Enhanced expression of epithelial sodium channels in the renal ...

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Enhanced expression of epithelial sodium channels in the renal medulla of Dahl S rats Md. Shahrier Amin, Erona Reza, Esraa El-Shahat, Hong-Wei Wang, Fre´de´rique Tesson, and Frans H.H. Leenen

Abstract: Inner medullary collecting duct (IMCD) cells from salt-sensitive (S) Dahl rats transport twice as much Na+ as cells from salt-resistant (R) rats, possibly related to dysregulation of the renal epithelial sodium channel (ENaC). The effect of a high-salt diet on ENaC expression in the inner medulla of S versus R rats has not yet been studied. Young, male S and R rats were placed on a regular-salt (0.3%) or high-salt (8%) diet for 2 or 4 weeks. mRNA and protein expression of ENaC subunits were studied by real-time PCR and immunoblotting. Intracellular distribution of the subunits in the IMCD was evaluated by immunohistochemistry. On regular salt, the abundance of the mRNA of b and gENaC was higher in the medulla of S rats than R rats. This was associated with a greater protein abundance of 90 kDa gENaC and higher immunoreactivity for both a and g ENaC. High salt did not affect mRNA abundance in either strain and decreased apical staining of bENaC in IMCD of R rats. In contrast, high salt did not affect the higher apical localization of aENaC and increased the apical membrane staining for b and gENaC in the IMCD of S rats. Expression of ENaC subunits is enhanced in the medulla of S vs. R rats on regular salt, and further increased on high salt. The persistent high expression of aENaC and increase in apical localization of b and gENaC may contribute to greater retention of sodium in S rats on a high-salt diet. Key words: kidney, salt, ENaC, real-time PCR, immunohistochemistry. Re´sume´ : Les cellules du tube collecteur me´dullaire interne (TCMI) des rats Dahl sensibles au sel (S) transportent deux fois plus de Na+ que celles des rats re´sistants au sel (R), probablement en raison d’un dysfonctionnement du canal sodique e´pithe´lial reńal (ENaC). L’effet d’une die`te hypersodeé sur l’expression du canal ENaC dans la partie me´dullaire interne des rats S versus R n’a a` ce jour fait l’objet d’aucune e´tude. Nous avons soumis de jeunes rats S et R maˆles a une die`te contenant une quantite´ normale (0,3 %) ou e´leveé (8 %) de sel pendant 2 ou 4 semaines. Nous avons examine´ l’expression de l’ARNm et des proteínes des sous-unite´s de ENaC par PCR en temps reél et immunobuvardage, et nous avons e´value´ par immunohistochimie la distribution intracellulaire des sous-unite´s dans le TCMI. En condition normosodeé, l’expression de l’ARNm des sous-unite´s b et gENaC a e´te´ plus e´leveé dans la partie me´dullaire des rats S que des rats R. Cet effet a e´te´ associe´ a` un taux plus e´leve´ des proteínes des sous-unite´s gENaC de 90 kDA et a` une plus forte immunoreáctivite´ tant pour ce qui est de b que de gENaC. La die`te hypersodeé n’a pas modifie´ la teneur en ARNm des rats S et R, et a diminue´ ` l’oppose´, la dose e´leveé de sel n’a pas influe´ sur la plus forte la coloration apicale de bENaC dans le TCMI des rats R. A localisation apicale de gENaC, et a elle augmente´ la coloration apicale de b et gENaC dans le TCMI des rats S. L’expression des sous-unite´s ENaC est stimuleé dans la partie me´dullaire des rats S versus R soumis a` une die`te normosodeé, et elle augmente davantage avec une die`te hypersodeé. L’expression e´leveé persistante de aENaC et l’augmentation de la localisation apicale de b et gENaC pourraient contribuer a` la plus forte re´tention de sodium chez les rats S soumis a` une die`te hypersodeé. Mots-cle´s : rein, sel, ENaC, PCR en temps reél, immunohistochimie. [Traduit par la Re´daction]

Introduction Blood pressure sensitivity to salt is seen in 40%–60% of patients with essential hypertension and in ~20%–30% of

the general population (Franco and Oparil 2006). Genetic analyses of human monogenic forms of hypertension and of common variants in essential hypertension have identified the epithelial sodium channel (ENaC) as being associated

Received 3 November 2010. Accepted 27 December 2010. Published on the NRC Research Press Web site at cjpp.nrc.ca on 18 February 2011. M.S. Amin, E. Reza, and F.H. Leenen.1 Hypertension Unit, University of Ottawa Heart Institute, Ottawa, ON K1Y 4W7, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada. E. El-Shahat. Hypertension Unit, University of Ottawa Heart Institute, Ottawa, ON K1Y 4W7, Canada; Laboratory of Genetics of Cardiac Disease, University of Ottawa Heart Institute, Ottawa, ON K1Y 4W7, Canada. H.-W. Wang. Hypertension Unit, University of Ottawa Heart Institute, Ottawa, ON K1Y 4W7, Canada. F. Tesson. Laboratory of Genetics of Cardiac Disease, University of Ottawa Heart Institute, Ottawa, ON K1Y 4W7, Canada; Faculty of Health Sciences, University of Ottawa, Ottawa, ON K1H 8M5, Canada. 1Corresponding

author (e-mail: [email protected]).

