Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel*

Background: Ubiquitination controls trafficking of the epithelial Na+ channel (ENaC) in the endocytic pathway. Results: USP8 deubiquitinated ENaC and blocked its degradation, resulting in increased ENaC abundance at the cell surface and increased current. Conclusion: USP8 regulates endocytic sorting of ENaC. Significance: Regulation of the ubiquitination state of ENaC is important for Na+ homeostasis and blood pressure control. Ubiquitination plays a key role in trafficking of the epithelial Na+ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na+ absorption.

Ubiquitination plays a key role in trafficking of the epithelial Na ؉ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na ؉ absorption.
The epithelial Na ϩ channel (ENaC), 2 a heterotrimer of ␣-, ␤-, and ␥-subunits, functions as a pathway for Na ϩ transport across epithelial cells (reviewed in Refs. 1 and 2). In the kidney collecting duct and connecting tubule, ENaC plays an important role in extracellular Na ϩ and volume homeostasis, which is critical for the control of blood pressure (3). In the lung, ENaC controls the volume and composition of airway surface liquid (4).
Na ϩ absorption is regulated in part by the ubiquitination state of ENaC subunits, which in turn controls ENaC traffick-ing. This concept was illuminated by work to understand the pathogenesis of Liddle syndrome, an inherited form of hypertension. This disorder is caused by mutations that disrupt PY motifs located in the C termini of ␤or ␥ENaC (5,6). These motifs function as binding sites for the E3 ubiquitin ligase Nedd4-2 (7)(8)(9). When bound to ENaC, Nedd4-2 catalyzes ubiquitination of lysines within the cytoplasmic domains of all three ENaC subunits (10,11). The interaction between ENaC and Nedd4-2 is a critical convergence point for pathways that regulate Na ϩ absorption. Aldosterone and vasopressin enhance Na ϩ absorption in part through serum-and glucocorticoidregulated kinase (SGK) and cAMP-dependent protein kinase (PKA), which phosphorylate Nedd4-2, reducing its binding to ENaC (12)(13)(14). Additional kinases (e.g. IB kinase-␤ (15) and AMP-activated kinase (16)) also modulate Nedd4-2 binding to ENaC.
Nedd4-2 reduces ENaC expression on the cell surface at two distinct sites in the endocytic pathway. First, Nedd4-2 increases the rate of ENaC endocytosis (17). Second, once in endosomes, Nedd4-2 enhances ENaC sorting to lysosomes for degradation, which reduces ENaC recycling back to the cell surface (11,17). In the endosome, ubiquitinated ENaC is detected and sorted to lysosomes by Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate). Together with STAM (signal-transducing adaptor molecule), Hrs forms a complex called ESCRT-0 (18,19). Disruption of this complex reduces ENaC degradation and increases its recycling to the cell surface (20).
The ubiquitination state of ENaC is also controlled by deubiquitinating enzymes (DUBs), which remove ubiquitin from ENaC subunits. Two DUBs that deubiquitinate ENaC have been identified. USP2-45 (ubiquitin-specific peptidase 2-45) is an aldosterone-induced protein that increases ENaC current by enhancing its abundance at the cell surface (21)(22)(23)(24). UCH-L3 (ubiquitin carboxyl-terminal hydrolase L3) also modulates ENaC trafficking; inhibition or knockdown of UCH-L3 increases ENaC current and surface expression (25). However, it is not known which trafficking steps are modulated by these DUBs and if additional DUBs play a role in ENaC trafficking. Previous work indicates that another DUB, USP8 (ubiquitinspecific peptidase 8) binds to the ESCRT-0 complex through an interaction with STAM (26). Thus, our goal in this work was to test whether USP8 modulates ENaC trafficking and to determine which trafficking steps are modulated by DUBs.
