1 Mario Negri Institute for Pharmacological Research and 2 Division of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, 24125 Bergamo, Italy
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ABSTRACT |
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Conjugated estrogens
shorten the prolonged bleeding time in uremic patients and are
similarly effective in a rat model of uremia. We have previously
demonstrated that the shortening effect of a conjugated estrogen
mixture or 17-estradiol on bleeding time was abolished by the nitric
oxide (NO) precursor L-arginine, suggesting that the effect
of these drugs on hemostasis in uremia might be mediated by changes in
the NO synthetic pathway. The present study investigated the
biochemical mechanism(s) by which conjugated estrogens limit the
excessive formation of NO. 17
-estradiol (0.6 mg/kg), given to rats
made uremic by reduction of renal mass, significantly reduced bleeding
time within 24 h and completely normalized plasma concentrations
of the NO metabolites, nitrites and nitrates, and of NO synthase (NOS)
catalytic activity, determined by NADPH-diaphorase staining in the
thoracic aorta. Endothelial NOS (ecNOS) and inducible NOS (iNOS)
immunoperoxidase staining in the endothelium of uremic aortas of
untreated rats was significantly more intense than in control rats,
while in uremic rats receiving 17
-estradiol staining was comparable
to controls. Thus 17
-estradiol corrected the prolonged bleeding time
of uremic rats and fully normalized the formation of NO by reducing the
expression of ecNOS and iNOS in vascular endothelium. These results
provide a possible biochemical explanation of the well-known effect of
estrogens on primary hemostasis in uremia, in experimental animals and humans.
chronic renal failure; conjugated estrogens; bleeding time; endothelium.
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INTRODUCTION |
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PATIENTS WITH RENAL FAILURE have an increased tendency to bleed (33), usually manifested by prolonged bleeding time, a global measure of platelet function and primary hemostasis that often correlates with clinical bleeding (57). Prolonged bleeding time and even clinical bleeding can be corrected by conjugated estrogens which, when given in adequate doses, (24, 35, 56, 61) have a lasting effect on primary hemostasis to the extent that they are widely used in clinical practice to protect uremics from the risk of bleeding after surgery (53). Despite extensive research, however (24, 37, 61, 62, 67), the mechanism(s) by which they normalize primary hemostasis in uremia remain elusive.
On the basis of recent evidence in rats made uremic by extensive
surgical ablation of renal mass, we suggested that prolonged bleeding
time in uremia might be the consequence of excessive formation of
nitric oxide (NO), an L-arginine derivative implicated in
vasodilatation and immune response, which also inhibits platelet function (19, 38, 42). Plasma concentrations of the stable NO metabolites, nitrites and nitrates
(NO2/NO3
), were higher than normal
in uremic rats with prolonged bleeding time (2). In
addition, N-monomethyl-L-arginine, a
competitive inhibitor of NO synthesis, normalized the prolonged
bleeding time of uremic rats and increased ex vivo platelet adhesion
(52). This effect was completely reversed by giving the
animals the NO precursor L-arginine (52). In
the same model we showed that the shortening effect of either
conjugated estrogen mixture or its active component, 17
-estradiol
(62), on bleeding time was also abolished by
L-arginine (64), which can be taken as
evidence that conjugated estrogens normalize uremic bleeding by
interfering with the pathway of NO synthesis.
The reaction by which L-arginine is converted to L-citrulline and NO is catalyzed by a family of NO synthase (NOS) enzymes which exist in at least three distinct isoforms: neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (ecNOS) (63). We used two histochemical approaches, NADPH-diaphorase, that locates NOS catalytic activity (4, 41, 59), and immunoperoxidase, that locates NOS isoenzymes (4, 41, 49, 59), to document an excess of NOS activity and a higher expression of ecNOS and iNOS in the endothelia of large vessels in uremic rats (2). These results were taken to indicate that in experimental uremia excessive systemic formation of NO is a direct consequence of upregulation of NOS genes in vascular endothelia, which generate higher than normal amounts of NOS isoenzyme proteins in vessels.
The present experiments were designed to explore whether the fact that conjugated estrogens normalize bleeding time and clinical bleeding and limit the excessive formation of NO in uremia was linked to the process leading to NOS isoenzyme formation from the corresponding genes.
