Dietary Sodium Intake Regulates the Ubiquitin-Protein Ligase Nedd4-2 in the Renal Collecting System
Dominique Loffing-Cueni*,||,
Sandra Y. Flores*,
Daniel Sauter,
Dorothée Daidié*,
Nicole Siegrist,
Pierre Meneton,
Olivier Staub* and
Johannes Loffing
* Department of Pharmacology & Toxicology, University of Lausanne, Lausanne, Switzerland; Institute of Anatomy, University of Zurich, Zurich, Switzerland; Unité 652, Institut National de la Santé et de la Recherche Médicale, Paris, France; and Units of; Anatomy and || Histology, Department of Medicine, University of Fribourg, Fribourg, Switzerland
Address correspondence to: Dr. Johannes Loffing, University of Fribourg, Department of Medicine–Anatomy, Route Albert Gockel 1, CH-1700 Fribourg, Switzerland. Phone: +41-26-300-8527; Fax: +41-26-300-9733; E-mail: johannes.loffing{at}unifr.ch
Received for publication June 27, 2005.
Accepted for publication February 8, 2006.
The activity of the epithelial sodium (Na+) channel (ENaC) inthe aldosterone-sensitive distal nephron (ASDN) needs to betightly regulated to match urinary Na+ excretion with dietaryNa+ intake. The ubiquitin-protein ligase Nedd4-2, which in vitrointeracts with ENaC subunits and reduces ENaC cell surface abundanceand activity by ubiquitylation of the channel, may participatein the control of ENaC. This study confirms in vivo by reverse–transcriptase–PCRthat Nedd4-2 is expressed throughout the nephron and is detectableby immunoblotting in kidney extracts. By immunohistochemistry,Nedd4-2 was found to be strongly expressed in the ASDN, withlow staining intensity in the late distal convoluted tubuleand early connecting tubule (where apical ENaC is high) andgradually increasing detection levels toward the collectingduct (CD; where apical ENaC is low). Compared with high-Na+diet (5% Na+), 2 wk of low-Na+ diet (0.01% Na+) drasticallyreduces Nedd4-2 immunostaining and increases apical ENaC abundancein ASDN. Reduced Nedd4-2 immunostaining is not dependent onincreased apical Na+ entry in the CD, because it is similarlyobserved in mice with intact and with suppressed apical ENaCactivity in the CD. Consistent with a role of mineralocorticoidhormones in the long-term regulation of Nedd4-2, 5-d treatmentof cultured CD (mpkCCDcl4) cells with 1 µM aldosteroneleads to reduction of Nedd4-2 protein expression. It is concludedthat Nedd4-2 abundance is regulated by Na+ diet, by a mechanismthat likely involves aldosterone. This regulation may contributeto adaptation of apical ENaC activity to altered Na+ intake.
Renal sodium (Na+) excretion has to match dietary Na+ intaketo maintain extracellular salt and volume homeostasis. The aldosterone-sensitivedistal nephron (ASDN), which comprises late distal convolutedtubule (DCT, or DCT2 [1]), connecting tubule (CNT), and corticaland medullary collecting duct (CCD and MCD, respectively) (2),mediates the final control of renal Na+ excretion (3,4). TheNa+ transport rate along this segment critically depends onthe activity and abundance of the epithelial Na+ channel (ENaC)in the luminal membrane. Consistently, ENaC is a main targetby which hormones and other factors control the Na+ transportin the ASDN (3,4).
Aldosterone profoundly stimulates ENaC activity and increasesNa+ transport in the ASDN and likely mediates many effects ofaltered Na+ intake on renal Na+ handling (for comprehensivereviews on ENaC regulation and/or aldosterone, action consult[3,4]). Exogenous aldosterone application and dietary Na+ restriction(increasing endogenous aldosterone) similarly augment the numberof active Na+ channels in the luminal membrane of the ASDN,which involves a redistribution of all three ENaC subunits (,, and ) from a cytoplasmic pool to the apical plasma membrane(2,5,6). The apical redistribution of ENaC shows a remarkableaxial heterogeneity along the ASDN. It is most pronounced inthe early ASDN portions (late DCT and early CNT) and progressivelydecreases along further downstream localized segments (lateCNT and CD) (2,6). This morphologically ascertainable gradientis consistent with numerous functional studies showing several-foldhigher transepithelial Na+ transport rates and ENaC activityin the CNT than in the CD (7–11). However, the underlyingmolecular mechanisms for this axial heterogeneity are unclear.
An important pathway that controls the abundance and activityof ENaC in the apical plasma membrane involves the ubiquitin-proteinligase Nedd4-2, an intracellular enzyme that is known to interactwith so-called PY motifs (xPPxY; x is any amino acid, P is proline,and Y is tyrosine) on the C-termini of each ENaC subunit (12–15).Nedd4-2 acts in concert with the ubiquitin-activating enzymeE1 and with the ubiquitin-activating enzyme UBE2E3 (16) to conjugateubiquitin peptides on the N-termini of the ENaC subunits (17).Such ubiquitylated channels are internalized and sorted fordegradation by lysosomal and proteasomal pathways (12,13,17).Moreover, recent evidence suggested that some of the ubiquitylatedchannels are released via exosomes into the tubular lumen ofthe renal collecting system (18). In Liddle’s syndrome,a rare form of monogenic hypertension, mutations in the - or-ENaC genes delete the PY motifs (19,20). Recently, it was shownthat the interaction between Nedd4-2 and ENaC is regulated byserum- and glucocorticoid-induced kinase 1 (Sgk1), a proteinkinase whose expression is regulated by aldosterone and thatis able to stimulate ENaC and transepithelial Na+ transport(21–23). It is thought that aldosterone stimulates transepithelialNa+ transport in part by increasing expression of Sgk1 and byphosphorylation of Nedd4-2. Nedd4-2 phosphorylation generatesNedd4-2 binding sites for 14-3-3 proteins, causing reduced Nedd4-2/ENaCinteraction and consequently reduced ubiquitylation and accumulationof ENaC at the plasma membrane (24–28).
