High dietary K+ intake inhibits proximal tubule transport.

The impact of chronic dietary K+ loading on proximal tubule (PT) function was measured using free-flow micropuncture along with measurements of overall kidney function, including urine volume, glomerular filtration rate, and absolute and fractional Na+ and K+ excretion in the rat. Feeding animals a diet with 5% KCl [high K+ (HK)] for 7 days reduced glomerular filtration rate by 29%, increased urine volume by 77%, and increased absolute K+ excretion by 202% compared with rats on a 1% KCl [control K+ (CK)] diet. HK did not change absolute Na+ excretion but significantly increased fraction excretion of Na+ (1.40% vs. 0.64%), indicating that fractional Na+ absorption is reduced by HK. PT reabsorption was assessed using free-flow micropuncture in anesthetized animals. At 80% of the accessible length of the PT, measurements of inulin concentration indicated volume reabsorption of 73% and 54% in CK and HK, respectively. At the same site, fractional PT Na+ reabsorption was 66% in CK animals and 37% in HK animals. Fractional PT K+ reabsorption was 66% in CK and 37% in HK. To assess the role of Na+/H+ exchanger isoform 3 (NHE3) in mediating these changes, we measured NHE3 protein expression in total kidney microsomes as well as surface membranes using Western blots. We found no significant changes in protein in either cell fraction. Expression of the Ser552 phosphorylated form of NHE3 was also similar in CK and HK animals. Reduction in PT transport may facilitate K+ excretion and help balance Na+ excretion by shifting Na+ reabsorption from K+-reabsorbing to K+-secreting nephron segments.NEW & NOTEWORTHY In rats fed a diet rich in K+, proximal tubules reabsorbed less fluid, Na+, and K+ compared with those in animals on a control diet. Glomerular filtration rates also decreased, probably due to glomerulotubular feedback. These reductions may help to maintain balance of the two ions simultaneously by shifting Na+ reabsorption to K+-secreting nephron segments.

Expression of ENaC subunits in epithelia.

The epithelial Na+ channel (ENaC) is a heterotrimeric protein whose assembly, trafficking, and function are highly regulated. To better understand the biogenesis and activation of the channel, we quantified the expression of individual subunits of ENaC in rat kidneys and colon using calibrated Western blots. The estimated abundance for the three subunits differed by an order of magnitude with the order γENaC ∼ ßENaC ≫ αENaC in both organs. Transcript abundance in the kidney, measured with digital-drop PCR and RNAseq, was similar for the three subunits. In both organs, the calculated protein expression of all subunits was much larger than that required to account for maximal Na+ currents measured in these cells, implying a large excess of subunit protein. Whole-kidney biotinylation indicated that at least 5% of ß and γ subunits in the kidney and 3% in the colon were expressed on the surface under conditions of salt restriction, which maximizes ENaC-dependent Na+ transport. This indicates a 10- to 100-fold excess of ßENaC and γENaC subunits at the surface relative to the requirement for channel activity. We conclude that these epithelia make much more ENaC protein than is required for the physiological function of the channel. This could facilitate rapid regulation of the channels at the cell surface by insuring a large population of inactive, recruitable subunits.

ENaC and ROMK channels in the connecting tubule regulate renal K+ secretion.

We measured the activities of epithelial Na channels (ENaC) and ROMK channels in the distal nephron of the mouse kidney and assessed their role in the process of K+ secretion under different physiological conditions. Under basal dietary conditions (0.5% K), ENaC activity, measured as amiloride-sensitive currents, was high in cells at the distal end of the distal convoluted tubule (DCT) and proximal end of the connecting tubule (CNT), a region we call the early CNT (CNTe). In more distal parts of the CNT (aldosterone-sensitive portion [CNTas]), these currents were minimal. This functional difference correlated with alterations in the intracellular location of ENaC, which was at or near the apical membrane in CNTe and more cytoplasmic in the CNTas. ROMK activity, measured as TPNQ-sensitive currents, was substantial in both segments. A mathematical model of the rat nephron suggested that K+ secretion by the CNTe predicted from these currents provides much of the urinary K+ required for K balance on this diet. In animals fed a K-deficient diet (0.1% K), both ENaC and ROMK currents in the CNTe decreased by ∼50%, predicting a 50% decline in K+ secretion. Enhanced reabsorption by a separate mechanism is required to avoid excessive urinary K+ losses. In animals fed a diet supplemented with 3% K, ENaC currents increased modestly in the CNTe but strongly in the CNTas, while ROMK currents tripled in both segments. The enhanced secretion of K+ by the CNTe and the recruitment of secretion by the CNTas account for the additional transport required for K balance. Therefore, adaptation to increased K+ intake involves the extension of robust K+ secretion to more distal parts of the nephron.

