Increased colonic sodium absorption in rats with chronic renal failure is partially mediated by AT1 receptor agonism

To test the hypothesis that colonic Na(+) transport is altered in the 5/6 nephrectomized rat model of chronic renal failure (CRF), we measured Na(+) fluxes across distal colon from control (CON), CRF, and CRF rats treated with the angiotensin II (ANG II) receptor antagonist losartan (+LOS). We also evaluated overall fluid and Na(+) balance and compared colonic protein and mRNA expression profiles for electroneutral [sodium-hydrogen exchanger (NHE)] and electrogenic Na(+) transport [epithelial sodium channel (ENaC)] in these groups. Consistent with a 60% enhancement in colonic Na(+) absorption in CRF, urinary Na(+) excretion increased by about 50% while serum Na(+) homeostasis was maintained. These CRF-induced changes in Na(+) handling were normalized by treatment with LOS. Net Na(+) absorption was also stimulated in in vitro tissues from CON rats following acute serosal addition of ANG II (10(-7) M), and this increase was blocked by AT(1) antagonism but not by an AT(2) antagonist. In CRF, colonic protein and mRNA expression variably increased for apical NHE2, NHE3, and ENaC alpha-, beta-, gamma-subunits, whereas expression of basolateral NHE1 and Na(+)-K(+)-ATPase (alpha-isoform) remained unaltered. Upregulation of the ENaC subunit mRNA was attenuated somewhat by LOS treatment. Previously, we showed that colonic AT(1) receptor protein is upregulated twofold in CRF, and here we find that AT(1) and AT(2) mRNA and AT(2) protein abundance is unchanged in CRF. We conclude that Na(+) absorption in CRF rat distal colon is increased due to elevated expression of proteins mediating electroneutral and electrogenic uptake and that it is partially mediated by AT(1) receptors.     

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.     

Identification of the roles of conserved charged residues in the extracellular domain of an epithelial sodium channel (ENaC) subunit by alanine mutagenesis

Epithelial sodium channels (ENaC) are composed of three homologous subunits whose extracellular domains (ECD) form a funnel that directs ions from the lumen into the pore of ENaC. To examine the roles of conserved charged residues (Asp, Glu, Arg, and Lys) on ECD, we mutated 16 residues in human α-ENaC to alanine. The modified cRNAs were expressed in Xenopus laevis oocytes together with wild-type β- and γ-ENaC. The effect of each mutation was examined on three parameters: amiloride-sensitive Na(+) conductance (assayed by the two-electrode voltage-clamp method), Na(+)-dependent self-inhibition of ENaC, and oocyte cell surface expression of ENaC (quantitated by confocal microscopy of yellow fluorescent protein linked to γ-ENaC). Mutation of 13 of 16 residues reduced the ENaC Na(+) conductance (to 40-80% of WT). Mutation of only six residues showed a significant effect on the Na(+) self-inhibition time constant (τ). All 16 mutants showed a strong correlation between ENaC activity and oocyte surface expression (r = 0.62). Exclusion of four mutants showing the greatest effect on self-inhibition kinetics (Glu250 and Arg350 with τ = ~30% of WT, and Asp393 and Glu530 with τ = ~170% of WT) increased the correlation to r = 0.87. In the ASIC1 homotrimeric model, the homologs of α-ENaC Asp400 and Asp446 are exposed on the protein surface far from the other two chains. The mutations of these two residues showed the strongest effect on cell surface expression but had no effect on self-inhibition. Control mutations to a homologous charged residue (e.g., Asp to Glu) did not significantly affect ENaC activity. Changes in the two parameters, Na(+) self-inhibition and oocyte surface expression level, accounted for the magnitude of reduction in ENaC activity as a result of the mutation to Ala. These results establish that while some conserved charged residues are part of the structure responsible for Na(+) self-inhibition, most are essential for transport to the oocyte cell surface.     

K(+) channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na(+) absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K(ATP) K(+) channel activities exerts sustained control in Na(+) transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K(+) channels, and 2) to determine the physiological impact of K(+) channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K(ATP) channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to K(ATP) activation by pinacidil. Conversely, pharmacological KvLQT1 and K(ATP) inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K(+) channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K(ATP) activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K(+) channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K(+) channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.     

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 βENaC 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.     

