Active Kallikrein Response to Changes in Sodium-Chloride Intake in Essential Hypertensive Patients1

Journal of the American Society of Nephrology 443

Active Kallikrein Response to Changes in Sodium-ChlorideIntake in Essential Hypertensive Patients1Claudio Fern,2 Cesare Bellini, Antonio Carbomagno, Giovambattista Desideri, and Anna Santucci

C. Fern, C. Bellini, A. Carlomagno, G. Desideri, Univer-

sily of Rome “La Sapienza,” Institute of I Clinica Med-ca, Andrea Cesalpino Foundation, Rome, Italy

A. Santucci, Universily of L’Aquila, Department of In-

ternal Medicine and Public Health, L’Aquila, Italy

(J, Am. Soc. Nephrol. 199#{243};7:443-453)

ABSTRACT

1To evaluate the behavior of active kallikrein excre-

tion in salt-sensitive and salt-resistant hypertensive pa-tients during changes in sodium-chloride (NaCI) in-take, 61 male, nonobese, nondlabetic outpatientsaffected by uncomplicated essential hypertensionwere given a diet that contained 140 mmol NaCI perday for 2 wk. Patients then received either a low- (20mmol NaCI/day) or a high- (320 mmol NaCI/day)sodium diet for 2 wk, according to a randomized,double-blind, cross-over protocol. Hypertensive pa-

tients were classified as salt sensitive when their dia-stolic blood pressure rose by at least 10 mm Hg afterthe high-sodium diet, and decreased by at least 10mm Hg after the low-sodium diet, considering as

baseline blood pressure values those that were takenat the end of the 140 mmol NaCI/day Intake period.

The remaining patients were classified as salt resistantor, when diastolic blood pressure increased by 10mm Hg or more after low-sodium intake, as counter-

regulating. Twenty-three patients were therefore clas-sified as salt sensitive, 28 as salt resistant, and 10 as

counterregulating. The baseline active kallikrein ex-cretion was significantly lower (P < 0.0001) in salt-

sensitive (0.62 ± 0.31 U/24 h) patients than in salt-resistant (1.39 ± 0.44 U/24 h) and counterregulatingpatients (1 .27 ± 0.38 U/24 h). Surprisingly, the kallikreinresponse to changes in sodium intake was similar inall subgroups, although enzyme excretion was al-ways at the lowest level in salt-sensitive hypertensivepatients. This buffer group also showed the highestplasma atrial natriuretic peptide levels (28.2 ± 8.5fmol/mL, P < 0.0001 versus salt-resistant and counter-regulating patients), and the greatest peptide incre-

ment with sodium load (P < 0.0001 versus salt-resistant

1 ReceIved November 29, 1994. Accepted September 15, 1995.

2 correspondence to Dr. c. Fern, Instituto dl I clinical Medica. Universita La

saplenza. Viale del Policlinico, 155, 00161 Rome, Italy.

1046.6673/0703-0443$03.00/0Journal of the AmerIcan Society of Nephrologycopyright C 1996 by the American Society of Nephrology

and counterregulating patients). Counterregulatingpatients showed the steepest increase in plasma re-

nm activity (from 0.24 ± 0.18 to 0.83 ± 0.21 ng/L per s,P < 0.001) and decrease of plasma atrial natriuretic

peptide (from 26.1 ± 6.3 to 6.8 ± 3.1 fmol/mL, P <

0.001) when switched from a high to a low-sodium

intake. In conclusion, salt-sensitive hypertensive pa-tients excrete less active kallikrein than do salt-resis-

tant and counterregulating patients, but maintain a

normal enzyme response to changes in dietary so-

dium intake. The exaggerated response of atrial na-

triuretic peptide to high-sodium intake that was ob-served in the same patients could be compensating

for an impaired renal capability to excrete a sodiumload.

Key Words: Sodium. hypertension. renin. atrial natriuretic

peptides, kidney

S alt sensitivity is defined as a significant rise In

blood pressure when individuals switch from alow- to a high-sodium (NaCl) intake and/or vice versa( 1-6). The frequency of salt sensitivity is stronglyinfluenced by age (2-4,7-9), body weight (2,3,8-13),

race (2,3,8,9, 14-16), gender (6, 17), and family history

of hypertension (2-4,18).

