Acute effects of aldosterone on the epithelial Na channel in rat kidney

Abstract

The acute effects of aldosterone administration on epithelial Na channels (ENaC) in rat kidney were examined using electrophysiology and immunodetection. Animals received a single injection of aldosterone (20 μg/kg body wt), which reduced Na excretion over the next 3 h. Channel activity was assessed in principal cells of cortical collecting ducts as amiloride-sensitive whole cell clamp current (I_Na). I_Na averaged 100 pA/cell, 20–30% of that reported for the same preparation under conditions of chronic stimulation. I_Na was negligible in control animals that did not receive hormone. The acute physiological response correlated with changes in ENaC processing and trafficking. These effects included increases in the cleaved forms of α-ENaC and γ-ENaC, assessed by Western blot, and increases in the surface expression of β-ENaC and γ-ENaC measured after surface protein biotinylation. These changes were qualitatively and quantitatively similar to those of chronic stimulation. This suggests that altered trafficking to or from the apical membrane is an early response to the hormone and that later increases in channel activity require stimulation of channels residing at the surface.

the adrenal-cortical steroid aldosterone is an important regulator of salt excretion in the urine. One of the primary actions of the hormone is stimulation of Na⁺ reabsorption through the epithelial Na⁺ channels (ENaC) in the late distal nephron including the late distal convoluted tubule (DCT), connecting tubules (CNT), and collecting ducts (16, 38). With chronic (∼1 wk) exposure to elevated hormone levels, either through direct administration or with a Na-deficient diet to promote endogenous secretion, the overall expression of the α subunit of ENaC is higher, but those of β- and γ-ENaC are not greatly affected (2, 6, 25). Under these conditions, surface expression of the β- and γ-subunits increases (9, 23). This suggests that a redistribution of channel protein from intracellular compartments to the apical membrane contributes to the final upregulation of transport. In addition, proteolytic cleavage of the α- and γ-ENaC subunits correlates with the chronic increases in activity (6, 9, 25).

Treatment of rats with aldosterone decreases Na excretion within 1–3 h (3, 19, 20, 27). This rapid response is associated with increased transcription of a number of genes including those coding for the serum and glucocorticoid-induced kinase SGK1 (4, 28), the leucine-zipper protein GILZ1 (33), and the deubiquitinase USP2-45 (7). These gene products are thought to be involved in the increase in ENaC function, although the precise mechanisms are not precisely known. Immunocytochemical approaches suggest that trafficking can be an early event in hormonal activation, at least in the earlier portion of the responding nephron (24). However, this has not been analyzed quantitatively. In this study, we use a combination of electrophysiology and surface labeling to examine the early effects of aldosterone in rat kidney.

METHODS

Animals.

All procedures using animals were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medical College. Female Sprague-Dawley rats (Charles Rivers Laboratories, Kingston, NY) weighing from 150–200 g were raised free of viral infections. Rats were fed a synthetic diet containing 1% NaCl and 1% K, mostly as Cl and phosphate salts (MP Biomedicals). In the morning of the day of the experiment, animals were injected subcutaneously with either aldosterone (20 μg/kg body wt) dissolved in PEG300, or with PEG300 alone. We did not measure the resulting plasma aldosterone concentrations. However, a comparable single intraperitoneal dose in mice resulted in a large transient elevation that peaked at 15 min and returned to baseline after 1 h (7). To measure urinary excretion of Na and K, animals were kept in metabolic cages with free access to drinking water but without food. Urine was collected twice, 1 and 3 h after injection. Na and K concentrations in the collected urine were measured using a flame photometer (Instrumentation Laboratory, model 943). In a few cases, animals were fed a Na-deficient diet containing 0.004% Na by weight (MP Biomedicals, Solon, OH) for 7 days.

Electrophysiology.

For whole cell clamp measurements, kidneys were sliced with a razor blade, and cortical collecting ducts (CCDs) were isolated with forceps and split open with a fine needle (30). The tubules were superfused with solutions prewarmed to 37°C containing the following (in mM): 135 Na methanesulfonate, 5 KCl, 2 Ca methanesulfonate, 1 MgCl₂, 2 glucose, 5 mM Ba methanesulfonate, and 10 HEPES adjusted to pH 7.4 with NaOH. The patch-clamp pipettes were filled with solutions containing the following (in mM): 7 KCl, 123 aspartic acid, 20 CsOH, 20 TEA·OH, 5 EGTA, 10 HEPES, and 3 MgATP, adjusted to pH 7.4 with KOH. The total concentration of K⁺ was 120 mM. Pipettes were pulled from hematocrit tubing, coated with Sylgard, and fire-polished with a microforge. Pipette resistances ranged from 3 to 6 MΩ. Amiloride-sensitive currents (I_Na) were measured as the difference in current with and without 10 μM amiloride in the bath solution.

