Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

Long-Term Follow-Up of Patients With Elevated Aldosterone-to-Renin Ratio but Negative Confirmatory Test: The Progression of Primary Aldosteronism Phenotypes.

BACKGROUND: About 10% of patients with arterial hypertension have a positive screening test for primary aldosteronism (PA) and 50% to 70% of them have a negative confirmatory test: the appropriate follow-up of these patients is currently unknown. We investigated the incidence of PA in patients with previous negative confirmatory testing, after at least a 2-year follow-up. METHODS: One hundred eighty-four patients with a previously elevated aldosterone-to-renin ratio followed by a negative confirmatory test were recruited in 2 hypertension centers (Torino and Munich). We repeated the screening test for PA and, if positive, the confirmatory test (seated saline infusion test or captopril challenge test). Primary end point of the study was the incidence of newly diagnosed overt PA, as defined by a positive confirmatory test. RESULTS: After a mean follow-up of 5 years, 20% of patients developed overt PA. When subtype diagnosis was offered systematically, one-third of patients displayed unilateral PA. Patients who developed PA showed worsening of blood pressure control and a higher rate of cardiac organ damage, despite similar implementation of antihypertensive therapy, compared with patients without PA. A mild progression of autonomous aldosterone secretion was evident even in patients without confirmed PA but with relatively stable control of blood pressure levels over time. CONCLUSIONS: About one-fifth of patients with a negative confirmatory test develop overt PA over time. A clinical follow-up of patients with a negative confirmatory test is advisable, along with the repetition of PA investigation, primarily in patients with worsening of blood pressure control.

Multiomics of Tissue Extracellular Vesicles Identifies Unique Modulators of Atherosclerosis and Calcific Aortic Valve Stenosis.

BACKGROUND: Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown. METHODS: Disease stage-specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells. RESULTS: Disease progression drove significant convergence (P<0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold (P<0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules FGFR2, PPP2CA, and ADAM17 in human carotid artery smooth muscle cells and WNT5A, APP, and APC in human aortic valvular interstitial cells significantly modulated calcification. CONCLUSIONS: The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.

Circulating extracellular vesicles derived from tumor endothelial cells hijack the local and systemic anti-tumor immune response: Role of mTOR/G-CSF pathway.

Circulating tumour-derived extracellular vesicles are supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumour-endothelial cells (TEVs) in the expansion of the metastatic bulk. We focus on the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model. 4T1 cells were intravenously injected and exposed to circulating TEVs from day 7. The lung, spleen, and bone marrow (BM) were recovered and analysed. We demonstrated that circulating TEVs boost lung metastasis and angiogenesis. FACS and immunohistochemically analyses revealed a significant enrichment of Ly6G+/F4/80+/CD11b+ cells and Ly6G+/F4/80-/CD11b+ in the lung and in the spleen, while Ly6G+/F4/80-/CD11b+ in the BM, indicating the occurrence of a systemic and local immune suppression. TEV immune suppressive properties were further supported by the increased expression of PD-L1, PD-1, and iNOS in the tumour mass. In addition, in vitro experiments demonstrated an increase of CD11+ cells, PD-L1+ myeloid and cancer cells, upregulation of LAG3, CTLA4 and PD-1 in T-cells, release of ROS and NOS, and impaired T-cell-mediated cytotoxic effect in co-culture of TEVs-preconditioned PBMCs and cancer cells. Granulocyte-colony stimulating factor (G-CSF) level was increased in vivo, and was involved in reshaping the immune response. Mechanistically, we also found that mTOR enriched TEVs support G-CSF release and trigger the phosphorylation of the S6 (Ser235/236) mTOR downstream target. Overall, we provided evidence that circulating TEVs enriched in mTOR supported G-CSF release thereby granting tumour immune suppression and metastasis outgrowth.

Prevalence of Cortisol Cosecretion in Patients With Primary Aldosteronism: Role of Metanephrine in Adrenal Vein Sampling.

