Aldosterone inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via a nongenomic pathway in medullary thick ascending limb

David W. Good1,2, Thampi George1, and Bruns A. Watts III1

Departments of 1 Medicine and 2 Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Rapid actions of aldosterone that are independent of transcription and translation have been described in a variety of cells; however, whether nongenomic pathways mediate aldosterone-induced regulation of renal tubule transport has not been determined. We report here that aldosterone induces rapid (<3.5 min) inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the medullary thick ascending limb (MTAL) of the rat. This inhibition is observed over the physiological range of hormone concentrations (IC50 sime  0.6 nM) and is not affected by pretreatment with actinomycin D (12.5 µg/ml), cycloheximide (40 µg/ml), or spironolactone (10 µM). The glucocorticoids dexamethasone, cortisol, and corticosterone (1 or 500 nM) did not affect HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the absence or presence of carbenoxolone. Thus the specificity of rapid aldosterone action is not dependent on 11beta -hydroxysteroid dehydrogenase activity. The inhibition by aldosterone is additive to inhibition by angiotensin II and vasopressin, indicating that these factors regulate MTAL transport through distinct pathways. These results demonstrate that aldosterone inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL via a pathway that is rapid, highly selective, independent of transcription and protein synthesis, and not mediated through the classic mineralocorticoid receptor. The results establish a role for nongenomic pathways in mediating aldosterone-induced regulation of transepithelial transport in the mammalian kidney. The novel action of aldosterone to inhibit luminal acidification in the MTAL may play a role in enabling the kidney to regulate acid-base balance independently of Na+ balance and extracellular fluid volume.

kidney; glucocorticoids; acid-base balance; mineralocorticoid receptor; 11beta -hydroxysteroid dehydrogenase


    INTRODUCTION
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INTRODUCTION
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ALDOSTERONE PLAYS AN IMPORTANT role in the regulation of Na+, K+, and acid-base balance through its effects on renal electrolyte excretion. Aldosterone influences ion transport in multiple nephron segments, including the collecting duct, thick ascending limb, and distal convoluted tubule (1, 11, 21, 31, 39, 45). In segments of the collecting duct, aldosterone acts directly to stimulate Na+ absorption, K+ secretion, and H+ secretion (1, 21, 39, 42). In collecting duct principal cells and other Na+-reabsorbing epithelia, aldosterone stimulates Na+ absorption by inducing gene transcription and the subsequent translation of new proteins. The aldosterone-induced proteins lead to increased Na+ absorption by increasing the activity and number of apical membrane Na+ channels (ENaC) and basolateral membrane Na+-K+-ATPase subunits (34, 39, 46). The transcriptional regulation of Na+ absorption, K+ secretion, and H+ secretion by aldosterone occurs after a latent period of 45 min-2 h and is mediated through binding of aldosterone to the classic (type 1) mineralocorticoid receptor (1, 21, 39, 42, 46). This receptor has equal affinity for aldosterone and glucocorticoids (cortisol and corticosterone) in cytosol preparations (15, 16). Specificity of aldosterone regulation in epithelial target tissues is achieved through the action of the enzyme 11beta -hydroxysteroid dehydrogenase type 2 (11beta -HSD2), which metabolizes cortisol and corticosterone to inactive analogs that do not bind the mineralocorticoid receptor (15, 17, 39).

In recent years, evidence has accumulated for rapid cellular actions of aldosterone that are not dependent on nuclear transcription or protein synthesis and occur through receptors other than the classic mineralocorticoid receptor (13, 28, 52). A prominent nongenomic effect of aldosterone is stimulation of Na+/H+ exchange activity. This stimulation is observed in a variety of tissues, including colonic epithelial cells and renal cell lines, and is mediated through activation of PKC (12, 13, 20, 28, 29, 33, 53). The rapid action of aldosterone on Na+/H+ exchange has been proposed to play a role in the regulation of epithelial Na+ absorption, intracellular pH-induced regulation of K+ channels, processing of aldosterone-induced proteins, and the rapid (5-10 min) effect of aldosterone on renal Na+ and K+ excretion in vivo (13, 18, 20, 28, 29, 52, 53). At present, however, it has not been determined whether nongenomic pathways are relevant to the regulation of transepithelial transport by aldosterone in the kidney. In addition, it is unknown whether aldosterone regulates Na+/H+ exchange activity or its related functions in mammalian renal tubules.

The medullary thick ascending limb (MTAL) of the loop of Henle performs a number of important renal transport functions, including reabsorption of NaCl that is essential for the maintenance of Na+ balance and the excretion of a dilute or concentrated urine (35). The MTAL also participates in the regulation of acid-base balance by reabsorbing most of the filtered HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that is not reabsorbed by the proximal tubule (23). In the MTAL, the H+ secretion necessary for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is mediated by the apical membrane Na+/H+ exchanger NHE3 (3, 4, 23, 27, 54). The MTAL also contains basolateral Na+/H+ exchange activity that plays a role in determining the rate of transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (49). Mineralocorticoid receptors are expressed in thick ascending limbs (6, 14, 43), and chronic changes in plasma aldosterone levels have been shown to alter both NaCl and NaHCO3 absorption by the loop of Henle in vivo (11, 41, 44, 54). However, whether aldosterone acts directly on the MTAL to regulate ion transport is unclear.