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doi:10.1139/Y11-005

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with salt sensitivity (Kreutz et al. 1997; Luft 2004; Iwai et al. 2002). Expression of ENaC in the kidneys is affected by dietary salt, and plays a major role in determining the way the body handles sodium (Rossier et al. 2002; Masilamani et al. 2002). ENaC are members of the DEG/ENaC family of cation selective ion channels (Canessa et al. 1995; Canessa et al. 1994). The functional channel is thought to be a heteromultimer of 3 subunits, a, b, and g, with a 2:1:1 (Firsov et al. 1998; Kosari et al. 1998) or 3:3:3 stoichiometry (Snyder et al. 1998). Individual ENaC subunits show tissue-specific expression and regulation. Channels with all 3 subunits show maximal function, but different combinations of subunits also form active channels and posttranslational modifications affecting one or more subunits can also affect Na+ transport (Duc et al. 1994; Escoubet et al. 1997; Farman et al. 1997). In the collecting ducts of the kidneys, all 3 subunits are expressed and ENaC mediates transport of Na+ along an electrochemical gradient created by basolateral Na+K+ATPase (Kudo et al. 1990; Palmer et al. 1980). The final rate-limiting steps of sodium reabsorbtion in the distal nephron segments are mediated by ENaC. The aldosteronemineralocorticoid receptor (MR) complex increases ENaC surface expression and activity by increasing transcription of the subunits and by increasing transcription of regulators, which in turn activate the channel (Snyder 2005; Snyder et al. 2002). Among these are the serum and glucocorticoid regulated kinase-1 (SGK1), an early aldosterone inducible protein kinase, which upregulates ENaC activity by phosphorylating neural precursor cells expressed developmentally downregulated-4 like gene (Nedd4L), thereby reducing the interaction between ENaC and Nedd4L (Snyder et al. 2002, 2004). In Dahl S rats, the expression and function of ENaC appear to be dysregulated. The conductive permeability of the apical membrane to Na+ and the rate of Na+ transport is higher in monolayers of cultured cells from inner medullary collecting ducts of S versus R rats, suggesting that ENaC is intrinsically different or differently regulated in S and R rats (Husted et al. 1996, 1997). Comprehensive screening did not show any variations in the ENaC genes between S and R rats (Shehata et al. 2007). However, high salt for 4 weeks caused the expected decrease in mRNA levels of aENaC in whole kidneys of R rats, but caused modest increases of a, b, and gENaC in S rats (Aoi et al. 2007). In a more recent study, high salt for 4 weeks did not affect aENaC and increased both mRNA and protein of b and gENaC in the cortex of Dahl S rats (Kakizoe et al. 2009). All previous studies on renal ENaC in Dahl rats only studied homogenates of whole kidneys or the renal cortex, and did not evaluate the medulla and distribution of the proteins. Whether mRNA, protein abundance, and distribution of ENaC and regulators such as SGK1 and Nedd4L are differently regulated in the renal medulla of S and R rats has not yet been studied. We hypothesized that enhanced ENaC expression is present in the renal medulla of S rats on regular salt intake and persists on high salt. To test this hypothesis, we evaluated the mRNA abundance, protein abundance, and intracellular distribution of ENaC subunits in the renal medulla of S and R rats on regular vs. high salt for 2 or 4 weeks.

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Materials and methods Male 3–4-week-old Dahl salt-sensitive (SS/Jr-Hsd: S) and Dahl salt-resistant (SR/Jr-Hsd: R) rats were purchased from Harlan Sprague–Dawley (Indianapolis, Ind., USA). The rats were housed 2 per cage under standard conditions on a 12 h light : 12 h dark cycle at 24 8C and were allowed a 3–5-day acclimatization period on normal rat chow and tap water before entering the study. All procedures were performed according to the guidelines of the Canadian Council on Animal Care and were approved by the University of Ottawa Animal Care Committee. Experimental protocol Dahl S and R rats were assigned randomly to either a regularsalt (120 mmol Na+/gm) or high-salt (1370 mmol Na+/gm) diet for 2 or 4 weeks (n = 6–8 per group). At the end of 2 or 4 weeks of the salt diet, BP was recorded and blood samples obtained for plasma aldosterone and plasma angiotensin II. Under pentobarbital anesthesia, the rats were perfused transcardially first with cold PBS (pH 7.4) to drain all the blood. This was followed by perfusion over 20– 30 min with ~500 mL per rat of 4% paraformaldehyde plus 0.05% glutaraldehyde in phosphate buffer (pH 7.4) for immunohistochemistry. For RNA and protein extraction, the rats were perfused with RNase-free cold PBS only (pH 7.4). mRNA studies Real-time PCR Total RNA was isolated from the kidney medulla as described previously (Amin et al. 2005). Specific primers for a, b, and gENaC and phosphoglycerate kinase1 (PGK1) were the same as described previously (Amin et al. 2005; Umemura et al. 2006). The amplicon length of the PCR products were PGK1 (263 bp), aENaC (429 bp), bENaC (220 bp), and gENaC (301 bp). Real-time PCR was performed with Roche Light Cycler using Fast Start DNA Master SYBR Green I dye (Roche Diagnostics, Que., Canada). The PCR conditions were set as follows: initial at 95 8C for 10 min to activate Taq polymerase and followed by 45 cycles of denaturation at 95 8C for 5 s; annealing for 10 s at 62 8C for PGK and aENaC, and 65 8C for b and gENaC. Extension time was determined by target amplicon length/25. The specificity of the real-time PCR products were determined by both melting curve analysis and agarose gel electrophoresis. External standard curves were created using serial dilutions of plasmids containing cDNA fragments for different genes with the same PCR conditions as described above. mRNA expression was normalized to PGK1 mRNA levels. Protein studies Characterization of the antibodies ENaC antibodies were generated as described previously (Ergonul et al. 2006; Masilamani et al. 1999). The aENaC antibody detected bands at ~80 (uncleaved) kDa. In addition, bands at 120, 50, and 37 kDa (aggregates or cleaved inactive fragments) were variably detected, but not quantifiable in most samples. aENaC protein was also not Published by NRC Research Press

