Ubiquitylation and Control of Renal Na Balance and Blood Pressure

Ubiquitylation is crucial for regulating numerous cellular functions. In the kidney, ubiquitylation regulates the epithelial Na channel ENaC. The importance of this process is highlighted in Liddle’s syndrome, where mutations interfere with ENaC ubiquitylation, resulting in constitutive Na reabsorption and hypertension. There is emerging evidence that NCC, involved in hypertensive diseases, is also regulated by ubiquitylation. Here, we discuss the current knowledge and recent findings in this field. Caroline Ronzaud and Olivier Staub Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland


Regulation of ENaC-Mediated Na ؉
Transport by the Ubiquitin-Ligase NEDD4-2 Ubiquitylation is a posttranslational modification process that is crucial for regulating many different cellular functions.Indeed, ubiquitylation is used as a signal for membrane protein internalization or degradation by the proteasome or lysosomes (52,99).Ubiquitin can be conjugated as monoubiquitin or polyubiquitin chains on the amino group of lysine side chains.Monoubiquitylation on single or several sites on target proteins is involved in numerous biological functions, including endocytosis and direction to lysosomal degradation (52).Also, polyubiquitin chains may have different roles, but the best characterized canonical function is the targeting to the proteasome via K48-linked ubiquitins (17,62).
Ubiquitylation is performed by several enzymes: an E1 ubiquitin-activating enzyme, E2 ubiquitinconjugating enzymes, and E3 ubiquitin-protein ligases.The ubiquitin molecule is first activated by the E1 enzyme using ATP, forming first a thioester with the active site of E1 and then to that of E2.The E3 enzymes, that recognize the target protein, interact with both E2 and the substrate and lead to the formation of an isopeptide bond between a lysine of the target protein and the COOH-terminal glycine of ubiquitin.Hundreds of E3 enzymes participate in the target protein recognition and thus provide the specificity to the cascade (65).
There are two main classes of E3 enzymes.The first class involves E3 ubiquitin-protein ligases that contain a RING motif (RING stands for really interesting new gene) that helps transfer the ubiquitin from E2 to the target protein (61).This class includes monomeric proteins, multimeric complexes comprising Cullin proteins as platforms that assemble E2 enzymes, RING motif-containing proteins, and adaptor proteins.KLHL3 and CUL3 belong to this class, whereby CUL3 represents the platform and KLHL3 an adaptor protein involved in target recognition.The second class is formed by the HECT (homologous to E6-AP COOH terminal) domain-containing E3 ubiquitin-protein ligases that form first a thioester with the ubiquitin before transferring it on the substrate (84).The NEDD4/ NEDD4 (neural precursor cell expressed developmentally downregulated protein)-like family belongs to the HECT E3 ligase class and regroups nine proteins: AIP4/ITCH, HecW1/NEDL1, and HecW2/ NEDL2, NEDD4, NEDD4-2/NEDD4L, SMURF1, SMURF2, WWP1/TIUL1, and WWP2 (79).All these proteins contain an NH 2 -terminal (Ca 2ϩ -dependent lipid binding) C2 domain for membrane binding (77), a central region containing two to four WW domains for interaction with the target protein (100), and a COOH-terminal HECT domain for binding to ubiquitin and transfer on the target (79,84).Ubiquitylation is a reversible process catalyzed by deubiquitylating enzymes (DUBs) (44,66).
The role of ubiquitylation in the regulation of cell surface expression and endocytosis of ion channels and transporters has emerged with the finding that the WW domains of the NEDD4/NEDD4-like E3 ligases bind to the proline-rich PY motifs of ENaC (core motif: PPXY, where P is a proline, Y is a tyrosine, and X is any amino acid), motifs that are deleted in Liddle's syndrome (96) (FIGURE 1B).In this study, the authors used the PY-containing region of the ␤-ENaC subunit as bait in a two-hybrid screen to identify proteins, which interact with ENaC, and identified NEDD4-2.It was then shown that each of the three ENaC subunits ␣, ␤, and ␥ contains a PY motif in the COOH terminus (85,93,96).Interestingly, NEDD4-2 is expressed in tissues that also express ENaC (37).It was proposed that the enhanced cell surface ENaC expression observed in Liddle's patients might be due to a defect in ubiquitylation and degradation of the channel (FIGURE 1C).This hypothesis was confirmed in 1999 by the finding that indeed the regulation of ENaC by NEDD4-2 was defective in Liddle's syndrome (2).Another study later showed that the NEDD4-2 WW3-4 domains regulate ENaC (25,37).