Electrophysiology in mpkCCD Cells-mpkCCD c14 cells were provided by Alain Vandewalle (INSERM). They were cultured on permeable filter supports (Millicell PCF, 0.4-m pore size, 12-mm diameter) in equal volumes of DMEM and Ham's F-12 medium with 60 nM sodium selenate, 5 mg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor, 5 mg/ml insulin, 20 mM D-glucose, 2% FCS, and 20 mM HEPES, pH 7.4, as described previously (33). The cells were transfected with USP8 or empty plasmid (2.8 g) using HVJ-E (GenomeONE) in medium lacking antibiotics. Six h after transfection, the cells were cultured in medium containing dexamethasone (50 nM) but not epidermal growth factor or insulin. Two days after transfection, shortcircuit current was measured in Ussing chambers (Warner Instruments). The apical and basolateral surfaces were bathed in 135 mM NaCl, 1.2 mM CaCl 2 , 1.2 mM MgCl 2 , 2.4 mM K 2 HPO 4 , 0.6 mM KH 2 PO 4 , and 10 mM HEPES, pH 7.4, at 37°C. Amiloride-sensitive short-circuit current was determined as the current difference with and without amiloride (10 M) in the apical bathing solution.
In some experiments, cells were cotransfected with ubiquitin-HA. The total cDNA concentration was held constant between groups using GFP cDNA. Following transfection, 10 M amiloride was added to the culture medium. Cells were harvested 48 h after transfection.
ENaC Endocytosis-HEK 293T cells transfected with ␣ Cl-2 ENaC-FLAG, ␤ENaC, and ␥ENaC with or without Nedd4-2 and either USP8 or USP2-45 were incubated with trypsin (5 g/ml) for 5 min at 37°C as described previously (17). The cells were washed three times with PBS-CM to remove trypsin, incubated at 37°C for times between 0 and 20 min to allow endocytosis of cleaved channels, and then placed on ice. Cleaved channels remaining at the cell surface were labeled with sulfo-NHS-biotin, isolated with NeutrAvidin beads, detected by immunoblotting, and quantitated by densitometry.
Degradation of Cell Surface ENaC-HEK 293T cells transfected with ␣-, ␤-, and ␥ENaC (one subunit with a FLAG epitope) with or without Nedd4-2 and USP8 or USP2-45 were biotinylated with sulfo-NHS-biotin at 4°C and then incubated at 37°C for 0 -120 min. Following lysis, the remaining biotinylated ENaC subunits were isolated with NeutrAvidin beads, detected by immunoblotting, and quantitated by densitometry as described previously (11,17).

RESULTS
USP8 Increases ENaC Current-To test the effect of USP8 on ENaC current, we coexpressed ␣-, ␤-, and ␥ENaC with or without USP8 in Xenopus oocytes and measured current by twoelectrode voltage clamp (holding potential of Ϫ60 mV). Fig. 1A shows representative current traces in the presence and absence of the ENaC blocker amiloride. In Fig. 1B, we quanti-tated the amiloride-sensitive ENaC current. We found that USP8 increased ENaC current by almost 2-fold. In contrast, ENaC current was not altered by a catalytically inactive USP8 mutant (C786S).
We also tested the effect of USP8 on endogenous ENaC in the mouse collecting duct cell line mpkCCD. Overexpression of USP8 in mpkCCD monolayers increased amiloride-sensitive short-circuit current (Fig. 1C). Thus, USP8 stimulates ENaC in both Xenopus oocytes and renal epithelia.