The present study was designed to examine directly the acute effects of aldosterone on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by the MTAL of rats. We found that aldosterone induces rapid inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption through a pathway that is highly selective, independent of transcription and translation, and not mediated through the classic mineralocorticoid receptor. These results establish a role for nongenomic pathways in mediating regulation of transepithelial transport by aldosterone in the mammalian kidney and identify a novel inhibitory effect of aldosterone on H+ secretion that may play a key role in enabling the kidney to regulate Na+ balance and extracellular fluid volume independently of acid-base balance.


    METHODS
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MTALs from male Sprague-Dawley rats (50-100 g; Taconic, Germantown, NY) were isolated and perfused in vitro as previously described (22, 25). The rats had free access to standard chow (NIH 31 diet, Ziegler) and distilled H2O up to the time of experiments. Tubules were dissected from the inner stripe of the outer medulla at 10°C in control bath solution (see below), transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipettes for perfusion at 37°C. In all experiments, the lumen and bath solutions contained (in mM) 146 Na+, 4 K+, 122 Cl-, 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 2.0 Ca2+, 1.5 Mg2+, 2.0 phosphate, 1.2 SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 1.0 citrate, 2.0 lactate, and 5.5 glucose (osmolality = 290 mosmol/kgH2O). Bath solutions also contained 0.2% fatty acid-free bovine albumin. All solutions were equilibrated with 95% O2-5% CO2 and were pH 7.45 at 37°C. Bath solutions were delivered to the perfusion chamber via a continuously flowing exchange system (22). Experimental agents were added to the bath solutions as described in RESULTS. Experiments using glucocorticoids were repeated in the absence of bath albumin to exclude possible effects of protein binding. Results did not differ with and without bath protein. Steroid hormones, spironolactone, and cycloheximide were prepared as stock solutions in ethanol and diluted into bath solutions to the final concentrations given in RESULTS. Actinomycin D was prepared as a stock solution in dimethyl sulfoxide. Equal concentrations of vehicle were added to control solutions in all protocols.

The protocol for study of transepithelial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption was as described (22, 25). In most experiments, tubules were equilibrated for 20-30 min at 37°C in the initial perfusion and bath solutions and the luminal flow rate (normalized per unit tubule length) was adjusted to 1.6-2.0 nl · min-1 · mm-1. One to three 10-min tubule fluid samples (sample volume 10 nl) were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 5-10 min after an experimental agent was added to or removed from the bath solution. In some experiments, longer treatment periods were used, as described in RESULTS. In one series of experiments, the luminal flow rate was increased to ~3.2 nl · min-1 · mm-1, the collected fluid volume was decreased to 5 nl, and sample collection was begun within 1 min after addition of aldosterone to the bath solution. With these modifications, samples could be obtained within 3.5 min of aldosterone exposure (see RESULTS). The absolute rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (pmol · min-1 · mm-1) was calculated from the luminal flow rate and the difference between total CO2 concentrations measured in perfused and collected fluids (22). Total CO2 concentrations were measured by microcalorimetry using a picapnotherm, as previously described (22). An average HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption rate was calculated for each period studied in a given tubule. When repeat measurements were made at the beginning and end of an experiment (initial and recovery periods), the values were averaged. Single-tubule values are presented in Figs. 1-6. Mean values ± SE (n = number of tubules) are presented in the text. Differences between means were evaluated by using Student's t-test for paired data, with P < 0.05 considered statistically significant.


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Fig. 1.   Aldosterone inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (JHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>) in the medullary thick ascending limb. A: absolute rate of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption was measured under control conditions (Cont) and after addition of 1 nM aldosterone (Aldo) to the bath solution. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. The P value is from paired t-test. Means ± SE are given in the text. B: effect of different aldosterone concentrations on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. Data show the aldosterone-induced inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption, expressed as percentage of control transport rate measured in the same tubule. Values are means ± SE for 3-6 experiments in each group. * P < 0.01, aldosterone vs. control.



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Fig. 2.   Actinomycin D (Act D) and cycloheximide (Cyclohex) do not prevent inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone. Tubules were bathed with 12.5 µg/ml actinomycin D for 90 min (A) or 40 µg/ml cycloheximide for 120 min (B) before aldosterone addition. Aldosterone (1 nM) was added to and removed from the bath in the presence of the inhibitors. <IT>J</IT><SUB>HCO<SUP>−</SUP><SUB>3</SUB></SUB>, data points, lines, and P values are defined as in Fig. 1A. Means ± SE are given in the text.



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Fig. 3.   Spironolactone (Spirono) does not block inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone. Tubules were bathed with 10 µM spironolactone for 100 min, and then 1 nM aldosterone was added to the bath solution. <IT>J</IT><SUB>HCO<SUP>−</SUP><SUB>3</SUB></SUB>, data points, lines, and P value are defined as in Fig. 1A. Means ± SE are given in the text.