In 2006, Knight et al. found out that Liddle's syndrome mutations enhance Na ϩ transport by acting on ENaC surface expression and open probability (41).One year later, Wiemuth et al. demonstrated that ENaC is multi-monoubiquitylated at the cell surface (104), and two other independent groups showed that the PY motifs on ENaC, that are mutated in Liddle's syndrome, regulate the internalization of the channel, its sorting, and its recycling (50) and that NEDD4-2 catalyzes the plasma membrane ENaC ubiquitylation and degradation (113).However, whether ENaC is mono-or polyubiquitylated remains controversial.Some groups have proposed that ENaC is polyubiquitylated and targeted to proteasomal degradation (53)(54)(55), whereas others suggested that the channel is either monoor multi-monoubiquitylated at the surface of the cell (81,104,113).One explanation might be that ENaC is polyubiquitylated in the endoplasmic reticulum (ER) in the context of ERAD (ER-associated degradation), where misfolded or unassembled proteins are degraded via ubiquitin-proteasome pathway (10), whereas the assembled channel expressed at the cell surface is likely degraded via endosomal/lysosomal degradation involving multimonoubiquitylation (97,112) (FIGURE 1).Direct evidence of ERAD-based regulation of ENaC has been provided by Brodsky and collaborators (10).
As mentioned above, ubiquitylation is a process that is reversible, catalyzed by DUBs (66).ENaC has been proposed to be regulated by several of these DUBs, including USP2-45, USP8, USP10, and UCH-L3 (8,11,21,114).Although deubiquitylation of ENaC appears to be an appealing concept for ENaC regulation, the physiological relevance of these observations remain to be proven, especially as a recent report of a Usp2 knockout model provides no evidence for the involvement of USP2 in the control of Na ϩ homeostasis or blood pressure control (71).
To elucidate the relationship between the regulation of ENaC and the control of Na ϩ balance and hypertension, an in vivo model for Liddle's syndrome was generated by mutating the ␤-subunit of the mouse ENaC, resulting in a truncated ␤-ENaC as found in Liddle's patients (73).Under high-Na ϩ diet, these Liddle mice presented the Liddle phenotype characterized by higher blood pressure, metabolic alkalosis, and hypokalemia, together with heart and kidney hypertrophy.The cell surface density of functional ENaC, but not the open probability, was enhanced in the kidneys of Liddle mice (14).In addition, whereas the ␥-ENaC subunit was rapidly internalized in control mice, it was retained at the plasma membrane of CNT in Liddle mice (73).Accordingly, measures in isolated perfused CCD and in confluent primary cultures of microdissected CCD isolated from Liddle mice exhibited higher ENaC activity (73).by 10.220.33.1 on April 27, 2017 http://physiologyonline.physiology.org/NEDD4-2-mediated ubiquitylation of ENaC is under the tight control of the mineralocorticoid hormone aldosterone, which prevents the interaction between the channel and the ubiquitin-protein ligase (FIGURE 1, A AND B).This is mediated by the serumand glucocorticoid-induced kinase 1 (SGK1): under aldosterone stimulus, SGK1 expression is induced and the kinase phosphorylates NEDD4-2, resulting in the recruitment of 14-3-3 proteins that disrupt the NEDD4-2/ENaC interaction and subsequent ubiquitylation and degradation of the channel (6,13,15,33,46,91,92) (FIGURE 1A).A number of alternative mechanisms of regulation by SGK1 or NEDD4-2 have also been suggested, including the direct phosphorylation of ENaC by SGK1 (16) or direct phosphorylation of ENaC by ERK kinase, which may facilitate the interaction with NEDD4-2 (86).Pearce and collaborators provided evidence for an ENaC regulatory complex that comprises NEDD4-2, SGK1, GILZ, RAF-1, and CNK3.It was proposed that the complex may integrate the activities of various signaling pathways involved in ENaC regulation, including those regarding steroid receptors, PI-3 kinase, mTOR, and RAF-MEK-ERK pathways (95).Very recently, another novel mode of regulation of ENaC, comprising the ubiquitin-like protein NEDD8, was also described (18,51).
