Proinflammatory/profibrotic effects of aldosterone in Gitelman's syndrome, a human model opposite to hypertension.

PURPOSE: Aldosterone proinflammatory/profibrotic effects are mediated by the induction of mononuclear leucocytes (MNL) to express oxidative stress (OxSt)-related proteins, such as p22phox, and by the activation of RhoA/Rho kinase pathway. Gitelman's syndrome (GS), an autosomal recessive tubulopathy, is an interesting opposite model to hypertension, being characterized by hypokalemia, activation of renin-angiotensin-aldosterone system yet normo/hypotension and lack of cardiovascular-renal remodeling. We aimed to evaluate the proinflammatory/profibrotic effect of aldosterone in MNL of 6 GS patients compared with 6 healthy subjects (HS). METHODS: p22phox expression and MYPT-1 phosphorylation status, a marker of RhoA/Rho kinase pathway activation, were evaluated in MNL of GS patients and HS at baseline and after incubation with aldosterone (1 × 10-8 M) alone or with canrenone (1 × 10-6 M). RESULTS: At basal condition, p22phox expression was significantly higher in HS than in GS patients (1.02 ± 0.05 densitometric unit (du) vs 0.40 ± 0.1 du, respectively). Aldosterone significantly increased p22phox expression in HS and this effect was reversed by coincubation with canrenone (1.4 ± 0.05 du and 1.09 ± 0.03 du, respectively). No significant change was reported in GS after incubation of MNL with aldosterone and/or canrenone compared with basaline. Even MYPT-1 phosphorylation was significantly higher in HS compared with GS patients at basal condition (1.16 ± 0.1 du vs 0.69 ± 0.07, respectively). Aldosterone significantly increased MYPT-1 phosphorylation only in HS (1.37 ± 0.1 du vs 0.83 ± 0.12 du in GS). CONCLUSIONS: GS patients seem to be protected by the OxSt status induced by aldosterone and revealed in HS. This human model could provide additional clues to highlight the proinflammatory/cardiovascular remodeling effects of aldosterone.

Is corifollitropin alfa effective in controlled ovarian stimulation among all poor ovarian responders? A retrospective comparative study.

Several studies have compared the effectiveness of corifollitropin alfa versus daily gonadotropins in poor ovarian responders (PORs) undergoing controlled ovarian stimulation (COS), showing conflicting results in terms of IVF outcomes. Given the heterogeneity of patients included in the classification of POR according to 'Bologna criteria', the aim of this study was to evaluate the impact of corifollitropin alfa in two different categories of POR distinguished according to patients' antral follicle count (AFC). We retrospectively evaluated 104 infertile POR, split into two groups according to AFC (Group A ≤ 5; Group B > 5) and subgroups according to the ovarian stimulation regimen (corifollitropin alfa plus daily gonadotropins (Subgroup 1) versus daily gonadotropins alone (Subgroup 2)). Outcome measures were total oocytes, MII oocytes, total embryos, follicular output rate (FORT), implantation rate (IR), clinical pregnancy rate (CPR), miscarriage rate (MR), and live birth rate (LBR). Subgroup A1 experienced a lower number of total oocytes, MII oocytes, total embryos, and FORT (p < .05) in comparison to Subgroup A2, while no difference was found when comparing Subgroups B1 and B2. No difference was found between subgroups even in terms of IR, CPR, MR, and LBR. In conclusion, corifollitropin alfa may be as effective as daily gonadotropins in POR with AFC > 5 undergoing COS, while it might be inferior to daily gonadotropins in POR with AFC ≤ 5.

The influence of thyroid autoimmunity on embryo quality in women undergoing assisted reproductive technology.

