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Endothelin-1 (ET-1) is a vasoactive peptide with vasoconstrictor and mitogenic properties. We investigated whether plasma levels of C-terminal pro-ET-1 (CT-proET-1), a newly described stable fragment of the ET-1 precursor, are associated with target-organ damage in hypertension.
Participants included 981 African Americans (65 ± 9 years, 71% women) and 812 non-Hispanic whites (61 ± 9 years, 54% women) ascertained from sibships with hypertension. We measured plasma CT-proET-1 by an immunoluminometric assay. Measures of target-organ damage included the ankle-brachial index (ABI) and urinary albumin:creatinine ratio (UACR). Multivariable regressions analyses were employed to assess whether plasma CT-proET-1 levels were independently associated with ABI and UACR.
In hypertensive African Americans, higher plasma levels of CT-proET-1 were significantly associated with lower ABI (P < 0.01) and higher UACR (P < 0.01). After adjustment for age, sex, body mass index, systolic blood pressure (SBP) and diastolic blood pressure (BP), diabetes, serum glucose, insulin use, estimated glomerular filtration rate (eGFR), history of smoking, total and high-density lipoprotein cholesterol, medication use, and previous history of myocardial infarction (MI) or stroke, higher plasma levels of CT-proET-1 remained significantly associated with lower ABI (P < 0.01) and higher UACR (P = 0.02). In non-Hispanic white hypertensives, higher plasma levels of CT-proET-1 were weakly associated with higher UACR (P = 0.02) and with lower ABI (P = 0.07). After adjustment for the relevant covariates, no statistically significant associations between CT-proET-1 and ABI or UACR were present in whites.
Plasma levels of CT-proET-1 were independently associated with lower ABI and greater UACR in African American but not non-Hispanic white adults with hypertension.
Endothelin (ET)-1 is a member of a vasoactive peptide family that includes the isoforms ET-1, ET-2, and ET-3.1 ET-1, the major active isoform, is expressed by vascular endothelial and smooth muscle cells, cardiac myocytes and glomerular endothelial and tubular cells.2,3 ET-1 is one of the most potent vasoconstrictor peptides known, has mitogenic properties, and stimulates the renin–angiotensin–aldosterone axis.1 ETs are derived from pre-pro-ETs that undergo cleavage by endopeptidases to form inactive precursors pro-ETs (big ETs), which are further cleaved by ET-converting enzyme to the active form of ET.4 ET-1 acts in a paracrine manner on two receptors, ETA and ETB. The activation of ETA and ETB on vascular smooth muscle results in vasoconstriction whereas the activation of ETB on the vascular endothelium results in vasodilation.4 Reliable measurement of plasma ET-1 has been hampered by its short half-life and its nonspecific binding to plasma proteins. Recently, a more stable C-terminal fragment of prepro-ET-1 (CT-proET-1) has been validated as a reliable measure of plasma ET gene products.5
Although a role for ET-1 in hypertension has been suggested in several previous studies,6,7 its association with measures of target-organ damage in hypertension remains unclear. In addition, ethnic differences in activity of ET-1 may exist, with enhanced ETA-dependent vasoconstrictor tone in African Americans.8 It is in this context that we aimed to investigate the associations of ET-1 in African American and non-Hispanic white hypertensives with two measures of target-organ damage: the ankle-brachial index (ABI), a measure of peripheral arterial disease, and urinary albumin–creatinine ratio (UACR), a measure of microalbuminuria and a surrogate for glomerular endothelial function.
Subjects included participants in the Genetic Epidemiology Network of Arteriopathy (GENOA) study, a multicenter community-based study to identify genes influencing blood pressure (BP) and development of target-organ damage due to hypertension.9 These cohorts are enriched for hypertension and thereby suitable for identifying markers associated with subclinical vascular disease.10 The African-American participants were recruited from Jackson in Hinds County, MS, whereas the non-Hispanic white participants were recruited from Rochester in Olmsted County, MN. Between 1 December 2000, and 31 October 2004, the phase I GENOA participants returned for a second study visit and underwent physical examination, provided blood samples and underwent characterization of measures of target-organ damage. The study was approved by the institutional review boards of the University of Mississippi Medical Center, Jackson, MS, and Mayo Clinic, Rochester, MN, and participants gave informed consent. The present study included 1,793 participants (981 African Americans and 812 non-Hispanic whites) who had hypertension (diagnosed as described below).
