The etiology of arterial hypertension (AHT) is unknown in more than 80-90% of cases, which is named essential hypertension (EH). One third of EH has been suggested to be associated with endocrine disorders (1). Primary aldosteronism (PA) is an endocrine disorder, currently identified as a broad-spectrum phenotype, spanning from normotension (4% prevalence) to hypertension (10% prevalence) (2–6). PA is characterized by an inappropriately high circulating aldosterone independent of known physiological regulators such as renin, angiotensin II, potassium, and sodium status (e.g., high saline intake) (7). The diagnosis of PA is relevant, not only for its association to high blood pressure but also for the harmful effects in extra-renal tissues, generally associated with the mineralocorticoid receptor (MR) activation by aldosterone which induces inflammation (8, 9), tissue remodeling, and fibrosis (8, 10–14), affecting the renal, heart, the vascular system (endothelial cells and smooth muscles cells), the immune system (15) and the adipose tissue (16).

Several studies have tied to advance in the identification of novel biomarkers for PA that support its early detection and also other reported effects as inflammation, endothelial dysfunction, renal damage, vascular remodeling and (17, 18), and oxidative stress (19, 20). Early “surrogate biomarkers” have been previously evaluated, such as high sensitive C-reactive protein (hs-CRP), Plasminogen inhibitor activator-1 (PAI-1), matrix metallopeptidase 9 (MMP-9) and malondialdehyde (MDA) (8, 9), free Cystatin-C (CysC), and neutrophil gelatinase associated lipocalin (NGAL or LCN2) (21–23). However, none of these biomarkers are currently available in clinical diagnoses for arterial hypertension or PA. Recent proteomic studies have shown that urinary and serum alpha-1-acid glycoprotein-1 (AGP1), also known as ORM1, have been proposed as prognostic biomarkers for inflammatory diseases such as chronic heart failure (24), some types of cancer (25), and lately for PA (26).

Experimental and clinical studies demonstrate that small extracellular vesicles (sEVs) or exosomes are potential biomarkers of disease (27), including in cancer, metabolic disorders, and cardiovascular diseases (28, 29). Urinary EVs originated mainly from cells lining the renal tubules carrying proteins, lipids, RNA, and miRNA, and have been recognized recently as a source of diagnostic biomarkers for different renal and endocrine pathologies (30–36), including primary aldosteronism (26).

MicroRNAs (miRNAs) are short non-coding RNA molecules genome-encoded, that are approximately 22 nucleotides in length and modulate downstream gene expression by post-transcriptional mechanisms, specifically by binding to the 3′ untranslated regions (UTR) of a target messenger RNA (mRNA), leading to mRNA degradation or repression of translation (37–39). Recent literature (30, 37, 40–46) proposes that microRNAs in exosomes are involved in physiological and pathophysiological processes correlated with hypertension (47) response to sodium intake (48) and PA (26, 30). miRNAs are packaged into EVs for transport into different biofluids (e.g., blood, urine) and change according to the metabolic microenvironment (e.g., inflammation) of the parent cell. In endocrine hypertension phenotypes, such as nonclassic-AME (31) and PA, have been identified in the differential expression of EV-associated miRNAs, such as miR-192, miR-204 (31), miRNA-21, and Let-7i. miR-21 has been found in EVs isolated from urine (43), plasma (44), and endothelial cells (45). Romero et al. reported on the protective role of miR-21 in the cardiac pathology triggered by excess aldosterone in the heart of mice and rats (49, 50). Let-7i negatively regulates cardiac inflammation and fibrosis in presence of angiotensin II or aldosterone (49–51). Similarly, Deccman et al. identified circulating miR-30e-5p, miR-30d-5p, miR-223-3p, and miR-7-5p in PA patients with bilateral adrenal hyperplasia (BAH) and aldosterone-producing adenoma (APA). Altogether, these reports highlight the potential role of both miRNA and EV-associated miRNAs as biomarkers or mediators of PA (46).

The current study aimed to evaluate lipocalins LCN2 and AGP1, and specific urinary extracellular vesicles miR-21-5p and Let-7i-5p as novel biomarkers of primary aldosteronism.

This study used the serum, plasma, and urine samples from a biobank obtained from a cohort of 206 adult Chilean subjects of both genders, between 18 and 65 years old. The subjects were recruited from outpatient centers associated with the UC-Christus Health Network in Santiago, Chile, following the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Faculty of Medicine of the Pontificia Universidad Católica de Chile (Certification CEC-MEDUC 12-207 and 14-268, and updated by CEC-MEDUC 190823001 and 200619004).

