ORIGINAL COMMUNICATIONS

Characterization of Angiotensin-(1-7) in theUrine of Normal and Essential HypertensiveSubjectsCarlos M. Ferrario, Nieves Martell, Carla Yunis, John M. Flack, Mark C. Chappell, K. Bridget Brosnihan,Richard H. Dean, Andrea Fernandez, Serguei V. Novikov, Carmen Pinillas, and Manolo Luque

A total of 31 healthy volunteers [39 6 7 (SD) years] and18 untreated essential hypertensive subjects [43 6 9years] collected urine for 24 h after a physicalexamination and laboratory tests. Radioimmunoassaymeasurements of angiotensin-(1-7) [Ang-(1-7)] in urineand plasma were done as described previously. Sittingsystolic and diastolic blood pressures (6 SD) averaged118 6 11/74 6 7 mm Hg and 146 6 16/96 6 8 mm Hg innormal and essential hypertensive subjects, respectively(P < .001), whereas 24 h urinary volume was notdifferent in normal and essential hypertensive subjects(P > .05). The concentration of Ang-(1-7) in the urine ofnormal subjects averaged 62.6 6 22.6 pmol/Lcorresponding to a urinary excretion rate of 98.9 6 44.7pmol/24 h. Concurrent measurements of plasma Ang-(1-7) showed that the content of Ang-(1-7) in urine was2.5-fold higher than that measured in the plasma. Incontrast, untreated essential hypertensive subjects hadlower concentrations and 24 h urinary excretion rates ofAng-(1-7) averaging 39.4 6 18.0 pmol/L and 60.2 6 14.6pmol/24 h, respectively, (P < .001). Differences in theexcretory rate of Ang-(1-7) between normal volunteersand essential hypertensive subjects were not modifiedby normalization of the data by urinary creatinine

excretion rates. Urinary concentrations of Ang-(1-7)correlated inversely with systolic, diastolic and meanarterial pressures (r 5 20.48, P < .001). Both urinaryAng-(1-7) [odds ratio of 0.92 (95% CI: 0.88–0.97)] andage were independent predictors of systolic bloodpressure. These studies demonstrated the presence ofAng-(1-7) in urine and the existence of reduced levels ofthe heptapeptide in individuals with untreatedessential hypertension. The relatively higherconcentrations of Ang-(1-7) in urine compared toplasma agrees with data that showed that Ang-(1-7)may contribute to the regulation of blood pressure. Theinverse association between Ang-(1-7) and arterialpressure provides a potential marker for thecharacterization of forms of essential hypertensionassociated with reduced production or activity ofvasodilator hormones. Am J Hypertens 1998;11:137–146 © 1998 American Journal ofHypertension, Ltd.

KEY WORDS: Angiotensin II, blood pressure, essentialhypertension, hypertensive mechanism, renalfunction, renin-angiotensin system, vasodilatorhormone.

Received February 3, 1997. Accepted August 19, 1997.From The Hypertension Center (CMF, CY, JMF, MCC, KBB,

RHD, AFS), The Bowman Gray School of Medicine of WakeForest University, Winston-Salem, North Carolina; and The Hy-pertension Division (NM, CP, ML), Universidad Complutense,Madrid, Spain.

This work was supported by grant HL-51952 from the NationalInstitutes of Health.

Address correspondence and reprint requests to Carlos M. Fer-rario, MD, Hypertension Center, The Bowman Gray School of Med-icine of Wake Forest University, Medical Center Boulevard, Win-ston-Salem, NC 27157; e-mail: cferrari@bgsm.edu

AJH 1998;11:137–146

© 1998 by the American Journal of Hypertension, Ltd. 0895-7061/98/$19.00Published by Elsevier Science, Inc. PII S0895-7061(97)00400-7

Accumulating evidence suggests that theheptapeptide angiotensin-(1-7) [Ang-(1-7)] regulates the pressor and proliferativeactions of angiotensin II (Ang II) through

tissue specific mechanisms that result in the produc-tion and release of nitric oxide, prostacyclin, orboth.1–4 Ang-(1-7) is generated from angiotensin I(Ang I) by two endopeptidases [neutral endopepti-dase 24.11 and prolyl-endopeptidase 24.26] present inthe blood, the brain, the kidney, and the vascularendothelium5; a third endopeptidase (metalloen-dopeptidase 24.15) converts Ang I into Ang-(1-7) invascular smooth muscle.6 Continuing research hasdemonstrated that Ang-(1-7) may function as an anti-hypertensive hormone acting to limit the vasopressorand hypertensive actions mediated by Ang II.7–9 Thisinterpretation has been derived from animal experi-ments that showed that endogenous neutralization ofAng-(1-7) raises blood pressure and antagonizes theantihypertensive action of angiotensin converting en-zyme inhibitors. Furthermore, Ang-(1-7) causes a dosedependent relaxation of coronary artery rings10,11 andpial arteries12 and a fall in blood pressure when giveninto a vein.13

