Title : Renal tubular SGK 1 deficiency causes impaired K + excretion via loss of regulation of NEDD 4-2 / WNK 1 and ENaC

The stimulation of postprandial K(+) clearance involves aldosterone-independent and -dependent mechanisms. In this context, serum- and glucocorticoid-induced kinase (SGK)1, a ubiquitously expressed kinase, is one of the primary aldosterone-induced proteins in the aldosterone-sensitive distal nephron. Germline inactivation of SGK1 suggests that this kinase is fundamental for K(+) excretion under conditions of K(+) load, but the specific role of renal SGK1 remains elusive. To avoid compensatory mechanisms that may occur during nephrogenesis, we used inducible, nephron-specific Sgk1(Pax8/LC1) mice to assess the role of renal tubular SGK1 in K(+) regulation. Under a standard diet, these animals exhibited normal K(+) handling. When challenged by a high-K(+) diet, they developed severe hyperkalemia accompanied by a defect in K(+) excretion. Molecular analysis revealed reduced neural precursor cell expressed developmentally downregulated protein (NEDD)4-2 phosphorylation and total expression. γ-Epithelial Na(+) channel (ENaC) expression and α/γENaC proteolytic processing were also decreased in mutant mice. Moreover, with no lysine kinase (WNK)1, which displayed in control mice punctuate staining in the distal convoluted tubule and diffuse distribution in the connecting tubule/cortical colleting duct, was diffused in the distal convoluted tubule and less expressed in the connecting tubule/collecting duct of Sgk(Pax8/LC1) mice. Moreover, Ste20-related proline/alanine-rich kinase phosphorylation, and Na(+)-Cl(-) cotransporter phosphorylation/apical localization were reduced in mutant mice. Consistent with the altered WNK1 expression, increased renal outer medullary K(+) channel apical localization was observed. In conclusion, our data suggest that renal tubular SGK1 is important in the regulation of K(+) excretion via the control of NEDD4-2, WNK1, and ENaC.


INTRODUCTION
Maintenance of plasma potassium (K + ) level within normal physiological range (3.5 to 5.0 mM) is crucial for proper function of excitable cells (e.g., neurons, skeletal and cardiac myocytes) and represents a significant homeostatic challenge that is mediated by the tight coordination of K + excretion via the kidney (and to a lesser extent colon), and K + storage occurring mainly in skeletal muscle (67).It is well accepted that control of K + balance by the kidney involves both aldosterone-dependent and -independent mechanisms (55,56,64).Renal K + secretion takes place essentially in the so-called aldosterone sensitive distal nephron (ASDN) as defined by Loffing et al. (35).One of the most prominent proteins stimulated by aldosterone in the ASDN is the otherwise ubiquitously expressed serum-and glucocorticoidinduced kinase 1 (SGK1) (28,34).Studies performed in cellular and animal models suggest that SGK1 controls renal Na + reabsorption through regulation of the epithelial Na + channel ENaC and Na + ,Cl --cotransporter NCC (3, 10, 17-19, 37, 65).This involves phosphorylation of S222, S246 and S328 residues of the ubiquitin-protein ligase NEDD4-2 (5,17,52), known to ubiquitylate and negatively regulate ENaC and NCC (1,3,22,26,45,51).On the other hand, it has also been suggested that SGK1 acts via phosphorylation of WNK kinases (12,44,48).
Several in vitro and in vivo reports confer to SGK1 a positive role in the regulation of K + secretion.In heterologous expression systems SGK1 is able to stimulate the K + -channel ROMK (KCNJ1) cell surface expression (38,66).Huang et al. (23) reported that mice harboring constitutive gene inactivation of Sgk1 (Sgk1 -/-) exhibited a defect in acute K + excretion after 30-60 min of intravenous K + load.At 6 days of high K + diet (HKD), Sgk1 -/-mice developed hyperkalemia but excreted K + similarly to control mice (23).Surprisingly, ROMK membrane expression was enhanced in Sgk1 -/-mice (23).The ubiquitous expression of SGK1 together with the involvement of the colon, the skeletal muscle and the liver in K + homeostasis, leave the possibility that the impaired ability of Sgk1 -/-mice to handle K + load was due to renal and extra-renal SGK1 (6,29).
To decipher the specific role of renal-tubular SGK1 in K + homeostasis, we took advantage of the previously described inducible, nephron specific Sgk1 Pax8/LC1 model (18).These mice showed a Na + losing phenotype when Na + supply was restricted, which correlated with decreased NCC and ENaC protein levels, and reduced NEDD4-2 phosphorylation (18).