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Fig. 4.   Dexamethasone (Dex) and cortisol do not affect HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. Dexamethasone (A) or cortisol (B) was added to the bath solution at 1 or 500 nM for up to 40 min. <IT>J</IT><SUB>HCO<SUP>−</SUP><SUB>3</SUB></SUB>, data points, lines, and P values are defined as in Fig. 1A. NS, not significant.



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Fig. 5.   Corticosterone (Corticost) has no effect on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the absence (A) or presence (B) of carbenoxolone (Carbenox). Corticosterone was added to the bath at 1 nM or 500 nM. B: tubules were bathed with 20 µM carbenoxolone for 100 min before corticosterone addition. <IT>J</IT><SUB>HCO<SUP>−</SUP><SUB>3</SUB></SUB>, data points, lines, and P values are defined as in Fig. 1A. NS, not significant.



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Fig. 6.   Inhibition by aldosterone is additive to inhibition by angiotensin II (Ang II) and vasopressin (AVP). Angiotensin II (10 nM: A) or AVP (10-10 M: B) was present in the bath throughout the experiments. Aldosterone was added to the bath at 1 nM. <IT>J</IT><SUB>HCO<SUP>−</SUP><SUB>3</SUB></SUB>, data points, lines, and P values are defined as in Fig. 1A. Means ± SE are given in the text.


    RESULTS
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Aldosterone rapidly inhibits HCO<UP><SUB>3</SUB><SUP><UP>−</UP></SUP></UP> absorption. Addition of 1 nM aldosterone to the bath decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by 28%, from 15.8 ± 0.5 to 11.4 ± 0.5 pmol · min-1 · mm-1 (P < 0.001; Fig. 1A). The inhibition was complete within 10-15 min of exposure to aldosterone (the time required for one sample collection), sustained for up to 100 min, and reversible. The aldosterone-induced decrease in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption was concentration dependent over the range 0.05-10 nM, with an IC50 of sime 0.6 nM (Fig. 1B). The HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption rate returned to its control value within 15 min after removal of aldosterone from the bath solution. These results demonstrate that aldosterone induces rapid inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption over the physiological range of hormone concentrations.

To define more precisely the rapidity of the aldosterone effect, additional tubules were studied in which the luminal flow rate was increased and the collected fluid volume was decreased to reduce the sample collection time (see METHODS). Under these conditions, bath addition of 50 nM aldosterone decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption from 21.1 ± 1.5 to 15.1 ± 1.2 pmol · min-1 · mm-1 within 3.5 min of aldosterone exposure (P < 0.01; n = 4).

Effect of furosemide. One possible explanation for the inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is that it occurs as the indirect result of an effect of aldosterone on transport pathways involved in transcellular NaCl absorption. Aldosterone has been reported to increase NaCl absorption by the loop of Henle of rats in vivo (41, 54). It is possible, therefore, that aldosterone-induced stimulation of apical membrane Na+-K+-2Cl- cotransport activity could increase intracellular Na+ activity, thereby reducing the driving force for apical Na+/H+ exchange and inhibiting HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. In MTAL perfused with 10-4 M furosemide to inhibit Na+-K+-2Cl- cotransport and net NaCl absorption, addition of 1 nM aldosterone to the bath decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption from 15.3 ± 0.7 to 10.3 ± 1.0 pmol · min-1 · mm-1 (P < 0.001; n = 3). Thus aldosterone inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption independently of net NaCl absorption.

Effects of actinomycin D and cycloheximide. MTALs were bathed with the transcription inhibitor actinomycin D (12.5 µg/ml) for 90 min or the protein synthesis inhibitor cycloheximide (40 µg/ml) for 120 min before the addition of aldosterone. Similar or less extensive treatments with these agents have been shown to block transcription and protein synthesis in multiple systems, including renal epithelial cell lines (8, 10, 30, 33, 37), and to inhibit genomic regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by vitamin D in the MTAL (Good D, unpublished observations). In tubules bathed with actinomycin D, addition of 1 nM aldosterone decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption from 15.7 ± 1.4 to 11.8 ± 1.1 pmol · min-1 · mm-1 (P < 0.025; Fig. 2A). In the presence of cycloheximide, 1 nM aldosterone decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption from 14.3 ± 0.9 to 9.5 ± 0.7 pmol · min-1 · mm-1 (P < 0.01, Fig. 2B). In both cases, the aldosterone-induced inhibition was rapid (<15 min) and reversible. Thus the inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone does not require gene transcription or protein synthesis. Actinomycin D and cycloheximide alone did not affect HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (data not shown).

Effect of spironolactone. MTALs were bathed initially for 100 min with 10 µM spironolactone, a competitive antagonist of the mineralocorticoid receptor. In the presence of spironolactone, addition of 1 nM aldosterone to the bath decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption from 15.1 ± 0.6 to 10.0 ± 0.6 pmol · min-1 · mm-1 (P < 0.001; Fig. 3). Thus the inhibition by aldosterone is not mediated through the classic mineralocorticoid receptor.

