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Am J Physiol Renal Physiol 286: F936-F944, 2004. First published March 2, 2004; doi:10.1152/ajprenal.00070.2003
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In vitro characterization of aldosterone and cAMP effects in mouse distal convoluted tubule cells

Daniel González-Núñez,1,2 Manuel Morales-Ruiz,2 Alberto Leivas,2 Steven C. Hebert,3 and Esteban Poch1

1Servicio de Nefrología, 2Laboratorio de Hormonología, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clínic, Universidad de Barcelona, 08036 Barcelona, Spain; and 3Departament of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8020

Submitted 19 February 2003 ; accepted in final form 29 December 2003


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The distal nephron plays a capital role in the fine regulation of sodium reabsorption. Compared with the cortical collecting duct, much less information is available on the hormonal regulation of sodium transporter genes in the distal convoluted tubule (DCT), where the thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major entry pathway for Na+. The purpose of this study was to characterize the in vitro effects of aldosterone (Aldo; 1 µM) and cAMP (8-BrcAMP; 0.5 mM) on mouse DCT (mDCT) by using an immortalized mDCT cell line. Western blot analysis and semiquantitative RT-PCR were performed to analyze the expression of genes involved in sodium transport. The mDCTcell line expressed the 11{beta}-hydroxysteroid dehydrogenase type 2 gene and both the mineralocorticoid and glucocorticoid receptor genes, suggesting Aldo responsiveness. In this sense, we found that mDCT cells expressed the amiloride-sensitive Na+ channel (ENaC) and responded to Aldo by upregulating the {alpha}-subunit protein. Similarly, {alpha}1 Na+-K+-ATPase protein was upregulated by Aldo and 8-BrcAMP. In addition, the Aldo intermediate gene sgk1 mRNA was increased in response to both Aldo and 8-BrcAMP, and the transcription factor HNF–3{alpha} mRNA was induced by 8-BrcAMP. With respect to NCC regulation, although Aldo induced NCC protein levels in mice in vivo, neither Aldo nor 8-BrcAMP significantly induced the NCC mRNA or protein levels in mDCT cells. These results suggest that in mDCT, Aldo and cAMP modulate some downstream mediators and effectors in vitro but do not influence the expression of NCC in this cell model.

sodium reabsoption; gene expression; mineralocorticoids; cell culture

THE DISTAL CONVOLUTED TUBULE (DCT) participates in the fine regulation of Na+ reabsortion because it reabsorbs ~7% of the filtered Na+ and is the specific site of action of thiazide diuretics (36). Although the amiloride-sensitive sodium channel (ENaC) is expressed in the late DCT, the major mediator of Na+ and Cl- transport is the thiazide-sensitive Na+-Cl- cotransporter (NCC), which is expressed at the apical membrane of DCT cells (2, 6, 34). This nephron segment is of capital importance in human diseases affecting blood pressure. On one hand, mutations in NCC cause Gitelman's syndrome, characterized by low blood pressure, hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria (39). On the other hand, NCC is the target of thiazide diuretics, which are one of the mainstays in the treatment of essential hypertension.

Na+ transport in the distal nephron is hormone regulated. Mineralocorticoid hormones are major contributors to Na+ homeostasis in the distal nephron, where the expression of the mineralocorticoid receptor (MR) is predominant (6, 43). In this regard, the most known action of mineralocorticoids is the induction of the Na+-K+-ATPase and ENaC activity in the cortical collecting duct (CCD) (18, 19, 48). In addition, there are evidences that aldosterone (Aldo) may also act in the DCT by stimulating Na+ transport (47) and the expression of NCC protein (27) in the rat, suggesting that the DCT may be Aldo sensitive. However, there are some controversies that may be species dependent. For instance, histochemical studies in mouse DCT (mDCT) found minor expression of 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}HSD2), the key enzime for selective aldosterone actions, suggesting that this nephron segment may lack Aldo sensitivity in the mouse (8). In contrast, receptor sites and metabolic effects of mineralocorticoids have been documented in DCT, connecting tubules, and medullary collecting ducts in rabbits and rats (15, 17). An important downstream mediator of Aldo is the serum- and glucocorticoid-regulated kinase-1 (sgk1), which is rapidly increased in response to mineralocorticoids in rat kidney collecting duct (5, 9, 31). Sgk1 integrates a variety of signals that modulate renal Na+ reabsorption (16, 49). It is not known whether sgk1 plays a role in DCT under Aldo stimulation because hormonal regulation in this cell type DCT has been less studied.

