In vivo activity of 11ß-hydroxysteroid dehydrogenase type 1 in man: effects of prednisolone and chenodesoxycholic acid.

The 11ß-hydroxysteroid dehydrogenases (11ß-HSDs) play a pivotal role in glucocorticoid (GC) action. 11ß-HSD1 is a predominant reductase, activating GCs from inert metabolites, whereas 11ß-HSD2 is a potent dehydrogenase inactivating GCs. Knowing the metabolic effects of GCs, a selective inhibition of 11ß-HSD1 represents a potential target for therapy of impaired glucose tolerance, insulin insensitivity and central obesity. In vitro, 11ß-HSD1 is selectively inhibited by chenodesoxycholic acid (CDCA) and upregulated under GC exposure. Therefore, we aimed to investigate the effects of CDCA and prednisolone on hepatic 11ß-HSD1 activity in vivo by measuring 11-reduction of orally given cortisone (E) acetate to cortisol (F). CDCA or placebo was given to 5 male healthy volunteers within a randomised cross-over trial before and after oral administration of 12.5 mg E acetate at 8:00 h. For measurement of in vivo effects of GCs on 11ß-HSD1 activity, hepatic reduction of 25 mg E acetate before and after treatment with prednisolone (30 mg for 6 days) was determined in 7 healthy males. Serum GC levels were determined using a fully automated liquid chromatographic system. CDCA had no effect on the activity of 11ß-HSD1 in vivo. Prednisolone therapy leads to a marked rise in serum F concentrations and an elevated F/E serum ratio. This proves GC-induced activation of hepatic 11ß-HSD1, which could not be extinguished by a parallel increase of IGF-1 under prednisolone. CDCA does not affect in vivo activity of 11ß-HSD1 when given in therapeutic dosages. During GC treatment, increased hepatic activation of E to F may aggravate metabolic side effects of GCs such as seen in the metabolic syndrome.

Drospirenone in combination with estrogens: for contraception and hormone replacement therapy.

Progesterone has a high affinity for the mineralocorticoid receptor and is an antagonist of aldosterone. Almost all synthetic progestogens are devoid of an antimineralocorticoid effect, and are unable to antagonize the salt-retaining effect of estrogens. This property could be one cause of the weight gain and increase in blood pressure associated with the use of combined oral contraceptives and, in some susceptible women, with postmenopausal hormone replacement therapy (HRT). This review illustrates the results of clinical studies with drospirenone, a new progestogen with antialdosterone activity. In normally menstruating women, 3 mg drospirenone per day, taken from day 5 to day 25 of the cycle, inhibits ovulation. A combination of 3 mg drospirenone with 30 microg ethinylestradiol (Yasmin, Schering AG, Berlin, Germany) is a highly effective and well-tolerated oral contraceptive, with an overall Pearl Index of 0.57 and an adjusted Pearl Index of 0.09. In addition, this combination leads to mild natriuresis and a slight compensatory activation of the renin-aldosterone system. This effect hasbeen clinically demonstrated: compared with an oral contraceptive containing 30 microg ethinylestradiol and 150 microg levonorgestrel, 30 microg ethinylestradiol/3 mg drospirenone, given over 6 months, led to slight decreases in body weight and blood pressure. For postmenopausal HRT, 2 mg drospirenone was combined with 1 mg 17beta-estradiol (Angeliq, Schering AG, Berlin, Germany) for continuous treatment. To evaluate the endometrial safety of this combination, data were assessed from 520 postmenopausal women receiving 1 mg 17beta-estradiol and drospirenone (1, 2 or 3 mg per day) for at least 100 weeks. There were no cases of hyperplasia or carcinoma during the entire study period, and 85-92% of women had an atrophic/inactive endometrium. Data from two studies, with a treatment duration of at least 6 months, demonstrated a decrease in mean systolic blood pressure of 1.8 mmHg and a decrease in mean diastolic blood pressure of 3.8 mmHg, after treatment with 1 mg 17beta-estradiol/2 mg drospirenonefor 28 weeks. However, in a subgroup of slightly hypertensive women (initial blood pressure of more than 140/90 mmHg), the mean decrease in systolic blood pressure after 28 weeks of treatment with 1 mg 17beta-estradiol/2 mg drospirenone was 12.5 mmHg, and the mean decrease in diastolic blood pressure was 9.4 mmHg. Since high blood pressure is a cardiovascular risk factor, the use of drospirenone may exert a beneficial effect in this regard. More prolonged studies need to be performed, in order to demonstrate whether negative cardiovascular end-points can be reduced by the new progestogen. In conclusion, drospirenone is not only a safe option for women using oral contraceptives or HRT, but it may offer new medical benefits, via its antimineralocorticoid and antialdosterone effects and its potential to decrease water retention and blood pressure.