In hypertensive subjects14 and spontaneously hy-pertensive rats8,9 the therapeutic effects of long-termadministration of converting enzyme inhibitors areassociated with increases in plasma levels of Ang-(1-7). Because Ang-(1-7) possess natriuretic activity thatis independent of changes in renal blood flow andglomerular filtration rate15 the possibility exists thatthe antihypertensive effects may be mediated in partby actions of this peptide in the kidneys. In keepingwith this interpretation, rat urine was recently re-ported to contain high levels of Ang I and Ang-(1-7),as compared with Ang II.16 Moreover, the data sug-gested the renal tubules as a site for the generation ofAng-(1-7) found in urine.16 Therefore, the presentstudy investigated whether Ang-(1-7) exists in theurine of human subjects and explored the existence ofpotential differences in untreated essential hyperten-sives.

METHODS

Patient Population A total of 31 white healthy vol-unteers and 18 untreated essential hypertension sub-jects, aged 23 to 58 years, were the subjects for thisstudy. Pregnant women were excluded. Informed con-sent was obtained from all subjects before their par-ticipation, and the study was approved by the Insti-tutional Review Board at each of the two centers.

Study Protocol Normal volunteers were recruitedfrom the staff of the respective clinics, whereas pa-tients were selected from the outpatient clinics afterprescreening for consideration for inclusion through a

standardized questionnaire given on their firstplanned visit. Eligible subjects were instructed of theprocedure for the collection of urine by the nursingstaff assigned to the study. Urine was collected for24 h beginning with the second urinary void the morn-ing after their first visit. Subjects returned to the clinicthe following day to deliver the urine collection, un-dergo medical examination, and have their bloodpressure measured with a mercury sphygmomanom-eter, following the guidelines described by the Amer-ican Heart Association. Blood pressure was deter-mined as the average of two readings obtained 5 minapart. The first reading was obtained 10 min after thesubjects assumed a sitting position. In addition, 16normotensive subjects provided a sample of venousblood (5 mL, antecubital vein) at the completion of the24-h urine collection period to assess plasma concen-trations of Ang-(1-7), as described later here and de-tailed elsewhere.3,17

The cohort of untreated hypertensive subjects in-cluded in this study was thoroughly characterized bymeans of clinical and laboratory procedures to excludesecondary causes of high blood pressure and comor-bid conditions. Essential hypertension was defined asthe average of two sitting systolic blood pressures .140 mm Hg or diastolic blood pressure . 90 mm Hgon two separate occasions determined at least 2 weeksapart. Patients with blood pressures $ 140/90 mm Hgwere considered eligible if they were not receivingblood pressure medication, had not been taken anti-hypertensive drugs for at least 12 weeks before thestudy, or were under any other medication for condi-tions unrelated to their blood pressure. At the time oftheir enrollment in the study hypertensive subjectsreceived an additional comprehensive physical andlaboratory examination and were also instructed inthe procedure for urine collection as previously de-scribed.

Laboratory Methods Blood sequential multiple anal-ysis (SMA) and urinalysis were performed by the hos-pital’s reference laboratories. Additional samples of ve-nous blood and the 24 h urine collection were processedfor determinations of plasma and urine concentrations ofAng-(1-7) by direct radioimmunoassay (RIA).

Collection of the urine specimen was accomplishedin a plastic container to which 20 mL of 6N HCl hadbeen added as a preservative. This acid treatment ofthe urine was found in preliminary experiments tocompletely inhibit the degradation of angiotensin pep-tides for over 36 h at ambient temperature. Subjectswere instructed to gently agitate the container aftereach urine void and to keep the container throughoutthe 24 h collection period inside a plastic ice chest. Theextraction procedure followed that of plasma angio-tensins (see later here) except for the volume of sample