Steroid specificity and effect of carbenoxolone. To determine the specificity of aldosterone action, we examined the effects of glucocorticoids. Addition of either dexamethasone or cortisol (1 or 500 nM) to the bath for up to 40 min had no effect on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (Fig. 4, A and B).

The thick ascending limb expresses 11beta -HSD2 at low levels (6, 32, 40). In vascular tissue, rapid effects of aldosterone to increase Na+/H+ exchange activity and intracellular pH could be reproduced by the addition of cortisol when the tissue was pretreated with carbenoxolone to inhibit 11beta -HSD2 activity (2). It was suggested that 11beta -HSD2 may be involved in mediating the specificity of rapid aldosterone effects. To test this in the MTAL, we examined the effects of corticosterone, the physiological glucocorticoid in rats, in the absence and presence of carbenoxolone. Tubules were bathed with 20 µM carbenoxolone for 100 min before corticosterone addition, a treatment sufficient to unmask rapid glucocorticoid-induced regulation in blood vessels (2). Addition of 1 or 500 nM corticosterone to the bath had no effect on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption either in the absence or presence of the inhibitor (Fig. 5, A and B). These data demonstrate further that the inhibitory pathway is highly selective for aldosterone but provide no evidence that 11beta -HSD2 plays a role in conferring the aldosterone specificity. Carbenoxolone alone did not affect HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (data not shown).

Interaction of aldosterone with angiotensin II and vasopressin. Angiotensin II and AVP inhibit HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL (22, 25). In addition, the plasma levels of these hormones are regulated in close association with aldosterone in response to changes in Na+ and volume balance. We therefore examined their interaction with aldosterone in the regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. In tubules bathed with 10 nM angiotensin II, addition of 1 nM aldosterone to the bath decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption from 11.5 ± 0.4 to 6.4 ± 0.6 pmol · min-1 · mm-1 (P < 0.005; Fig. 6A). In the presence of 10-10 M AVP, 1 nM aldosterone decreased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption from 10.8 ± 1.0 to 6.1 ± 0.8 pmol · min-1 · mm-1 (P < 0.01; Fig. 6B). Thus the inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone is additive to inhibition by angiotensin II and AVP, indicating that these factors regulate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption through different pathways.


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INTRODUCTION
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Aldosterone regulates Na+, K+, and net acid excretion in the mammalian kidney by binding to nuclear receptors and stimulating the production of transcriptionally induced regulatory proteins (1, 21, 39, 42, 46). In recent years, rapid effects of aldosterone on ion transport and signaling pathways that are not dependent on gene transcription or protein synthesis have been described in multiple systems, including epithelial cells (13, 52). However, whether nongenomic pathways are relevant for aldosterone-induced regulation of renal tubule function has not been determined. The results of the present study demonstrate that aldosterone inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL of the rat via a pathway that is rapid (<3.5 min), highly selective, independent of transcription and translation, and not mediated through the classic mineralocorticoid receptor. This inhibition is observed in tubules from adrenal-intact animals on a normal Na+ intake and occurs over the physiological range of hormone concentrations (0.05-1.0 nM). In addition, the inhibition by aldosterone is additive to inhibition by angiotensin II and vasopressin, indicating that these factors regulate MTAL transport through distinct pathways. In contrast, glucocorticoids did not affect HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. These results are the first to establish a role for nongenomic pathways in the regulation of transepithelial transport by aldosterone in the mammalian nephron. The novel inhibitory action of aldosterone on acid secretion in the MTAL may be important in the ability of the kidneys to regulate acid-base balance independently of Na+ balance and extracellular fluid volume and may contribute to changes in renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption induced by disorders of K+ balance.

Mineralocorticoid and glucocorticoid receptors are expressed in thick ascending limbs (6, 11, 14, 43). At least two observations indicate that the classic mineralocorticoid receptor does not mediate the aldosterone-induced inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. First, pretreatment with the mineralocorticoid receptor antagonist spironolactone at a concentration 10,000 times that of aldosterone did not prevent the transport inhibition. Second, corticosterone at high concentrations had no effect on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the presence of carbenoxolone, a condition in which glucocorticoids can bind to and activate the mineralocorticoid receptor (17, 39). The latter experiment also demonstrates that the specificity of rapid aldosterone action in the MTAL is not dependent on 11beta -HSD2 activity. The lack of effect of cortisol, corticosterone, and dexamethasone on HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption excludes a role for glucocorticoid receptors in mediating the aldosterone-induced regulation and shows that the inhibition by aldosterone is not the result of nonspecific steroid effects, such as on membrane fluidity (52).