CONTEXT: Adrenal venous sampling (AVS) is the gold standard procedure for subtype diagnosis in patients with primary aldosteronism (PA). Cortisol is usually adopted for the normalization of aldosterone levels in peripheral and adrenal samples. However, asymmetrical cortisol secretion can potentially affect the lateralization index, leading to subtype misdiagnosis. OBJECTIVE: We aimed to assess the prevalence of asymmetrical cortisol secretion in patients undergoing AVS and whether variations in adrenal vein cortisol might influence AVS interpretations. We then evaluated the use of metanephrines for the normalization of aldosterone levels for lateralization index. METHODS: We retrospectively included 101 patients with PA who underwent AVS: 49 patients underwent unstimulated AVS, while 52 patients underwent both unstimulated and cosyntropin-stimulated AVS. Eighty-eight patients had bilateral successful AVS according to metanephrine ratio. We assessed the prevalence of asymmetrical cortisol secretion through the cortisol to metanephrine (C/M) lateralization index (LI). We then evaluated whether the use of aldosterone to metanephrine (A/M) LI can improve the diagnostic accuracy of AVS compared with aldosterone to cortisol (A/C) LI. RESULTS: Asymmetrical cortisol secretion is present in 18% of patients with PA. Diagnosis with A/M LI and A/C LI is discordant in 14% of patients: 9% had a diagnosis of unilateral PA with A/M LI instead of bilateral PA with A/C LI and 5% had a diagnosis of bilateral PA with A/M LI instead of unilateral PA. CONCLUSION: The assessment of metanephrine levels in AVS is useful for the determination of selectivity and lateralization, allowing an accurate diagnosis, especially in patients with asymmetrical cortisol secretion.

Aldosterone as a Mediator of Cardiovascular Damage.

Besides the physiological regulation of water, sodium, and potassium homeostasis, aldosterone modulates several physiological and pathological processes in the cardiovascular system. At the vascular level, aldosterone excess stimulates endothelial dysfunction and infiltration of inflammatory cells, enhances the development of the atherosclerotic plaque, and favors plaque instability, arterial stiffness, and calcification. At the cardiac level, aldosterone increases cardiac inflammation, fibrosis, and myocardial hypertrophy. As a clinical consequence, high aldosterone levels are associated with enhanced risk of cardiovascular events and mortality, especially when aldosterone secretion is inappropriate for renin levels and sodium intake, as in primary aldosteronism. Several clinical trials showed that mineralocorticoid receptor antagonists reduce cardiovascular mortality in patients with heart failure and reduced ejection fraction, but inconclusive results were reported for other cardiovascular conditions, such as heart failure with preserved ejection fraction, myocardial infarction, and atrial fibrillation. In patients with primary aldosteronism, adrenalectomy or treatment with mineralocorticoid receptor antagonists significantly mitigate adverse aldosterone effects, reducing the risk of cardiovascular events, mortality, and incident atrial fibrillation. In this review, we will summarize the major preclinical and clinical studies investigating the cardiovascular damage mediated by aldosterone and the protective effect of mineralocorticoid receptor antagonists for the reduction of cardiovascular risk in patients with cardiovascular diseases and primary aldosteronism.

Role of Extracellular Vesicles in the Pathogenesis of Vascular Damage.

Extracellular vesicles (EVs) are nanosized membrane-bound structures released by cells that are able to transfer nucleic acids, protein cargos, and metabolites to specific recipient cells, allowing cell-to-cell communications in an endocrine and paracrine manner. Endothelial, leukocyte, and platelet-derived EVs have emerged both as biomarkers and key effectors in the development and progression of different stages of vascular damage, from earliest alteration of endothelial function, to advanced atherosclerotic lesions and cardiovascular calcification. Under pathological conditions, circulating EVs promote endothelial dysfunction by impairing vasorelaxation and instigate vascular inflammation by increasing levels of adhesion molecules, reactive oxygen species, and proinflammatory cytokines. Platelets, endothelial cells, macrophages, and foam cells secrete EVs that regulate macrophage polarization and contribute to atherosclerotic plaque progression. Finally, under pathological stimuli, smooth muscle cells and macrophages secrete EVs that aggregate between collagen fibers and serve as nucleation sites for ectopic mineralization in the vessel wall, leading to formation of micro- and macrocalcification. In this review, we summarize the emerging evidence of the pathological role of EVs in vascular damage, highlighting the major findings from the most recent studies and discussing future perspectives in this research field.

