Aldosterone blunts tubuloglomerular feedback by activating macula densa mineralocorticoid receptors

Chronic aldosterone administration increases glomerular filtration rate, whereas inhibition of mineralocorticoid receptors (MRs) markedly attenuates glomerular hyperfiltration and hypertension associated with primary aldosteronism or obesity. However, the mechanisms by which aldosterone alters glomerular filtration rate regulation are poorly understood. In the present study, we hypothesized that aldosterone suppresses tubuloglomerular feedback (TGF) via activation of macula densa MR. First, we observed the expression of MR in macula densa cells isolated by laser capture microdissection and by immunofluorescence in rat kidneys. Second, to investigate the effects of aldosterone on TGF in vitro, we microdissected the juxtaglomerular apparatus from rabbit kidneys and perfused the afferent arteriole and distal tubule simultaneously. Under control conditions, TGF was 2.8±0.2 μm. In the presence of aldosterone (10(-8) mol/L), TGF was reduced by 50%. The effect of aldosterone to attenuate TGF was blocked by the MR antagonist eplerenone (10(-5) mol/L). Third, to investigate the effect of aldosterone on TGF in vivo, we performed micropuncture, and TGF was determined by maximal changes in stop-flow pressure P(sf) when tubular perfusion rate was increased from 0 to 40 nL/min. Aldosterone (10(-7) mol/L) decreased ΔP(sf) from 10.1±1.4 to 7.7±1.2 mm Hg. In the presence of l-NG-monomethyl arginine citrate (10(-3) mol/L), this effect was blocked. We conclude that MRs are expressed in macula densa cells and can be activated by aldosterone, which increases nitric oxide production in the macula densa and blunts the TGF response.     

A highly sensitive immunofluorometric assay for the measurement of aldosterone in small sample volumes: validation in mouse serum

Data on the involvement of aldosterone in the regulation of the renin-angiotensin-aldosterone system (RAAS) in rodents are still scarce, partly due to the high sample volumes needed by commercially available assays and to the very low aldosterone concentrations present. We have developed a highly sensitive and non-isotopic immunoassay, requiring a volume of only 50 microl serum for a duplicate measurement, employing a highly specific monoclonal antibody against aldosterone. The assay was validated in human and mouse samples and exhibited a linear working range from 10 to 1000 pg/ml. Values obtained after a chromatographic purification step correlated significantly to the dichloromethane extraction ordinarily used. Basal aldosterone values were measured in 75 mouse hybrids and found within the linear range (173+/-21 pg/ml), with no significant difference between males and females. Additionally, we show an increase in serum aldosterone in mice from 3 to 11 weeks of age. Mice of the same genetic background were treated with dexamethasone intraperitoneally (n=7), resulting in significantly decreased concentrations (35+/-3 vs 114+/-33 pg/ml in controls; P<0.001). In contrast, adrenocorticotropic hormone resulted in significantly increased serum aldosterone (603+/-119 pg/ml; n=7; P<0.001), as did the physiological stimulation of the RAAS by a high K(+)/low Na(+) diet (1369+/-703 vs 172+/-36 pg/ml). In conclusion, we have developed and validated an extremely sensitive assay for determination of aldosterone concentrations from very small serum samples, which could be especially useful in pharmacological intervention studies in rodent models.     

Receptor binding and biological activity of 18-hydroxycortisol

Recently, 18-hydroxycortisol (11 beta,17 alpha,18,21-tetrahydroxy-4-pregnene-3,20-dione) was isolated and identified from extracts of urine and adrenal incubates of patients with primary aldosteronism. The receptor-binding activity to the renal gluco- and mineralocorticoid receptors and its biological activity as a glucocorticoid and mineralocorticoid were investigated using synthetic 18-hydroxycortisol. The ability of 18-hydroxycortisol to compete with [3H]aldosterone for renal binding to the receptor was 0.13% that of unlabeled aldosterone. The addition of a specific glucocorticoid, RU-26988 (11 beta,17-dihydroxy-21-methyl-17 alpha-pregna-1,4,6-triene-20-yn-3-one) decreased the competing ability to 0.02%, indicating significant binding to the glucocorticoid receptor. The ability to compete with [3H]dexamethasone for the renal cytoplasmic glucocorticoid receptor was 0.1% that of unlabeled dexamethasone. The mineralocorticoid activity of 18-hydroxycortisol was undetectable. Its glucocorticoid activity using an in vitro bioassay based on the induction of tyrosine aminotransferase in the HTC cell was detectable at 10(-5) M, but was too low for adequate quantification. In a second in vitro glucocorticoid bioassay, inhibition of cell growth of the L929 fibroblast, 18-hydroxycortisol also showed minimal activity. In summary, it is unlikely that 18-hydroxycortisol plays a role in the metabolic syndrome in those patients who produce it in excess due to its inactivity as a gluco- or mineralocorticoid.     

Does aldosterone upregulate the brain renin-angiotensin system in rats with heart failure?