In addition to mineralocorticoids, other hormones and autacoids also regulate Na+ transport in the distal nephron. One of the most studied has been vasopressin and cAMP analogs, which stimulate Na+ reabsorption in rat CCD (14, 22, 35, 44). However, little is known about the actions of cAMP in DCT at the molecular level. Thus the purpose of the present study was to determine the effects of Aldo and cAMP on mDCT in vitro and whether these cells expressed and modulated physiological mediators of these stimuli.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. DCT cells were kindly provided by Dr. Peter A. Friedman. The cells had been previously isolated and immortalized from mice by Pinzzonia et al. (33) and functionally characterized by Friedman and Gesek (20, 21). Cells were grown on 100-mm dishes (Techno Plastic Products) in Dulbecco's modified Eagle's medium/Ham's F-12 media (1:1; Sigma, St. Louis, MO) supplemented with 5% heat-inactivated fetal calf serum (Sigma), 2 mM L-glutamine (GIBCO BRL), 0.5 µg/ml streptomycin, 0.5 µg/ml penicilin, and 1 µg/ml neomycin (GIBCO BRL) in a humidified atmosphere of 5% CO2-95% air at 37°C. In some experiments (indicated in RESULTS and in corresponding figure legends), mDCT cells were grown on the apical chamber of rat-tail collagen-coated 24-mm-diameter Transwells (Corning-Costar, Cambridge, MA) at 70,000 cells/well in the same conditions as above and were trypsinized at confluence before protein isolation.

To examine effects of the hormonal treatment, cells were grown to confluence and monolayers were transferred to serum-free media for 24 h. The monolayers were then subsequently placed in either serum-free media supplemented with 1 µM aldosterone (Sigma), 0.5 mM 8-BrcAMP (Sigma), or both. Serum-free media with 0.032% ethanol was used as the vehicle control. The concentration of Aldo used is the one recognized to elicit the maximum short-circuit current in mouse CCD cells, with both the MR and glucocorticoid receptor (GR) expected to be activated (37, 40). The dose of 8-BrcAMP (0.5 mM) used corresponds to the one employed in published experiments of Na+ transport regulation in cultured cells by other groups (1, 12, 42).

Animal treatment. The work with animals was approved by the local ethical committee for the care of animals. Seven-week-old C57BL/6J mice (20–23 g; n = 8) with free access to a normal diet (0.3% Na+) were treated with 150 µg·kg-1·day-1 Aldo (Clinalpha) or vehicle for 5 days. The mice were killed, and whole kidney membrane proteins were isolated as described previously (34).

RNA isolation and RT-PCR. Cells were washed three times with 3 ml of PBS, and total RNA was then carefully extracted using 1.5 ml of TRIzol Reagent (GIBCO BRL). An aliquot was treated with DNA-free (Ambion, Austin, TX), eliminating potential contaminating DNA from RNA preparations. RNA concentration was then determined spectrophotometrically by ultraviolet absorption at 260 nm, and 3 µg were resolved by denaturing (formamide/formaldehyde) agarose gel electrophoresis to inspect for degradation.

For RT, 2.5 µg of total RNA were transcribed using an AMV-cDNA synthesis kit (Boehringer Mannheim). cDNA synthesis was carried out in a PTC-200 DNA Thermocycler (MJ Research) for 1 h at 42°C and stopped by heating at 95°C for 5 min. RT reagents were checked for amplicon contamination by the inclusion of a control without RNA. Different PCR were performed using specific primers (summarized in Table 1) and conducted in 25 µl containing 20 mM Tris·HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, 0.8 µM of each primer, 1 U Taq polymerase (Boehringer Mannheim), and 2.5 µl of cDNA (diluted 1:16 for GAPDH and grp58). Mouse HNF-3{alpha} primers were designed by Vaisse et al. (45), and GAPDH primers were described by Rocco et al. (38). Mouse MR and GR genes primers have been described previously (4, 25). Each PCR was within the linear range of amplification (see Table 1).


