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-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol
1Endocrine Section and Research Service, G. V. (Sonny) Montgomery Veterans Affairs Medical Center, 2Division of Endocrinology, and 3Department of Pathology, The University of Mississippi Medical Center, Jackson, Mississippi 39216; and Departments of 4Medicine and 5Veterinary Biomedical Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
Submitted 16 September 2002 ; accepted in final form 6 April 2003
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ABSTRACT |
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-hydroxysteroid dehydrogenase (11HSD) type 1
(11HSD1) enzyme is an NADP+-dependent oxidoreductase, usually
reductase, of major glucocorticoids. The NAD+-dependent type 2
(11HSD2) enzyme is an oxidase that inactivates cortisol and
corticosterone, conferring extrinsic specificity of the mineralocorticoid
receptor for aldosterone. We reported that addition of a reducing agent to
renal homogenates results in the monomerization of 11HSD2 dimers and a
significant increase in NAD+-dependent corticosterone conversion.
Estrogenic effects on expression, dimerization, and activity of the kidney
11HSD1 and -2 enzymes are described herein. Renal 11HSD1 mRNA and
protein expressions were decreased to very low levels by estradiol
(E2) treatment of both intact and castrated male rats; testosterone
had no effect. NADP+-dependent enzymatic activity of renal
homogenates from E2-treated rats measured under nonreducing
conditions was less than that of homogenates from intact animals. Addition of
10 mM DTT to aliquots from these same homogenates abrogated the difference in
NADP+-dependent activity between E2-treated and control
rats. In contrast, 11HSD2 mRNA and protein expressions were
significantly increased by E2 treatment. There was a marked
increase in the number of juxtamedullary proximal tubules stained by the
antibody against 11HSD2 after the administration of E2.
Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone
excreted in the urine nor their ratio differed between E2- and
vehicle-treated rats. NAD+-dependent enzymatic activity in the
absence or presence of a reducing agent demonstrated that the increase in
11HSD2 protein was not associated with an increase in in vitro activity
unless the dimers were reduced to monomers. hypertension; aldosterone
-hydroxysteroid dehydrogenase (11HSD) type 2
(7,
8). This enzyme converts
corticosterone and cortisol to their inactive metabolites
11-dehydrocorticosterone and cortisone, respectively, before they can interact
with the MR (2,
7,
8,
31). The
11-18-hemiacetal of aldosterone prevents it from being a substrate for
the 11HSD2.
Two 11HSD isozymes have been cloned and characterized. 11HSD1
is NADP+ dependent, has a high Km of 1–3
µM for corticosterone and cortisol, and is bidirectional, although in vivo
and in intact cells it is primarily a reductase. It does not colocalize with
the MR in the kidney (19,
30,
32). 11HSD2 is
NAD+ dependent, acts only as a dehydrogenase, has a
Km for corticosterone and cortisol low enough to be
relevant to circulating levels of free glucocorticoids (4–14 nM), and is
colocalized with the MR in aldosterone target tissues
(15,
31). 11HSD2 has been
cloned from kidney cDNA libraries of the human
(2), sheep
(1), rabbit
(23), mouse
(6), cow
(29), and rat
(42). In addition to classical
aldosterone target cells, data from in situ hybridization studies indicate
that 11HSD2 mRNA is expressed in several other tissues, including the
female rat reproductive system and the central nervous system
(26–28).
The direction of 11HSD1 activity, reduction or oxidation, in vitro is
dependent on substrate concentration, cofactor, and type of preparation.
Before the mid-1990s, when the existence of a second 11HSD was confirmed
(2), there was confusion about
the bidirectionality of the 11HSD enzyme in different tissues under
different experimental conditions. Significant 11HSD2 activity in the
rat kidney was difficult to demonstrate until the recent report that the
addition of the reducing agent dithiothreitol (DTT) to the incubation mixture
of renal homogenates in the presence of NAD+ resulted in
significant conversion of tritiated corticosterone to tritiated
11-dehydrocorticosterone (11).