Membrane-bound receptors have been proposed to mediate specific, nongenomic effects of aldosterone on the basis of several lines of evidence: 1) radioligand-binding studies in plasma membranes from pig kidney identified specific, high-affinity aldosterone binding sites exhibiting kinetic and pharmacological properties consistent with the nongenomic actions of aldosterone on cell functions (9, 52); 2) rapid stimulation of Na+/H+ exchange by aldosterone in Madin-Darby canine kidney (MDCK) cells is elicited by using an aldosterone-albumin conjugate that prevents aldosterone entry into the cells (20); and 3) membrane receptors that mediate rapid cellular effects have been partially characterized for other steroid hormones, including vitamin D and estrogen (13, 38, 48, 52). In addition to membrane-bound receptors, nonclassic receptor mechanisms for steroids may include binding to enzymes or signaling proteins, or the rapid activation of signal transduction pathways through binding to classic receptors (7, 13, 28, 38). Recent work suggests that PKC isoforms may act as specific aldosterone receptors (28); however, as discussed below, PKC is unlikely to mediate rapid inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone in the MTAL. A membrane receptor specific for aldosterone has not yet been molecularly identified. However, our studies show that the MTAL contains a highly specific aldosterone receptor that is distinct from the classic mineralocorticoid receptor and plays a physiological role in regulating the transport function of this nephron segment.

Although the mechanism by which aldosterone inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL remains to be identified, our studies provide new insights into regulatory actions of aldosterone on renal cells. Absorption of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by the MTAL is mediated virtually completely by the apical membrane Na+/H+ exchanger NHE3, and the regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is achieved through regulation of this apical exchanger (3-5, 27, 50, 51). Thus our findings suggest strongly that aldosterone rapidly inhibits NHE3 activity in the MTAL. Aldosterone has been shown to stimulate Na+/H+ exchange activity in multiple systems (13, 28). However, inhibition of Na+/H+ exchange has not been reported as a physiological aldosterone effect. Thus inhibition of Na+/H+ exchange activity in the MTAL may be a novel cellular action of aldosterone. Further direct studies of the effects of aldosterone on the activity and kinetics of the apical Na+/H+ exchanger, independent of effects on other transporters, will be required to test this hypothesis.

Rapid regulation of Na+/H+ exchange activity by aldosterone in a variety of cell types is mediated through activation of PKC (12, 20, 28, 33, 53). At least two lines of evidence suggest that PKC does not mediate aldosterone-induced inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL: 1) activation of PKC does not inhibit MTAL HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (24); and 2) PKC inhibitors do not block the aldosterone-induced transport inhibition (26). The inhibition by aldosterone is additive to inhibition by AVP, which inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via cAMP (22), and angiotensin II, which inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via cytochrome P-450 (25). Thus it is unlikely that cAMP- or cytochrome P-450-dependent pathways are involved in mediating inhibition by aldosterone. In recent preliminary studies, we found that the inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone is nearly eliminated by inhibitors of the ERK signaling pathway (26). Further studies are required to define the role of ERK in mediating aldosterone-induced transport inhibition in the MTAL. Recent evidence supports the involvement of ERK in aldosterone regulation of Na+/H+ exchange activity in MDCK cells (19).

Rapid effects of aldosterone have been previously described in renal cells. In the mouse cortical collecting duct cell line M-1 and in MDCK cells, which share transport properties with collecting duct cells, aldosterone increased Na+/H+ exchange activity via nongenomic pathways involving Ca2+ and PKC, as noted above (20, 29). In principal cells isolated enzymatically from cortical collecting ducts, aldosterone induced a rapid increase in Na+ channel (ENaC) activity that was not blocked by spironolactone, consistent with nongenomic regulation (55). However, in the latter study, the stimulation of Na+ channel activity was induced with a pharmacological concentration of aldosterone, specificity of the transport effect for aldosterone was not established, and the transport stimulation was observed in principal cells from rabbits but not from rats (55). Thus the significance of nongenomic pathways for aldosterone-induced regulation of collecting duct Na+ transport remains to be determined. These studies suggest, however, that the collecting duct may be a target for nongenomic aldosterone regulation, in addition to the MTAL.

Previous information on regulation of thick ascending limb acid secretion by aldosterone has been obtained from studies of adrenalectomized rats. Adrenalectomy reduced net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by the loop of Henle in vivo (44) and the MTAL perfused in vitro (23). Absorption by the loop segment in vivo was restored to normal by replacement of the adrenalectomized animals with physiological levels of dexamethasone or high levels of aldosterone, suggesting that both mineralocorticoids and glucocorticoids may influence thick ascending limb HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (44). Interpretation of these results is complicated, however, because adrenalectomy and corticosteroid replacement lead to changes in systemic factors, renal hemodynamics, and intrarenal factors, such as luminal Na+ delivery, that may secondarily influence MTAL HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption (11). In addition, the stimulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone in the loop of Henle and MTAL of adrenalectomized rats was induced with high doses of the hormone (23, 44). Thus the precise role of aldosterone in long-term regulation of thick ascending limb HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption remains unclear. The data in adrenalectomized rats suggest, however, that chronic changes in adrenal corticosteroid levels may induce changes in MTAL HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption that differ from the acute effects of aldosterone in tubules from adrenal-intact animals. Our present results show that the decrease in MTAL HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption induced by acute aldosterone addition is comparable in magnitude to the change induced chronically by adrenalectomy (23); thus the nongenomic regulation is quantitatively as important as the adaptive regulation. The overall, integrated control of MTAL transport may depend on the interaction of nongenomic and genomic pathways, as proposed for other systems (52).

