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Aldosterone and ENaC: From Genetics to Physiology
1Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio 78229-3900; 2Department of Chemistry and Biochemistry, Texas State University, San Marcos, Texas 78666; and 3Epithelial Pathobiology Group, Department of Surgery, University of Cincinnati, Cincinnati, Ohio 45219
Submitted 26 September 2003 ; accepted in final form 18 February 2004
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ABSTRACT |
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epithelial sodium channel; downstream effectors; Ras; serum and glucocorticoid-induced kinase; hypertension
Aldosterone, as mentioned above, is a critical endocrine modulator of ENaC activity. Studies from our laboratory (9, 25), as well as that of Verrey et al. (13, 21, 22), demonstrated that aldosterone increases K-RasA levels in A6 epithelial cells through a mechanism dependent on transcriptional control of the K-ras gene. We also made similar observations in cardiac fibroblasts from adult rats (24). Aldosterone not only increased K-RasA levels in A6 cells but also increased the amount of active GTP-complexed K-RasA (9). Induction of K-RasA is necessary and possibly sufficient for aldosterone action in some instances, especially in A6 cells (25). In addition, overexpression of constitutively active K-RasA with ENaC in Xenopus laevis oocytes increases channel open probability (13).
Aldosterone activates two established downstream effector-signaling pathways of K-RasA, the mitogen-activating protein kinase (MAPK)1/2 and phosphatidylinositol 3-kinase (PI3-K) cascades, in renal A6 epithelial cells (2, 9, 17, 27). Similar to K-RasA, PI3-K signaling is necessary for Na+ transport in this model (2, 17, 27). In contrast, activation of the MAPK1/2 cascade independent of aldosterone suppresses Na+ transport by targeting ENaC subunits for degradation (3). The downstream PI3-K target Sgk is also an aldosterone-induced protein with aldosterone signaling, in addition, activating this kinase in a PI3-K-dependent manner (6, 15, 27).
Although the MAPK1/2 and PI3-K signaling cascades, as well as a common initiator of both cascades, K-RasA, are known modulators of Na+ transport, the relationship between these cascades and their possible common activator in aldosterone-activated epithelial cells has not been investigated. In addition, a direct link between PI3-K and ENaC activity has not been established. Possible signaling convergence involving the aldosterone-induced proteins K-RasA and Sgk, moreover, has not been investigated. The current studies confirm that aldosterone activates both PI3-K and MAPK1/2 signaling in A6 cells but importantly demonstrate that it can do so through induction of K-RasA. In addition, we demonstrate cross-talk between these two cascades in response to aldosterone, as well as signaling convergence between Sgk and K-RasA. Finally, and we believe most importantly, the current study demonstrates directly that PI3-K activates ENaC with the PI3-K-dependent signaling cascade, but not the MAPK1/2 cascade, being a powerful mediator of aldosterone action on Na+ transport in native epithelia.
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METHODS |
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-, 
-, and 
-hENaC cDNA in the pMT3 plasmid have been described previously (14, 19) and were a gift from Dr. P. Snyder (Univ. of Iowa College of Medicine, Iowa City, Iowa). The adenovirus major late promoter drives expression of each cDNA. These constructs were used without modification. The construct encoding constitutive active PI3-K (p110-) was from BD Transduction Laboratory. Dominant-negative Ras (DNRasN17) was a kind gift from Dr. G. Firestone (Univ. of California at Berkeley, Berkeley, CA) and has been used previously by our laboratory (9). The glutathione S-transferase (GST)-Sgk fusion protein used in the current study was created by subcloning X. laevis Sgk in a frame behind GST in the pGEX-KG plasmid. The donor xSgk plasmid and the pGEX-KG plasmid were kind gifts from Drs. D. Pearce (Univ. of California at San Francisco, San Francisco, CA) and A. Firulli (Univ. of Texas Health Science Center at San Antonio, San Antonio, TX), respectively. Phosphorothioate antisense and sense K-ras oligonucleotides have been described previously (9, 25) and were from the Emory Univ. (Atlanta, GA) microchemical facility. The rabbit polyclonal anti-MAPK1/2 and anti-PI3-K p110 antibodies and mouse monoclonal anti-PI3-K p85 antibody were from Upstate Biotechnology. The sheep polyclonal anti-SGK antibody was also from Upstate Biotechnology. This antibody has been discontinued. The mouse monoclonal anti-c-Raf-1 antibody was from Transduction Laboratories. The mouse monoclonal anti-v-Ha-Ras antibody was from Oncogene. The rabbit polyclonal anti-K-Ras2A and goat polyclonal anti-PKB kinase (PDK1) antibodyies were from Santa Cruz Biotechnology. All phospho-specific rabbit polyclonal antibodies were from Cell Signaling Technologies, except anti-phospho-MBP antibody, which was from Upstate Biotechnology. All secondary horseradish peroxidase-conjugated antibodies were from Kirkegaard & Perry Laboratories.