USP8 Increases ENaC Cell Surface Abundance-To examine the mechanism by which USP8 increased ENaC current, we tested whether USP8 alters ENaC abundance. We expressed ␣-, ␤-, and ␥ENaC in HEK 293 cells with or without USP8. We labeled the cell surface fraction of ENaC with sulfo-NHS-biotin, isolated biotinylated channels through binding to immobilized NeutrAvidin, and then detected the biotinylated channels by immunoblotting. We found that USP8 increased the abundance of ␣-, ␤-, and ␥ENaC at the cell surface ( Fig. 2A, upper panel; the data are quantitated in Fig. 2B). For ␣and ␥ENaC, we observed two bands, which correspond to the full-length (immature) and proteolytically cleaved (mature) forms, respectively (␤ENaC does not undergo cleavage) (29,35); USP8 selectively increased surface expression of the cleaved forms. We did not detect USP8 or USP2-45 in the biotinylated fraction because they are cytoplasmic proteins (supplemental Fig. S1). In contrast to its effects on ENaC cell surface abundance, USP8 did not increase ENaC abundance in the total cell lysate ( Fig.  2A, middle panel), which principally reflects the fraction of ENaC in the biosynthetic pathway. Thus, USP8 selectively enhances ENaC at the cell surface without altering its total abundance, consistent with an alteration in ENaC trafficking.
USP8 Does Not Alter ENaC Endocytosis-In previous work, we found that ubiquitination controls ENaC cell surface expression at two distinct steps in the endocytic pathway; ubiquitination increases both ENaC endocytosis and lysosomal targeting for degradation (11,17). To determine which step is modulated by USP8, we first quantitated the endocytosis rate of mature proteolytically cleaved ENaC using a method we previ-   FEBRUARY 22, 2013 • VOLUME 288 • NUMBER 8 ously described (17). In the Golgi and at the cell surface, the extracellular domain of ␣ENaC is proteolytically cleaved by furin at two sites, which increases channel activity (35). We prevented proteolytic cleavage by mutating the two furin consensus sites (␣ Cl-2 ENaC). As a result, only full-length ␣-subunits were detected at the cell surface in HEK 293 cells expressing ␣ Cl-2 ENaC with ␤and ␥ENaC. We generated a pool of cleaved channels (65-kDa band) at the cell surface by briefly treating with trypsin. Following trypsin removal, we incubated the cells at 37°C for 0 -20 min to allow endocytosis of cleaved channels and then labeled and detected channels remaining at the cell surface with sulfo-NHS-biotin (Fig. 3A). The full-length band reflects a steady state between endocytosis and exocytosis of newly synthesized channels. In the absence of USP8, the cleaved pool of ␣ENaC was rapidly removed from the cell surface ( Fig. 3A; the data are quantitated in Fig. 3B), consistent with our previous results (17). USP8 did not alter the rate of ENaC endocytosis (Fig. 3, A and B).

USP8 Regulates ENaC Trafficking
It is possible that USP8 failed to alter ENaC endocytosis because the basal ubiquitination state of ENaC was low. To enhance ubiquitination of the cell surface pool of ENaC, we cotransfected cells with Nedd4-2; by catalyzing ENaC ubiquitination, Nedd4-2 accelerates ENaC endocytosis (17). As shown in Fig. 3 (C and D), ENaC endocytosis was very rapid in the presence of Nedd4-2; the majority of ENaC was removed from the cell surface by the 5-min time point. However, USP8 did not slow the rate of ENaC endocytosis. Together, the data indicate that USP8 increases ENaC cell surface abundance by modulating a trafficking step other than endocytosis.
USP8 Reduces ENaC Degradation in the Endocytic Pathway-Once ENaC undergoes endocytosis, either it is targeted to lysosomes for degradation, or it recycles back to the cell surface to participate in Na ϩ absorption. This endosomal sorting decision is governed in part by Nedd4-2-mediated ubiquitination of ENaC; ubiquitination promotes lysosomal degradation and reduces recycling (11,17). To test whether USP8 modulates this trafficking step, we quantitated the degradation rate of the cell surface pool of ENaC. In HEK 293 cells expressing ENaC with or without USP8, we pulse-labeled the cell surface fraction of ENaC with sulfo-NHS-biotin, incubated the cells at 37°C for 0 -120 min, and then quantitated the remaining (non-degraded) biotinylated channels at each time point. In the absence of USP8, biotinylated ␣ENaC was degraded with a half-life of ϳ70 min (Fig. 4, A and B). We found that USP8 abolished degradation of biotinylated ␣ENaC (Fig. 4, A and B). Similarly, USP8 also prevented degradation of biotinylated ␤ENaC (Fig. 4, C and D).