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  		Table 1. Primer sequences for RT-PCR

 

Protein isolation and Western blot analysis. mDCT cells were washed twice with cold PBS and scraped with 2 ml of PBS. Cells were transferred to a clean tube and centrifuged at 1,000 g for 5 min.

For isolation of membrane protein, the supernatant was removed and cells were homogenized in 500 µl ice-cold homogenization buffer [0.32 M sucrose, 5 mM Tris·HCl (pH 7.5), and 2 mM EDTA] plus protease inhibitors (2 mM Pefabloc SC and 70 µl Complete Protease inhibitor X7, Roche) following established protocols (23, 34). Thereafter, cells were sonicated for 60 s (4 cycles of 15 s) at 20 kHz. The cell lysate (homogenate) was subjected to centrifugation at 4,000 g for 5 min at 4°C, and the supernatant was further centrifuged at 100,000 g for 75 min at the same temperature. The 100,000-g supernatant was regarded as the cytosolic fraction, and the 100,000-g pellet as the membrane fraction, which was resuspended in 250 µl buffer containing 5 mM Tris·HCl (pH 7.5) and 2 mM EDTA with protease inhibitors plus 0.5% of SDS and stirred at 4°C for 1 h. Protein content of all samples was determined using the Bio-Rad (Hercules, CA) protein assay based on the Bradford method (7) with bovine serum albumin as a standard. Proteins (50 µg) of the subcellular fractions were denatured in SDS-sample lysis buffer [2% SDS, 62.5 mM Tris·HCl (pH 6.8), 30% glycerol, 5% {beta}-mercaptoethanol, and 0.1% bromophenol blue] at 37°C for 5 min. Samples were then subjected to SDS-PAGE (28) on 7.5% polyacrylamide gels before electrophoretic transfer to polyvinylidene difluoride membrane (Immobilon-P, Millipore). Preliminary 7.5% SDS-polyacrylamide gels were run and stained with Coomassie blue to confirm equality of loading in each lane. After the polyvinylidene difluoride membranes were blocked with 3% milk in TBS (150 mM NaCl and 10 mM Tris) with 0.1% Tween 20 for 60 min, membranes were incubated at either a 1:600 final dilution of a rabbit polyclonal antiserum against mouse TSC (kindly provided by Dr. D. H. Ellison) or a 1:1,000 final dilution of rabbit polyclonal antiserum against rat TSC for 60 min. The latter serum, used thereafter for the remainder of experiments, had been previously used and characterized by Plotkin et al. (34) and Hoover et al. (23). The immunocomplexes were detected by binding with 1:4,000 diluted secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, Amersham) using the enhanced chemiluminescence method (Amersham). A mouse monoclonal antibody specific to the Na+-K+-ATPase {alpha}1-subunit was obtained from a commercial source (Upstate Biotechnology, Lake Placid, NY). Membranes were incubated at a 1:2,000 final dilution for 60 min and secondary antibody at 1:2,000 anti-mouse IgG. A polyclonal antibody against rat {alpha}-ENaC was obtained from Affinity BioReagents and used at 1:1,000 dilution. The blots were quantified by densitometry (Non Linear Dynamics, Newcastle upon Tyne, UK). Densitometric values were expressed as arbitrary units.

Statistical analysis. Data are presented as means ± SE of densitometric arbitrary units. Comparisons between treatments were determined by a two-sided Mann-Whitney U-test for unpaired data. The 5% probability level was regarded as significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of 11{beta}HSD2 and steroid receptors (MR and GR). To characterize the steroid responsiveness of the mDCT cell line, RT-PCR was conducted with specific primers against mouse 11{beta}HSD2 and MR and GR cDNA (Table 1). Figure 1 shows that mDCT cells express all three genes under basal conditions. As a positive control, mouse kidney RNA was used. The identity of the bands was confirmed by the sequencing method.



