(Hypertension. 2001;37:645.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Effects of Estradiol and Its Metabolites on Glomerular Endothelial Nitric Oxide Synthesis and Mesangial Cell Growth
From the Center for Clinical Pharmacology, Departments of Medicine (D.G.G., E.K.J., R.K.D.) and Pharmacology (E.K.J.), University of Pittsburgh, Penn; Department of Physiology (S.X., C.B.), West Virginia University, Morgantown; and Clinic for Endocrinology, Department of Obstetrics and Gynecology (R.K.D.), University Hospital Zurich, Switzerland.
Correspondence to Dr Raghvendra K. Dubey, D217, NORD-1, Clinic for Endocrinology, Department of Obstetrics and Gynecology, Frauenklinik, Zurich 8091, Switzerland. E-mail rag{at}fhk.usz.ch
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Abstract |
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Reduced nitric oxide synthesis by glomerular endothelial cells and increased proliferation of glomerular mesangial cells is associated with glomerular remodeling that leads to accelerated glomerulosclerosis. Estradiol induces nitric oxide synthesis and slows the progression of renal disease. Because the estradiol metabolites 2-hydroxyestradiol and 2-methoxyestradiol are more potent than estradiol in inhibiting growth of vascular smooth muscle cells, which are phenotypically similar to mesangial cells, we compared the effects of estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol on growth of glomerular mesangial cells and on basal nitric oxide synthesis by glomerular endothelial cells. In human glomerular mesangial cells, estradiol and its metabolites concentration-dependently (1 nmol/L to 10 µmol/L) inhibited serum (2.5%)-induced DNA synthesis, cell proliferation, and collagen synthesis with the order of potency being 2-methoxyestradiol > 2-hydroxyestradiol > estradiol. ICI182780 (100 µmol/L, an estrogen receptor antagonist) blocked the growth inhibitory effects of estradiol but not 2-hydroxyestradiol or 2-methoxyestradiol. Treatment with estradiol, but not 2-hydroxyestradiol and 2-methoxyestradiol, induced nitric oxide synthesis (P<0.05, assayed by the formation of 3H-L-citrulline from 3H-L-arginine) in human glomerular endothelial cells, and these effects were blocked by ICI182780 and L-NMA (a nitric oxide synthesis inhibitor). In conclusion, estradiol may attenuate glomerulosclerosis by inducing nitric oxide synthesis via an estrogen receptor–dependent mechanism and by conversion to 2-hydroxyestradiol and 2-methoxyestradiol, which inhibit glomerular mesangial cell proliferation independent of estrogen receptors.
Key Words: 2-hydroxyestradiol • nitric oxide • 2-methoxyestradiol • glomerulosclerosis • renal disease, end-stage • postmenopause
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Introduction |
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Estradiol may induce protective effects on the kidney. Compared with age-matched men, the rate of progression of renal disease in premenopausal women is decreased.1 2 However, with the onset of menopause and decreased synthesis of 17ß-estradiol (estradiol), the progression of renal diseases accelerates, and estradiol replacement therapy slows this process.1 2 3
Although estradiol induces protective effects on the kidney, the mechanisms via which it induces its effects remain undefined. Estradiol is known to protect the vasculature by inhibiting processes that initiate or mediate vascular remodeling associated with neointima formation and the vaso-occlusive process.1 In this regard, estradiol stimulates endothelial cell–derived nitric oxide (NO) synthesis and inhibits vascular smooth muscle cell growth. Analogous to the vasculature, decreased NO synthesis by damaged and dysfunctional glomerular endothelial cells (GECs)4 and abnormal growth of glomerular mesangial cells (GMCs), a cell phenotypically similar to smooth muscle cells, is associated with the pathogenesis of renal diseases, such as glomerulosclerosis.4 5 It is feasible that, similar to the effects on the vasculature, estradiol may protect against glomerulosclerosis and reduce the rate of progression of renal disease by stimulating NO synthesis by GECs and inhibiting growth of GMCs.