All subjects had a sodium diet ad libitum and declared that they did not ingest any herbal products or extreme diets during the month prior to the analysis. Subjects with a BMI >30 kg/m², kidney disease, diabetes mellitus, liver, and heart failure were excluded. Subjects using glucocorticoids, contraceptives, or some interfering drugs, such Ag-II-receptor blockers (ARB), ACE-Inhibitors (ACEI), and spironolactone (MR antagonist), were also excluded.

After exclusion criteria were applied, 132 subjects were included in the study. The subjects were classified as normotensive controls (CTL), have clinical and biochemical parameters in the normal range, essential hypertensives (EH) according to the 2017 ACC/AHA Guidelines for High Blood Pressure (52), and subjects having a positive screening for PA (Aldosterone >9ng/dl, PRA <1 ng/ml*h), according to The Endocrine Society 2016 guidelines (53) and Vaidya et al. (5, 54–56). All studied subjects (PA, EH, and CTL) have a clinical record including medical history and physical examination, as well a biochemical profile, creatinine, electrolytes, aldosterone, plasma renin activity (PRA), serum, and 24-hour and morning urine samples. Aldosterone and PRA were measured by immunoassay using a commercial kit (DiaSorin, Stillwater, MN). Urine samples for uEV isolation were stored at -80C with a 1X protease inhibitor cocktail (Roche, USA).

In the present study, we evaluated the concentration of lipocalins AGP1 and LCN2, and the expression of miR-21-5p and Let-7i-5p in uEVs as potential biomarkers of PA. We observed a higher concentration of AGP1 in PA subjects, which is associated with the critical variables used to screen PA, as plasma aldosterone, renin, and ARR. Further to these novel findings, we noted a low expression of miR-21-5p in PA subjects, which is interesting since it supports a combinate model for the identification of PA conditions. We suggest that both AGP1 and miR-21-5p are associated with the pathogenic course of the primary aldosteronism and can be useful in the design of a novel diagnostic algorithm for PA. There is also a widely accepted consensus regarding a positive screening for PA is an ARR >30 ng/dL per ng/mL/h, with suppressed renin (PRA<1.0 ng/mL*h or DRC <10 uUI/ml) and an aldosterone concentration >15 ng/dL. Some studies have been identified that can improve the detection of milder forms of primary aldosteronism when using less conservative ARR thresholds with suppressed renin activity and plasma aldosterone levels >9 ng/dL (5, 53, 54), which is in agreement with the outcome of this study.

We found similar levels of hs-CRP and IL-6 as markers of inflammation in PA subjects, which were similar to EH and control groups, according to previous studies (12, 64). Similarly, endothelial damage markers (PAI-1, MMP9, and MMP2) and renal function markers (Urinary albumin (UACR)) do not show any significant changes in PA compared with EH and controls. It suggests these subjects, currently classified as subclinical PA (54, 65) do not have chronic inflammation, vascular compromise, or renal function impairment as is seen in overt or classic PA. Hence, is highly necessary novel and sensitive biomarkers aimed to detect subclinical PA and avoid complications associated with the renal and extra-renal effects reported in classic PA.

This perspective is the first to report findings that show a higher serum AGP1 concentration in PA than EH and controls subjects ( Figure 4 ). We also observed a significant association of AGP1 with classic screening parameters for PA (e.g., aldosterone, PRA, and ARR) ( Figure 5 ). Moreover, we found by discriminative analyses by ROC curves that AGP1 can identify PA from HE&CTL subjects with high sensitivity and specificity. All these results suggest that circulating AGP1 protein is a novel and potential biomarker of PA, which was also suggested for AGP1 protein in urinary exosomes (26). Since AGP1 is a protein from the family lipocalin associated with the acute phase response with immunomodulatory properties (66, 67), affected by glucocorticoids (68–70) and mineralocorticoids (71), we suggest that AGP1 expression is modified by high aldosterone levels through MR activation, having a dual role as a potential biomarker of PA, and possible mediator of the tissue response to high aldosterone. Further clinical and animal model or in-vitro studies using MR antagonists should be performed to support this hypothesis.

Similarly, we measured free LCN2 and LCN2 conjugated with matrix metalloproteinase 9 protein as a potential biomarker of PA (72). We found an increase only in total LCN2 (the sum of free and complexed LCN2) in EH, but it did not reach a significant difference in PA when compared with the control or EH. LCN2 is a proinflammatory molecule upregulated in obese individuals or patients with cardiometabolic syndrome, as also has been described in classic PA (73, 74) and is suggested as an MR sensitive protein (75, 76). LCN2 expression is influenced by several factors including obesity, salt intake, aging, infection, and inflammatory status (72, 74, 77–79). Since these subjects have a middle or subclinical PA, with no clear evidence of inflammation, renal/vascular damage ( Figure 3 ), or concomitant cardiometabolic disease, we hypothesize the LCN2 fails to increase in these PA subjects since they require a concomitant hit as inflammation (78, 79), obesity (high adipose tissue) (74), or high salt intake (72) to increase the circulating LCN2 levels.