Height was measured by stadiometer, weight by electronic balance, and body mass index was calculated as weight in kilograms divided by the square of height in meters. Diabetes was considered present if the participant was being treated with insulin or oral agents or had a fasting glucose level ≥126 mg/dl. “Ever” smoking was defined as having smoked >100 cigarettes. Information about the use of medications was obtained from the participants at the time of the study visit. Each prescription drug recorded at the study visit was assigned a code number corresponding to its therapeutic action. BP-lowering medications were classified as: diuretics, β-blockers, calcium-channel blockers, or renin–angiotensin–aldosterone system inhibitors.
Blood was drawn by venipuncture after an overnight fast. Serum total and high-density lipoprotein cholesterol were measured by standard enzymatic methods. The glomerular filtration rate was estimated (eGFR) using the Modification of Diet in Renal Disease equation as previously described.11 Resting systolic BP (SBP) and diastolic BP (DBP) were measured by random zero sphygmomanometer (Hawksley and Sons, London, UK) after participants had rested for at least 10 min in the supine position. Three measures at least 2 min apart were taken and the average of the second and third measurements was used. The diagnosis of hypertension was established based on BP levels measured at the study visit (≥140/90 mm Hg) or a prior diagnosis of hypertension and current treatment with antihypertensive medications.
Plasma was collected at the time of blood sampling in plastic vials containing EDTA. Samples were placed on ice and then centrifuged at 3,000g and frozen at −80 °C until assayed. CT-proET-1 was measured by a novel commercial assay (BRAHMS Aktiengesellschaft, Hennigsdorf, Germany) in the chemiluminescence/coated tube-format, as previously described.5,12
At each center, the ABI was measured by examiners who had undergone training in Mayo Clinic’s noninvasive vascular laboratory in Rochester, MN. An identical, standardized protocol was used at both centers. Following a 5-min rest, subjects were evaluated in the supine position. Appropriately sized BP cuffs were placed on each arm and ankle, and a Doppler ultrasonic instrument (Medisonics, Minneapolis, MN) was used to detect arterial signals. The cuff was inflated to 10 mm Hg above SBP and deflated at 2 mm Hg/s. The first reappearance of the arterial signal was taken as the SBP. To calculate the ABI, the SBP at each ankle site (posterior tibial and dorsalis pedis arteries) was divided by the higher of the two brachial pressures. The lower of the average ABIs from the two legs was used in the analyses. Subjects with ABI >1.3 (n = 90) were excluded from the analyses as they may have noncompressible arteries due to medial arterial calcification.
The first voided urine was collected on the morning of the study visit and stored at −80 °C until analyzed. Urine albumin, urine creatinine, and serum creatinine concentrations were measured by standard methods on a Hitachi 911 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, IN), and UACR was expressed as milligrams of albumin per gram of creatinine. To minimize confounding, subjects with chronic kidney disease as defined by creatinine >2.5 mg/dl (n = 6) or UACR >3,000 mg/g (n = 4) were excluded from the analyses.
Statistical analyses were carried out using SAS v 9.1 (SAS Institute, Cary, NC). Because of sibships in the sample, we used generalized estimating equations to account for intrafamilial correlations.13 Continuous variables were expressed as mean ± s.d. or median (quartile). Categorical variables were expressed as number (percentage). Values for plasma CT-proET-1, eGFR, and UACR were log transformed (after adding 1 in the case of UACR) to minimize skewness. Because of significant differences in age and the proportion of women between the two ethnic groups, ethnic differences in participant characteristics were compared after adjustment for age and sex. We constructed multiple regression models adjusting for age, sex, body mass index, SBP, DBP, smoking history, diabetes, total and high-density lipoprotein cholesterol, eGFR, medication (BP-lowering, statin, and aspirin) use, previous history of myocardial infarction (MI) or stroke. Age and sex were forced into all multivariable regression models. Backward elimination was performed to identify the set of variables independently associated with each measure of target-organ damage in each ethnic group. A two-sided P value of <0.05 was deemed statistically significant.