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  		Fig. 1. Expression of 11{beta}-hydroxysteroid dehydrogenase type 2 (11{beta}HSD2), the mineralocorticoid receptor (MR), and the glucocorticoid receptor (GR) in mouse distal convoluted tubule (mDCT) cells. RT-PCR with specific primers (Table 1) against 11{beta}HSD2 (A), the mineralocorticoid receptor (B), and the glucocorticoid receptor genes (C) was performed with total RNA of mouse kidney as positive control (C+), whereas PCR tube without cDNA was performed in each reaction to discard environmental contamination of PCR reaction (C-; shown in A). Specific bands of 442, 280, and 298 bp were obtained, purified, and sequenced to confirm their identity. M, membrane fraction.

 

Regulation of NCC. We characterized NCC mRNA and protein expression in mDCT cells. Figure 2, A and B, shows two independent blots with two different antisera against NCC (see METHODS). With either antiserum, a predominant band of ~110 kDa was seen in the membrane fraction and in the total cell extract, but not in the cytosolic fraction. To improve the molecular size estimation of the NCC band, a biotinylated protein ladder was used and confirmed the size of the core, unglycosylated NCC protein (Fig. 2C) as reported previously (23, 34). In Fig. 2C, a larger band representing glycosylated NCC can be seen. As previously demonstrated (23), the glycosylated band appears fainter in cells or oocytes than in native tissue, this probably representing differences in protein processing. Expression of NCC in mDCT cells was further determined by the detection of a specific NCC 205-bp band by RT-PCR (Fig. 2D), which was confirmed by sequencing.



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  		Fig. 2. Thiazide-sensitive Na+-Cl- cotransporter (NCC) expression in mDCT cells. Two different specific antisera were used to confirm NCC protein expression in the different cellular subfractions. Fifty micrograms of cytosolic fraction (CIT), membrane (M) fraction, and total protein (TP) were loaded in each lane. A: Western blot analysis of polyvinylidene difluoride (PVDF) membranes probed with a rabbit anti-mouse NCC antiserum. B: Western blot analysis of PVDF membranes probed with a rabbit anti-rat NCC antiserum. C: NCC detection with the rat antibody using a biotinylated protein ladder to improve the molecular size estimation of the NCC band. The size of the core unglycosylated NCC protein (110 kDa) was confirmed as previously reported. D: mRNA-NCC expression by RT-PCR using specific primers. A specific band (205 bp) in lanes 1, 2 (mDCT cells-cDNA), and 3 (mouse kidney cDNA) is detected. Lane 4 is a ladder, and lane 5 is a negative control.

 

For the hormonal regulation experiments, the anti-rat NCC immune serum (Fig. 2B) was employed. As shown in Fig. 3, A and B, no differences in NCC protein level were detected between treatments: neither Aldo nor 8-BrcAMP significantly induced NCC protein in mDCT cells. This lack of effect was also observed at up to 72 h (data not shown). Only the combination of treatments (Aldo plus 8-BrcAMP) produced a slight, nonsignificant, increase in NCC protein at 24 h (vehicle: 34.2 ± 3.8 vs. combined treatment: 45.0 ± 4.6; P = 0.09; n = 8). This lack of effect was observed on both the 110-kDa band (unglycosylated) and on the larger band (glycosylated and physiological more relevant). In addition, NCC mRNA levels were analyzed by semiquantitative RT-PCR and, as occurred with protein abundance, no differences in mRNA were detected among treatments (data not shown). Because these negative results were seen in the mDCT cells grown on plastic, independent experiments were performed with cells grown on collagen-coated filters to better mimic physiological conditions. As shown in Fig. 3C, no differences in NCC protein expression were detected in response to Aldo or cAMP under these conditions. Finally, NCC regulation in response to Aldo was evaluated in mice in vivo, and the results are displayed in Fig. 4. In contrast to what occurred in the in vitro experiments, Aldo did induce NCC protein levels in mice in vivo (vehicle 133 ± 27, Aldo 233 ± 21; P < 0.03; n = 8). Although the effect was mostly due to increases in the 110-kDa band density, there was a trend toward an increase in the glycosylated band as well.



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  		Fig. 3. Effect of aldosterone (Aldo) and 8-BrcAMP on NCC protein levels in mDCT cells. Top: Aldo, 8-BrcAMP, or combined treatment effects at 6 (A) and 24 h (B). Western blot analysis with specific rat NCC antiserum shows a specific band of 110 kDa. The top band represents the glycosylated form of NCC. Preliminary 7.5% SDS-polyacrylamide gels were run and were stained with Coomassie blue to confirm equality of loading in each lane. Values are means ± SE in densitometric arbitrary units. C: representative semiquantitative immunoblots (Western blots) probed with specific rat NCC antiserum. Proteins were obtained from cells seeded and grown on rat-tail collagen-coated 24-mm-diameter Transwells and treated with vehicle (Veh), Aldo, or 8-BrcAMP.