In situ biotinylation.

Biotinylation of membrane proteins followed a modification of a protocol described previously (9). Rats were anesthetized with 90 mg/kg ketamine plus 4 mg/kg ip xylazine and placed on ice. The abdominal cavity was opened and the aorta was cannulated below the renal arteries. The kidneys were perfused by gravity with ice-cold solutions at a rate of ∼10 ml/min. The vena cava was punctured to allow fluid outflow. The aorta was then ligated above the renal arteries, as were the superior mesenteric and hepatic arteries. The animal was killed by opening the chest and cutting the cardiac apex. The abdominal cavity was kept at <7°C with a steady stream of ice-cold 150 mM NaCl solution over the left kidney. Flow rates through the urethra were 0.2–0.5 ml/min.

The kidneys were perfused first for 5–10 min with PBS (in mM): 140 NaCl, 4.5 KCl, 10 Na₂HPO₄, at pH 8.0 and containing 50 USP U heparin/dl, then for 20–25 min with PBS + 0.5 mg/ml sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (sulfo-NHS-biotin; Campbell Science, Rockville, IL), and finally for 30 min with TBS (in mM): 120 NaCl, 4.5 KCl, 25 Tris at pH 8.0 to quench the reaction of the reagent with the tissue and remove excess biotin.

The left kidney was removed, minced with a razor blade, and homogenized with a tight-fitting Dounce in 8 ml of lysis buffer containing (in mM) 250 sucrose, 10 triethanolamine HCl, 1.6 ethanolamine and 0.5 EDTA, and 60 μl protease inhibitors cocktail (Sigma) at pH 7.4. The homogenate was sieved with a 100-μm nylon mesh to separate intact cells. The filtrate was centrifuged at 100,000 g for 2 h to sediment a total membrane fraction. This pellet was resuspended in 2 ml of lysis buffer, aliquoted, and frozen at −80°C. Protein concentrations were measured with a BCA kit (Pierce). For isolation of biotinylated proteins, 3 mg of protein were solubilized in 1.5 ml of solubilization buffer containing (in mM) 100 NaCl, 50 TRIS, 5 EDTA, 3% Triton X-100, 1 μg/ml leupeptin (Sigma), 0.1 mg/ml PMSF (Sigma) at pH 7.4 along with 0.4 ml of a 50% suspension of UltraLink Neutravidin beads (Pierce Chemical) and gently rocked overnight at 4°C. The beads were washed twice with solubilization buffer with 1% Triton X-100, twice with high-salt solubilization buffer containing (in mM) 500 NaCl, 50 TRIS, 5 EDTA, 0.1% Triton X-100, and twice with no-salt buffer containing (in mM) 10 TRIS, at pH 7.4. After a 2-min centrifugation, the fluid over the beads was aspirated and the proteins were eluted from the beads with 60 μl of 500 mM DTT at 85°C for 15 min. The eluate was collected after centrifugation at 3,000 rpm for 5 min and mixed with 20 μl of 4× sample buffer. Forty microliters of this mixture were loaded into one lane of a polyacrylamide gel for electrophoresis.

Whole kidney samples were prepared for electrophoresis with 0.2–0.5 mg of protein in 65-μl lysis buffer, 25-μl LDS sample buffer, and 10-μl sample reducing agent and heated at 85°C for 8 min. Each lane of the gel was loaded with 40 μg (β- and γ-ENaC) or 75 μg (α-ENaC) total protein.

Immunoblotting.

Whole kidney and cell surface samples were electrophoresed on 4–12% Bis-TRIS gels (Invitrogen) and the proteins were transferred electrophoretically to PVDF membranes. After being blocked, membranes were incubated overnight at 4°C with primary antibodies of α-, β-, or γ-ENaC at 1:1,000 dilution as described in Ref. 6. Anti-rabbit IgG conjugated with alkaline phosphatase was used as a secondary antibody. Bound antibody was visualized on autoradiography film (HyBlot CL, Denville Scientific) using a chemiluminescence substrate (Western Breeze, Invitrogen). Semiquantitative densitometry of protein bands was performed with background subtraction using AdobePhotoshop CS5.

Antibodies.