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Fig. 1. Protein abundance in the inner medulla and immunoreactivity to a, b, and g epithelial sodium channel (ENaC) in the collecting ducts in Wistar rats after 2 weeks of a regular- vs. low-salt diet. Diffuse and mostly cytoplasmic staining was noted in the collecting ducts of Wistar rats on a regular-salt diet. Low salt increased apical immunoreactivity in the collecting ducts (arrows) and abundance of 80 kDa aENaC, 85 kDa bENaC, and 70 kDa gENaC proteins in the inner medulla.

consistently detectable in all inner medulla samples. The bENaC antibody detected a major band at 85 kDa and the gENaC antibody detected bands at ~90 (uncleaved) and 70 (cleaved active fragment) kDa. These bands were abolished by preadsorbtion with the immunizing peptides. Omission of the primary antibody or coincubation with the immunizing peptide also abolished immunostaining. As a positive control, the effects of a low- (10 mmol Na+/gm) vs. regular-salt diet for 2 weeks were studied in Wistar rats. Consistent with previous studies (Frindt et al. 2007; Masilamani et al. 1999), a low-salt diet for 2 weeks increased the abundance of 80 kDa aENaC by ~40%, bENaC by ~70%, and 70 kDa band of gENaC by ~20% in the inner medulla, and also increased the apical immunoreactivity in the collecting ducts (Fig. 1). Immunoblotting Whole protein was extracted from macroscopically dissected inner medullas and homogenized in ice-cold solution (pH 7.5) containing 300 mmol/L sucrose and 10 mmol/L Triethanolamine (ENaC) containing 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Mo., USA). The membranes were probed with the respective primary antibodies overnight at 4 8C (aENaC and Nedd4L — 1:1000, b and gENaC — 1:5000, SGK1 — 1:2000), followed by incubation in goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:5000, Santa Cruz Biotechnology, Santa Cruz, Calif., USA). The membranes were stripped and re-probed with anti-b-actin antibody (Sigma-Aldrich). Signal was developed with ECL+ system (PerkinElmer, Waltham, Mass., USA) and visualized using the Alpha-Ease system. The results are expressed in arbitrary units (percent

change of normalized densitometry of target gene vs. b-actin from control). Immunohistochemistry Sagittal sections were cut on a rotary microtome at a thickness of 7 mm. Antigen retrieval was carried out by heating the sections with 1.0 mmol/L Tris (pH 8.0) at 90 8C for 25 min and incubation in 1% sodium borohydride for 30 min. Nonspecific IgG binding and endogenous peroxidase activity were blocked by incubation in 50 mmol/L NH4Cl for 30 min and 0.5% H2O2 in 20% methanol in PBS for 30 min. Sections were blocked with a cocktail of 1% normal goat serum, 0.02% gelatin, 0.05% Triton X-100, and 1% BSA in PBS (pH 7.4) for 2 h. Endogenous biotin was blocked with avidin-biotin blocking solution (Vector Laboratories, Burlington, Ont., Canada). The sections were then incubated for 12 h with the respective primary antibodies diluted appropriately in the blocking solution (aENaC 1:250; bENaC and gENaC 1:100). Further processing was done with the Vectastain Elite ABC kit for rabbit IgG and visualized using the DAB kit with Nickel enhancement (Vector Laboratories). Slides were counterstained with Vector hematoxylin, dehydrated, cleared, and coated with Permount (Fisher Scientific, Ottawa, Ont., Canada). Images were captured using a Spot digital camera attached to a high resolution bright-field transmitted light microscope (Olympus BX60). The inner medullary collecting ducts (IMCD) were identified based on predominance of cuboidal cells, centrally located nucleus with clear outline, and a large-sized lumen (Mills 2008). Tubules that had uniformity of staining and were sectioned in the same plane were Published by NRC Research Press

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Can. J. Physiol. Pharmacol. Vol. 89, 2011 Table 1. Blood pressure, plasma aldosterone, and angiotensin II levels in Dahl R and S rats on a regular- or high-salt diet for 2 or 4 weeks. Dahl R MAP (mm Hg) Aldosterone (pg/mL) Ang II (pg/mL)

2 4 2 4 4

Dahl S

Reg 114±5 121±3 378±76 426±68 12.1±2.6

wks wks wks wks wks

High 113±3 125±4 329±48 257±44* 5.4±1.8*

Reg 119±4 134±4 720±89{ 627±83{ 4.4±0.8{

High 148±2* 192±10* 447±74* 340±80* 1.3±0.4

Note: Values are means ± SEM (n = 6–9 per group). MAP, mean arterial pressure. *p < 0.05 vs. reg salt. { p < 0.05 vs. Dahl R.