The influence of thyroid autoimmunity in assisted reproductive technology (ART) outcome in euthyroid women is still controversial. In this study, we retrospectively evaluated embryo quality in 123 euthyroid women undergoing ART with or without thyroid autoantibodies (TAA). Embryo quality was assessed in 119 embryos of 29 infertile patients with TAA and in 394 embryos of 94 infertile patients without TAA. Our results showed not statistically significant differences in age, body mass index, anti-Müllerian hormone, follicle stimulating hormone, free triiodothyronine, and free thyroxine levels between cases and controls. Thyroid stimulating hormone was within the normal range, but significantly higher in TAA patients compared with the controls (2.4 ± 0.8 vs. 2 ± 0.9 mIU/L, respectively, p < .01). The number of oocytes picked up and fertilized was comparable between the two groups. Embryo quality was significantly impaired in women with at least one autoantibody (p < .001). Implantation rate, pregnancy rate, and ongoing pregnancy rate were comparable in the two groups. These results suggest a negative impact of thyroid autoimmunity in embryo quality in women undergoing ART even when thyroid function is normal.

Spironolactone and intermenstrual bleeding in polycystic ovary syndrome with normal BMI.

PURPOSE: Spironolactone (SP) is an effective treatment for polycystic ovary syndrome (PCOS), but it is often associated with menstrual abnormalities whose mechanism is still under investigation. In this study, we investigated the serum sex steroids and endometrial thickness in 30 PCOS patients, before and after one-month 100 mg SP treatment. METHODS: Serum FSH, LH, estradiol, progesterone and endometrial thickness were evaluated at the 14th and 16th day of the menstrual cycle, before and during short-term SP treatment. According to the presence (15 cases) or absence (15 cases) of menstrual bleeding at the 14th day during SP, the patients were divided into two groups, which were then compared using a two-tailed Student's t test. RESULTS: Serum estradiol and endometrial thickness were lower than pretreatment at both determinations in all patients, but patients with bleeding had significantly lower estradiol values than non-bleeding ones, both before and after therapy. Endometrial thickness was significantly lower in the bleeding group compared with non-bleeding group only at the 16th day of the cycle. These differences were significant, even though the values of estradiol and endometrial thickness remained in the normal range. CONCLUSIONS: SP therapy can reduce the values of estradiol and the endometrial thickness in patients with PCOS compared with pretreatment, but PCOS patients with bleeding had pretreatment estradiol values lower than the patients who did not complain of this side effect. Intermenstrual abnormalities may represent the low estrogen impregnation of endometrium due to SP, whose mechanism is complex, involving several factors, such as the effects of some metabolites of SP on estradiol and progesterone production, on their receptors, and the individual metabolism of SP in vivo.

Towards generalised reference condition models for environmental assessment: a case study on rivers in Atlantic Canada.

Evaluation of the ecological status of river sites in Canada is supported by building models using the reference condition approach. However, geography, data scarcity and inter-operability constraints have frustrated attempts to monitor national-scale status and trends. This issue is particularly true in Atlantic Canada, where no ecological assessment system is currently available. Here, we present a reference condition model based on the River Invertebrate Prediction and Classification System approach with regional-scale applicability. To achieve this, we used biological monitoring data collected from wadeable streams across Atlantic Canada together with freely available, nationally consistent geographic information system (GIS) environmental data layers. For the first time, we demonstrated that it is possible to use data generated from different studies, even when collected using different sampling methods, to generate a robust predictive model. This model was successfully generated and tested using GIS-based rather than local habitat variables and showed improved performance when compared to a null model. In addition, ecological quality ratio data derived from the model responded to observed stressors in a test dataset. Implications for future large-scale implementation of river biomonitoring using a standardised approach with global application are presented.

Endogenous reactive oxygen species content and modulation of tyrosine phosphorylation during sperm capacitation.

Generation of controlled amounts of reactive oxygen species (ROS) and phosphorylation of protein tyrosine (Tyr) residues are two main cellular changes involved in sperm capacitation. This study examined the relationship between tyrosine-phosphorylation (Tyr-P) and endogenous ROS production during sperm capacitation, and correlated them with both sperm motility and functionality expressed as acrosome-reacted cells. Immediate ROS generation was observed to peak after a 45-min incubation, followed by a rapid decrease in ROS content and successive regeneration of the ROS peak in 3 h and later. These two peaks were directly correlated with both the Tyr-P process involving sperm heads and tails, and the acrosome reaction (69 ± 8% and 65 ± 4%, respectively). The period of low-ROS content resulted in low Tyr-P patterns, located exclusively in the cell midpiece, and drastic reduction in acrosome-reacted cells. Ascorbic acid addition inhibited both Tyr-P patterns and acrosome reactions, whereas NADPH induced high ROS generation, with Tyr-P patterns located only on sperm tails, and prevented the acrosome reaction. Sperm hyperactivation was insensitive to ROS content. This is an important parameter for evaluation of sperm capacitation, which is achieved only when both ROS generation reaches a peak and Tyr-P involves the sperm head.