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METHODS |
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Chemicals.
Sodium nitrate, sodium nitrite, sulphanilic acid,
n-(1-naphthyl)-ethylendiamine dihydrochloride, sodium borate,
17-estradiol, N-nitro-L-arginine,
-NADPH,
nitroblue tetrazolium, Triton X-100, diphenyleneiodonium (DPI),
cadmium powder (100 mesh), and all other chemicals were from Sigma (St.
Louis, MO). Phosphoric acid, ZnSO4 × 7H2O, and diaminobenzidine tablets were from Merck
(Darmstadt, Germany). [3H]L-arginine (1.6 TBq/mmol) was purchased from New England Nuclear (Boston, MA). A
low-nitrate diet was prepared from commercial standard diet (Rieper,
Bolzano, Italy) by extracting the nitrate in water. Briefly, the
pulverized diet was resuspended in distilled water at 37°C and
whipped, then filtered and dried up. Because water-soluble vitamins and
minerals were also lost, the low-nitrate diet was supplemented with
minerals, vitamins, choline, DL-methionine, and
L-arginine, in amounts recommended by the American
Institute of Nutrition.
Experimental design. Male Sprague-Dawley rats (Charles River Italy, Calco, Italy), 275-300 g, (n = 29) were made uremic by surgical removal of the right kidney and ligation of two or three branches of the left renal artery according to Olson et al. (47). Seventeen rats were sham operated with manipulation of pedicles and served as controls (CTR). Procedures involving animals and their care are conducted in conformity with the institutional guidelines that are in compliance with national (D.L. no. 116, G.U., Suppl. 40, 1992, Circolare No. 8, G.U., 1994) and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, 1987; Guide for the Care and Use of Laboratory Animals. Washington, DC: National Research Council, 1996).
Animals with renal mass reduction (RMR) and controls were studied three mo after the surgical procedure. Renal function, measured as serum creatinine, urinary protein excretion, bleeding time, and systolic blood pressure were assayed in all RMR and CTR rats. To obtain an indirect in vivo indicator of systemic NO synthesis, NO2NO2/NO3
in plasma and urines.
NO2
/NO3
levels were measured
semi-automatically by using a HPLC (model 421A, Beckman Instruments,
Berkeley, CA) coupled with a 163 variable wavelength detector (Beckman
Instruments) and a Shimadzu C-R3A Chromatopack Recorder-Integrator
(Kyoto, Japan), according to the method of Green et al.
(18) with some modifications. Briefly, samples were
treated with zinc sulphate (60 µM final concentration) and
centrifuged at 14,000 g to eliminate proteins. Supernatants
were eluted onto a Dowex AG 50 WX-8 column (Bio-Rad, Hercules,
California) followed by a cadmium column which catalyzes the reduction
of nitrate to nitrite (eluent: borate buffer, pH 8.5; flow rate 0.5 ml/min). The post-column eluate reacted with Griess reagent [1 vol of
5% o-H3PO4, 1% sulphanilamide, and
one volume of 0.1% of N-(1-naphthyl)-ethylendiamine
dihydrochloride; flow rate 0.5 ml/min] to form a purple azo dye and
the colour was analyzed with a ultraviolet visible detector at 504 nm. The absorbance peak area was measured and the
NO2
/NO3
concentration in the sample
was calculated by extrapolation from a standard nitrate curve. Values
were corrected for recovery which averaged 80% (range 70-90%) as
determined by the addition of known amounts of standard nitrate to an
additional aliquot of each plasma or urine sample (2).
Results were expressed as nmol
(NO2
/NO3
)/ml plasma and nmol
(NO2
/NO3
)/24-h urines, respectively.