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.

Pharmacodynamics and pharmacokinetics of synthetic mineralocorticoids and glucocorticoids: receptor transactivation and prereceptor metabolism by 11beta-hydroxysteroid-dehydrogenases.

Glucocorticoid (GC) and mineralocorticoid (MC) action in target tissues is determined by prereceptor metabolism by 11beta-hydroxysteroid-dehydrogenases (HSDs) and receptor transactivation. We characterized these parameters for steroids often used in clinical practice. HSD activity was examined in human liver (HSD1) and kidney microsomes (HSD2) and in CHO cells stably transfected with both enzymes. GC and MC transcriptional activity was tested by luciferase assay in CV-1 cells transfected with human GC or MC receptor expression vectors. The 11-hydroxy-group is necessary for GC and MC receptor transactivation. As HSD2 oxidizes 11-hydroxysteroids to inactive 11-dehydrosteroids, GC and MC activity in HSD2-expressing tissues (kidney, colon) is regulated by this enzyme. As 9alpha-fluorination (such as in 9alpha-fluorocortisol) decreases oxidation by HSD2 and increases both GC and MC receptor transactivation, this modification leads to optimal, but non-selective transactivation of both receptors. Increased GC receptor and decreased MC receptor transactivation leading to more selective GC activity is reached using the following substituents: 16beta-methyl (in betamethasone), 16alpha-methyl (in dexamethasone) and triangle up 1-dehydro-configuration (in prednisolone). Whereas the modifications in position 16 decrease oxidation by HSD2, the triangle up 1-dehydro-configuration increases HSD2-activity leading to an enhanced inactivation of prednisolone compared to all other steroids. 9alpha-fluorocortisol, the most frequently used substance for MC-substitution, seems to be the best choice of available steroids for this purpose. Whereas GC selectivity can be improved by hydrophobic substituents in position 16 and the triangle up 1-dehydro-configuration, maximal GC activity needs additional fluorination in position 9alpha (such as in dexamethasone). For GC therapy directed to HSD2-expressing organs, widely used prednisolone does not seem to be the optimal recommendation.

Renal inactivation, mineralocorticoid generation, and 11beta-hydroxysteroid dehydrogenase inhibition ameliorate the antimineralocorticoid effect of progesterone in vivo.

Progesterone (P) is a strong mineralocorticoid receptor (MR) antagonist in vitro. The high P concentrations seen in normal pregnancy only moderately increase renin and aldosterone concentrations. In previous in vitro studies we hypothesized that this may be explained by intrarenal conversion of P to less potent metabolites. To investigate the in vivo anti-MR potency of P, we performed an infusion study in patients with adrenal insufficiency (n = 8). They omitted 9alpha-fluorocortisol for 4 d and hydrocortisone for 0.5 d before a continuous iv infusion of aldosterone for 8.5 h, with an additional iv P infusion commenced at 4 h. During aldosterone infusions the initially elevated urinary sodium to potassium ratio decreased significantly. Despite the 1000-fold excess of P over aldosterone, the urinary sodium to potassium ratio and urinary sodium excretion increased only slightly after 3 h of P infusion. We detected inhibition of renal 11beta-hydroxysteroid dehydrogenase type 2 by P, thus giving cortisol/prednisolone access to the MR. Urinary and plasma concentrations of 17alpha-hydroxyprogesterone, a major metabolite of renal P metabolism, and those of serum androstenedione and deoxycorticosterone, a mineralocorticoid itself, increased significantly during P infusion. This supports the hypothesis of an effective protection of the MR from P by efficient extraadrenal downstream conversion of P.

The human kidney is a progesterone-metabolizing and androgen-producing organ.