Cleavage state of γENaC in mouse and rat kidneys.

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the γ subunit. Here, we investigated the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in Fisher rat thyroid cells by coexpressing the apical membrane-bound protease channel-activating protease 1 (CAP1; prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney, we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest COOH-terminal fragment of γENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in Fisher rat thyroid cells. Similar cleaved peptides were observed in total and cell surface fractions of the rat kidney. This outcome suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using nonreducing gels, in which the NH2- and COOH-terminal fragments of γENaC are linked by a disulfide bond. Under these conditions, the major cleaved form in the rat kidney had an apparent molecular mass of 56 kDa, ∼4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant γENaC species in the apical membrane of rat and mouse kidneys on a low-Na diet is the twice-cleaved, presumably activated form.NEW & NOTEWORTHY We have identified the major aldosterone-dependent cleaved form of the epithelial Na channel (ENaC) γ subunit in the kidney as a twice-cleaved peptide. This form appears to be identical in size with a subunit cleaved in vitro by the extracellular protease channel-activating protease 1 (prostasin). In the absence of reducing agents, it has an overall molecular mass less than that of the intact subunit, consistent with the excision of an inhibitory domain.

Aldosterone-dependent and -independent regulation of Na+ and K+ excretion and ENaC in mouse kidneys.

We investigated the regulation of Na+ and K+ excretion and the epithelial Na+ channel (ENaC) in mice lacking the gene for aldosterone synthase (AS) using clearance methods to assess excretion and electrophysiology and Western blot analysis to test for ENaC activity and processing. After 1 day of dietary Na+ restriction, AS-/- mice lost more Na+ in the urine than AS+/+ mice did. After 1 wk on this diet, both genotypes strongly reduced urinary Na+ excretion, but creatinine clearance decreased only in AS-/- mice. Only AS+/+ animals exhibited increased ENaC function, assessed as amiloride-sensitive whole cell currents in collecting ducts or cleavage of αENaC and γENaC in Western blots. To assess the role of aldosterone in the excretion of a K+ load, animals were fasted overnight and refed with high-K+ or low-K+ diets for 5 h. Both AS+/+ and AS-/- mice excreted a large amount of K+ during this period. In both phenotypes the excretion was benzamil sensitive, indicating increased K+ secretion coupled to ENaC-dependent Na+ reabsorption. However, the increase in plasma K+ under these conditions was much larger in AS-/- animals than in AS+/+ animals. In both groups, cleavage of αENaC and γENaC increased. However, Na+ current measured ex vivo in connecting tubules was enhanced only in AS+/+ mice. We conclude that in the absence of aldosterone, mice can conserve Na+ without ENaC activation but at the expense of diminished glomerular filtration rate. Excretion of a K+ load can be accomplished through aldosterone-independent upregulation of ENaC, but aldosterone is required to excrete the excess K+ without hyperkalemia.

Ubiquitination of renal ENaC subunits in vivo.

Ubiquitination of the epithelial Na+ channel (ENaC) in epithelial cells may influence trafficking and hormonal regulation of the channels. We assessed ENaC ubiquitination (ub-ENaC) in mouse and rat kidneys using affinity beads to capture ubiquitinated proteins from tissue homogenates and Western blot analysis with anti-ENaC antibodies. Ub-αENaC was observed primarily as a series of proteins of apparent molecular mass of 40-70 kDa, consistent with the addition of variable numbers of ubiquitin molecules primarily to the NH2-terminal cleaved fragment (~30 kDa) of the subunit. No significant Ub-ßENaC was detected, indicating that ubiquitination of this subunit is minimal. For γENaC, the protein eluted from the affinity beads had the same apparent molecular mass as the cleaved COOH-terminal fragment of the subunit (~65 kDa). This suggests that the ubiquitinated NH2 terminus remains attached to the COOH-terminal moiety during isolation through disulfide bonds. Consistent with this, under nonreducing conditions, eluates contained material with increased molecular mass (90-150 kDa). In mice with a Liddle syndrome mutation (ß566X) deleting a putative binding site for the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-2, the amount of ub-γENaC was reduced as expected. To assess aldosterone dependence of ubiquitination, we fed rats either control or low-Na+ diets for 7 days before kidney harvest. Na+ depletion increased the amounts of ub-αENaC and ub-γENaC by three- to fivefold, probably reflecting increased amounts of fully cleaved ENaC. We conclude that ubiquitination occurs after complete proteolytic processing of the subunits, contributing to retrieval and/or disposal of channels expressed at the cell surface. Diminished ubiquitination does not appear to be a major factor in aldosterone-dependent ENaC upregulation.

