Impact of Dietary Na+ on Glycyrrhetinic Acid-Like Factors (Kidney 11β-(HSD2)-GALFs) in Human Essential Hypertension
Our previous studies have shown that human urine contains glycyrrhetinic acid-like factors (GALFs) that possess inhibitory activity against kidney 11β-hydroxysteroid dehydrogenase isoform 2 (HSD2). The present studies were undertaken to determine the impact of dietary Na+ intake on the levels of kidney 11β(HSD2)-GALFs. The excretion of kidney 11β(HSD2)-GALFs in 24-hour urine samples of 30 unmedicated subjects (10 normotensive and 10 high/normal-renin and 10 low-renin essential hypertensive subjects) on both 200- and 10-mmol Na+ diets was studied. No differences in the urinary levels of kidney 11β(HSD2)-GALFs were observed among the three groups on the high-Na+ diet. However, with a low-Na+ diet, the levels of kidney 11β(HSD2)-GALFs were significantly increased in hypertensive subjects but not in normal subjects. Levels increased from 8.3±1.4 to 17.3±2.9 and 6.7±1.3 to 10.6±1.4 carbenoxolone sodium units/d in high/normal-renin (P=.01) and low-renin hypertensive subjects (P=.07), respectively; normal subjects changed from 8.0±1.9 to 10.6±2.4. The levels of kidney 11β(HSD2)-GALFs were significantly higher in the high/normal-renin hypertensive subjects than in either the control normotensive subjects or the low-renin hypertensive subjects when challenged with the low-Na+ diet (P<.05 by Wilcoxon rank-sum test). The greater response of the high/normal-renin hypertensive subjects indicated that they may utilize kidney 11β(HSD2)-GALFs when challenged with a low-Na+ diet, whereas the low-renin essential hypertensive subjects do not.
- 11 β-hydroxysteroid dehydrogenase
- glycyrrhetinic acid
- glycyrrhetinic acid-like factors
- CBX = carbenoxolone sodium
- GALF = glycyrrhetinic acid-like factor
- HSD1, HSD2 = hydroxysteroid dehydrogenase isoform 1, isoform 2
- MR = mineralocorticoid receptor
Our earlier experiments had suggested the presence of GALFs in human urine that by inhibiting the steroid-inactivating enzyme 11β-HSD1 permit glucocorticoids to act as mineralocorticoids and augment overall Na+ retention. Subsequent studies confirmed that human urine also contains 11β(HSD2)-GALF inhibitory substances and that these likewise are elevated in pregnancy.2 Edwards et al3 and Funder et al4 proposed that in vivo renal MRs display apparent aldosterone-specificity because the enzyme 11β-HSD metabolizes cortisol and corticosterone unidirectionally to cortisone and 11-dehydrocorticosterone, respectively. These 11-dehydro products, which have low binding affinities for MRs,4,5⇓ do not elicit mineralocorticoid-like effects. After the identification of 11β-HSD2 in MR containing cortical-collecting duct cells in kidney,6 the 11β-HSD isoform 2 of this enzyme was cloned and shown to be the major isoform of 11β-HSD in sheep and human kidney.7,8⇓
Shortly thereafter, the hypertensive children with the syndrome of apparent mineralocorticoid excess who display excessive Na+ retention, K+ wasting, and markedly increased blood pressure together with altered peripheral metabolism of the glucocorticoid cortisol9–11⇓⇓ were shown to exhibit mutations of this unidirectional NAD-dependent, low Km for corticosterone (≈10 to 20 nmol/L) 11β-HSD2.12,13⇓ These and more recent findings14 have offered considerable evidence supporting the “guardian” role of the renal enzyme 11β-HSD2, which confers mineralocorticoid specificity on MR-mediated mechanisms of Na+ handling. The role of 11β(HSD1)-GALF inhibitory substance is less clear at this time.
To explore the possible interaction of GALFs and Na+ homeostasis and blood pressure, we have now measured kidney 11β(HSD2)-GALF inhibitory substances in normotensive and essential hypertensive subjects and measured the impact of low dietary Na+ intake on the levels in these individuals.
The patients were enrolled at the Clinical Research Centers of the Brigham and Women’s Hospital in Boston, Mass. The study was reviewed and approved by the institutional review board, and all patients gave informed written consent before enrollment.