Here, we show that even though SGK1 is expressed in several organs involved in K + homeostasis, the action of renal-tubular SGK1 is crucial for proper renal K + excretion and is mediated mainly by ENaC.Our data suggest that NEDD4-2 and WNK1 are key players in SGK1-mediated K + homeostasis.

MATERIAL AND METHODS
Induction of renal-tubule specific SGK1 mutant mice and the verification of the renal tubular specificity.Inducible, renal-tubule specific SGK1 flox/flox /Pax8-rTA/LC1 KO (SGK1 Pax8/LC1 ) or homozygous littermate controls Sgk1 Pax8 or Sgk1 LC1 mice were generated as previously described (18).Mice were housed in a temperature-controlled facility (19-22°C) with a 12:12-h light-dark cycle.To induce gene deletion, 21 to 24 day old mice were treated with doxycycline (2 mg/ml in 2% sucrose in drinking water) for 12 days.Male mice were used in the study after 1 to 3 weeks following doxycycline treatment.Genotype was identified by PCR performed on ear biopsies as described previously (18).Experimental protocols were designed with respect to the Swiss Animal Welfare Act and approved by the veterinary administration of the Canton de Vaud, Switzerland, authorization number is 2590.
Plasma and organs collection.Mice were anesthetized by Ketamine/Xylazine intraperitoneal injection; blood was collected by exsanguination from the retro-orbital plexus in SARSTEDT heparin contained micro tubes and plasma was separated according to manufacturer's instructions.Mice were then humanely euthanized by cervical dislocation.
Metabolic cages, urine and plasma analysis.After 2 days of adaptation in metabolic cages (Indulab, Cat.#3600M021), data related to bodyweight, food and water intake were registered and 24h urine samples were collected as described previously (18).Urine analysis (Na + , K + , Ca 2+ , Mg 2+ , PO4 3-, Creatinine, Urea) was performed by the Laboratory of Clinical Chemistry at the Lausanne Hospital (CHUV) using a Modular Analytics System (Roche Diagnostics).Plasma Na + and K + levels were measured with a flame photometer (Cole-Palmer Instrument).Plasma aldosterone measurement was performed at the Service of Nephrology of the CHUV using the radioimmunoassay kit ALDOSTERONE-RIACT according to the manufacturer's instructions.
Immunoblots were carried out and proteins quantified as previously described (45).
Generation and characterization of ROMK antibody.Anti-ROMK antibodies are directed against a sequence of 49 residues (342-391) of rat ROMK protein (NFGKTVEVETPHCAMCLYNEKDARARMKRGYDNPNFVLSEVDETDDTQM).Guinea pigs were immunized by GST fusion proteins containing the ROMK peptide.Antibodies were generated at Cocalico Biologicals.Inc. (Reamstown, PA, USA).Sera of the immunized animals were collected and affinity-purified using the Maltose Binding Fusion Protein (MBP) system.
The specificity of the antibodies was validated by Western blotting of lysates of HEK293 cell transfected with a plasmid encoding ROMK and kidneys from WT and ROMK knockout mice (Kcnj1 KO) (36) kept under HKD for 5 days to enhance ROMK protein expression (Fig. 5).
Immunohistochemistry. Mice were anesthetized by Ketamine/Xylazine i.p. injection.Cardiac perfusions with PBS followed by PFA 4% were performed.Before freezing, fixed kidneys were kept in 30% sucrose in PBS solution at 4°C overnight.Immunostaining was performed on 5µ cryosections using the primary antibodies listed in the antibody section below.

Sgk1 Pax8/LC1 mice display suppression of SGK1 in the kidney, but not in the liver.
The present model, Sgk1 Pax8/LC1 , was described previously; recombination of genomic DNA was not only reported for the kidney, but also the liver (18), consistent with Pax8 promotor activity as reported previously (57).We therefore analyzed Sgk1 mRNA and SGK1 protein expression in the liver and compared it to the kidney (Fig. 1).Whereas there was recombination as seen previously in both organs (Fig. 1A), mRNA and protein levels of SGK1 (using an anti pT256-SGK1 antibody) were not affected in the liver, whereas they were strongly reduced in the kidney (Fig. 1B-D).We therefore conclude that SGK1 is primarily deleted in the nephron; we cannot exclude, however, that there is some suppression in a minor subpopulation of liver cells.
Severe hyperkalemia correlating with diminished K + excretion in Sgk1 Pax8/LC1 mice under challenging conditions.