In the present study, we investigated whether estradiol stimulates NO synthesis in human GECs and inhibits growth of GMCs. Although some of the biological effects of estradiol are estrogen receptor (ER) mediated,6 recent findings suggest that ER-independent effects may also play a role in mediating the protective effects of estradiol.7 8 Because 2-hydroxyestradiol and 2-methoxyestradiol, major endogenous metabolites of estradiol with no affinity for ERs,9 possess biological activity and are more potent than estradiol in inhibiting vascular smooth muscle cell growth,10 we also investigated the effects of estradiol metabolites on NO synthesis by human GECs and on growth of GMCs. Moreover, using ICI182780, an ER antagonist,1 we investigated the role of ERs in mediating the effects of estradiol and its metabolites. Because estradiol is used in combination with progestins, we also evaluated how the effects of estradiol on NO synthesis and GMC growth are influenced by natural and synthetic progestins.
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Methods |
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Endothelial and Mesangial Cell Culture
Human GECs and GMCs were obtained from Cell System Corporation (Kirkland, Wash). Cells were cultured in multiwell plates under standard cell culture conditions. GECs (passage 4 to 7) were maintained in endothelium growth medium (EGM), and GMCs (passage 3) were grown in phenol red-free DMEM/F12 medium supplemented with 10% FCS (steroid free) and antibiotics.
Determination of Nitric Oxide Synthase
Activity
Confluent monolayers of GECs were fed EGM
supplemented with charcoal-stripped FCS (steroid free) that contained
or lacked the following treatments and were dissolved in sterile DMSO
or H2O: estradiol, 2-hydroxyestradiol,
2-methoxyestradiol, estradiol plus ICI182780, 2-hydroxyestradiol plus
ICI182780, 2-methoxyestradiol plus ICI182780, estradiol plus L-NMA,
estradiol plus EDTA, and estradiol plus progesterone or
medroxyprogesterone. The controls were treated with
an equal volume of vehicle (DMSO, 0.1% vol/vol). After 1 hour or 24
hours, NOS activity was determined by measuring the conversion of
L-[3H]arginine
to
L-[3H]citrulline
according to the method of Davda et
al,11 with minor
modifications. Briefly, after 1 or 24 hours of treatment, the medium
was removed and cells were incubated with 0.5 mL Krebs-HEPES buffer
that contained (in mmol/L): 131 NaCl, 5.5 KCl, 2.5
CaCl2, 1.0 MgCl2, 25.0
NaHCO3, 1.0
Na2HPO4, 5.5 D-glucose,
20.0 HEPES and 0.05 L-arginine and 2 µCi
L-[3H]arginine
for 1 hour at 37°C. The experiment was terminated by removing the
medium and rapidly washing the cells 3 times with ice-cold phosphate
buffer solution (PBS) that contained 10 mmol/L unlabeled
L-arginine. Cells were
solubilized in 0.5 mL 1% Triton X-100, and 50-µL aliquots were taken
for determination of total uptake of
L-[3H]arginine
in a scintillation counter. A portion of the cell
homogenate (0.3 mL) was added to 0.7 mL of 50% Dowex 50WX8
(Na+ form) to remove unconverted
L-[3H]arginine.
These samples were vortex mixed for 3 minutes, centrifuged at
2000g for 2 minutes, and the radioactivity of an aliquot (0.5 mL) of
the supernatant was measured in a liquid scintillation counter to give
the activity due to
L-[3H]citrulline.
This method of separating
L-[3H]arginine
and
L-[3H]citrulline
has previously been validated by HPLC and thin-layer
chromatography. The background radioactivity was
determined by the addition of
L-[3H]arginine
to the cell medium, which was immediately removed and the cells
solubilized. To validate our technique, we initially measured NOS
activity in the GEC in response to several known agonists and
antagonists, ie, bradykinin, calcium ionophore (A23187),
IL-1ß+IFN
+LPS, dexamethasone, and L-NMA. Each
experiment was repeated at least 3 times. NOS activity, expressed as
picomoles of arginine converted to citrulline/min per milligram of
protein was calculated as follows: NOS activity (pmol/min per milligram
protein)={[(Radioactivity of
L-citrulline-background)÷total
radioactivity in assay buffer]xtotal
L-arginine concentration in
assay buffer÷protein content per well}/incubation
time.