Notably, these concepts have been derived mainly from experimentsin heterologous expression systems or cell lines as model systemsfor the ASDN. Little is known about the in vivo regulation ofNedd4-2. Previous studies addressed the short-term effect ofaldosterone on Nedd4-2 in the CD but did not examine the morechronic effects of altered dietary Na+ intake (24). In thisstudy, we used two Nedd4-2–specific affinity-purifiedNedd4-2 antisera, directed against different epitopes withinNedd4-2, to provide a comprehensive analysis of the distributionand regulation by dietary Na+ intake of Nedd4-2 along the nephron.
Animal Experiments
Animal studies were in accordance with the Guide for Care andUse of Laboratory Animals (Institute of Laboratory Animal Resources,National Academy of Science, Bethesda, MD). Experiments wereperformed with 6- to 8-wk-old male NMRI mice (Iffa Credo, Arbresle,France), male Sprague Dawley rats (Harlan, Horst, Netherlands),and kidneys from previously described male Scnn1aloxlox andScnn1aloxloxCre mice (29).
Series 1.
Thirty NMRI mice (body weight between 25 and 30 g) were randomlydivided in two groups of 15 mice each. Group 1 received for2 wk a diet with high Na+ contents (5.0%), whereas group 2 receiveda diet with low Na+ (0.01%) contents (UAR, Epinay-S/Orge, France).During the last 4 d of the experiment, mice were housed individuallyin metabolic cages to record 24-h urinary volume and urinaryNa+ and K+ excretion. Urinary Na+ and K+ concentrations weremeasured with an indirect potentiometer (Beckman model E2A,Villepinte/Orsy, France). Plasma levels of aldosterone weremeasured by RIA (Aldo RIA; Sanofi Diagnostics, Marnes-La-Coquette,France) in blood that was taken from the retrobulbar venousplexus under anesthesia with a combination of ketamine (Narketan10, 80 mg/kg body wt; Chassot, Belp, Switzerland) and xylazine(Rompun, 33 mg/kg body wt; Bayer, Leverkusen, Germany).
Series 2.
Ten Sprague Dawley rats (body weights between 200 and 220 g)were divided in two groups of five rats each and received eitherthe standard diet (0.3% Na+) or a low-Na+ (0.01%) diet for 2wk.
Series 3.
Rubera et al. (29) generated by CreLox technology mice withtargeted inactivation of -ENaC specifically in the CD. Micewith "floxed" but intact -ENaC alleles (Scnn1aloxlox) are similarto wild-type mice. Mice with CD-specific -ENaC deletion (Scnn1aloxloxCre)lack any apical ENaC activity in the CD but are otherwise normal.The remaining ENaC activity in connecting tubules seems to besufficient to keep the animals in Na+ balance even when challengedby 6 d of Na+ restriction (29). Kidneys of four Scnn1aloxloxand four Scnn1aloxloxCre mice that were kept on standard-Na+(0.3%) diet and four Scnn1aloxlox and five Scnn1aloxloxCre micethat were kept for 6 d on low-Na+ (0.01%) diet were used forthis study.
Reverse Transcriptase–PCR on Microdissected Tubules
Nephron segments were obtained by microdissection from threenormal mice as described previously (30). After anesthesia (Nembutal,5 mg/100 g body wt), the left kidney was perfused with collagenasethat was dissolved in microdissection medium. Thin pieces oftissue were postincubated in the microdissection solution thatcontained collagenase (1 mg/ml). Glomeruli, proximal tubules(PT), medullary and cortical thick ascending limbs (mTAL andcTAL, respectively), and CCD were isolated. Total RNA were extractedfrom isolated glomeruli (5–10) and nephron segments (40mm each) using a microtechnique (30) adapted from the methodof Chomczynski and Sacchi (31). Reverse transcriptase (RT) wasperformed using Superscript II (Invitrogen, Carlsbad, CA) with5x RT buffer (4 µl), 100 mM dithiothreitol (2 µl),50 µM random hexamers (1 µl; Applied Biosystems,Foster City, CA), 20 mM dNTP (1 µl; GE Healthcare, Munich,Germany), H2O (1 µl), and 10 µl of tubules RNA.For analysis of Nedd4-2 expression along the nephron, standardRT-PCR was carried out on total RNA from 1 mm of isolated nephronsegments of three normal mice using the Titan one-tube RT-PCRsystem (Roche, Rotkreuz, Switzerland) with the following Nedd4-2primers: Sense 5'-ccattctacaagatgatgctgggg-3', antisense 5'-gctctattgtaaacagctgaggacc-3';35 cycles of PCR. Amplified DNA fragments were visualized onan agarose gel that was stained with ethidium bromide. For quantitativereal-time RT-PCR (7500 Fast Real-Time PCR System; Applied Biosystems),reverse transcription was performed on total RNA from microdissectedmouse CCD tubules, using Superscript II (Invitrogen). For thequantitative real-time PCR, a standard curve (five dilutions)was done with cDNA from mpkCCDcl4 cells (32). The reaction wasperformed in a total volume of 20 µl according to themanufacturer’s instructions; each reaction was done intriplicate. The primers were from Applied Biosystems, correspondingto the following order numbers: glyceraldehyde-3-phosphate dehydrogenase(mouse GAPDH), Mm99999915_g1; mSGK1, Mm00441380_m1; and mNedd4-2,Mm00459584_m1. The data were analyzed using the software providedwith the instrument, and results were normalized to GAPDH andto the low-Na+ condition.
