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. Author manuscript; available in PMC: 2010 Jul 1.
Published in final edited form as: J Hypertens. 2010 Jul;28(7):1422–1428. doi: 10.1097/HJH.0b013e3283392673

Urinary angiotensinogen is correlated with blood pressure in men (Bogalusa Heart Study)

Hiroyuki Kobori a, Maki Urushihara a, Ji H Xu b, Gerald S Berenson b, Luis Gabriel Navar a
PMCID: PMC2891987  NIHMSID: NIHMS203729  PMID: 20375906

Abstract

Background

The Bogalusa Heart Study is a long-term study on cardiovascular disease and has followed a biracial (black/white) population from childhood. Risk factor data pertaining to many patients have been collected over 35 years, and the time course of hypertension has been documented by repeated examinations and measurements. Considerable sex and racial differences have been found to be related to cardiovascular disease. Urinary angiotensinogen (UAGT) is a novel biomarker for the intrarenal activity of the renin–angiotensin system in hypertension and kidney disease. We aimed to determine the relationship of UAGT with traditional cardiovascular disease risk factors in asymptomatic young adults in this biracial population.

Method

We recruited 251 individuals and collected a single random spot urine sample from each one. Because UAGT is significantly increased in diabetic patients and the use of antihypertensive drugs affects UAGT levels, we excluded patients who had diabetes, who were receiving antihypertensive treatment, or both. Consequently, 190 participants were included for this analysis.

Results

UAGT levels did not differ with race or sex, but were significantly correlated with SBP (r = +0.23, P = 0.0015) and DBP (r = +0.24, P = 0.0012). Moreover, high correlations were shown in men, especially in black men (SBP, r = +0.85, P = 0.0005 and DBP, r = +0.72, P = 0.0079). Thus, UAGT is correlated with blood pressure in men, even when they do not show overt proteinuria or albuminuria.

Conclusion

The biomarker, UAGT, may facilitate the identification of individuals that are at increased risk for the development of hypertension and early asymptomatic renal disease.

Keywords: angiotensinogen, clinical study, ELISA, hypertension, renin-angiotensin system, urine

Introduction

Uncontrolled hypertension induces structural and functional alterations in the kidney, which can eventually lead to end-stage renal disease [1]. Effective control of blood pressure (BP) retards the progression of renal failure and reduces the morbidity and mortality rates associated with hypertensive vascular disease [2–5]. Identification of individuals at risk of developing hypertension and renal disease is of cardinal importance.

The Bogalusa Heart Study [6–9] is a long-term study of cardiovascular disease (CVD) and has followed a biracial (black/white) population from childhood. Risk factor data on many patients have been collected over 35 years, and the time course of hypertension has been documented by repeated examinations and measurements. Considerable sex and racial contrasts have been noted in relation to CVD [6–9].

Recent findings related to the renin–angiotensin system (RAS), one of the most important mechanisms of BP regulation and water–electrolyte homeostasis, have provided an improved understanding of the pathophysiology of hypertension [10,11]. In recent years, the focus of interest on the RAS has shifted to the role of the local/tissue RAS in specific tissues [12]. Many studies have reported the importance of the tissue RAS in the brain [13], heart [14], adrenal glands [15], vasculature [16,17], and kidneys [10,11]. Further, there is substantial evidence that the major fraction of angiotensin II present in renal tissues is locally generated from angiotensinogen (AGT) delivered to the kidney as well as from AGT locally produced by the proximal tubule cells [18]. Renin secreted by the juxtaglomerular apparatus cells into the renal interstitium and vascular compartment also results in the local generation of angiotensin I [19]. Angiotensin-converting enzyme is abundant in the kidney and is present in the proximal tubules, distal tubules, and collecting ducts [20]. Angiotensin I delivered to the kidney can also be converted to angiotensin II [21]. Therefore, all of the components necessary to generate intrarenal angiotensin II are present along the nephron [10,11].

AGT is the only known substrate for renin, the rate-limiting enzyme of the RAS. Because the level of AGT is close to the Michaelis–Menten constant for renin, changes in AGT levels as well as renin levels can control the activity of the RAS, and upregulation of AGT levels may lead to increases in the angiotensin peptide levels and in BP [22,23]. Recent studies [24–32] on experimental animal models and transgenic mice have documented the involvement of AGT in the activation of the RAS and development of hypertension. Genetic manipulations that lead to overexpression of AGT have consistently been shown to cause hypertension [33,34]. In human genetic studies, a linkage has been established between the AGT gene and hypertension [35–38]. Thus, AGT plays an important role in BP regulation.