We have suggested previously that the MTAL plays a key role in enabling the kidney to maintain acid-base balance during changes in Na+ and volume balance (23, 25). The results of the present study identify nongenomic regulation by aldosterone as a mechanism that may contribute to this process. Activation of the renin-angiotensin-aldosterone system in response to Na+ and extracellular fluid volume depletion promotes renal Na+ retention but also results in multiple transport effects that act cooperatively to increase urinary net acid excretion and promote metabolic alkalosis. The latter include stimulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by angiotensin II in proximal and distal tubules (1, 47), stimulation of production and secretion of ammonium by angiotensin II in the proximal tubule (36), and stimulation of H+ secretion by aldosterone in segments of the collecting duct (1, 42). We suggest that these effects in proximal and distal tubule segments are opposed by the direct action of aldosterone to inhibit H+ secretion and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the MTAL. The impact of this aldosterone effect would be magnified by its additivity to inhibition by angiotensin II. In this way, nongenomic regulation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption by aldosterone in the MTAL would act to minimize changes in H+ excretion and stabilize acid-base balance during changes in Na+ balance while permitting regulated changes in Na+ excretion that are necessary to control extracellular fluid volume and blood pressure. An additional possibility is that the nongenomic regulation may play a role in mediating interactions between K+ balance and renal acid excretion. For example, increased acid secretion by the MTAL in response to a reduced plasma aldosterone level may contribute to the increased renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorptive capacity induced by hypokalemia (1).

In addition to the physiological roles outlined above, the nongenomic pathway for aldosterone identified here has other important physiological and clinical implications for renal function. A defect in nongenomic pathway(s) may contribute to mineralocorticoid resistance in cases of pseudohypoaldosteronism with no evidence of abnormality in the mineralocorticoid receptor gene (16, 52). In addition, the nongenomic pathway regulating MTAL function is highly aldosterone selective. Thus this pathway may provide a mechanism for mineralocorticoid specificity independent of 11beta -HSD2 activity, mediate aldosterone-specific regulation of electrolyte transport or other cell functions in nephron segments in which mineralocorticoid receptor expression is low or absent, and lead to novel therapeutic targets and strategies for modifying renal corticosteroid action.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38217.


    FOOTNOTES

Address for reprint requests and other correspondence: D. W. Good, 4.200 John Sealy Annex, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, Texas 77555-0562 (E-mail: dgood{at}utmb.edu).

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.

June 4, 2002;10.1152/ajprenal.00133.2002

Received 10 April 2002; accepted in final form 25 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alpern, RJ. Renal acidification mechanisms. In: The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 2000, vol. 1, p. 455-519.

2.   Alzamora, R, Michea L, and Marusic ET. Role of 11beta -hydroxysteroid dehydrogenase in nongenomic aldosterone effects in human arteries. Hypertension 35: 1099-1104, 2000[Abstract/Free Full Text].

3.   Amemiya, M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, and Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney Int 48: 1206-1215, 1995[ISI][Medline].

4.   Biemesderfer, D, Rutherford PA, Nagy T, Pizzonia JH, Abu-Alfa AK, and Aronson PS. Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney. Am J Physiol Renal Physiol 273: F289-F299, 1997[Abstract/Free Full Text].

5.   Borensztein, P, Juvin P, Vernimmen C, Poggioli J, Paillard M, and Bichara M. cAMP-dependent control of Na+/H+ antiport by AVP, PTH, and PGE2 in rat medullary thick ascending limb cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F354-F364, 1993[Abstract/Free Full Text].

6.   Bostonjoglo, M, Reeves WB, Reilly RF, Velazquez H, Robertson N, Litwack G, Morsing P, Dorup J, Bachmann S, and Ellison DH. 11Beta-hydroxysteroid dehydrogenase, mineralocorticoid receptor, and thiazide-sensitive Na-Cl cotransporter expression by distal tubules. J Am Soc Nephrol 9: 1347-1358, 1998[Abstract].

7.   Buitrago, C, Vazquez G, De Boland AR, and Boland RL. Activation of Src kinase in skeletal muscle cells by 1,1,25-(OH2)-vitamin D3 correlates with tyrosine phosphorylation of the vitamin D receptor (VDR) and VDR-Src interaction. J Cell Biochem 79: 274-281, 2000[ISI][Medline].

8.   Christ, M, Günther A, Heck M, Schmidt BMW, Falkenstein E, and Wehling M. Aldosterone, not estradiol, is the physiological agonist for rapid increases in cAMP in vascular smooth muscle cells. Circulation 99: 1485-1491, 1999[Abstract/Free Full Text].

9.   Christ, M, Sippell K, Eisen C, and Wehling M. Nonclassical receptors for aldosterone in plasma membranes from pig kidneys. Mol Cell Endocrinol 99: R31-R34, 1994[ISI][Medline].

10.   Cooper, GJ, and Hunter M. Role of de novo protein synthesis and calmodulin in rapid activation of Na+-H+ exchange by aldosterone in frog diluting segment. J Physiol 491: 219-223, 1996[Abstract].