Cell culture and cell transfection.
Renal A6 cells were maintained in culture as described previously (9, 25). For Western blot analysis and assessment of open-circuit current, cells were grown to confluence on polycarbonate supports (Transwell-Clear Inserts, pore size 0.4 µM, 4.7 cm2; Costar, Cambridge, MA) in the presence of serum and aldosterone (1.5 µM). Two days before experiments, cells were maintained in minimal media depleted of serum and steroids. Chinese hamster ovary (CHO) cells were maintained in tissue culture as described previously (4). For patch-clamp analysis, cells were plated on coverglass chips treated with 0.01% poly-lysine and transfected using the PolyFect reagent (Qiagen, Valencia, CA) per the manufacturer's recommendations. Initially, cells 
20–60% confluent were treated with 2.0 µg total plasmid cDNA [0.5, 0.5, 0.5, 0.5 µg of -, -, and -hENaC, and green fluorescent protein (GFP) cDNA]. To investigate PI3-K regulation, cells were transfected with 0.1, 0.1, 0.1, 1.0, and 0.5 µg of -, -, -hENaC, PI3-K, and GFP cDNA. Cells were used for up to 72 h after transfection and were maintained in culture in the presence of 10 µM amiloride replenished daily. Plasmid maxiprep cDNA was prepared using anion-exchange resin and isoproponal precipitation (Qiagen) with cDNA solubilized in water.
Western blot analysis.
Western blot analysis closely followed methods described previously (3, 9, 25). In brief, cells were extracted with gentle lysis buffer [76 mM NaCl, 50 mM Tris·HCl, 2 mM EGTA, plus 1% Nonidet P-40, 10% glycerol (pH 7.4), and 1 mM phenylmethylsulfonyl fluoride] with equal amounts of total cellular protein (80 µg) loaded per lane. In some instances, whole cell lysates were prepared in the presence of standard protein phosphatase inhibitors (in mM): 0.1 NaPPi, 0.5 NaF, 0.1 Na2MoO4, 0.1 ZnCl2, and 0.04 Na3VO4. For immunoprecipitation experiments, starting samples had equal amounts of total cellular protein (400 µg). The methodology used in the current study for pulldown experiments using the Ras-binding-domain of Raf (RBD) and the in vitro MAPK1/2 activity assay was identical to that described previously (9). All Western blot analysis was performed using material and reagents from Pierce (Rockford, IL) and Bio-Rad (Hercules, CA).
Electrophysiology. Open-circuit current across A6 cell monolayers was quantified as described previously (3, 25). Activity of ENaC reconstituted in CHO cells was quantified using voltage-clamp analysis in the whole cell configuration as described previously (4). For these experiments, the major cations in the bath and pipette solutions were Na+ and Cs+, respectively, with symmetrical Cl–. For bi-ionic selectivity studies, bath and pipette solutions were 50 mM NaCl + 100 mM NMDGCl, and 150 mM NMDGCl, respectively, with bath Na+ completely replaced with Cs+, K+, and Li+.
Statistics.
Data are reported as means ± SE. Statistical significance (P 
0.05) was determined using the t-test for differences in mean values.
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RESULTS |
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We next asked whether aldosterone-activated K-RasA influenced the PI3-K cascade in A6 cells. Shown in Fig. 1D are typical Western blots containing the anti-p110 PI3-K (top), anti-Ras (middle), and anti-p85 PI3-K (bottom) precipitants from the same whole cell lysates of A6 cell monolayers in the absence and presence of aldosterone for 30 and 60 min. The top, middle, and bottom blots were probed with anti-K-RasA, anti-p85, and anti-p85 antibody, respectively. The results demonstrate that K-RasA associated with the catalytic p110 subunit of PI3-K in an aldosterone-dependent manner. Interestingly, we also observed an aldosterone-dependent decrease in the amount of regulatory p85 subunit associated with K-RasA and presumably p110.