In middle-aged Caucasian populations, approxi-mately 50% of hypertensive patients can be included

within the salt-sensitive subgroup (1-3,5,6,17,19). In

the remaining patients, blood pressure does not

change or may even increase during bow-sodium In-

take ( 1-3,20-22), making the potential efficacy of

health care programs that are directed toward Indis-

criminate sodium restriction debatable (2,3,21-26).

As a consequence, a number of studies attempted to

define genetic (8,27), familial (8), racial (8,9,14-16),

sexual (6, 1 7), ethnic ( 1 6), environmental (28), renal

(14,29,30), endocrine (19,27,31-33), and cell-mem-

brane (34-36) characteristics related to salt sensltlv-

ity, and to indicate one or more parameters that would

be able to predict the individual blood-pressure re-

sponse to sodium.In this regard, the work of B#{246}nner et a!. with nor-

motensive patients (37,38) and our group’s work with

hypertensive patients (39) have recently suggested

that a bow-kalbikrein excretion might determine the

salt sensitivity of blood pressure. In particular, we

demonstrated that a reduced kallikrein excretion at

baseline was present in 84% of salt-sensitive and 22%

of salt-resistant patients (39). Therefore, we suggested

an inability of the kidney to modulate kalbikrein pro-

duction during changes in sodium intake as the cause

for sodium retention and the consequent bbood-pres-

sure rise (39).

I STUDY PROTOCOL I

- ..NORMAL LOW

#{182}‘Blood pressure and hormonal evaluation

Kallikrein and Salt Sensitivity

444 Volume 7 - Number 3 ‘ 1996

Although fascinating, this theory was merely spec-

ulative, because we did not evaluate the kalllkrein

response to changes in dietary sodium intake (39).

Furthermore, we excluded from the study the so-

called “counterregulating hypertensives,” i.e. , thosepatients whose blood-pressure levels increase with

sodium restriction (and/or decrease with sodium

load) (20-22,39-44). This fact limited the pathoge-

netic as well as therapeutic implications of our find-

ings . Indeed, counterregulating hypertensive patients

are those patients who could report a potential incre-

ment of cardiovascular risk after severe sodium re-

striction (3,20-22).

Herewith we present a randomized double-blind

cross-over study that alms to Investigate the behavior of

urinary active kallikrein during changes in dietary sod-

um-chboride intake in a barge number of nonobese,nondiabetic hypertensive men, classified according to

individual blood-pressure response to changes in so-

dium intake as salt-sensitive, salt-resistant or counter-

regulating hypertensive patients. In this study, we also

analyzed the relationship between kalllkrein and other

sodium-rebated hormonal changes. Because reproduc-

Ibifity Is a crucial point in salt-sensitivity evaluation

(3,6, 18,45), and the entire existence of salt sensitivity

has been attributed to artifacts and / or casual variations

of blood pressure (2,6,45), we also tested the reproduc-

ibility of our procedure 6 months after the end of the

study.

PATIENTS AND METHODS

Patients

The study was approved by the Ethics Committee of the

Andrea Cesalpino Foundation and stated in 79 never-treated outpatients who had a body mass index > 1 9 and <

27 kg/m2. Patients were between the ages of 30 to 55 yr(mean, 47.8 ± 6.3 yr), and were affected by uncomplicatedessential hypertension. All patients were white Caucasians.Because the menstrual cycle strongly Influences the behav-ior of several of the hormones that are Involved in renalsodium handling (46), and because urine collection for kal-

hlkreln and electrolyte determinations Is not feasible In men-struating women, only male patients were recruited.

In all patients, diastolic blood pressure (DBP) was requiredto be >95 mm Hg and < 1 1 4 mm Hg at three consecutivevisits performed at 1 -wk intervals before definitive enroll-

ment in the study (Figure 1 ). Serum creatinine concentrationwas < 100 �tmol/L: proteinurla was absent, microalbumin-uria was <20 �.tg/mIn on two consecutive 24-h urine collec-tions, ‘ � ‘ I-OrthoIodohyppurate scintirenographies were nor-

mal. All patients had a normal glucose tolerance, asevaluated by oral glucose tolerance tests (75 g D-glucose),and had no cardiovascular alterations as evaluated on thebasis of clinical and ultrasound studies. The secondaryforms of hypertension were excluded by clinical and labora-tory assessments.

