Spironolactone-treated Group Research Articles

Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.

Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes. Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation. We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 μL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group. GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.

Anti-stress effects of combined block of glucocorticoid and mineralocorticoid receptors in the paraventricular nucleus of the hypothalamus.

Mineralocorticoid receptors (MRs), glucocorticoid receptors (GRs) and corticotropin-releasing factor (CRF) in the paraventricular nucleus of hypothalamus (PVN) are involved in the response to stress. The present study investigated the role of GRs and MRs in the PVN in regulating depressive and anxiety-like behaviours. To model chronic stress, rats were exposed to corticosterone treatment via drinking water for 21 days, and GR antagonist RU486 and MR antagonist spironolactone, alone and combined, were directly injected in the PVN daily for the last 7 days of corticosterone treatment. Behavioural tests were run on days 22 and 23. Depressive- and anxiety-like behaviours were evaluated in forced swim test, sucrose preference test, novelty-suppressed feeding test and social interaction test. The expression of GRs, MRs and CRF were detected by western blot. Rats exposed to corticosterone exhibited depressive- and anxiety-like behaviours. The expression of GRs and MRs decreased, and CRF levels increased in the PVN. The intra-PVN administration of RU486 increased the levels of GRs and CRF without influencing depressive- or anxiety-like behaviours. The spironolactone-treated group exhibited an increase in MRs without influencing GRs and CRF in the PVN and improved anxiety-like behaviours. Interestingly, the intra-PVN administration of RU486 and spironolactone combined restored expression of GRs, MRs and CRF and improved depressive- and anxiety-like behaviours. In this rat model of stress, the simultaneous restoration of GRs, MRs and CRF in the PVN might play an important role in the treatment of depression and anxiety.

The hemodynamic and metabolic effects of spironolactone treatment in acute kidney injury assessed by hyperpolarized MRI.

Renal ischemia-reperfusion injury (IRI) is one of the most common types of acute kidney injury. Spironolactone has shown promising kidney protective effects in renal IRI in rats. We investigated the hemodynamic and metabolic effects of spironolactone (100 mg/kg) administered immediately after 40 min unilateral kidney ischemia in rats. Hyperpolarized MRI using co-polarized [1-13 C]pyruvate and [13 C,15 N2 ]urea as well as 1 H dynamic contrast-enhanced (DCE) MRI was performed 24 h after induction of ischemia. We found a significant decrease in renal blood flow (RBF) in the ischemic kidney compared with the contralateral one measured using DCE and [13 C,15 N2 ]urea. The RBF measured using [1-13 C]pyruvate and [13 C,15 N2 ]urea was significantly altered by spironolactone. The RBFs in the ischemic kidney compared with the contralateral kidney were decreased similarly as measured using both [13 C,15 N2 ]urea and [1-13 C]pyruvate in the spironolactone-treated group. Spironolactone treatment increased the perfusion-corrected pyruvate metabolism by 54% in both the ischemic and contralateral kidney. Furthermore, we showed a correlation between vascular permeability using a histological Evans blue analysis and the ratio of the volumes of distribution (VoDs), ie VoD-[13 C,15 N2 ]urea/VoD-[1-13 C]pyruvate. This suggests that [13 C,15 N2 ]urea/[1-13 C]pyruvate VoD ratio may be a novel indicator of renal vascular permeability associated with renal damage in rodents.

Spironolactone reduces oxidative stress in living donor kidney transplantation: a randomized controlled trial.

Mineralocorticoid receptor antagonism prevents acute kidney injury induced by ischemia-reperfusion in rodent and pig preclinical models. In a pilot study, we showed that spironolactone (25 mg) reduced oxidative stress after 5 days of kidney transplant (KT). In the present study, we investigated the effects of higher doses (50 and 100 mg) of spironolactone on kidney function, tubular injury markers, and oxidative stress in living donor KT recipients. We included KT recipients aged 18 yr or older who received immunosuppression therapy with IL-2 receptor antagonist, mycophenolate mofetil, corticosteroids, and tacrolimus with negative cross-match, and compatible blood group. Patients were randomized to receive placebo (n = 27), spironolactone (50 mg, n = 25), or spironolactone (100 mg, n = 25). Treatment was given from 3 days before and up to 5 days after KT. Serum creatinine, K+, urine neutrophil gelatinase-associated lipocalin-2, heat shock protein 72, and 8-hydroxy-2-deoxyguanosine levels were assessed. As expected, kidney function was improved after KT. Serum K+ remained in the normal range along the study. There was no significant effect of spironolactone on urinary neutrophil gelatinase-associated lipocalin-2 levels, whereas the increase in urinary heat shock protein 72 levels tended to be less intense in the 100 mg spironolactone-treated group (P = 0.054). In the placebo-treated group, urinary 8-hydroxylated-guanosine levels increased on days 3 and 5 after transplantation. This effect was prevented in patients that received spironolactone. In conclusion, spironolactone reduces the acute increase in urinary oxidative stress in living donor KT recipients.