Subjects were not preselected for the study. Hypertension was defined as a history of hypertension with a diastolic blood pressure ≥100 mm Hg with no medications, a diastolic blood pressure ≥90 mm Hg with one antihypertensive agent, or the need for two or more antihypertensive medications at the time of the screening visit. A group of normotensive subjects served as control subjects. Secondary forms of hypertension were excluded by history and physical examination, and, when indicated, biochemical testing. All antihypertensive medications were discontinued at least 2 weeks before the study. Exclusion criteria included diabetes mellitus, obesity (body mass index >33 for men and >31 for women), renal insufficiency, or other significant medical problems.
Each subject consumed high (200 mmol/d)-low (10 mmol/d)-sodium diets which were prepared in the metabolic kitchens of each of the study sites and then consumed in the outpatient setting. Dietary compliance was ensured by measurement of urinary sodium and creatinine in a 24-hour urine collection after at least 3 days of consuming the high-salt diet and 7 days for the low-salt diet. The high-salt diet was consumed first, and immediately followed by the low-salt diet for 7 days. Twenty-four-hour urine collections were obtained from 20 white patients with essential hypertension and 10 normotensive white subjects who met the above criteria.
Ten of the essential hypertensive patients (group 2) had high or normal plasma renin values (>2.4 ng/ml per hour) and 10 (group 3) had low plasma renin values (<2.4 ng/ml per hour). Samples for renin assays were obtained from all subjects after 60 minutes of upright posture following overnight recumbency, after 7 days of the low-Na+ diet (10 mmol/d). Assays were performed as previously described.15
Patients were admitted on the last day of each diet to enable standardization of blood pressure measurement. Blood pressure was measured using a Dinamap blood pressure monitor (Critikon Inc) with the patient in the supine position after overnight recumbency and in the fasting state. Blood pressure was reported as the mean of three separate measurements taken at least 5 minutes apart.
Urine was stored without preservatives or additives at −20°C until assay. A commercial kit was used to measure plasma renin activity (Incstar Corporation). Whole blood was collected in EDTA and spun at room temperature, and the plasma was quick-frozen and stored at −20°C until assay. Sodium and potassium in urine were measured by direct potentiometry with an ion-selective electrode (NOVA analyzer I, Nova Biochemical). Creatinine was measured in urine on a Beckman creatinine analyzer (model II).
Preparation of Urine Extracts
Urine samples were desalted and partially purified by the Sep-Pak C18 solid-phase extraction method, which we have previously described.1,2⇓ Briefly, Sep-Pak C18 cartridges (Waters Chromatography Division, Millipore Co) were primed with successive washes of 5 ml of methanol and 5 ml of water. Aliquots of 5 ml of urine were then passed through the cartridges. After elution of unbound solutes with 5 ml of water, the compounds of interest were eluted with 3 ml of 100% methanol. These methanolic eluates were dried under reduced pressure in Savant Speed-Vac system (Savant Instruments Inc) and then redissolved in water to one fifth of the original volume. These aqueous preparations were designated U0 and were stored at 4°C until used.
The abilities of these extracts to inhibit 11β-HSD activity in microsomal preparations from sheep kidney (11β-HSD2) were measured in duplicate as previously described.2 For the assay of 11β-HSD2 enzymatic activity, sheep kidney microsomes (4 to 30 μg of protein) were incubated at 37°C for 10 minutes with 50 nmol/L [3H]-corticosterone (1 μCi) as tracer and substrate in 50 mmol/L Tris-HCl buffer (pH 8.5), containing 200 μmol/L NAD+ in a total volume of 0.25 ml. For all assays, an aliquot of either water (control subjects), urine extracts, or aqueous solutions of known quantities of CBX were added. The CBX was used for the construction of standard curves of inhibitory activity against which the inhibitory activities of the urine extracts could be compared. The reactions were terminated by addition of 0.75 ml of 100% methanol.
