[Show abstract][Hide abstract] ABSTRACT: Salt and water retention is a hallmark of nephrotic syndrome (NS). In this study, we test for changes in the abundance of urea transporters, aquaporin 2 (AQP2), Na-K-2Cl cotransporter 2 (NKCC2), and Na-Cl cotransporter (NCC), in non-pair-fed and pair-fed nephrotic animals. Doxorubicin-injected male Sprague-Dawley rats (n = 10) were followed in metabolism cages. Urinary excretion of protein, sodium, and urea was measured periodically. Kidney inner medulla (IM), outer medulla, and cortex tissue samples were dissected and analyzed for mRNA and protein abundances. At 3 wk, all doxorubicin-treated rats developed features of NS, with a ninefold increase in urine protein excretion (from 144 ± 21 to 1,107 ± 165 mg/day; P < 0.001) and reduced urinary sodium excretion (from 0.17 to 0.12 meq/day; P < 0.001). Urine osmolalities were reduced in the nephrotic animals (1,057 ± 37, treatment vs. 1,754 ± 131, control). Unlike animals fed ad libitum, UT-A1 protein abundance was unchanged in nephrotic pair-fed rats. Glycosylated AQP2 was reduced in the IM base of both nephrotic groups. Abundances of NKCC2 and NCC were consistently reduced (71 ± 7 and 33 ± 13%, respectively) in both nephrotic pair-fed animals and animals fed ad libitum. In pair-fed nephrotic rats, we observed an increase in the cleaved form of membrane-bound γ-epithelial sodium channel (ENaC). However, α- and β-ENaC subunits were unaltered. NKCC2 and AQP2 mRNA levels were similar in treated vs. control rats. We conclude that dietary protein intake affects the response of medullary transport proteins to NS.
Preview · Article · Mar 2012 · AJP Renal Physiology
[Show abstract][Hide abstract] ABSTRACT: Amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC) play a crucial role in Na(+) transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na(+) transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends upon a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits, alpha, beta, and gamma. The C-terminal domain of all three subunits is intracellular and contains a proline rich motif (PPxY). Mutations or deletion of this PPxY motif in the beta and gamma subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein (Nedd4-2) to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when MDCK cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected epithelial cells (A6) expressing endogenous ENaC, ENaC appears to be degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by the rate of Nedd4-2-mediated ENaC ubiquitination. Controlling the rate of degradation is apparently important enough to have multiple, redundant pathways to control Nedd4-2 and ENaC ubiquitination.
Full-text · Article · Feb 2010 · Proceedings of the American Thoracic Society
[Show abstract][Hide abstract] ABSTRACT: The epithelial sodium channel (ENaC) is regulated by epidermal growth factor (EGF). We investigate whether ENaC is regulated by another EGF receptor (EGFR) ligand, transforming growth factor-alpha (TGF-alpha). We show that chronic (24 h) treatment with TGF-alpha inhibits ENaC in Xenopus laevis kidney cells 20 times more strongly than EGF. By using single-channel measurements, we show that TGF-alpha significantly reduces the number of ENaC per patch. The open probability (P(o)) is unchanged by 24-h treatment with TGF-alpha. alpha-, beta-, and gamma-ENaC mRNA levels are significantly reduced by TGF-alpha or EGF. TGF-alpha or EGF reduces alpha- and gamma-ENaC proteins in the membrane; however, beta-ENaC is unchanged. TGF-alpha or EGF inhibits ENaC by activating EGFR since the EGFR inhibitor AG1478 blocks the effects of both. The MAPK 1/2 inhibitor U0126 also blocks the effect of TGF-alpha or EGF on ENaC, indicating that the MAPK1/2 pathway is involved in the TGF-alpha- or EGF-induced inhibition of ENaC. Interestingly, acute treatment (<1 h) with TGF-alpha or EGF does not inhibit ENaC current; it enhances ENaC activity by increasing P(o). Pretreatment of the cells with U0126 potentiates the acute TGF-alpha- or EGF-induced stimulation of ENaC. This TGF-alpha- or EGF-induced increase in sodium current is abolished by a phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, LY294002, suggesting that PI-3 kinase is involved in the activation of sodium transport. In conclusion, chronic treatment with TGF-alpha or EGF inhibits ENaC by decreasing the number of channels in the membrane transcriptionally through MAPK1/2 pathways, but acute treatment with TGF-alpha or EGF activates ENaC by increasing P(o) via PI-3 kinase.
