Urinary angiotensinogen as a marker of intrarenal Angiotensin II activity in adolescents with primary hypertension

Elżbieta Kuroczycka-Saniutycz1, Anna Wasilewska1, Agnieszka Sulik2, Robert Milewski3

 

Department of Pediatrics and Nephrology, Medical University of Bialystok, Poland

Department of Rheumatology and Internal Medicine, Medical University of Bialystok, Poland

Department of Statistics and Medical Informatics, Medical University of Bialystok, Poland

 

Head of Department: Prof. dr hab. Anna Wasilewska

Department of Pediatrics and Nephrology

Medical University of Białystok

17 Waszyngtona Street,

15-274 Białystok

POLAND

 

Corresponding author: Anna Wasilewska, Medical University of Białystok, Department of Pediatrics and Nephrology, 15-274 Białystok, ul. Waszyngtona 17, POLAND.

Tel. 0048 85 7450-825, Fax. 0048 85 7421-838, e-mail – annwasil@interia.pl

 

Abstract:

Background: Experimental and epidemiological studies have demonstrated that urinary angiotensinogen (AGT) is a novel biomarker for the intrarenal activity of the renin-angiotensin system in hypertension. Several large-scale epidemiological studies reported that elevated serum uric acid level is associated with hypertension (HT). We aimed to assess the urinary angiotensinogen excretion and its correlation with serum uric acid level, lipid profile, and Z-score BMI in hypertensive adolescents.

Patients and methods: Participants were divided into two groups: HT – 55 subjects with primary hypertension and R – reference group – 33 subjects with white-coat hypertension. Immunoenzymatic ELISA commercial kit was used to determine AGT urinary concentration.

Results: The AGT/ cr. ratio in HT subjects was significantly higher when compared to the reference group (p<0.01). The AGT/ cr. ratio showed strong positive correlation with serum uric acid (r=0.47, p< 0.01). The relationship between AGT/ cr. levels and serum uric acid levels after controlling for age, gender and BMI-Z-score continued to show significant association.

Conclusion: In adolescents with primary hypertension, an increased urinary excretion of angiotensinogen is observed. It seems that hyperuricemia is a factor which significantly increases excretion of angiotensinogen in adolescents with primary hypertension, although large, multicentre studies are needed to confirm this observation.

 

Supplement:

Epidemiologic and experimental data suggest that uric acid could play a contributory role in the pathogenesis of elevated blood pressure. Feig et al. reported that increased uric acid level above 5.5 mg/ dL was observed in 90% of adolescents with newly diagnosed primary hypertension. Saito et al. have confirmed that the renin–angiotensin system (RAS) is also strongly related to elevated serum uric acid levels in hypertensive subjects. The authors found that uric acid stimulated proliferation, angiotensin II production, and oxidative stress in vascular smooth muscle cells (VSMC) through tissue RAS. In recent years, the role of the intrarenal RAS in the pathophysiology of both hypertension and renal injury has become a focus of interest. A considerable attention has been paid to the significance of the local/ tissue RAS in different tissues including brain, heart, adrenal glands, vasculature and kidneys. Urinary angiotensin II is unstable and, therefore, cannot be used as a reliable marker of intrarenal RAS activity in clinical studies. Experimental studies have demonstrated that angiotensinogen levels in renal tissues reflect the activity of intrarenal RAS.

Most recently, Katsurada et al. have developed a sandwich enzyme-linked immunosorbent assay (ELISA) system to directly measure urinary human angiotensinogen. It has been shown that urinary angiotensinogen level was highly correlated with intrarenal angiotensinogen and angiotensin II levels and has been suggested as a reliable marker for intrarenal RAS activity.

The aim of this study was to assess the urinary angiotensinogen excretion in hypertensive adolescents and to evaluate its correlation with serum uric acid, lipid profile and Z-score BMI.