To increase ENaC ubiquitination and degradation, we cotransfected cells with Nedd4-2. This shortened the half-life of biotinylated ␤ENaC to Ͻ40 min (Fig. 4, E and F). USP8 reversed this effect, abolishing degradation of biotinylated ␤ENaC (Fig. 4, E and F). Thus, USP8 increases ENaC cell surface abundance by modulating endosomal sorting rather than by altering ENaC trafficking at the cell surface.
USP8 Interacts with ENaC-To further explore the mechanism by which USP8 modulates ENaC trafficking, we asked whether USP8 interacts with the ENaC channel complex. As shown in Fig. 5A, we expressed ENaC (␣, ␤, and ␥), USP8, or both together. When we immunoprecipitated ␣ENaC, we detected coprecipitated USP8 by immunoblotting only in cells that coexpressed both proteins. Likewise, we detected coprecipitation between USP8 and ␤ENaC (Fig. 5B) and ␥ENaC (Fig.  5C). These results indicate that there is an interaction between USP8 and ENaC.
USP8 Deubiquitinates ENaC-USP8 could regulate ENaC trafficking by deubiquitinating ENaC or through deubiquitination of another protein. To distinguish between these possibilities, we tested whether USP8 can deubiquitinate ENaC. In Fig.  6A, we expressed ENaC in HEK 293 cells with or without Nedd4-2 and USP8. As shown in the upper panel, we immunoprecipitated HA-tagged ubiquitin and then detected ubiquitinated ␣ENaC by immunoblotting. The other panels show immunoblots to confirm expression of ␣ENaC and USP8 and to detect ␤-actin as a control. Nedd4-2 catalyzed monoubiquitination and polyubiquitination of ␣ENaC, as we reported previously (11). We found that USP8 reversed the effect of Nedd4-2, reducing ␣ENaC ubiquitination (Fig. 6A; the data are quanti- tated in Fig. 6D). In Fig. 6 (B and C), we tested whether USP8 would deubiquitinate ␤and ␥ENaC as well. Similar to ␣ENaC, Nedd4-2 enhanced ubiquitination of ␤and ␥ENaC, and USP8 reversed this effect, reducing ubiquitination (the data are quantitated in Fig. 6D). These findings indicate that USP8 can function as a DUB for all three ENaC subunits.

JOURNAL OF BIOLOGICAL CHEMISTRY 5393
USP8 on wild-type ENaC (Fig. 7, A and B). Thus, ENaC lysines contribute to regulation by USP8.
USP2-45 Regulates ENaC Endocytosis-It is intriguing that Nedd4-2 regulates ENaC at both the endocytosis and degradation steps of endocytic trafficking, but USP8 selectively regulates ENaC degradation. We therefore hypothesized that another DUB might regulate ENaC endocytosis. Previous work indicated that USP2-45 reduces ENaC ubiquitination and increases ENaC abundance at the cell surface (21). We therefore tested whether USP2-45 regulates ENaC endocytosis.
We examined the effect of USP2-45 on ENaC abundance in HEK 293 cells (Fig. 8). USP2-45 increased the quantity of ␣ENaC at the cell surface (biotinylated fraction) (Fig. 8A, upper panel; the data are quantitated in Fig. 8B) but had no effect on ␣ENaC in the total cell lysate (Fig. 8A, lower panel). Because USP2-45 is a cytoplasmic protein, we did not detect it in the biotinylated fraction (supplemental Fig. S1). USP2-45 also increased the cell surface abundance of ␤and ␥ENaC (Fig. 8, A  and B).