Distribution of ion transport mRNAs throughout murine nose and lung

Evidence of absorptive or secretory ion transport in different respiratory regions of the mouse was sought by assessing the regional distribution of alpha-, beta-, and gamma-epithelial sodium channel (ENaC; Na(+) absorptive), cystic fibrosis transmembrane conductor regulator (CFTR), and Na(+)-K(+)-2Cl(-) cotransporter mRNAs. High levels of ENaC subunit expression were found in nasal surface epithelium and gland ducts. CFTR was expressed in both superficial nasal respiratory epithelium and glands. These results are consistent with basal amiloride-sensitive Na(+) absorption and cAMP-dependent Cl(-) secretion in murine nasal epithelia. Expression of all three ENaC subunits increased progressively from trachea to terminal bronchioles. Intermediate levels of CFTR and cotransporter expression in bronchial epithelium diminished in bronchioles. The low abundance of CFTR mRNA throughout murine pulmonary epithelium is consistent with functional data that attributes Cl(-) secretion predominantly to an alternative Cl(-) channel. alpha-ENaC as the only mRNA found in all regions of airway epithelia is consistent with the alpha-subunit as requisite for Na(+) absorption, and the increased expression of alpha-, beta-, and gamma-ENaC in distal airways suggests a greater absorptive capability in this region.     

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.     

Mechanisms of diarrhea in the interleukin-2-deficient mouse model of colonic inflammation

Colitis in interleukin-2-deficient (IL-2(-/-)) mice resembles ulcerative colitis in humans. We studied epithelial transport and barrier function in IL-2(-/-) mice and used this model to characterize mechanisms of diarrhea during intestinal inflammation. (22)Na(+) and (36)Cl(-) fluxes were measured in proximal colon. Net Na(+) flux was reduced from 4.0 +/- 0.5 to 0.8 +/- 0.5 micromol.h(-1).cm(-2), which was paralleled by diminished mRNA and protein expression of the Na(+)/H(+) exchanger NHE3. Net Cl(-) flux was also decreased from 2.2 +/- 1.6 to -2.7 +/- 0.6 micromol.h(-1).cm(-2), indicating impaired Na(+)-Cl(-) absorption. In distal colon, aldosterone-induced electrogenic Na(+) absorption was 6.1 +/- 0.9 micromol.h(-1).cm(-2) in controls and was abolished in IL-2(-/-) mice. Concomitantly, mRNA expression of beta- and gamma-subunits of the epithelial sodium channel (ENaC) was reduced. Epithelial barrier was studied in proximal colon by impedance technique and mannitol fluxes. In contrast to ulcerative colitis, epithelial resistance was increased and mannitol fluxes were decreased in IL-2(-/-) mice. This was in accord with the findings of reduced ion transport as well as increased expression of tight junction proteins occludin and claudin-1, -2, -3, and -5. In conclusion, the IL-2(-/-) mucosa exhibits impaired electroneutral Na(+)-Cl(-) absorption and electrogenic Na(+) transport due to reduced mRNA and protein expression of NHE3 and ENaC beta- and gamma-subunit mRNA. This represents a model of early intestinal inflammation with absorptive dysfunction due to impaired transport protein expression/function while epithelial barrier is still intact. Therefore, this model is ideal to study regulation of transporter expression independent of barrier defects.     

cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2

The epithelial Na(+) channel (ENaC) functions as a pathway for epithelial Na(+) transport, contributing to Na(+) homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na(+) transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na(+) transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na(+) transport.     

Intestinal NaCl transport in NHE2 and NHE3 knockout mice

Sodium/proton exchangers [Na(+)/H(+) (NHEs)] play an important role in salt and water absorption from the intestinal tract. To investigate the contribution of the apical membrane NHEs, NHE2 and NHE3, to electroneutral NaCl absorption, we measured radioisotopic Na(+) and Cl(-) flux across isolated jejuna from wild-type [NHE(+)], NHE2 knockout [NHE2(-)], and NHE3 knockout [NHE3(-)] mice. Under basal conditions, NHE(+) and NHE2(-) jejuna had similar rates of net Na(+) (approximately 6 microeq/cm(2) x h) and Cl(-) (approximately 3 microeq/cm(2) x h) absorption. In contrast, NHE3(-) jejuna had reduced net Na(+) absorption (approximately 2 microeq/cm(2) x h) but absorbed Cl(-) at rates similar to NHE(+) and NHE2(-) jejuna. Treatment with 100 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA) completely inhibited net Na(+) and Cl(-) absorption in all genotypes. Studies of the Na(+) absorptive flux (J) indicated that J in NHE(+) jejunum was not sensitive to 1 microM EIPA, whereas J in NHE3(-) jejunum was equally sensitive to 1 and 100 microM EIPA. Treatment with forskolin/IBMX to increase intracellular cAMP (cAMP(i)) abolished net NaCl absorption and stimulated electrogenic Cl(-) secretion in all three genotypes. Quantitative RT-PCR of epithelia from NHE2(-) and NHE3(-) jejuna did not reveal differences in mRNA expression of NHE3 and NHE2, respectively, when compared with jejunal epithelia from NHE(+) siblings. We conclude that 1) NHE3 is the dominant NHE involved in small intestinal Na(+) absorption; 2) an amiloride-sensitive Na(+) transporter partially compensates for Na(+) absorption in NHE3(-) jejunum; 3) cAMP(i) stimulation abolishes net Na(+) absorption in NHE(+), NHE2(-), and NHE3(-) jejunum; and 4) electroneutral Cl(-) absorption is not directly dependent on either NHE2 or NHE3.     