Na restriction activates epithelial Na channels in rat kidney through two mechanisms and decreases distal Na+ delivery.

KEY POINTS: Dietary Na restriction, through the mineralocorticoid aldosterone, acts on epithelial Na channels via both fast (24 h) and slow (5-7 days) mechanisms in the kidney. The fast effect entails increased proteolytic processing and trafficking of channel protein to the apical membrane. It is rapidly reversible by the mineralocorticoid receptor antagonist eplerenone and is largely lost when tubules are studied ex vivo. The slow effect does not require increased processing or surface expression, is refractory to acute eplerenone treatment, and is preserved ex vivo. Both slow and fast effects contribute to Na retention in vivo. Increased Na+ reabsorption in the proximal tubule also promotes Na conservation under conditions of chronic dietary Na restriction, reducing Na+ delivery to the distal nephron. ABSTRACT: Changes in the activity of the epithelial Na channel (ENaC) help to conserve extracellular fluid volume. In rats fed a low-salt diet, proteolytic processing of ENaC increased within 1 day, and was almost maximal after 3 days. The rapid increase in the abundance of cleaved αENaC and γENaC correlated with decreased urinary Na+ excretion and with increased ENaC surface expression. By contrast, ENaC activity, measured ex vivo in isolated cortical collecting ducts, increased modestly after 3 days and required 5 days to reach maximal levels. The mineralocorticoid receptor antagonist eplerenone reversed the increase in cleaved γENaC and induced natriuresis after 1 or 3 days but failed to alter either ENaC currents or Na+ excretion after 7 days of Na restriction. We conclude that Na depletion, through aldosterone, stimulates ENaC via independent fast and slow mechanisms. In vivo, amiloride-induced natriuresis increased after 1 day of Na depletion. By contrast, hydrochlorothiazide (HCTZ)-induced natriuresis decreased gradually over 7 days, consistent with increased ability of ENaC activity to compensate for decreased Na+ reabsorption in the distal convoluted tubule. Administration of amiloride and HCTZ together increased Na+ excretion less in Na-depleted compared to control animals, indicating decreased delivery of Na+ to the distal nephron when dietary Na is restricted. Measurements of creatinine and Li+ clearances indicated that increased Na reabsorption by the proximal tubules is responsible for the decreased delivery. Thus, Na conservation during chronic dietary salt restriction entails enhanced transport by both proximal and distal nephron segments.

Responses of distal nephron Na+ transporters to acute volume depletion and hyperkalemia.

We assessed effects of acute volume reductions induced by administration of diuretics in rats. Direct block of Na+ transport produced changes in urinary electrolyte excretion. Adaptations to these effects appeared as alterations in the expression of protein for the distal nephron Na+ transporters NCC and ENaC. Two hours after a single injection of furosemide (6 mg/kg) or hydrochlorothiazide (HCTZ; 30 mg/kg) Na+ and K+ excretion increased but no changes in the content of activated forms of NCC (phosphorylated on residue T53) or ENaC (cleaved γ-subunit) were detected. In contrast, amiloride (0.6 mg/kg) evoked a similar natriuresis that coincided with decreased pT53NCC and increased cleaved γENaC. Alterations in posttranslational membrane protein processing correlated with an increase in plasma K+ of 0.6-0.8 mM. Decreased pT53NCC occurred within 1 h after amiloride injection, whereas changes in γENaC were slower and were blocked by the mineralocorticoid receptor antagonist spironolactone. Increased γENaC cleavage correlated with elevation of the surface expression of the subunit as assessed by in situ biotinylation. Na depletion induced by 2 h of furosemide or HCTZ treatment increases total NCC expression without affecting ENaC protein. However, restriction of Na intake for 10 h (during the day) or 18 h (overnight) increased the abundance of both total NCC and of cleaved α- and γENaC. We conclude that the kidneys respond acutely to hyperkalemic challenges by decreasing the activity of NCC while increasing that of ENaC. They respond to hypovolemia more slowly, increasing Na+ reabsorptive capacities of both of these transporters.