The brain renin-angiotensin system (RAS) contributes to increased sympathetic drive in heart failure (HF). The factors upregulating the brain RAS in HF remain unknown. We hypothesized that aldosterone (ALDO), a downstream product of the systemic RAS that crosses the blood-brain barrier, signals the brain to increase RAS activity in HF. We examined the relationship between circulating and brain ALDO in normal intact rats, in adrenalectomized rats receiving subcutaneous infusions of ALDO, and in rats with ischemia-induced HF and sham-operated controls. Brain ALDO levels were proportional to plasma ALDO levels across the spectrum of rats studied. Compared with sham-operated controls rats, HF rats had higher plasma and hypothalamic tissue levels of ALDO. HF rats also had higher expression of mRNA and protein for angiotensin-converting enzyme and angiotensin type 1 receptors in the hypothalamus, increased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide generation in the paraventricular nucleus of the hypothalamus, increased excitation of paraventricular nucleus neurons, and increased plasma norepinephrine. HF rats treated for 4 weeks with intracerebroventricular RU28318 (1 microg/h), a selective mineralocorticoid receptor antagonist, had less hypothalamic angiotensin-converting enzyme and angiotensin type 1 receptor mRNA and protein, less reduced nicotinamide-adenine dinucleotide phosphate-induced superoxide in the paraventricular nucleus, fewer excited paraventricular nucleus neurons, and lower plasma norepinephrine. RU28318 had no effect on plasma ALDO or on angiotensin-converting enzyme or angiotensin type 1 receptor expression in brain cortex. The data demonstrate that ALDO of adrenal origin enters the hypothalamus in direct proportion to plasma levels and suggest that ALDO contributes to the upregulation of hypothalamic RAS activity and sympathetic drive in heart failure.     

Reversal of acute ("malignant") myelosclerosis by allogeneic bone marrow transplantation

A 28-yr-old woman with acute malignant myelosclerosis received, as primary treatment, ablative chemotherapy and total body radiation therapy followed by bone marrow transplantation from her histocompatible brother. The patient is now well more than 15 mo after bone marrow transplantation, with normal peripheral blood counts, a normal bone marrow, no evidence of graft-versus-host disease, and is on no therapy. In light of the poor results obtained with conventional chemotherapy in this disease, bone marrow transplantation may represent the treatment of choice for patients who have an appropriate donor.     

Integrin alpha 4 beta 1 and glycoprotein IV (CD36) are expressed on circulating reticulocytes in sickle cell anemia

The abnormal adherence of red blood cells, especially circulating reticulocytes (erythrocyte precursors), to the endothelium is believed to contribute to vascular occlusion observed in patients with sickle cell disease. Although several plasma proteins including von Willebrand factor and fibronectin have been proposed to mediate this adhesion, the mechanism of sickle cell adhesion to the endothelium remains unknown. Using flow cytometry, we screened sickle red blood cells with monoclonal antibodies (MoAbs) against known adhesion receptors and detected integrin subunits alpha 4 and beta 1 and the nonintegrin glycoprotein IV on reticulocytes but not on erythrocytes. No reactivity was detected against integrin subunits alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 2, beta 3, integrin alpha IIb beta 3, or the nonintegrin glycoprotein Ib. Immunoprecipitation of reticulocytes with either alpha 4- or beta 1-specific antibodies identified the alpha 4 beta 1 complex (alpha 4(70) and alpha 4(80) forms), a receptor for fibronectin and vascular cell adhesion molecule-1. An antibody against glycoprotein IV, a receptor reported to bind thrombospondin and collagen, immunoprecipitated an 88-kD protein consistent with its reported M(r). MoAbs against alpha 4 and glycoprotein IV bound to an average of 4,600 and 17,500 sites per reticulocyte, respectively. Identification of alpha 4 beta 1 and glycoprotein IV on reticulocytes suggests both plasma-dependent and independent mechanisms of reticulocyte adhesion to endothelium and exposed extracellular matrix.     

Glucocorticoid remediable aldosteronism: low morbidity and mortality in a four-generation italian pedigree

Glucocorticoid remediable hyperaldosteronism (GRA) is a monogenic form of inherited hypertension caused by a chimeric gene originating from an unequal cross-over between the 11 beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes. GRA is characterized by high plasma levels of aldosterone (regulated by ACTH) with suppressed plasma renin activity and the production of two rare steroids, 18hydroxycortisol and 18oxocortisol. Affected patients usually show severe hypertension and an elevated frequency of stroke at a young age. Affected women have a high risk of developing preeclampsia during pregnancy. Here, we describe a 5-generation pedigree from Sardinia in which the presence of the chimeric gene is demonstrated in 4 generations. This family displays a mild phenotype with average blood pressure levels of 131/86 mm Hg for GRA+ patients. The occurrence of stroke is very low, and preeclampsia was not observed in 29 pregnancies from 8 GRA+ mothers. We investigated whether the cross-over site (between the CYP11B1 and CYP11B2 genes) or biochemical characteristics could explain this phenotype. The cross-over site was located at the end of intron 3, in the same region as described in other families. We found a significant correlation between blood pressure and 18hydroxycortisol, 18oxocortisol, and plasma aldosterone levels, but not with kallikrein. However, none of the biochemical or genetic parameters investigated could explain the mild phenotype of the family.     

Multicenter trial of the proficiency of smart quantitative sensation tests

Introduction: We assessed proficiency (accuracy and intra‐ and intertest reproducibility) of smart quantitative sensation tests (smart QSTs) in subjects without and with diabetic sensorimotor polyneuropathy (DSPN). Methods: Technologists from 3 medical centers using different but identical QSTs independently assessed 6 modalities of sensation of the foot (or leg) twice in patients without (n = 6) and with (n = 6) DSPN using smart computer assisted QSTs. Results: Low rates of test abnormalities were observed in health and high rates in DSPN. Very high intraclass correlations were obtained between continuous measures of QSTs and neuropathy signs, symptoms, or nerve conductions (NCs). No significant intra‐ or intertest differences were observed. Conclusions: These results provide proof of concept that smart QSTs provide accurate assessment of sensation loss without intra‐ or intertest differences useful for multicenter trials. Smart technology makes possible efficient testing of body surface area sensation loss in symmetric length‐dependent sensorimotor polyneuropathies. Muscle Nerve 49 : 645–653, 2014