cAMP increases density of ENaC subunits in the apical membrane of MDCK cells in direct proportion to amiloride-sensitive Na(+) transport

Antidiuretic hormone and/or cAMP increase Na(+) transport in the rat renal collecting duct and similar epithelia, including Madin-Darby canine kidney (MDCK) cell monolayers grown in culture. This study was undertaken to determine if that increment in Na(+) transport could be explained quantitatively by an increased density of ENaC Na(+) channels in the apical membrane. MDCK cells with no endogenous ENaC expression were retrovirally transfected with rat alpha-, beta-, and gammaENaC subunits, each of which were labeled with the FLAG epitope in their extracellular loop as described previously (Firsov, D., L. Schild, I. Gautschi, A.-M. Mérillat, E. Schneeberger, and B.C. Rossier. 1996. PROC: Natl. Acad. Sci. USA. 93:15370-15375). The density of ENaC subunits was quantified by specific binding of (125)I-labeled anti-FLAG antibody (M2) to the apical membrane, which was found to be a saturable function of M2 concentration with half-maximal binding at 4-8 nM. Transepithelial Na(+) transport was measured as the amiloride-sensitive short-circuit current (AS-I(sc)) across MDCK cells grown on permeable supports. Specific M2 binding was positively correlated with AS-I(sc) measured in the same experiments. Stimulation with cAMP (20 microM 8-p-chlorothio-cAMP plus 200 microM IBMX) significantly increased AS-I(sc) from 11.2 +/- 1.3 to 18.1 +/- 1.3 microA/cm(2). M2 binding (at 1.7 nM M2) increased in direct proportion to AS-I(sc) from 0.62 +/- 0.13 to 1.16 +/- 0.18 fmol/cm(2). Based on the concentration dependence of M2 binding, the quantity of Na(+) channels per unit of AS-I(sc) was calculated to be the same in the presence and absence of cAMP, 0.23 +/- 0.04 and 0.21 +/-0.05 fmol/microA, respectively. These values would be consistent with a single channel conductance of approximately 5 pS (typically reported for ENaC channels) only if the open probability is <0.02, i.e., less than one-tenth of the typical value. We interpret the proportional increases in binding and AS-I(sc) to indicate that the increased density of ENaC subunits in the apical membrane can account completely for the I(sc) increase produced by cAMP.     

Tissue kallikrein activation of the epithelial Na channel

Epithelial Na Channels (ENaC) are responsible for the apical entry of Na(+) in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K(+) or low-Na(+) diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (I(Na)) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity.     

Activation of the epithelial sodium channel by the metalloprotease meprin β subunit

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion.     

Sildenafil reduces polyuria in rats with lithium-induced NDI

Lithium (Li)-treated patients often develop urinary concentrating defect and polyuria, a condition known as nephrogenic diabetes insipidus (NDI). In a rat model of Li-induced NDI, we studied the effect that sildenafil (Sil), a phosphodiesterase 5 (PDE5) inhibitor, has on renal expression of aquaporin-2 (AQP2), urea transporter UT-A1, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-K(+)-2Cl(-) cotransporter (NKCC2), epithelial Na channel (ENaC; α-, β-, and γ-subunits), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase. We also evaluated cGMP levels in medullary collecting duct cells in suspension. For 4 wk, Wistar rats received Li (40 mmol/kg food) or no treatment (control), some receiving, in weeks 2-4, Sil (200 mg/kg food) or Li and Sil (Li+Sil). In Li+Sil rats, urine output and free water clearance were markedly lower, whereas urinary osmolality was higher, than in Li rats. The cGMP levels in the suspensions of medullary collecting duct cells were markedly higher in the Li+Sil and Sil groups than in the control and Li groups. Semiquantitative immunoblotting revealed the following: in Li+Sil rats, AQP2 expression was partially normalized, whereas that of UT-A1, γ-ENaC, and eNOS was completely normalized; and expression of NKCC2 and NHE3 was significantly higher in Li rats than in controls. Inulin clearance was normal in all groups. Mean arterial pressure and plasma arginine vasopressin did not differ among the groups. Sil completely reversed the Li-induced increase in renal vascular resistance. We conclude that, in experimental Li-induced NDI, Sil reduces polyuria, increases urinary osmolality, and decreases free water clearance via upregulation of renal AQP2 and UT-A1.     