African Americans were older and there were greater proportion of women in both African American and non-Hispanic white cohorts (Table 1). The proportion of participants with an eGFR <60 ml/min/1.73 m2 was 22.9% (n = 221) for African Americans and 43.3% (n = 314) for non-Hispanic whites. After adjustment for age and sex, African Americans had a higher prevalence of diabetes, lower use of statins, and higher eGFR, SBP, and DBP, lower ABI, and greater UACR than their non-Hispanic white counterparts. Plasma levels of CT-proET-1 were higher in African Americans than in non-Hispanic whites (Table 1).
African Americans in the highest quartile for CT-proET-1 levels had significantly lower ABI compared to those in the lowest quartile (mean ABI: 0.94 vs. 1.01; P < 0.01) (Figure 1). After adjustment for age, sex, conventional risk factors, serum glucose, insulin therapy, and other medication use, higher plasma levels of CT-proET-1 remained significantly associated with lower ABI (P < 0.01) in African Americans (Table 2). In this multivariable regression model, lower BMI, other variables associated with a lower ABI in African Americans included greater age, history of smoking, higher total cholesterol, and diuretic use.
African Americans in the highest quartile for CT-proET-1 levels had significantly higher UACR compared to those in the lowest quartile (UACR: 105.53 vs. 29.80 mg/g creatinine) (P < 0.01) (Figure 1). After adjustment for age, sex, conventional risk factors, and medication use, higher plasma levels of CT-proET-1 were significantly associated with higher UACR (P < 0.01) (Table 2). In this multivariable regression model, the other variables associated with higher UACR in African Americans included diabetes, higher serum glucose, higher total cholesterol, higher SBP, history of MI or stroke, and use of a calcium-channel blocker.
In non-Hispanic whites, participants in the highest quartile for CT-proET-1 levels had significantly lower ABI and higher UACR compared to those in the lowest quartile (mean ABI: 1.09 vs. 1.12; P = 0.05); UACR: 21.21 vs. 6.37 mg/g creatinine, P = 0.01 (Figure 1). However, plasma levels of CT-proET-1 were not significantly associated with either ABI or UACR after adjustment for age, sex, conventional risk factors, and medication use. In non-Hispanic whites, male sex, greater age, history of MI or stroke, higher serum glucose levels, and smoking history were all significant determinants of lower ABI; whereas higher SBP and serum glucose level were associated with higher UACR.
We report significant associations of plasma CT-proET-1 with lower ABI and greater UACR in African-American adults with hypertension, after adjustment for age, sex, conventional risk factors, eGFR, and medication use. In contrast, the corresponding associations in non-Hispanic whites were not statistically significant after adjustment for relevant covariates. Our findings suggest that higher plasma CT-proET-1 levels may be a contributor toward target-organ damage in African-American adults with hypertension but not in whites.
The association between plasma levels of CT-proET-1 with ABI in non-Hispanic whites was modest in our study (P = 0.07) and not significant after adjustment for relevant confounding factors (P = 0.22). In contrast, two prior studies reported a strong correlation between ET-1 and extent of atherosclerotic vascular disease in this ethnic group.14,15 The first study included 40 patients with symptomatic atherosclerotic vascular disease and demonstrated a significant correlation between plasma ET-1 levels and the number of sites of atherosclerotic disease (r = 0.89, P < 0.01).14 The second study of 24 patients with peripheral arterial disease showed a significant correlation between plasma ET-1 levels and number of the arterial obstructive lesions (r = 0.698; P < 0.01).15 This apparent discrepancy may be attributable to differences in the study populations. Previous studies were conducted in patients with peripheral arterial disease, while the prevalence of peripheral arterial disease in the non-Hispanic white participants in our study was relatively low.
