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Division of Nephrology and Hypertension, Department of Medicine, University Hospital of Berne, Inselspital, 3010 Berne, Switzerland
Address all correspondence and requests for reprints to: Felix J. Frey, M.D., Division of Nephrology, Freiburgstrasse 3, Inselspital, 3010 Berne, Switzerland. E-mail: felix.frey{at}insel.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Furosemide was the only inhibitor. Its inhibition constant
(Ki) was 30 µmol when extracts from COS-1 cells
transfected with human 11ß-OHSD2 were used as an enzyme source. The
type of inhibition was competitive. To establish whether furosemide
inhibits 11ß-OHSD2 and 11ß-OHSD1 in the renal target tissue,
isolated tubular segments from rats were analyzed. Furosemide decreased
the oxidative activity of 11ß-OHSD2 in intact distal tubules and
11ß-OHSD1 in proximal convoluted tubules. For the assessment of
furosemide on the excretion of corticosterone metabolites in
vivo, rats were given furosemide ip, and the ratio of
tetrahydrocorticosterone plus 5
-tetrahydrocorticosterone to
11-dehydrotetrahydrocorticosterone was determined in urine. This ratio
increased after the administration of furosemide in all animals,
indicating inhibition of the oxidative activity of 11ß-OHSD. Thus,
furosemide inhibits the 11ß-OHSD2 enzyme in the target tissue and
might by that mechanism enhance the mineralocorticoid effect of
11ß-hydroxyglucocorticoids.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several endo- and xenobiotics have been found to modulate the activity of 11ß-OHSD (15, 16, 17, 18, 19, 20, 21). We have previously shown that furosemide inhibits 11ß-OHSD activity, as assessed by using kidney and liver microsomal enzyme preparations or extracts from COS-1 cells transfected with 11ß-OHSD1 (22). Furthermore, the effect of furosemide was assessed by determining the concentration ratio of prednisolone to its 11-ketometabolite prednisone in vivo (22). Based on these results, it was speculated that the urinary loss of potassium in patients treated with furosemide might at least partly be attributable to the activation of mineralocorticoid receptors by glucocorticoids. In the meantime, it became clear that 11ß-OHSD1 did not fulfill the prerequisites for the protection of the mineralocorticoid receptor, and subsequently, the relevant candidate 11ß-OHSD isozyme, 11ß-OHSD2, was cloned (1, 6). Therefore, the main purpose of the present investigation was to establish whether furosemide inhibits 11ß-OHSD2. Furthermore, we attempted to determine whether furosemide inhibits 11ß-OHSD activity at the site of action, i.e. in the distal tubule, using isolated segments to assess the activity of 11ß-OHSD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Androstane-3,17-diol,
stigmasterol, and cholesteryl butyrate were purchased from Steraloids
(Wilton, NH).
Test substances
The following diuretics were assessed with respect to their
inhibitory capacity on 11ß-OHSD: acetazolamide (10/100 µg/ml;
45/450 µM), amiloride hydrochloride (10/100 ng/ml;
0.04/0.4 µM), bendroflumethiazide (0.1/1 µg/ml;
0.24/2.4 µM), buthiazide (12/120 ng/ml; 0.035/0.35
µM), chlorthalidone (8/80 µg/ml; 23.6/236
µM), furosemide (6/60 µg/ml; 18.2/180
µM), hydrochlorothiazide (0.26/2.6 µg/ml; 0.87/8.7
µM), indapamide (0.14/1.4 µg/ml; 0.38/3.8
µM), piroxicam (2/20 µg/ml; 6.04/60.4
µM), spironolactone (0.2/2 µg/ml; 0.48/4.8
µM), torasemide (2.7/27 µg/ml; 7.75/77.5
µM), and triamterene (4/40 µg/ml; 15.8/158
µM).
Transfection
COS-1 cells were cultured in DMEM supplemented with 10% FCS at
37 C in a CO2 incubator. Transfection of cells with the
complementary DNA (cDNA) of rat 11ß-OHSD1 and human 11ß-OHSD2 in
pcDNA3 was performed using the diethylaminoethyl-dextran method (22).
Forty-eight hours after transfection, cells were either harvested and
extracted with the appropriate buffer (see below) or incubated in
situ for 1 h with the indicated drug concentrations in the
presence of 10 µM (for 11ß-OHSD1) or 10 nM
(for 11ß-OHSD2) corticosterone and 100 respectively, 50 nCi
[3H]corticosterone. After sonication, protein
determination was performed using the BCA reagent. For measuring the
activity of 11ß-OHSD, approximately 10 µg total protein from
transfected cells was used to obtain a conversion rate of 30–50%.
Assay for 11ß-OHSD1
The assay was performed as previously described by Monder et al.