L-arginine in plasma. L-arginine was analyzed by HPLC with fluorescence detection after precolumn derivatization with o-phthaldialdehyde (OPA; fluoraldehyde, Pierce, Rockford, IL) according to the method of Jones and Gilligan (26) with minor modifications. Briefly, 10 µl of a 100 µg/ml homoserine solution (internal standard) were added to 200 µl of plasma and vortex mixed, and 25 µl of sample were then mixed vigorously with acetonitrile (75 µl). After centrifugation, 10 µl aliquots were derivatized with OPA and analyzed. The HPLC system consisted of a model 334 liquid chromatograph (Beckman) equipped with a programmable fluorescence detector (Chrompack, Middelburg, Netherlands) set to an excitation wavelength of 338 nm and an emission wavelength of 425 nm. Samples were chromatographed on a C-18 reverse-phase column (Chromsep 5 µm, 200 mm × 3 mm ID, Chrompack) by using a linear gradient from 100% solvent A (0.025 M KH2PO4 at pH 7.2/methanol: 75/25 plus 0.8 ml/l tetrahydrofuran) to 25% solvent B (methanol) in 10 min at a flow rate of 0.5 ml/min.
Determination of [3H]L-citrulline formation from [3H]L-arginine. Incubations were stopped by adding one volume of ice-cold 15% trichloroacetic acid (TCA). TCA-treated samples were centrifuged at 10,000 g to precipitate proteins. The supernatant was extracted with ether, vacuum lyophilized and resuspended in 2 ml HEPES, pH 5.5, and purified on 2 ml wet bed volumes of Dowex AG 50 WX-8 (100-200 mesh, Li+ form), as described (2). [3H] L-citrulline was quantitated by liquid scintillation counting in the 4-ml column effluent and identified as described (2).
Tissue preparation for histochemistry. The thoracic aorta was surgically dissected and fixed by immersion in 4% p-formaldehyde in PBS overnight at 4°C. A portion of each sample was processed for conventional paraffin inclusion, and the remainder was treated with 10% sucrose in PBS for cryoprotection, then frozen in liquid nitrogen.
NADPH-diaphorase.
Frozen sections 3 µm thick were cut on a cryostat (HM500-O, Microm,
Zeiss Oberkochen, Germany). Sections were air-dried, then washed in PBS
0.05 M pH 7.4 for 5 min at room temperature and permeabilized by
immersion in 0.3% Triton X-100/PBS 0.01 M, pH 7.2, at 4°C for 30 min
(2). For the NADPH-diaphorase reaction slides were
incubated with 1 mM -NADPH/0.2 mM nitroblue tetrazolium/100 mM
Tris · HCl buffer, pH 8.0, containing 0.2% Triton X-100 for 1 h at 37°C. The reaction was stopped by rinsing sections in PBS 0.05 M, pH 7.4.
Immunoperoxidase. Rabbit polyclonal antibody directed against mouse macrophage iNOS (Transduction Laboratories, Exeter, UK) and mouse monoclonal antibody against human ecNOS (Transduction Laboratories) were used. Both antibodies recognize rat NOS antigens [(58, 59) and Transduction Laboratories catalogue]. The specificity of anti-ecNOS and anti-iNOS antibodies was first verified by immunofluorescence. Human microvascular endothelial cells [SV-40 transfected immortalized endothelial cell line (HMEC) (1)], which constitutively express ecNOS but not iNOS, were grown on glass coverslips, fixed in 2% paraformaldehyde and stained with anti-ecNOS or anti-iNOS antibodies at different dilutions (anti-ecNOS, 1:150 and 1:300; anti-iNOS, 1:25 and 1:100) followed by secondary antibody [FITC-conjugated goat anti-mouse IgG 1:50, Jackson ImmunoResearch Laboratories, West Grove, PA, for ecNOS; and indocarbocyanine (Cy3) conjugated goat anti-rabbit IgG 1:100, Jackson ImmunoResearch Laboratories, for iNOS]. Fixed cells were permeabilized for 4 min with Triton X-100 0.1% before anti-iNOS staining. As positive controls, rat peritoneal macrophages were incubated for 18 h at 37°C with lipopolysaccharide (LPS) (20 µg/ml) (to upregulate iNOS) in culture dishes containing glass coverslips, then fixed and stained as described above. The slides were observed with an Olympus BH-2 epifluorescence microscope. Anti-ecNOS antibody stained HMEC at the optimal dilution of 1:150, while no signal was found on peritoneal macrophages. At 1:25 dilution anti-iNOS brightly stained rat peritoneal macrophages, but also HMEC, indicating a cross reaction with ecNOS. At 1:100 dilution the antibody still brightly stained macrophages without cross-reacting with ecNOS, as indicated by a very faint staining on HMEC.