Effect of spironolactone for 1 yr on endothelial function and vascular inflammation biomarkers in renal transplant recipients.

Kidney transplantation is associated with increased cardiovascular risk. Endothelial dysfunction and vascular inflammation contribute to negative outcome. In experimental models, mineralocorticoid receptor antagonists improved endothelial function and reduced inflammation. The present study tested the hypothesis that the mineralocorticoid receptor antagonist spironolactone improves endothelial function and reduces vascular inflammation in renal transplant patients. Eighty prevalent renal transplant patients from an ongoing, double-blind randomized placebo-controlled trial were included. Paired plasma samples before and after 1 yr of treatment (n = 39 in the spironolactone-treated group and 41 in the placebo-treated group) were used to determine markers of endothelial dysfunction (nitrite, nitrate, cGMP, arginine, citrulline, ornithine, asymmetric dimethylarginine, symmetric dimethylarginine, NG-monomethyl-l-arginine, von Willebrand factor, tissue-type plasminogen activator antigen, and plasminogen activator inhibitor 1 antigen) and markers of inflammation (intercellular adhesion molecule, vascular adhesion molecule, high-sensitivity C-reactive protein, and serum amyloid protein A). The median time since the transplantation was 4.6 (0.12-22.3) yr in the spironolactone-treated group and 2.1 (0.17-13.9) yr in the placebo-treated group (P > 0.05). Spironolactone increased plasma aldosterone (P < 0.001) and K+ (P < 0.001). Blood pressure did not change significantly. No significant differences were detected between groups in any of the measured markers of endothelial dysfunction or inflammation except in the subgroup analysis of patients with diabetes, where spironolactone decreased nitrite compared with placebo. In this study, mineralocorticoid receptor antagonism did not improve biomarkers of endothelial dysfunction or vascular inflammation in prevalent renal transplant patients. Further studies are needed to evaluate the potential beneficial effect of early or late mineralocorticoid receptor antagonism on vascular outcomes in renal transplant patients.

Clinical benefit of spironolactone in patients with acute decompensated heart failure and severe renal dysfunction: Data from the Korean Heart Failure Registry

We investigated the relationship between spironolactone use and all-cause mortality in acute decompensated heart failure (ADHF) patients with severe renal dysfunction. The clinical benefit of spironolactone in the treatment of heart failure (HF) has been described in several large randomized clinical trials. However, its clinical benefits have not been studied in hospitalized ADHF patients with severe renal dysfunction (estimated glomerular filtration rate [eGFR] <45 mL/min per 1.73 m(2)). We retrospectively analyzed data from the Korean Heart Failure Registry. We included 1,035 ADHF patients with severe renal dysfunction. In Kaplan-Meier survival analysis, all-cause mortality in the spironolactone-treated group was significantly lower than that in the nonspironolactone group (18.1% vs 24.9%, respectively, log rank P = .028). However, spironolactone use was not an independent predictor after adjusting other HF risk factors (hazard ratio 0.974, 95% CI 0.681-1.392, P = .884) and after propensity score matching (P = .115). In subgroup analysis, the clinical benefit of spironolactone use was preserved in women, prehospital spironolactone use, the chronic kidney disease stage 3b (eGFR 30-44 mL/min per 1.73 m(2)), and the appropriate spironolactone use (eGFR ≥30 mL/min per 1.73 m(2) and K ≤5.0 mmol/L). The spironolactone therapy was not beneficial in ADHF patients with severe renal dysfunction after multivariable adjusting and propensity score matching. However, we reassured the current HF guidelines for spironolactone use and the clinical benefit in chronic kidney disease stage 3b should be assessed in future clinical trial.