To investigate aldosterone action on the downstream PI3-K effector Sgk in amphibian A6 cells, we first characterized a commercially available anti-Sgk antibody using a GST-xSgk fusion protein (Fig. 2A). The blot on the left contains the glutathione-agarose precipitant from bacterial lysate from cells transformed with empty pGEX-EZ plasmid (lane 1) and this plasmid containing the cDNA encoding xSgk positioned in frame behind GST (pSgk: lane 2). This Western blot was probed with anti-GST antibody. Next, we took lysate from bacteria expressing pSgk precipitated with glutathione-agarose and then exposed an aliquot of this precipitant to thrombin to cleave xSgk from GST. Shown in the Western blot on the right are the uncleaved (lane 1) and cleaved (lane 2) products from this experiment. This blot was probed with anti-Sgk antibody. These results demonstrate that the antibody used in this study recognizes amphibian Sgk.
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Figure 2, C and D, tested the effects of aldosterone on Sgk. These representative Western blots contain the whole cell lysate from A6 cell monolayers treated with aldosterone from 0 to 8 h. Lysates were prepared in the presence of phosphatase inhibitors. The blots were probed with anti-Sgk antibody. Clearly shown is that aldosterone increases Sgk levels by 1 h with levels tending downward after 2 h. Similar to other groups (16, 27), we were able to distinguish phosphorylated (slower migrating band) from unphosphorylated (faster migrating band) Sgk in our experiments. This is particularly apparent in Fig. 2D. The bottom blot in Fig. 2D shows a loading control and is the top blot (probed with anti-Sgk antibody) stripped and subsequently reprobed with anti-MAPK antibody. Aldosterone preferentially increased phosphorylated Sgk consistent with activation of the upstream PI3-K-PDK1 cascade (see Ref. 16). These results demonstrate that aldosterone has two effects on Sgk, increasing absolute levels as well as active levels, a finding similar to aldosterone action on K-RasA (25, 27).
Western blots in Fig. 3A further tested whether aldosterone activates the MAPK1/2 cascade in a K-RasA-sensitive manner. These typical blots contained whole A6 cell lysate from cells treated with aldosterone (4 h) following a 24-h pretreatment with sense and antisense K-ras oligonucleotides (10 µM) identical to the area around the translation start site of X. laevis K-ras. Use of these oligonucleotides has been described previously (9, 25). In figure 3A, blot 1, blot 2, blot 3, and blot 4 were probed with anti-K-RasA, phospho-c-Raf, phospho-MAPK1/2, and MAPK1/2 antibodies, respectively. Blot 4 is simply blot 3 stripped of anti-phospho-MAPK1/2 and reprobed with anti-MAPK1/2. Consistent with our previous findings (9), suppression of K-RasA levels with antisense oligonucleotide decreased aldosterone-sensitive increases in K-RasA levels, as well as active (phospho)-MAPK1/2 levels, but not total MAPK1/2 levels. Interestingly, we also saw K-RasA-sensitive phosphorylation of c-Raf at Ser259. Akt is known to phosphorylate this site on active c-Raf, resulting in negative regulation of c-Raf kinase (18, 33). Thus measuring phosphorylation of c-Raf on Ser259 is an indirect measurement of Akt activity. We interpret these results as demonstrating that aldosterone simultaneously activates both the MAPK1/2 and PI3-K signaling cascades with cross talk between these cascades happening at the level of c-Raf and Akt. The results in the blot 1 of Fig. 3C are consistent with this interpretation.
Results shown in Fig. 3B were from experiments testing whether aldosterone increased active levels of Sgk in a K-RasA- and PI3-K-dependent manner. The typical blot in Fig. 3B probed with anti-Sgk (top) antibody contains whole A6 cell lysate from untreated cells (CON) and monolayers treated with aldosterone in the absence and presence of the PI3-K inhibitor LY-294002 (50 µM) for 4 h, as well as lysates from cells pretreated with sense and antisense K-ras oligonucleotides (24 h) followed by a 4-h aldosterone treatment. Figure 3B, bottom, is a loading control containing the same lysates and probed with anti-MAPK1/2 antibody. These results showing that, in the presence of antisense K-ras and LY-294002, aldosterone has less of an effect to increase phospho-Sgk levels are consistent with both K-RasA and PI3-K signaling being necessary for steroid-dependent activation of Sgk.