Table 2. mRNA and protein expression of epithelial sodium channel (ENaC) subunits in the inner medulla of Dahl R and S rats on a regular- or high-salt diet. Dahl R aENaC

mRNA mRNA

bENaC 85 kDa protein gENaC

mRNA 90 kDa protein 70 kDa protein

2 4 2 4 2 4 2 4 2 4 2 4

wks wks wks wks wks wks wks wks wks wks wks wks

Dahl S

Reg salt 1.7±0.2 1.7±0.1 3.1±0.4 2.3±0.2 100±22 100±32 1.8±0.1 2.5±0.3 100±31 100±08 100±12 100±13

High salt 1.4±0.1 1.5±0.1 3.6±0.2 2.1±0.1 97±27 117±49 2.4±0.2 2.7±0.3 154±35* 93±16 97±14 123±33

Reg salt 1.4±0.1 1.4±0.2 3.0±0.1 3.3±0.2{ 71±17 105±73 2.0±0.1 4.0±0.4{ 157±18{ 204±12{ 167±17{ 241±33{

High salt 1.4±0.2 1.3±0.1 3.5±0.2 3.9±0.4{ 93±17 129±49 2.0±0.1 4.8±0.3{ 220±22{ 132±5*,{ 147±32{ 157±30

Note: Values are means ± SEM (n = 5–6 per group for mRNA and 4–7 per group for protein). mRNA expression was normalized to PGK1 levels. Values for protein are normalized to b-actin and compared with Dahl R on a regular-salt diet (100%). ENaC, renal epithelial sodium channel. *p < 0.05 vs. reg salt. { p < 0.05 vs. Dahl R.

used for analysis. Immunostaining was assessed blindly by two individuals (MSA and ER). A score of 1 (70% cells stained with strong staining intensity) was given for immunostaining in the apical membranes and in the cytoplasm. Statistical analysis Values are presented as means ± SEM. All comparisons between groups were determined by two-way analysis of variance (ANOVA) followed by the Student Newman–Keuls test where applicable. A value of p < 0.05 was considered statistically significant.

Results Blood pressure, plasma aldosterone, and angiotensin II (Table 1) On regular salt, mean arterial pressure (MAP) tended to be higher in S compared with R rats. High salt intake did not affect MAP in R rats but increased MAP moderately after 2 weeks and markedly after 4 weeks in S rats. On regular

salt, R rats had higher plasma Ang II but lower plasma aldosterone than S rats. On high-salt intake, plasma Ang II was decreased particularly in R rats and plasma aldosterone in S rats. ENaC expression in the renal medulla aENaC Abundance of aENaC mRNA was not significantly different in the renal medulla of S and R rats on a regular-salt diet (Table 2). However, apical immunoreactivity in IMCDs was greater in S rats on a regular-salt diet at both 2 and 4 weeks and cytoplasmic reactivity higher at 4 weeks (Table 3, Fig. 2). The high-salt diet did not affect aENaC mRNA expression in either strain, and immunoreactivity remained increased in S rats (Tables 2 and 3, Fig. 2). bENaC The abundance of bENaC mRNA was similar in the medulla of both strains after 2 weeks, but significantly higher in S rats after 4 weeks; bENaC protein abundance and cytoplasmic staining were similar (Tables 2 and 3, Fig. 3). On high salt, the higher mRNA abundance persisted in S rats. High salt did not affect bENaC protein in either strain, but Published by NRC Research Press

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Table 3. Immunoreactivity to the epithelial sodium channel (ENaC) subunits in the inner medullary collecting ducts of Dahl R and S rats on a high- vs. regular-salt diet, as assessed by relative abundance in the apical membrane and cytoplasm. Dahl R

Dahl S

Reg salt Apical

Cytoplasm

a b g a b g

2 wks 1.4±0.2 2±0.4 1.8±0.3 1.8±0.2 1.2±0.3 1±0.01

High salt 4 wks 1.7±0.5 2.3±0.2 1.8±0.3 2.3±0.3 1.2±0.3 1±0.01

2 wks 1.6±0.2 1.5±0.3 2±0.4 1.7±0.2 1.3±0.3 1.5±0.3

Reg salt 4 wks 2±0.4 1.3±0.3* 2±0.4 2.3±0.5 1.3±0.3 1.5±0.3

2 wks 2.5±0.3{ 1.8±0.2 1.8±0.3 1.3±0.3 1.5±0.2 1±0.01

High salt 4 wks 2.3±0.3{ 1.5±0.2 1.8±0.3 3±0.01{ 1.5±0.2 1±0.01

2 wks 2.6±0.2{ 2.3±0.2* 3±0.01*,{ 1.2±0.2 1.3±0.2 2.5±0.3*

4 wks 2.5±0.3{ 1.8±0.2 3±0.01*,{ 3±0.01{ 1.3±0.2 2.5±0.3*

Note: Assessment of immunoreactivity: 1 if 70%. Values are means ± SEM (n = 4–7 per group). *p < 0.05 vs. reg salt group. { p < 0.05 vs. Dahl R.