Recently, we reported that urinary excretion rates of AGT provide a specific index of the intrarenal RAS status in angiotensin II-dependent hypertensive rats [39–43]. Further, we recently developed a direct quantitative method to measure urinary AGT (UAGT) using human AGT ELISA [44]. This method has recently been used to determine the urinary excretion rates of AGT, and these rates have been found to be a novel biomarker for the intrarenal activity of the RAS in chronic kidney disease [45,46], hypertension [47], and diabetic nephropathy [48,49]. The current study was performed to determine the relationships of UAGT with traditional CVD risk factors in asymptomatic young adults in the biracial populations of the Bogalusa Heart Study.

Methods

Study design and participant recruitment

The protocol of this observational study was approved by the Institutional Review Board and by the Clinical and Translational Research Center at Tulane University. Two hundred and fifty-one individuals were recruited in Tulane University Health Sciences Center and associated clinics, and samples were obtained from them after acquiring written informed consent. It has been reported that UAGT is significantly increased in diabetic patients [46], and the use of antihypertensive drugs also affects UAGT levels [47]. Therefore, we excluded patients who had diabetes, those who were receiving antihypertensive therapy, or both, from further analyses. Consequently, 190 samples were analyzed. A random spot urine sample and a blood sample were obtained from the participants when they visited our clinic. Height and body weight were also recorded on the same day. BP was measured as previously described [46,50]. Briefly, participants sat quietly for at least 5 min prior to BP measurements in their right arms. We used an automated manometer (Omron Corporation, Kyoto, Japan) to make at least three BP measurements, 2 min apart. The mean BP values were calculated from the last two SBP and DBP readings that fell within 5 mmHg of each other.

Measurements

Serum concentrations of sodium, creatinine, and cystatin C, and urinary concentrations of sodium and protein were measured in the clinical laboratory in the Tulane University Health Sciences Center with an AU 400 Auto-analyzer (Olympus, Melville, New York, USA). Urinary concentrations of albumin and creatinine were measured with a DCA 2000 Analyzer (Bayer AG, Leverkusen, Germany). Urinary concentrations of AGT were measured with human AGT ELISA kits as previously described [44]. The coefficients of variation of the intraassay and of the interassay of this AGT ELISA were 4.4 and 4.3%, respectively [44].

Statistical analyses

Pearson correlation coefficients and Spearman correlation coefficients were used for parametric data and nonparametric data, respectively. The standard least squares method was used for multiple regression analysis. Comparisons of continuous variables between groups were performed using one-way analysis of variance and Dunnett’s test. All data are presented as means ± SEM. A P value of less than 0.05 was considered significant. All computations, including data management and statistical analyses, were performed with the JMP software (SAS Institute, Cary, North Carolina, USA).

Results

Participants’ profiles and laboratory data

The demographics and baseline laboratory data of the study participants are summarized in Tables 1 and 2, respectively. The important finding is that serum creatinine levels, urinary protein–creatinine ratio, fractional excretion of sodium, serum cystatin C levels, or urinary albumin–creatinine ratio are not different and thus renal functions were similar among the groups in this study.

Table 1.

Participants’ profiles

Black women Black men White women White men
Parameters N = 29 N = 12 N = 67 N = 82 P
Age (year) 38.62 ± 1.06 40.25 ± 1.37 40.84 ± 0.57 40.52 ± 0.44 0.1769
Height (cm) 164.21 ± 1.31 178.33 ± 2.60 163.89 ± 0.76 178.13 ± 0.67 <0.0001*
Body weight (kg) 83.16 ± 4.08 100.13 ± 8.59 82.11 ± 2.61 93.80 ± 1.81 0.0005*
BMI 30.56 ± 1.24 31.17 ± 2.17 30.52 ± 0.93 29.51 ± 0.52 0.6864
SBP (mmHg) 113.91 ± 1.73 131.64 ± 6.06 115.38 ± 1.63 118.84 ± 1.55 0.0009*
DBP (mmHg) 75.21 ± 1.48 87.94 ± 3.70 77.36 ± 1.00 81.05 ± 1.16 0.0002*
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*

P <0.05.

Table 2.