This was a prospective cohort study of hypertensive adolescents. The study included 88 subjects (33 girls and 55 boys) aged 11 – 18 years, who were appointed to our unit (Department of Pediatrics and Nephrology, The Medical University of Białystok, Poland) between May 2010 and September 2011 in order to confirm or rule out hypertension. The majority of subjects were referred to our Department by general practitioners after finding the elevated causal BP in primary care office. On the basis of ABPM, the examined adolescents were divided into two groups: HT – subjects with confirmed primary hypertension and R – reference group – subjects with white-coat hypertension. In addition, a group of adolescents with HT was subsequently divided into two subgroups: HT HU (+) – subjects with hypertension accompanied by hyperuricemia and HT HU (-) i.e. subjects with hypertension and normal serum uric acid levels.

The study group (HT) consisted of 55 hypertensive subjects (43 with hyperuricemia – serum uric acid level ≥ 5.5 mg/ dL and 12 with serum uric acid level < 5.5 mg/ dL).

The reference group consisted of 33 subjects in whom at the moment of examination hypertension was excluded on the basis of ABPM.

For all subjects, careful clinical histories were taken and physical examinations were performed. After 12 – hours of night fasting, urine and blood samples were taken for the measurement of urinary concentration of angiotensinogen and 24 – hour microalbuminuria, plasma renin activity, plasma aldosterone level, CRP, basal glucose level, lipid profile, serum creatinine, urea and uric acid (SUA) levels, morphology of peripheral blood.

The urinary concentration of angiotensinogen (AGT) was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (USCN) according to the manufacturer’s instructions. In brief, a monoclonal antibody specific for AGT was used to detect AGT in the urine samples. Horseradish peroxidase conjugated streptavidin was added, followed by colour – forming peroxidase substrate containing tetramethylbenzidine. The colour was then measured at 450 nm by a microtiter plate reader and compared with a standard curve. Urinary AGT levels were expressed in nanograms per milliliter (ng/ mL).

Urinary creatinine concentration was used to normalize the AGT measurements to account for the influence of urinary dilution on its concentration. The urinary levels of creatinine were analyzed by Jaffé’s method. The AGT levels were expressed as urinary AGT/ cr. ratio in nanograms per milligram creatinine (ng/ mg cr.).

Ambulatory blood pressure monitoring (ABPM) was performed using the oscillometric Spacelabs Medical. Hypertension (HT) on the basis of ABPM were defined as mean systolic or diastolic daytime or nighttime BP levels that are ≥ 95th percentile and LSBP or LDBP daytime or nighttime levels more than 30%. The values were adjusted by gender and body height according to the reference values provided by Wühl et al..

The demographic and clinical data for each group are summarized in Table 1. Ambulatory blood pressure monitoring and laboratory results were successfully collected from 88 adolescents. Of all studied subjects, 55 were hypertensive (HT) and 33 subjects with white-coat hypertension (reference group). Effect size between the hypertensive and reference groups was (Es): 0.925326 – power test 96%. The median age did not differ between the groups. Males were more frequently affected with HT than females, consistently with the available reports in the area. In the examined group, 46 subjects (83.6%) were males (M) and 9 (16.4%) were females (F), whereas more girls (n=24, 72.7%) than boys (n=9, 27.3%) were found in the reference group.

The body height, weight and BMI of HT adolescents were higher comparing to those in the reference (p< 0.01). Median BMI Z – score in the examined group was 2.12 (range: -1.11 – 9.48) and was higher in comparison with the references median (p< 0.01). Thirty seven subjects (67.3%) from HT group were classified as overweight or obese.

We found significant differences in AGT/ cr. ratio, plasma renin activity, plasma aldosterone level, serum level of uric acid, creatinine, urine, triglycerides and also in eGFR between HT subjects and healthy reference.

The median SUA concentration in HT subjects was significantly higher compared to reference group (HT: 6.49 mg/ dL (3.26 – 8.89 mg/ dL) vs. R: 4.6 mg/ dL (2.19 – 5.48 mg/ dL) (p< 0.01)). Serum uric acid exceeded 5.5 mg/ dL in 78.2% of HT subjects.

We performed single regression and correlation analyses of AGT/ cr. with clinical parameters.