To understand the involvement of renal-tubular SGK1 in K + homeostasis, we assessed metabolic parameters of control and inducible Sgk1 Pax8/LC1 mice under normal (ND; containing 0.3 % K + ) and high K + diet (HKD, 5% of K + ; K + 3-citrate) after 48 hours.We sacrificed the animals at the end of the daylight period, in order to have maximal aldosterone levels and assure highest difference between Sgk1 Pax8/LC1 and controls.No significant differences in urine and plasma electrolytes were observed between the genotypes under ND (Fig. 2 and Table 1).After 2 days of HKD, Sgk1 Pax8/LC1 mice developed severe hyperkalemia and mild hyponatremia (Fig. 2A, B), accompanied with elevated aldosterone levels (Fig. 2C), but no change of mRNA levels of renin 1 in the kidney (Fig. 2F).K + excretion was enhanced upon the consumption of HKD in both genotypes, but this increase was less important in Sgk1 Pax8/LC1 mutants (Fig. 2D).No significant difference in Na + excretion was observed between the two groups (Fig. 2E).After 5 days of HKD consumption, the mutant mice were capable to increase their urinary K + secretion to levels similar to the one observed in control mice, as no significant difference in K + excretion was observed between control (1664.2+/-71 mmol/day) and mutant (1319.5 +/-124 mmol/day) mice after 5 days of HKD intake, suggesting that they come back into balance.
Taken together, these data suggest that basal levels of K + excretion do not require the renal-tubular SGK1, whereas the kinase appears to be crucial for the adjustment of K + elimination after a high K + load for 48 hours.Moreover, the finding that Sgk1 Pax8/LC1 show higher circulating aldosterone but no change in kidney renin 1 mRNA levels under basal and HKD conditions, suggests that the increased aldosterone levels are primarily a compensation for disturbed K + regulation.

Aberrant ENaC cleavage, expression and membrane localization in Sgk1
Pax8/LC1 mice under HKD It was previously described that Sgk1 -/-mice develop hyperkalemia when subjected to HKD (23).Electrophysiological measurements in isolated perfused cortical collecting ducts (CCDs) from Sgk1 -/-animals showed lower absolute and amiloride-sensitive transepithelial potential differences, suggesting a defect in Na + ,K + -ATPase and/or ENaC (23).We therefore assessed ENaC expression and localization including full-length and proteolytically processed forms, representing ENaC maturation and activation (24,46).Under ND, ENaC showed similar protein expression and patterns in Sgk1 Pax8/LC1 versus control mice (data now shown).However, after 2 days of HKD, the cleavage product of αENaC, and both full-length and cleaved γENaC were decreased with no change in βENaC (Fig. 3A,B); this difference was accompanied by a decrease in αENaC apical localization and a reduction in intracellular and apical γENaC expression (Fig. 3C).mRNA levels of the ENaC subunits did not change (not shown).These observations suggest that under HKD, the deficiency of renal epithelial SGK1 causes a defect in ENaC regulation.
Sgk1 Pax8/LC1 mice display alterations in NEDD4-2/WNK1pathway under HKD As outlined above, it is well documented that SGK1 phosphorylates NEDD4-2, thereby interfering with NEDD4-2 dependent inhibition of ENaC and NCC (3,5,8,17,52).Because membrane localization of α-and γENaC was altered in Sgk1 Pax8/LC1 mice under HKD, we evaluated the phosphorylation status of NEDD4-2 and observed a reduction in S222 and S328 phosphorylation in Sgk1 Pax8/LC1 mice (Fig. 4A,B).Interestingly, total NEDD4-2 expression was also decreased (Fig. 4A-C) without any change in mRNA levels (not shown).No alteration in NEDD4-2 phosphorylation or protein levels was observed in the Sgk1 Pax8/LC1 mice under normal diet (not shown).We have recently demonstrated that WNK1 is ubiquitylated and negatively controlled by NEDD4-2.Proteins levels of the "long" kinase active isoform (L-WNK1) protein levels were increased in Neddl Pax8/LC1 animals kept under high Na + diet and decreased in Sgk1 Pax8/LC1 mice under low Na + diet (47).Under the present conditions (48h of HKD), Western blot analysis did not reveal a significant decrease in WNK1 in the Sgk1 Pax8/LC1 mice (Fig. 4A,B), but we observed important changes by immunofluorescence analysis.The stainings in control mice showed that in the DCT (co-localization with NCC), WNK1 exhibited a punctate pattern (Fig. 4D).Such localization profile had been previously described for WNK4 (55).On the other hand, in the CNT/CD (co-localization with AQP2) of these mice, WNK1 was homogenously diffused within the cells (Fig. 4E).This segment-specific pattern of WNK1 may be related to differences in the signaling pathway associated to WNK1 in each segment.In contrast, in the Sgk1 Pax8/LC1 mice, WNK1 in the DCT displayed a diffuse localization (Fig. 4D).In addition, its expression was reduced in the CNT/CD compared to control littermates (Fig. 4E).These deregulations were related to a decrease in WNK activity in both DCT and CNT/CD of mutant compared to control mice as evidenced by the reduced staining with a p382WNK1/WNK4 (Pan-pWNK) antibody, an indicator of WNK1/WNK4 activity (Fig. 5).These data suggest that SGK1 controls NEDD4-2 phosphorylation also under HK conditions (as was the case with a low Na + diet ( 18)), and influences the subcellular localization/expression and activity of WNK1.