Growth Studies
DNA and collagen synthesis and cell
proliferation studies were performed under the following treatments:
(1) estradiol; (2) estradiol plus ICI182780; (3) estradiol plus
progesterone; (4) estradiol plus
medroxyprogesterone (MPA, a synthetic progestin);
(5) 2-hydroxyestradiol; (6) 2-hydroxyestradiol plus ICI182780; (7)
2-methoxyestradiol; (8) 2-methoxyestradiol plus ICI182780; (9)
2-hydroxyestradiol plus progesterone; (10) 2-methoxyestradiol plus
progesterone; (11) progesterone, and (12) ICI182780. All the test
agents were dissolved in DMSO; for comparisons, the controls in each
experiment were treated with DMSO (0.1% final
concentration).
Incorporation studies of 3H-thymidine and 3H-proline were performed as a measure of DNA and collagen synthesis, respectively. For DNA synthesis studies, GMCs were grown to subconfluence. For collagen synthesis studies, GMCs were grown to confluence, and cell counting was performed in cells treated in parallel to the cells used for the collagen synthesis, and the data were normalized to cell number. Monolayers of GMCs were growth arrested by feeding DMEM that contained 0.4% bovine serum albumin (BSA) for 48 hours. Growth was stimulated by treating growth-arrested GMCs with DMEM supplemented with 2.5% FCS and containing or lacking the various treatments as described above. For DNA synthesis, after 20 hours of incubation, the cells were pulsed with 3H-thymidine (1 µCi/mL) for an additional 4 hours. For collagen synthesis, cells were treated for 36 hours in the presence of 3H-L-proline (1 µCi/mL). The experiments were terminated by washing cells twice with PBS and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized in 500 µL of 0.3 N NaOH and 0.1% SDS after incubation at 50°C for 2 hours. Aliquots from 4 wells for each treatment with 10 mL of scintillation fluid were counted in a liquid scintillation counter, and each experiment was conducted using 4 separate cultures. 3H-thymidine studies were conducted in subconfluent monolayers.
Cell counting was performed as a direct measure of cell proliferation. Trypsinized GMCs were suspended in DMEM/F12 that contained 10% FCS and plated in a 24-well culture dish at a density of 1x104 cells/well. After incubation for 18 hours, the cells were fed DMEM that contained 0.4% BSA for 48 hours to growth arrest the cells. GMCs were then treated every 24 hours for 4 or 8 days with DMEM supplemented with 2.5% FCS and containing or lacking various treatments as described above. The treatments were terminated on day 5 or 9, and the cells were dislodged with trypsin-EDTA, diluted in Isoton-II, and counted with a Coulter counter. Aliquots from 3 wells were counted for each group, with 4 separate cultures. The total cellular protein was determined by the Bio-Rad detergent method, which uses a modification of the Lowry assay with BSA as a standard.
Statistics
Results are expressed as mean±SEM. Statistical
analysis was performed with ANOVA, Student’s
t test, or Fishers least
significant difference test, as appropriate. Values of
P<0.05 were considered to be
significantly different.