Effects of Angiogenesis Inhibition by Spironolactone on Isolated Vas Deferens Contractility in an Experimental Varicocele Model in Rats

To investigate the effect of spironolactone, as an angiogenesis inhibitor, on the isometric contractile responses in isolated vas deferens strips from left varicocele-induced rats. Twenty-four adult (12-14 months) male Wistar albino rats were randomly assigned to 4 groups (n = 6 in each): (1) Control group, (2) sham-operated group, (3) experimental left varicocele group, and (4) Spironolactone (20 mg/kg/d)-treated experimental left varicocele group. Histopathologic and immunohistochemical (CD31 staining) findings in the rat testis and functional findings in the rat isolated vas deferens were investigated. Angiogenesis increased in the varicocele group and the spironolactone inhibited angiogenesis in the spironolactone-treated group. Spironolactone seemed to change phenylephrine and serotonin responses in the left vas deferens. It is possible that by inhibiting angiogenesis, spironolactone treatment negatively impairs testicular morphology and functional (vas deferens) pathways. Varicocele formation seems to elicit an increase to 5-HT sensitivity in rat vas deferens, and this process is prevented by spironolactone pretreatment.

Spironolactone and Enalapril Differentially Up-Regulate the Expression of VEGF and Heme Oxygenase-1 in the Neonatal Rat Kidney

Both the renin-angiotensin-aldosterone system (RAAS) and hypoxia are vital physiological factors involved in the control of nephrogenesis and vascularization. We investigated the relationship between RAAS and hypoxia in the developing kidney. The expression of VEGF and heme oxygenase (HO)-1 related with the oxygen was analyzed in the enalapril- or spironolactone-treated neonatal rat kidneys. Enalapril (30 mg/kg/d) or spironolactone (200 mg/kg/d) was administered to newborn rat pups for 7 d. The newborn rats were injected i.p. with pimonidazole (200 mg/kg), a marker of severe tissue hypoxia, 1 h before killing. VEGF and HO-1 protein expression was significantly increased by immunoblots and immunohistochemistry in both the enalapril- and spironolactone-treated kidneys, compared with the controls (p < 0.05). HO-1 mRNA expression was increased in the spironolactone-treated group (p < 0.05). The immunoactivity of pimonidazole was not different from that of the controls in the enalapril-treated group, whereas it was increased in the spironolactone-treated group. The results of this study indicate that aldosterone blockade or angiotensin II inhibition in the developing rat kidney up-regulated renal VEGF and HO-1 expression regardless of the hypoxic conditions and may differentially modulate VEGF and HO-1 production.

Aldosterone Blockade in Diabetic Nephropathy

In the 1950s, electrocortin, as aldosterone was then known,1 was a relatively novel hormone, the importance of which in human pathophysiology was established by reports of cases of aldosterone excess by Conn in 1955.2 Despite more than 50 yr of study, the breadth of many actions of aldosterone continues to unfold in basic, translational, and clinical milieus. From a basic aspect, aldosterone has typical genomic effects. After receptor binding, aldosterone exposure initiates transcriptional processes, which increase protein synthesis in pathways that enhance sodium retrieval from tubular lumens, salivary and sweat glands, and colon. Nongenomic aspects have been reported in which aldosterone infusion reduces the caliber of isolated afferent and efferent arterioles3 through the activation of systems that inhibit endothelial nitric oxide action.4 From a translational aspect, the synthetic pathways detailing aldosterone production offer the opportunity for studying genes such as CYP11B2 , which regulates production of 11β-hydroxylase, a key enzyme in corticosteroid production.5 Population studies of polymorphisms in the CYP11B2 gene suggest that compensation for reduced activity of CYP11B2 , through modest increases in the release of ACTH, results in upregulation of other …

Aldosterone regulates cellular turnover and mitogen‐activated protein kinase family expression in the neonatal rat kidney

Growing evidence indicates that aldosterone is a potent mitogenic signal regulating genes involved in antiapoptosis, cell proliferation and growth. We investigated the role of endogenous aldosterone in renal development, cell proliferation and apoptosis, and mitogen-activated protein kinase (MAPK) family expression. Newborn rats were treated with either spironolactone (200 mg/kg/d) in olive oil or only olive oil for 7 days. TUNEL assay and proliferating cell nuclear antigen (PCNA) stain were performed on kidney sections. Immunoblots, immunohistochemical (IHC) stain, and reverse transcriptase-PCR for MAPKs were performed. PCNA-positive proliferating cells decreased and apoptotic cells increased significantly with spironolactone (P < 0.05). In the spironolactone-treated group, c-jun N-terminal kinase (JNK)-2 expression increased, whereas extracellular signal regulated kinase (ERK)-2 and p38 expressions decreased in immunoblots (P < 0.05) and IHC stain. ERK-2 and p38 mRNA expressions increased in the spironolactone-treated group (P < 0.05). This study demonstrates that aldosterone blockade in the developing kidney decreases cellular proliferation, increases apoptosis, and modulates the expressions of JNK-2, ERK-2, and p38. Aldosterone possibly participates in renal development and MAPK family may serve as, in part, the signaling intermediate through the mineralocorticoid receptor (MR) in the developing kidney. J. Cell. Physiol. 219: 724-733, 2009. (c) 2009 Wiley-Liss, Inc.

