Serum Alpha-1-Acid Glycoprotein-1 and Urinary Extracellular Vesicle miR-21-5p as Potential Biomarkers of Primary Aldosteronism

Abstract

Primary aldosteronism (PA) is the most common cause of secondary hypertension and reaches a prevalence of 6-10%. PA is an endocrine disorder, currently identified as a broad-spectrum phenotype, spanning from normotension to hypertension. In this regard, several studies have made advances in the identification of mediators and novel biomarkers of PA as specific proteins, miRNAs, and lately, extracellular vesicles (EVs) and their cargo.

Aim: To evaluate lipocalins LCN2 and AGP1, and specific urinary EV miR-21-5p and Let-7i-5p as novel biomarkers for PA.

Subjects and methods: A cross-sectional study was performed in 41 adult subjects classified as normotensive controls (CTL), essential hypertensives (EH), and primary aldosteronism (PA) subjects, who were similar in gender, age, and BMI. Systolic (SBP) and diastolic (DBP) blood pressure, aldosterone, plasma renin activity (PRA), and aldosterone to renin ratio (ARR) were determined. Inflammatory parameters were defined as hs-C-reactive protein (hs-CRP), PAI-1, MMP9, IL6, LCN2, LCN2-MMP9, and AGP1. We isolated urinary EVs (uEVs) and measured two miRNA cargo miR-21-5p and Let-7i-5p by Taqman-qPCR. Statistical analyses as group comparisons were performed by Kruskall-Wallis, and discriminatory analyses by ROC curves were performed with SPSS v21 and Graphpad-Prism v9.

Results: PA and EH subjects have significantly higher SBP and DBP (p <0.05) than the control group. PA subjects have similar hs-CRP, PAI-1, IL-6, MMP9, LCN2, and LCN2-MMP9 but have higher levels of AGP1 (p <0.05) than the CTL&EH group. The concentration and size of uEVs and miRNA Let-7i-5p did not show any difference between groups. In PA, we found significantly lower levels of miR-21-5p than controls (p <0.05). AGP1 was associated with aldosterone, PRA, and ARR. ROC curves detected AUC for AGP1 of 0.90 (IC 95 [0.79 - 1.00], p <0.001), and combination of AGP1 and EV-miR-21-5p showed an AUC of 0.94 (IC 95 [0.85 - 1.00], p<0.001) to discriminate the PA condition from EH and controls.

Conclusion: Serum AGP1 protein was found to be increased, and miR-21-5p in uEVs was decreased in subjects classified as PA. Association of AGP1 with aldosterone, renin activity, and ARR, besides the high discriminatory capacity of AGP1 and uEV-miR-21-5p to identify the PA condition, place both as potential biomarkers of PA.

Keywords: AGP1; Alpha-1-acid glycoprotein-1; biomarker; extracellular vesicles; lipocalin; miR-21-5p; primary aldosteronism (PA).

Figure 1

Clinical characteristics of subjects with PA. (A) Age (years old). (B) Body mass index (BMI; kg/m²) (C) Systolic blood pressure (SBP; mmHg). SBP was higher in PA and EH subjects in the CTL group. (D) Diastolic blood pressure (DBP; mmHg). DBP was higher in PA and EH subjects in the CTL group. Comparison between groups was performed by unpaired one-way ANOVA or Kruskal-Wallis test. Data are presented as median and interquartile range [Q1-Q3], N.S: No significative difference, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Biochemical characteristics of subjects with PA. (A) Serum aldosterone concentration (ng/dL). Serum aldosterone levels were higher in PA subjects in the EH group. (B) Plasmatic renin activity (PRA; ng/mL*h). Plasmatic renin activity was lower in PA subjects in both the EH and CTL groups. (C) Aldosterone to renin ratio (ARR). ARR was higher in PA subjects in both the EH and CTL groups. Comparison between groups was performed by unpaired one-way ANOVA or Kruskal-Wallis test. Data are presented as median and interquartile range [Q1-Q3], N.S, No significative difference; ***p < 0.001, ****p < 0.0001.

Figure 3

Evaluation of parameters associated with inflammation, endothelial and renal dysfunction in PA subjects. (A) High sensitivity C reactive protein (hsPCR; mg/L). (B) Plasminogen activator inhibitor – 1 (PAI-1; ng/mL). (C) Metalloproteinase 9 (fold change activity). (D) Metalloproteinase 2 (fold change activity). No differences of parameters associated with inflammation, endothelial and renal dysfunction were found between groups. Comparison between groups was performed by unpaired one-way ANOVA or Kruskal-Wallis test. Data are presented as a median and interquartile range [Q1-Q3], N.S, No significative difference.

Figure 4

Determination of serum AGP1, LCN2, LCN2-MMP9, and in PA subjects. (A) Serum AGP1 concentration (µg/mL). We detected higher levels of AGP1 in PA subjects in both EH and CTL groups. (B) Serum LCN2 concentration (ng/mL). LCN2 concentration was similar between groups (C) Serum LCN2-MMP9 concentration. LCN2-MMP9 concentration was similar between groups (ng/mL). (D) Serum LCN2 + LCN2-MMP9 concentration (ng/mL). Serum levels of LCN2 + LCN2-MMP9 were higher in EH subjects in the CTL group. LCN2 + LCN2-MMP9 concentration was similar between PA and EH subjects and PA and CTL subjects. Comparison between groups was performed by unpaired one-way ANOVA or Kruskal-Wallis test. Data are presented as a median and interquartile range [Q1-Q3]. N.S, No significative difference, *p < 0.05, **p < 0.01.

Figure 5

Heat map of AGP1 associations with serum Aldosterone, PRA, and ARR in PA, EH, and CTL subjects. Positive associations are presented in blue gradient with the respective ρ (rho) value. Similarly, negative associations are presented in the red gradient. We observed a significant association between AGP1 concentration and the 3 relevant biochemical parameters in primary aldosteronism screening. Association studies were performed by Spearman test, p < 0.05.

Figure 6

Characterization and quantification of urinary EVs. (A). Identification of uEVs by Transmission Electron Microscopy (TEM) (indicated by black arrows). (B) Representative size distribution plot from uEVs using a NanoSight NS300 instrument. (C). Western blot of classic extracellular vesicles markers TSG101 and CD9.

Figure 7

Quantification of uEVs by NTA. (A) Urinary creatinine normalized uEVs concentration (uEVs particles/mg creatinine). (B) Mean diameter of uEVs particle size distribution (nm). (C) Mode diameter of uEVs particle size distribution (nm). uEVs concentration and diameter were similar between groups. Comparison between groups was performed by unpaired one-way ANOVA or Kruskal-Wallis test. Data are presented as a median and interquartile range [Q1-Q3]. N.S: No significant difference.

Figure 8

Expression of miR-21-5p and Let-7i-5p in uEVs. (A) miR-Let7i-5p expression in uEVs normalized by urinary creatinine (RU/mg creatinine). No differences in miR-Let7i-5p levels were found between groups. (B) miR-21-5p expression in uEVs normalized by urinary creatinine (RU/mg creatinine). uEVs miR-21-5p expression was higher in PA and EH subjects respect CTL group. Comparison between groups was performed by unpaired one-way ANOVA or Kruskal-Wallis test. Data are presented as a median and interquartile range [Q1-Q3]. N.S: No significative difference, *p < 0.05, **p < 0.01.

Figure 9

Regression model and Receiver operating characteristic (ROC) curve. ROC curve for serum AGP1 levels (black) and serum AGP1 levels + uEVs associated miR-21-5p (red) can discriminate the PA condition from EH and CTL groups.