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Results |
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Treatment of GECs with estradiol induced NOS activity in a concentration-dependent fashion (Figure 1). Physiological concentrations (10-10 mol/L) of estradiol induced NOS activity in GECs from 16.8 to 24.6 pmol/min per milligram protein. The lowest concentration of estradiol that significantly induced NOS activity was 10-12 mol/L. In GECs treated with estradiol (10-10 mol/L) for 1 hour and 24 hours, the NOS activity was stimulated by 19% and 48%, respectively, indicating that the stimulatory effects of estradiol were dependent on the time of exposure. Basal as well as estradiol-stimulated NOS activity was blocked by L-NMA (Figure 1). Neither 2-hydroxyestradiol nor 2-methoxyestradiol altered NOS activity (Figure 1). The stimulatory effects of estradiol on NOS activity were blocked in a concentration-dependent manner by ICI182780 (Figure 2, top ). Moreover, the effects of 2-hydroxyestradiol and 2-methoxyestradiol on NOS activity remained unaltered in the presence of ICI182780 (Figure 2, bottom). The stimulatory effects of estradiol on NOS activity were significantly abrogated by both progesterone and medroxyprogesterone (Figure 3).
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In GMCs, FCS stimulated
3H-thymidine and
3H-proline incorporation and cell number by
5- to 7-fold (P<0.05).
Estradiol inhibited FCS-induced 3H-thymidine
incorporation in a concentration-dependent manner
(Figure 4). In this regard, significant inhibition occurred
even with physiological concentrations of estradiol
(1 nmol/L), and a 50% decrease was observed at 3 to 4 µmol/L of
estradiol
(Figure 4). Estradiol also inhibited FCS-induced
3H-proline incorporation and cell number in
a concentration-dependent fashion
(Figures 5 and 6, respectively). The lowest concentration of
estradiol that significantly inhibited FCS-induced increases in cell
number and proline incorporation was 1 nmol/L, and this concentration
inhibited cell number by 23±3% and proline incorporation by
12.6±2.6%. The inhibitory effects of estradiol on
FCS-induced 3H-thymidine and
3H-proline incorporation and cell number
were completely blocked by ICI182780 (100 µmol/L).
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Regarding the inhibition of FCS-induced DNA synthesis (Figure 4), collagen synthesis (Figure 4), and cell proliferation (Figure 5), 2-hydroxyestradiol and 2-methoxyestradiol were more potent than estradiol. The order of potency was 2-methoxyestradiol > 2-hydroxyestradiol > estradiol. At physiological concentrations (1 nmol/L), estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol inhibited cell proliferation by 23%, 31%, and 37%, respectively. A 50% decrease in proline incorporation in GMCs by estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol was observed at 8 µmol/L, 0.15 µmol/L, and 0.03 µmol/L, respectively. The inhibitory effects of estradiol (1 nmol/L) on thymidine incorporation (Figure 6A), proline incorporation (Figure 6B), and cell proliferation (Figure 6C) were completely reversed by ICI182780 (100 µmol/L; Figure 6 A through C). In contrast, the inhibitory effects of 2-hydroxyestradiol (1 nmol/L) and 2-methoxyestradiol (1 nmol/L) on FCS-induced thymidine incorporation, proline incorporation, and cell proliferation were not blocked by ICI182780 (100 µmol/L; Figure 6 A through C).
Treatment of GMCs for 4 days with 100 nmol/L progesterone alone inhibited 2.5% FCS-induced cell proliferation by 16%, and similar inhibitory effects were observed in GMCs treated with 100 nmol/L medroxyprogesterone (Figure 6D, right panel). The inhibitory effects of 1 nmol/L estradiol on GMC growth were reduced from 24% to 19% by progesterone and to 18% by MPA; however, these decreases did not attain statistical significance (Figure 6D, left panel).