 


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  		Fig. 4. Effects of Aldo on NCC protein expression in mice in vivo. Seven-week-old C57BL/6J mice (20–23 g; n = 8) under free access to a normal diet (0.3% sodium) were treated with 150 µg·kg-1·day-1 Aldo (Clinalpha) or vehicle for 5 days. Mice were killed, and whole kidney membrane proteins were isolated as described. Proteins were run on 7.5% SDS-PAGE and transferred to PVDF membranes. Membranes were probed with rat NCC antiserum. Top: representative Western blot analysis showing NCC bands under vehicle or Aldo. Bottom: quantitative analysis. Vehicle 133 ± 27, Aldo 233 ± 21, P < 0.03, n = 8. Values are means ± SE in densitometric arbitrary units.

 

Regulation of {alpha}1 Na+-K+-ATPase and {alpha}-ENaC proteins. The effect of hormonal treatments on {alpha}1 Na+-K+-ATPase subunit and {alpha}-ENaC protein levels is shown in Fig. 5. Both Aldo and 8-BrcAMP induced {alpha}1 Na+-K+-ATPase protein level in late time courses as previously described (18). Whereas induction was not significant at 6 h (vehicle 50.8 ± 2.4, Aldo 54.5 ± 7.9, 8-BrcAMP 63.8 ± 8.9, and combined treatment 70.0 ± 12.6; P = not significant), the differences were statistically significant at 24 h (vehicle 52.8 ± 5.7, Aldo 81.8 ± 10.0, P <= 0.03 vs. vehicle, 8-BrcAMP 99.5 ± 7.9, P <= 0.01 vs. vehicle, and combined treatment 97.0 ± 9.2, P <= 0.01 vs. vehicle). In the basal state, mDCT cells expressed the {alpha}-, {beta}-, and {gamma}-subunits of ENaC at the mRNA level (data not shown). As extensively demonstrated by others in CCD cells and in vivo (18), Aldo also induced {alpha}-ENaC protein in the mDCT cell line (Fig. 5C).



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  		Fig. 5. Effects of Aldo and 8-BrcAMP on 1 Na+-K+-ATPase and {alpha}-amiloride-sensitive Na+ channel (ENaC) in mDCT cells. Cells were treated with Aldo, 8-BrcAMP, or combined treatment as described above. Top: protein levels of Na+-K+-ATPase {alpha}1-subunit with different treatments at 6 (A) and 24 h (B). Western blot analysis with a specific mouse Na+-K+-ATPase {alpha}1-antibody (Upstate Biotechnology, Lake Placid, NY) at 1:2,000 dilution resulted in a 100-kDa band (top band). Preliminary 7.5% SDS-polyacrylamide gels were run and stained with Coomassie blue to confirm equality of loading in each lane. Values are means ± SE (n = 8) in densitometric arbitrary units. *P <= 0.03 and **P <= 0.01 vs. vehicle (n = 5). Bottom: representative semiquantitative immunoblots. C: Western blot analysis showing the effect of Aldo on {alpha}-EnaC in mDCT cells at 24 h. A polyclonal antibody against rat {alpha}-ENaC (Affinity BioReagents) was used at 1:1,000 dilution and shows a specific band under Aldo treatment or vehicle (Veh). Values are means ± SE in densitometric arbitrary units.

 

Expression and regulation of sgk1, grp58, and HNF-3{alpha} mRNA. We next evaluated the expression and regulation of several intracellular mediators known to be involved in the control of Na+ uptake in the distal nephron.