11.   Dietl, P, Good D, and Stanton B. Adrenal corticosteroid action on the thick ascending limb. Semin Nephrol 10: 350-364, 1990[ISI][Medline].

12.   Ebata, S, Muto S, Okada K, Nemoto J, Amemiya M, Saito T, and Asano Y. Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms. Kidney Int 56: 1400-1412, 1999[ISI][Medline].

13.   Falkenstein, E, Tillmann HC, Christ M, Feuring M, and Wehling M. Multiple actions of steroid hormones-a focus on rapid, nongenomic effects. Pharmacol Rev 52: 513-555, 2000[Abstract/Free Full Text].

14.   Farman, N, Oblin ME, Lombes M, Delahaye F, Westphal HM, Bonvalet JP, and Gasc JM. Immunolocalization of gluco- and mineralocorticoid receptors in rabbit kidney. Am J Physiol Cell Physiol 260: C226-C233, 1991[Abstract/Free Full Text].

15.   Farman, N, and Rafestin-Oblin M-E. Multiple aspects of mineralocorticoid selectivity. Am J Physiol Renal Physiol 280: F181-F192, 2001[Abstract/Free Full Text].

16.   Funder, JW. Glucocorticoid and mineralocorticoid receptors: biology and clinical relevance. Annu Rev Med 48: 231-240, 1997[ISI][Medline].

17.   Funder, JW, Pearce PT, Smith R, and Smith IA. Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242: 583-585, 1988[ISI][Medline].

18.   Ganong, WF, and Mulrow PJ. Rate of change in sodium and potassium excretion after injection of aldosterone into the aorta and renal artery of the dog. Am J Physiol 195: 337-341, 1958[Abstract/Free Full Text].

19.   Gekle, M, Freudinger R, Mildenberger S, Schenk K, Marschitz I, and Schramek H. Rapid activation of Na+/H+ exchange in MDCK cells by aldosterone involves MAP-kinases ERK1/2. Pflügers Arch 441: 781-786, 2001[ISI][Medline].

20.   Gekle, M, Silbernagl S, and Wünsch S. Nongenomic action of the mineralocorticoid aldosterone on cytosolic sodium in cultured kidney cells. J Physiol 511: 255-263, 1998[Abstract/Free Full Text].

21.   Giebisch, G, Malnic G, and Berliner RW. Control of renal potassium excretion. In: The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 2000, vol. 1, p. 455-519.

22.   Good, DW. Inhibition of bicarbonate absorption by peptide hormones and cyclic adenosine monophosphate in rat medullary thick ascending limb. J Clin Invest 85: 1006-1013, 1990[ISI][Medline].

23.   Good, DW. The thick ascending limb as a site of renal bicarbonate reabsorption. Semin Nephrol 13: 225-235, 1993[ISI][Medline].

24.   Good, DW. PGE2 reverses AVP inhibition of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in rat MTAL by activation of protein kinase C. Am J Physiol Renal Fluid Electrolyte Physiol 270: F978-F985, 1996[Abstract/Free Full Text].

25.   Good, DW, George T, and Wang DW. Angiotensin II inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via a cytochrome P450-dependent signaling pathway in rat medullary thick ascending limb. Am J Physiol Renal Physiol 276: F726-F736, 1999[Abstract/Free Full Text].

26.   Good, DW, George T, and Watts BA, III. Aldosterone inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption via a nongenomic pathway in rat medullary thick ascending limb (Abstract). J Am Soc Nephrol 12: 4A, 2001.

27.   Good, DW, and Watts BA, III. Functional roles of apical membrane Na+/H+ exchange in rat medullary thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 270: F691-F699, 1996[Abstract/Free Full Text].

28.   Harvey, BJ, Condliffe S, and Doolan CM. Sex and salt hormones: rapid effects in epithelia. News Physiol Sci 16: 174-177, 2001[Abstract/Free Full Text].

29.   Harvey, BJ, and Higgins M. Nongenomic effects of aldosterone on Ca2+ in M-1 cortical collecting duct cells. Kidney Int 57: 1395-1403, 2000[ISI][Medline].

30.   Horie, S, Moe OW, Miller RT, and Alpern RJ. Long-term activation of protein kinase C causes chronic Na/H antiporter stimulation in cultured proximal tubule cells. J Clin Invest 89: 365-372, 1992[ISI][Medline].

31.   Kim, GH, Masilamani S, Turner R, Mitchell C, Wade JB, and Knepper MA. The thiazide-sensitive Na-Cl cotransporter is an aldosterone-induced protein. Proc Natl Acad Sci USA 95: 14552-14557, 1998[Abstract/Free Full Text].

32.   Kyossev, Z, Walker PD, and Reeves WB. Immunolocalization of NAD-dependent 11beta -hydroxysteroid dehydrogenase in human kidney and colon. Kidney Int 49: 271-281, 1996[ISI][Medline].

33.   Maguire, D, MacNamara B, Cuffe JE, Winter D, Doolan CM, Urbach V, O'Sullivan GC, and Harvey BJ. Rapid responses to aldosterone in human distal colon. Steroids 64: 51-63, 1999[ISI][Medline].