Laboratory data

Black women Black men White women White men
Parameters N = 29 N = 12 N = 67 N = 82 P
Serum Na (mEq/l) 138.59 ± 0.35 139.08 ± 0.56 138.56 ± 0.23 138.59 ± 0.26 0.8835
Serum Creatinine (mg/dl) 0.78 ± 0.03 1.09 ± 0.05 0.78 ± 0.01 1.02 ± 0.02 <0.0001*
UNa/UCre (mEq/g) 106.55 ± 14.24 69.91 ± 10.16 111.46 ± 7.08 93.47 ± 7.29 0.1134
UPro/UCre (g/g) 0.06 ± 0.01 0.07 ± 0.03 0.08 ± 0.01 0.06 ± 0.01 0.5314
FENa (%) 0.59 ± 0.07 0.54 ± 0.08 0.61 ± 0.04 0.67 ± 0.05 0.6314
Serum cystatin C (mg/l) 1.21 ± 0.09 1.27 ± 0.14 1.10 ± 0.05 1.27 ± 0.05 0.1617
UAlb/UCre (mg/g) 11.92 ± 4.08 7.62 ± 2.52 9.02 ± 1.24 8.30 ± 2.01 0.7519
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FENa, fractional excretion of sodium; UAlb/UCre, urinary albumin–creatinine ratio; UNa/UCre, urinary sodium–creatinine ratio; UPro/UCre, urinary protein–creatinine ratio.

*

P <0.05.

Single regression and correlation analyses

We performed single regression and correlation analyses of UAGT–creatinine ratio (UAGT/UCre) with clinical parameters. The UAGT/UCre levels were not correlated with age, height (r = −0.03, P = 0.6481), body weight (r = +0.00, P = 0.9745), BMI, serum sodium levels, serum creatinine levels (r = −0.11, P = 0.131), serum cystatin C levels, or fractional excretion of sodium. However, the UAGT/UCre levels were significantly and positively correlated with the SBP (Fig. 1a, r = +0.23, P = 0.0015), DBP (Fig. 1b, r = +0.24, P = 0.0012), urinary sodium–creatinine ratio (r = +0.16, P = 0.0342), urinary albumin–creatinine ratio (r = +0.39, P <0.0001), and urinary protein–creatinine ratio (r = +0.47, P <0.0001).

Fig. 1.

Fig. 1

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(a and b) Single regression analyses for urinary angiotensinogen–creatinine ratio levels. UAGT/UCre levels are not correlated with age, height (r = −0.03, P = 0.6481), body weight (r = +0.00, P = 0.9745), BMI, serum sodium levels, serum creatinine levels (r = −0.11, P = 0.131), serum cystatin C levels, or fractional excretion of sodium. However, UAGT/UCre levels are significantly and positively correlated with SBP (a; r = +0.23, P = 0.0015), DBP (b; r = +0.24, P = 0.0012), urinary sodium–creatinine ratio (r = +0.16, P = 0.0342), urinary albumin–creatinine ratio (r = +0.39, P <0.0001), and urinary protein–creatinine ratio (r = +0.47, P <0.0001). (c) Multiple regression analyses for UAGT/UCre levels. Only three parameters (DBP, urinary sodium–creatinine ratio, and urinary protein–creatinine ratio) can account for almost 30% of variation of UAGT/UCre levels (r = 0.54, R2 = 0.29, P <0.0001). (d–f) Relationship of UAGT/UCre levels with blood pressure in populations. UAGT/UCre levels were not different among race or sex (d). However, as described in this figure, UAGT/UCre levels were significantly correlated with SBP and DBP. Moreover, high correlations were shown in men, especially in black men in SBP (e; r = +0.85, P = 0.0005) and DBP (f; r = +0.72, P = 0.0079). UAGT/UCre; urinary angiotensinogen–creatinine ratio (in μg/g).

Multiple regression analysis

Factors that were found to have a significant correlation with the UAGT/UCre levels in the single regression analyses (SBP, DBP, urinary sodium–creatinine ratio, urinary albumin–creatinine ratio, and urinary protein–creatinine ratio) were used as explanatory variables in the multiple regression analysis. To reduce the impact of multicollinearity, we selected explanatory variables such that the mean sum of the squares of the residual would be minimal in the multiple regression analysis. As a result, SBP and urinary albumin–creatinine ratio were excluded from the multiple regression analysis (Table 3). The remaining three parameters were subjected to multiple regression analysis by stepwise method for UAGT/UCre. These three parameters accounted for almost 30% of the variations in UAGT/UCre levels (Fig. 1c; r = 0.54, R2 = 0.29, P <0.0001).

Table 3.