Progesterone (P) is a potent antagonist of the human mineralocorticoid receptor (MR) in vitro. We have previously demonstrated effective downstream metabolism of P in the kidney. This mechanism potentially protects the MR from P action. Here, we have investigated the expression and functional activity of steroidogenic enzymes in human kidney. RT-PCR analysis demonstrated the expression of 5 alpha-reductase type 1, 5 beta-reductase, aldo-keto-reductase (AKR) 1C1, AKR1C2, AKR1C3, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) type 2, and 17 alpha-hydroxylase/17,20-lyase (P450c17). The presence of 3 beta-HSD type 2 and P450c17 indicated that conversion of pregnenolone to dehydroepiandrosterone (DHEA) and to androstenedione may take place effectively in kidney. To investigate this further, we incubated kidney subcellular fractions with radiolabeled pregnenolone. This resulted in efficient formation of DHEA from pregnenolone, indicating both 17 alpha-hydroxylase and 17,20-lyase activities exerted by P450c17. Radiolabeled DHEA was converted via androstenedione, androstenediol, and testosterone, indicating both 3 beta-HSD type 2 activity and 17 beta-HSD activity. In addition, the conversion of testosterone to 5 alpha-dihydrotestosterone was detectable, indicating 5 alpha-reductase activity. In conclusion, we verified the expression and functional activity of several enzymes involved in downstream metabolism of P and androgen synthesis in human kidney. These findings may be critical to the understanding of water balance during the menstrual cycle and pregnancy and of sex differences in hypertension.

Enhanced 11beta-hydroxysteroid dehydrogenase type 1 activity in stress adaptation in the guinea pig.

The 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) convert cortisol to its inactive metabolite cortisone and vice versa. 11beta-HSD type 1 (11beta-HSD-1) functions as a reductase in vivo, regulating intracellular cortisol levels and its access to the glucocorticoid receptor. In contrast, 11beta-HSD-2 only mediates oxidation of natural glucocorticoids, and protects the mineralocorticoid receptor from high cortisol concentrations. We investigated the in vivo and in vitro effects of ACTH on the recently characterized 11beta-HSDs in guinea pig liver and kidney. Tissue slices of untreated guinea pigs were incubated with (3)H-labelled cortisol or cortisone and ACTH(1-24) (10(-10) and 10(-9) mol/l). The 11beta-HSD activities in liver and kidney slices were not influenced by in vitro incubation with ACTH(1-24). In addition, guinea pigs were treated with ACTH(1-24) or saline injections s.c. for 3 days. Liver and kidney tissue slices of these animals were incubated with (3)H-labelled cortisol or cortisone. In vivo ACTH treatment significantly increased reductase and decreased oxidase activity in liver and kidney. Furthermore, 11beta-HSD-1 activity assessed by measurement of the urinary ratio of (tetrahydrocortisol (THF)+5alphaTHF)/(tetrahydrocortisone) was significantly increased after ACTH treatment compared with the control group. Plasma levels of cortisol, cortisone, progesterone, 17-hydroxyprogesterone and androstenedione increased significantly following in vivo ACTH treatment. The enhanced reductase activity of the hepatic and renal 11beta-HSD-1 is apparently caused by cortisol or other ACTH-dependent steroids rather than by ACTH itself. This may be an important fine regulation of the glucocorticoid tonus for stress adaptation in every organ, e.g. enhanced gluconeogenesis in liver.

Expression of the progesterone receptor and progesterone- metabolising enzymes in the female and male human kidney.

Due to high binding affinity of progesterone to the human mineralocorticoid receptor (hMR), progesterone competes with the natural ligand aldosterone. In order to analyse how homeostasis can be maintained by mineralocorticoid function of aldosterone at the MR, especially in the presence of elevated progesterone concentrations during the luteal phase and pregnancy, we investigated protective mechanisms such as the decrease of free progesterone by additional binding sites and progesterone metabolism in renal cells. As a prerequisite for sequestration of progesterone by binding to the human progesterone receptor (hPR) we demonstrated the existence of hPR expression in female and male kidney cortex and medulla at the level of transcription and translation. We identified hPR RNA by sequencing the RT-PCR product and characterised the receptor by ligand binding and scatchard plot analysis. The localisation of renal hPR was shown predominantly in individual epithelial cells of distal tubules by immunohistology, and the isoform hPR-B was detected by Western blot analysis. As a precondition for renal progesterone metabolism, we investigated the expression of steroid-metabolising enzymes for conversion of progesterone to metabolites with lower affinity to the hMR. We identified the enzyme 17alpha-hydroxylase for renal 17alpha-hydroxylation of progesterone. For 20alpha-reduction, different hydroxysteroid dehydrogenases (HSDs) such as 20alpha-HSD, 17beta-HSD type 5 (3alpha-HSD type 2) and 3alpha-HSD type 3 were found. Further, we detected the expression of 3beta-HSD type 2 for 3beta-reduction, 5alpha-reductase (Red) type 1 for 5alpha-reduction, and 5beta-Red for 5beta-reduction of progesterone in the human kidney. Therefore metabolism of progesterone and/or binding to hPR could reduce competition with aldosterone at the MR and enable the mineralocorticoid function.