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added to the column and the aliquoting of the eluate.Urine was extracted using Sep-Pak columns (WatersAssociates, Watford, Hertsofordshire, England) acti-vated with 5 mL sequential washes of a mixture ofethanol:water:4% acetic acid (83:13:4), methanol, ultrapure water, and 4% acetic acid. The sample was ap-plied to the column, washed with ultrapure water andacetone, and eluted with 2-1 mL and 1-1.5 mL washesof a mixture of ethanol:water:4% acetic acid. Theweight of the eluate was recorded and from the totaleluate two 2 mL aliquots were transferred into coni-cal bottom polystyrene tubes and dried. The elutedsample was reconstituted into a Tris buffer with0.1% bovine serum albumin. Ang-(1-7) was measuredusing the antibody described by us previously.18,19

Samples were corrected for recoveries as described byus elsewhere.17

Chromatographic analysis of urinary Ang-(1-7) wasachieved by high performance liquid chromatography(HPLC) using the heptafluorobutyric acid (HFBA, Se-quanal Grade, Pierce, Rockford, IL) solvent system.6

This system consisted of 0.1% HFBA, pH 3.0 (mobilephase A) and 80% acetonitrile/0.1% HFBA (mobilephase B). The analysis was performed on an AppliedBiosystems 400 HPLC (Foster City, CA) equippedwith a narrow bore Nova-Pak C18 column (Waters,Milford, MA, 2.1 3 150 mm) and an Aquapore C8guard column (Applied Biosystems, 3.2 3 15 mm).Samples (0.5 mL in 20% mobile phase B) were chro-matographed under the following conditions: 25%mobile phase B for 2 min; 25% to 45% mobile phase Bfor 20 min (linear gradient) and 45% mobile phase Bfor 10 min at a flow rate of 0.35 mL/min at ambienttemperature. Fractions were collected at 1 min inter-vals and completely evaporated in a Savant vacuumcentrifuge (Savant Instruments, Holbrook, NY) beforeanalysis with the Ang-(1-7) RIA.20 In addition, 500fmol of synthetic Ang-(1-7) was added to identify therelation between the sample immunoreactive peakand the corresponding retention time of the Ang-(1-7)standard. Peptide standards were monitored at 220nmol/L (0.1 AUFS, Applied Biosystems 783 Spectro-flow detector).

Plasma Ang-(1-7) concentrations were evaluated us-ing techniques developed by our laboratory,21 or mod-ified by us, as described elsewhere.20 Briefly, venousblood was collected in a cocktail of protease inhibitors(25 mmol/L ethylenediaminetetraacetic acid [EDTA],0.44 mmol/L o-phenanthroline, and 0.12 mmol/Lpepstatin A) that prevents degradation and artifactualproduction of peptides during drawing and collectionof the sample.17 Plasma was extracted in Sep-Pak col-umns as described previously for urine samples.

As documented in detail by us elsewhere,17,19 theAng-(1-7) antibody showed no cross-reactivity withAng I or Ang II, whereas it cross-reacted with Ang-

(2-7) by 100% and , 0.01% with the Ang-(3-7) frag-ment. The minimum detectable levels of the assaywere 2.5 pg/tube for Ang-(1-7). The intraassay coeffi-cient of variation averaged 8%.

Statistical Analysis For the purposes of this reportparticipants were stratified in two groups: normoten-sive subjects (SBP , 140 and DBP , 90 mm Hg) anduntreated hypertensives (SBP $ 140 mm Hg or DBP $90 mm Hg and taking no blood pressure medication).Initial analyses included descriptive statistics for thetwo groups using the Student’s t test for continuousvariables and x2 or Fisher’s exact test for categoricalvariables. Values are reported as means 6 1 SD, unlessdenoted otherwise.

The relationship between urinary Ang-(1-7) levelsand hypertensive status was investigated by: a) ana-lyzing the relationship between urinary Ang-(1-7) andblood pressure by linear regression; and b) calculatingthe relationship between urinary Ang-(1-7) and dis-ease status with a logistic regression model wherehypertension was the dependent variable and urinaryAng-(1-7) was the main predictor variable, after ad-justment for potential confounders (age, body massindex, gender, and smoking status). Odds ratios and95% confidence intervals were calculated from modelparameters by Woolf’s method.22 Results were ex-pressed as odds ratios and were interpreted as thelikelihood for the prevalence of hypertension amongindividuals with high levels of Ang-(1-7) comparedwith the prevalence of high blood pressure amongthose subjects with lower levels of Ang-(1-7). Simpleand multiple linear regression analysis was used todescribe the association between urinary Ang-(1-7)and mean blood pressure levels, before and after ad-justment for potential confounding variables. The re-lationship between urinary Ang-(1-7) and hyperten-sion was assessed by a logistic regression technique.SAS software (SAS Institute, Cary, NC) was used toperform all analysis. A P # .05 was considered statis-tically significant.