The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel

The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.     

High sodium intake increases HCO(3)- absorption in medullary thick ascending limb through adaptations in basolateral and apical Na+/H+ exchangers

A high sodium intake increases the capacity of the medullary thick ascending limb (MTAL) to absorb HCO(3)(-). Here, we examined the role of the apical NHE3 and basolateral NHE1 Na(+)/H(+) exchangers in this adaptation. MTALs from rats drinking H(2)O or 0.28 M NaCl for 5-7 days were perfused in vitro. High sodium intake increased HCO(3)(-) absorption rate by 60%. The increased HCO(3)(-) absorptive capacity was mediated by an increase in apical NHE3 activity. Inhibiting basolateral NHE1 with bath amiloride eliminated 60% of the adaptive increase in HCO(3)(-) absorption. Thus the majority of the increase in NHE3 activity was dependent on NHE1. A high sodium intake increased basolateral Na(+)/H(+) exchange activity by 89% in association with an increase in NHE1 expression. High sodium intake increased apical Na(+)/H(+) exchange activity by 30% under conditions in which basolateral Na(+)/H(+) exchange was inhibited but did not change NHE3 abundance. These results suggest that high sodium intake increases HCO(3)(-) absorptive capacity in the MTAL through 1) an adaptive increase in basolateral NHE1 activity that results secondarily in an increase in apical NHE3 activity; and 2) an adaptive increase in NHE3 activity, independent of NHE1 activity. These studies support a role for NHE1 in the long-term regulation of renal tubule function and suggest that the regulatory interaction whereby NHE1 enhances the activity of NHE3 in the MTAL plays a role in the chronic regulation of HCO(3)(-) absorption. The adaptive increases in Na(+)/H(+) exchange activity and HCO(3)(-) absorption in the MTAL may play a role in enabling the kidneys to regulate acid-base balance during changes in sodium and volume balance.     

Electroneutral sodium absorption and electrogenic anion secretion across murine small intestine are regulated in parallel

Electrolyte transport processes of small intestinal epithelia maintain a balance between hydration of the luminal contents and systemic fluid homeostasis. Under basal conditions, electroneutral Na(+) absorption mediated by Na(+)/H(+) exchanger 3 (NHE3) predominates; under stimulated conditions, increased anion secretion mediated by CFTR occurs concurrently with inhibition of Na(+) absorption. Homeostatic adjustments to diseases that chronically affect the activity of one transporter (e.g., cystic fibrosis) may include adaptations in the opposing transport process to prevent enterosystemic fluid imbalance. To test this hypothesis, we measured electrogenic anion secretion (indexed by the short-circuit current) across NHE3-null [NHE3(-)] murine small intestine and electroneutral Na(+) absorption (by radioisotopic flux analysis) across small intestine of mice with gene-targeted disruptions of the anion secretory pathway, i.e., CFTR-null [CFTR(-)] or Na(+)-K(+)-2Cl(-) cotransporter-null [NKCC1(-)]. Protein expression of NHE3 and CFTR in the intestinal epithelia was measured by immunoblotting. In NHE3(-), compared with wild-type small intestine, maximal and bumetanide-sensitive anion secretion following cAMP stimulation was significantly reduced, and there was a corresponding decrease in CFTR protein expression. In CFTR(-) and NKCC1(-) intestine, Na(+) absorption was significantly reduced compared with wild-type. NHE3 protein expression was decreased in the CFTR(-) intestine but was unchanged in the NKCC1(-) intestine, indicating that factors independent of expression also downregulate NHE3 activity. Together, these data support the concept that absorptive and secretory processes determining NaCl and water movement across the intestinal epithelium are regulated in parallel to maintain balance between the systemic fluid volume and hydration of the luminal contents.     

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.     

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.