Surprisingly, however, in the hyperkalemic Sgk1 -/-KO mice, apical localization of ROMK was increased (23).One possible explanation for this apparent contradiction might be compensatory mechanisms taking place during nephrogenesis.We generated a novel anti-ROMK antibody in guinea pigs as described in Material and Methods, and validated the antibody by Western blotting (Fig. 6A,B).The antibody recognized transfected ROMK in HEK293 cells and in total kidney membrane extracts, whereas the corresponding bands were not visible in non-transfected cells or in kidneys from ROMK KO mice (Kcnj1 KO mice).We then analyzed ROMK in Sgk1 Pax8/LC1 kidneys from mice kept under HKD and found also in this model a slight increase in the fully-glycosylated form of ROMK in Sgk1 Pax8/LC1 animals (Fig. 6C, D).
Immunofluorescence analysis further revealed that ROMK protein abundance and membrane expression were enhanced both in the DCT and CNT/CD of Sgk1 Pax8/LC1 animals as indicate the co-localization images of ROMK with either NCC or αENaC (Fig. 6E, F).This suggests that the hyperkalemia triggered by renal SGK1 deletion is independent of ROMK.
NCC and SPAK phosphorylation is altered in Sgk1 Pax8/LC1 mice NCC dephosphorylation under K + load has been described in several reports (42,(53)(54)(55)58).To analyze the response of NCC to the observed hyperkalemia in Sgk1 Pax8/LC1 mice, we analyzed NCC phosphorylation and expression both under ND and after 2 days of HKD.We found that under ND, NCC phosphorylation and expression were similar in Sgk1 Pax8/LC1 and control mice (not shown).Under HKD, NCC phosphorylation levels were much weaker in the mutant mice and no difference in total NCC expression was observed (Fig. 7A-C).In addition, immunofluorescence analysis of kidney sections revealed that NCC was less localized to the apical membrane in the KO mice (Fig. 7C).We then asked if this weak NCC phosphorylation correlated with changes in SPAK phosphorylation, a kinase known to phosphorylate and activate NCC (43).SPAK is phosphorylated by WNK kinases on threonine (T233) in the Tloop of the kinase domain that is crucial for SPAK activation (2,61).Our IF analyses revealed that similarly to WNK1, total SPAK exhibited a punctuate pattern in the distal convoluted tubule (DCT) of control animals, which was less pronounced in Sgk1 Pax8/LC1 mice (Fig. 7D).
Phosphorylation at T233 was reduced in the DCT as compared to control animals consistent with the reduced NCC phosphorylation (Fig. 7E).Together, these data show that under HKD, NCC phosphorylation and membrane localization are both diminished in the Sgk1 Pax8/LC1 mice, coinciding with a reduction in SPAK activation.