The effect of spironolactone on experimental periodontitis in rats

Elevated levels of tumour necrosis factor (TNF) have been found in patients with adult periodontitis. Animal studies have shown that TNF plays an important role in the pathogenesis of periodontitis. New findings suggest that the aldosterone-inhibitor spironolactone possesses an anti-TNF effect. The purpose of the study was to determine the anti-TNF effect of spironolactone in an endotoxic shock rat model and to disclose the effect of oral administration of spironolactone on the development of experimental periodontitis in rats. The study was divided in two parts. Part 1: oral administration of spironolactone (100 mg/kg) followed by intravenous lipopolysaccharide (1 mg/kg) infusion 45 min later. Blood samples were taken before and 90 min after lipopolysaccharide infusion to determine the TNF levels in spironolactone treated and non-treated rats. Part 2: oral administration of spironolactone [100 mg/(kg day)] starting 2 days prior to induction of experimental periodontitis established by peridental ligatures. Morphometrical and radiographical registrations of alveolar bone destruction were carried out to determine the effect of spironolactone on the progression of experimental periodontitis. In part 1 the endotoxic shock model showed a significant reduction in TNF levels in the spironolactone-treated group compared to the non-treated group, suggesting that spironolactone acts as a TNF inhibitor. In part 2 spironolactone-treated rats did not demonstrate significantly less alveolar bone destruction compared to non-treated rats. The insignificant effect of spironolactone treatment could be explained by the fast metabolism of spironolactone and that spironolactone does not completely inhibit TNF production in rats. Moreover, many other cytokines and mediators involved in alveolar bone destruction may account for the lacking response to spironolactone.

Spironolactone in Combination with Cilazapril Ameliorates Proteinuria and Renal Interstitial Fibrosis in Rats with Anti-Thy-1 Irreversible Nephritis

Blockade of the renin-angiotensin system has been established as a treatment for heart failure with hypertension and left ventricular hypertrophy, and for progressive kidney diseases. The present study was conducted to examine whether spironolactone, a mineralocorticoid receptor antagonist, alone or in combination with cilazapril, an angiotensin converting enzyme (ACE) inhibitor, ameliorates proteinuria and renal lesions in an immune-initiated progressive nephritis model. Wistar rats were uninephrectomized 7 days before injection of anti-Thy-1 monoclonal antibody 1-22-3 to induce progressive glomerulonephritis. The nephritic rats were untreated or treated with spironolactone (400 mg/kg body weight/day), cilazapril (1 mg/kg body weight/day), or both for 10 weeks. Proteinuria was increased in the untreated rats 1 week after nephritis induction and was maintained throughout the experiment. Compared with the untreated animals (212.9+/-49.2 mg/day), proteinuria was significantly reduced in the spironolactone-treated group (62.0+/-4.0 mg/day, p=0.0046) and the cilazapril-treated group (71.8+/-26.0 mg/day, p=0.0048) on day 70 after antibody injection. Further reduction of proteinuria (42.4+/-4.5 mg/day, p=0.0019 vs. the untreated group) and less renal cortex interstitial fibrotic change (fibrosis score: 142.0+/-18.4 vs. 80.3+/-18.5 in the untreated group, p=0.0123) were detected in the spironolactone plus cilazapril-treated group. Blood pressure did not differ among the three treatment groups. In conclusion, spironolactone ameliorates proteinuria to the same degree as cilazapril, and concomitant use of spironolactone and an ACE inhibitor further suppresses renal disease progression. These data suggest that concomitant treatment with spironolactone and an ACE inhibitor has beneficial effects on immune-initiated progressive kidney disease.