Western Blot Analysis
For each mouse, 70 µg of protein extracts from homogenizedrenal cortex was separated by SDS-PAGE (7% polyacrylamide gels)and blotted on polyvinylidene difluoride membranes (Bio-Rad,Reinach, Switzerland). Membranes were blocked with dry milk(5%) in a Tris-NaCl-Tween buffer and incubated with either affinity-purifiedrabbit anti-mouse Nedd4-2 antisera A26 (24) diluted 1:500 orA27 (33) diluted 1:200. Bound Nedd4-2 antibodies were revealedwith horseradish peroxidase–conjugated sheep anti-rabbitIgG (Amersham, Pharmaceutica, Otelfingen, Switzerland) followedby enhanced chemiluminescence (ECL; Amersham) and exposure toBiomax XAR films (Kodak, Ro-chester, NY). Subsequently, blotswere exposed to mouse anti–-actin antibodies (Sigma, St.Louis, MO; dilution 1:5000) and horseradish peroxidase–conjugatedgoat anti-mouse IgG (Amersham). Signal intensities of recognizedNedd4-2–related bands were quantified using a molecularimager FX (Bio-Rad, Hercules, CA) and normalized to the -actincontrol. In competition experiments, the primary antibodieswere preincubated with the GST fusion proteins (100 µg/ml)that were used for the immunization of the rabbits.
Immunohistochemistry
The kidneys of anesthetized mice and rats were fixed by vascularperfusion with 3% paraformaldehyde/0.05% picric acid and processedfor immunohistochemistry as described previously (6). Serialcryosections (4 µm thick) were incubated with one of thefollowing primary antibodies: Affinity-purified rabbit anti-mouseNedd4-2 (A26; [24]) diluted 1:500, affinity-purified rabbitanti-mouse Nedd4-2 (A27 [33]) diluted 1:200, rabbit anti-rat-ENaC diluted 1:500 (29), rabbit anti-rat -ENaC diluted 1:500(29), mouse anti-bovine H-ATPase diluted 1:4 (34), or mouseanti-chicken calbindin D28K diluted 1:20,000 (Swant, Bellinzona,Switzerland). The binding sites of the primary antibodies wererevealed by Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearchLaboratories, West Grove, PA) and FITC-conjugated goat anti-mouseIg (Jackson ImmunoResearch Laboratories) diluted 1:1000 and1:40, respectively. All antibody dilutions were in PBS-1% BSA.The specificity of the Nedd4-2 antibodies was confirmed by preincubationswith the GST fusion proteins (100 µg/ml) that had beenused for the immunization of the rabbits.
Evaluation of Immunofluorescence
The sections were studied with a Zeiss fluorescence microscopeby three investigators (D.L.C., D.S., and J.L.), who were blindedto the treatment or genotype of the animals. Tubular segmentswere identified according to standard morphologic criteria.CNT and CCD were distinguished on the basis of their localizationin the cortical labyrinth and in the medullary rays, respectively.Qualitative judgments regarding immunostainings were similarfor all investigators. For further confirmation of qualitativeratings about Nedd4-2 immunofluorescent intensities, micrographsof the stained cryosections were taken and analyzed by determiningthe mean pixel intensities (MPI) in at least three randomlyselected CCD profiles of each animal. MPI were determined withNational Institutes of Health Imaging software, normalized tothe background fluorescence in nuclei of PT, and expressed inarbitrary units as described previously (24)
Cell Culture
Experiments were carried out in the mpkCCDc14 cell line, a cloneof principal cells that has been derived from microdissectedCCD of an SV-PK/Tag transgenic mouse (32). Cells were culturedon plastic tissue-culture flasks as described previously (35).For studying the effect of aldosterone on Nedd4-2 expression,cells were grown on collagen-coated filters (Transwell 0.4-µmpore, 4.7 cm2). At confluence, cells were maintained in modifiedHam’s F12 medium without EGF, transferrin, and FCS. Cellsthen were kept for 5 d in the presence of vehicle or 1 µMaldosterone. The medium was replaced daily, and fresh aldosteronewas added. Cells were lysed in lysis buffer (50 mM HEPES [pH7.5], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, and1% Triton X-100) that contained protease inhibitors (1 mM PMSF,10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10µg/ml aprotinin). Samples were analyzed by SDS-PAGE/Westernblotting as described above.
Statistical Analyses
The data are given as means ± SEM. Statistical differencesbetween means of groups were evaluated by t test or by ANOVAfollowed by Bonferroni multiple comparisons test using standardcomputer software. Differences were considered statisticallysignificant at P > 0.05.
Effect of Na+ Diets on Urinary Na+ and K+ Excretion and Plasma Levels of Aldosterone
To establish experimental conditions in which to study the influenceof Na+ diet on Nedd4-2 expression, we confirmed first the effectof Na+ diets on urinary Na+ and K+ excretion and on plasma levelsof aldosterone. Mice were kept either on a high (5% Na+) ora low (0.01% Na+) dietary Na+ intake. After 2 wk on the dietaryregimens, urinary Na+ and K+ excretion was determined by urinesampling during the last 24-h period. Mice with high dietaryNa+ intake excreted significantly higher amounts of Na+ (2935± 179 µmol Na+/d; n = 15 mice) and water (9.75± 1.31 ml/d water; n = 15 mice) than mice that fed alow-Na+ diet (12 ± 5 µmol Na+/d; 1.01 ±0.46 ml/d water; n = 15 mice). The high water excretion on highNa+ intake is likely explained by salt-induced thirst. The urinaryK+ excretion was not affected (372 ± 28 [high Na+] versus368 ± 37 µmol/d K+ [low Na+]; n = 15 mice per group).In mice that were fed for 2 wk the high-Na+ diet, the plasmalevel of aldosterone was 46.4 ± 3.5 pg/ml (approximately0.1 nM), and in mice that were fed for 2 wk the low-Na+ diet,the level was 414.7 ± 23.7 pg/ml (approximately 1.0 nM).