Polyclonal antibodies against the α-, β-, and γ-subunits of rat ENaC were based on short peptide sequences in the NH₂ terminus of α-ENaC and the COOH termini of β-ENaC and γ-ENaC as described previously (6, 25). Antisera were purified using peptide-linked agarose bead affinity columns (Sulfolink Kit, Pierce Biotechnology). A second antibody against the N-terminus of mouse α-ENaC was a generous gift of Prof. Johannes Loffing (U. Zürich). The two anti-α-ENaC antibodies gave similar results.

Statistics.

Statistical significance between two groups was assessed by unpaired Student's t-tests. P < 0.05 was considered significant.

RESULTS

Acute aldosterone effects on Na and K excretion.

To acutely increase levels of circulating aldosterone, we treated rats with a single subcutaneous injection of the hormone. The overall physiological responses to this treatment were assessed as the rates of excretion of Na and K in the urine over the next 3 h. Results are shown in Fig. 1. The natriferic effects of the hormone were superimposed on falling rates of Na excretion over this period (from 8:30 to 11:30 AM) observed even in control animals (Fig. 1A). This presumably reflects diurnal patterns in food intake (mostly during the night) and excretion of this Na load (12, 18, 39). The decrease was variable, probably depending on the timing of overnight food consumption. However, animals treated with aldosterone excreted significantly less Na during the period 1–3 h after the injection, in accord with previous findings (19, 27).

Fig. 1.

Effects of acute aldosterone administration on urinary Na and K excretion. Urine was collected overnight, and then 1 and 3 h after injection of aldosterone (2 μg/100 g sc) or diluent at ∼8:30 AM. A: Na excretion. During the AM, Na excretion decreased from overnight levels in both control and treated animals. The fall in the animals receiving aldosterone was greater and more consistent. B: K excretion. K excretion was maintained at 50–70% of the overnight rate. There was no significant difference between control and aldosterone-treated groups. Data are expressed as means ± SE for 9 animals in each group. *Statistical significance compared with control.

Baseline K excretion did not decrease as much as that of Na, presumably reflecting the intracellular buffering and subsequent slow release of absorbed K (Fig. 1B). Aldosterone did not significantly alter K excretion in this protocol. A similar lack of an acute kaliuretic effect of the hormone has been observed previously (31).

The fall in Na excretion presumably reflects, at least in part, activation of ENaC in the late distal nephron. It is also possible, however, that other transporters, such as the thiazide-sensitive NaCl cotransporter NCC (5) or the Na-dependent Cl-HCO₃ exchanger SLC4A8 (22), contribute to the response.

Acute effects of aldosterone on ENaC activity.

We next assessed the effects of acute aldosterone administration on ENaC activity. After the 3-h exposure to the hormone, kidneys were harvested and CCDs were isolated and opened. Currents were measured under whole cell clamp conditions in the absence and presence of amiloride (Fig. 2). Representative traces are shown in Fig. 2, A and C. In 12 of 13 cells in tubules from the treated animals, we observed a clear effect of the blocker, defined as an abrupt decrease in inward current at the cell holding potential of 0 mV and a substantial decline of inward but not outward currents as the potential varied from −100 to +60 mV (Fig. 2B). Most cells from control animals did not respond to amiloride; only 1 of 14 showed a clear response to the drug. Representative traces and current-voltage (I–V) relationships are shown in Fig. 2, C and D. The average I_Na at −100 mV was 110 ± 25 pA/cell in aldosterone-treated animals (Fig. 2E), with a range of 46–374 pA/cell. This is 1/3 to 1/8 the value reported for chronically stimulated animals (i.e., 1 wk of hormone administration or dietary Na restriction) measured under the same in vitro experimental conditions (8, 11, 13). The amiloride-insensitive current was variable and in some cases included a significant component through the seal between the pipette and the cell.

Fig. 2.

Currents through Na⁺ channels after acute aldosterone administration. Cortical collecting ducts (CCDs) from rats injected with aldosterone or diluent were isolated and tubules were split open for whole cell patch-clamp recording. Currents were measured in principal cells before and after addition of 10 μM amiloride to the bath. A: typical current traces with and without amiloride in a cell from an aldosterone-treated animal. B: current-voltage (I–V) relationships from the cell in A. The curve marked I_Na represents the difference in current with and without amiloride. C: typical current traces with and without amiloride in a cell from a control animal. D: I–V relationships from the cell in C. The curve marked I_Na represents the difference in current with and without amiloride E: mean values of I_Na measured at −100 mV. Data are means ± SE for 13 cells (aldosterone) and 14 cells (controls). *Statistical significance compared with control.