Multiple regression analysis by stepwise method for urinary angiotensinogen–creatinine ratio

Parameters Estimate F ratio P
Intercept −6.41 0.00 1.0000
UPro/UCre (g/g) 29.41 51.66 0.0000*
DBP (mmHg) 0.11 10.92 0.0012*
UNa/UCre (mEq/g) 0.01 4.71 0.0314*
UAlb/UCre (mg/g) 0.00 2.41 0.1221
SBP (mmHg) 0.00 1.18 0.2798
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UAlb/UCre, urinary albumin–creatinine ratio; UNa/UCre, urinary sodium–creatinine ratio; UPro/UCre, urinary protein–creatinine ratio.

*

P <0.05.

Relationship of urinary angiotensinogen–creatinine ratio levels with blood pressure in the study populations

The average UAGT/UCre levels did not differ with race or sex (Fig. 1d). However, as described above, the UAGT/UCre levels were significantly correlated with the SBP and DBP. Moreover, these levels were strongly correlated in men, especially in black men with the SBP (Fig. 1e; r = +0.85, P = 0.0005) and DBP (Fig. 1f; r = +0.72, P = 0.0079).

Discussion

In this study, we recruited men and women without any bias in the selection process. Accordingly, there were some differences among the participant groups in terms of height, body weight, and serum creatinine levels (Tables 1 and 2). However, most of these differences may have stemmed from well recognized sex-related differences, and we emphasize here that none of these parameters (height, body weight, and serum creatinine levels) were correlated with the UAGT/UCre levels. Therefore, it seems unlikely that the above differences affected the final results reported herein.

Although most of the circulating AGT is produced and secreted by the liver, the kidneys also produce AGT [11]. Intrarenal AGT mRNA and protein have been localized in the proximal tubule cells, indicating that intratubular angiotensin II may be derived from locally formed and secreted AGT [51,52]. In the proximal tubule cells, AGT appears to be directly secreted into the tubular lumen; in addition, some AGT is broken down intracellularly to produce metabolites, which are then secreted into the tubular lumen [53]. Proximal tubular AGT concentrations in anesthetized rats have been reported to be in the range of 300–400 nmol/l and greatly exceed the free angiotensin I and II tubular fluid concentrations [10]. Because of its molecular size (50–60 kDa), little plasma AGT is expected to filter across the glomerular membrane, which further supports the concept that proximal tubular cells secrete AGT directly into the tubules [54].

To determine whether circulating AGT is a source of UAGT, human AGT was infused into hypertensive and normotensive rats, and it was found that circulating human AGT was not detectable in the urine [42]. The failure to detect human AGT in the urine indicates limited glomerular permeability, tubular degradation, or both. These findings support the hypothesis that UAGT is derived from the AGT that is formed and secreted by the proximal tubules and not from plasma AGT.

As described previously, we reported that UAGT excretion rates provide a specific index of the intrarenal RAS status in angiotensin II-dependent hypertensive rats [39–43]. To determine whether the increase in UAGT excretion was simply a nonspecific consequence of the proteinuria and hypertension, further studies were performed in a rat model of hypertension established by administering a deoxycorticosterone acetate salt and a high-salt diet to the rats [42]. Although the urinary protein excretion rates in the deoxycorticosterone acetate salt-induced volume-dependent hypertensive rats were increased to the same or greater extent, the UAGT levels were significantly lower in the volume-dependent hypertensive rats than in angiotensin II-dependent hypertensive rats and were not greater than the levels in control rats [42].

Similar observations were made in patients with chronic kidney disease [46]. The UAGT/UCre levels in patients with type 2 diabetic nephropathy (795.67 ± 296.03 μg/g) or with membranous nephropathy (503.53 ± 297.68 μg/g) were much higher than the overall average levels in patients with chronic kidney disease (273.17 ± 62.22 μg/g) [46]. Importantly, activation of the intrarenal RAS has been reported to be involved in the progression of renal injury in human patients with diabetic nephropathy [55,56] or membranous nephropathy [57]. In contrast, the UAGT/UCre levels in patients with minimal change disease (8.28 ± 3.70 μg/g) were similar to those in control individuals (10.78 ± 3.42 μg/g), even though the patients with minimal change had severe proteinuria [46]. The findings in hypertensive animals as well as those in patients with chronic kidney disease support the hypothesis that the enhanced UAGT observed in this study was not a nonspecific consequence of proteinuria.