Western blots under nonreducing conditions indicated that most of the
11HSD2 was present as an inactive dimer
(11). With this in mind, we
restudied the effect of estrogens on the mRNA, protein, and activity of
11HSD1 and 11HSD2 in vitro and in vivo by performing ribonuclease
protection assays and Western blots and measuring oxidation of
[3H]corticosterone by kidney homogenates with and without DTT, as
well as by measuring the 24-h urinary excretion of adrenal cortical steroids.
In addition, changes in expression and distribution of the 11HSD2 were
assessed by immunohistochemistry.
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MATERIALS AND METHODS |
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Animals. All animal experiments were performed under protocols approved by the University of Missouri-Columbia Animal Care and Use Committee and the Harry S Truman Memorial Veterans Committee on Animal Studies. Sprague-Dawley rats from Harlan Industries (Indianapolis, IN) were housed under conventional husbandry conditions in American Association for Accreditation of Laboratory Animal Care-accredited facilities. Orchiectomies were done with animals under isoflurane anesthesia delivered in oxygen by a veterinary anesthesia machine with use of standard aseptic surgical procedures through a midline skin incision in the scrotum. The tunicae vaginalis were obliterated to prevent intestinal herniation and entrapment. There were 10 male Sprague-Dawley rats in each group for the urinary steroid excretion studies. Intact and castrated male rats received subcutaneous injections of control oil, 5 mg of estradiol valerate, or 5 mg of testosterone propionate. Injections were given 2 days after surgery, before the animals were placed in metabolism cages without urine collection funnels. After 4 days of acclimatization to the metabolism cages, the rats received a second injection of estradiol valerate, testosterone, or vehicle, and urine was collected for 48 h. On removal from the metabolism cages, the rats received a third injection and were placed in their home cages. The rats were anesthetized with isoflurane 48 h later, and blood was taken by cardiac puncture before tissue harvest. Two rats from each group were gravity-perfused under anesthesia with heparinized saline, followed by Streck's Tissue Fixative (STF; Streck laboratories, La Vista, NE), and their kidneys were used for immunocytochemistry. One kidney from each of the other rats was cut in half, and the pieces were frozen separately in liquid nitrogen and stored at -80°C for RNA and protein extraction at a later date. The other kidney was homogenized and used for enzymatic activity measurements. The mRNA and protein expression and enzyme activity studies were repeated with 6, 4, or 3 rats per group; statistical comparisons were made within each group.
Ribonuclease protection assay. Total RNA was prepared using
UltraSpec RNA isolation solution from Biotecx (Houston, TX). Biotinylated RNA
probes were made as follows. PCR products of 11HSD1 (305 bp),
11HSD2 (163 bp), and GAPDH (282 bp) were individually cloned into pCR3
plasmid, and the positive clones with the correct orientation were identified
by restriction digestion and sequencing. The plasmids were linearized and
transcribed with T7 RNA polymerase by use of a BrightStar BIOTINscript in
vitro transcription kit from Ambion (Austin, TX) to make the cRNA probes. The
RNA probes were gel purified by denaturing polyacrylamide gel. Twenty
micrograms of total RNA from three individual rats of each group were
hybridized with 11HSD1 or 11HSD2 probes, as well as with a GAPDH
probe as an internal control for the amount of RNA. The ribonuclease
protection assay (RPA) was performed according to the instruction manuals from
Ambion (RPA II and the BrightStar BioDetect kits). The films were scanned and
quantified with a Kodak Image Station 440 CF by use of Kodak ds 1D image
analysis software. Enzyme mRNA densitometry values were normalized for sample
loading differences by dividing them by the densitometry values of the GAPDH
signals run concomitantly and were expressed as arbitrary units (au). Each
lane represents RNA from a different individual rat.
Western blots for 11HSD2 and 11HSD1.
Rat kidney microsomes were isolated by differential centrifugation, as
described before (21).
Microsomes were solubilized using Laemmli buffer with or without 10 mM
-mercaptoethanol (16)
and run in a 12% PAGE, transferred by semidry blot to a polyvinylidene
membrane, dried, blocked with 5% nonfat milk, and incubated with the
11HSD2 antibody raised against the recombinant rat 11HSD2 protein
in sheep (11) or with a rabbit
anti-11HSD1 antibody [kindly provided by Dr. Mathew Hardy from
Rockefeller University (17)].