High-Performance Liquid Chromatography Assay of Enzyme Activity
The conversion of corticosterone to 11-dehydrocorticosterone was measured by separating the compounds by high-performance liquid chromatography and detecting and quantitating them with an on-line system β-detection system (Radiomatic model FLO-ONE\Beta radiochromatography detector; Packard Instrument Co). Aliquots of the methanol extracts from the incubation media were diluted with water to 45% methanol and chromatographed on DuPont Zorbax C8 reverse-phase columns at 44°C using isocratic 62% aqueous methanol. The percent conversion was calculated and used as a measure of enzyme activity. Inhibitory activity was expressed as the percent reduction in enzyme activity resulting from the presence of either known amounts of CBX or known volumes of urine extract in the incubation mixture. The percent inhibition produced by varying quantities of CBX was plotted against its concentrations as a reference curve, and the inhibitory activity of the urine aliquots was calculated (from the CBX standard curve) as the micrograms of CBX producing the same amount of inhibition under the standardized conditions. The amount of inhibtiory activity present per 24-hour urine sample was calculated and expressed as equivalent milligrams of CBX per 24 hours. The range used for the CBX standard curve for sheep kidney 11β-HSD2 is 200 to 3200 ng/ml. The GALF units represent only the relative inhibitory potencies of the GALFs in the samples compared with CBX. They are not an absolute measure of the quantities of the inhibitory substances. It should be noted that similar patterns of inhibitory activity were found when [3H]cortisol was used as a substrate to determine 11β-HSD2 activity.
Measurement of Kidney 11β(HSD2)-GALFs in U0 Extracts from Essential Hypertensive and Normotensive Subjects on the 200-mmol Na+ Diet
The U0 extracts from 10 high/normal-renin (group 2) and 10 low-renin (group 3) essential hypertensive patients were measured for the inhibitory activities against sheep kidney 11β-HSD2 and compared with the U0 extracts from 10 normoteinsive (group 1) control subjects. When urine samples were obtained from subjects maintained on the 200-mmol Na+ diet, the amounts of 11β(HSD2)-GALF inhibitory activity found in the urine extracts of the two groups of essential hypertensive subjects were not significantly different (P>.23) from those in the control normotensive subjects (Table).
Measurement of Kidney 11β(HSD2)-GALFs in U0 Extracts from Essential Hypertensive and Control Normotensive Subjects on the Low (10 mmol) Na+ Diet
When 24-hour urine samples were obtained from subjects maintained on the low (10 mmol/d)-Na+ diet, the amounts of 11β(HSD2)-GALF inhibitory activity were significantly greater (P<.05; P<.02, respectively) in the high/normal-renin (group 2) hypertensive patients when compared with both normotensive (group 1) and low-renin hypertensive subjects (group 3) (Table 1). No differences were observed between the levels of the low-renin hypertensive and the control normotensive subjects (P>.4).
The impact of a low-Na+ diet challenge was most noticeable in the essential hypertensive patients (but not in the normotensive subjects). The amounts of 11β(HSD2)-GALFs excreted in the urine of both groups of essential hypertensive patients were significantly elevated when patients were challenged with a low-sodium diet (Table and Fig.). Of particular importance, the levels of kidney 11β(HSD2)-GALFs in the high/normal-renin (group 2) patients were markedly increased from 8.3±1.4 to 17.3±2.9 (mean±SE, P=.01) when challenged by the low-Na+ diet. The levels in the urine of the low-renin (group 3) patients increased from 6.7±1.3 to 10.6±1.4 (P=0.07; Table). The urinary levels of kidney 11β(HSD2)-GALFs were significantly higher in the high/ normal-renin essential hypertensive patients than in the lowrenin essential hypertensive patients (P<.02) on the low-Na+ diet. When the normotensive control subjects were challenged with the low-Na+ diet, no significant increase was observed (P>.4).
In each case the statistical comparisons were made using the wilcoxon two sample rank-sum (Mann-Whitney test) analysis.