In Vivo NEDD4-2 Knockout Models: New Insights in the Role of NEDD4-2-Mediated Regulation of Na ؉ Transport and Blood Pressure As described above, genetic mutations in human and in vitro biochemical and overexpression studies suggested that the interaction between NEDD4-2  and ENaC is crucial for regulating Na ϩ balance and blood pressure.Moreover, single nucleotide polymorphisms (SNPs) in human Nedd4L (that encodes NEDD4-2) have been linked to hypertension (19).To validate the role of NEDD4-2 in vivo, several groups have generated in the last few years mouse knockout models of Nedd4L (for summary of these mouse models, see Table 1).The first model was developed by the group of Baoli Yang.In these Nedd4L knockout mice, the exons 6 -8 of the Nedd4L gene were removed in a constitutive manner (Nedd4L-⌬6-8 mice) (87).Surprisingly, despite the predicted function of NEDD4-2 as a major regulator of ENaC, the Nedd4L-⌬6-8 knockout mice were viable and only displayed cardiac hypertrophy and a mild salt-sensitive hypertensive phenotype.The extracellular volume and Na ϩ /K ϩ balance were not impaired (87).The protein expression of the three subunits of ENaC was enhanced in the knockout mice, and the increased blood pressure under high-Na ϩ diet could be treated by injecting amiloride into the mice, suggesting overactive ENaC.However, the plasma aldosterone levels were normal under standard and high-Na ϩ diet, indicating a rather mild ENaC-overactivation phenotype.
Another Nedd4L total knockout model has been generated by Sharan Kumar's group, by deleting exons 15 and 16 of the Nedd4L gene in the mouse (7).In contrast to Yang's mice, the model of Kumar displayed perinatal lethality resulting from premature fetal lung fluid clearance as a consequence of increased ENaC expression and activity in embryonic lungs (7).Expression of the three ENaC subunits ␣, ␤, and ␥ was increased in lungs and kidneys of E18.5 embryos, and the amiloridesensitive ENaC-mediated current was strongly enhanced in embryonic knockout lung cells.These data clearly indicate that NEDD4-2 is important for the regulation of ENaC in the kidney and that it is a key regulator of ENaC function in the lung, leading to lethality when deleted (7).These data also confirmed what was observed in the ␤-ENaC Liddle mice, in which the mutation in ␤-ENaC prevented the downregulation of the channel by NEDD4-2.Indeed, these  (40).This study showed that ablating the exon 15 and the downstream region of the Nedd4L gene in mice leads to a phenotype resembling lung cystic fibrosis, with airway mucus obstruction, intense inflammation, and lethality at the age of 3 wk.These observations were accompanied with increased protein abundance of the three ENaC subunits, enhanced ENaC currents and amiloridesensitive dehydration, and increased dryness of the lungs (40).These results confirm what was observed in transgenic mice overexpressing ENaC (56) and confirm the crucial role of lung NEDD4-2 in regulating ENaC expression and activity by ubiquitylation.Interestingly, Kimura et al. could rescue the lung defects by nasally administrating amiloride to the newly born, lung-specific Nedd4L knockout mice, suggesting that the observed lung phenotype resulted from a defect in ENaC degradation due to NEDD4-2 ablation (40).
Very recently, Ronzaud et al. published a new Nedd4L knockout mouse model, where exons 6 -8 of the Nedd4L gene have been removed [using the same floxed allele as Shi et al. (87)] in an inducible and renal tubule-specific manner (Nedd4L Pax8/LC1 knockout mice) using a combination of the Cre-loxP and Tet-On systems (78).As expected, these mice displayed salt-sensitive hypertension, decreased plasma aldosterone levels, and increased renal ␤and ␥-ENaC protein expression.However, the urine and plasma Na ϩ and K ϩ levels were normal in the knockout mice.In addition, the cellular localization of ␤and ␥-ENaC was mainly cytoplasmic, and ␥-ENaC was found in its uncleaved form.Surprisingly, the Nedd4L Pax8/LC1 knockout mice showed reduced ␣ENaC mRNA levels and proteolytic cleavage, suggesting downregulation of the channel (FIGURE 1D).Interestingly, the ENaC downregulation that accompanied the reduced plasma aldosterone could be rescued by giving aldosterone to the knockouts.Thus these data confirmed that NEDD4-2 is important for mediating the degradation of ␤and ␥-ENaC, that aldosterone is crucial for regulating renal ␣-ENaC expression (4), and that ␣-ENaC is needed for apical trafficking of the two other ENaC subunits (80).Of interest, no mutation in the human ␣ENaC gene was found to cause Liddle's syndrome.In addition, injection of amiloride in Nedd4L Pax8/LC1 knockout mice did not reduce the elevated blood pressure, indicating that the hypertensive phenotype is not due to increased ENaC function.Therefore, the Nedd4L Pax8/LC1 knockout mice question the previous in vitro and in vivo studies that showed the implication of NEDD4-2-mediated ubiquitylation in the regulation of ␣-ENaC.