Oxidative stress-related proteins in a Conn's adenoma tissue. Relevance for aldosterone's prooxidative and proinflammatory activity.

BACKGROUND AND AIM: Angiotensin II (Ang II) induces oxidative stress (OxSt), which is essential for cardiovascular remodeling. Aldosterone also induces fibrosis and remodeling through direct effect on non-classical mineralocorticoid (MR) target tissues. However, studies on the role of aldosterone on OxSt and related factors in humans are lacking. MATERIALS AND METHODS: We assessed gene and protein expression of p22phox (RT-PCR and Western blot), NAD(P)H oxidase subunit essential for superoxide production and gene expression of transforming growth fator (TGF) beta, plasminogen activator inhibitor (PAI)-1, and heme oxygenase (HO)-1, effectors of OxSt (RT-PCR), in a Conn's adenoma, removed from a patient with primary hyperaldosteronism. Ang II type 1 (AT1R) and MR receptors expression were also evaluated (RT-PCR). The normal adrenal tissue adjacent to the adenoma was used as control. RESULTS: p22phox gene and protein expression were higher (31% and 53%, respectively) in the adrenal adenoma. TGFbeta, PAI-1, and HO-1 gene expression were also higher (25%, 129%, and 25%, respectively) in the adrenal adenoma while AT1R gene expression was similar (8%). The expression of MR in the adenoma was documented. CONCLUSIONS: This report demonstrates in a human model that the increased aldosterone production has effects on enzyme systems related to OxSt, enhancing the systemic fibrogenic effects of aldosterone excess through TGFbeta and PAI-1 expression which was previously demonstrated only indirectly in vitro and in animal models. The presence of MR expression in the adenoma may link the hormone with the adenoma growth. Therefore, the results of this study derived from a single case might represent an important working hypothesis for further research in a larger number of cases to clarify the role of aldosterone overproduction on OxSt and its clinical relevance.

Some considerations about evolution of idiopathic primary aldosteronism.

The prevalence of primary aldosteronism has increased since many patients who were previously considered as being affected by low renin essential hypertension are actually satisfying the new diagnostic criteria using plasma aldosterone/ plasma renin activity (PRA) ratio. Many of these cases could be classified as subclinical hyperaldosteronism, having normal aldosterone and low PRA, or in alternative the normal range of aldosterone should be revised. Idiopathic hyperaldosteronism can, in many cases, be considered as an evolutive disease: it can be hypothesized that the biochemical picture can be preceded by essential hypertension and that, after several years, primary aldosteronism can evolve back to essential hypertension due to age-related reduced vascular and adrenal sensitivity to angiotensin II. This effect is also evident after longterm treatment with aldosterone receptors blockers and therefore it possible that aldosterone-receptors blockers are able to normalize the sensitivity of glomerulosa to angiotensin II even after long-term withdrawal. The use of aldosterone receptors blockers prevents cardiovascular complications due to local aldosterone effect at the level of endothelium and mononuclear leukocytes; therefore, these drugs should be also considered for therapy of patients with hypertension. It is not excluded that aldosterone receptor blockers could prevent the onset of idiopathic hyperaldosteronism and its complications in patients with hypertension without primary hyperaldosteronism. From all these considerations it follows that the concept of normal range of aldosterone should be revised and the use of aldosterone receptor blockers should be revisited.

Effect of canrenone and amiloride on the prooxidative effect induced by aldosterone in human mononuclear leukocytes in vitro.