Fig. 2. Immunoreactivity to a epithelial sodium channel (aENaC) in the inner medulla of Dahl R and S rats after 2 or 4 weeks on a regularsalt (RS) or high-salt (HS) diet. In R rats, the staining is modest at 2 and 4 weeks, and similar on both salt diets. In contrast, distinct apical immunoreactivity is notable in the IMCD of S rats at 2 weeks and is not affected by a high-salt diet. The higher abundance of ENaC subunits in S rats is more apparent after 4 weeks, although at this point the apical immunoreactivity is less contrasting due to the increased cytoplasmic staining. Arrows show apical immunoreactivity in IMCDs in S rats.

decreased apical immunoreactivity in IMCDs of R rats and transiently increased apical staining in S rats (Tables 2 and 3, Fig. 3). gENaC On regular salt, gENaC mRNA abundance was similar in the 2 strains at 2 weeks, but S rats had greater abundance of the mRNA at 4 weeks and of both protein fractions at both 2 and 4 weeks (Tables 2 and 3, Fig. 4). Apical membrane staining was similar in the IMCDs of the 2 strains on regular salt. In R rats, high salt intake caused a modest, transient increase in the 90 kDa fraction, but not in the active 70 kDa band or in staining of gENaC. In contrast, high salt intake

did not affect the elevated mRNA and protein of S rats, and clearly increased both cytoplasmic and apical immunoreactivity in IMCDs of S rats (Tables 2 and 3, Fig. 4).

Discussion The current study shows, as major new findings, that on a regular-salt diet, abundance of mRNA (b and gENaC), protein (gENaC), and apical staining (aENaC) of ENaC is higher in the medulla of S rats. On a high-salt diet, in S rats these higher abundances persist, and in addition, apical membrane staining for bENaC was increased after 2 weeks and for gENaC after both 2 and 4 weeks. Published by NRC Research Press

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Fig. 3. b Epithelial sodium channel (bENaC) abundance and immunoreactivity in the renal medulla of Dahl R and S rats after 2 or 4 weeks on a regular-salt (RS) or high-salt (HS) diet. (A) Abundance of bENaC protein in the medulla. Both strains had similar abundance of the protein, which was not affected by high salt. (B) Distribution of bENaC in the IMCDs. Apical staining was decreased in the IMCDs of Dahl R on a high-salt diet, but became more prominent in Dahl S. Arrows show immunoreactivity in IMCDs.

Renal ENaC on regular salt ENaC subunits are expressed predominantly along the aldosterone-sensitive distal nephron (Duc et al. 1994) and are the major Na+ transporter in the IMCD (Frindt et al. 2007; Volk et al. 1995). ENaC expression and distribution in the IMCD of R and S rats have not been studied before. In the medulla of S rats, b and gENaC mRNA abundance was

higher and associated with more gENaC protein and higher apical membrane immunoreactivity to aENaC in the IMCD. Increased abundance at the cell surface of active aENaC alone has been shown to be sufficient for increased ENaC activity (Rotin et al. 1994). These expression data and previous in vitro studies showing an increased rate of Na+ transport in monolayers of cultured IMCD in S vs. R rats Published by NRC Research Press

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Fig. 4. g Epithelial sodium channel (gENaC) protein abundance and immunoreactivity in the inner medulla of Dahl R and S rats after 2 or 4 weeks on a regular (RS) or high (HS)-salt diet. (A) Abundance of gENaC protein in the medulla. Dahl S have greater abundance of the protein at both 2 and 4 weeks. (B) Distribution of gENaC in the inner medullary collecting ducts (IMCDs). Modest apical and cytoplasmic staining can be seen in the IMCDs, which is significantly increased by high salt in Dahl S.


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(Husted et al. 1996, 1997) suggest that in Dahl S, higher ENaC expression may contribute to increased Na+ reabsorption, even on a regular-salt diet. Renal ENaC on high salt In Wistar or Sprague–Dawley rats, a high-salt diet causes time-dependent, modest decreases in the abundance of 1 or more subunits in the medulla (Farjah et al. 2003; Loffing et al. 2000; Masilamani et al. 1999, 2002). In R rats, Aoi et al. (2006, 2007) showed that high salt for 4 weeks decreased mRNA levels of aENaC in the whole kidney. Our results in R rats show only modest changes by high salt, which may reflect at the most a minor decrease in ENaC activity in different nephron segments. The magnitude of the changes appears similar to those in Wistar rats, and is substantially less than low vs. regular salt (Fig. 1), suggesting that regular salt intake already causes most of the decrease. In S rats, Aoi et al. (2006, 2007) reported increased mRNA expression of all 3 subunits in whole kidneys after 4 weeks high salt. In contrast, Kakizoe et al. (2009) reported no change in aENaC mRNA, but an increase in b and gENaC mRNA and protein in the cortex. These studies (Aoi et al. 2006, 2007; Kakizoe et al. 2009) did not evaluate expression in the medulla, nor intracellular distribution. In the present study, on a high-salt diet, the higher mRNA and protein expression of b and g subunits in the medulla of S rats and abundance of aENaC in the apical membrane of IMCDs persisted, whereas apical staining of b and gENaC increased. Increased apical staining in the medulla of S vs. R rats — for aENaC only on regular salt and for all 3 subunits on high salt — may reflect enhanced transport to the membrane and (or) decreased removal from the membrane. These changes are distinctly different from those of saltresistant strains and indicate that high salt in S rats affects mechanisms that regulate subcellular distribution and therefore, presumably, activity of the subunits and Na+ transport. Regulators of renal ENaC The absence of differences in the regulatory and coding sequences of ENaC genes between S and R rats (Shehata et al. 2007) suggests that the greater abundance on a regularsalt diet and the opposite response — i.e., persistence or increase with high salt — in kidneys of Dahl S are due to differential regulation of the ENaC subunits. Aldosterone, acting through the mineralocorticoid receptor, is a major regulator of ENaC. While studies in the early 1990s showed lower levels for plasma aldosterone in S vs. R (Cover et al. 1995), recent studies show similar or higher levels in S rats (Manger et al. 2009; Takeda et al. 2007). Plasma aldosterone concentration decreased with high salt in both strains, consistent with previous studies (Aoi et al. 2007; Farjah et al. 2003; Zhu et al. 2009), but remained somewhat higher in S. This suggests a similar regulation of plasma aldosterone by high salt. In contrast, the activity of the intrarenal renin–angiotensin–aldosterone system is increased by high salt intake in S rats (Bayorh et al. 2005; Zhu et al. 2009), and may contribute to increased ENaC expression in Dahl S on high salt. Classically, aldosterone increases expression of aENaC in the kidney and of b and gENaC in the colon (Duc et al. 1994). This pattern may be disrupted in S rats, and aldosterone contributes to the higher expression of b and