We studied the urinary extracellular vesicles as a source of potential biomarkers for PA (26, 30). In the present study, uEVs showed similar particle concentration and size in PA subjects with respect to EH and controls ( Table 4 ). Previous studies in PA show similar findings in uEVs concentration (26) but differ from studies in circulating EVs in PA (75, 76), where they reported an increased concentration of circulating EVs in the serum of PA patients when compared with essential hypertensives and attributed it to an enhanced biological response of the endothelium to aldosterone in vivo (75), which has also been observed in vitro (80–82). These differences could be related to the PA classification, overt PA versus subclinical PA, and also the different biofluids used to quantify the impact of high aldosterone in EV concentration, serum versus urine, which is associated with distinct mechanisms and the rates of EV shedding that have different tissues (e.g., vascular endothelium vs renal epithelia).

Based on previous reports, we measured the expression of two miRNA in urinary EVs, miR-21-5p, and Let-7i-5p, as potential biomarkers of PA. We observed that uEV-associated miR-21-5p expression in uEVs from PA were lower than controls ( Figure 8 ) and similar to EH, however a trend to lower levels was observed in PA. This result suggests that uEV-miR-21-5p is downregulated and associated with pathophysiological mechanisms depending on both high BP and PA conditions. miR-21-5p expression is regulated by cytokines, inflammatory modulators (e.g., NF1, AP1), and steroids. Downregulation of miR-21-5p would affect the downstream target genes related with inflammation (83) as IL-1B gene, aldosterone effect as NEDD4, YWHAZ, SCL12A2 genes, and fibrotic processes (42, 84) as COL1A and COL4A1 genes ( Table 6 ). Prospective animal models and in vitro studies with miR-21-5p are necessary to gain depth of understanding about the role of this miRNA in high aldosterone conditions in renal epithelia, as occurs in PA.

With respect to uEV-associated Let-7i-5p, we did not observe any differences in Let-7i-5p expression in all groups. Let-7i has been found in either urine (31) and plasma exosomes (44) and is associated with RAAS, mediating inflammation and fibrosis, in both in vitro models and experimental models of kidney disease (51, 85). Let-7i regulates downstream target genes TGFBR1, IL6, IL10, COL1A1, COL3A1, DNMT3A, NEDD4, ORM1, VIM, FN1, ACTIN, SCL12A1, and YWHAZ, among others (85–87) ( Table 6 ). In the current study, we did not find differences in inflammation parameters, and were unable to measure other important parameters related to fibrosis in these PA subjects, such as the procollagen type 1 protein (PINP, COL1A1).

The ROC curves analyses with AGP1 and miR-21-5p as significant variables associated with PA subjects, support a simple (AGP1) or combinate model (AGP1 + miR-21-5p) to discriminate PA with significant AUC of 90% or 94%, respectively. This AUC is similar to previous reports on AGP1 in uEVs (92%), which support free or uEV-associated AGP1 as potential biomarkers of PA (26).

In summary, we found higher levels of serum AGP1 and lower uEV-miR-21-5p expression in subjects classified as PA with respect to EH and controls. Besides the high discriminatory capacity identified by ROC curves, the association of AGP1 with aldosterone, PRA, and ARR, place both as potential biomarkers of PA. Further studies examining the possible role of AGP1 and miR-21-5p as a mediator of the pathogenic course of PA are encouraged.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Unidad de Ética y Seguridad de la Investigación, CEC-SaludUC, Pontificia Universidad Católica de Chile. The patients/participants provided their written informed consent to participate in this study.

CC, AT-C, and CF contributed to conception and design of the study. CC and CF provide with the contribution of patients or study material. CC, AT-C, JP performed the collection and/or obtaining of results, organized the database, and performed the statistical analysis. CC and AT-C achieve the analysis and interpretation of the results. CC wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.

This study was supported partially by grants ANID-FONDECYT 1212006 (CC) and 3200646 (AT-C); CONICYT-FONDEQUIP EQM150023 (CC); ANID–Millennium Science Initiative Program- IMII P09/016-F, ICN09_016 (CF); SOCHED 2019-09 (CC).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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