Clinical studies have demonstrated that aldosterone receptor antagonists do improve the survival of patients with chronic heart diseases and in vitro studies have shown that canrenone blocks the proinflammatory effect of aldosterone in mononucler leukocytes (MNL). The aim of the study was to compare, in the model of human MNL, the effect of potassium-sparing diuretics amiloride and canrenone, on the protein expression of p22phox, a NADPH-oxidase system subunit, that is a principal marker of production of superoxide anions. MNL were isolated from 10 informed healthy volunteers (5 males and 5 females, age range 24-36 yr) and the proteins extracted. p22phox protein expression was evaluated by Western blot and quantified using a densitometric semiquantitative analysis. The experiments showed that aldosterone (10(-8) M) enhances the protein expression of p22phox and that its effect is reversed by co-incubation with canrenone (10(-6) M), while incubation with amiloride (10(-6) M) reduced the prooxidative effect of aldosterone at a significantly lower extent than canrenone. Co-incubation with canrenone, amiloride, and aldosterone together produced the same effect as aldosterone plus canrenone. Incubation with cortisol (40(-8) M) was not effective. These data confirm the prooxidative effect of aldosterone in MNL. The addition of aldosterone-receptor antagonist canrenone produced a higher inhibition than sodium channel blocker amiloride on the effect of aldosterone on p22phox protein expression.

The mechanism of mineralocorticoid action of carbenoxolone.

The principal side effects of the drug carbenoxolone (Biogastrone; 18 beta-glycyrrhetinic acid sodium hemisuccinate) are sodium retention, hypokalemic alkalosis, suppressed plasma renin, and hypertension. In previous animal studies, carbenoxolone appeared not to have intrinsic mineralocorticoid activity but, rather, to enhance aldosterone action by displacing it from nonspecific binding sites. We here report studies showing that carbenoxolone has demonstrable affinity for rat kidney mineralocorticoid receptors, intrinsic mineralocorticoid activity in the adrenalectomized rat at doses consistent with its receptor affinity, and, in addition, a powerful action of amplifying the electrolyte effects of near-maximal doses of aldosterone.

Aldosterone receptors in different types of primary hyperaldosteronism.

The number of mineralocorticoid-binding sites on mononuclear leukocytes and plasma aldosterone (aldo) concentrations were measured in patients with different types of primary hyperaldosteronism. Patients with unilateral adenoma and patients with bilateral adrenal hyperplasia had a significantly lower (P less than 0.001) mean number of binding sites for aldo [144 +/- 36 (+/- SD; n = 6) and 140 +/- 28 sites/cell (n = 4), respectively] compared with normal subjects (292 +/- 110 sites/cell; n = 25). In four patients with dexamethasone-suppressible hyperaldosteronism, mineralocorticoid-binding sites in mononuclear leukocytes were normal (291 +/- 108 sites/cell). In all patients undergoing surgery for unilateral adenoma, the receptors normalized 3 months after the operation. In two patients the reduction in receptors persisted for a short time after surgery even though the plasma aldo level had already normalized. We conclude that mineralocorticoid excess produces down-regulation of mineralocorticoid receptors, which, in turn, might contribute to the genesis of the aldo escape phenomenon.

No alteration in the primary structure of the mineralocorticoid receptor in a family with pseudohypoaldosteronism.

We have studied the molecular structure of the mineralocorticoid receptor (MR) complementary DNA (cDNA) in a kindred affected by pseudohypoaldosteronism (PHA). In this family, the clinical symptoms included salt wasting and failure to thrive, accompanied by high urinary levels of sodium despite hyponatremia, hyperkalemia and metabolic acidosis, elevation of PRA, and high plasma aldosterone levels. The patients were resistant to mineralocorticoid administration, but their symptoms ameliorated after a period of sodium supplementation, which was discontinued in older patients. Binding studies performed on mononuclear leukocytes of the members of the family have shown the absence of MR in two siblings and a marked reduction in another sibling of the father, suggesting either the absence of MR or a defect of the ligand-binding domain of the MR in these patients. Southern analysis of patient's DNA did not show any major rearrangement of the MR gene. To search for point mutations in the cDNA of the MR, we performed amplification of the MR cDNA by the polymerase chain reaction and direct sequencing of amplified products. No mutation was found in the entire coding sequence of the MR in patients affected by PHA. Although these results do not exclude a molecular abnormality present on the MR gene, they indicate that PHA in this family is not related to a modification of the MR primary structure.