SGK1-dependent ENaC processing and trafficking in mice with high dietary K intake and elevated aldosterone.

We examined renal Na and K transporters in mice with deletions in the gene encoding the aldosterone-induced protein SGK1. The knockout mice were hyperkalemic, and had altered expression of the subunits of the epithelial Na channel (ENaC). The kidneys showed decreased expression of the cleaved forms of the γENaC subunit, and the fully glycosylated form of the ßENaC subunits when animals were fed a high-K diet. Knockout animals treated with exogenous aldosterone also had reduced subunit processing and diminished surface expression of ßENaC and γENaC. Expression of the three upstream Na transporters NHE3, NKCC2, and NCC was reduced in both wild-type and knockout mice in response to K loading. The activity of ENaC measured as whole cell amiloride-sensitive current (INa) in principal cells of the cortical collecting duct (CCD) was minimal under control conditions but was increased by a high-K diet to a similar extent in knockout and wild-type animals. INa in the connecting tubule also increased similarly in the two genotypes in response to exogenous aldosterone administration. The activities of both ROMK channels in principal cells and BK channels in intercalated cells of the CCD were unaffected by the deletion of SGK1. Acute treatment of animals with amiloride produced similar increases in Na excretion and decreases in K excretion in the two genotypes. The absence of changes in ENaC activity suggests compensation for decreased surface expression. Altered K balance in animals lacking SGK1 may reflect defects in ENaC-independent K excretion.

Regulation of ENaC trafficking in rat kidney.

The epithelial Na channel (ENaC) forms a pathway for Na(+) reabsorption in the distal nephron, and regulation of these channels is essential for salt homeostasis. In the rat kidney, ENaC subunits reached the plasma membrane in both immature and fully processed forms, the latter defined by either endoglycosidase H-insensitive glycosylation or proteolytic cleavage. Animals adapted to a low-salt diet have increased ENaC surface expression that is specific for the mature forms of the subunit proteins and is similar (three- to fourfold) for α, ß, and γENaC. Kidney membranes were fractionated using differential centrifugation, sucrose-gradient separation, and immunoabsorption. Endoplasmic reticulum membranes, isolated using an antibody against calnexin, expressed immature γENaC, and the content decreased with Na depletion. Golgi membranes, isolated with an antibody against the cis-Golgi protein GM130, expressed both immature and processed γENaC; Na depletion increased the content of processed γENaC in this fraction by 3.8-fold. An endosomal compartment isolated using an antibody against Rab11 contained both immature and processed γENaC; the content of processed subunit increased 2.4-fold with Na depletion. Finally, we assessed the content of γENaC in the late endocytic compartments indirectly using urinary exosomes. All of the γENaC in these exosomes was in the fully cleaved form, and its content increased by 4.5-fold with Na depletion. These results imply that stimulation of ENaC surface expression results at least in part from increased rates of formation of fully processed subunits in the Golgi and subsequent trafficking to the apical membrane.

Acute effects of aldosterone on the epithelial Na channel in rat kidney.

The acute effects of aldosterone administration on epithelial Na channels (ENaC) in rat kidney were examined using electrophysiology and immunodetection. Animals received a single injection of aldosterone (20 µg/kg body wt), which reduced Na excretion over the next 3 h. Channel activity was assessed in principal cells of cortical collecting ducts as amiloride-sensitive whole cell clamp current (INa). INa averaged 100 pA/cell, 20-30% of that reported for the same preparation under conditions of chronic stimulation. INa was negligible in control animals that did not receive hormone. The acute physiological response correlated with changes in ENaC processing and trafficking. These effects included increases in the cleaved forms of α-ENaC and γ-ENaC, assessed by Western blot, and increases in the surface expression of ß-ENaC and γ-ENaC measured after surface protein biotinylation. These changes were qualitatively and quantitatively similar to those of chronic stimulation. This suggests that altered trafficking to or from the apical membrane is an early response to the hormone and that later increases in channel activity require stimulation of channels residing at the surface.

mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity.