RESULTS

Findings in Normal Healthy Volunteers A total of31 healthy volunteers (15 from Spain and 16 from theUS) provided 24 h urine collections for the character-ization of urinary concentrations of Ang-(1-7). Themean age of the group (6 SD), comprised of 17 menand 14 women, was 39 6 7 years. Body mass indexaveraged 25.2 6 4.3 kg/m2. As indicated in Table 1,the mean office systolic and diastolic blood pressurevalues (6 SD) following completion of the 24 h urinecollection were 118 6 11 mm Hg and 74 6 7 mm Hg,respectively. Group values for urinary volume andurinary excretory function in the 31 normotensivehealthy volunteers are documented in Table 1. These

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values are within the range defined for normal sub-jects.23 In addition, subanalysis of potential differencesin age, gender, weight, blood pressure, and renal ex-cretory function revealed no statistical differences be-tween subjects recruited from either clinics.

Urine from healthy normotensive individuals wereprocessed for the identification of Ang-(1-7) by HPLC.A single peak with a retention time coinciding withthe elution of the synthetic Ang-(1-7) standard wasdetected in all urine samples (Figure 1). For the groupas a whole the mean (6 SD) urinary levels of Ang-(1-7)averaged 0.06 6 0.02 pmol/mL, a value that yields aurinary concentration of 62.6 6 22.6 pmol/L. Twenty-four hour urinary excretion of Ang-(1-7) averaged98.9 6 44.7 (SD) pmol/24 h. Urinary excretion ofAng-(1-7), expressed as a function of 24 h urinaryexcretion of creatinine, was 12.2 6 9.2 pmol/mmol ofcreatinine.

Concurrent measurements of the concentration ofAng-(1-7) in plasma were obtained in 16 of the 31normal healthy volunteers. Mean values (6 SD) forurinary Ang-(1-7) concentration and excretion, in thissubset of the 31 normal volunteers, averaged 0.05 60.01 pmol/mL and 70.0 6 29.2 pmol/24 h, respec-tively. These values were not statistically differentfrom those determined for the group of normotensivesubjects as a whole (P . .05). In addition, 24 h urinevolume (1568 6 943 (SD) mL) and urinary creatinineexcretion (11.5 6 4.6 mmol/24 h) did not differ signif-icantly from the values determined for the group as awhole (Table 1). In contrast, plasma concentrations ofAng-(1-7) averaged 22.9 6 8.8 (SD) pmol/L, a valuethat was 2.75-fold lower than the concentration of theheptapeptide in the urine (63 6 17 [SD] pmol/L).These data showed that Ang-(1-7) is present in the

urine of normal subjects at a concentration signifi-cantly higher than that recorded in the plasma.

Angiotensin-(1-7) in Essential Hypertension A totalof 18 white subjects, 12 men and six women, met thecriteria for inclusion (newly diagnosed or no antihy-pertensive treatment for at least 12 weeks). As illus-trated in Table 1, the average age of the essentialhypertensive subjects was not different than that de-termined in the group of normotensive individuals(P 5 .116). Although mean body weight was slightlyhigher in hypertensive subjects (P 5 .22) than in nor-motensive controls (Table 1), only their body massindex attained a statistical significant difference (P 5.005; Table 1) when compared with the values ob-tained in normotensive volunteers. At the time of theurine collection mean values (6 SD) for systolic anddiastolic blood pressures were 146 6 16 mm Hg and96 6 8 mm Hg, respectively. These values were sta-tistically higher than those determined in normal vol-unteers (Table 1).

Urine volume was not different in essential hyper-tensive subjects compared with the control group (Ta-ble 1). Although urinary excretion of creatinine washigher (P 5 .02) in the untreated essential hyperten-sive subjects, this difference was not statistically sig-nificant after correction for body weight (P 5 .08;Table 1). Twenty-four-hour urinary sodium and po-tassium volumes were also not different in hyperten-sive and normotensive subjects (Table 1).