Cyproheptadine and mineralocorticoid effector mechanisms.

Cyproheptadine has recently been reported to blunt the furosemide-induced rise in PRA in normal subjects and to acutely lower plasma aldosterone levels in patients with hyperaldosteronism due to bilateral adrenal hyperplasia; both actions have tentatively been ascribed to the antiserotoninergic action of the drug. We here describe receptor studies showing cyproheptadine to occupy mineralocorticoid, but not glucocorticoid, receptors in rat and mouse kidney. On bioassay, cyproheptadine is a partial mineralocorticoid agonist/predominant antagonist.

Mineralocorticoid effector mechanism in preeclampsia.

Mineralocorticoid effector mechanisms were evaluated in 29 patients with preeclampsia and in 25 uncomplicated pregnancies by measurement of plasma aldosterone, levels of mineralocorticoid receptor (MR) in mononuclear leucocytes, and subtraction potential difference (SPD; rectal minus oral values). Mean values for plasma aldosterone were not different between the two groups, but significant differences were observed for MR (preeclampsia, 81 +/- 44 receptors/cell; controls, 306 +/- 168) and SPD (preeclampsia, 65 +/- 7 mV; controls, 12 +/- 5 mV). In six cases we determined MR, plasma aldosterone, and SPD in patients with preeclampsia before and 3 months after delivery. MR were reduced before delivery (96 +/- 27 receptors/cell), and SPD increased (64 +/- 8 mV), with both parameters normalizing after delivery (MR, 242 +/- 79; SPD, 14.0 +/- 4 mV). Aldosterone levels returned to normal nonpregnant values after delivery. These data suggest an important role for abnormalities in mineralocorticoid effector mechanisms in the etiology of preeclampsia and could be an useful marker for diagnosis.

Clinical and hormonal effects of the 5 alpha-reductase inhibitor finasteride in idiopathic hirsutism.

Hyperactivity of 5 alpha-reductase in the skin is considered a major mechanism of excessive hair growth in hirsute women with normal levels of serum androgens (idiopathic hirsutism). Preventing the conversion of testosterone to dihydrotestosterone by inhibiting 5 alpha-reductase activity could thus be the most rational and effective treatment in this condition. The present study evaluated the effects of the oral administration of finasteride (5 mg once daily) for 6 months in 17 young women with idiopathic hirsutism, 5 of whom were also given an oral contraceptive. The degree of hirsutism (graded by a modified Ferriman-Gallwey score), serum sex hormone levels, and serum and urinary 5 alpha-metabolism steroid profiles were determined basally and periodically during the treatment period. The modified Ferriman-Gallwey score showed a remarkable reduction after 6 months of finasteride treatment (5.9 +/- 0.6 vs. 11.7 +/- 1.3; P < 0.01). Serum 5 alpha-dihydrotestosterone and 3 alpha-androstanediol glucuronide levels were decreased, and urinary C19 and C21 5 beta/5 alpha metabolite ratios were increased compared with pretreatment values. No significant adverse effect was reported. In women treated with finasteride and oral contraceptive, clinical efficacy was slightly more pronounced. In conclusion, the 5 alpha-reductase inhibitor finasteride is well tolerated and seems to be a useful tool in the treatment of idiopathic hirsutism.

Pseudohypoaldosteronism in eight families: different forms of inheritance are evidence for various genetic defects.