Detection of Nedd4-2 mRNA in Microdissected Nephron Portions
Previous studies on rat (24) and human (36) kidneys indicatedthat Nedd4-2 is expressed in several nephron portions. To confirmthese observations for mouse kidneys, we performed RT-PCR experimentson microdissected tubules from mice (n = 3) that were on thestandard-Na+ diet (0.8% Na+). Nedd4-2 mRNA expression was foundin PT, mTAL and cTAL, and CCD (Figure 1). The strongest signalsfor Nedd4-2 mRNA were seen in samples from PT and CCD. The signalintensities of samples from mTAL and cTAL were usually lessintense and for mTAL often close to the detection limit (seeFigure 1). Nedd4-2 mRNA was not detectable in microdissectedglomeruli.
Figure 1. Detection of Nedd4-2 mRNA in microdissected nephron portions from mouse kidney. Glomeruli, proximal tubules (PT), medullary and cortical thick ascending limbs (mTAL and cTAL, respectively), and cortical collecting ducts (CCD) were microdissected from murine kidney; RNA was isolated; and reverse transcriptase–PCR (RT-PCR) was performed using primers specific for Nedd4-2 as described in Materials and Methods. The amplified fragments were verified by sequencing.
Characterization of Nedd4-2 Antibodies on Mouse Tissue Samples
For Western blot analysis and immunohistochemistry, we usedaffinity-purified antisera A26 (24) and A27 (33) that were raisedagainst different epitopes and that specifically recognize mouseNedd4-2 but not Nedd4 (33). In Western blot analysis of mousekidney cortex homogenates, both antibodies showed similar bindingpatterns with one major band at approximately 120 kD and severaladditional bands at lower molecular weights (Figure 2). Themultiple bands likely reflect the expression of several Nedd4-2variants as a result of alternative promoters and splicing (37–39).Alternative promoters and splicing also may explain the slightdifferences in the banding patterns between both antibodies,because the antibodies recognized different epitopes, whichare not necessarily present in all Nedd4-2 variants. Preincubationof the antibodies with the Nedd4-2 GST fusion protein that wasused for immunization completely abolished the binding of theantibody to the membrane (Figure 2).
Figure 2. Characterization of affinity-purified anti–Nedd4-2 antisera A26 and A27 by Western blot analysis. Homogenates of kidney cortex of normal mice (700 µg of protein loaded per gel in a single wide slot) were subjected to SDS-PAGE analysis and transferred to polyvinylidene difluoride membrane. Using the multiscreen apparatus of BioRad, separated lanes on the same membrane were incubated with affinity-purified anti–Nedd4-2 antisera A26 and A27 as indicated. Preincubation of the affinity-purified antisera with the corresponding fusion protein that was used for immunization (FP26 and FP27, respectively) abolished the specific binding of the antisera to the membrane. The left (A26) and right (A27) halves of the same membrane are shown at different exposure times for enhanced chemiluminescence detection.
In immunohistochemical experiments, both anti–Nedd4-2antibodies showed similar binding patterns with a strong cytoplasmiclabeling in CD profiles. Preincubation of the antibodies withthe corresponding but not with the noncorresponding Nedd4-2GST fusion protein abolished the immunostaining (Figure 3A).Compared with ASDN segments, PT and distal tubules revealedweak Nedd4-2–related immunostaining, which became clearlyapparent only at high antibody concentrations (Figure 3B). Co-immunostainingwith a mAb against calbindin-D28k confirmed Nedd4-2 immunostainingin calbindin-positive DCT and CNT and in calbindin-negativePT (Figure 3B).
Figure 3. (A) Medullary rays in mouse renal cortex. Immunostaining for Nedd4-2 with affinity-purified antisera A26 (diluted 1:500) and A27 (dilution 1:200) shows a strong cytoplasmic staining in CD. Weak immunostaining also is seen in adjacent thick ascending limb (T). Preincubation of the affinity-purified antisera with the corresponding fusion protein that was used for immunization (FP26 and FP27, respectively) abolished the specific binding of the antisera to the section. Each row shows consecutive cryosections. (B) Cortical labyrinth of mouse kidney. Co-immunostaining with affinity-purified antiserum A26 (diluted 1:300) and mouse anti-calbindin D28k (CB; diluted 1:20,000). Nedd4-2 is seen in CB-positive distal convoluted tubules (D) and connecting tubules (CN), as well as in CB-negative proximal tubules (P). The presence of CB-negative intercalated cells (IC) and the slightly weaker CB staining distinguishes the CN from D. Note the diffuse intracellular localization of Nedd4-2 in the cytoplasm of CN and most P and the more subapical localization of Nedd4-2 in D and few P. Nedd4-2 immunostaining in profiles of P is heterogeneous and ranges from strong labeling of all epithelial cells (P), via labeling of only single cells (P') to very weak immunostaining (P"). The strong immunofluorescent labeling of tubular basement membranes and interstitial cells in the right panel of B (calbindin D28k) is due to binding of the secondary FITC-labeled anti-mouse IgG to endogenous immunoglobulins. Bar = approximately 50 µm.