Agonistic and antagonistic properties of progesterone metabolites at the human mineralocorticoid receptor.

OBJECTIVE: Progesterone binds to the human mineralocorticoid receptor (hMR) with nearly the same affinity as do aldosterone and cortisol, but confers only low agonistic activity. It is still unclear how aldosterone can act as a mineralocorticoid in situations with high progesterone concentrations, e.g. pregnancy. One mechanism could be conversion of progesterone to inactive compounds in hMR target tissues. DESIGN: We analyzed the agonist and antagonist activities of 16 progesterone metabolites by their binding characteristics for hMR as well as functional studies assessing transactivation. METHODS: We studied binding affinity using hMR expressed in a T7-coupled rabbit reticulocyte lysate system. We used co-transfection of an hMR expression vector together with a luciferase reporter gene in CV-1 cells to investigate agonistic and antagonistic properties. RESULTS: Progesterone and 11beta-OH-progesterone (11beta-OH-P) showed a slightly higher binding affinity than cortisol, deoxycorticosterone and aldosterone. 20alpha-dihydro(DH)-P, 5alpha-DH-P and 17alpha-OH-P had a 3- to 10-fold lower binding potency. All other progesterone metabolites showed a weak affinity for hMR. 20alpha-DH-P exhibited the strongest agonistic potency among the metabolites tested, reaching 11.5% of aldosterone transactivation. The agonistic activity of 11beta-OH-P, 11alpha-OH-P and 17alpha-OH-P was 9, 5.1 and 4.1% respectively. At a concentration of 100 nmol/l, progesterone, 17alpha-OH-P and 20alpha-DH-P inhibit nearly 75, 40 and 35% of the transactivation by aldosterone respectively. All other progesterone metabolites tested demonstrate weaker affinity, and agonistic and antagonistic potency. CONCLUSIONS: The binding affinity for hMR and the agonistic and antagonistic activity diminish with increasing reduction of the progesterone molecule at C20, C17 and at ring A. We assume that progesterone metabolism to these compounds is a possible protective mechanism for hMR. 17alpha-OH-P is a strong hMR antagonist and could exacerbate mineralocorticoid deficiency in patients with congenital adrenal hyperplasia.

Antimineralocorticoid activity of a novel oral contraceptive containing drospirenone, a unique progestogen resembling natural progesterone.

Sex hormones are known to interfere with the renin-angiotensin-aldosterone system (RAAS) in two ways. First, estrogens strongly stimulate the production of renin substrate (angiotensinogen), leading to increased levels of angiotensin and aldosterone, and sodium retention. Second, progesterone is a potent aldosterone antagonist, which acts on the mineralocorticoid receptor to prevent sodium retention. In combined oral contraceptives, progestogens devoid of antimineralocorticoid and antiandrogenic activity are unable to counteract the sodium-retaining effect of the ethinylestradiol component. As a consequence, these preparations may increase fluid retention, and promote related symptoms such as edema and body weight. Drospirenone is a new progestogen with antimineralocorticoid and antiandrogenic activity. The relationship between the progestogenie and antimincralocorticoid potency of drospirenone is similar to that of endogenous progesterone. At a dosage that suppresses ovulation, drospirenone induces mild natriuresis, which is followed by compensatory stimulation of the RAAS (comparable to a low sodium diet). An oral contraceptive containing 3 mg drospirenone and 30 microg ethinylestradiol. (Yasmin, Schering AG, Berlin, Germany) provides reliable contraception and, due to a lack of sodium retention, may counteract cyclical weight gain and other symptoms related to estrogen-induced fluid retention.

Therapeutic strategies in adrenal insufficiency.

Severe chronic adrenal insufficiency (primary or secondary) is a potentially lethal disorder, unless the patient is regularly substituted with glucocorticoids, usually with hydrocortisone (15-25 mg/day) and with 9 alpha-fluor-hydrocortisone (0.05-0.2 mg/day) in addition in patients with the primary adrenal disorder (Addison's disease). In stressful situations and in febrile disorders, the glucocorticoid dosage must be increased prophylactically in order to prevent an "adrenal crisis". Most women with adrenal insufficiency will profit from the additional substitution of dehydroepiandrosterone (DHEA) with regard to well-being and sexual function. A patient with acute adrenal insufficiency will die if the diagnosis is missed and high-dose glucocorticoid treatment is not instituted immediately. Acute adrenal insufficiency developing de novo in an intensive care patient (e.g. from adrenal hemorrhage or adrenal vein thrombosis) is a most challenging diagnosis. In these patients, however, survival not only depends on glucocorticoid substitution but also on the underlying disease.