We tested whether USP2-45 increases ENaC surface abundance by altering endocytosis using the methods described above for Fig. 3. Following trypsin cleavage, Nedd4-2 caused a rapid decrease in the pool of cleaved channels at the cell surface (Fig. 9, A and B). In contrast, the rate of decrease was much slower in cells coexpressing USP2-45 (Fig. 9, A and B). Thus, USP2-45 reduces the rate of ENaC endocytosis.
As shown in Fig. 9 (C and D), we tested whether USP2-45 modulates ␤ENaC endosomal sorting and degradation. Following pulse labeling of the cell surface pool of ENaC with sulfo-NHS-biotin, we quantitated the degradation rate of biotinylated ␤ENaC as described above for Fig. 4. We found that USP2-45 did not alter ␤ENaC degradation (Fig. 9, C and D). Similarly, USP2-45 did not reduce ␣ENaC degradation (Fig. 9, E  and F). Together, the data indicate that USP2-45 increases ENaC surface expression by reducing endocytosis but not by altering ENaC trafficking in endosomes.

DISCUSSION
The ubiquitination state of ENaC plays a key role in its trafficking in the endocytic pathway. This in turn regulates epithelial Na ϩ absorption. The E3 ubiquitin ligase Nedd4-2 catalyzes ENaC ubiquitination and reduces Na ϩ absorption by altering ENaC trafficking at two distinct steps in the endocytic pathway; it enhances ENaC endocytosis, and it sorts ENaC to lysosomes for degradation, reducing ENaC recycling to the cell surface (Fig. 10). Both reduce epithelial Na ϩ absorption. DUBs reduce ENaC ubiquitination and have the opposite effect on Na ϩ absorption. In this study, we identified selective roles for two such DUBs in this pathway. USP8 deubiquitinated ENaC and reduced ENaC degradation ( Fig. 10) but had no effect on ENaC endocytosis. In contrast, USP2-45 reduced ENaC endocytosis but had no effect on ENaC degradation (Fig. 10). Thus, USP8 and USP2-45 both increase ENaC cell surface abundance and ENaC current, but they do so at different locations within the endocytic pathway.
Our finding that USP8 regulates ENaC trafficking selectively at the endocytic sorting step is consistent with earlier work localizing USP8 to endosomes, where it interacts with STAM, a component of the ESCRT-0 complex (26). In a previous study, we found that Hrs, another component of ESCRT-0, detects ubiquitinated ENaC and directs it to the multivesicular body/ lysosomal compartment for degradation (20). Nedd4-2 also interacts with Hrs and, by catalyzing ENaC ubiquitination, enhances the interaction between ENaC and Hrs (20). This suggests a model in which ENaC endocytic sorting is determined by the concerted actions of a complex of ubiquitin ligases, DUBs, and ubiquitin-interacting proteins, which titrate the ENaC ubiquitination state and hence determine its fate. An important focus for future work will be to identify the mechanisms that coordinate the opposing actions of Nedd4-2 and USP8 within this trafficking complex.
It is interesting that USP2-45 slowed ENaC endocytosis but had no effect on its degradation. This finding indicates there is selective control of these trafficking steps. The endosomal sorting step is rate-limiting for ENaC degradation because it is much slower than ENaC endocytosis. This allows USP2-45 to selectively regulate endocytosis. It seems likely that the selective effects of USP2-45 and USP8 are based on compartmentalization. However, it is also possible that USP2-45 and USP8 target different subsets of ENaC lysines for deubiquitination. In this scenario, different lysines might mediate ENaC endocytosis and degradation. Additional work will be necessary to address these questions.  We found that in ENaC, cytoplasmic lysines play an important role in regulation by USP8; mutation of the lysines reduced the ability of USP8 to increase ENaC cell surface abundance. This was expected because USP8 functions as a ubiquitin peptidase, removing ubiquitin from cytoplasmic lysines. However, we were surprised to find that mutation of the lysines did not completely abolish the effect of USP8 on ENaC surface abundance; USP8 increased surface abundance of lysine mutant ENaC but to a lesser extent compared with wild-type ENaC. This contrasts with earlier work in which ENaC regulation by USP2-45 was abolished by lysine mutations (24). Our findings raise the intriguing possibility that USP8 regulates ENaC trafficking through two different mechanisms. First, it deubiquitinates ENaC, reducing its degradation by blocking targeting to lysosomes. Second, USP8 alters trafficking in part through an additional mechanism that is independent of ENaC deubiquitination.