Nedd4-2 Is Expressed in Segment-Specific and Intercalated Cells
The epithelium that lines the ASDN is heterogeneous and composedof segment-specific DCT, CNT, and CD cells and of intercalatedcells (IC). The latter are subdivided further in proton-secretingtype A IC with apical localization of vacuolar H+-ATPase andbicarbonate-secreting type B IC with basolateral localizationof H+-ATPase (40). A third form (non-A–non-B) with diffuselabeling for the H+-ATPase also is frequent in mice (40). Co-immunostainingfor Nedd4-2 and vacuolar H+-ATPase revealed that Nedd4-2 ishighly abundant in the segment-specific ASDN cells and in ICwith predominant baso-lateral and/or diffuse staining with theH+-ATPase antibody (Figure 4), suggesting that among IC Nedd4-2is predominantly expressed in non-A IC.
Figure 4. CCD profile in kidney of a mouse on a high sodium (Na+) intake. Double immunofluorescence on the same cryostat section with affinity-purified rabbit anti–Nedd4-2 antiserum A26 and mouse mAb against vacuolar H+-ATPase. Nedd4-2 immunostaining is seen in H+-ATPase–negative principal cells and in some (arrow) but not all (arrowhead) H+-ATPase–positive IC. The labeling of tubular basement membranes and interstitial cells in the right panel (H+-ATPase) is due to binding of the secondary FITC-labeled anti-mouse IgG to endogenous immunoglobulins. Bar = approximately 20 µm.
Quantitative RT-PCR and Western Blot Analysis Do not Detect Any Significant Effect of Altered Na+ Intake on Nedd4-2 Expression in the Kidney
In heterologous expression systems, the cell surface densityof ENaC is inversely correlated to the expression of Nedd4-2(12,14,26). In rodents, high dietary Na+ intake reduces andlow dietary Na+ intake increases the apical plasma membranelocalization of ENaC (5,6). To test the hypothesis that dietaryNa+ intake may mediate its effects on the subcellular localizationof ENaC by changes in the expression levels of Nedd4-2, we usedfirst quantitative RT-PCR to determine Nedd4-2 expression levelsin CD that were microdissected from the kidneys of mice on differentNa+ diets. The measured Nedd4-2 mRNA expression levels did notdiffer significantly between groups (99 ± 20 [high Na+]versus 100 ± 12 arbitrary units [low Na+], mean ±SEM; P = 0.48 [t test]; n = 6 mice per group; values were normalizedto GAPDH and the low-Na+ condition). Aldosterone-induced Sgk1was significantly increased with dietary Na+ restriction (22± 10 [high Na+] versus 100 ± 13 arbitrary units[low Na+], mean ± SEM; P = 0.0003; n = 6 mice per group;values were normalized to GAPDH and low Na+), consistent withprevious data (41) and confirming the validity of our RT-PCRmethod. Previous studies indicated that aldosterone may havean impact on the abundance of some proteins by posttranscriptionalmechanisms. For example, it has been shown repeatedly that theexpression of the thiazide-sensitive NaCl co-transporter isinduced by aldosterone only at the protein but not at the mRNAlevel (42,43). To determine the effect of dietary Na+ intakeon Nedd4-2 protein levels, we performed immunoblotting withrenal cortex homogenates from mice on different Na+ diets. Wedid not detect significant difference in the abundance of Nedd4-2protein between both groups of mice as demonstrated by densitometricquantification of protein bands that were obtained with theA26 antibody (10.9 ± 2.0 [high Na+] versus 7.6 ±1.0 arbitrary units [low Na+], mean ± SEM; n = 9 miceper group; values were normalized to -actin).
Nedd4-2 Immunostaining Varies in Response to Altered Dietary Na+ Intake and along the ASDN
PT contribute more than 70% to the fractional tubular volumeof the renal cortex. It is conceivable that the "background"expression of Nedd4-2 in PT may prevent the detection of differencesin Nedd4-2 expression in ASDN as long as tissue homogenatesare analyzed. Therefore, we used immunohistochemistry to assessqualitatively the abundance of Nedd4-2 in the ASDN under thedifferent conditions. In the kidneys of mice that were keptfor 2 wk on a high dietary Na+ intake, Nedd4-2 was easily detectablein the cytoplasm of all -ENaC–positive CNT and CCD profiles.A low dietary Na+ intake for 2 wk drastically reduced Nedd4-2immunostaining in the ASDN segments. The intensity of Nedd4-2immunostaining varied considerably along the ASDN segments.It was weak in late DCT and early CNT and increased progressivelyalong the CNT and CCD. The axial heterogeneity of Nedd4-2 stainingwas seen with both Nedd4-2 antisera and is well evident whenCNT and CD profiles are compared (Figure 5). The generally weakcytoplasmic Nedd4-2 immunostaining in some PT and distal tubulesdid not differ between mice that were on a low and a high dietaryNa+ intake (Figure 5).
Figure 5. Overviews on renal cortex of mice that were kept for 2 wk on either a high-Na+ (5%) or a low-Na+ (0.01%) diet. Immunofluorescence with rabbit antisera against the subunit of the epithelial Na+ channel (-ENaC) and Nedd4-2 (affinity-purified sera A26 and A27) on triplets of consecutive cryostat sections is shown. Compared with the situation on high Na+ intake, dietary sodium restriction increases the apical localization of -ENaC in connecting tubules (CN) and, although to less extent, in the CD, whereas it decreases the staining intensity with both Nedd4-2 antisera in the same tubules. Note the different Nedd4-2 staining intensity in CN and CD profiles. The weak cytoplasmic Nedd4-2 immunostaining in some proximal tubules (P) is not affected by dietary Na+ intake. Bar = approximately 50 µm.