34.   Masilamani, S, Kim GH, Mitchell C, Wade JB, and Knepper MA. Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. J Clin Invest 104: R19-R23, 1999[ISI][Medline].

35.   Masilamani, S, Knepper MA, and Burg MB. Urine concentration and dilution. In: The Kidney, edited by Brenner BM.. Philadelphia, PA: Saunders, 2000, vol. 1, p. 595-635.

36.   Nagami, GT. Effect of angiotensin II on ammonia production and secretion by mouse proximal tubules perfused in vitro. J Clin Invest 89: 925-931, 1992[ISI][Medline].

37.   Nahm, O, Woo SK, Handler JS, and Kwon HM. Involvement of multiple kinase pathways in stimulation of gene transcription by hypertonicity. Am J Physiol Cell Physiol 282: C49-C58, 2002[Abstract/Free Full Text].

38.   Norman, AW, Ishizuka SI, and Okamura WH. Ligands for the vitamin D endocrine system: different shapes function as agonists and antagonists for genomic and rapid response receptors or as a ligand for the plasma vitamin D binding protein. J Steroid Biochem Mol Biol 76: 49-51, 2001[ISI][Medline].

39.  Rossier BC and Palmer LG. Mechanisms of aldosterone action on sodium and potassium transport. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. New York: Raven, 1992, p. 1373-1409.

40.   Smith, RE, Li KXZ, Andrews RS, and Krozowski Z. Immunohistochemical and molecular characterization of the rat 11beta -hydroxysteroid dehydrogenase type II enzyme. Endocrinology 138: 540-547, 1997[Abstract/Free Full Text].

41.   Stanton, BA. Regulation by adrenal corticosteroids of sodium and potassium transport in loop of Henle and distal tubule of rat kidney. J Clin Invest 78: 1612-1620, 1986[ISI][Medline].

42.   Stone, DK, Crider BP, and Xie XS. Aldosterone and urinary acidification. Semin Nephrol 10: 375-379, 1990[ISI][Medline].

43.   Todd-Turla, KM, Schnermann J, Fejes-Toth G, Naray-Fejes-Toth A, Smart A, Killen PD, and Briggs JP. Distribution of mineralocorticoid and glucocorticoid receptor mRNA along the nephron. Am J Physiol Renal Fluid Electrolyte Physiol 264: F781-F791, 1993[Abstract/Free Full Text].

44.   Unwin, R, Capasso G, and Giebisch G. Bicarbonate transport along the loop of Henle: effects of adrenal steroids. Am J Physiol Renal Fluid Electrolyte Physiol 268: F234-F239, 1995[Abstract/Free Full Text].

45.   Velazquez, H, Bartiss A, Bernstein P, and Ellison DH. Adrenal steroids stimulate thiazide-sensitive NaCl transport by rat renal distal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 270: F211-F219, 1996[Abstract/Free Full Text].

46.   Verrey, F. Early aldosterone action: toward filling the gap between transcription and transport. Am J Physiol Renal Physiol 277: F319-F327, 1999[Abstract/Free Full Text].

47.   Wang, T, and Giebisch G. Effects of angiotensin II on electrolyte transport in the early and late distal tubule in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 271: F143-F149, 1996[Abstract/Free Full Text].

48.   Watson, CS, and Gametchu B. Membrane estrogen and glucocorticoid receptors---implications for hormonal control of immune function and autoimmunity. Int Immunol 1: 1049-1063, 2001.

49.   Watts, BA, III, George T, and Good DW. Nerve growth factor inhibits HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in renal thick ascending limb through inhibition of basolateral membrane Na+/H+ exchange. J Biol Chem 274: 7841-7847, 1999[Abstract/Free Full Text].

50.   Watts, BA, III, and Good DW. Apical membrane Na+/H+ exchange in rat medullary thick ascending limb: pHi-dependence and inhibition by hyperosmolality. J Biol Chem 269: 20250-20255, 1994[Abstract/Free Full Text].

51.   Watts, BA, III, and Good DW. Hyposmolality stimulates apical membrane Na+/H+ exchange and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in renal thick ascending limb. J Clin Invest 104: 1593-1602, 1999[Abstract/Free Full Text].

52.   Wehling, M. Specific, nongenomic actions of steroid hormones. Annu Rev Physiol 59: 365-393, 1997[ISI][Medline].

53.   Winter, DC, Schneider MF, O'Sullivan GC, Harvey BJ, and Geibel JP. Rapid effects of aldosterone on sodium-hydrogen exchange in isolated colonic crypts. J Membr Biol 170: 17-26, 1999[ISI][Medline].

54.   Work, J, and Jamison RL. Effect of adrenalectomy on transport in the rat medullary thick ascending limb. J Clin Invest 80: 1160-1164, 1987[ISI][Medline].

55.   Zhou, ZH, and Bubien JK. Nongenomic regulation of ENaC by aldosterone. Am J Physiol Cell Physiol 281: C1118-C1130, 2001[Abstract/Free Full Text].


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