It is difficult to reconcile the different phenotypes obtained with all these Nedd4L knockout mouse models, since they differ at several levels: genetic background, part of the Nedd4L gene that was deleted, targeted tissue, and knockout strategy (constitutive or inducible).However, their comparison opens new questions regarding NEDD4-2 function and tissue specificity.First, it should be noted that Shi et al. did not specify whether the observed enhanced ␣-, ␤-, and ␥-ENaC subunits were expressed apically or within the cell.In addition, the absence of any decrease in plasma aldosterone is in contrast to what is observed in Liddle's patients.It has been proposed that the absence of lung phenotype and lethality in the Shi et al.'s model could be explained by the presence of a shorter Nedd4L splice isoform (7).Indeed, the region of the Nedd4L gene that has been deleted in the Shi et al.'s model is known to be subjected to differential splicing (34).However, using different antibodies against NEDD4-2, Ronzaud et al. were not able to detect any shorter NEDD4-2 isoform in the Nedd4L Pax8/LC1 knockout mice, at least in the kidney (78).It is also plausible that other NEDD4 family members might compensate for the loss of NEDD4-2 in the lung, although Ronzaud et al. did not observe any increase in NEDD4-1, the closest family member (78).Another possibility could be that the mixed genetic background used in the Shi et al.'s model contributes to the milder phenotype of these mice compared with those developed by Kumar and Rotin (7,40).
Moreover, it is also difficult to reconcile the phenotype of the Nedd4L Pax8/LC1 knockout mice with what has been observed in mouse models of Liddle's syndrome, where the activity of ENaC is increased despite the low plasma aldosterone and where the three ENaC subunits are accumulating in the plasma membrane (72).It is likely that loss of the regulator NEDD4-2 affects other downstream factors like NCC upregulation and consequent compensatory mechanisms, or other factors involved in the control of ENaC function or trafficking (99), whereas mutations in the ENaC subunits would affect the channel directly.Thus it would be of interest to generate Nedd4L knockout mice specifically in the CNT and CD, without targeting the DCT, to avoid the effects of NEDD4-2 loss on NCC expression and function, and thus see the impact on ENaC only.(68).Later, it was shown that the WNK pathway is implicated in both NCC phosphorylation and trafficking (59).WNK1, WNK4, and a third family member (WNK3) all activate SPAK/OSR1 that phosphorylate NCC and stimulate its activity (69,76).In addition, WNK4 downregulates NCC abundance at the cell surface by promoting the lysosome-mediated degradation of the cotransporter (26,42,103,108).However, it has long remained unclear how NCC trafficking to the cell surface is regulated.Based on two independent in vitro studies, it has been proposed that WNK4 could divert NCC to the lysosomal pathway, possibly by regulating the interaction between the cotransporter and the lysosome-targeting receptor sortilin (111) and the AP3 adaptor complex (101).In PHAII patients, resulting from mutations in both WNK1 and WNK4 kinases, enhanced NCC phosphorylation and cell surface expression are observed, leading to increased Na ϩ reabsorption and hypertension (83,109,110).In contrast, WNK4overexpressing transgenic mice develop characteristics of Gitelman's syndrome characterized by decreased NCC expression and function (45).Intriguingly, total WNK4 knockout mice characterized by the group of Gerardo Gamba showed rather an incomplete form of Gitelman's syndrome with decreased NCC expression, phosphorylation, and activity, but without any changes in blood pressure and urinary calcium excretion (12).