Isoenzyme specificity of anti-iNOS and anti-ecNOS antibodies was confirmed by Western blot analysis of HMEC and rat macrophage extracts, following the manufacturer's instructions. On the basis of these results, in the immunoperoxidase studies described below anti-ecNOS and anti-iNOS antibodies were used at dilutions of 1:150 and 1:100, respectively. Three-µm paraffin sections from aortic tissue were processed for light microscopy immunohistochemistry by using an avidin-biotin horseradish peroxidase complex technique [avidin-biotin-complex (ABC) method, ABC-Elite, Vector Laboratories, Burlingame, CA] (2). Briefly, the sections were dewaxed, rehydrated, and incubated for 1 h with 0.3% H2O2 in methanol to quench endogenous peroxidase. Tissue was permeabilized in 0.1% Triton X-100 in PBS 0.01 M, pH 7.2, for 30 min and aspecificities were blocked by 30 min incubation with nonimmune sera (goat serum for anti-iNOS, horse serum for anti-ecNOS). All the above steps were carried out at room temperature. Slides were then incubated overnight at 4°C in a moist chamber with the primary antibody (anti-iNOS 1:100, anti-ecNOS 1:150) in PBS/1% BSA (Miles, Bayer, Milan, Italy), followed by the secondary antibody (biotinylated goat anti-rabbit IgG, or biotinylated horse anti-mouse IgG), ABC solution, and developed with diaminobenzidine as described. The sections were counterstained with Harris hematoxylin (Biooptica, Milan, Italy). Negative controls were obtained by omitting the primary antibody on a second section present on all the slides. The slides were observed under the light microscope by a pathologist blind to the nature of the experiment.Analysis of NADPH-diaphorase and immunohistochemical data. Multiple sections from each animal for two separate NADPH-diaphorase or immunoperoxidase reactions were examined by one investigator who was blinded to the identity of sample. Each section was scored for intensity of immunostaining and NADPH-diaphorase staining (absent, faint, moderate, intense, very intense: 0 through 4). These values were then compared with those in appropriate, concurrently run negative controls. At least 8-10 fields per section were examined. The modal value for each section was determined and the mean for each group calculated, as previously described (45).
Western blotting.
Western blots were performed as previously described (40).
After pulverization, frozen arteries were resuspended in 0.5 ml lysis
buffer (50 mM glicerolphosphate, 2 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 0.5% NP-40, 1 mM DTT, 1 mM pefabloc, 20 µM pepstatin, 20 µM leupeptin, 1,000 U/ml aprotinin) and sonicated. The
whole lysate was stored at
70°C. Protein concentration was determined by using the Bradford method (Bio-Rad). The proteins (20 µg for each lane) were separated on denaturating sodium dodecyl sulfate 7.5% polyacrylamide gel by electrophoresis and then blotted on
nitrocellulose membrane by wet electroblotting for 90 min. Blots were
blocked overnight at 4°C with 5% nonfat dry milk in Tris-buffered
saline (TBS) at pH 7.5 (20 mM Tris base, 137 mM NaCl, 0.1% Tween 20)
and then incubated for 2.5 h with anti-iNOS (1:1,000) or
anti-ecNOS (1:250) followed by the secondary antibody (biotinylated
goat anti-rabbit IgG, or biotinylated horse anti-mouse IgG), ABC
solution, and finally developed with diaminobenzidine (Vector Laboratories).
Urinary protein excretion. Proteinuria was determined in 24-h urines by the modified Coomassie blue G-dye binding assay, with BSA as standard (2).
Serum creatinine. Serum was obtained by leaving 1 ml portions of native blood, collected from the tail vein of anesthetized animals, at 37°C for 30 min. Creatinine was measured by the alkaline picrate method (2).
Bleeding time. Bleeding time was determined as previously described (52). Briefly, nonanesthetized rats were placed in a plastic cylinder with several openings, from one of which the tail emerged. Bleeding time was measured by using a standardized Simplate II device (General Diagnostic, Milan, Italy) and expressed in s from the moment the tail was incised until bleeding stopped completely (no rebleeding within 30 s).
Statistical analysis.