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Discussion |
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Glomerular remodeling associated with glomerulosclerosis and glomerular injury is a complex process that involves dysfunction/damage to GECs and abnormal growth of GMCs.4 5 The abnormal growth processes that lead to the dynamic changes in glomerular structure involve hypertrophic/hyperplastic growth of GMCs and modulation of the amount of extracellular matrix proteins, such as collagen.4 Under normal conditions, GECs play a critical role in maintaining homeostasis by generating a battery of both growth-inhibitory and growth-stimulatory factors, as well as relaxing and contracting factors.1 4 5 Moreover, endothelial damage or dysfunction triggers a cascade of events that often lead to increased GMC proliferation and extracellular matrix synthesis.4 5 In this regard, decreased synthesis of nitric oxide by damaged/dysfunctional GECs as well as abnormal growth of GMCs is associated with glomerulosclerosis.5 Estradiol stimulates recovery and NO synthesis in damaged/dysfunctional vascular endothelial cells1 and inhibits mitogen as well as injury-induced growth of vascular smooth muscle cells.7 8 12 Because GECs and GMCs are phenotypically similar to vascular endothelial and smooth muscle cells, respectively,4 we hypothesize that the protective effects of estradiol against the progression of renal disease in females may in part be mediated by its stimulatory effects on NO synthesis by GECs and its inhibitory effects on GMC growth. Moreover, because endogenous metabolites of estradiol (methoxyestradiols) with no binding affinity for ERs inhibit growth of vascular smooth muscle cells,10 13 a cell phenotypically similar to GMCs, we hypothesize that estradiol metabolites may prevent glomerulosclerosis by inhibiting abnormal growth of GMCs.
The findings of the present study demonstrate that estradiol upregulates the synthesis of NO by GECs and that the stimulatory effects of estradiol on NO synthesis are blocked by ICI182780, an ER antagonist, suggesting that the stimulatory effects of estradiol on NO synthesis are ER mediated. In contrast to estradiol, the major active endogenous metabolites of estradiol (2-hydroxyestradiol and 2-methoxyestradiol) do not influence basal NO synthesis by GECs. Moreover, progesterone and medroxyprogesterone (a synthetic progestin used in combination with estradiol for hormone replacement therapy14 ) abrogated the stimulatory effects of estradiol on NO synthesis.
We also demonstrate that estradiol inhibits GMC DNA and collagen synthesis and GMC proliferation. In contrast to the effects on NO synthesis, the inhibitory effects of estradiol on GMCs are mimicked by 2-hydroxyestradiol and 2-methoxyestradiol. The relative potencies of estradiol and its metabolites to inhibit GMC growth are 2-methoxyestradiol > 2-hydroxyestradiol > estradiol, and this relative potency does not match their relative affinities for ERs.13 Progesterone also inhibited GMC growth and attenuated the inhibitory effects of estradiol. The inhibitory effects of estradiol, but not 2-hydroxyestradiol and 2-methoxyestradiol, on GMC growth are blocked by ICI182780, a specific ER antagonist. Taken together, our findings provide the first evidence that estradiol induces NO synthesis in GECs via an ER-dependent mechanism. Moreover, estradiol and its endogeous metabolites inhibit GMC growth, and these antimitigenic effects are mediated via ER-dependent and ER-independent mechanisms, respectively.
The finding that physiological concentrations of estradiol induce NOS activity in GECs by almost 50% suggests that estradiol may protect against glomerular remodeling processes associated with glomerulosclerosis. This contention is supported by the fact that inhibition of NO leads to glomerular injury and glomerulosclerosis.1 5 Indeed, in the kidney, NO generated by endothelial constitutive NOS participates in physiological regulation of glomerular hemodynamics by modifying the tone of afferent arterioles and mesangial cells and maintaining glomerular capillary blood pressure, glomerular plasma flow, and the glomerular ultrafiltration.5 NO synthesized under basal conditions has multiple antiglomerulosclerotic effects; for example, NO contributes to the antithrombogenic properties of the endothelium by preventing platelet aggregation and adhesion; improves the barrier function of endothelial cells within capillaries, thereby preventing glomerular monocyte/macrophage infiltration; and inhibits proliferation and extracellular matrix synthesis in GMCs.4 5 The fact that estradiol reduces the rate of progression of end-stage renal disease15 and the finding that NO synthesis is reduced in patients with end-stage renal disease16 suggests that estradiol may induce its protective effects by upregulating NOS activity.
The stimulatory effects of estradiol on NOS activity are
blocked by ICI182780, which suggests that GECs possess functional ERs.