Higher magnification of consecutive cryosections that were stainedeither with antibodies against ENaC or antibodies against Nedd4-2demonstrated a close inverse correlation between the apicallocalization of ENaC and the abundance of Nedd4-2 (Figure 6).On a high dietary Na+ intake, -ENaC was diffusely distributedwithin the cytoplasm of CNT, CCD, and outer medullary CD (OMCD)cells and Nedd4-2 was highly abundant in the cytoplasm of theepithelial cells of the same tubule. Dietary Na+ restrictionincreased the apical localization of -ENaC and significantlyreduced the cytoplasmic immunostaining for Nedd4-2, when comparedwith the corresponding tubular segments of a mouse that wason a high-Na+ diet. In mice that were on a low dietary Na+ intake,apical localization of ENaC was most prominent in the CNT anddecreased along the CCD toward the OMCD, whereas Nedd4-2 immunostainingexhibited an increasing gradient from CNT to OMCD (Figure 6A).The inverse correlation of apical ENaC localization and Nedd4-2abundance also was seen in rats that were kept on a standard-or a low-Na+ diet (Figure 7A), confirming that the diet changesof Nedd4-2 immunostaining are not species specific. The qualitativeobservations on altered Nedd4-2 immunostaining intensities inresponse to altered dietary Na+ intake were substantiated furtherby measurements of MPI in Nedd4-2 immunostained CD (Figures 6Band 7B).
Figure 6. (A) CNT, CCD, and outer medullary CD (OMCD) profiles from mice that were kept for 2 wk on either a high-Na+ (5%) or a low-Na+ (0.01%) diet. Immunofluorescence with rabbit antisera against -ENaC and Nedd4-2 (affinity-purified sera A26) on pairs of consecutive cryostat sections is shown. On a high Na+ intake, -ENaC is seen predominantly at intracellular sites in CNT, CCD, and OMCD, and Nedd4-2 immunostaining is strong in the same tubules. On a low Na intake, -ENaC is shifted toward the apical plasma membrane, and Nedd4-2 immunostaining is decreased. Note the decreasing intensity of apical -ENaC immunostaining from CNT to CD and the clear inverse relationship of apical ENaC localization and Nedd4-2, which is most evident on low-Na+ diet. Bar = approximately 20 µm. (B) Mean pixel intensities (MPI) of Nedd4-2 immunostaining (affinity-purified sera A26) in CD of mice that were kept on the indicated sodium diets. At least three randomly selected CCD were analyzed per mouse as described (24). Each dot represents the MPI value for an individual mouse. The horizontal bars are the means for each group. The difference between the means is statistically significant (P = 0.003, unpaired t test).
Figure 7. (A) CCD profiles from rats kept for 2 wk on either a standard-Na+ (0.3%) or a low-Na+ (0.01%) diet. Immunofluorescence with rabbit antisera against -ENaC and Nedd4-2 (affinity-purified sera A26 and A27) on triplets of consecutive cryostat sections is shown. Note the predominant intracellular localization of -ENaC, the strong cytoplasmic staining for Nedd4-2 on a standard Na+ intake, the distinct apical localization of -ENaC, and the weak staining for Nedd4-2 on a low Na+ intake. Bar = approximately 20 µm. (B) MPI of Nedd4-2 immunostaining (affinity-purified sera A26) in CD of rats kept on the indicated sodium diets. At least three randomly selected CCD were analyzed per rat as described (24). Each dot represents the MPI value for an individual rat. The horizontal bars are the means for each group. The difference between the means is statistically significant (P = 0.001, unpaired t test).
Intensity of Nedd4-2 Immunostaining Does not Depend on ENaC Activity
The data reported thus far support the hypothesis that dietaryNa+ restriction reduces Nedd4-2 expression in ASDN cells, whichin turn may result in an enhanced cell surface abundance ofENaC. However, it also is conceivable that the increased Na+entry as a result of enhanced ENaC activity is responsible forthe reduced Nedd4-2 immunostaining in response to dietary Na+restriction. To test the latter hypothesis, we took advantageof a recently developed transgenic mouse line with targetedinactivation of -ENaC in the CD (29). The loss of -ENaC in theCD impairs the cell surface targeting of ENaC channels, andthese mice consistently lack any ENaC activity in the apicalplasma membrane of the CD. If the increased ENaC activity isresponsible for the reduced Nedd4-2 expression, then we expectedto see that lowered Na+ intake reduces Nedd4-2 only in CD fromwild-type mice but not in CD from transgenic mice. However,dietary Na+ restriction similarly reduced Nedd4-2 immunostainingin CD profiles of mice of both genotypes (Figure 8). Therefore,apical ENaC activity seems not to interfere with the expressionof Nedd4-2, as determined by immunohistochemistry.
Figure 8. (A) CCD profiles from mice without (Scnn1aloxlox) and with (Scnn1aloxloxCre) targeted inactivation of -ENaC in the CD. The generation and phenotypic characterization of these mice were described previously (29). Mice were kept either on standard-Na+ diet (0.3%) or on dietary Na+ restriction (Na+ 0.01%) for 6 d. Immunofluorescence with rabbit antisera against -ENaC and Nedd4-2 (affinity-purified sera A26) on pairs of consecutive cryostat sections is shown. Dietary Na+ restriction induces -ENaC and increases the apical localization -ENaC in the Scnn1aloxlox mice, whereas -ENaC is virtually absent from the CCD of the Scnn1aloxloxCre mice on both diets. Nedd4-2 immunostaining is well detectable in the CCD profiles of the mice on standard Na+ (0.3%) intake and is similarly weak in the CCD profiles of mice of both genotypes on low-Na+ diet. Bar = approximately 20 µm. (B) MPI of Nedd4-2 immunostaining (affinity-purified sera A26) in CD of mice of the four indicated experimental groups. At least three randomly selected CCD were analyzed per mouse as described (24). Each dot represents the MPI value for an individual mouse. The horizontal bars are the means for each group. Differences between diets but not between the genotypes for each diet were statistically significant (ANOVA followed by Bonferroni multiple comparisons test).