Results are expressed as means ± SE. Bleeding times,
NO2/NO3
plasma concentrations and
urinary excretion, plasma L-arginine, and the production of
[3H]L-citrulline in aortic tissue were
analyzed by the Mann-Whitney U-test, Wilcoxon's rank sum
test, or Kruskal-Wallis test, as appropriate. Data on NADPH-diaphorase
staining and ecNOS and iNOS staining were analyzed by the
Kruskal-Wallis test. The statistical level of significance was defined
as P < 0.05.
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RESULTS |
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Three months after surgery RMR rats had proteinuria (Table
1, P < 0.01 vs. CTR
rats) and renal insufficiency (Table 1, P < 0.01 vs.
CTR rats).
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Bleeding time of RMR rats was markedly longer than in sham-operated CTR
animals (Table 1, P < 0.01). Plasma concentrations of
the NO metabolites (NO2/NO3
) were
also significantly higher in RMR than control (CTR) rats (42.85 ± 7.34 vs. 17.60 ± 3.88 nmol/ml, P < 0.01, Fig.
1). At variance,
NO2
/NO3
urinary excretion was lower
in RMR than in CTR rats (1,857.8 ± 233.9, n = 6, vs. 5,370.3 ± 3,217.6 nmol/24 h, n = 6, P < 0.05), likely due to reduced renal synthesis of
NO, as previously reported (2).
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The effect of 17-estradiol on bleeding time of uremic rats is shown
in Fig. 2. 17
-estradiol iv
significantly shortened bleeding time within 24 h (before:
275 ± 15, 24 h: 157 ± 14 s, P < 0.01) without significant changes in renal function (24 h
17
-estradiol: 1.76 ± 0.22, 24 h vehicle: 1.77 ± 0.32 mg creatinine/100 ml serum), proteinuria (24 h 17
-estradiol:
287.67 ± 55.04, 24 h vehicle: 270.31 ± 82.72 mg/24 h),
and systolic blood pressure (24 h 17
-estradiol: 171 ± 3, 24 h vehicle: 174 ± 4 mmHg). In the same animals
17
-estradiol almost completely normalized plasma
NO2
/NO3
(before: 48.42 ± 14.50, 24 h: 17.47 ± 3.81 nmol/ml, P < 0.05, Fig. 3), while vehicle was
ineffective (before: 37.29 ± 3.77, 24 h: 41.31 ± 15.09 nmol/ml, Fig. 3). Urinary excretion of
NO2
/NO3
was slightly reduced by
17
-estradiol (before 1,857.8 ± 233.9, 24 h after
17
-estradiol: 1,354.2 ± 387.4 nmol/24 h, P = ns).
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The plasma concentration of the NO precursor
L-arginine was not significantly different in RMR and
CTR rats (CTR: 12.87 ± 2.19, RMR: 12.44 ± 0.82 µg/ml,
Fig. 4) (7). As shown in
Fig. 4, 17-estradiol numerically, but not significantly reduced
plasma L-arginine in RMR rats (before: 11.97 ± 0.82, 24 h: 9.28 ± 1.59 µg/ml, Fig. 4).
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To evaluate the effect of conjugated estrogens on vascular NOS activity
ex vivo, we measured the conversion of
[3H]L-arginine to
[3H]L-citrulline in aorta rings from RMR and
CTR rats incubated for 24 h in the presence of autologous plasma.
Aorta rings from RMR rats produced more
[3H]L-citrulline compared with CTR aortas
(RMR: 964 ± 150, n = 5, CTR: 475 ± 70 dpm
[3H]L-citrulline/mg tissue, n = 5, P < 0.05). Addition of 17-estradiol to RMR
aorta rings during the 24-h incubation period almost completely normalized [3H]L-citrulline production
(641 ± 181 dpm [3H]L-citrulline/mg
tissue, n = 5, P = ns vs. CTR aortas).
