-κB pathway can be classically activated by bacterial products such as LPS via activation of Toll-like receptor 4 (TLR4) located in the apical membrane, by cytokines such as TNFα that binds to basolateral receptors, or by aldosterone via mineralocorticoid receptors. Activation of NF-κB through the classic pathway requires activation of IKKβ that phosphorylates IκBα that indices its dissociation from p65 NF-κB and its degradation by the proteasome. After dissociation from IκBα, NF-κB complexes are translocated to the nucleus where they modulate transcription of target genes including proinflammatory cytokines. Increased intracellular Na⁺ concentration induces dissociation of a protein complex containing IκBα, p65 NF-κB subunit and PKAc. This cascade leads to PKA activation and increased cell surface expression and activity of Na⁺,K⁺-ATPase.

We further analyzed the coupling between apical Na⁺ entry through ENaC and basolateral exit via Na⁺,K⁺-ATPase in CD principal cells.⁵⁹ In vivo experiments revealed that in the presence of a saturating concentration of aldosterone that is expected to maximally stimulate ENaC cell surface expression, increasing luminal Na⁺ availability induces an increase in Na⁺,K⁺-ATPase protein abundance and maximal hydrolytic activity in isolated rat CCD (Figure 5). Rigorous analysis of CD sections from normal and adrenalectomized rats by immuno-electron microscopy revealed an extensive gold labeling of the basolateral membrane, including basal infoldings. We did not detect any pool of intracellular vesicles displaying Na⁺,K⁺-ATPase antibody labeling although some intracellular organelles resembling multivesicular-bodies were significantly labeled with anti-Na⁺,K⁺-ATPase antibodies. This morphologic analysis indicated that the presence of an intracellular Na⁺,K⁺-ATPase storage compartment, such as exocytotic vesicles, is very unlikely. However, these labeled multivesicular bodies suggested that plasma membrane Na⁺,K⁺-ATPase is constitutively internalized and then degraded.

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Figure 5.

Na⁺,K⁺-ATPase expression and activity varies in parallel with apical Na⁺ delivery in rat CCD. Rats were subcutaneously implanted with an osmotic minipump delivering aldosterone for 7 days. After 3 days of low sodium diet, rats were separated into two groups and given either low sodium diet (L) or normal sodium diet (N) for 4 additional days. Rats were then euthanized and CCD were isolated by microdissection from collagenase-treated kidneys. Na⁺,K⁺-ATPase α-subunit expression was assessed by Western blot on pools of 50 CCDs and Na⁺,K⁺-ATPase hydrolytic activity was determined by a radiochemical assay in isolated CCD. Results show that increased Na⁺ entry following reintroduction of sodium in the diet is associated with increased Na⁺,K⁺-ATPase abundance and activity in CCD.

We tested the hypothesis that variations of apical Na⁺ entry via ENaC induce parallel changes in Na⁺,K⁺-ATPase basolateral membrane expression independent of a hormonal influence (Figure 6). In cultured mCCD_cl1 principal cells overexpressing γ−ENaC in a doxycyclin-inducible manner grown on semipermeable filters, 48-hour doxycyclin treatment increased the amiloride-sensitive transepithelial current, i.e., a measurement of ENaC-dependent Na⁺ transport, by about 2-fold. These results confirmed that γ-ENaC is rate limiting for the activity of this channel in CD principal cells.^60,61 The increase in ENaC mediated apical Na⁺ entry was associated with a parallel increase in total and cell-surface expression of basolateral Na⁺,K⁺-ATPase.⁵⁹ These results were confirmed using mpkCCD_cl4 cells, where a decrease of luminal Na⁺ availability at constant osmolality for 24 hours decreased both total and cell surface Na⁺,K⁺-ATPase expression levels.⁵⁹ In addition, overexpression of a constitutively active mutant β-ENaC in mpkCCD_cl4 cells⁶² increased both total and cell surface Na⁺,K⁺-ATPase expression compared with cells overexpressing wild-type β-ENaC. We analyzed whether increased Na⁺,K⁺-ATPase abundance in response to a sustained increase in ENaC activity relies on increased protein biosynthesis or decreased degradation. Analysis of Na⁺,K⁺-ATPase subunit mRNA did not reveal significant differences between cells overexpressing the γ−ENaC or not. Moreover, biosynthesis of the Na,K-ATPase α subunit, assessed by pulse labeling newly synthesized protein, remained unchanged (V. Leroy and E. Feraille, personal communication). In contrast, pulse-chase labeling of cell surface protein, used as a means to assess Na⁺,K⁺-ATPase internalization, and inhibition of lysosomal protein degradation revealed that increased apical Na⁺ entry via ENaC inhibits constitutive Na⁺,K⁺-ATPase endocytosis and its subsequent lysosomal degradation. This effect was specific since neither internalization rate nor degradation of E-cadherin, another basolateral transmembrane protein, was altered. These results indicate that Na⁺,K⁺-ATPase can be regulated through modulation of its degradation in ion transporting epithelia.

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Figure 6.