The blots were then incubated with a peroxidase-labeled second antibody and
developed using West Pico Chemiluminescence substrate from Pierce Chemical
(Rockville, IL). The films were scanned and quantified with a Kodak Image
Station 440 CF using Kodak ds 1D image analysis software. For the
quantification of the 11HSD2, a group of four control and
estradiol-treated rat renal microsomal protein isolates were electrophoresed,
blotted as above, developed using SuperSignal West Dura Extended Duration
Substrate (Pierce Chemical), and quantified directly using the Kodak Image
Station.
Enzyme activity assays. Kidney homogenates were prepared in ice-cold Tris buffer 0.1 M, pH 7.6, 0.25 M sucrose, and 5 mM magnesium chloride solution with short bursts of a polytron homogenizer. Microsomes were separated by differential centrifugation, as previously described (21), and then incubated at 37°C for 30 min with 0.5 mM NAD+ or NADP+, 0 or 10 mM DTT, and 10 nM [3H]corticosterone (NAD+) or 500 nM [3H]corticosterone (NADP+).
Measurements of urinary corticosterone and
11-dehydrocorticosterone. A 1-ml aliquot of urine mixed with
[3H]corticosterone (
3,000 dpm) was extracted with 25%
dichloromethane in hexane, washed with water, evaporated under air, and
reconstituted in ELISA buffer. An aliquot was used for measurement of
recovery. ELISAs for corticosterone and 11-dehydrocorticosterone were
performed using antibodies raised in sheep against corticosterone- and
11-dehydrocorticosterone-3-carboxymethoxylamine-chicken serum albumin
conjugates and methods developed by us as previously described
(9,
20). The anti-corticosterone
antibody has a cross-reactivity of 0.83% with 11-dehydrocorticosterone, 2.7%
with cortisol, 12.5% with deoxycorticosterone, 2.5% with progesterone, and
0.083% with 18-OH-deoxycorticosterone. The antibody against
11-dehydrocorticosterone has a cross-reactivity of 0.81% with corticosterone,
<0.09% with cortisol, 0.75% with deoxycorticosterone, 0.11% with cortisone,
and 0.1% with progesterone. Two consecutive 24-h urine collections were pooled
after an acclimatization period to achieve an average 24-h excretion over 2
days.
Immunohistochemistry. The kidneys from perfused animals were fixed
overnight at room temperature in STF, embedded in paraffin, and cut in 6-µm
slices. Immunocytochemistry was performed within 2 wk of sectioning the tissue
by use of an antibody we raised in sheep against a recombinant 11HSD2
enzyme (11) at a dilution of
1:2,000, followed by an anti-sheep affinity-purified biotin-labeled antibody
detected using a streptavidin-peroxidase system (Zymed Laboratories, S. San
Francisco, CA) and diaminobenzidene (Sigma), and counterstained with Gil
hematoxylin.
Statistical analysis. Differences in the measured variables between control and treated samples were evaluated by ANOVA, followed by a Bonferroni test where appropriate, with use of a STATISTICA 6.0 (StatSoft) package. Results are expressed as means ± SE.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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HSD1 mRNA, measured by RPA
(Fig. 1, top), were
unaffected by castration (0.324 ± 0.035 vs. 0.387 ± 0.039),
decreased by estradiol valerate (E2) treatment in both intact
(0.324 ± 0.035 vs. 0.069 ± 0.014; P = 0.000025) and
castrated rats (0.387 ± 0.039 vs. 0.043 ± 0.008; P =
0.000003), and increased in testosterone-treated castrated rats (0.387
± 0.039 vs. 0.484 ± 0.024; P < 0.05).
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Western blot (Fig. 1,
bottom) showed that 11HSD1 protein expression in castrated male
rats was about two-thirds that of intact animals, 117.8 ± 9.4 and 79.8
± 1.2 au, respectively, a change that did not attain significance.