The effect of conjugated estrogens on vascular NOS activity in vivo was
evaluated by NADPH-diaphorase technique, which detects catalytic NOS
activity irrespective of the enzyme isoform. Specific NADPH-diaphorase staining was found on endothelial cells with few traces on the vessel
wall both in CTR and RMR rats. Endothelial staining appeared to be more
intense in the aorta section from vehicle-treated RMR rats than in CTR
rats. Semiquantitative analysis of thoracic aorta specimens showed that
in uremic rats given 17
-estradiol endothelial diaphorase staining
was significantly weaker than in vehicle-treated RMR rats
(P < 0.01) and even than in control rats
(P < 0.05, Table 2),
indicating that 17
-estradiol treatment reduced endothelial vascular
NOS activity in RMR rats.
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Expression of the endothelial and inducible isoforms of NOS was
evaluated on thoracic aorta of CTR and 17-estradiol or
vehicle-treated RMR rats, by using immunoperoxidase with specific
antibodies. Moderate to intense immunostaining for ecNOS and iNOS
(semiquantitative scores ranged from 1 to 3 for both), was evident
on the endothelium of thoracic aortas from vehicle-treated RMR
rats (Fig. 5, B and E, Table 2), and vascular smooth muscle cells showed weak,
focal staining. Expression of iNOS was very faint or absent in the
thoracic endothelium of CTR rats (Fig. 5D).
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Statistical analysis indicated that ecNOS and iNOS staining in the
endothelium of uremic aortas was significantly more intense than in CTR
rats (P < 0.05 for both ecNOS and iNOS, Fig. 5,
A, B, D, E, Table 2),
consistent with the diaphorase results. In RMR rats receiving
17-estradiol ecNOS and iNOS endothelial staining was normal
(P < 0.01 vs. RMR vehicle, Fig. 5, C and
F, Table 2). iNOS staining in vascular smooth muscle cells
did not differ in 17
-estradiol-treated and untreated uremic rats.
When the primary antibodies were omitted no staining was observed in adjacent sections in all experiments (Fig. 5, G and H).
Western blot analysis with anti-iNOS antibody of aorta homogenates from
RMR rats treated with vehicle showed an immunoreactive band at 130 kDa
(Fig. 6), by contrast no signal was found
in samples from RMR rats treated with 17-estradiol and from CTR rats
(Fig. 6), which is consistent with immunoperoxidase data. With
anti-ecNOS antibody a faint immunoreactive band was evident at 140 kDa
in aorta homogenates from RMR rats treated with vehicle and from CTR
rats, by contrast no band was found in samples from
17
-estradiol-treated animals (not shown).
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DISCUSSION |
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In this study uremic rats with prolonged bleeding time had higher
plasma concentrations of the NO metabolites
(NO2/NO3
) than CTR rats. These
findings are consistent with evidence obtained in experimental animals
(2, 52) and in humans (46), that bleeding
abnormalities in uremia were associated with excessive systemic NO synthesis.
We also showed that 17-estradiol significantly shortened the
bleeding time of uremic rats without significant changes in renal
function or proteinuria. In the same animals 17
-estradiol almost
completely normalized plasma
NO2
/NO3
. These results indicate
that estrogens normalize primary hemostasis in uremia (24, 35,
56, 61) by correcting the abnormalities in NO synthesis, in line
with previous reports that the NO precursor L-arginine
eliminated the effect of 17
-estradiol by shortening bleeding time in
uremic rats (66).
Estrogens can reduce the availability of L-arginine by
inducing the synthesis of ornithine decarboxylase (30, 48)
which by converting ornithine to putrescine, activates the enzyme
arginase, which degrades L-arginine in the urea cycle
(25). To test whether estrogens' effects on the NO
pathway in uremia were actually related to lowering
L-arginine, we measured L-arginine in RMR rats
before and after injection of 17-estradiol. 17
-estradiol did not
cause significant reduction in plasma L-arginine so it is
extremely unlikely that the effects of 17
-estradiol on primary
hemostasis and NO synthesis in RMR rats were dependent on
L-arginine.
Alternatively, estrogens may act on vascular NO synthesis. Relevant to
this possibility are in vitro data that physiological concentrations of
estrogens stimulate (5, 10, 21), and very high doses of
estrogens inhibit (6, 21, 27) NOS activity and NO release
in vascular endothelium. Here we used a histochemical approach, with
NADPH-diaphorase, which detects catalytic NOS activity in cells and
tissues irrespective of the enzyme isoform (4, 41, 59), to
check in vivo whether the fact that estrogens limited NO formation in
uremia was related to an inhibitory effect on endothelial NOS activity
in vessels. We found that 17-estradiol dramatically reduced
endothelial vascular NOS activity, and indeed endothelial diaphorase
staining was significantly weaker in uremic rats given 17
-estradiol
than in vehicle-treated RMR rats and even than in control rats.