Long-Term Aldosterone Treatment Reduces Nedd4-2 Abundance in mpkCCDcl4 Cells
Because ENaC activity does not regulate Nedd4-2 expression,we wondered whether it is aldosterone that affects Nedd4-2 expression.Chronic effects of aldosterone are difficult to analyze in vivo,because long-term treatment with aldosterone without any concomitantdietary Na+ restriction causes extracellular volume expansion,hypokalemia, and alkalosis, which may confound the interpretationof results. We therefore chose an in vitro approach and stimulatedmpkCCDcl4 cells that were grown on collagen-coated filters forup to 5 d with aldosterone and followed the expression of Nedd4-2by immunoblotting of cell lysates (Figure 9). When cells werecultured without aldosterone, Nedd4-2 remained constant. However,when aldosterone (1 µM) was added, a decrease in Nedd4-2expression was observed starting at day 2 (Figure 9) and progressedup to day 5. The observed suppression of Nedd4-2 expressionwith aldosterone is consistent with the observed weak immunostainingfor Nedd4-2 in animals with low-Na+ diet, a condition in whichplasma aldosterone is high.
Figure 9. Immunoblot of mouse CCD mpkCCD cells that were treated for the indicated time intervals with either vehicle (–Aldo) or 1 µM aldosterone (+Aldo). Membranes were incubated with affinity-purified anti–Nedd4-2 antiserum A26 and subsequently probed with mouse anti–-actin to confirm equal protein loading and transfer. For each lane, cells from three different filter cups were pooled. The shown blot is representative for the results from four independent experiments. Note the time-dependent decrease of Nedd4-2 protein abundance in aldosterone-treated cells that is not seen in vehicle-treated cells.
Our study provides a comprehensive analysis of the localizationand regulation by dietary Na+ intake of Nedd4-2 in rodent kidneys.We show that Nedd4-2 is expressed along the nephron, with thehighest expression levels in ENaC-positive ASDN cells. Moreover,we reveal by immunohistochemistry a close reciprocal relationshipof Nedd4-2 immunostaining intensity and apical ENaC localizationin the kidney in vivo. This inverse relationship is seen bothalong the axis of the ASDN and in response to altered dietaryNa+ intake. Furthermore, we show by immunoblotting that long-termtreatment with aldosterone downregulates Nedd4-2 protein levelsin a mouse CCD cell line. Taken together with previous in vitrodata, our study supports the hypothesis that Nedd4-2 is involvedin the control of apical ENaC localization in the mammaliankidney.
Nedd4-2 expression is found in various tissues and cell types,suggesting that Nedd4-2 has multiple cellular functions. Northernblot analysis revealed Nedd4-2 mRNA expression in brain, heart,lung, liver, and kidney (13,15,33). Within the human kidney,Nedd4-2 mRNA was reported to be expressed along the entire nephron(36). This study confirms these data for the mouse nephron andextends them to the protein level by demonstrating localizationof Nedd4-2 protein in several tubular portions. The highestexpression levels of Nedd4-2 protein were seen in ASDN segments,consistent with the proposed regulatory role of Nedd4-2 forENaC function. However, Nedd4-2 also was seen in tubular segmentsthat do not express ENaC. In particular, PT cells showed a considerableamount of Nedd4-2 mRNA expression and protein immunostaining.The functional role and the molecular targets for Nedd4-2 innon-ASDN tubular segments is unclear. The ClC5 Cl– channelsof PT and the -subunit of the ClCk chloride channel of thickascending limbs, barttin, were suggested to be putative targetsfor Nedd4-2 (44–46). ClC5 and barttin have PY motifs withintheir amino acid sequence, and in the Xenopus oocyte expressionsystem, Nedd4-2 profoundly diminishes protein abundance andactivity of co-expressed ClC5 and barttin via a PY motif–dependentmechanism (44–46).
Several previous in vitro experiments showed that Nedd4-2 decreasesthe abundance and activity of ENaC at the cell surface (12,14,26).Our study suggests that the same may hold true in the ASDN invivo. As presented in Figures 4 through 7, we found a strikinginverse relationship between the Nedd4-2 immunostaining intensityand the apical ENaC localization in ASDN cells of mice and rats.Whereas Nedd4-2 immunostaining increased along the axis of theASDN, apical ENaC localization decreased. The axial gradientof apical ENaC localization was seen in several species andwas evident also in microperfusion, micropuncture, and patch-clampstudies that showed much higher ENaC activity in early ASDN(i.e., the CNT) than in late ASDN segments (i.e., the CD) (2,7–11,29).Differences in the expression levels of mineralocorticoid receptor11-HSD2 and SGK1 likely do not account for this axial heterogeneity,because these ENaC-regulating molecules seem to be equally abundantalong the CNT and CCD (reviewed in [(47]). To our knowledge,Nedd4-2 is the first known ENaC-regulatory protein that showsan axial gradient of expression along the ASDN that could helpto explain the axial gradient of apical ENaC.