DISCUSSION
This study shows that mice with inducible deletion of renal-tubular SGK1 exhibit reduced K + excretion as compared to control animals, leading to severe hyperkalemia when subjected to HKD.The reduced K + excretion is correlated to altered NEDD4-2 and WNK1 in addition to reduced apical membrane expression of ENaC.Our observations confirm partly the previous report by Huang et al. demonstrating that total Sgk1 -/-mice display normal K + handling when fed standard diet, but develop hyperkalemia, although without a difference in K + excretion, when challenged by HKD (23).We also observed elevated aldosterone levels (but no difference in renin 1 mRNA expression in the kidney) in Sgk1 Pax8/LC1 mice both under standard and K + rich diets similarly to Sgk1 -/-mice (23).Given the similar K + content in the HKD used in both studies (5% K + ), the lower K + excretion observed under chronic HKD in Sgk1 Pax8/LC1 but not Sgk1 -/-mice suggests that renal-tubular SGK1 plays a critical role in the regulation of K + excretion under HKD and its role may be attenuated by compensatory mechanisms triggered during nephrogenesis in Sgk1 -/-mice.Extra-renal SGK1 had been shown previously to play a role in K + homeostasis as suggested by experiments performed in Sgk1 -/- animals demonstrating that hepatic SGK1 regulates kalemia via its role in insulin-stimulated K + uptake (6).In our mice, we found no evidence for suppression of SGK1 in the liver; although we cannot exclude suppression of SGK1 in a subpopulation of liver cells, we consider it as unlikely that hepatic SGK1 plays a major role in this model.Finally, we cannot exclude that strain background differences may contribute to the alterations as well.
Our molecular analyses suggest that the effect of SGK1 on K + balance is mediated at least in part by the positive regulation of ENaC, implicating phosphorylation and consequent interference with NEDD4-2 action (5, 17-19, 21, 25, 31, 52).After 2 days of HKD, NEDD-2 phosphorylation and total expression were reduced in Sgk1 Pax8/LC1 with no alteration of its mRNA level.Similar observations were made in Sgk1 Pax8/LC1 animals under low salt diet showing reduced NEDD4-2 phosphorylation on both S222 and S328 sites (18).However, the decrease in NEDD4-2 total expression found here might be due to the hyperkalemia and/or to increased aldosterone levels.Indeed, van der Lubbe et al. had shown that NEDD4-2 expression was decreased in rats fed with HKD (59).Moreover, Loffing-Cueni et al. had described that low Na + diet caused reduction in NEDD4-2 expression, and provided evidence that it was aldosterone dependent inhibition of NEDD4-2 levels (33).Taken together, this suggests that aldosterone inhibits NEDD4-2 action via 1) SGK1-dependent phosphorylation (likely a rapid mechanism of regulation) and 2) lowering its expression by an uncharacterized posttranscriptional mechanism (slow or late mode).NEDD4-2 phosphorylation at S328 interferes with regulation of ENaC (17,52).Consistent with this, we observed under HKD lower expression of γENaC, as well as decreased cleavage of αand γENaC, features that had been associated with increased ubiquitylation of ENaC (24,27,49,50).Our findings are in line with previous reports showing the decrease in γENaC cleavage in Sgk1 -/-mice treated by aldosterone for 7 days (19).These Sgk1 -/-mice exhibited a Na + losing phenotype under low salt diet, but intriguingly an increase in amiloride-sensitive Na + currents, as measured by whole-cell patchclamping in the CCD.In contrast, low amiloride-sensitive transepithelial potential differences (a marker for ENaC activity) in isolated CCDs from Sgk1 -/-mice under HKD were reported by Huang et al. (23).Similarly to the Sgk1 -/-mice, Sgk1 Pax8/LC1 did not exhibit Na + loss compared to control mice when fed HKD despite the observed decrease in α and γENaC inactivation and NCC.One possible explanation is an increase in Na + reabsorption in the proximal tubules as previously reported in Sgk1 -/-under low salt diet (65).Interestingly, transgenic mice with specific deletion of αENaC in renal-tubules (39) or only in the CNT (13,41)  mice under our conditions, we did observe a striking alteration in the WNK1 localization pattern in both the DCT and the CNT/CD.Taken together with the observation that WNK1 phosphorylation was reduced under HKD conditions, our data suggest that SGK1 regulates NCC phosphorylation via WNK kinases, and the NEDD4-2/WNK1/SPAK pathway participates in this process.Another mechanism for NCC regulation was proposed by Rozansky & collaborators, who suggested that phosphorylation of WNK4 by SGK1 on S1196 inhibits WNK4 dependent downregulation of NCC (48).However, regulation of NCC by WNK4 is complex, and can be both inhibitory and stimulatory, depending on the physiological context (9).To our knowledge, no antibodies against this WNK4 phosphorylation site are currently available (32,44), hence we were not able to test this possibility in our model.It was also reported that intracellular Cl -is able to inhibit WNK kinases (4,40) and evidence was presented that low plasma K + leads to lower intracellular [Cl -] and stimulation of WNK kinase activity (i.e.WNK1, WNK3 and WNK4 (7,54,55)).Consequently, hyperkalemia may cause an increase in intracellular [Cl -] and inhibition of the WNK kinases.Taken together, the deregulation in NCC apical localization and phosphorylation in Sgk1 Pax8/LC1 mice under HKD may be due to decreased NEDD4-2 phosphorylation and reduced L-WNK1 expression and activity, exacerbated by hyperkalemia.Moreover, it is important to note that the reduced phosphorylation levels of NCC is likely a secondary effect of the hyperkalemia and not the cause of it.