(24). Oxidation or reduction at C-11 was determined by measuring the
rate of conversion of corticosterone to 11-dehydrocorticosterone in the
presence of NADP or the rate of conversion of dehydrocorticosterone to
corticosterone in the presence of NADPH. Transfected COS-1 cells with
the cDNA of 11ß-OHSD1 were extracted with 10 mM Tris-HCl
(pH 7.5), 5 mM EDTA (pH 8), 1% Triton X-100, 2
mM phenylmethylsulfonylfluroide, and 10 µg total protein
were used for the reaction. The assay was performed in 0.25
mM NADP, 100 mM Tris (pH 8.3), 100 nCi
[3H]corticosterone, and 5 µM corticosterone
in the presence or absence of the mentioned drug. Samples were
incubated for 1 h at 37 C, the reaction was stopped on ice, and
steroids were extracted with 1 vol ethyl acetate. The organic layer was
separated by centrifugation at 13,000 rpm and evaporated under a stream
of nitrogen. The steroid residue was dissolved in 20 µl methanol
containing a mixture of 20 µg each of unlabeled corticosterone and
dehydrocorticosterone. This was quantitatively transferred to thin
layer plates and developed in chloroform-methanol (90:10, vol/vol). The
spots corresponding to the steroids were located under a UV lamp, cut
out, transferred to scintillation vials, and counted in scintillation
fluid in a Tricarb 2000 CA (Canberra Packard, Zurich, Switzerland)
fluid scintillation counter. Specific activity was expressed as
micromoles of product formed per µg of protein/h.
For activity measurements in single nephron segments, the assay was performed in a final volume of 10 µl, incubated for 1 h at 37 C. To the supernatant, 20 µg each of unlabeled corticosterone and dehydrocorticosterone were added, and the mixture was transferred directly onto the TLC plate without prior ethyl acetate extraction.
Assay for 11ß-OHSD2
The assay was performed as previously described by Albiston
et al. (6). COS-1 cells transfected with the cDNA of
11ß-OHSD2 were homogenized in a buffer containing 250 mM
sucrose and 10 mM Tris-HCl, pH 7.5. Ten micrograms of
protein extract were incubated for 1 h at 37 C with 1
mM NAD, 10 nM corticosterone, and 50 nCi
[3H]corticosterone in 500 µl homogenization buffer in
the presence or absence of the above-mentioned drugs. The subsequent
steps were the same as those described for 11ß-OHSD1.
For activity measurements in single nephron segments, the assay was performed with the same buffers and incubation conditions, but in a volume of 10 µl.
Experimental animals
The protocol was approved by the ethics committee at our
institution. Male Wistar rats, weighing 200–230 g, were kept in a
temperature-, humidity-, and light (12-h light, 12-h dark
cycle)-controlled room and maintained on a normal chow diet without
fluid restriction. On the morning of the experiment, rats were
separated into four groups of four animals each. The animals received
ip furosemide (400 mg/kg), torasemide (20 mg/kg), glycyrrhetinic acid
(400 mg/kg), or the solvent dimethylsulfoxide.
Preparation of tubular segments
One hour after administration of furosemide, rats were
anesthetized with 10 mg/kg pentobarbital and perfused via the aorta
with an ice-cold perfusion solution (containing 137 mM
NaCl, 5 mM KCl, 0.8 mM MgSO4, 0.33
mM Na2HPO4, 0.44 mM
KH2PO4, 1 mM MgCl2, 1
mM CaCl2, 5 mM glucose, and 10
mM Tris-HCl, pH 7.4) followed by perfusion with a
collagenase solution (similar solution containing, in addition, 10
mg/ml collagenase) (23, 24, 25). At the end of the perfusion, the kidney
was removed, cut into thin pyramid pieces, and incubated at 30 C for 15
min in a perfusion solution containing 5 mg/ml collagenase and 0.1%
BSA. The corresponding solutions without collagenase were used during
microdissection under a stereoscope as previously described (26, 27).
Tubular length was measured using a millimeter scale placed under the
microdissection dish. The same concentrations of furosemide were added
into the solutions for the dissection of the kidneys from the animals
treated with furosemide to minimize the loss of furosemide from the
tubule during manipulation procedures. Tubular integrity was assessed
using the dye exclusion method (28).
For activity measurements of 11ß-OHSD1 and 11ß-OHSD2 in single nephron segments, the assay was performed with the same buffers and incubation conditions as those mentioned above, but in a volume of 10 µl. Briefly, 2.5 mm of proximal convoluted tubule (PCT) were incubated for 2 h for measurement of 11ß-OHSD1 activity, and 1 mm of cortical collecting duct (CCT) was incubated for 30 min for measurement of 11ß-OHSD2 activity. In preliminary experiments lengths of tubules and incubation time were chosen to obtain a conversion rate of the steroidal substrates of about 25%.