First, Fig. 6 shows the expression level of sgk1 mRNA by using semiquantitative RT-PCR. Sgk1 was stimulated by Aldo and 8-BrcAMP within 1 h of treatment and reached a peak at 3 h [vehicle 0.93 ± 0.06, Aldo 1.57 ± 0.09, 8-BrcAMP 1.71 ± 0.14, and combined treatment 1.92 ± 0.10 (P <= 0.01 vs. vehicle)]. Sgk1 levels decreased with time but were still significantly higher up to 24 h after treatments. The values at 9 h were vehicle 0.56 ± 0.07, Aldo 0.97 ± 0.14 (P <= 0.03 vs. vehicle); 8-BrcAMP 1.23 ± 0.10 (P <= 0.01 vs. vehicle); and combined treatments 1.34 ± 0.16 (P <= 0.01 vs. vehicle). At 24 h, values were vehicle 0.45 ± 0.05, Aldo 0.93 ± 0.13 (P < 0.03 vs. vehicle); 8-BrcAMP 0.99 ± 0.06 (P < 0.01 vs vehicle); and combined treatments 1.20 ± 0.19 (P < 0.03 vs. vehicle). Moreover, we tested the effect of serum (FCS) on sgk1 mRNA levels. In this sense, confluent cells were maintained for 24 h in the presence or absence of 5% serum-supplemented medium. As shown in Fig. 7, sgk1 transcript levels were stimulated by the presence of serum (serum free 0.56 ± 0.06 vs. FCS 0.94 ± 0.05; P <= 0.02; n = 6).



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  		Fig. 6. Effect of Aldo and 8-BrcAMP on sgk1 in mDCT cells. Expression levels of sgk1 mRNA with different treatments at 3 (A) and 24 h (B) of treatment. GAPDH was amplified to standardize for the amount of RNA subjected to reverse transcription. Values are means ± SE (n = 5) in densitometric arbitrary units. *P <= 0.03 and **P <= 0.01 vs. vehicle. A representative gel with sgk1 band (top) and GAPDH housekeeping band (bottom) is shown.

 


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  		Fig. 7. Serum induction of sgk1 transcripts in mDCT cells. Cells were treated in the presence (FCS+) or absence (FCS-) of 5% of fetal calf serum for 24 h before RNA extraction. Values are means ± SE (n = 5) in densitometric arbitrary units. **P <= 0.02.

 

Second, grp58 has been recently described as a protein that binds to the NCC in vivo and that influences the functional properties of NCC (51). RT-PCR with specific primers demonstrated expression of grp58 in mDCT cells. However, as shown in Fig. 8, there were no differences in the expression level of grp58 mRNA between different treatments. At 3 h the values were vehicle 1.05 ± 0.09, Aldo 1.00 ± 0.20, 8-BrcAMP 0.90 ± 0.18, and combined treatments 0.92 ± 0.19 (P = not significant); and at 9 h vehicle 1.12 ± 0.15, Aldo 1.03 ± 0.09, 8-BrcAMP 0.93 ± 0.16, and combined treatments 1.23 ± 0.07 (P = not significant). The lack of induction was also observed at a longer (24 h) time course (data not shown).



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  		Fig. 8. Effects of Aldo and 8-BrcAMP on Grp58 mRNA levels. mRNA grp58/GAPDH values are represented at 3 (A) and 9 h (B) of treatment. GAPDH was amplified to standardize for the amount of RNA subjected to reverse transcription. Values are means ± SE (n = 5) in densitometric arbitrary units. A representative gel with grp58 band (bottom) and GAPDH housekeeping band (top) is also shown.

 

Finally, recent evidence identified HNF-3{alpha} as a vasopressin-induced gene in a mouse clonal CCD principal cell line (37). As shown in Fig. 9, HNF-3{alpha} mRNA was stimulated (2-fold increase) by 8-BrcAMP without any effect observed for Aldo. The values observed at 3 h were vehicle 0.83 ± 0.09, Aldo 0.89 ± 0.13 (P = not significant vs. vehicle), 8-BrcAMP 1.82 ± 0.3 (P <= 0.01 vs. vehicle), and combined treatments 1.42 ± 0.16 (P <= 0.01 vs. vehicle), and at 9 h vehicle 0.68 ± 0.12, Aldo 0.74 ± 0.17, 8-BrcAMP 1.56 ± 0.29 (P <= 0.03 vs. vehicle), and combined treatments 1.34 ± 0.16 (P <= 0.03 vs. vehicle). HNF-3{alpha} mRNA levels were still enhanced up until 24 h after treatments (data not shown). The differences between 8-BrcAMP alone and in combination with Aldo were not significant. The effect of serum on the expression of HNF-3{alpha} mRNA was tested, but no difference was observed with or without serum (data not shown).