Differential diagnosis of polyuric/polydipsic syndromes with the aid of urinary vasopressin measurement in adults.

OBJECTIVE: A water deprivation test or a hypertonic saline infusion test with the measurement of plasma osmolality and plasma vasopressin are the gold standard tests in the differential diagnosis of polyuric syndromes. Because commercially available vasopressin kits are too insensitive for this approach, and the concentration of vasopressin in urine is much higher than in plasma, urinary vasopressin measurements may be an alternative to the more difficult plasma vasopressin measurement. DESIGN: The diagnostic value of the measurement of urinary vasopressin with a rather insensitive commercially available vasopressin kit was compared with plasma vasopressin measurement by a highly sensitive radioimmunoassay (RIA). PATIENTS AND METHODS: Thirteen normal subjects and 27 patients with polyuria/polydipsia were examined by an 8-h fluid deprivation test. In all blood samples (0800 h, 1200 h, 1400 h and 1600 h) and in all urine collections (2-hourly fractions), osmolality as well as vasopressin were measured. RESULTS: Using plasma vasopressin measurement with a highly sensitive RIA as gold standard test, nine patients were classified as having primary polydipsia, whereas 18 had partial or complete cranial diabetes insipidus. Whereas the substitution of plasma vasopressin measurement by urinary vasopressin measurement alone did not provide 100% separation between both groups, the product of urinary vasopressin and urinary osmolality related to plasma osmolality completely separated the patients with primary polydipsia from those with diabetes insipidus. Urinary measurement of vasopressin and osmolality alone, which was recommended as a noninvasive diagnostic procedure in children, was too insensitive for exact differential diagnosis in our adult patients. CONCLUSIONS: The simultaneous measurement of plasma vasopressin and plasma osmolality in a dehydration test is the most powerful diagnostic tool in the differential diagnosis of polyuria/polydipsia. However, if highly sensitive assays for plasma vasopressin measurements are not available, the measurement of urinary vasopressin with commercially available, less sensitive RIAs may be a diagnostic alternative, which showed nearly the same sensitivity as plasma vasopressin measurement in our study population.

Clinical implications of glucocorticoid metabolism by 11beta-hydroxysteroid dehydrogenases in target tissues.

11beta-Hydroxysteroid dehydrogenases (11beta-HSD) are microsomal enzymes that catalyze the conversion of active glucocorticoids (GC) to their inactive 11-dehydro products and vice versa. Two isoenzymes of 11beta-HSD have been characterized and cloned in human tissues. The tissue-specific metabolism of GC by these enzymes is important for mineralocorticoid (MC) and GC receptor occupancy and seems to play a crucial role in the pathogenesis of diseases such as apparent MC excess syndrome, and may play roles in hypertension, obesity and impaired hepatic glucose homeostasis. This article reviews the literature and examines the role and importance of 11beta-HSD in humans.

Enzyme-mediated protection of the mineralocorticoid receptor against progesterone in the human kidney.

Progesterone (P) is a mineralocorticoid (MC)-antagonist in vitro. During pregnancy, plasma P concentrations exceed aldosterone concentrations at least 50-fold, but plasma aldosterone increases only 4-8-fold in a compensatory manner. Since the in vivo anti-MC activity of P seems to be only moderate, we hypothesized that P is metabolized by enzymes of MC target tissue similar to the way cortisol is metabolized by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2 in order to protect the MC receptor. We, therefore, examined P metabolism using 4-(14)C-P in subcellular fractions of human postmenopausal and male kidneys, and in homogenates of one premenopausal kidney. We found that P is converted effectively, even at high P concentrations (10(-6) mol/l), to various metabolites: 20alpha-dihydro(DH)-P; 17alpha-OH-P; 17alpha-OH,20alpha-DH-P; 5alpha-DH-P; 3beta,5alpha-tetrahydro(TH)-P; and 20alpha-DH,5alpha-DH-P. Homogenates of premenopausal kidney also showed conversion to 3alpha- and 5beta-reduced P metabolites. These results confirm the existence of an efficient renal enzyme system as a possible mechanism of an enzyme-mediated MC receptor selectivity.