The urine AGT/ cr. levels were correlated positively with height (r=0.32, p< 0.01), body weight (r=0.34, p< 0.01), BMI Z -score (r=0.24, p< 0.05), serum uric acid (r=0.47, p< 0.01) and triglycerides levels (r=0.29, p< 0.05). In further analysis we checked the relationships between AGT/ cr. and plasma renin activity, plasma aldosterone level. In HT subjects, AGT/ cr. ratio correlated positively with plasma aldosterone level (r=0.40, p< 0.01) and plasma renin activity (r=0.30, p< 0.05). In the reference group we didn’t find statistically significant correlations between these parameters.

Because of the known gender, age and parameters of physical development differences in SUA, multivariable linear regression was performed to control for gender, age, BMI-Z-score, and serum creatinine, triglycerides and cholesterol levels. To reduce the impact of multicollinearity, we selected explanatory variables such that the mean sum of squares of the residual would be minimal in the multiple regression analysis. As a result gender, BMI Z –score and serum creatinine level were excluded (p= 0.682; p=0653; p=0.712, respectively). The remaining four parameters (serum uric acid, triglycerides, cholesterol and age) accounted for more than 41% of the variations in AGT/ cr. ratio levels (r=0.642, p=0.00028).

The relationship between AGT/ cr. levels and serum uric acid levels after controlling for age, gender and BMI Z-score continued to show significant association.

According to the 24 h ABPM, we found that all the ABPM parameters, except night-time DBP load were significantly higher in adolescents with primary hypertension than in the reference group.

We also analysed the relationship between AGT/ cr. and the parameters derived from ABPM. We found a positive correlation between AGT/ cr. and mean SBP/ 24 h (r=0.24, p< 0.05), AGT/ cr. and mean SBP during the daytime (r=0.27, p< 0.05), and AGT/ cr. and daytime SBP loads (r=0.31, p< 0.05).

Finally, we found that AGT/ cr., serum creatinine and triglycerides levels were significantly higher in subjects with HT and hyperuricemia than in subjects with HT and normouricemia.

In the group of subjects with HT accompanied by hyperuricemia AGT/ cr. ratio was positively correlated with plasma renin activity (r=0.35, p< 0.05)and plasma aldosterone level (r=0.39, p< 0.05).

In summary, our investigations into this area are still ongoing, however the preliminary results allow us to draw the following conclusions: 1. In children and adolescents with primary hypertension, an increased urinary excretion of angiotensinogen is observed, 2. It seems that hyperuricemia is a factor which significantly increases excretion of angiotensinogen in adolescents with primary hypertension, although large, multicentre studies are needed to confirm this observation. Finally, a number of important limitations need to be considered. Firstly, the reference group was not a group of healthy teenagers, but a group of patients in whom hypertension was not confirmed in 24-hour blood pressure monitoring, so they are considered white coat hypertension group. Secondly, there were the evident differences between the studied groups: hypertensive group consisted of adolescent males mainly with high BMI Z-score and reference group included white coat hypertension group of slim females. However, as it was shown in multivariate analysis neither the gender nor the Z-score BMI influenced the relationship between AGT/ cr. ratio levels and serum uric acid levels.

However, despite the limitation of this study, we believe, that the unique position of angiotensinogen in the renin – angiotensin system and its function gives the meaning to this protein as attractive target in developing new effective strategies for the management of many diseases, particularly hypertension, atherosclerosis and obesity. At present, there is still no information in the literature about the role of angiotensinogen produced in local tissues in the pathophysiology, nor on the correlation of angiotensinogen produced in situ with the circulating pool.

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Figure 1. Comparison of AGT/ cr. ratio between the HT subjects and the reference group.

 

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Figure 2. Exponential regression analysis demonstrating the relationship between AGT/ cr. and SUA in all subjects.

 

Anna Wasilewska-3

Figure 3. Comparison of the values of AGT/ cr. ratio for the HT subjects with hyperuricemia (HT HU (+)), HT subjects with normal serum uric acid level (HT HU (-)) and the reference group.

 

Table 1. Anthropometric, clinical and metabolic characteristics of examined group (HT) and reference group (R).

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Table 2. Multivariate regression analysis of AGT/ cr. ratio and serum uric acid.

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