11HSD1 protein in the kidneys was decreased by E2 treatment,
in intact rats from 117.8 ± 9.4 to 24.97 ± 14.0 au (P =
0.0008) and in castrated rats from 79.8 ± 12.0 to 23.5 ± 10.0 au
(P = 0.032). 11HSD1 protein in castrated and
testosterone-treated castrated rats was 79.8 ± 12.0 and 116.3 ±
28.0 au, respectively; the difference was not statistically significant.
NADP+-dependent enzymatic activity in renal homogenates from intact
E2-treated rats was 45% less than in those from intact,
untreated animals when enzymatic activity was measured in incubations without
DTT or in the presence of 1 mM DTT (Fig.
2). The addition of 10 mM DTT to the incubations produced a small,
but significant, increase in the activity of NADP+-dependent
11HSD activity in kidney homogenates from control animals, 27.17
± 1.51%, compared with incubations of control homogenates without DTT,
21.83 ± 0.52%. The activity in kidney homogenates from
E2-treated rats measured in the presence of 10 mM DTT, 25.11
± 0.67%, was greater than that in aliquots of the same homogenates
incubated with 0 or 1 mM DTT, but not significantly different from that in any
of the control homogenates (Fig.
2).
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Expression of 11HSD2 mRNA (Fig.
3) was increased by castration, from 0.66 ± 0.05 in intact
males to 1.00 ± 0.01 (P = 0.03). Testosterone replacement in
the castrated males did not alter expression significantly (1.08 ±
0.06). E2 treatment increased 11HSD2 mRNA in both intact,
from 0.66 ± 0.05 to 1.47 ± 0.1 (P = 0.000004), and
castrated, from 1.00 ± 0.01 to 1.64 ± 0.05 (P =
0.00003), male rats. Figure 4
depicts eletrophoresis gels of kidney proteins with and without 10 mM of the
reducing agent -mercaptoethanol. The first, in which no reducing agent
was present in the buffer and 30% less protein from the E2-treated
animals was loaded on the gel than for the controls, demonstrates the effect
of E2 on 11HSD2 protein expression (n = 4). In the
second, -mercaptoethanol was used as the reducing agent, and graded
amounts of protein from the E2-treated animals were loaded to
demonstrate the E2-induced increase in 11HSD2 protein
expression (n = 2). The dimer-to-monomer ratio was 1.3 for control
rats, which was not significantly different from the ratio for E2
treatment, 1.6. In a similar experiment in which microsomal protein was
extracted with the reducing agent -mercaptoethanol, n = 4, the
optical density quantified directly (±SE) was 7.0 ± 1.3 au for
control and 35.2 ± 5.2 au for E2-treated rats; P =
0.0018.
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11HSD2 activity was very low for both treatment groups when the
kidney microsomes were incubated in buffer alone, and it increased
progressively when increasing concentrations of DTT were added to the
incubation media (Fig.
5A). Although there was almost no
NAD+-dependent activity when 20 µg of kidney microsomes from
control and estrogen-treated rats were incubated in buffer without DTT,
NAD+-dependent activity in all samples was increased in the
presence of 1 and 10 mM DTT. To better appreciate the difference in activity
in the different treatment groups, kidney microsomal protein was decreased to
5 µginthe incubation (Fig.
5B). Under these conditions, NAD+-dependent
activity of kidneys from E2-treated rats was increased by 71%
compared with control rats, 26.7 ± 2.6 compared with 15.6 ± 1.1%
conversion of [3H]corticosterone. The 24-h excretion of
corticosterone and 11-dehydrocorticosterone, or the ratio of corticosterone to
11-dehydrocorticosterone in the urine, a measurement of 11HSD activity
in vivo, was not significantly altered by castration or treatment of male rats
with estradiol (Fig. 6).
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The pattern of expression of the 11HSD2 enzyme in the kidneys of
control rats as assessed by immunohistochemistry was similar to that described
by others (34) and consistent
between animals of a given treatment group.