The epithelial Na+ channel (ENaC) is essential for Na+ homeostasis, and dysregulation of this channel underlies many forms of hypertension. Recent studies suggest that mTOR regulates phosphorylation and activation of serum/glucocorticoid regulated kinase 1 (SGK1), which is known to inhibit ENaC internalization and degradation; however, it is not clear whether mTOR contributes to the regulation of renal tubule ion transport. Here, we evaluated the effect of selective mTOR inhibitors on kidney tubule Na+ and K+ transport in WT and Sgk1-/- mice, as well as in isolated collecting tubules. We found that 2 structurally distinct competitive inhibitors (PP242 and AZD8055), both of which prevent all mTOR-dependent phosphorylation, including that of SGK1, caused substantial natriuresis, but not kaliuresis, in WT mice, which indicates that mTOR preferentially influences ENaC function. PP242 also substantially inhibited Na+ currents in isolated perfused cortical collecting tubules. Accordingly, patch clamp studies on cortical tubule apical membranes revealed that mTOR inhibition markedly reduces ENaC activity, but does not alter activity of K+ inwardly rectifying channels (ROMK channels). Together, these results demonstrate that mTOR regulates kidney tubule ion handling and suggest that mTOR regulates Na+ homeostasis through SGK1-dependent modulation of ENaC activity.

Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the γ-subunit by a second protease.

During maturation, the α- and γ-subunits of the epithelial Na+ channel (ENaC) undergo proteolytic processing by furin. Cleavage of the γ-subunit by furin at the consensus site γRKRR143 and subsequent cleavage by a second protease at a distal site strongly activate the channel. For example, coexpression of prostasin with ENaC increases both channel function and cleavage at the γRKRK186 site. We generated a polyclonal antibody that recognizes the region 144-186 in the γ-subunit (anti-γ43) to determine whether prostasin promotes the release of the intervening tract between the putative furin and γRKRK186 cleavage sites. Anti-γ43 precipitated both full-length (93 kDa) and furin-processed (83 kDa) γ-subunits from extracts obtained from oocytes expressing αßHA-γ-V5 channels, but only the full-length (93 kDa) γ-subunit from oocytes expressing αßHA-γ-V5 channels and either wild-type or a catalytically inactive prostasin. Although both wild-type and catalytically inactive prostasin activated ENaCs in an aprotinin-sensitive manner, only wild-type prostasin bound to aprotinin beads, suggesting that catalytically inactive prostasin facilitates the cleavage of the γ-subunit by an endogenous protease in Xenopus oocytes. As dietary salt restriction increases cleavage of the renal γ-subunit, we assessed release of the 43-mer inhibitory tract on rats fed a low-Na+ diet. We found that a low-Na+ diet increased γ-subunit cleavage detected with the anti-γ antibody and dramatically reduced the fraction precipitated with the anti-γ43 antibody. Our results suggest that the inhibitory tract dissociates from the γ-subunit in kidneys from rats on a low-Na+ diet.

Inhibition of ROMK channels by low extracellular K+ and oxidative stress.

We tested the hypothesis that low luminal K⁺ inhibits the activity of ROMK channels in the rat cortical collecting duct. Whole-cell voltage-clamp measurements of the component of outward K⁺ current inhibited by the bee toxin Tertiapin-Q (ISK) showed that reducing the bath concentration ([K⁺]o) to 1 mM resulted in a decline of current over 2 min compared with that observed at 10 mM [K⁺]o. However, maintaining tubules in 1 mM [K⁺]o without establishing whole-cell clamp conditions did not affect ISK. The [K⁺]o-dependent decline was not prevented by increasing cytoplasmic-side pH or by inhibition of phosphatase activity. It was, however, abolished by the inclusion of 0.5 mM DTT in the pipette solution to prevent oxidation of the intracellular environment. Conversely, treatment of intact tubules with the oxidant H2O2 (100 µM) decreased ISK in a [K⁺]o-dependent manner. Treatment of the tubules with the phospholipase C inhibitor U73122 prevented the effect of low [K⁺]o, suggesting the involvement of this enzyme in the process. We examined these effects further using Xenopus oocytes expressing ROMK2 channels. A 50-min exposure to the permeant oxidizing agent tert-butyl hydroperoxide (t-BHP; 500 µM) did not affect outward K⁺ currents with [K⁺]o = 10 mM but reduced currents by 50% with [K⁺]o = 1 mM and by 75% with [K⁺]o = 0.1 mM. Pretreatment of the oocytes with U73122 prevented the effects of t-BHP. Under conditions of low dietary K intake, K⁺ secretion by distal nephron segments may be suppressed by a combination of low luminal [K⁺]o and oxidative stress.