To relate the changes in NOS catalytic activity to differences in NOS
isoenzyme expression, we evaluated ecNOS and iNOS protein expression in
the thoracic aorta of 17-estradiol or vehicle-treated RMR rats, by
immunohistochemical analysis. ecNOS and iNOS staining in the
endothelium of uremic aortas was significantly stronger than in control
rats. By contrast, in RMR rats given 17
-estradiol ecNOS and iNOS
endothelial staining was normal.
These data demonstrate that conjugated estrogens, at the doses needed
to correct primary hemostasis in experimental uremia, markedly reduce
vascular endothelial expression of ecNOS and iNOS protein, which may
explain the fact that these compounds limit excessive NO synthesis in
the systemic circulation. Although the effect of 17-estradiol on
ecNOS and iNOS expression in microvessels was not evaluated in this
present study, we observed that ecNOS and iNOS expression was lower in
vasa vasorum of aortas from rats receiving the 17
-estradiol compared
with untreated uremic rats (not shown), indicating that the drug
reduced endothelial NOS isoenzymes expression also in small vessels.
Surprisingly, 17-estradiol did not modify systolic blood pressure in
uremic rats, despite the impressive reduction of vascular NOS
isoenzymes. It is well known, however, that estrogens are potent
inhibitors of several vasoconstrictor systems, and they inhibit the
release of endothelin by endothelial cells (3), reduce
plasma angiotensin converting enzyme activity (8), and blunt the vascular pressor response to phenylephrine (23).
It is tempting to speculate that in RMR the inhibitory effect of 17
-estradiol on vasoconstrictors might have counterbalanced the increase in vascular tone due to reduction of vascular NO.
Endothelial cells of bovine (21) and rabbit (12) origin appear to have estrogen receptors. In human endothelial cells estrogen receptors were found in cultures from umbilical vein (21) and in aorta (9). Although the ecNOS promoter does not contain a canonical estrogen cis-regulatory element, the left palindromic sites of an estrogen responsive element (GGTCA) and the right half sites (TGACC) were identified in bovine (60) and human (39) ecNOS genes which might be one mechanism by which estrogens regulate ecNOS gene expression.
That this may be the case is confirmed by a recent report by Hall et
al. (20) who studied the effect of 17-estradiol on transcription of the human ecNOS gene, by using transient transfections of a series of luciferase/reporter constructs, nuclear run-off, and
Northern blot analysis. The maximally active construct for human ecNOS
in endothelial cells and minimal Sp-1-dependent core promoter was
markedly repressed by 17
-estradiol at concentrations higher than 1 nM. Thus the 5' half partial sequences may have functional roles as
estrogen responsive elements in ecNOS.
Previous findings on how estrogens affect ecNOS are controversial
(21, 27) and recent observations showed increased
expression and/or activity of ecNOS in 17-estradiol-treated cultured
endothelial cells (11, 21, 31, 32, 36). Similarly,
elevated plasma levels of NO2
/NO3
were found in postmenopausal women given 17
-estradiol as hormone replacement therapy (5, 55). It was suggested that
physiological amounts of estrogens stimulate NO production by vascular
endothelium which may contribute to their beneficial cardiovascular
actions (13, 34, 64), although alternative, not
NO-dependent mechanisms have also been proposed (16, 64).
One possible explanation for the apparent inconsistency between the
above studies and our present data is based on two observations: 1) the
dose of conjugated estrogens necessary to improve primary hemostasis
and reduce clinical bleeding in uremia in experimental animals
(62, 67) and humans (35, 56, 61) is
approximately fifty times the dose conventionally prescribed for
contraception (44), postmenopausal replacement
(15), or prevention of osteoporosis (44); 2)
as shown by Hall et al. (20), transcriptional regulation of ecNOS by 17-estradiol has a biphasic response: limited amounts of
17
-estradiol enhanced, while increasing amounts of ligand inhibited
ecNOS transcription in endothelial cells. One can therefore reasonably
assume that the effect of estrogens on vascular ecNOS expression are
complex and depend very much on the concentration of the hormone:
physiologic amounts enhance vascular endothelial ecNOS expression
whereas pharmacological doses, as high as the ones needed to improve
primary hemostasis and correct bleeding time, inhibit the expression of
vascular ecNOS. This possibility is consistent with in vivo findings in
rats (6) that low-dose estrogens increased vascular NO
release, while high doses significantly reduced basal release of NO and
acetylcholine-induced relaxation.