Further, important evidence was recently reported in relation to juvenile patients with type 1 diabetes [49]. The UAGT/UCre levels were compared between patients with type 1 diabetes and the age and sex-matched control individuals. Neither the urinary albumin–creatinine ratio nor the urinary protein–creatinine ratio was found to be increased in patients with type 1 diabetes compared with the control individuals, suggesting that these patients were in the premicroalbuminuric phase of diabetic nephropathy. However, the UAGT/UCre levels were significantly higher in the patients with type 1 diabetes than in the control individuals. These data indicate that in patients with type 1 diabetes, the increase in UAGT levels precedes the increase in urinary albumin levels. Taken together, these data indicate that augmented UAGT excretion is not simply a nonspecific consequence of proteinuria.

Hypertension is a multi-etiological disease and multiple factors are involved [1,58]. Our data suggest that in men, especially in black men, the pathway described in Fig. 2 is a major contributor to the pathogenesis of hypertension. In human genetic studies, a linkage has been established between the AGT gene and hypertension [35–38,59]. Also, oxidative stress-induced intrarenal AGT enhancement is reported to play an important role in the development of hypertension in animal models [60–62] and in clinical studies [48,55]. In addition, it is also reported that metabolic disorders augment AGT expression [63–66]. These factors may lead to increases in kidney/UAGT levels. Renin is secreted by the juxtaglomerular apparatus [67,68] and by principal cells of collecting ducts [54,69–72]. Angiotensin-converting enzyme is also abundant in the kidney and is present in the proximal tubules, distal tubules, and collecting ducts [20]. Because the level of AGT is close to the Michaelis–Menten constant for renin [22,23], increases in kidney/UAGT levels may lead to the increases in kidney/urinary angiotensin I and thus angiotensin II levels. The augmented kidney/urinary angiotensin II levels are reported to enhance salt sensitivity and lead to BP increases via salt retention [73–75]. Although it is not possible to address these mechanistic issues in this observational study, our results are consistent with this perspective. Obviously, further studies, including interventional studies and a randomized clinical trial, are required to confirm its applicability.

Fig. 2.

Fig. 2

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Perspectives based on the findings in this observational study. SNP, single-nucleotide polymorphisms

The limitation of this study comes from the nature of the observational study using one-point sample collection. Although these data would establish a foundation of the normal range allowing comparison with data from patients with various diseases, we believe that a larger multicenter, randomized, control study is required to extend these observations. A prospective study to determine the relationship between the effect of RAS blockade on UAGT and CVD events will be helpful in assessing the clinical significance of the decrease in UAGT with RAS blockade. On the basis of these findings, a randomized clinical trial has been projected to establish the clinical significance of the decrease in UAGT with RAS blockade.

In summary, in asymptomatic young adults taken from a biracial population of the Bogalusa Heart Study, UAGT/UCre levels were significantly and positively correlated with the SBP, DBP, urinary sodium–creatinine ratio, urinary albumin–creatinine ratio, and urinary protein–creatinine ratio. They were not correlated with age, height, body weight, BMI, serum sodium levels, serum creatinine levels, serum cystatin C levels, or fractional excretion of sodium, and did not vary with race or sex.

However, these levels were strongly correlated with SBP and DBP, especially in black men. Because UAGT originates from the AGT that is formed and secreted by the proximal tubules and not from plasma AGT, the enhanced UAGT may not be just a nonspecific consequence of proteinuria, but rather indicate activation of the intrarenal RAS. These data suggest that UAGT excretion is correlated with the BP in men, even when they do not show overt proteinuria or albuminuria. Although a longer observation period is required to study long-term clinical outcomes such as cardiac events for which UAGT acts as a biomarker, UAGT may facilitate the identification of individuals at increased risk for the development of hypertension, and early asymptomatic renal disease.

Acknowledgments

This study was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK072408 to H.K.); the National Center for Research Resources (P20RR017659 to L.G.N.); the National Heart, Lung, and Blood Institute (R01HL026371 to L.G.N.); the National Institute of Aging (R01AG016592 to G.S.B.); Louisiana Board of Regents (Research Commercialization and Educational Enhancement Program for Louisiana State University/Tulane University Health Sciences Centers’ Clinical and Translational Research Education and Commercialization Program to H.K.), Tulane University Research Enhancement Fund (Phase I to H.K.), and Tulane University Research Enhancement Fund (Phase II to H.K.). H.K. is the recipient of the Oliver Fund Scholar Award.

Abbreviations

AGT

angiotensinogen

CVD

cardiovascular disease

RAS

renin–angiotensin system

UAGT

urinary angiotensinogen

UAGT/UCre

urinary angiotensinogen–creatinine ratio

Footnotes

There are no conflicts of interest.

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