gENaC in the medulla of S rats. Alternatively, other regulators, such as vasopressin, may contribute to the dysregulated expression of ENaC subunits in S vs. R rats on a high-salt diet. High salt does not affect plasma vasopressin levels in R rats, but increases vasopressin levels by ~2.6 fold in S rats (Wainford and Kapusta 2010). Vasopressin affects posttranscriptional expression of the a and gENaC subunits (Perlewitz et al. 2010) and increases amiloride blockable luminal Na+ transport across the IMCD (Kudo et al. 1990). Perspectives Expression of ENaC subunits is enhanced in the IMCD of S vs. R rats on a regular-salt diet and is further enhanced by a high-salt diet. Since Dahl S rats were not found to have any mutations in ENaC genes, it remains to be established whether increases in aldosterone–MR activation or other regulators are responsible for these changes. Functional studies are needed to assess whether this higher expression is associated with increased ENaC activity and leads to greater Na+ reabsorbtion in S rats. Unraveling the mechanisms that contribute to more apical localization of ENaC in salt-sensitive individuals may provide new strategies for preventing and treating hypertension. Ongoing genetic studies in humans may provide insights into putative factors affecting ENaC expression and function and thereby to salt sensitivity of BP in humans (Leenen et al. 2011).

Acknowledgement The authors would like to acknowledge Ms. Danielle Oja for her excellent skills in assisting in the preparation and formatting of this article. Dr. Leenen holds the Pfizer Chair in Hypertension Research, an endowed chair supported by Pfizer Canada, the University of Ottawa Heart Institute Foundation, and Canadian Institutes of Health Research. Sources of funding: this research was supported by operating grant FRN: MOP-74432 from the Canadian Institutes of Health Research. Md. Shahrier Amin was supported by a Pfizer/CIH/Canadian Hypertension Society doctoral research award and program grant PRG5275 from the Heart and Stroke Foundation of Ontario.

References Amin, M.S., Wang, H.W., Reza, E., Whitman, S.C., Tuana, B.S., and Leenen, F.H. 2005. Distribution of epithelial sodium channels and mineralocorticoid receptors in cardiovascular regulatory centers in rat brain. Am. J. Physiol. Regul. Integr. Comp. Physiol. 289(6): R1787–R1797. PMID:16141309. Aoi, W., Niisato, N., Sawabe, Y., Miyazaki, H., and Marunaka, Y. 2006. Aldosterone-induced abnormal regulation of ENaC and SGK1 in Dahl salt-sensitive rat. Biochem. Biophys. Res. Commun. 341(2): 376–381. doi:10.1016/j.bbrc.2005.12.194. PMID: 16426574. Aoi, W., Niisato, N., Sawabe, Y., Miyazaki, H., Tokuda, S., Nishio, K., et al. 2007. Abnormal expression of ENaC and SGK1 mRNA induced by dietary sodium in Dahl salt-sensitive hypertensive rats. Cell Biol. Int. 31(10): 1288–1291. doi:10.1016/j. cellbi.2007.03.036. PMID:17485228. Bayorh, M.A., Ganafa, A.A., Emmett, N., Socci, R.R., Eatman, D., and Fridie, I.I. 2005. Alterations in aldosterone and angiotensin II levels in salt-induced hypertension. Clin. Exp. Hypertens. 27(4): 355–367. PMID:15921072. Published by NRC Research Press