Acute effects of aldosterone on ENaC expression, processing, and surface expression.

Finally, we examined changes in ENaC expression and trafficking during the acute response to aldosterone (Figs. 3, 4, 5). Overall expression levels for the full-length subunits were not appreciably altered by the 3-h time treatment (Fig. 6A). In particular, the full-length form of α-ENaC, which increases two- to threefold with chronic stimulation (6, 25), did not significantly change (Figs. 3 and 6A). This marks a clear qualitative difference between acute and chronic effects of the hormone.

Fig. 3.

Effects of acute aldosterone administration on expression of α-epithelial Na channel (ENaC). The Western blot assays show protein extracted from kidneys of rats treated with aldosterone or diluent. Each lane was loaded with 75 μg protein from a homogenate of kidneys from a different animal. Stained bands represent full-length (85 kDa) and cleaved (25–30 kDa) forms of the protein. The anti-α-ENaC antibody was a gift of Dr. J. Loffing.

Fig. 4.

Effects of acute aldosterone administration on total and surface expression of β-ENaC. The Western blots assay protein extracted from kidneys of rats treated with aldosterone or diluent. For total expression, each lane was loaded with 40 μg protein from a microsomal pellet of a different animal. For surface expression, each lane was loaded with 40 μl of eluate from neutravidin beads.

Fig. 5.

Effects of acute aldosterone administration on total and surface expression of γ-ENaC. The Western blots assay protein extracted from kidneys of rats treated with aldosterone or diluent. For total expression, each lane was loaded with 40 μg protein from a microsomal pellet of a different animal. For surface expression, each lane was loaded with 40 μl of eluate from neutravidin beads. Stained bands represent full-length (80 kDa) and cleaved (65 kDa) forms of the protein.

Fig. 6.

Effects of acute aldosterone administration on total and surface expression of ENaC: quantitation of band densities for Figs. 3–5. α85 And α30 represent full-length and cleaved forms of α-ENaC, respectively. γ80 And γ65 represent full-length and cleaved forms of γ-ENaC, respectively. A: total expression in microsomes. B: surface expression. Data are means ± SE for 8 animals (A) or 4 animals (B) in each group. *Statistical significance compared with control.

In contrast, the induction of cleaved forms of α (Fig. 3) and γ (Fig. 5) ENaC, also biochemical markers of chronic aldosterone stimulation (6, 8), was clearly observed after a 3-h exposure to the hormone. These truncated subunits are thought to arise from the proteolytic action of furin in the Golgi apparatus during movement of the channel protein to the cell surface (21). This suggests that increased trafficking of the channels to, or decreased rates of retrieval from, the apical membrane contributes to the early effects of aldosterone.

To test this more directly, we measured the surface expression of β- and γ-ENaC using in situ biotinylation of surface proteins of the kidney. A low signal/noise ratio in the eluates precluded the quantitative assessment of α-ENaC (9). γ-ENaC at the surface was primarily in the cleaved form, as reported previously (9). Quantitatively, β- and γ-ENaC increased by 1.7- and 2.4-fold, respectively. These changes were similar in magnitude to those observed under more chronic conditions (9, 15). In an additional matched set of rats kept on a low-Na diet for 1 wk and processed identically to the control and aldosterone-injected animals, γ-ENaC in the surface fraction increased by 2.7-fold, not significantly different from the acutely stimulated group. Thus, it seems that trafficking of ENaC protein to the apical membrane is an early action of aldosterone.

DISCUSSION

Na and K excretion.

Aldosterone decreased Na excretion, as expected. Documentation of this effect was complicated by a strong diurnal pattern of excretion, as has been noted previously (12, 18, 39). We measured excretion rates between 8:30 and 11:30 AM, after the period of maximal food (and Na) intake. As the animals completed the elimination of the overnight Na load excretion fell. The mechanisms underlying this pattern have not been delineated. Aldosterone did not significantly increase K excretion in this protocol. Although the hormone is generally considered to be kaliuretic, similar negative results with acute hormone application have been reported previously (31). Since activation of transport via Na⁺ channels in the late distal nephron should increase the driving force for K⁺ secretion, it is unclear why K excretion was not stimulated. Possible factors that could compensate for this effect include inhibition of apical K⁺ channels (40), or decreased Na⁺ delivery to the K⁺-secreting segments through stimulation of upstream Na reabsorption.

Effects on channel activity.