Figure 3C shows results from experiments testing aldosterone-dependent cross talk between the PI3-K and MAPK1/2 signaling cascades. The top two blots in Fig. 3C contain the same lysate and are actually the same blot cut in half and probed with anti-phospho(Ser259)-Raf (blot 1) and anti-phospho-MAPK1/2 (blot 2) antibodies. The bottom two blots in Fig. 3C are the same blot probed first with anti-phospho(Ser259)-Raf antibody (blot 3) and then stripped and reprobed with anti-MAPK (blot 4) antibody as a loading control. Whole A6 cell lysates were from monolayers treated with vehicle and aldosterone in the absence and presence of Mek 1/2 inhibitors (10 µM PD-98059; 0.5 µM U-0126), an inactive analog of the Mek 1/2 inhibitor U-0126 (0.5 µM U-0124), LY-294002 (50 µM), and this PI3-K inhibitor plus the U-0126 Mek 1/2 inhibitor for 4 h. Aldosterone activated (phosphorylated) MAPK1/2 only in the presence of uninhibited Mek 1/2 signaling. Similarly, aldosterone resulted in Akt-dependent phosphorylation of c-Raf only in the presence of functional PI3-K. These results support the idea that aldosterone activates both MAPK1/2 and PI3-K signaling in A6 cells with cross talk between these cascades at the level of Akt and c-Raf. As a whole, results in Figs. 1–3 are consistent with the signal transduction scheme depicted in Fig. 1A.
We next determined the contributions of K-RasA, MAPK1/2, and PI3-K signaling to aldosterone-induced Na+ transport across A6 cell monolayers. Figure 4A shows the relative (time 4 h/time 0) open-circuit current across A6 cell monolayers in response to treatment with aldosterone in the absence and presence of Mek 1/2, c-Raf (1 µM ZM-336372), and PI3-K inhibitors. Although aldosterone significantly increased current at this time point, as expected, only inhibition of PI3-K signaling affected this increase, suggesting that this pathway and not the MAPK1/2-dependent pathway plays a role in the positive actions of aldosterone on transport. The results in Fig. 4B determined whether K-RasA was necessary for aldosterone action on transport. This figure reports the aldosterone-sensitive (4 h) open-circuit current from A6 cell monolayers pretreated with sense and antisense K-ras oligonucleotide. As observed previously (25), suppression of K-RasA expression reduces the ability of steroid to induce the current. Interestingly, subsequent treatment of the antisense group with the PLA2 inhibitor aristolochic acid (200 µM; 5 min) significantly increased the current to levels similar to that observed in the aldosterone-treated sense group. Because this was a rapid response and inhibition of PLA2 has been reported previously to stimulate transport (5, 29), we interpret these results as showing that suppression of K-RasA signaling does not overtly affect expression levels of ENaC but merely reduces the activity of this channel. In summary, the results in Fig. 4 demonstrate that K-RasA and PI3-K but not MAPK1/2 signaling are necessary for aldosterone stimulation of transport in A6 cells.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The current study also addressed the second question. We demonstrate directly for the first time that PI3-K affects ENaC activity. Patch-clamp experiments demonstrate that ENaC reconstituted in CHO cells is about three times more active in the presence of constitutively active PI3-K. The mechanisms by which PI3-K stimulates ENaC in native epithelia and the reconstituted system used in the current study remain to be definitively determined. At this moment, two mechanisms appear most attractive. PI3-K signaling could sustain ENaC in the membrane as others suggested through the actions of Sgk (7, 20). However, as shown here and previously by others (6, 15, 27), Sgk is induced and activated by aldosterone in a transient manner peaking between 1 and 2 h leading to the supposition that PI3-K signaling must affect ENaC activity through multiple pathways, one of which can function in a sustained manner coupling PI3-K activity to ENaC activity over a prolonged period. The recent findings that ENaC is activated by anionic phospholipids (12, 32) are exciting and lead to the possibility that ENaC activity in some manner directly reflects the cellular levels of the phospholipids produced by PI3-K.