Ubiquitylation and Regulation of NCC-Mediated Na
Aldosterone has been shown to increase NCC protein expression without affecting the NCC mRNA levels (1,39,63), suggesting a posttranslational mechanism.In the past few years, many in vitro studies suggested that NCC ubiquitylation plays an important role in the regulation of the cotransporter.Ko et al. showed for the first time that NCC is internalized via a dynamin-dependent mechanism (42) and that this process is regulating NCC, resulting in a decreased activity of the cotransporter (43).They showed that the increased NCC ubiquitylation and decreased activity, observed after treating mDCT cells that endogenously express NCC with phorbol-ester, could be prevented using an E1 ubiquitin-activating enzyme inhibitor.Interestingly, a recent study showed that NCC phosphorylation prevents its ubiquitylation (31).In addition, Heise et al. showed that WNK1 and WNK4 phosphorylate NEDD4-2 on the same sites as SGK1 (30), suggesting that phosphorylation and ubiquitylation could regulate NCC concomitantly.Supporting this hypothesis, Arroyo et al. showed in vitro that NEDD4-2 co-immunoprecipitates with NCC and induces its ubiquitylation, decreasing the expression of the cotransporter at the plasma membrane and its activity.Moreover, like for ENaC regulation, SGK1 prevented the NEDD4-2 effect on NCC (3).There is also in vivo evidence that SGK1 is involved in NCC regulation (22,23).The implication of NEDD4-2 in NCC regulation has now been confirmed in vivo with the generation of the Nedd4L Pax8/LC1 knockout mouse model.Ronzaud et al. showed that NCC protein expression and phosphorylation were elevated in the Nedd4L Pax8/LC1 knockout mice, as well as NCC function since the high blood pressure and hypercalciuria observed in the knockouts could be corrected by thiazide treatment (78).Taken together with the decreased ␣-ENaC and the absence of amiloride effect on the increased blood pressure, it was proposed that the elevated NCC-mediated Na ϩ reabsorption resulting from NEDD4-2 ablation is compensated by reduced ENaC function.Similarly, the increased NCC observed in the kidney-specific ks-wnk1 knockout mice was also compensated by downregulation of the three ENaC subunits (28).The observation that NEDD4-2 is involved in NCC regulation is also supported by recent studies of a SNP in the NEDD4-2 gene, which appears to affect thiazide sensitivity (60).
It has been shown that NEDD4 and NEDD4like proteins can bind to and ubiquitylate target proteins that have no PY motif using PY-motifcontaining adaptors (32,47,64).Arroyo et al. showed that, in contrast to ENaC for which the NEDD4-2-mediated inhibition can be prevented when SGK1 phosphorylates NEDD4-2 on S328, the inhibition of NCC by the SGK1-NEDD4-2 pathway can be prevented by phosphorylation of NEDD4-2 on both S222 and S328 sites (3).The facts that NCC and ENaC are regulated by the SGK1-NEDD4-2 pathway in a different manner and that NCC does not contain any PY motif suggest that NEDD4-2 controls NCC indirectly.Whether NCC and NEDD4-2 interact via an adaptor and its identity represent questions for further investigation.
Interestingly, the Nedd4L Pax8/LC1 knockout mice did not show all the PHAII characteristics, despite the strong NCC upregulation.This contrasts with other mouse models for PHAII, like the PHAII-Wnk4 (Q562E) transgenic mice developed by Lalioti et al. (45).In these mice, the elevated NCC and other PHAII features were abolished after breeding with NCC knockouts (45).Similarly, thiazide treatment of the Wnk4-D561A mutant mice developed by Yang et al. could prevent the increased NCC expression and phosphorylation and the associated hypertension and hyperkalemia (110).More and more studies suggest that PHAII cannot result from overactive NCC only but rather from the misregulation of other channels and transporters.For instance, the ks-wnk1 knockouts described above display some but not all PHAII features (28).Moreover, NCC-overexpressing mice showed no by 10.220.33.1 on April 27, 2017 http://physiologyonline.physiology.org/Downloaded from increase in blood pressure or Ca 2ϩ excretion (58).Finally, the increased ROMK protein expression in the Nedd4L Pax8/LC1 knockouts (78) may result in elevated K ϩ excretion, thus preventing the hyperkalemia normally observed in PHAII patients (28).
Very interestingly, two other groups have recently identified another ubiquitin-protein ligase pathway affecting NCC cell surface abundance and involved in the pathology of PHAII, thus further supporting the importance of ubiquitylation in NCC regulation.Using exome sequencing, Boyden et al. identified mutations in two genes encoding the KLHL3 and CUL3 proteins that led to PHAII (9).These two proteins are part of a Cullin-RING E3 ubiquitin-protein ligase complex and, very interestingly, are expressed in DCT like NCC (9,70).The same findings were reported by an independent group: Louis-Dit-Picard et al. also used combined whole-exome sequencing with linkage analysis and identified mutations in Kelch3 that encodes the KLHL3 protein (48).They confirmed the coexpression of KLHL3 and NCC, and showed in vitro that KLHL3 downregulates NCC expression at the cell surface and that the reverse is observed after Kelch3 silencing.These recent findings strongly support the relevance of ubiquitylation in the control of NCC function.