The regulation of ROMK by SGK1 is still under debate.Several in vitro reports suggest a positive regulation of the channel by SGK1 (11,38,66).However, in vivo studies do not confirm this hypothesis (23,56).Our data show that ROMK protein levels and membrane localization are increased in Sgk1 Pax8/LC1 mice under HKD, indicating that a deregulation of ROMK cannot account for the decrease in K + excretion in Sgk1 Pax8/LC1 animals.This is in agreement with the observation made in Sgk1 -/-and aldosterone synthase-KO mice, which exhibit hyperkalemia together with increased ROMK membrane expression when fed with HKD (23,56).Importantly, in vitro and in vivo evidences suggest that L-WNK1 inhibits ROMK by stimulating its endocytosis (14,30,60,62).Consequently, the increased ROMK levels are compatible with the observed decrease of WNK1 expression and activity in our mouse model.
We note that the findings regarding ROMK regulation in our mice indicate that the mechanisms of K + excretion, affected by SGK1, may also involve other channels/transporters such as the BK (15), Kir4.1 (Kcnj10) (70) channels or the H + ,K + -ATPase ( 16).This will be addressed by future studies.
Taken together, the renal-tubular SGK1 plays a crucial role in K + homeostasis via the regulation of NEDD4-2 mediated ENaC inhibition.Sgk1 Pax8/LC1 mice exhibit an alteration of WNK1 puncta in the DCT and a decrease of WNK1 staining in the CNT/CD.This is correlated with decreased SPAK/NCC phosphorylation and increased ROMK expression/apical localization in the DCT/CNT/CD.Normal diet (0.3% K) high K diet (5% K) WT ( 12) KO ( 13) WT ( 12) KO (13) Body weight (g)  1: Metabolic parameters of control and SGK1 Pax8/LC1 KO mice under normal diet and after 2 days of high K + diet.No significant differences were observed in these parameters between control and mutant mice.(n=12 WT and 13 SGK1 Pax8/LC1 KO mice in both diets).Note that water intake 24h/body weight was significantly different between ND and HKD with no significant difference in urine volume between both diets which may result from the high variability due to the evaporation in metabolic cages.*= P < 0.05.

Fluor- 546
conjugated to donkey anti-sheep (Thermo Fisher Scientific; Cat #A21098) were used.Images in the Figures are representatives of data obtained from 3 control and 2 Sgk1 Pax8/LC1 mice.
exhibit hyperkalemia and decreased K + excretion.In view of what precedes, we consider it as very likely that the low ENaC cleavage and membrane localization in the Sgk1 Pax8/LC1 mice account at least in part for the observed decrease in K + excretion, by limiting the electrogenic driving force for K + secretion.We also observed that Sgk1 Pax8/LC1 mice exhibited decreased phosphorylation and apical localization of NCC after 2 days of HKD.Vallon et al., had shown that Sgk1 -/-mice fed with HKD (5% K + ) for 7 days exhibit decreased NCC phosphorylation and total protein expression(58).The longer exposure to HKD may explain this more severe NCC alteration observed by these authors.Our data suggest that the decrease in NCC apical localization in Sgk1 Pax8/LC1 mice is likely an effect of reduced NEDD4-2 phosphorylation leading to increased activity towards NCC(3).The deregulation of NCC in our model was accompanied by reduced phosphorylation of WNK1/4 S382 and SPAK T233.Recently, Roy et al. have reported that L-WNK1 kinase, known to stimulate NCC via SPAK/OSR1 kinase, is a target of NEDD4-2 (47) and the expression of L-WNK1 was reduced in Sgk Pax8/LC1 mice fed with a low Na + diet.Though we did not detect a statistically significant reduction in WNK1 protein expression in SgkPax8/LC1

Fig. 1 .
Fig. 1.Sgk1 mRNA and SGK1 protein levels are decreased in the kidney, but not in the liver of Sgk1 Pax8/LC1 mutant mice.A: PCR on genomic DNA extracted from total kidney and liver

Fig. 7 .
Fig. 7. Alteration in SPAK and NCC phosphorylation and cellular localization after 2 days of HKD.A: WB analysis of NCC in control and Sgk1 Pax8/LC1 mice showing reduced NCC