We demonstrated a significant independent association between plasma levels of CT-proET-1 with lower ABI in African Americans. The reasons for the ethnicity-specific association of plasma levels of CT-proET-1 with the ABI are unclear. Previous reports demonstrated a significant difference in ET receptor density and the magnitude of response to ET-1 between African Americans and whites.8,16,17 This may, in part, account for the increased vascular tone and peripheral vasoconstriction in response to stressful stimuli, and lower vasodilatation in response to nitric oxide in African Americans compared to their non-Hispanic white counterparts.18,19
Albuminuria in systemic hypertension may occur with or without a decline in GFR, is a marker of target-organ damage, and is independently associated with cardiovascular morbidity and mortality.20 ET-1 has been implicated in the development and progression of proteinuria in several animal studies,21,22 possibly by promoting mesangial cell fibrosis, podocyte injury, reactive oxygen species production and inflammation.23 ET-1 decreases GFR by constricting both afferent and efferent arterioles and decreasing renal flow.24 Multiple small (n < 40) studies demonstrated that plasma ET-1 levels are elevated in the presence of proteinuria in patients with diabetes and hypertension.25-27 In a prior study of 279 diabetic subjects, plasma ET-1 was associated with urinary albumin excretion after controlling for age, sex, body mass index, BP, hemoglobin A1c, and total cholesterol (r = 0.436; P < 0.01).28 Our study included a much larger sample size than the previous studies and demonstrated a significant association of plasma CT-proET-1 with UACR in African-American adults with hypertension after adjustment for age, sex, conventional risk factors, eGFR, serum glucose, insulin therapy, and other medication use.
In non-Hispanic whites, a significant association between plasma CT-proET-1 and UACR was noted, but this was not independent of relevant covariates. CT-proET-1 levels were lower in non-Hispanic whites than in African Americans whereas eGFR was higher in the latter. Previous studies have shown that CT-proET-1 levels are increased in chronic kidney disease due to decreased renal clearance and increased renal production of ET-1.29,30 However, African Americans in our study had significantly higher eGFR and yet higher levels of CT-proET-1 compared to the non-Hispanic whites (Table 1). Thus nonrenal mechanisms likely underlie higher CT-proET-1 levels in African Americans.
The strength of our study is the large biethnic cohort of adults with hypertension and the use of uniform protocols including questionnaires, anthropometric, and laboratory measurements. Plasma levels of CT-proET-1 were measured using a novel immunoassay, allowing a more reliable assessment of plasma ET-1 levels. Because of the cross-sectional nature of the study, inferences about causality cannot be made. Moreover, our study included older, hypertensive subjects and the results cannot be generalizable to younger individuals and those without hypertension. Our results are based on a single CT-proET-1 measurement. There may have been a degree of selection bias in our study as the African-American participants belonged to a population with a high rate of adverse cardio vascular events.31 Finally, as with all observational studies, confounding by unmeasured variables cannot be excluded.
Plasma levels of CT-proET-1 were independently associated with lower ABI and greater UACR in African Americans with hypertension. In whites, plasma CT-proET-1 levels were weakly associated with lower ABI and higher UACR and the association was not significant after adjustment for conventional confounders. Although these data raise the possibility that measurement of CT-proET-1 levels may aid in the early detection of target-organ damage, additional investigations are needed to validate our results and to evaluate the screening characteristics of this peptide. Whether blockade of ET-1 will retard target-organ damage also merits further study.
This work was supported by grants HL-81331 and M01 RR00585 from the National Institutes of Health.
The first two authors contributed equally to this work.
Disclosure: N.G.M., J.S., and A.B. are employed by BRAHMS Aktiengesellschaft, which developed the assay that we used for the measurement of CT-proET-1 in this study. No other author has a conflict of interest.