The close inverse relationship between Nedd4-2 immunostainingintensity and apical ENaC localization also was seen in responseto altered dietary Na+ intake, suggesting that changes in Nedd4-2abundance contribute to the adaptation of ENaC cell surfaceactivity to long-term changes in dietary Na+ intake. Althoughquantitative RT-PCR with microdissected CCD and immunoblottingwith kidney cortex homogenates failed to provide further conclusivesupport for this conclusion, the inverse correlation of Nedd4-2immunostaining intensity and apical ENaC localization was observedconsistently in mouse and rat kidneys and was seen with twoNedd4-2 antibodies that were directed against two different,separated epitopes. This makes it unlikely but does not ruleout that the observed changes in Nedd4-2 immunostaining intensitiessimply reflect altered Nedd4-2 conformations or protein–proteininteractions that may limit access of the antibodies to theepitopes. While this article was in revision, Umemura et al.(48) found by using quantitative RT-PCR and in situ hybridizationsignificantly higher Nedd4-2 mRNA expression levels in kidneysof Dahl salt-resistant rats that were kept on a 8% NaCl dietthan in the kidneys of their littermates that were kept on a0.3% NaCl diet. Although these results differ from our findingsin mice, in which we did not see any apparent effect on Nedd4-2mRNA levels, the data provide additional evidence that dietaryNa+ intake may have an impact on Nedd4-2 abundance. Remarkably,the authors also described that the diet-induced changes inNedd4-2 mRNA expression were not observed in Dahl salt-sensitiverats, which led to the speculation that disturbed Nedd4-2 regulationin this model may contribute to the salt-sensitive phenotypeof the rats.
It is interesting that dietary Na+ restriction reduced Nedd4-2immunostaining not only in the segment-specific ASDN cells butalso in the IC. IC do not contribute directly to transepithelialNa+ transport, indicated by the lack of ENaC expression (6)and the very low levels of Na+-K+-ATPase in their basolateralplasma membranes (49). IC are involved mainly in the urinaryacidifying mechanism by secretion of protons (type A IC) orbicarbonate (type B IC). Moreover, they may limit renal K+ lossas a result of K+ reabsorption via an apical H+,K+-ATPase (50).Recent studies suggested that the non-A IC also may be importantfor transepithelial transport of Cl–via pendrin, a Cl–/HCO3–exchanger in the apical plasma membrane of these IC (51,52).Because dietary NaCl restriction equally reduces urinary Na+and Cl+ excretion and because Nedd4-2 immunostaining is seenpredominantly in the non-A IC, it is tempting to speculate thatNedd4-2 may have an impact on both Na+ transport in the segment-specificDCT, CNT, and CD cells and Cl– transport in IC, whichwould contribute to simultaneous maintenance of Na+ and Cl–balance.
What might be the underlying mechanism for the NaCl diet–inducedchanges in Nedd4-2 immunostaining? Our findings in mice, withtargeted inactivation of -ENaC in the CD, suggest that the observedchanges in Nedd4-2 expression were not secondary to alteredENaC function. The findings in mpkCCDcl4 cells rather suggestedthat long-term changes in aldosterone levels may regulate Nedd4-2protein abundance. Remarkably, the downregulatory effect ofaldosterone was apparent only after more than 1 d of aldosteronetreatment, suggesting that changes in Nedd4-2 protein levelsare not part of the early response to aldosterone. Consistentwith these conclusions, we recently showed in the same CCD cellline and in kidneys of adrenalectomized rats that aldosteronedoes not change the level of Nedd4-2 protein within the first2 h after aldosterone application (24). Within this early timeframe, aldosterone, however, increased Nedd4-2 phosphorylation,which is thought to impair the interaction of Nedd4-2 with ENaC(see beginning of article). Taken together, these observationssuggest a model in which the rapid effects of aldosterone onENaC are mediated by phosphorylation of Nedd4-2, whereas thechronic effects of aldosterone involve altered Nedd4-2 proteinlevels. Whether the phosphorylation status of Nedd4-2 also maychange with chronically altered Na+ intake or aldosterone levelswas not assessed in our study. Furthermore, the cellular andmolecular mechanisms by which aldosterone modulates Nedd4-2protein abundance and the role of other hormones that possiblyare involved in the regulation of ENaC by dietary Na+ intake(e.g., angiotensin II) remain to be elucidated.
Our study shows a reciprocal relationship between Nedd4-2 immunostainingand apical ENaC localization along the ASDN. This study supportsthe important role of Nedd4-2 in renal Na+ handling and suggeststhat aldosterone-dependent regulation of Nedd4-2 protein abundancemay contribute to the long-term adaptation of apical ENaC activityto altered Na+ transport requirements. Dysfunctions of theseregulatory processes could either cause renal salt losing orpredispose to salt-sensitive hypertension.
Acknowledgments
This work was supported by the Swiss National Science Foundationto O.S. (3100A0-103779/1) and to J.L. (3200B0-105769/1), theRoche Research Foundation in Basel (to O.S.), the CloëttaFoundation (to J.L.), the EMDO Foundation (to J.L.), and theSwiss Diabetes Foundation (to J.L.). D.L.-C. is supported bya Marie-Heim Vögtlin Fellowship of the Swiss National ScienceFoundation.
We thank Drs. H. Abriel, D. Eladari, D. Firsov, J.-D. Horisberger,B. Kaissling, L. Schild, P. Shaw, and B. Rossier for commentson the manuscript and M. Carrel and M. Bloch-Faure for experttechnical assistance. We are grateful to Dr. D. Ackermann andN. Gresko for valuable help with the microdissection of CCDfrom mice on different Na+ diets. The ENaC antibodies and thekidneys from Scnn1aloxlox and Scnn1aloxloxCre were kindly providedby Drs. B. Rossier and E. Hummler. Samples of microdissectedmouse glomeruli and renal tubules were from D. Firsov.
Footnotes
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Top
Abstract
Introduction
, 
, and 
) from a cytoplasmic pool to the apical plasma membrane (2,5,6). The apical redistribution of ENaC shows a remarkable axial heterogeneity along the ASDN. It is most pronounced in the early ASDN portions (late DCT and early CNT) and progressively decreases along further downstream localized segments (late CNT and CD) (2,6). This morphologically ascertainable gradient is consistent with numerous functional studies showing several-fold higher transepithelial Na+ transport rates and ENaC activity in the CNT than in the CD (7–11). However, the underlying molecular mechanisms for this axial heterogeneity are unclear. 