Effect of furosemide on urinary steroid metabolites in rats
Animals were kept in metabolic cages for urine collection. On
the morning of the experiment, rats were separated and administered ip
furosemide, torasemide, glycyrrhetinic acid, or dimethylsulfoxide. For
the assessment of steroid metabolite excretion in urine, gas
chromatography was performed according to the method of Shackelton (29, 30). Briefly, the analytical procedure consisted of hydrolysis, solid
phase extraction, derivatization, and purification by gel filtration.
For each measurement, 2.5 ml urine mixed with 0.5 ml acetate buffer
(0.5 M) were hydrolyzed at 55 C for 3 h with Sigma
type I powdered Helix promatia enzyme (12 mg) and 0.0125 ml
ß-glucuronidase/arylsulfatase (Boehringer Mannheim) liquid enzyme.
After extraction on a Sep-Pak cartridge (Waters Corp., Milford,
MA) and adding the internal standards (5-androstane-3,17-diol,
stigmasterol, and cholesteryl butyrate), the samples were derivatized
to form the methyloxime-trimethylsilyl ethers. After purification by
gel filtration on Lipidex-5000 columns, the samples were analyzed on a
Shimadzu GC-9A with an integrator Chromatopac C-R2A (Shimadzu Corp.,
Kyoto, Japan). A temperature programed run of 210–270 C was
used over a 40-min period. Using this method, the following metabolites
were determined in urine: tetrahydrocorticosterone (THB),
5-tetrahydrocorticosterone (5-THB), and
11-dehydrotetrahydrocorticosterone (THA).
Analysis of data
The effect of furosemide on 11ß-OHSD2 was assessed by
analyzing the concentration-response curves and Lineweaver-Burk linear
transformations of the Michaelis-Menten equation. The
Ki was calculated by unweighted linear regression
analysis with the mean values of at least three experiments. Results
are given as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The concentrations of furosemide
required to exhibit half-maximal inhibition of 11ß-OHSD2 were 30–40
µM. A Ki value of similar magnitude
was recently described for 11ß-OHSD1 (22). Double reciprocal plots of
the concentration-dependent oxidation of corticosterone in the presence
of increasing concentrations of furosemide are depicted in Fig. 2. The data converged on the ordinate in
a pattern consistent with competitive inhibition.
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, upper
panel). Whereas a strong inhibition of the activity was observed
when enzyme extracts were used, the inhibition was much less pronounced
in the intact cells. A similar difference was observed between the
effect of furosemide in intact cells and enzyme extracts when
11ß-OHSD1 was considered (Fig. 4, upper panel). To exclude that this difference between the
inhibitory effect of furosemide on the two types of enzyme source was
restricted to furosemide, glycyrrhetinic acid was added to intact cells
and enzyme extracts (Figs. 3 and 4, lower part). A similar
difference between the dose-response curve in intact cells and cell
extracts was found with this model compound for the inhibition for the
11ß-OHSD enzymes.
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). CCT segments express a high activity
of 11ß-OHSD2 (23). In these segments furosemide at high
concentrations inhibited the dehydrogenase activity (Fig. 5). When CCT
were incubated with high concentrations of piretanide or torasemide,
two structurally different loop diuretics, no inhibition of 11ß-OHSD2
was observed (results not shown).
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-THB)/THA was analyzed. Furosemide increased this ratio in all four
rats investigated (Fig. 6). When another
loop diuretic, torasemide (20 mg/kg), was given ip, no changes in the
urinary ratio of (THB+5-THB)/THA were observed, indicating that the
furosemide dose-associated changes in the pattern of steroid
metabolites excreted in urine were not due to the enhanced diuresis
induced by the diuretic. As a positive control, four rats given 400
mg/kg glycyrrhetinic acid, ip, showed a significant increase in the
ratio of (THB plus 5-THB)/THA in all animals (mean ±SD
values at baseline, 1.22 ± 0.11; 24 h after treatement,
1.75 ± 0.13).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10 nM) in cell extracts from transfected
COS cells was in the same range as that found by Albiston et
al. (6) using CHOP-transfected cells as a source of enzymes (50
nM). When furosemide was added to intact transfected cells,
the inhibition of 11ß-OHSD2 was less pronounced. A similar
discrepancy between inhibition of 11ß-OHSD obtained from microsomal
preparations and intact cells was previously described by Monder
et al. (32). These researchers observed a 75% inhibition in
the presence of 20 nM glycyrrhetinic acid when microsomal
preparations were used and a 60% inhibition in the presence of 100
µM of glycyrrhetinic acid when intact cells were used.