Amin et al. Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.D., and Rossier, B.C. 1994. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature, 367(6462): 463–467. doi:10.1038/367463a0. PMID: 8107805. Canessa, C.M., Horisberger, J.D., Schild, L., and Rossier, B.C. 1995. Expression cloning of the epithelial sodium channel. Kidney Int. 48(4): 950–955. doi:10.1038/ki.1995.376. PMID: 8569104. Cover, C.M., Wang, J.-M., St. Lezin, E., Kurtz, T.W., and Mellon, S.H. 1995. Molecular variants in the P450c11AS gene as determinants of aldosterone synthase activity in the Dahl rat model of hypertension. J. Biol. Chem. 270(28): 16555–16560. doi:10. 1074/jbc.270.28.16555. PMID:7622461. Duc, C., Farman, N., Canessa, C.M., Bonvalet, J.P., and Rossier, B.C. 1994. Cell-specific expression of epithelial sodium channel a, b, and g subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J. Cell Biol. 127(6): 1907–1921. doi:10.1083/jcb.127.6.1907. PMID:7806569. Ergonul, Z., Frindt, G., and Palmer, L.G. 2006. Regulation of maturation and processing of ENaC subunits in the rat kidney. Am. J. Physiol. Renal Physiol. 291(3): F683–F693. doi:10.1152/ ajprenal.00422.2005. PMID:16554417. Escoubet, B., Coureau, C., Bonvalet, J.P., and Farman, N. 1997. Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone. Am. J. Physiol. 272(5 Pt 1): C1482–C1491. PMID:9176138. Farjah, M., Roxas, B.P., Geenen, D.L., and Danziger, R.S. 2003. Dietary salt regulates renal SGK1 abundance: relevance to salt sensitivity in the Dahl rat. Hypertension, 41(4): 874–878. doi:10.1161/01.HYP.0000063885.48344.EA. PMID:12642512. Farman, N., Talbot, C.R., Boucher, R., Fay, M., Canessa, C., Rossier, B., and Bonvalet, J.P. 1997. Noncoordinated expression of a-, b-, and g-subunit mRNAs of epithelial Na+ channel along rat respiratory tract. Am. J. Physiol. 272(1 Pt 1): C131–C141. PMID:9038819. Firsov, D., Gautschi, I., Merillat, A.M., Rossier, B.C., and Schild, L. 1998. The heterotetrameric architecture of the epithelial sodium channel (ENaC). EMBO J. 17(2): 344–352. doi:10.1093/ emboj/17.2.344. PMID:9430626. Franco, V., and Oparil, S. 2006. Salt sensitivity, a determinant of blood pressure, cardiovascular disease and survival. J. Am. Coll. Nutr. 25(3 Suppl): 247S–255S. PMID:16772636. Frindt, G., Ergonul, Z., and Palmer, L.G. 2007. Na channel expression and activity in the medullary collecting duct of rat kidney. Am. J. Physiol. Renal Physiol. 292(4): F1190–F1196. doi:10. 1152/ajprenal.00399.2006. PMID:17200158. Husted, R.F., Takahashi, T., and Stokes, J.B. 1996. IMCD cells cultured from Dahl S rats absorb more Na+ than Dahl R rats. Am. J. Physiol. 271(5 Pt 2): F1029–F1036. PMID:8945997. Husted, R.F., Takahashi, T., and Stokes, J.B. 1997. The basis of higher Na+ transport by inner medullary collecting duct cells from Dahl salt-sensitive rats: implicating the apical membrane Na+ channel. J. Membr. Biol. 156(1): 9–18. doi:10.1007/ s002329900182. PMID:9070459. Iwai, N., Baba, S., Mannami, T., Ogihara, T., and Ogata, J. 2002. Association of a sodium channel a subunit promoter variant with blood pressure. J. Am. Soc. Nephrol. 13(1): 80–85. PMID: 11752024. Kakizoe, Y., Kitamura, K., Ko, T., Wakida, N., Maekawa, A., Miyoshi, T., et al. 2009. Aberrant ENaC activation in Dahl saltsensitive rats. J. Hypertens. 27(8): 1679–1689. doi:10.1097/HJH. 0b013e32832c7d23. PMID:19458538.