In addition to aldosterone stimulating PI3-K signaling and inducing and activating Sgk, aldosterone induces and activates K-RasA as well as its downstream effector MAPK1/2 cascade (9, 13, 21, 22, 25). Interestingly, PI3-K is one of only a few well-established first effectors of Ras signaling proteins with these small G proteins directly interacting with the p110 subunit of this kinase to stimulate activity (10, 28, 30, 31). Thus aldosterone-activated K-RasA could sit at the top of a bifurcating signaling pathway, leading to stimulation of both MAPK1/2 and PI3-K cascades. Our results are consistent with aldosterone activating such a cascade in A6 cells. Two questions arose from this aldosterone response during the course of the current study. Was there cross talk between these cascades, and which cascade played a major role in mediating aldosterone action on Na+ transport and ENaC activity? The current results demonstrate that there is cross talk between aldosterone-sensitive PI3-K and MAPK1/2 signaling at the level of Akt and Raf with the prior phosphorylating the latter at a site established to be involved in negative regulation of Raf (18, 33). Activation of MAPK1/2 signaling independent of aldosterone is known to decrease ENaC activity by promoting degradation of ENaC subunit protein (3). This is a slowly developing response to MAPK1/2 activation leading to decreased activity after 6 h. The current results demonstrate that aldosterone only stimulates transport in the presence of uninhibited PI3-K signaling and that MAPK1/2 signaling plays little overt role in the positive actions this steroid has on transport. With these results in mind and considering our earlier findings regarding suppression of ENaC activity by MAPK1/2, we propose that during aldosterone signaling to ENaC, the PI3-K cascade plays a major stimulatory role at the same time as suppressing negative feedback via aldosterone-activated MAPK1/2 signaling.
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GRANTS |
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FOOTNOTES |
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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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REFERENCES |
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ENaC is required for normal channel gating. J Biol Chem 278: 41367–41379, 2003.- and -subunits of the human epithelial sodium channel. Am J Physiol Cell Physiol 268: C1157–C1163, 1995.This article has been cited by other articles:
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  M. W. Musch, A. Lucioni, and E. B. Chang Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+ pump activity Am J Physiol Gastrointest Liver Physiol, November 1, 2008; 295(5): G909 - G919. [Abstract] [Full Text] [PDF]
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 L. Y. Teos, A. Zhao, Z. Alvin, G. G. Laurence, C. Li, and G. E. Haddad Basal and IGF-I-dependent regulation of potassium channels by MAP kinases and PI3-kinase during eccentric cardiac hypertrophy Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H1834 - H1845. [Abstract] [Full Text] [PDF]
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 O. Pochynyuk, Q. Tong, J. Medina, A. Vandewalle, A. Staruschenko, V. Bugaj, and J. D. Stockand Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel J. Gen. Physiol., September 24, 2007; 130(4): 399 - 413. [Abstract] [Full Text] [PDF]
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 R. Gros, Q. Ding, S. Armstrong, C. O'Neil, J. G. Pickering, and R. D. Feldman Rapid effects of aldosterone on clonal human vascular smooth muscle cells Am J Physiol Cell Physiol, February 1, 2007; 292(2): C788 - C794. [Abstract] [Full Text] [PDF]
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 F. Lang, C. Bohmer, M. Palmada, G. Seebohm, N. Strutz-Seebohm, and V. Vallon (Patho)physiological Significance of the Serum- and Glucocorticoid-Inducible Kinase Isoforms. Physiol Rev, October 1, 2006; 86(4): 1151 - 1178. [Abstract] [Full Text] [PDF]
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 O. Pochynyuk, Q. Tong, A. Staruschenko, H.-P. Ma, and J. D. Stockand Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides Am J Physiol Renal Physiol, May 1, 2006; 290(5): F949 - F957. [Abstract] [Full Text] [PDF]
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 J. Gorelik, Y. Zhang, D. Sanchez, A. Shevchuk, G. Frolenkov, M. Lab, D. Klenerman, C. Edwards, and Y. Korchev Aldosterone acts via an ATP autocrine/paracrine system: The Edelman ATP hypothesis revisited PNAS, October 18, 2005; 102(42): 15000 - 15005. [Abstract] [Full Text] [PDF]
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N. Markadieu, R. Crutzen, D. Blero, C. Erneux, and R. Beauwens Hydrogen peroxide and epidermal growth factor activate phosphatidylinositol 3-kinase and increase sodium transport in A6 cell monolayers Am J Physiol Renal Physiol, June 1, 2005; 288(6): F1201 - F1212. [Abstract] [Full Text] [PDF]
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 J. D. Stockand Preserving salt: in vivo studies with Sgk1-deficient mice define a modern role for this ancient protein Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R1 - R3. [Full Text] [PDF]
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Q. Tong and J. D. Stockand Receptor tyrosine kinases mediate epithelial Na+ channel inhibition by epidermal growth factor Am J Physiol Renal Physiol, January 1, 2005; 288(1): F150 - F161. [Abstract] [Full Text] [PDF]
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