Channel activity assessed after removal of kidneys from the animal increased in response to aldosterone, although this acute response was considerably smaller than that observed after chronic stimulation either with aldosterone administration or dietary Na restriction. The average Na current was ∼100 pA/cell in this study as opposed to 300–800 pA/cell with chronic aldosterone elevation (8, 11, 13). This could result from a shorter exposure to the hormone; others have suggested the existence of “early” and “late” responses to aldosterone based on experiments with model systems such as toad urinary bladders (1, 34, 37). In addition, average circulating hormone concentrations may be lower under the acute conditions; aldosterone will be cleared from the body after its injection and the concentration in plasma will vary over the course of the experiment (7).

Effects on channel biochemistry.

From these electrophysiological data, it is not possible to say whether the acute response in channel activity is quantitatively or a qualitatively different from the long-term one. To examine this point, we examined ENaC expression, processing, and trafficking with acute stimulation.

Chronic dietary Na restriction or aldosterone administration increases the abundance of α-ENaC mRNA (2) and protein (6, 25). This was not observed with acute stimulation, indicating that it is a delayed or late response to the hormone. We previously found that increased α-ENaC protein induced by chronic administration of the glucocorticoid dexamethasone was not accompanied by increased channel activity (14). Thus, a greater abundance of this subunit is neither necessary nor sufficient to augment channel function.

Chronic elevation of aldosterone also promotes the cleavage of both α-ENaC and γ-ENaC (6, 25). Previous experiments indicated that cleavage of γ-ENaC occurred after overnight dietary Na depletion (11), but earlier time points have not been investigated. Here, we report a robust increase in the abundance of cleaved subunits after a 3-h hormone challenge, suggesting that this is part of the “early” response.

Surface expression of ENaC protein increases in response to chronic aldosterone administration or dietary Na depletion, indicating that trafficking of channels to (or from) the apical membrane constitutes a major response to the hormone (9, 15). However, the increase in surface protein (∼3-fold for γ-ENaC, and somewhat less for β-ENaC) was smaller than that of increased channel activity. Cells from fully activated CCDs have mean currents of 300–800 pA/cell (8, 11, 13), while those from control CCDs have currents below the detection level of 10–20 pA/cell. This could be explained by dual effects on surface expression and activation of channels at the surface. Indeed, these numbers suggest that the latter effect, accomplished by the recruitment of silent channels and an increase in open probability is larger than the increase in surface expression. We initially guessed that channel activation might occur more quickly. In fact, we observed a strong increase in ENaC surface expression after a hormone exposure of only 3 h. This shows that channels move to the apical membrane as part of the early response to aldosterone. In fact, the increase in surface abundance during this period was comparable to that observed under more chronic conditions.

In conclusion, the α- and γ-subunits of ENaC are cleaved as part of the early–within 3 h–response to aldosterone. This process is due in part to processing by the proteolytic enzyme furin in the Golgi apparatus (21). At the same time, increased amounts of γ-ENaC appear at the surface, and this increase is entirely in the cleaved form of the subunit. Taken together, these findings suggest that one early effect of the hormone is to promote trafficking of the channels through the Golgi to the apical membrane, or the retention of the processed channels at the surface.

The findings of a nearly complete effect of trafficking after 3 h of hormone stimulation but an incomplete physiological response imply a later action that can increase channel open probability, or an all-or-none conversion from inactive to active forms. The mechanisms underlying these responses remain unresolved but may involve methylation of membrane proteins or lipids (32, 35) or lipid signaling pathways (17, 36).

Spatial heterogeneity in the responses might also contribute to the discrepancy. A recent study demonstrated variation in the responses of the late DCT, CNT, and CCD to mineralocorticoids (29). Furthermore, a rapid response to aldosterone specific to the CNT was observed using immunocytochemistry (24). In this scenario, channels in the early portion of the aldosterone-responsive part of the nephron such as the CNT would respond first. Because ENaC is abundant in this part of the kidney (13), it will contribute much of the biochemical signal and changes in processing and surface expression would be seen quickly. A slower response in more distal segments could explain the submaximal channel activity observed in the CCD. In any case, channel trafficking correlates with the most acute renal response to the hormone.

GRANTS

This study was supported by National Institutes of Health Grant RO1-DK27847.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: G.F. and L.G.P. conception and design of research; G.F. and L.G.P. performed experiments; G.F. and L.G.P. analyzed data; G.F. and L.G.P. interpreted results of experiments; G.F. and L.G.P. edited and revised manuscript; G.F. and L.G.P. approved final version of manuscript; L.G.P. prepared figures; L.G.P. drafted manuscript.