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  		Fig. 9. Effects of Aldo and 8-BrcAMP on HNF-3{alpha} mRNA levels. mRNA HNF-3{alpha}/GAPDH values are represented at 3 (A) and 9 h (B) of treatment. GAPDH was amplified to standardize for the amount of RNA subjected to reverse transcription. Values are means ± SE (n = 5) in densitometric arbitrary units. *P <= 0.03 and **P <= 0.01 vs. vehicle. A representative gel with HNF-3{alpha} band (bottom) and GAPDH housekeeping band (top) is also shown.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Whereas the effects of Aldo on CCD cells have been extensively studied, fewer data are available about the effects of this hormone in the DCT. In this study, we show that Aldo modulates some of established downstream mediators in a mDCT cell line. For instance, in resting conditions, mDCTcells expressed both the MR and the GR as well as the 11{beta}HSD2, a key enzyme in Aldo selectivity. When the cells were stimulated with Aldo, sgk1, an important mediator in Aldo actions, was induced at early stages. In addition, basolateral {alpha}1 Na+-K+-ATPase and apical {alpha}-ENaC, effectors of Aldo in CCD, were also stimulated by this hormone in mDCT cells. Moreover, highlighting the hormonal responsiveness of these cells, we show that they were also capable of regulating cAMP-induced genes as well.

In the DCT, the major pathway for apical Na+ and Cl- entry is the NCC (36). Several studies have shown that rat NCC is regulated by Aldo in vivo. Indeed, the responsiveness of the DCT to hormonal stimuli has been evaluated previously in vivo by several authors (reviewed in Ref. 36). These studies have been performed mainly in the rat by evaluating Na+ and Cl- transport in in vivo microperfusion experiments and by analyzing the binding of [3H]metolazone as an estimation of the number of NCC units (10, 11). Evidence for the action of Aldo, glucocorticoids, ANG II, and calcitonin, among others on the rate of NaCl transport and [3H]metolazone binding in rat DCT has been provided in the past (36). More recently, the cloning of NCC from several species has allowed the study of the regulation of this gene at the molecular level. In this sense, Kim et al. (27) showed that Aldo infusion in the rat induced an increase in NCC protein abundance. This effect has been shown to take place without concomitant changes in NCC mRNA levels (30, 36, 50). Because of the lack of data about the regulation of NCC in vitro, one of the goals of the study was to evaluate the effects of Aldo and cAMP on this cotransporter in a mDCT cell model. The human and rat NCC promoter has recently been cloned and characterized (29, 41), and its sequence contains glucocorticoid-responsive elements and cAMP-responsive elements. In contrast to the in vivo studies performed in rats, we were not able to see an induction of NCC protein or mRNA after challenging mDCT cells with Aldo. This lack of effect was also observed when cells were seeded and grown on collagen-coated Transwells. There may be several explanations for this discrepancy. To rule out the possibility of species differences, we tested the effects of Aldo on NCC in mice in vivo. As has been demonstrated in the rat, Aldo also upregulated NCC protein levels in mice in vivo. Therefore, the lack of effect of Aldo on NCC in vitro could be alternatively explained because cells in culture may not mimic the conditions present in vivo. In this sense, similar dicrepancies have been described for another electroneutral Na+ cotransporter, namely, the bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2/BSC1). While dexamethasone induces NKCC2 in rat medullary thick ascending limb in vivo, it is unable to induce this gene in cultured medullary thick ascending limb cells unless vasopressin or cAMP is present in the medium at physiological concentrations (1).

It has been proposed that the mechanism of mineralocorticoid upregulation of NCC protein could be indirect, as a consequence of the observed discrepancies between mRNA and protein levels (30, 50). Therefore, the activation of additional mediators that regulate NCC protein abundance through other mechanisms (mRNA translation, NCC protein half-life) might be in play. In addition, Aldo may enhance transporter activity by increasing its half-life at the plasma membrane or by translocating transporters to the plasma membrane (48). In our experiments, the coadministration of Aldo and cAMP only produced a slight increase in NCC protein abundance in mDCT, which was not statistically significant compared with nontreated cells. Other potential mediators have been recently identified. Specifically, a protein termed grp58 has been shown to interact with and affect NCC activity in rats (51). To test whether this gene could be an Aldo-induced gene, we measured the effects of Aldo and cAMP on grp58 mRNA and found that this gene was not affected by these treatments.