Feedback inhibition of ENaC during acute sodium loading in vivo.

The epithelial Na(+) channel (ENaC) is tightly regulated by sodium intake to maintain whole body sodium homeostasis. In addition, ENaC is inhibited by high levels of intracellular Na(+) [Na(+)](i), presumably to prevent cell Na(+) overload and swelling. However, it is not clear if this regulation is relevant in vivo. We show here that in rats, an acute (4 h) oral sodium load decreases whole-cell amiloride-sensitive currents (I(Na)) in the cortical collecting duct (CCD) even when plasma aldosterone levels are maintained high by infusing the hormone. This was accompanied by decreases in whole-kidney cleaved α-ENaC (2.6 fold), total ß-ENaC (1.7 fold), and cleaved γ-ENaC (6.2 fold). In addition, cell-surface ß- and γ-ENaC expression was measured using in situ biotinylation. There was a decrease in cell-surface core-glycosylated (2.2 fold) and maturely glycosylated (4.9 fold) ß-ENaC and cleaved γ-ENaC (4.7 fold). There were no significant changes for other apical sodium transporters. To investigate the role of increases in Na(+) entry and presumably [Na(+)](i) on ENaC, animals were infused with amiloride prior to and during sodium loading. Blocking Na(+) entry did not inhibit the effect of resalting on I(Na). However, amiloride did prevent decreases in ENaC expression, an effect that was not mimicked by hydrochlorothiazide administration. Na(+) entry and presumably [Na(+)](i) can regulate ENaC expression but does not fully account for the aldosterone-independent decrease in I(Na) during an acute sodium load.

Effects of insulin on Na and K transporters in the rat CCD.

We tested the effects of insulin (2 nM, 30-60 min) on principal cells of isolated split-open rat cortical collecting ducts (CCD) using whole-cell current measurements. Insulin addition to the superfusate of the tubules enhanced Na pump (ouabain-sensitive) current from 18 ± 3 to 31 ± 3 pA/cell in control and from 74 ± 9 to 126 ± 11 pA/cell in high K-fed animals. It also more than doubled ROMK (tertiapin-Q-sensitive) K(+) currents in control CCD from 320 ± 40 to 700 ± 80 pA/cell, although it did not affect this current in tubules from K-loaded rats. Insulin did not induce the appearance of amiloride-sensitive Na(+) current in control animals, while in high K-fed animals the currents were similar in the presence (140 ± 30) and the absence (180 ± 70 pA/cell) of insulin. Intraperitoneal injection of insulin plus hypertonic dextrose decreased Na excretion, as previously reported. However, injection of dextrose alone, or the nonmetabolized sugar mannose, had similar effects, suggesting that they were largely the result of vascular volume depletion rather than specific actions of the hormone. In summary, we find no evidence for acute upregulation of the epithelial Na channel (ENaC) by physiological concentrations of insulin in the mammalian CCD. However, the hormone does activate both the Na/K pump and apical K(+) channels and could, under some conditions, enhance renal K(+) secretion.

Regulation of epithelial Na+ channels by adrenal steroids: mineralocorticoid and glucocorticoid effects.