However, the observations that KLHL3 and CUL3 proteins downregulate NCC expression at the cell surface do not prove the direct effect of these regulators on the cotransporter.Khan et al. recently showed that phosphorylation of NCC controls its ubiquitylation and might play a role in NCC expression at the cell surface (31).In addition, some light has very recently been shed on the possible involvement of KLHL3/CUL3 in WNK1 or WNK4 ubiquitylation (67,88,102,106).It has been shown in vitro that the KLHL3/CUL3 complex interacts with WNK1 FIGURE 2. Proposed model for the differential effect of ubiquitylation via NEDD4-2 or via KLHL3/CUL3 on NCC regulation in normal and pathological conditions A: in conditions of RAAS activation, the WNK kinases phosphorylate and activate NCC and at the same time SGK1, and possibly the WNKs, inhibit the NEDD4-2-mediated ubiquitylation of the cotransporter, thus maintaining sufficient NCC expression at the cell surface.B: in conditions of RAAS inactivation, NCC is inactivated via two pathways: the KLHL3/CUL3 complex ubiquitylates the WNKs, thus decreasing WNK expression and resulting in decreased NCC phosphorylation and expression at the cell surface; in parallel, NCC is ubiquitylated by NEDD4-2, which decreases its cell surface expression.C: in PHAII patients, mutations in either the WNKs or the KLHL3/CUL3 complex inhibit the interaction between the WNKs and the KLHL3/CUL3.This results in a rise of WNK expression, leading to elevated NCC phosphorylation and expression.This leads to elevated Na ϩ reabsorption and blood pressure, RAAS inactivation, hypercalciuria, and hyperkalemia.D: in case of NEDD4-2 ablation, NCC cannot be ubiquitylated and degraded, resulting in increased NCC surface expression and function, elevated Na ϩ reabsorption and blood pressure, RAAS inactivation, and hypercalciuria.and WNK4, but not with NCC, and ubiquitylates and degrades the two WNK isoforms.Very interestingly, the WNK region, which interacts with KLHL3, also mutated in PHAII (67,88).
Taking all these data together, we propose a model for the differential effect of ubiquitylation via NEDD4-2 or via KLHL3/CUL3 on NCC regulation in normal and pathological conditions (FIGURE 2).In conditions of RAAS activation (increased angiotensin II and aldosterone), NCC could be activated by phosphorylation via the WNKs, and NEDD4-2-mediated ubiquitylation of NCC would be inhibited by SGK1 and possibly by the WNK kinases, thus maintaining sufficient NCC expression at the cell surface.In conditions of RAAS inactivation (decreased angiotensin II and aldosterone), NCC could be inactivated via two pathways: the KLHL3/CUL3 complex could interact with and ubiquitylate the WNKs, thus decreasing WNK expression and resulting in decreased NCC phosphorylation and expression at the cell surface; in parallel, NEDD4-2 would ubiquitylate NCC and thus decrease its cell surface expression (FIGURE 2B).In PHAII patients, mutations in either the WNKs or the KLHL3/CUL3 complex inhibit the interaction between the WNKs and the KLHL3/CUL3.As a result, WNK expression rises, leading to elevated NCC phosphorylation and expression.This results in elevated Na ϩ reabsorption, high blood pressure, RAAS inactivation, hypercalciuria, and hyperkalemia.How the KLHL3/CUL3 and NEDD4-2 pathways interact with each other remains unknown.One hypothesis could be that the WNKs inhibit NEDD4-2 either directly or via SGK1 activation (30, 107) (FIGURE 2C).Finally, in the case of NEDD4-2 ablation, NCC cannot be ubiquitylated and degraded, resulting in increased NCC surface expression and function, elevated Na ϩ reabsorption, high blood pressure, RAAS inactivation, and hypercalciuria.In the Nedd4L Pax8/LC1 knockout mice, the increased NCC was phosphorylated, indicating that the WNK pathway is activated (78) (FIGURE 2D).How the KLHL3/CUL3 and NEDD4-2 pathways interact with each other remains unknown.