No preview · Article · Mar 2009 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: Amiloride-sensitive epithelial Na+ channels (ENaC) play a crucial role in Na+ transport and fluid reabsorption in the kidney, lung, and colon. The magnitude of ENaC-mediated Na+ transport in epithelial cells depends on the average open probability of the channels and the number of channels on the apical surface of epithelial cells. The number of channels in the apical membrane, in turn, depends on a balance between the rate of ENaC insertion and the rate of removal from the apical membrane. ENaC is made up of three homologous subunits: alpha, beta, and gamma. The COOH-terminal domain of all three subunits is intracellular and contains a proline-rich motif (PPxY). Mutations or deletion of this PPxY motif in the beta- and gamma-subunits prevent the binding of one isoform of a specific ubiquitin ligase, neural precursor cell-expressed, developmentally downregulated protein (Nedd4-2), to the channel in vitro and in transfected cell systems, thereby impeding ubiquitin conjugation of the channel subunits. Ubiquitin conjugation would seem to imply that ENaC turnover is determined by the ubiquitin-proteasome system, but when Madin-Darby canine kidney cells are transfected with ENaC, ubiquitin conjugation apparently leads to lysosomal degradation. However, in untransfected renal cells (A6) expressing endogenous ENaC, ENaC is indeed degraded by the ubiquitin-proteasome system. Nonetheless, in both transfected and untransfected cells, the rate of ENaC degradation is apparently controlled by Nedd4-2 activity. In this review, we discuss the role of the ubiquitin conjugation and the alternative degradative pathways (lysosomal or proteasomal) in regulating the rate of ENaC turnover in untransfected renal cells and compare this regulation to that of transfected cell systems.
Preview · Article · Jul 2006 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: Hypertension is an increase of blood pressure to levels greater than normal that arises because of a mismatch between the
volume of the vascular tree and the volume of blood. Blood volume depends on total body sodium content, which is a balance
between sodium intake and output. Total body sodium is controlled by variable excretion of sodium by the kidneys. To regulate
sodium balance, the primary variable that the kidney monitors is not total body sodium, but rather systemic blood pressure.
Renal regulation of blood pressure is via the release of the peptide hormone, renin from specialized renal cells. Release
of renin ultimately leads to the production of angiotensin II. Angiotensin II increases total peripheral resistance and blood
pressure and also leads to an increase in aldosterone. Aldosterone is a steroid hormone that increases sodium reabsorption
in the distal nephron by activating epithelial Na channels (ENaCs). Thus, Hypertension is a defect in one of these elements
that control total body sodium balance.
[Show abstract][Hide abstract] ABSTRACT: Several studies have shown that nitric oxide (NO) inhibits Na(+) transport in renal and alveolar monolayers. However, the mechanisms by which NO alters epithelial Na(+) channel (ENaC) activity is unclear. Therefore, we examined the effect of applying the NO donor drug l-propanamine 3,2-hydroxy-2-nitroso-1-propylhidrazino (PAPA-NONOate) to cultured renal epithelial cells. A6 and M1 cells were maintained on permeable supports in medium containing 1.5 microM dexamethasone and 10% bovine serum. After 1.5 microM PAPA-NONOate was applied, amiloride-sensitive short-circuit current measurements decreased 29% in A6 cells and 44% in M1 cells. This differed significantly from the 3% and 19% decreases in A6 and M1 cells, respectively, treated with control donor compound (P < 0.0005). Subsequent application of PAPA-NONOate to amiloride-treated control (no NONOate) A6 and M1 cells did not further decrease transepithelial current. In single-channel patch-clamp studies, NONOate significantly decreased ENaC open probability (P(o)) from 0.186 +/- 0.043 to 0.045 +/- 0.009 (n = 7; P < 0.05) without changing the unitary current. We also showed that aldosterone significantly decreased NO production in primary cultures of alveolar type II (ATII) epithelial cells. Because inducible nitric oxide synthase (iNOS) coimmunoprecipitated with the serum- and glucocorticoid-inducible kinase (SGK1) and both proteins colocalized in the cytoplasm (as shown in our studies in mouse ATII cells), SGK1 may also be important in regulating NO production in the alveolar epithelium. Our study also identified iNOS as a novel SGK1 phosphorylated protein (at S733 and S903 residues in miNOS) suggesting that one way in which SGK1 could increase Na(+) transport is by altering iNOS production of NO.