Urine from untreated essential hypertensive sub-jects also contained Ang-(1-7) that was verified byHPLC to elute with a retention time identical to that ofthe synthetic Ang-(1-7) standard. The mean (6 SD)urinary levels of Ang-(1-7) in essential hypertensive

TABLE 1. CLINICAL AND LABORATORY CHARACTERISTICS OF STUDY PARTICIPANTS

Variable Normotensive Group Hypertensive Group

Number of subjects 31 18Age (years) 39 6 7 43 6 9Sex (M/F) 17/14 12/6Proportion of men (%) 54.8 50.0Body weight (kg) 73 6 15 79 6 18Body mass index (kg/m2) 25.2 6 4.3 27.8 6 4.8†Arterial pressure (mm Hg)

Systolic blood pressure 118 6 11 146 6 16*Diastolic blood pressure 74 6 7 96 6 8*Mean blood pressure 89 6 8 112 6 9*

Renal functionUrine volume (mL/24 h) 1644 6 725 1789 6 836Urinary creatinine excretion (mmol/24 h) 9.8 6 4.1 12.9 6 5.2‡Urinary creatinine excretion (mmol/kg/24 h) 0.14 6 0.05 0.16 6 0.05Urinary sodium excretion (mEq/24 h) 289 6 188 200 6 116Urinary potassium excretion (mEq/24 h) 65.7 6 21.0 66.2 6 20

Values are means 6 1 SD. Statistical differences in P values compared with the normotensive group are, * P , .05; † P , .005; ‡ P , .02.

AJH–FEBRUARY 1998–VOL. 11, NO. 2140 FERRARIO ET AL

subjects averaged 39.4 6 18.0 pmol/L. Twenty-four-hour urinary excretion of Ang-(1-7) averaged 60.2 614.6 (SD) pmol/24 h, whereas urinary excretion ofAng-(1-7), expressed as a function of 24 h urinaryexcretion of creatinine, was 5.78 6 3.91 (SD) pmol/mmol of creatinine. Both urinary Ang-(1-7) concentra-tion (P 5 .0005) and excretion (P 5 .0001) in essentialhypertensive subjects were significantly lower thanthe values determined in normal volunteers (Figure 2).The values for Ang-(1-7) concentration in the urine of

untreated essential hypertensive subjects were 37%(P , .001) lower than those found in healthy volun-teers. Comparison of the differences in the urinaryexcretion of Ang-(1-7) after correction for urinary ex-cretion of creatinine yielded an even greater differenceamounting to 52% (Figure 2). Thus, these data showedthat urine from essential hypertensive subjects con-tained Ang-(1-7) in quantities significantly less thanthose found in normal volunteers. As plasma con-centrations of Ang-(1-7) were not measured in thisgroup of subjects, we do not known whether the lowerlevels of urinary Ang-(1-7) would be associated withcomparative reductions in the circulating levels ofAng-(1-7).

Association Between Urinary Ang-(1-7) and Essen-tial Hypertension The presence of Ang-(1-7) in theurine of normal volunteers and untreated essentialhypertensive subjects prompted a further examinationof potential relationships among the concentration ofthe peptide in the urine and hemodynamic and renalexcretory variables. A multiple correlation analysiswas performed among all recorded variables for thegroup of normal volunteers and untreated essentialhypertensive subjects, separately and in combination.

Plasma concentrations of Ang-(1-7) in normotensivecontrols correlated significantly with both urinaryAng-(1-7) concentration (r 5 0.50, P , .05) and excre-tion (r 5 0.49, P , .05). Moreover, both urinary con-centration and excretion rates of Ang-(1-7) showed anegative correlation with systolic (r 5 20.60, P , .05),diastolic (r 5 20.50, P , .05), and mean arterial pres-sure (r 5 20.60, P , .05). Finally, urinary excretion ofAng-(1-7) was also correlated significantly with both24 h urinary sodium (r 5 0.49, P , .05) and potassiumexcretion (r 5 0.77, P , .05).

Correlations between either urinary concentrationor excretion of Ang-(1-7) and blood pressure or elec-trolyte excretion were not statistically significant inuntreated essential hypertensive subjects. In the over-all study group (normotensive and hypertensives), astatistically significant inverse correlation was foundbetween urinary levels of Ang-(1-7) and body massindex (r 5 20.30, P , .05); as found in the group ofnormal subjects, a statistically significant inverse cor-relation (Figure 3) was also established among urinaryexcretion of Ang-(1-7) and systolic (r 5 20.41, P 5.003), diastolic (r 5 20.47, P 5 .0007) and mean 20.47,P , .001) arterial pressures.