Figure 7 is a composite of
photomicrographs of two representative kidneys, one from a control rat and the
other from an E2-treated rat, at x100 and x200
magnifications, respectively. 11HSD2 immunoreactivity is seen primarily
in the distal convoluted tubules and collecting tubules, with expression
disappearing as the tubules penetrate the papilla. E2 treatment
markedly increased the number of both tubules and cells stained within a cross
section of tubule, as well as the intensity of staining. Kidneys of the
E2-treated, but not of control, animals have 11HSD2
immunoreactive cells in the straight segments of the proximal tubules in the
medullary rays and outer strip of the medulla. There is considerable
variability in the intensity of staining, even in adjacent cells in the
proximal straight tubule, with one-third to more than three-fourths of the
cells staining very strongly, and the rest staining weakly or not at all. The
staining has a finely granular to reticulated appearance and is equally
distributed throughout the cytoplasm. No 11HSD2 immunoreactivity is seen
in glomeruli or proximal convoluted tubules within the cortical labyrinth in
any kidneys.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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HSD enzymes in the kidney
(11) that was only partially
resolved by the discovery that there were two distinct members of this enzyme
family. Although the colocalization of the 11HSD2 with the MR limits
access of glucocorticoids to this receptor, both 11HSD enzymes modulate
glucocorticoid access to the glucocorticoid receptor (GR)
(35,
41), including GR of the
hypothalamo-pituitary-adrenal feedback system
(33).
In the present study, as in others
(18), 11HSD1 mRNA and
protein are decreased to almost undetectable levels in the kidneys of animals
treated with estradiol. However, in vitro NADP+-dependent activity
decreased by <50% in incubations under nonreducing conditions, but not when
the incubations were performed with 10 mM DTT in the media. This is further
evidence that NADP+-dependent 11HSD activity in the kidney
may be mediated by an enzyme different from the 11HSD1 or -2 isozyme, as
previously suggested by us and others
(12,
18). Measurements of the
11HSD1 in microsomes are usually done by determining the enzymatic
activity of the dehydrogenase reaction, because the reductase activity is very
unstable (19). Even in liver
microsomes, the dehydrogenase reaction is easily measured, whereas the
reductase has been difficult to measure. 11HSD1 acts exclusively as a
reductase in intact hepatocytes in primary culture
(13). Incubation of the kidney
microsomes in the presence of 500 nM corticosterone and NADP+ makes
it unlikely that the activity seen is due to a contribution of the
11HSD2, since the activity of this enzyme is very low in the absence of
NAD+. In addition, a high level of substrate produces substrate
inhibition of the 11HSD2 enzyme
(21).
Estradiol treatment of both intact and castrated male rats resulted in a
large increase in the mRNA and protein expressions of the 11HSD2 enzyme.
The demonstration by immunocytochemistry of a marked increase in 11HSD2
expression induced by estradiol in proximal tubules of the corticomedullary
junction corroborates the results of Western blot analysis. Despite this
increase, when enzymatic activity was measured in the absence of a reducing
agent, no significant activity in microsomes from either control or treated
animals was seen, as previously reported
(11)
(Fig. 5A). Even in the
presence of 10 mM DTT, which we have shown to optimize 11HSD2 activity
in kidney homogenates (11),
estradiol treatment results in only a 73% increase in
NAD+-dependent conversion of cortisol to 11-dehydrocorticosterone
(Fig. 5B). It has been
proposed that activity of the 11HSD2 may be regulated by the formation
of inactive dimers that act as a latent form of the enzyme
(4).
Castration of male rats and estradiol treatment produced no change in the
urinary excretion pattern or amounts of corticosterone and
11-dehydrocorticosterone. The increase in 11HSD2 enzyme expression
produced by estradiol treatment is not adequately represented by an increase
in in vitro and in vivo enzymatic activity. Activity of 11HSD1 in
microsomes was similar in estradiol-treated and control rats when optimal
reducing amounts of DTT were added. Estradiol may promote the formation of
dimers of both the 11HSD1 and -2 enzymes in an unknown manner. If
regulated, dimerization or monomerization of the 11HSDs would serve as a
mechanism for rapid modulation of enzymatic activity, and thus MR- and
GR-mediated events, at the cellular level.