It is now generally considered that 11β-HSD2 acts in the distal nephron as a “protective mechanism,” which prevents glucocorticoids from accessing renal MRs. Hypertensive children with the syndrome of apparent mineralocorticoid excess were shown to have homozygous mutations in the 11β-HSD2 gene, which results in diminished 11β-HSD2 activity.12,13⇓ The salt retention and increased blood pressure associated with excessive licorice ingestion is also thought to be due to inhibition of renal 11β-HSD2 by glycyrrhetinic acid, the active principal in licorice.16 These findings have offered considerable support for the “guardian” role of renal 11β-HSD2. 11β-HSD1 is present predominantly in liver and lung,17 is bidirectional, and principally uses NADP+ as a cofactor and has a higher Km for glucocorticoids (2 μmol/L). It’s functional role is less clear at this time. A role for vascular smooth muscle 11β-HSD1 is emerging because it is also bidirectional, and uses NADP+ but, significantly, has a 10-fold smaller Kmm (200 nmol/L) than hepatic 11β-HSD1.18,19⇓ It has been known for several years that glucocorticoids enhance the vasoconstrictive actions of both catecholamines and angiotensin II in aortic ring preparations.20 We have recently reported that inhibition of vascular smooth muscle 11β-HSD1 by glycyrrhetinic acid, CBX, and other potent inhibitors all amplify the above enhancement of vasoconstriction by corticosterone.18,19⇓ Thus, vascular smooth muscle 11β-HSD1 may play an important protective mechanism regulating the local concentrations of glucocorticoids in vascular smooth muscle and enhance vascular tone.
We have previously suggested that endogenous substances (GALFs) exist in humans and other mammalian species, which, like the liquorice derivative glycyrrhetinic acid, inhibit these steroid-inactivating enzymes.12 We have confirmed our initial findings and shown that urine samples from both normotensive men and women do indeed contain reproducibly measurable GALF inhibitory activity against kidney 11β-HSD2 as well as vascular smooth muscle 11β-HSD1. Their levels are elevated and increase throughout human pregnancy, and their presence in humans suggests that they may play a role in Na+ homeostasis and blood pressure.2 Their chemical identity(ies) is not known at this time but their chemical and high-performance liquid chromatography characteristics suggest that several of the GALFs are steroidal.
In the present study we have examined the levels of urinary kidney 11β(HSD2)-GALFs in patients with essential hypertension and compared them with normotensive control subjects. No significant differences in the levels of kidney 11β(HSD2)-GALFs were observed between two groups of essential hypertensive and normotensive control subjects. The group of essential hypertensive patients with high/normalrenin levels and the group of low-renin essential hypertensive patients excreted similar amounts of kidney 11β(HSD2)-GALFs in 24-hour urine samples. However, when these groups of individuals were challenged with a low-Na+ diet (10 mmol of Na+), the impact on the excretion of urinary kidney 11β(HSD2)-GALFs was significant in the hypertensives only. Both groups of hypertensive individuals showed a marked increase in the level of 11β(HSD2)-GALFs in their urine compared with the normotensive control subjects. In particular, when on the low-Na+ diet, the group of high/ normal-renin essential hypertensive patients displayed the largest increase in the urinary levels of kidney 11β(HSD2)-GALFs.
The levels of urinary kidney 11β(HSD2)-GALFs in patients with high/normal-renin essential hypertension (when on the low-Na+ diet) were significantly greater than both the normotensive individuals and the low-renin essential hypertensive subjects. The impact of the low-Na+ diet challange to the normotensive control individuals is not clear at this time with some of the individuals showing an increase and others showing a decrease in kidney 11β(HSD2)-GALF levels. Further experiments with a larger number of normotensive subjects are now necessary to better understand the physiological role of kidney 11β(HSD2)-GALFs and sodium homeostasis. It should be noted that an earlier report by Takeda et al21 showed increased levels of 11β(HSD2)-GALF inhibitory substances in urine of patients with low-renin essential hypertension when stimulated by increased dietary Na+. The differences between the present data and those reported by Takeda et al are not understood at this time.
A statistical analysis of all 30 individuals involved in the present study showed a significant increase (P<.05) when they were challenged with a low-Na+ diet. The finding that high/normal-renin essential hypertensive patients increase their urinary kidney 11β (SHD2)-GALF levels when challenged with a low-Na+ diet suggests that this group of essential hypertensive patients, in part, may use glucocorticoids to increase their overall mineralocorticoid activity. Low-renin essential hypertensive patients do not. However, further experiments are necessary not only to isolate and chemically identify GALF substances, but also to determine their role in sodium homeostasis in the hypertensive population.
This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute Grant HL-52972 (D.J.M.), by the Miriam Hospital Research Foundation, by National Institutes of Health Grant HL55000 (G.H.W. and W.R.L.) and by GCRC Grant 5M01 RR002635 (G.H.W.). We thank Elizabeth Gifford for excellent secretarial assistance and Jennifer Bowen and Judith Everett as study co-ordinators for this work.
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