Clinical Score and Machine Learning-Based Model to Predict Diagnosis of Primary Aldosteronism in Arterial Hypertension.

[Figure: see text].

Assessment of Anti-Hypertensive Drug Adherence by Serial Aldosterone-To-Renin Ratio Measurement.

Reduced or absent compliance to anti-hypertensive treatment is a major obstacle to the achievement of blood pressure target in patients with arterial hypertension. Current available methods for therapeutic adherence assessment display low accuracy, limited applicability in clinical practice and/or high costs. We designed a prospective study to evaluate the accuracy of serial measurement of ARR to assess the therapeutic compliance to RAAS inhibitors. We prospectively enrolled 80 subjects: 40 patients with arterial hypertension and 40 normotensive controls. The ARR was evaluated at baseline and 2 and 8 week after initiation of a RAAS inhibitor in patients with hypertension, and at baseline and 2 weeks for the control group. Adherence to the prescribed therapy was confirmed by therapeutic drug monitoring. We observed a significant increase of renin levels and reduction of aldosterone levels after RAAS inhibitors initiation, with consequent reduction of ARR. Delta ARR (ΔARR), defined as relative change in ARR before and after treatment initiation, provided high accuracy for determination of therapeutic compliance, with an AUC of 0.900 at 2 weeks and 0.886 at 8 weeks. A cut-off of -48% of ΔARR provided 90% sensitivity and 75% specificity, at 2 and 8 weeks. In conclusion, the measurement of ΔARR is a powerful test, cheap and widely available to accurately identify the non-adherence to RAAS inhibitors treatment. Herein we propose the implementation of ΔARR in clinical practice through a multi-step flow-chart for the management of patients with uncontrolled blood pressure, with identification of those suspected of non-adherence, reserving therapeutic drug monitoring for non-adherence confirmation.

Mineralocorticoid Receptor Antagonist Effect on Aldosterone to Renin Ratio in Patients With Primary Aldosteronism.

CONTEXT: Although current international guidelines recommend to avoid mineralocortcoid receptor antagonists in patients undergoing screening test for primary aldosteronism, a recent report suggested that mineralocorticoid receptor antagonist treatment can be continued without significant influence on screening results. OBJECTIVE: We aimed to evaluate the effect of mineralocorticoid receptor antagonists on the aldosterone to renin ratio in patients with primary aldosteronism. METHODS: We prospectively enrolled 121 patients with confirmed primary aldosteronism who started mineralocorticoid receptor antagonist (canrenone) treatment. Eighteen patients (11 with unilateral and 7 with bilateral primary aldosteronism) constituted the short-term study cohort and underwent aldosterone, renin, and potassium measurement after 2 and 8 weeks of canrenone therapy. The long-term cohort comprised 102 patients (16 with unilateral and 67 with bilateral primary aldosteronism, and 19 with undetermined subtype) who underwent hormonal and biochemical re-assessment after 2 to 12 months of canrenone therapy. RESULTS: Renin and potassium levels showed a significant increase, and the aldosterone to renin ratio displayed a significant reduction compared with baseline after both a short- and long-term treatment. These effects were progressively more evident with higher doses of canrenone and after longer periods of treatment. We demonstrated that canrenone exerted a deep impact on the diagnostic accuracy of the screening test for primary aldosteronism: the rate of false negative tests was raised to 16.7%, 38.9%, 54.5%, and 72.5% after 2 weeks, 8 weeks, 2 to 6 months, and 7 to 12 months of mineralocorticoid receptor antagonist treatment, respectively. CONCLUSION: Mineralocorticoid receptor antagonists should be avoided in patients with hypertension before measurement of renin and aldosterone for screening of primary aldosteronism.

Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model.

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE-/- mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Quality of life in primary aldosteronism: A prospective observational study.

BACKGROUND: Previous studies suggested that patients affected by primary aldosteronism (PA) have impaired quality of life (QOL) compared to the general population, but a direct comparison with patients affected by essential hypertension (EH) has never been performed. The aim of the study was to compare the QOL of patients affected by PA to the QOL of patients affected by EH. MATERIAL AND METHODS: We designed a prospective observational study comparing the QOL of patients with PA and carefully matched patients with EH before and after treatment. We recruited 70 patients with PA and 70 patients with EH, matched for age, sex, blood pressure levels and intensity of antihypertensive treatment. We assessed QOL at baseline and after specific treatment for PA or after optimization of medical therapy for patients with EH. RESULTS: Patients with PA displayed impaired QOL compared with the general healthy population, but similar to patients with EH. Both laparoscopic adrenalectomy and treatment with mineralocorticoid receptor antagonist allowed an improvement of QOL in patients with PA, that was more pronounced after surgical treatment. Optimization of blood pressure control by implementation of antihypertensive treatment (without MR antagonists) allowed a minimal improvement in only one of eight domains in patients with EH. CONCLUSIONS: Patients with PA have impaired QOL, which is likely caused by uncontrolled hypertension and the effects of intensive antihypertensive treatment. Surgical and medical treatment of PA allows a significant improvement of QOL, by amelioration of blood pressure control and, after surgical treatment, by reduction of antihypertensive treatment.

Development of a Prediction Score to Avoid Confirmatory Testing in Patients With Suspected Primary Aldosteronism.

CONTEXT: The diagnostic work-up of primary aldosteronism (PA) includes screening and confirmation steps. Case confirmation is time-consuming, expensive, and there is no consensus on tests and thresholds to be used. Diagnostic algorithms to avoid confirmatory testing may be useful for the management of patients with PA. OBJECTIVE: Development and validation of diagnostic models to confirm or exclude PA diagnosis in patients with a positive screening test. DESIGN, PATIENTS, AND SETTING: We evaluated 1024 patients who underwent confirmatory testing for PA. The diagnostic models were developed in a training cohort (n = 522), and then tested on an internal validation cohort (n = 174) and on an independent external prospective cohort (n = 328). MAIN OUTCOME MEASURE: Different diagnostic models and a 16-point score were developed by machine learning and regression analysis to discriminate patients with a confirmed diagnosis of PA. RESULTS: Male sex, antihypertensive medication, plasma renin activity, aldosterone, potassium levels, and the presence of organ damage were associated with a confirmed diagnosis of PA. Machine learning-based models displayed an accuracy of 72.9%-83.9%. The Primary Aldosteronism Confirmatory Testing (PACT) score correctly classified 84.1% at training and 83.9% or 81.1% at internal and external validation, respectively. A flow chart employing the PACT score to select patients for confirmatory testing correctly managed all patients and resulted in a 22.8% reduction in the number of confirmatory tests. CONCLUSIONS: The integration of diagnostic modeling algorithms in clinical practice may improve the management of patients with PA by circumventing unnecessary confirmatory testing.

Use of Steroid Profiling Combined With Machine Learning for Identification and Subtype Classification in Primary Aldosteronism.