Pseudohypoaldosteronism is a rare hereditary disorder presenting in early infancy with renal salt loss leading to hyponatremia and hyperkalemia despite high levels of plasma aldosterone. The patients are insensitive to mineralocorticoids; however, sodium supplementation is able to correct electrolyte abnormalities. Absent or greatly diminished type I aldosterone receptors in peripheral mononuclear leucocytes have been recently demonstrated and explain the lack of response to mineralocorticoids. We have studied the mode of inheritance in eight families with a total of nine patients. There was evidence for an autosomal recessive form of inheritance in four families, while the other four families appeared to have an autosomal dominant mode of transmission. In three families the autosomal recessive form was characterized by normal receptor as well as hormone data in both parents, while in one family receptor levels in both parents were greatly reduced, but hormone levels were normal. In the four families with an autosomal dominant mode of transmission there was always one parent with reduced receptor binding in peripheral mononuclear leucocytes and elevated serum hormone levels. These parents were entirely asymptomatic. In an extended family we were able to study an aunt and her newborn daughter, who were both also biochemically affected but clinically asymptomatic. It, therefore, appears that this dual pattern of genetic transmission may indicate differing genetic defects which cause the same clinical picture of pseudohypoaldosteronism.

Mineralocorticoid hypertension due to a nasal spray containing 9 alpha-fluoroprednisolone.

The finding of hypokalemia and of low plasma renin activity (PRA) in a hypertensive patient suggests a diagnosis of primary hypermineralocorticoidism. Medications containing compounds with mineralocorticoid-like activity (licorice, carbenexolone) may also cause the same syndrome. Recently, we carried out detailed studies on 10 patients with severe hypertension and hypokalemic alkalosis, suppressed PRA and low aldosterone levels. Plasma levels of cortisol and ACTH were suppressed in most of the cases. Measurement of deoxycorticosterone and corticosterone (and in some patient of 18-hydroxydeoxycorticosterone and 18-hydroxycorticosterone) was not significantly higher than normal. Therapeutic trials of dexamethasone and aminoglutethimide were ineffective. In contrast, spironolactone and amiloride treatment resulted in substantial but incomplete amelioration of both hypertension and hypokalemia. All of the patients share a common history of chronic rhinitis and habitual use of large doses of nasal spray containing 9 alpha-fluoroprednisolone and vasoconstrictor agents. Withdrawal resulted in a complete remission of hypokalemia in one to two weeks in all patients. The hypertension and depressed levels of PRA, aldosterone and cortisol took longer to return to normal, varying from case to case; in all but one patient, the values returned to normal within two months. This report reveals another cause of factitious mineralocorticoid excess which may be considered in the differential diagnosis of hypokalemic hypertensive syndromes.

Corticosteroid receptors in mononuclear leucocytes of obese subjects.

Abnormalities of the hypothalamus-pituitary-adrenal axis and hypersensitivity to corticosteroids have been suggested as major determinants of the development of visceral obesity. Since at the cellular level most effects of corticosteroids are mediated by specific receptors, we evaluated the number of type I and type II corticosteroid receptors in mononuclear leucocytes of 26 obese and 13 control subjects. We also studied the relationship between corticosteroid receptors, measured by radioreceptor assay, and abdominal visceral fat, evaluated by computed tomography scan, plasma and urine corticosteroid hormone concentrations and overall glucose metabolism, assessed by euglycaemic-hyperinsulinaemic clamp. We observed a decrease in type II receptors in the obese subjects (1746 +/- 160 vs 2829 +/- 201 per cell; P < 0.0001), with no change in type I receptors. Type II receptors decreased in relation to body mass index (r = -0.53; P < 0.005) and total glucose disposal (r = 0.51; P < 0.01). Abdominal visceral fat did not correlate with type II receptor number, but did correlate with total glucose disposal (r = -0.35; P < 0.05); the rate of glucose disposal was lower in obese subjects (3.3 +/- 0.3 vs 7.4 +/- 0.4 mg/kg per min; P < 0.001). Plasma and urine cortisol did not differ between the two groups. However, a direct correlation between type II receptor number and both plasma (r = 0.43; P < 0.02) and urine cortisol concentrations (r = 0.60; P < 0.05) was observed. In conclusion, the number of type II corticosteroid receptors in mononuclear leucocytes was found to be lower in obese subjects. This abnormality appears to be related to the degree of adiposity and to the main endocrine-metabolic features of the obesity syndrome, further supporting the hypothesis of involvement of hypothalamus-pituitary-adrenal axis hyperactivity in the pathophysiology of obesity.