The existence of statistically significant correla-tions between blood pressure and urinary levels ofAng-(1-7) led us to explore further the potentialeffect of Ang-(1-7) as an independent predictor ofblood pressure levels using the following linear re-gression model:

FIGURE 1. High pressure liquid chromatography characteriza-tion of angiotensin-(1-7) in a pool of urine collected from normo-tensive subjects. The peak of human immunoreactive angiotensin-(1-7) (bottom panel, solid bars) corresponds with the peak reten-tion time of the synthetic angiotensin-(1-7) standard (top panel,solid bars). Chromatographic conditions are described in the text.

AJH–FEBRUARY 1998–VOL. 11, NO. 2 ANGIOTENSIN-(1-7) IN HUMANS 141

SBP 5 a 1 b1/[urinary Ang-(1-7)] 1 b2/BMI) 1

b3/Male) 1 b4/Age) 1 b5/Smoking Status)

Table 2 shows that urinary Ang-(1-7) and age weresignificant independent predictors of systolic bloodpressure with an estimated 6 mm Hg lower systolicblood pressure for each 1 SD increase in urinary Ang-(1-7). The probability of hypertension associated withurinary Ang-(1-7) levels was calculated using a logis-tic regression model that included parameters for uri-nary Ang-(1-7), age, sex, body mass index, and smok-ing status as independent variables. As illustrated inTable 3, urinary levels of Ang-(1-7) was the singlesignificant predictor with an odds ratio of 0.92 (95% CI0.88 to 0.97, P , .003), whereas age was of borderlinesignificance (P 5 .058). The direction of the odds ratiossuggest that individuals with higher Ang-(1-7) excre-tion rates are less likely to be hypertensives.

DISCUSSION

These studies demonstrate for the first time that Ang-(1-7) is a regular constituent of human urine existingat concentrations higher in normal than in untreatedessential hypertensive subjects. In normal subjects andin the study group as a whole, urinary concentrationand excretion of Ang-(1-7) were significantly and in-versely correlated with arterial blood pressure andpositively correlated with 24 h excretion of sodium

and potassium. Another important finding of thisstudy is that urinary concentrations of Ang-(1-7) in thenormotensive subjects were positively correlated withplasma levels of the peptide. The demonstration oflower levels of Ang-(1-7) in the urine of untreatedessential hypertensive subjects is a finding worthy offurther investigation, as verification of this initial ob-servation in a larger group of subjects may provide anovel tool for a noninvasive assessment of the role ofthe angiotensin system in the evolution of primaryessential hypertension.

This cross-sectional study included both normoten-sive individuals and a group of subjects that werecharacterized clinically as having primary hyperten-sion. Hemodynamic and renal excretory values werecomparable in the subjects recruited from either clinic,a finding that excluded a possible effect of the studiedpopulation in the interpretation of the findings. To ourknowledge these are the first studies that determinedthe existence of significant differences in the concen-trations of Ang-(1-7) in the plasma and urine of nor-mal subjects. The higher concentrations of Ang-(1-7) inthe urine compared with plasma are in agreementwith previous studies done by us in the rat.16 Thesedata suggest, but obviously do not prove, that thekidneys may be an important source for the produc-tion of urinary Ang-(1-7). Although plasma levels ofAng-(1-7) were not measured in the group of un-

FIGURE 2. Twenty-four-hour urinary excretion rates of angiotensin-(1-7) corrected for creatinine in normal volunteers anduntreated essential hypertension subjects.

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treated hypertensive subjects, a previous study fromour laboratory14 reported plasma Ang-(1-7) levels av-eraging 42 6 40 (SD) fmol/mL in untreated essentialhypertensive subjects. These data would suggest thatessential hypertension might be associated withhigher plasma concentrations of Ang-(1-7). Therefore,the presence of reduced concentrations of Ang-(1-7) in

the urine of essential hypertensive subjects suggestsimpaired filtration, reduced tubular secretion, or acombination of both.

The finding that Ang-(1-7) is present in humanurine extends the study of Chappell et al16 in normalrats. These investigators found high concentration andexcretion rates for both Ang I and Ang-(1-7) in the

FIGURE 3. Scattergram depicting the relationship between systolic (top panel) or diastolic (bottom panel) arterial pressure andurinary concentrations of angiotensin-(1-7) in 31 normotensive and 18 untreated hypertensive subjects. Confidence intervals (95%) forthe slope of the relation between angiotensin-(1-7) concentration and systolic blood pressure are 20.87 to 20.18 pmol/L mm Hg.Corresponding 95% confidence intervals for the slope of the relation between angiotensin-(1-7) concentration and diastolic blood pressureare 21.35 to 20.39 pmol/L mm Hg.