167 Kosari, F., Sheng, S., Li, J., Mak, D.O., Foskett, J.K., and Kleyman, T.R. 1998. Subunit stoichiometry of the epithelial sodium channel. J. Biol. Chem. 273(22): 13469–13474. doi:10. 1074/jbc.273.22.13469. PMID:9593680. Kreutz, R., Struk, B., Rubattu, S., Hubner, N., Szpirer, J., Szpirer, C., et al. 1997. Role of the a-, b-, and g-subunits of epithelial sodium channel in a model of polygenic hypertension. Hypertension, 29(1 Pt 1): 131–136. PMID:9039092. Kudo, L.H., van Baak, A.A., and Rocha, A.S. 1990. Effect of vasopressin on sodium transport across inner medullary collecting duct. Am. J. Physiol. 258(5 Pt 2): F1438–F1447. PMID: 2159725. Leenen, F.H.H., Amin, M.S., Stewart, A.F.R., and Tesson, F. 2011. Genetic basis of salt-sensitive hypertension in humans. In Genes and Cardiovascular Function. Edited by B. Ostadal, M. Nagano, N.S. Dhalla. Springer Science+Business Media, Inc. In press. Loffing, J., Pietri, L., Aregger, F., Bloch-Faure, M., Ziegler, U., Meneton, P., et al. 2000. Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and lowNa diets. Am. J. Physiol. Renal Physiol. 279(2): F252–F258. PMID:10919843. Luft, F.C. 2004. Present status of genetic mechanisms in hypertension. Med. Clin. North Am. 88(1): 1–18, vii. doi:10.1016/ S0025-7125(03)00118-4. PMID:14871048. Manger, W.M., Simchon, S., Stokes, M.B., Reidy, J.J., Kumar, A.R., Baer, L., et al. 2009. Renal functional, not morphological, abnormalities account for salt sensitivity in Dahl rats. J. Hypertens. 27(3): 587–598. doi:10.1097/HJH.0b013e32831ffec7. PMID:19330919. Masilamani, S., Kim, G.H., Mitchell, C., Wade, J.B., and Knepper, M.A. 1999. Aldosterone-mediated regulation of ENaC a, b, and g subunit proteins in rat kidney. J. Clin. Invest. 104(7): R19– R23. doi:10.1172/JCI7840. PMID:10510339. Masilamani, S., Wang, X., Kim, G.H., Brooks, H., Nielsen, J., Nielsen, S., et al. 2002. Time course of renal Na-K-ATPase, NHE3, NKCC2, NCC, and ENaC abundance changes with dietary NaCl restriction. Am. J. Physiol. Renal Physiol. 283(4): F648–F657. PMID:12217855. Mills, S.E. 2008. Histology for pathologists. 3rd ed. Lippincott Williams and Wilkins, Philadelphia. Palmer, L.G., Edelman, I.S., and Lindemann, B. 1980. Current-voltage analysis of apical sodium transport in toad urinary bladder: effects of inhibitors of transport and metabolism. J. Membr. Biol. 57(1): 59–71. doi:10.1007/BF01868986. PMID:6256553. Perlewitz, A., Nafz, B., Skalweit, A., Fahling, M., Persson, P.B., and Thiele, B.J. 2010. Aldosterone and vasopressin affect aand g-ENaC mRNA translation. Nucleic Acids Res. 38(17): 5746–5760. doi:10.1093/nar/gkq267. PMID:20453031. Rossier, B.C., Pradervand, S., Schild, L., and Hummler, E. 2002. Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors. Annu. Rev. Physiol. 64(1): 877–897. doi:10.1146/annurev.physiol.64. 082101.143243. PMID:11826291. Rotin, D., Bar-Sagi, D., O’Brodovich, H., Merilainen, J., Lehto, V.P., Canessa, C.M., et al. 1994. An SH3 binding region in the epithelial Na+ channel (a rENaC) mediates its localization at the apical membrane. EMBO J. 13(19): 4440–4450. PMID:7925286. Shehata, M.F., Leenen, F.H., and Tesson, F. 2007. Sequence analysis of coding and 3’ and 5’ flanking regions of the epithelial sodium channel a, b, and g genes in Dahl S versus R rats. BMC Genet. 8(1): 35. doi:10.1186/1471-2156-8-35. PMID:17592634. Snyder, P.M. 2005. Minireview: regulation of epithelial Na+ channel trafficking. Endocrinology, 146(12): 5079–5085. doi:10. 1210/en.2005-0894. PMID:16150899. Published by NRC Research Press

168 Snyder, P.M., Cheng, C., Prince, L.S., Rogers, J.C., and Welsh, M.J. 1998. Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits. J. Biol. Chem. 273(2): 681–684. doi:10.1074/jbc.273.2.681. PMID:9422716. Snyder, P.M., Olson, D.R., and Thomas, B.C. 2002. Serum and glucocorticoid-regulated kinase modulates Nedd4–2-mediated inhibition of the epithelial Na+ channel. J. Biol. Chem. 277(1): 5–8. doi:10.1074/jbc.C100623200. PMID:11696533. Snyder, P.M., Olson, D.R., Kabra, R., Zhou, R., and Steines, J.C. 2004. cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na+ channel through convergent phosphorylation of Nedd4–2. J. Biol. Chem. 279(44): 45753– 45758. doi:10.1074/jbc.M407858200. PMID:15328345. Takeda, Y., Zhu, A., Yoneda, T., Usukura, M., Takata, H., and Yamagishi, M. 2007. Effects of aldosterone and angiotensin II receptor blockade on cardiac angiotensinogen and angiotensinconverting enzyme 2 expression in Dahl salt-sensitive hypertensive rats. Am. J. Hypertens. 20(10): 1119–1124. doi:10.1016/j. amjhyper.2007.05.008. PMID:17903697.

Can. J. Physiol. Pharmacol. Vol. 89, 2011 Umemura, M., Ishigami, T., Tamura, K., Sakai, M., Miyagi, Y., Nagahama, K., et al. 2006. Transcriptional diversity and expression of NEDD4L gene in distal nephron. Biochem. Biophys. Res. Commun. 339(4): 1129–1137. doi:10.1016/j.bbrc.2005.11. 120. PMID:16338225. Volk, K.A., Sigmund, R.D., Snyder, P.M., McDonald, F.J., Welsh, M.J., and Stokes, J.B. 1995. rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct. J. Clin. Invest. 96(6): 2748–2757. doi:10.1172/ JCI118344. PMID:8675644. Wainford, R.D., and Kapusta, D.R. 2010. Hypothalamic paraventricular nucleus Gaq subunit protein pathways mediate vasopressin dysregulation and fluid retention in salt-sensitive rats. Endocrinology, 151(11): 5403–5414. doi:10.1210/en.2010-0345. PMID: 20861238. Zhu, A., Yoneda, T., Demura, M., Karashima, S., Usukura, M., Yamagishi, M., and Takeda, Y. 2009. Effect of mineralocorticoid receptor blockade on the renal renin-angiotensin system in Dahl salt-sensitive hypertensive rats. J. Hypertens. 27(4): 800– 805. doi:10.1097/HJH.0b013e328325d861. PMID:19516179.