Importance: Most patients with primary aldosteronism, a major cause of secondary hypertension, are not identified or appropriately treated because of difficulties in diagnosis and subtype classification. Applications of artificial intelligence combined with mass spectrometry-based steroid profiling could address this problem. Objective: To assess whether plasma steroid profiling combined with machine learning might facilitate diagnosis and treatment stratification of primary aldosteronism, particularly for patients with unilateral adenomas due to pathogenic KCNJ5 sequence variants. Design, Setting, and Participants: This diagnostic study was conducted at multiple tertiary care referral centers. Steroid profiles were measured from June 2013 to March 2017 in 462 patients tested for primary aldosteronism and 201 patients with hypertension. Data analyses were performed from September 2018 to August 2019. Main Outcomes and Measures: The aldosterone to renin ratio and saline infusion tests were used to diagnose primary aldosteronism. Subtyping was done by adrenal venous sampling and follow-up of patients who underwent adrenalectomy. Statistical tests and machine-learning algorithms were applied to plasma steroid profiles. Areas under receiver operating characteristic curves, sensitivity, specificity, and other diagnostic performance measures were calculated. Results: Primary aldosteronism was confirmed in 273 patients (165 men [60%]; mean [SD] age, 51 [10] years), including 134 with bilateral disease and 139 with unilateral adenomas (58 with and 81 without somatic KCNJ5 sequence variants). Plasma steroid profiles varied according to disease subtype and were particularly distinctive in patients with adenomas due to KCNJ5 variants, who showed better rates of biochemical cure after adrenalectomy than other patients. Among patients tested for primary aldosteronism, a selection of 8 steroids in combination with the aldosterone to renin ratio showed improved effectiveness for diagnosis over either strategy alone. In contrast, the steroid profile alone showed superior performance over the aldosterone to renin ratio for identifying unilateral disease, particularly adenomas due to KCNJ5 variants. Among 632 patients included in the analysis, machine learning-designed combinatorial marker profiles of 7 steroids alone both predicted primary aldosteronism in 1 step and subtyped patients with unilateral adenomas due to KCNJ5 variants at diagnostic sensitivities of 69% (95% CI, 68%-71%) and 85% (95% CI, 81%-88%), respectively, and at specificities of 94% (95% CI, 93%-94%) and 97% (95% CI, 97%-98%), respectively. The validation series yielded comparable diagnostic performance. Conclusions and Relevance: Machine learning-designed combinatorial plasma steroid profiles may facilitate both screening for primary aldosteronism and identification of patients with unilateral adenomas due to pathogenic KCNJ5 variants, who are most likely to show benefit from surgical intervention.

Annexin A1-dependent tethering promotes extracellular vesicle aggregation revealed with single-extracellular vesicle analysis.

Extracellular vesicles (EVs) including plasma membrane-derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)-dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level. Single-EV microarray and proteomics revealed increased ANXA1 primarily on aggregating and calcifying microvesicles. ANXA1 vesicle aggregation was suppressed by calcium chelation, altering pH, or ANXA1 neutralizing antibody. ANXA1 knockdown attenuated EV aggregation and microcalcification formation in human cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings explain why microcalcifications are more prone to form in vulnerable regions of plaque, regulating critical cardiovascular pathology, and likely extend to other EV-associated diseases, including autoimmune and neurodegenerative diseases and cancer.

Primary Aldosteronism and Obstructive Sleep Apnea: Casual Association or Pathophysiological Link?

The coexistence of aldosterone oversecretion and obstructive sleep apnea is frequently observed, especially in patients with resistant hypertension, obesity, and metabolic syndrome. Since aldosterone excess and sleep apnea are both independently associated with an increased risk of cardiovascular disease, to investigate whether their coexistence might be attributed to common predisposing conditions, such as metabolic disorders, or to an actual pathophysiological interconnection appears of great importance. Fluid overload and metabolic abnormalities relating to aldosterone oversecretion may be implicated in obstructive sleep apnea development. Nocturnal intermittent hypoxia may in turn exacerbate renin-angiotensin-aldosterone system activity, thus leading to hyperaldosteronism. Furthermore, fat tissue excess and adipocyte secretory products might predispose to both sleep apnea and aldosterone oversecretion in subjects with obesity. Consistent with these evidences, obstructive sleep apnea frequently affects patients with primary aldosteronism. Conversely, whether primary aldosteronism is more prevalent in individuals affected by obstructive sleep apnea compared to the general population remains controversial.

The spectrum of low-renin hypertension.