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urine of Sprague-Dawley rats. In contrast, Ang II ex-cretion rates were much lower than those determinedfor both Ang I and Ang-(1-7).16 In addition, studies ofthe metabolic degradation of Ang I, Ang II, and Ang-(1-7) in rat urine showed that the kidney was a site forthe generation of urinary Ang-(1-7).16 In keeping withthis interpretation, we found that addition of 125I-AngI to human urine did not result in the production ofAng-(1-7) during 60 min of incubation at ambienttemperature. Nevertheless, we took the precaution ofexcluding the activity of urinary peptidases form con-tributing to the formation of Ang-(1-7) during samplecollection, storing, and processing. Harvesting of theurine in 6N HCl acid and storing the samples duringcollection in a chilled container (;20°C) were verifiedin preliminary experiments to inhibit proteolysis. Fur-thermore, HPLC analysis of the immunoreactive ma-terial found in the urine of both normal and essentialhypertensive subjects coeluted with the Ang-(1-7)standard and excluded degradation of the peptideinto the smaller fragments [Ang-(2-7) and Ang-(3-7)]during sample collection or storage. The minor un-identified peaks present in chromatogram contributednegligibly (, 5%) to the assay and did not correspondto any of the known fragments of angiotensins.

The cost of performing multiple assays did not al-low for concurrent measurements of Ang I and Ang II

in the urine obtained from our subjects. There is evi-dence, however, that in humans, as in rats, Ang II maybe excreted in quantities significantly lower than thoseof Ang-(1-7). Boer et al24 determined the urinary ex-cretion rates of Ang II in normal subjects. From theirdata we calculated that the urinary excretion of Ang IIwas about one-half the urinary excretion rates re-ported in our healthy volunteers for Ang-(1-7). Thesedata agree with our previous observation of high con-centrations of Ang-(1-7) but not Ang II in rat urine.16

A renal renin-angiotensin system may play an impor-tant role in the regulation of renal function and thepathogenesis of arterial hypertension.25,26 Our findingsthat plasma levels of Ang-(1-7) were significantly lowerthan corresponding values of Ang-(1-7) in the urine inhealthy volunteers suggest, but obviously did not prove,that the content of Ang-(1-7) in human urine may reflectlocal production of the peptide in a kidney compart-ment. The enzymes required for the processing of Ang-(1-7) from Ang I are abundant in renal tissue, especiallyin the brush border of proximal tubules.5 Moreover,Ang-(1-7) is the major product of the metabolism of AngI during its passage through the renal circulation (un-published observations), a finding that was first de-scribed by Admiraal et al27,28 in patients with renovas-cular hypertension. On the other hand, the existence of asignificant correlation between plasma and urine Ang-(1-7) in normal subjects suggest the presence in humansof a mechanism for either selective filtration or tubularsecretion of Ang-(1-7) from plasma. Although circulatingpeptides are readily filtered in the glomerulus, it hasbeen reported that Ang II is rapidly destroyed at this siteor may even undergo rapid uptake in the brush borderof proximal tubules.29,30 The presence of proline at theC5terminus of Ang-(1-7) makes the peptide more resis-tant to degradation; this may protect Ang-(1-7) throughits passage in the urinary tract. Therefore, our studies inhuman subjects suggest that a portion of the Ang-(1-7)found in urine may originate in the circulation. Thisinterpretation does not negate, however, the possibilitythat Ang-(1-7) may also be either formed or secreted intothe tubular fluid. It has been reported that Ang II wasrecovered essentially intact in the renal pelvis followinginjection distal to the brush border region of the proxi-mal tubule.29–31 These data provides additional evidencefor the existence of a tissue system capable of formingangiotensin peptides in the distal components of thenephron.32–34 Although there are no previous studies ofthe formation and catalytic metabolism of Ang-(1-7)in human renal tubules, the data obtained in thisstudy suggest that the kidneys may be a principal sourcefor the presence of Ang-(1-7) in human urine. Furtherwork, however, will be required to determine the mech-anisms that contribute to the presence of Ang-(1-7) inhuman urine.

TABLE 2. LINEAR MULTIPLE REGRESSIONSUMMARY FOR URINARY ANG-(1-7) LEVELS AND

SYSTOLIC BLOOD PRESSURE ADJUSTED FORPOTENTIAL CONFOUNDERS

Parameter Coefficient P

b1, Urinary Ang-(1-7) (pmol/L) 20.38 .0001b2, Body mass index (kg/m2) 0.84 .0080b3, Male 0.33 .5426b4, Age (years) 2.27 .6434b5, Smoker 24.90 .5679

Linear regression coefficients derived from a model that included urinaryangiotensin-(1-7) and adjusted for potential confounders. F value by anal-ysis of variance 5 4.08; P 5 .0043.

