Recent studies in hypertensive populations that have used the serum aldosterone (SA) to plasma renin activity (PRA) ratio as a screening test have demonstrated a high prevalence of primary aldosteronism (PA). This frequency is higher than that previously described when hypokalemia was used as a screening tool. However, other factors, such as the characteristics of hypertensive disease, could also influence the prevalence of PA. We studied 609 essential hypertensive patients, classified according to the Joint National Committee VI (JNC VI), in 3 different stages depending on the severity of their hypertensive disease. We measured SA and PRA and calculated the SA-PRA ratio for all patients. An SA-PRA ratio >25 was detected in 63 of 609 patients, and the fludrocortisone test confirmed the PA diagnoses in 37 of 609 (6.1%) cases. PA prevalence according to hypertension stage was as follows: stage 1, 6 of 301 cases (1.99%); stage 2, 15 of 187 cases (8.02%); and stage 3, 16 of 121 cases (13.2%). PA patients were slightly younger than the other hypertensive patients (48.4±10.5 vs 53.6±10.2 years; P <0.05). Serum potassium levels were normal in 36 of 37 PA patients; only 1 patient had minor hypokalemia. Computed tomography scans showed bilateral adrenal enlargement in 7 and an adrenal nodule in 2 cases. In summary, we found a high frequency of PA in essential hypertensives classified in stages 2 and 3 according to the JNC VI. The low frequency of computed tomography scan abnormalities and hypokalemia suggests that the diagnosis for most PA patients corresponds to attenuated forms of the disease.

Excessive production of aldosterone leads to the development of hypertension and cardiovascular disease by generating an inflammatory state that can be promoted by T cell immunity. Because nature and intensity of T cell responses is controlled by dendritic cells (DCs), it is important to evaluate whether the function of these cells can be modulated by aldosterone. In this study we show that aldosterone augmented the activation of CD8⁺ T cells in a DC-dependent fashion. Consistently, the mineralocorticoid receptor was expressed by DCs, which showed activation of MAPK pathway and secreted IL-6 and TGF-β in response to aldosterone. In addition, DCs stimulated with aldosterone impose a Th17 phenotype to CD4⁺ T cells, which have recently been associated with the promotion of inflammatory and autoimmune diseases. Accordingly, we observed that aldosterone enhances the progression of experimental autoimmune encephalomyelitis, an autoimmune disease promoted by Th17 cells. In addition, blockade of the mineralocorticoid receptor prevented all aldosterone effects on DCs and attenuated experimental autoimmune encephalomyelitis development in aldosterone-treated mice. Our data suggest that modulation of DC function by aldosterone enhances CD8⁺ T cell activation and promotes Th17-polarized immune responses, which might contribute to the inflammatory damage leading to hypertension and cardiovascular disease.

Significance Statement: Urinary extracellular vesicles (uEVs) are a promising noninvasive source of kidney biomarkers, but the optimal approaches for normalization, quantification, and characterization in spot urines are unclear. To address the hypothesis that urine creatinine can be used as a normalization variable, urine particles were quantified in dilute and concentrated urines (water deprivation–loading study) and randomly from healthy subjects and patients with kidney disease. In these various settings, urine creatinine was highly correlated with particle counts, suggesting it can be used as a normalization variable. Additional findings relevant for future uEV studies include interference of Tamm-Horsfall protein with nanoparticle tracking analysis, excretion of larger uEVs in dilute urine, and the ability to treat uEVs with detergent to enhance intracellular epitope recognition. Background: Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear. Methods: Urine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization. Results: Urine particle and creatinine concentrations were highly correlated in the water-loading study (R ² 0.96) and in random spot urines from healthy subjects (R ² 0.47–0.95) and patients (R ² 0.41–0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers. Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment–specific EVs.

Objective Primary aldosteronism (PA) is the most common secondary cause of hypertension and recently has been implicated as a cause of impaired glucose tolerance. We investigated the glucose insulin sensitivity and insulin secretion in patients with idiopathic primary aldosteronism. Design Thirty PA patients and 60 essential hypertensive (EH) patients as controls were included, matched (1: 2) by their body mass index (BMI) (29.9 ± 4.3 versus 29.8 ± 5.8 m/kg²), age (53.7 ± 9.4 versus 59.9 ± 8.6 years old) and gender (male/female: 8/22 versus 17/43). In all patients, we measured insulin, total cholesterol, triglycerides, C-peptide and fasting glucose levels. Homeostasis model assessment for insulin resistance (HOMA-IR) and HOMA of pancreatic β-cell function (HOMA-βF) indexes were calculated. We also evaluated the response to spironolactone in 19 PA patients. Results PA patients had higher levels of glucose (5.2 ± 0.7 versus 4.9 ± 0.7 mmol/l; P = 0.017). Insulin levels (10.7 ± 6.5 versus 11.5 ± 5.8 uUI/ml, P = 0.525) and HOMA-IR (2.51 ± 1.59 versus 2.45 ± 1.29 uUI/ml × mmol/l, P = 0.854) were similar in both groups. HOMA-βF index (138.9 ± 89.8 versus 179.8 ± 100.2%, P = 0.049) and C-peptide (0.83 ± 0.63 versus 1.56 ± 0.84 ng/dl, P = 0.0001) were lower in PA patients. Potassium was normal in both groups. Negative correlations between serum aldosterone/plasma renin activity (SA/PRA) ratio and HOMA-βF, and between C-peptide and SA levels were found in all patients. After the spironolactone treatment, we found an increase of C-peptide and insulin levels without changes in HOMA-IR or HOMA-βF. Conclusion Our results showed differences in glucose metabolism between PA patients and those with hypertension suggesting that these findings could probably be determined by a lower β-cell function influenced by aldosterone. These findings highlight the importance of aldosterone in glucose metabolism.