Influence of spironolactone on urinary prostaglandin E 2 and kinin excretion in rats

Spironolactone was administered to spontaneously hypertensive rats (SHRs) in order to examine the urinary excretions of prostaglandin E 2 (PGE 2) and kinin. Thirteen SHRs were divided into 2 groups: 0.1 ml of sesame oil was administered to one group (the spironolactone-lactone-untreated group, n = 6) and 20 mg of spironolactone in 0.1 ml of sesame oil was administered to the other group (the spironolactone-treated group, n = 7) by the subcutaneous route for 10 days in succession. Determinations were then made of the body weight, blood pressure, urine volume, and excretion levels of Na, K, kinin and PGE 2 in the 24-hour urine. After the animals had been killed by decapitation, blood samples were drawn for determination of the plasma renin activity (PRA). The results obtained indicated decreased blood pressure and increased urinary Na excretion in the spironolactone-treated group. On the other hand, the PGE 2 excretion level in the 24-hour urine decreased markedly immediately after administration of spironolactone (p<0.05) and was maintained at lower levels up to the end of the experiment. However, the 24-hour urinary kinin levels showed similar changes in both the spironolactone-treated group and the untreated group with no significant difference between them. These findings suggest that spironolactone has a suppressive effect on urinary PGE 2 excretion, the activity of which is not mediated by kinin production in the kidneys but is the result of a direct action of spironolactone itself.

Influence of spironolactone on urinary prostaglandin E2 and kinin excretions in spontaneously hypertensive rats

Spironolactone was administered to spontaneously hypertensive rats (SHRs) in order to examine the urinary excretions of prostaglandin E2 (PGE2) and kinin. Thirteen SHRs were divided into 2 groups, and 0.1 ml of sesame oil was administered to one of 2 groups (spironolactone-untreated group) (n = 6) and 20 mg of spironolactone in 0.1 ml of sesame oil was administered to the other group (spironolactone-treated group) (n = 7) by the subcutaneous route for 10 days in succession, followed by the determination of body weight, blood pressure, urine volume, excretion levels of Na, K, kinin and PGE2 in 24-hour urine. Blood samples were drawn after these animals were killed by decapitation for determining plasma renin activity (PRA). In consequence, decreased blood pressure and increased urinary Na excretion were observed in the spironolactone-treated group. On the other hand, PGE2 excretion level in 24-hour urine markedly decreased immediately after administration of spironolactone (p less than 0.05) and was maintained at lower levels up to the end of experiment. However, 24-hour urinary kinin levels showed similar changes in the spironolactone-treated group and the untreated group with no significant difference between the two groups. These results indicate that spironolactone has a suppressive effect on urinary PGE2 excretion, the activity of which is not mediated by kinin production in the kidneys but produced by a direct action of spironolactone itself.

Long term spironolactone and the adrenal cortex in essential hypertension.

In view of recent evidence that spironolactone may inhibit synthesis of corticosteroids by a direct effect on the adrenal cortex, adrenocortical function was studied in eight patients with essential hypertension who had been treated with spironolactone from 3 months to 14 years. Their 24 h renal excretion of adrenocorticoid metabolites and the responses of cortisol, aldosterone and 18-hydroxy-11 -deoxycorticosterone (18-OH-DOC) to an incremental infusion of tetracosactrin (1-24 ACTH) were compared with those in eight patients with recently diagnosed essential hypertension who had received no spironolactone. The spironolactone-treated group had a significantly higher excretion of aldosterone, whilst the excretion of other adrenocorticoid metabolites did not differ. The same group also required less tetracosactrin to stimulate a detectable rise of plasma cortisol and 18-OH-DOC, they had greater plasma 18-OH-DOC responses at all infusion rates and, at the lowest infusion rates, had greater aldosterone responses. These results indicate that long-term spironolactone therapy does not inhibit adrenocortical function and may have some stimulatory effects.

Pathophysiology of spironolactone-induced gynecomastia.

Peripheral blood levels of testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone and the metabolic clearance rates of testosterone and estradiol, as well as the peripheral conversion of testosterone into estradiol, were measured in 16 patients with hypertension. Six of these patients were treated with spironolactone and developed gynecomastia. The other 10 patients served as control subjects. The blood testosterone level in the spironolactone-treated group (2.7 +/- 0.5 ng/ml) was significantly less (P less than 0.02) than in the control group (4.4 +/- 0.4 ng/ml). On the other hand, blood estradiol levels in the spironolactone group (30 +/- 4 pg/ml) were significantly greater (P less than 0.01) than in the control group (13 +/- 2 pg/ml). These changes were primarily due to significant increases in the metabolic clearance rate of testosterone (P less than 0.02) and in the rate of peripheral conversion of testosterone into estradiol (P less than 0.001) in the spironolactone-treated group. Thus, spironolactone does alter the peripheral metabolism of testosterone resulting in changes in the ratio of testosterone to estradiol, which could contribute to the production of gynecomastia.