One can speculate on the possible mechanism of the effect of
17-estradiol on iNOS taking into account the fact that estrogens share with glucocorticoids a steroidal tridimensional structure, a
receptor-mediated mechanism of action and certain biological activities
(30, 44 ,48) including the capacity to reduce uremic bleeding (66). Recent studies in macrophages
(43) and endothelial cells in vitro (50), and
in vivo in endotoxin-treated rats (51), have shown that
dexamethasone potently inhibits iNOS expression by blocking the
transcription of the enzyme. Therefore 17
-estradiol may control iNOS
content in uremic vessels by a similar mechanism. This is consistent
with recent findings that 17
-estradiol in vitro inhibited
LPS-stimulated iNOS expression and NO production in a murine macrophage
cell line (22) and in rat alveolar macrophages (54). An estrogen receptor antagonist blocked the effect
of 17
-estradiol on iNOS (22), indicating that
17
-estradiol inhibited the induction of iNOS by a classic
receptor-mediated mechanism. In another study estradiol reduced iNOS
expression and NO production in rat aortic endothelial cells stimulated
with interleukin-1
(65). The effect was evident at
doses as low as 0.1 nM, indicating that, at variance with ecNOS,
estrogens inhibit iNOS expression also at physiological concentrations.
Consistently, in isolated rat aorta rings, 17
-estradiol caused
concentration-dependent inhibition of interleukin-1
-induced iNOS
expression and NO production and restored vasoconstriction
responsiveness (28). In addition, Kauser et al. showed
that administration of 17
-estradiol in vivo to ovariectomized rats
attenuated the endotoxin-induced elevation of plasma nitrite levels
(29), demonstrating that 17
-estradiol can inhibit
excessive NO production by iNOS in vivo.
Like estrogens, the hemostatic effect of dexamethasone in uremic rats
was eliminated by L-arginine (66) which
further suggests that the two compounds have similar mechanisms of
action. Because of a Km of around 30 µM, the
iNOS isoenzyme is totally dependent on the availability of
extracellular L-arginine (38, 42). Presumably
the addition of the NO precursor in uremic rats overcomes the action of
17-estradiol and dexamethasone of normalizing iNOS expression in
vascular endothelium, by maximizing the catalytic activity of residual
iNOS molecules either in the endothelium or in smooth muscle cells.
In conclusion, in uremic rats 17-estradiol, at a dose that
normalizes the prolonged bleeding time, fully corrects the excessive formation of NO by markedly reducing the expression of both ecNOS and
iNOS in vascular endothelium. These results provide a possible biochemical explanation for the well-known effect of estrogens on
primary hemostasis in uremics. Further studies are needed to provide
definitive prove of the cause-and effect relationship between the
activity of estrogens on NO biology and uremic bleeding and to sort out
the relative importance of ecNOS vs. iNOS and to sort out the relative
contribution of iNOS and ecNOS on uremic bleeding.
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ACKNOWLEDGEMENTS |
---|
The authors thank Mrs. Judy Baggott for editing the manuscript.
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FOOTNOTES |
---|
Part of this paper was presented as an abstract at the 31st Annual Meeting of the American Society of Nephrology (October 25-27, 1998, Philadelphia, PA). Sergio Zappella is a recipient of a fellowship from "Fondazione A., A. Valenti." Samantha Bonazzola is a fellow of the Rotary Club Bergamo Nord.
Address for reprint requests and other correspondence: M. Noris, Mario Negri Institute for Pharmacological Research, Via Gavazzeni, 11, 24125 Bergamo, Italy (E-mail, noris{at}irfmn.mnegri.it).
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.
Received 22 June 1999; accepted in final form 24 May 2000.
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