[Show abstract][Hide abstract] ABSTRACT: Amiloride-sensitive epithelial sodium channels (ENaC) are responsible for transepithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, and gamma). In Madin-Darby canine kidney (MDCK) cells and Xenopus laevis oocytes transfected with all three ENaC subunits, neural precursor cell-expressed developmentally downregulated protein (Nedd4-2) promotes ubiquitin conjugation of ENaC. For native proteins in some cells, ubiquitin conjugation is a signal for their degradation by the ubiquitin-proteasome pathway, whereas in other cell types ubiquitin conjugation is a signal for endocytosis and lysosomal protein degradation. When ENaC are transfected into MDCK cells, ubiquitin conjugation leads to lysosomal degradation. In this paper, we characterize the involvement of the ubiquitin-proteasome proteolytic pathway in the regulation of functional ENaC in untransfected renal A6 cells expressing native ENaC subunits. In contrast to transfected cells, we show that total cellular alpha-, beta-, and gamma-ENaC subunits are polyubiquitinated and that ubiquitin conjugation of subunits increases when the cells are treated with a proteasome inhibitor. We show that Nedd4-2 is associated with alpha- and beta-subunits and is associated with the apical membrane. We also show the Nedd4-2 can regulate the number of functional ENaC subunits in the apical membrane. The results reported here suggest that the ubiquitin-proteasome proteolytic pathway is an important determinant of ENaC function in untransfected renal cells expressing endogenous ENaC.
Preview · Article · Aug 2005 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: We investigated the mechanisms of endogenous nitric oxide (NO) modulation of lung sodium (Na(+)) transport. C57BL/6 mice injected intraperitoneally with the specific inducible NO synthase (iNOS) inhibitor 1400W (10 mg/kg every 8 h for 72 h) exhibited decreased alveolar nitrite levels and Na(+)-dependent amiloride-sensitive alveolar fluid clearance as compared with mice injected with vehicle. Similarly, pretreatment of mouse tracheal epithelial cells with 1400W abolished the inhibitory effects of amiloride on their Na(+) short circuit currents. On the other hand, mouse tracheal epithelial cells pretreated with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of guanylate cyclase, had lower levels of cGMP, but normal values of amiloride-sensitive Na(+) currents. Amiloride also inhibited whole-cell Na(+) currents across A549 cells treated with vehicle (K(i) = 249 nM), but had no effect in A549 cells treated with 1400W. Western blotting studies showed significantly lower levels of alpha and gammaENaC in lung tissues and alveolar type II (ATII) cells from iNOS(-/-) as well as iNOS(+/+) mice treated with 1400W, as compared with the corresponding values from vehicle-treated iNOS(+/+) mice. Similar values for ratios of alpha, beta, and gammaenac to gapdh were obtained by real-time polymerase chain reaction for iNOS(+/+) mice and iNOS(-/-) mice. We concluded that NO derived from iNOS under basal conditions is necessary for amiloride-sensitive Na(+) transport across lung epithelial cells and modulates the amount of alpha and gammaENaC via post-transcriptional, cGMP-independent mechanisms.
Preview · Article · Jun 2004 · American Journal of Respiratory Cell and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: We studied the cellular phosphatase inhibitors okadaic acid (OKA), calyculin A, and microcystin on the epithelial sodium channel (ENaC) in A6 renal cells. OKA increased the amiloride-sensitive current after approximately 30 min with maximal stimulation at 1-2 h. Fluctuation analysis of cell-attached patches containing a large number of ENaC yielded power spectra with corner frequencies in untreated cells almost two times as large as in cells pretreated for 30 min with OKA, implying an increase in single channel open probability (P(o)) that doubled after OKA. Single channel analysis showed that, in cells pretreated with OKA, P(o) and mean open time approximately doubled. Two other phosphatase inhibitors, calyculin A and microcystin, had similar effects on P(o) and mean open time. An analog of OKA, okadaone, that does not inhibit phosphatases had no effect. Pretreatment with 10 nM OKA, which blocks protein phosphatase 2A (PP2A) but not PP1 in mammalian cells, had no effect even though both phosphatases are present in A6 cells. Several proteins were differentially phosphorylated after OKA, but ENaC subunit phosphorylation did not increase. We conclude that, in A6 cells, there is an OKA-sensitive phosphatase that suppresses ENaC activity by altering the phosphorylation of a regulatory molecule associated with the channel.