Several inactivating mutations in the gene for 11HSD2 have been
described in the syndrome of apparent mineralocorticoid excess (AME). Lack of
11HSD2 activity allows the more abundant endogenous glucocorticoids
access to the MR in mineralocorticoid target tissues, increasing both the
ratio of cortisol to cortisone metabolites in plasma and urine and the
half-life of cortisol and producing clinical signs of mineralocorticoid excess
(22,
40). Similar, though less
severe, differences in cortisol metabolite excretion have been found in a
subset of essential hypertensive subjects compared with control subjects
(38). In a third of these
patients, the half-life of cortisol exceeded 2 SD of the mean, although no
11HSD2 gene mutation was found. This milder form of AME could be due to
posttranslational changes in the protein-altering enzyme activity, perhaps by
increased inactivation through dimer formation. Results of studies using cells
transfected with the cDNA encoding the defective 11HSD2 of patients with
AME have been puzzling. There was small, but measurable 11HSD2 activity
in the intact cells, but no activity in homogenates
(39). The mutations in these
patients might not only produce enzymes that are less effective but may also
promote the formation of inactive dimers that are more apparent when cells are
homogenized.
Levels of 11HSD1 and 11HSD2 are independently regulated in the
placenta and fetal tissues throughout gestation
(32). The interconversion of
cortisol and corticosterone differs in the mother, fetus, and placenta at
midterm in baboons, as well as in other mammals
(37). Estrogens, rather than
adrenocorticoid substrate or product, were found to regulate this difference
(24,
36). 11HSD2 in the
placenta maintains normal fetal blood glucocorticoid levels in the face of
elevated maternal glucocorticoids early in the pregnancy, and relatively high
11HSD2 levels in the fetal brain protect the developing brain
throughout, when 11HSD2 in the placenta decreases significantly near
term, allowing maternal glucocorticoids, crucial for fetal lung maturation, to
enter the fetal circulation (5,
25).
The data presented herein support the possibility that, in addition to the
genomic regulation of 11HSD enzyme expression by estrogens, increasing
levels of estrogenic steroids exert a posttranscriptional regulation of
11HSD2. Estrogens increase near term. 11HSD2 dimerization in some
organs, but not in others, in response to periparturient elevations of
estrogens might be a mechanism to ensure that glucocorticoids pass to the
maturing fetal lungs without loss of 11HSD2 protection of MR where still
needed, for example, in the kidney and brain.
In conclusion, both message and protein for the NADP+-dependent
11HSD1 were reduced to very low levels by estradiol in both intact and
castrated male rats. However, enzymatic activity did not change, particularly
when the reducing agent DTT was added to the incubation media. These data
support the suggestion that another NADP+-dependent enzyme with
11HSD activity exists. There have been other reports of 11HSD
activity that does not appear to be due to either 11HSD1 or -2, but as
yet no other enzyme has been isolated or gene cloned
(10,
12,
18). Despite a large increase
in message and protein measured by both Western blot and immunocytochemistry
for the 11HSD2 enzyme, in vivo activity, as measured by the ratio of
urinary corticosterone to 11-dehydrocorticosterone, did not change in
estradiol-treated rats. We have previously reported that reducing agents
separate inactive 11HSD dimers into active monomers. In this study,
NADP+-dependent enzyme activity in renal microsomes from
estradiol-treated rats was lower than that of controls under nonreducing
conditions but was restored to the level of control kidneys by the addition of
DTT. NAD+-dependent activity in microsomes from control or
estradiol-treated kidneys was minimal, and no difference between the treated
and untreated groups could be appreciated unless DTT was added to the
incubation media. We suggest that, in addition to transcriptional regulation,
11HSD activity may be regulated in vivo by dimer formation or other
posttranslational modification. The significance of the large increase in
11HSD2 expression in proximal tubules of the cortical medullary junction
induced by estradiol without a significant change in vivo or in vitro activity
is yet unknown.
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DISCLOSURES |
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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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 H. C. Thiesson, B. L. Jensen, C. Bistrup, P. D. Ottosen, A. D. McNeilly, R. Andrew, J. Seckl, and O. Skott Renal sodium retention in cirrhotic rats depends on glucocorticoid-mediated activation of mineralocorticoid receptor due to decreased renal 11beta-HSD-2 activity Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R625 - R636. [Abstract] [Full Text] [PDF]
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