Epithelial Na+ channels (ENaC) can be regulated by both mineralocorticoid and glucocorticoid hormones. In the mammalian kidney, effects of mineralocorticoids have been extensively studied, but those of glucocorticoids are complicated by metabolism of the hormones and cross-occupancy of mineralocorticoid receptors. Here, we report effects of dexamethasone, a synthetic glucocorticoid, on ENaC in the rat kidney. Infusion of dexamethasone (24 µg/day) for 1 wk increased the abundance of αENaC 2.26 ± 0.04-fold. This was not accompanied by an induction of Na+ currents (I(Na)) measured in isolated split-open collecting ducts. In addition, hormone treatment did not increase the abundance of the cleaved forms of either αENaC or γENaC or the expression of ßENaC or γENaC protein at the cell surface. The absence of hypokalemia also indicated the lack of ENaC activation in vivo. Dexamethasone increased the abundance of the Na+ transporters Na+/H+ exchanger 3 (NHE3; 1.36 ± 0.07-fold), Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2; 1.49 ± 0.07-fold), and Na-Cl cotransporter (NCC; 1.72 ± 0.08-fold). Surface expression of NHE3 and NCC also increased with dexamethasone treatment. To examine whether glucocorticoids could either augment or inhibit the effects of mineralocorticoids, we infused dexamethasone (60 µg/day) together with aldosterone (12 µg/day). Dexamethasone further increased the abundance of αENaC in the presence of aldosterone, suggesting independent effects of the two hormones on this subunit. However, I(Na) was similar in animals treated with dexamethasone+aldosterone and with aldosterone alone. We conclude that dexamethasone can occupy glucocorticoid receptors in cortical collecting duct and induce the synthesis of αENaC. However, this induction is not sufficient to produce an increase in functional Na+ channels in the apical membrane, implying that the abundance of αENaC is not rate limiting for channel formation in the kidney.

Regulation and dysregulation of epithelial Na+ channels.

Epithelial Na(+) channels (ENaC) form a highly regulated pathway for the reabsorption of Na(+) from urine. This regulation can take place at a number of different levels, including synthesis of channel protein, trafficking of the protein between the surface and internal membranes, proteolytic cleavage and channel gating. This article reviews the role of these different modes of regulation under physiological conditions and considers the possible contributions of dysregulation of these processes in disease states, particularly hypertension.

Conservation of Na+ vs. K+ by the rat cortical collecting duct.

Regulation of transport by principal cells of the distal nephron contributes to maintenance of Na(+) and K(+) homeostasis. To assess which of these ions is given a higher priority by these cells, we investigated the upregulation of epithelial Na(+) channels (ENaC) in the rat cortical collecting duct (CCD) during Na depletion with and without simultaneous K depletion. ENaC activity, assessed as whole cell amiloride-sensitive current in split-open tubules, was 260 ± 40 pA/cell in K-repleted but virtually undetectable (3 ± 1 pA/cell) in K-depleted animals. This difference was confirmed biochemically by the reduced amounts of the cleaved forms of both the α-ENaC and γ-ENaC subunits measured in immunoblots. In contrast, in K-depleted rats, simultaneously reducing Na intake did not affect the activity of ROMK channels, assessed as tertiapin-Q-sensitive whole cell currents, in the CCDs. The lack of Na current in K-depleted animals was the result of reduced levels of aldosterone in plasma, rather than a reduced sensitivity to the hormone. However, rats on a low-Na, low-K diet for 1 wk did not excrete more Na than those on a low-Na, control-K diet for the same period of time. Immunoblot analysis indicated increased levels of the thiazide-sensitive NaCl cotransporter and the apical Na-H exchanger NHE3. This suggests that with reduced K intake, Na balance is maintained despite reduced aldosterone and Na(+) channel activity by upregulation of Na(+) transport in upstream segments. Under these conditions, Na(+) transport by the aldosterone-sensitive distal nephron is reduced, despite the low-Na intake to minimize K(+) secretion and urinary K losses.

Magnesium modulates ROMK channel-mediated potassium secretion.

The ability of intracellular and extracellular Mg(2+) to block secretory K(+) currents through ROMK channels under physiologic conditions is incompletely understood. We expressed ROMK2 channels in Xenopus oocytes and measured unitary currents in the inside-out and cell-attached modes of the patch-clamp technique. With 110 mM K(+) on both sides of the membrane, 0.2 to 5 mM Mg(2+) on the cytoplasmic side reduced outward currents, but not inward currents, at V(m) > 0. With 11 or 1.1 mM extracellular K(+) ([K(+)](o)), ≥0.2 mM Mg(2+) blocked outward currents in the physiologic V(m) range (0 to -60 mV). With decreasing [K(+)](o), the apparent dissociation constant of the blocker decreased, but the voltage dependence of block did not significantly change. Whole-cell recordings from principal cells of rat cortical collecting ducts revealed similar inhibitory effects of intracellular Mg(2+). Mg(2+) added to the extracellular solution also reduced single-channel currents with an affinity that increased as [K(+)](o) decreased. In conclusion, physiologic concentrations of intracellular and extracellular Mg(2+) can influence secretory K(+) currents through ROMK channels. These effects could play a role in the modulation of K(+) transport under conditions of K(+) and/or Mg(2+) depletion.