Preview · Article · Dec 2002 · American journal of physiology. Renal physiology
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane lipid found in all eukaryotic cells, which regulates many important cellular processes, including ion channel
activity. In this study, we used inside-out patch clamp technique, immunoprecipitation, and Western blot analysis to investigate
the effect of PIP2 on epithelial sodium channel activity in A6 cells. A6 cells were cultured in media supplemented with 1.5 μm aldosterone. Single sodium channel activity in excised, inside-out patches was increased by perfusion of the bath solution
with 30 μm PIP2 plus 100 μm GTP (NP
o = 1.34 ± 0.14) compared with the paired control (NP
o = 0.09 ± 0.02). However, neither 30 μmPIP2 (NP
o = 0.11 ± 0.02) nor 100 μm GTP (NP
o = 0.10 ± 0.02) alone stimulated the sodium channels. The PIP2-stimulated channel activity was abolished by application of 10 nm G protein βγ subunits (NP
o = 0.14 ± 0.05). However, 10 nm Gαi-3 + 30 μmPIP2 increased both NP
o. The stimulating effect of 10 nmGαi-3 + 30 μm PIP2 is similar to that of 30 μm PIP2 plus 100 μm GTP. Immunoprecipitation and Western blot analysis show that both Giα-3 and PIP2 bind β and γ epithelial Na+ channels (ENaC), but not α ENaC. These results indicate that PIP2 increases ENaC activity by direct interaction with β or γ xENaC in the presence of Gαi-3.
Full-text · Article · May 2002 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Aldosterone maintains total organism sodium balance in all higher vertebrates. The level of sodium reabsorption is primarily determined by the action of aldosterone on epithelial sodium channels (ENaC) in the distal nephron. Recent work shows that, in an aldosterone-sensitive renal cell line (A6), aldosterone regulates sodium reabsorption by short- and long-term processes. In the short term, aldosterone regulates sodium transport by inducing expression of the small G-protein, K-Ras2A, by stimulating the activity of methyl transferase and S-adenosyl-homocysteine hydrolase to activate Ras by methylation, and, possibly, by subsequent activation by K-Ras2A of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K), which ultimately activates ENaC. In the long term, aldosterone regulates sodium transport by altering trafficking, assembly, and degradation of ENaC.
No preview · Article · Jan 2002 · Journal of Membrane Biology
[Show abstract][Hide abstract] ABSTRACT: Amiloride-sensitive epithelial Na+ channels (ENaC) are responsible for trans-epithelial Na+ transport in the kidney, lung, and colon. The channel consists of three subunits (α, β, γ) each containing a proline rich
region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions
with the ubiquitin ligaseNeural precursor cell-expresseddevelopmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading
to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC
channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132,
increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules
by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal
function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important
regulator of ENaC function.
Preview · Article · May 2001 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Regulation of epithelial Na+channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit
levels, differentially decreasing the levels of both β and γ, but not αENaC. Temporal regulation of β and γENaC by PKC differed;
γENaC decreased with a time constant of 3.7 ± 1.0 h, whereas βENaC decreased in 13.9 ± 3.0 h. Activation of PKC also resulted
in a decrease in trans-epithelial Na+reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning
within 4 h. Both β and γENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC
inhibitors attenuated the effects of PMA on ENaC subunit levels and Na+ transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing
the levels of β and γ but not αENaC protein. These results imply a PKC-dependent, long term decrease in Na+ reabsorption.
[Show abstract][Hide abstract] ABSTRACT: Amiloride-sensitive Na+ transport by lung epithelia plays a critical role in maintaining alveolar Na+ and water balance. It has been generally assumed that Na+ transport is mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) because molecular biology studies have confirmed the presence of ENaC subunits alpha, beta, and gamma in lung epithelia. However, the predominant Na+-transporting channel reported from electrophysiological studies by most laboratories is a nonselective, high-conductance channel that is very different from the highly selective, low-conductance ENaC reported in other tissues. In our laboratory, single-channel recordings from apical membrane patches from rat alveolar type II (ATII) cells in primary culture reveal a nonselective cation channel with a conductance of 20.6 +/- 1.1 pS and an Na+-to-K+ selectivity of 0.97 +/- 0.07. This channel is inhibited by submicromolar concentrations of amiloride. Thus there is some question about the relationship between the gene product observed with single-channel methods and the cloned ENaC subunits. We have employed antisense oligonucleotide methods to block the synthesis of individual ENaC subunit proteins (alpha, beta, and gamma) and determined the effect of a reduction in the subunit expression on the density of the nonselective cation channel observed in apical membrane patches on ATII cells. Treatment of ATII cells with antisense oligonucleotides inhibited the production of each subunit protein; however, single-channel recordings showed that only the antisense oligonucleotide targeting the alpha-subunit resulted in a significant decrease in the density of nonselective cation channels. Inhibition of the beta- and gamma-subunit proteins alone or together did not cause any changes in the observed channel density. There were no changes in open probability or other channel characteristics. These results support the hypothesis that the alpha-subunit of ENaC alone or in combination with some protein other than the beta- or gamma-subunit protein is the major component of lung alveolar epithelial cation channels.
No preview · Article · Jul 1999 · The American journal of physiology