Conclusions
The recent studies in the field of ubiquitylation revealed the importance of this process in the regulation of Na ϩ reabsorption and blood pressure by the kidney.However, the importance of well known ubiquitylated targets like ENaC has probably to be redefined, and novel targets like NCC are now emerging.
Indeed, as discussed very recently by Ellison (20), there is more and more evidence suggesting that the absence of NEDD4-2-mediated ubiquitylation is not a prerequisite for upregulating ENaC (5,78).The recent data in this field rather suggest that 1) the ␣-ENaC abundance (and thus ENaC function) is mainly regulated by aldosterone and 2) the principal effect of NEDD4-2 on ENaC might be the regulation of the channel degradation inside the cell and not its removal from the cell surface.This last proposal should, however, be taken with caution since others have suggested that ubiquitylation plays an important role in driving ENaC endocytosis (35).
In fact, the recent studies have rather demonstrated the critical role of ubiquitylation in renal physiology by modulating the abundance of NCC.However, the underlying mechanisms of how NCC phosphorylation and ubiquitylation interact with each other to regulate NCC remain to be clarified.The observation that NEDD4-2 regulates NCC activity by ubiquitylation and reduction of its surface expression (3,78), whereas KLHL3/CUL3 seems to target WNK1/4 (67,88,102,106), may lead to the hypothesis that NEDD4-2 regulates NCC activity via direct ubiquitylation, whereas KLHL3/CUL3 may act indirectly by ubiquitylating the NCC regulators, namely the WNKs, thus decreasing NCC phosphorylation.Importantly, inhibiting the NEDD4-2-NCC pathway appears not to be sufficient for resulting in a PHAII phenotype, whereas ablating the KLHL3/ CUL3-WNK-NCC pathway does.Of interest, the work of Melanie Cobb has shown in vitro that the WNKs can inhibit NEDD4-2 either directly or via SGK1 activation (30,107).How exactly and in which physiological conditions the HECT ubiquitin-ligase NEDD4-2 pathway and the RING ubiquitin-ligases KLHL3 and CUL3 pathway are involved in the modulation of NCC represent important questions for

FIGURE 1 .
FIGURE 1. Model of NEDD4-2-mediated ubiquitylation of ENaC in normal and pathological conditionsA: in conditions of RAAS activation, aldosterone induces SGK1 expression that leads to phosphorylation and inhibition of NEDD4-2, thus disrupting the NEDD4-2/ENaC interaction and subsequent ubiquitylation and degradation of the channel.In parallel, misfolded or unassembled polyubiquitylated ENaC channels are degraded via the ubiquitin-proteasome pathway in the ER in the context of ERAD (ER-associated degradation).The deubiquitinating enzymes (DUBs) are also involved in deubiquitylation and recycling of ENaC.B: in conditions of RAAS inactivation, aldosterone and SGK1 are low, whereas NEDD4-2 is highly expressed.NEDD4-2 ubiquitylates ENaC and leads either to degradation of the channel or to its recycling.It remains unclear whether NEDD4-2-mediated ENaC ubiquitylation happens on the channel at the cell surface or on the intracellular pool.C: in Liddle patients, mutations in the PY motifs of ENaC result in absence of interaction between ENaC and NEDD4-2 and thus defect in ubiquitylation and degradation of the channel, leading to permanently enhanced cell surface ENaC expression.D: in NEDD4L Pax8/LC1 KO mice generated by Ronzaud et al.(78), it was shown that absence of NEDD4-2 protein expression leads to increased intracellular expression of ␤-ENaC and ␥-ENaC, but not ␣-ENaC.It was proposed that ␣-ENaC is regulated at the transcriptional level by aldosterone, thus leading to diminished ENaC activity to compensate increased NCC-mediated Na ϩ reabsorption (see FIGURE2).

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220.33.1 on April 27, 2017 http://physiologyonline.physiology.org/ ؉ Reabsorption and Blood Pressure: An Emerging Field Given the relevance of NCC activity in human hypertension (90), many studies have focused on how by 10.220.33.1 on April 27, 2017 http://physiologyonline.physiology.org/Downloaded from NCC is regulated.It was first described in 2006 by Pacheco-Alvarez et al. that NCC phosphorylation on T53, T58, and S71 is crucial for its activity