Although human kidneys express both ER
and
ERß,1 whether these
receptors are expressed in the GECs is not known. Therefore, the
present findings do not allow deductions in regard to the relative
importance of ER versus ERß in mediating the stimulatory effects
of estradiol on NOS activity. Additionally, recent findings that
estradiol induces NO synthesis acutely in vascular
endothelial cells via a nongenomic mechanism that
involves ER localized within the cell
membrane17 suggest that
estradiol may similarly induce NOS activity in GECs.
Analogous to the vascular remodeling process in atherosclerosis, abnormal growth of GMCs after capillary or GEC damage/dysfunction contributes to the glomerular remodeling process associated with glomerulosclerosis.4 5 The abnormal growth process involves increased proliferation of GMCs and increased production and deposition of extracellular matrix proteins, such as collagen. Moreover, multiple autocrine/paracrine/endocrine factors are involved in inducing the structural changes in the glomeruli.4 5 Because FCS contains a battery of growth factors that may contribute to the remodeling process, we thought it important to evaluate the effects of estradiol and its metabolites on FCS-induced growth of GMCs to elucidate the growth regulatory effects of estrogens under more physiological conditions. The finding that estradiol, its metabolites, and progesterone inhibit FCS-induced GMC growth provides evidence that these hormones are important modulators of GMC growth.
Physiological concentrations (1 nmol/L) of estradiol inhibit cell proliferation and collagen synthesis by 13% and 11%, respectively. Moreover, 1 nmol/L of 2-hydroxyestradiol and 2-methoxyestradiol inhibit cell proliferation and collagen synthesis by 19% and 17%, respectively, and by 32% and 25%, respectively. This suggests that physiological concentrations of estradiol inhibit GMC growth and collagen synthesis and that the inhibitory effects of estradiol in vivo may be considerably higher owing to the presence of estradiol metabolites. In addition, our findings suggest that the protective effects of estradiol on the kidney may vary and be dependent on the metabolic capability of the individual.
The stimulatory effects of estradiol on NOS activity in GECs are inhibited by progesterone and medroxyprogesterone, a synthetic progestin.14 This finding is consistent with our previous observation that in postmenopausal women receiving hormone replacement therapy, the circulating NO levels are increased during treatment with estradiol alone but not during treatment with estradiol plus medroxyprogesterone.18 In contrast, the inhibitory effects of estradiol on GMC growth are not significantly influenced by progesterone and medroxyprogesterone. Thus, progestins may reduce the beneficial effects of estradiol mediated by NO generated by GECs, but progestins may not attenuate the beneficial effects of estradiol mediated by inhibition of GMC growth. Because combined administration of a progestin with an estrogen is currently the preferred method of hormone replacement therapy in nonhysterectomized postmenopausal women, our findings suggest that progestins may reduce the full benefits of estradiol within the glomerulus.
In summary, our findings indicate that estradiol, but not its metabolites, induce NOS activity in GECs and that these effects are ER-mediated. The effects of estradiol on NOS activity are abrogated by progesterone (natural progestin) and medroxyprogesterone (synthetic progestin). The inhibitory effects of estradiol on GMC growth and collagen synthesis are mimicked by 2-hydroxyestradiol and 2-methoxyestradiol, endogenous metabolites of estradiol with no binding affinity to ERs. The antimitogenic effects of estradiol, but not 2-hydroxyestradiol and 2-methoxyestardiol, are blocked by ICI182780. Together our findings indicate that estradiol may protect against the progression of renal disease by inducing NO synthesis in GECs and inhibiting GMC growth and that both ER-dependent and ER-independent pathways may participate in mediating these effects.
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Acknowledgments |
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This study was supported by the Swiss National Science Foundation (grants 32-54172.98 and 32-64040.00); by the National Institutes of Health (grants HL-35909 and HL-5314); and by an unconditional educational grant from Schering (Schweiz AG).
Received October 25, 2000; first decision December 11, 2000; accepted December 19, 2000.
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