TABLE 3. HYPERTENSION ODDS RATIOS AND95% CONFIDENCE INTERVALS FOR SELECTED

COVARIABLES

CovariableOddsRatios 95% CI P

Urinary Ang-(1-7) (pmol/L) 0.92 0.88–0.97 .003Age (years) 1.10 0.99–1.22 .058Body mass index (kg/m2) 1.09 0.93–1.27 .295Male 1.39 0.27–6.67 .680Smoker 0.56 0.04–7.12 .650

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The finding that subjects with untreated essentialhypertension had significantly lower levels and excre-tion rates of Ang-(1-7) was not accounted for by dif-ferences in urine volume or renal excretory capacity.Thus, the lower levels of Ang-(1-7) excretion mayreflect reduced synthesis, filtration, or tubular secre-tion of Ang-(1-7) in the kidney of untreated essentialhypertensive subjects. On the other hand, it can not beexcluded whether the differences in urinary Ang-(1-7)in essential hypertension may be a consequence oflower glomerular filtration rates. Reduced urinary ex-cretion of Ang II has been reported in essential hyper-tensive subjects by Fukuchi.35 Because the status of therenin-angiotensin system was not characterized in oursubjects, it can not be ascertained whether the lowerlevels of urinary Ang-(1-7) were accounted for by areduced renin activity. Further characterization of therelation between urinary Ang-(1-7) and plasma reninand Ang II concentrations will be required to ascertainthe mechanism responsible for the presence of lowerconcentrations of Ang-(1-7) in the urine of essentialhypertensive subjects.

That excretion rates of Ang-(1-7) were inversely cor-related with arterial pressure in both normal volun-teers and after combined analysis of both normal andhypertensive subjects is worthy of further investiga-tion. Taken in concert with previous studies of thefunction of Ang-(1-7),1–4 we hypothesize that this cor-relation may be an indicator of an important role ofthe peptide in the control of arterial pressure. Previousstudies showed that Ang-(1-7) acts as a endogenousvasodilator2 and natriuretic agent.15 Moreover, con-centrations of plasma Ang-(1-7) are significantly re-duced in a transgenic model of renin-dependent hy-pertension,19 whereas endogenous neutralization ofAng-(1-7) with a selective antibody elevates bloodpressure in both normal and hypertensive rats.7 Stud-ies in humans14 and in animal models of hyperten-sion3 indicate that the antihypertensive action of an-giotensin converting enzyme inhibitors is associatedwith increases in Ang-(1-7) production. Although thedemonstration of an inverse correlation betweenblood pressure and urinary Ang-(1-7) levels cannot beinterpreted as providing evidence for a cause-effectrelationship, these new data suggest a potential forthis variable to serve as an indicator of the activity ofthe angiotensin system and, perhaps, a marker forassessing the therapeutic effectiveness of antihyper-tensive drugs.

The results obtained by analysis of the data with alogistic regression model and the assessment of theratio of the probability of the occurrence of low levelsof urinary Ang-(1-7) with hypertension are in keepingwith this interpretation. Our sample estimate for anodd ratio of 0.92 indicate that hypertensive subjectswere less likely to excrete high amounts of urinary

Ang-(1-7) when compared with healthy volunteers. Inthis context, the observation that body mass index wasinversely correlated with urinary Ang-(1-7) is an in-teresting finding, as obesity is associated with elevatedblood pressure, reduced vasodilator capacity, andhigher dose requirements of angiotensin convertingenzyme therapy.36–39 These intriguing associations arecompatible with the hypothesis that reduced produc-tion or activity of Ang-(1-7) may contribute to theevolution of hypertension. Further studies will be re-quired to validate these observations.

In summary, the present report identifies, but doesnot explain, the mechanism for the existence of signif-icant differences in the excretion of urinary Ang-(1-7)among normal and essential hypertensive subjects.Although the lower concentrations of urinary Ang-(1-7) in essential hypertension were not accounted forby differences in renal excretory capacity betweennormotensive and hypertensive subjects, renin sup-pression in these subjects could also explain our find-ings.

ACKNOWLEDGMENTS

We thank Ms. Margaret King for her important contributionin the performance of the radioimmunoassays.

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