Variation of apical Na⁺ entry via ENaC induces parallel changes in Na⁺,K⁺-ATPase expression in cultured CD cells. Total and amiloride-sensitive (amilo) short-circuit currents were measured on cells treated (spotted boxes) or not (plain boxes) with 10⁻⁶ M amiloride for 15 minutes, whereas Na⁺,K⁺-ATPase α-subunit (NKA-α) was detected by Western blot performed on total cellular extracts and after pull-down of biotinylated cell-surface proteins by streptavidin-agarose beads. GAPDH, a cytosolic protein, was used as a loading control for total cellular extract and its absence was taken as a control of quality for pull-down of cell-surface proteins. (A) mpkCCD_cl14 cells grown to confluence on polycarbonate filters were incubated for 24 hours in presence of 150 or 30 mM apical Na⁺ concentration before measurement of transepithelial current and labeling of cell-surface proteins. (B) mpkCCD_cl14 cells overexpressing a constitutively active mutant βENaC (βGY) or wild-type βENaC (βG525) were grown to confluence on polycarbonate filters before measurement of transepithelial current and labeling of cell-surface proteins. (C) γCCD-TetOn cells, conditionally overexpressing a wild type ENaC γ subunit in response to 5 days of doxycycline treatment, were grown to confluence on polycarbonate filters before measurement of transepithelial current and labeling of cell-surface proteins. Results show that under conditions of increased transcellular Na⁺ transport assessed by increased total and amiloride-sensitive currents, total and cell-surface expression of Na,K-ATPase were increased.

Experimental evidence supports a role for cell swelling in the intracellular Na⁺ concentration-dependent modulation of Na⁺,K⁺-ATPase cell surface expression. Early experiments performed in isolated mouse CCDs showed that the increased number of specific ³H-ouabain binding sites in response to increased intracellular Na⁺ concentration was dependent on cell swelling.⁶³ In cultured CD cells (mpkCCD_cl4 cells) we showed that extracellular hypotonicity increases both cell volume and Na⁺,K⁺-ATPase cell surface expression.⁶⁴ In contrast, cell shrinkage in response to extracellular hypertonicity did not alter cell surface expression of Na⁺,K⁺-ATPase. The observed effect of hypotonicity was not mediated by a cell volume-dependent activation of ENaC, but relies on proteasomal-dependent PKA activation reminiscent of the effect of increased intracellular Na⁺.⁶⁵ Along the same lines, cell swelling in response to increased apical sodium entry is likely a key initiating event in tubuloglomerular feedback signaling by macula densa cells.⁶⁶

Early experiments have shown that basal levels of aldosterone are necessary to observe an increase in active Na⁺,K⁺-ATPase units in response to increased intracellular Na⁺ concentration in mammalian CD.^20,57 This was interpreted as a requirement for aldosterone to synthesize an intracellular pool of Na⁺,K⁺-ATPase units that can be recruited to the plasma membrane. In the light of our recent results, this observation rather suggests that aldosterone and intracellular Na⁺ concentration both control the rate of Na⁺,K⁺-ATPase degradation. Activation of the PKA pathway may play a major role in controlling Na⁺,K⁺-ATPase expression at the cell surface. Increased Na⁺,K⁺-ATPase cell surface expression in response to AVP, cAMP and elevated intracellular Na⁺ concentration is dependent on PKA activity.^54,58 This effect relies at least in part on cAMP-independent PKA activation following dissociation of a PKAc/IκBα/p65 complex leading on the one hand to PKA activation and on the other hand to NF-κB activation following proteasomal degradation of IκBα (Figure 4).^58,65 Activation of the NF-κB pathway inhibits transepithelial Na⁺ transport by CD principal cells via decreased expression of α−ENaC in response to SGK1 transcriptional inhibition⁶⁷; aldosterone activates NF-κB via the mineralocorticoid receptor both in vivo and in cultured CD principal cells.⁶⁸ Therefore, activation of the NF-κB pathway may represent a negative feedback mechanism that prevents overstimulation of transepithelial Na⁺ transport by CD principal cells challenged with aldosterone and/or increased apical Na⁺ entry.

We have documented a key role of p38 kinase, a member of the MAP kinase family, in the inhibition of Na⁺,K⁺-ATPase endocytosis and degradation in response to an increase in Na⁺ entry via ENaC.⁵⁹ Overexpression of γ-ENaC is associated with inhibition of p38 kinase activity, as assessed by its decreased phosphorylation. Aldosterone, which also increases Na⁺,K⁺-ATPase cell surface expression, similarly inhibits p38 kinase activity.⁶⁸ The key role of p38 kinase was confirmed by its pharmacological inhibition or activation that increased or inhibited internalization of cell surface Na⁺,K⁺-ATPase.⁵⁹ In contrast with results obtained in MDCK cells, that suggested that the major metabolic sensor AMPK may control Na⁺,K⁺-ATPase internalization,⁶⁹ we were not able to demonstrate any effect of AMPK in cultured CD principal cells.⁵⁹ In addition, salt-induced protein kinase that was shown to modulate Na⁺,K⁺-ATPase in PT cells did not alter Na⁺,K⁺-ATPase trafficking in CD principal cells (E. Féraille, personal communication).

In conclusion, increased apical Na⁺ influx through ENaC induces a coordinated increase in basolateral Na,K-ATPase expression and activity. Signaling through PKA and p38 kinase plays a key role in this process that may rely on the modulation of a Na⁺ sensor that remains to be identified.