(32). On the other hand, Bujalska et al. (33) observed a
significant inhibition of 11ß-OHSD2 at much lower concentrations of
glycyrrhetinic acid (100–1000 nM) in intact cells when
fetal kidney 293 cells transfected with 11ß-OHSD2 were analyzed.
These researchers did not analyze the impact of glycyrrhetinic acid on
extracts of 11ß-OHSD enzymes from the transfected 293 cells.
With respect to inhibition of 11ß-OHSD isoenzymes, we have screened a
number of diuretics other than furosemide, including acetazolamide,
amiloride hydrochloride, bendroflumethiazide, butizide, chlorthalidone,
indapamide, piroxicam, spironolactone, torasemide, piretanide,
thiazides, and triamterene. None of these diuretics inhibited
11ß-OHSD enzymes (results not shown), indicating that the inhibitory
effect of furosemide is a particular feature of this loop diuretic. The
inhibition of 11ß-OHSD1 in PCT and that of 11ß-OHSD2 in CCT by
furosemide are consistent with a direct effect of this diuretic in the
kidney tubule. The renal effect of furosemide is furthermore supported
by the changes in the urinary ratio of (THB plus 5-THB)/THA, an
observation consistent with inhibition of 11ß-OHSD dehydrogenase
activity. The impact of furosemide on the urinary excretion of
glucocorticoid metabolites is not a nonspecific consequence of the
enhanced diuresis, because rats given another loop diuretic
(torasemide) did not exhibit a similar change in the urinary excretion
of the steroid metabolites.
The role of 11ß-OHSD2 inhibition in cortical collecting tubules by furosemide remains speculative. The kaliuretic effect of furosemide is attributed to the inhibition of the Na+/K+/2Cl cotransporter in the thick ascending limb of Henle (34), resulting in an increased delivery of potassium to the distal nephron. Microperfusion studies showed no conclusive direct effect of furosemide in the distal nephron (35). Interestingly, these researchers observed an increasing potassium concentration in the furosemide perfusions not completely explained by the slightly higher potassium concentration in the furosemide perfusion solution, suggesting tubular secretion of potassium. Distal tubular secretion of potassium is enhanced by mineralocorticoids. Glucocorticosteroids such as cortisol act as mineralocorticoids whenever the enzyme 11ß-OHSD2 is inhibited. As the concentrations of furosemide required to show a significant inhibition of 11ß-OHSD2 were of the same magnitude as those found after a moderate dose of iv or oral furosemide in humans and the concentrations of furosemide increase along the kidney tubule, furosemide might enhance the urinary excretion of potassium (36, 37, 38).
Hypokalemia from furosemide is traditionally attributed to 1) increased delivery of sodium to the distal tubule, so that a larger fraction of sodium is available for exchange with potassium; 2) a shift of potassium into the cells because of alkalosis; and 3) secondary hyperaldosteronism due to volume depletion (39, 40). Inhibition of 11ß-OHSD might be a fourth mechanism to account for the increased urinary loss of potassium in subjects treated with furosemide. Indeed, the apparent absence of the activity of 11ß-OHSD or its inhibition by glycyrrhetinic acid leads to the activation of mineralocorticoid receptors by cortisol with urinary loss of potassium (1, 2, 41, 42). Such an activation of the mineralocorticoid receptors, however, should cause sodium retention and hypertension, an effect apparently overcome by the potent inhibition of the Na+/K+/2Cl cotransporter in the thick ascending limb of the loop of Henle. Alternatively, glucocorticoids might induce kaliuresis without concomitant sodium retention, as recently shown in patients with Addison’s disease (43). The latter effect might not be mediated through mineralocorticoid receptors.
One might speculate that some of the hitherto poorly understood side-effects of furosemide, such as insulin resistance or increased lipid levels, are attributable to increased access of cortisol to glucocorticoid receptors in the presence of furosemide (44). This mechanism could, however, account only for part of these furosemide-related unwanted effects, as other diuretics, such as thiazides, do not inhibit 11ß-OHSD, but also induce insulin resistance and high lipid concentrations. 11ß-OHSD2 is expressed in placental tissue (1). Evidence is growing that it plays a pivotal role in fetal physiology by excluding maternal glucocorticoids from the fetal circulation. Thus, inhibition of the 11ß-OHSD in hypertensive pregnant women by furosemide might be hazardous, a hypothesis in line with the fact that many authorities discourage the use of furosemide during pregnancy (45).
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Acknowledgments |
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Footnotes |
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Received March 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß enhance the cortisone/cortisol shuttle. J Exp
Med 186:189–198- and
11ß-hydroxyprogesterone, potent inhibitors of 11ß-hydroxysteroid
dehydrogenase, possess hypertensinogenic activity in the rat.
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