USP8 has been shown to deubiquitinate additional membrane proteins, although the functional effects of deubiquitination are complex. USP8 overexpression reduces degradation of the EGF receptor (36) and KCa3.1 (37), similar to our results with ENaC. However, other work reported that USP8 is required for lysosomal degradation of the EGF receptor (38), as well as chemokine receptor 4 (39) and protease-activated receptor 2 (40). These seemingly incongruous findings can be explained by the observation that USP8 can deubiquitinate cargo at multiple steps in the endocytic pathway. In early endosomes, ubiquitination sorts cargo between recycling and degradation pathways. Deubiquitination at this step can rescue cargo from degradation and increase recycling to the cell surface. In the case of ENaC, USP8 appears to function at this step. Later in the trafficking pathway, after cargo is committed to the multivesicular bodies for degradation, ubiquitin is removed prior to delivery to intraluminal vesicles and lysosomal fusion. Reducing deubiquitination at this step can decrease cargo degradation. A second DUB, AMSH, can also participate in deubiquitination at these endosomal trafficking steps (41). Ubiquitination can also contribute to the assembly and stability of trafficking proteins in the endocytic pathway (42). Thus, USP8 can play multiple roles in endosomal cargo sorting.  (1 g each), with Nedd4-2 (0.5 g), and with or without USP2-45 (3 g). The cells were treated with 5 g/ml trypsin for 5 min, incubated at 37°C for 0 -15 min, and then biotinylated. FL, full-length, and Cl, cleaved ␣ENaC. B, quantitation of the cleaved ␣ENaC band relative to 0 min (mean Ϯ S.E., n ϭ 3). *, p Ͻ 0.05 versus the ϪUSP2-45 group. C, immunoblot (anti-FLAG) of biotinylated ␤ENaC-FLAG in HEK 293 cells transfected with ␣ENaC, ␤ENaC-FLAG, and ␥ENaC (1 g each), with Nedd4-2 (0.5 g), and with or without USP2-45 (3 g). Cell surface proteins were pulse-labeled at 4°C with sulfo-NHS-biotin, and the cells were incubated at 37°C for 0 -120 min. D, biotinylated ␤ENaC at each time point was quantitated relative to 0 min (mean Ϯ S.E., n ϭ 3). E, immunoblot (anti-FLAG) of biotinylated ␣ENaC-FLAG in HEK 293 cells transfected with ␣ENaC-FLAG-, ␤ENaC, and ␥ENaC (1 g each), with Nedd4-2 (0.5 g), and with or without USP2-45 (3 g). Cell surface proteins were pulse-labeled at 4°C with sulfo-NHS-biotin, and the cells were incubated at 37°C for 0 or 80 min. F, biotinylated ␣ENaC at each time point was quantitated relative to 0 min (mean Ϯ S.E., n ϭ 3). *, p Ͻ 0.05 versus the ϪUSP2-45 group. n.s. indicates that there was no significant difference between the 80-min time points. In summary, USP8 has an important role in modulating the ubiquitination state of ENaC, functioning selectively at the endosomal sorting step of ENaC trafficking. This complements the activity of USP2-45, which functions at the endocytosis step. The balance between ubiquitination and deubiquitination is critical in the regulation of epithelial Na ϩ absorption. A more detailed understanding of the signaling pathways that control this balance will provide new insights into mechanisms that maintain Na ϩ homeostasis and into the pathogenesis of hypertension and other diseases.