Low-renin hypertension (LRH) is a frequent condition in patients with arterial hypertension, accounting for 30% of patients. Monogenic forms can cause LRH in a minority of cases. However, in the large majority of patients, LRH is caused by the combined effects of congenital and acquired factors, comprising dietary habits. Several genetic variants have been proposed as co-factors in the pathogenesis of LRH with normal-low serum aldosterone. Emerging evidences support the hypothesis that a large proportion of LRH with normal-high serum aldosterone is associated with subclinical primary aldosteronism (PA). The recent identification of aldosterone-producing cell clusters (APCCs) as the possible cause of subclinical PA, further supported the concept of a continuous spectrum of autonomous aldosterone secretion, from subclinical forms towards overt PA. In this review we describe the main aspects of LRH, focusing on molecular basis, clinical risk profile and patients' management.

Prevalence of Hypokalemia and Primary Aldosteronism in 5100 Patients Referred to a Tertiary Hypertension Unit.

Primary aldosteronism (PA) was considered a rare disorder almost always associated with hypokalemia. The widespread screening of patients with hypertension unveiled an increased prevalence of PA with normokalemic hypertension the prevailing phenotype. Many studies have reported the prevalence of hypokalemia in patients with PA; conversely, the prevalence of PA in patients with hypokalemia is unknown. In this retrospective observational study, we define the prevalence of hypokalemia in referred patients with hypertension and the prevalence of PA in patients with hypokalemia and hypertension. Hypokalemia was present in 15.8% of 5100 patients with hypertension, whereas 76.9% were normokalemic, and 7.3% hyperkalemic. The prevalence of PA in patients with hypokalemia was 28.1% and increased with decreasing potassium concentrations up to 88.5% of patients with spontaneous hypokalemia and potassium concentrations <2.5 mmol/L. A multivariate regression analysis demonstrated the association of hypokalemia with the occurrence of cardiovascular events independent of PA diagnosis. An association of PA with the occurrence of cardiovascular events and target organ damage independent of hypokalemia was also demonstrated. In conclusion, our results confirm that PA is a frequent cause of secondary hypertension in patients with hypokalemia, and the presence of hypertension and spontaneous hypokalemia are strong indications for PA diagnosis. Finally, we show that PA and hypokalemia are associated with an increased risk of cardiovascular events.

Renal damage in primary aldosteronism: a systematic review and meta-analysis.

OBJECTIVES: In experimental animal models, exogenous aldosterone excess has been linked to the progression of renal disease. However, the evidence of an increased risk of renal damage in patients affected by primary aldosteronism remains controversial. We aimed at evaluating the association between primary aldosteronism and renal damage through a meta-analysis. METHODS: We performed a quantitative review of studies evaluating parameters of renal function in patients affected by primary aldosteronism compared with hypertensive patients without primary aldosteronism and in patients affected by primary aldosteronism before and after treatment. We searched MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials from January 1960 up to April 2019. RESULTS: Forty-six studies including 6056 patients with primary aldosteronism and 9733 patients affected by arterial hypertension without primary aldosteronism were included. After 8.5 years from hypertension diagnosis, patients with primary aldosteronism had an increased estimated glomerular filtration rate (eGFR) compared with hypertensive patients without primary aldosteronism [by 3.37 ml/min IQR (0.82-5.93)] and a more severe albuminuria [standard mean difference 0.55 (0.19-0.91)], resulting into an association with microalbuminuria [odds ratio (OR) 2.09 (1.40; 3.12)] and proteinuria [OR 2.68 (1.89;3.79)]. Following primary aldosteronism treatment, after a median follow-up of 12 months, a reduction in eGFR was observed [by -10.69 ml/min (-13.23; -8.16)], consistent in both medically and surgically treated patients. Similarly, a reduction in albumin excretion and an increase in serum creatinine were observed after treatment. CONCLUSION: Patients affected by primary aldosteronism, compared with patients affected by arterial hypertension without primary aldosteronism, display a more pronounced target organ damage, which can be mitigated by the specific treatment.