It is interesting that besides the lack of upregulation of NCC protein, these cells seem to express the basic machinery of mineralocorticoid sensitivity. The expression of 11{beta}HSD2 is correlated with localization and catalytic activity studies, which show that this enzyme is expressed, albeit at low and variable levels, in mDCT, being the most clear signal present in the final part of DCT, next to the connecting tubule (8, 26). The expression of the MR in mDCT cells is also correlated with localization studies in mice (6). In addition, we also observed upregulation of established mediators and effectors of Aldo in cultured mDCT cells. For instance, sgk1, {alpha}1 Na+-K+-ATPase, and {alpha}-ENaC are induced in response to Aldo in the mDCT cell line at time courses typically found in other cell systems (48). Sgk1 mRNA has been found by other authors to increase early (1 h) in response to Aldo and to decrease in time even in the presence of Aldo (9, 31), making our finding in agreement with these observations. In vivo experiments in mice have shown that sgk1 mRNA is upregulated in response to Aldo in both cortex and outer medulla (24). With respect to {alpha}1 Na+-K+-ATPase, this gene responds late (>6 h) to Aldo, with levels still being elevated at late time points (24 h) (reviewed in Ref. 18). Finally, {alpha}-ENaC is also upregulated at late time points in mDCT, as observed in CCD (3, 37).

Previous studies have shown that the DCT is responsive to cAMP, which mediates the actions of a number of hormones (vasopressin, glucagon, PTH, calcitonin). Van Huyen et al. (46) have shown that vasopressin stimulated Cl- transport in a mouse transimmortalized cell line from the DCT. Similarly, glucagon and vasopressin have been shown to stimulate Mg2+ transport in the same mDCT cell line used in our study (13). We show that cAMP clearly induced {alpha}1 Na+-K+-ATPAse protein abundance in mDCT. The effects of vasopressin or cAMP on activity and expression of Na+-K+-ATPase have been previously described both in vitro and in vivo in CCD (14, 18). The DCT is the nephron segment where Na+-K+-ATPase activity is most abundant (36), and to our knowledge no previous data about in vitro regulation of this gene by cAMP have been reported in the mouse. The mechanism of action of cAMP in these cells is not evident from our experiments. However, we observed that cAMP increased the expression of the transcription factor HNF-3{alpha}, which had been shown to be strongly induced by vasopressin in a CCD cell line (37). In addition, although cAMP has been shown to increase the activity of sgk1 expressed in COS cells (32), the induction of sgk1 by this mediator has not been previously reported in DCT.

In summary, we have shown that Aldo induces the upregulation of known mediators (sgk1) and effectors ({alpha}1 Na+-K+-ATPase and {alpha}-ENaC) in mDCT cells. In addition, mDCT cells respond to cAMP, with upregulation of known mediators (HNF-3{alpha}, sgk1) and effectors ({alpha}1 Na+-K+-ATPase) of their actions. In contrast, although Aldo upregulated mouse NCC in vivo, no effects of this hormone were seen in the mDCT cell model in vitro. Further studies to search for additional mediators will be necessary to elucidate the mechanisms of NCC regulation by Aldo.


   ACKNOWLEDGMENTS
 
We are indebted to Dr. P. Friedman for providing us with the mDCT cell line and to Dr. D. H. Ellison for the anti-mouse NCC antiserum. We also thank Silvia F. Boj and Alicia de Diego for help in the animal studies.

GRANTS

This work was supported by Grant FIS 01/1151 from the Fondo de Investigaciones Sanitarias. D. González-Núñez is a recipient of a grant from the Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain. M. Morales-Ruiz is an established investigator of the Programa Ramón y Cajal (MCyT).


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Poch, Servicio de Nefrología, Hospital Clinic, Universidad de Barcelona, Villarroel 170, 08036 Barcelona, Spain (E-mail: epoch{at}medicina.ub.es).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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