At the first of the three screening visits, patients answered

standard questions concerning family history of hyperten-sion, according to a previously described methodology(39,47). In brief, family information was first obtained byeach patient and then confirmed by the family physician and,when possible, by other patient’s relatives (wife, brethren,

I MU�kS 2W((KS I 2(EKS I 2W�K3 I a UEKS I

I PHASE 1 I PHASE 2 IFigure 1 . Schematic representing the study protocol. Re-cruited patients were followed-up for 4 wk, during which theenrollment criteria were evaluated. Then, after the definitiveenrollment, patients followed a normal sodium diet (140mmol NaCI/day) for 2 wk to obtain the same baselineconditions. After baseline blood and urine samplings, pa-tients were randomized to one of two different sequences: 32patients followed the high-sodium diet (320 mmol/day),then the normal-sodium diet, and ended with the low-sodium diet (20 mmol/day); 33 patients followed the oppo-site sequence (LOW-NORMAL-HIGH). Blood-pressure mea-surements for the salt sensitivity assessment and blood andurine samplings for hormonal assays were repeated at theend of each diet period.

children, etc.). A positive family history was defined as the

presence of at least one first-degree relative who was affected

by essential hypertension. To avoid bias, the family historyinformation was collected by a separate group of researchers,who were unaware of the study purpose and results.

During the recruitment period, all patients were on aconstant sodium Intake ( 140 mmol NaC1 per day). Eachpatient received a scheme ofa diet prepared by our dietitians,and containing approximately 1 g/kg protein, 2.5 g/kg car-bohydrates, 0.7 g/kg fat, 35 mmol calcium, 20 mmol NaCl,and 70 mmol potassium per day. A total amount of 120 mmol

NaC1 was added to the diet by means of six capsules per day

(each capsule containing 20 mmol of NaC1), to be takenorally. Each patient was given oral and written instructionsand detailed information on the way to cook their meals

without added salt. To simulate as closely as possible theabsorption of salt with food, according to the Italian habit,patients were advised to take three capsules at lunch andthree at dinner.

The total number of calories remained identical during theentire study period and was calculated on the basis ofindividual needs. All patients were advised to drink 1 .5 L

water/day. Adherence to the diet was assessed by 24-h urinecollections on the last two consecutive days of each week.Participants were instructed by our medical staff on how to

collect the 24-h urine sample. Sodium-chloride excretionwas required to be between 100 mmol/24 h and 140

mmol/24 h. On these same occasions, potassium and cal-cium excretions were also measured. In particular, urinarypotassium was required to be between 50 and 70 mmol/24 hto allow patients’ continued participation in the study.

Fern et al


Of the Initial cohort of 79 patIents, 14 dId not enter thesalt-sensitivity assessment phase because of noncompliance

with the diet (N = 7), DBP <95 mm Hg (N = 4), or > 1 14 mmHg (N = 1 ) requirements, and because of hack of return tofollow-up visits (N = 2). Therefore, only 65 patients were

definitively enrolled in the following phase of the study.Salt-Sensitivity Assessment Phase. At the end of the

recruitment phase, blood samples for plasma renin activity(PRA), aldosterone (PAC), atrIaj natriuretic peptide (ANP),

and digoxin-like substance (DLS) levels were taken after 1 hat rest in a supine position. Two consecutive 24-h urinecollections were requested from each patient, to determinethe urinary active kallikreln excretion. After blood and urinesamples were taken, patients were assigned in a randomdouble-blind manner to a high- (320 mmoh NaC1 per day for2 wk) or a low- (20 mmol NaCl per day for 2 wk) sodiumintake (Figure 1). Thirty-two patients received first the highand then the low-sodium diet. The remaining patients (N =

33) followed the reverse order. To have a similar baseline dietbefore switching to low or high-sodium intake, a period of 2wk on a diet that contained 140 mmol NaCl per day wasInterposed during the cross-over phase (Figure 1).

Individual sodium Intake was obtained by changing theNaC1 content of the capsules. During the high-sodium intakeperiod, each capsule contained 50 mmol NaC1. During thelow-sodium Intake period, each capsule contained a placebo(dextrose). Throughout the study, potassium intake re-malned unvarled (70 mmol potassium per day).

During the evaluation of salt sensitivity, compliance wasalso controlled by measuring 24-h sodium excretion levels.Patients were considered compliant when their 24-h sodiumexcretion level was >270 mmol and <40 mmol In all urinecollections obtained during the high- and low-sodium Intakeperiods, respectively. Urinary potassium levels were requiredto be between 50 mmol/24 h and 70 mmol/24 h. Fouradditional patients were noncomphiant, all during the high-sodium diet period. and the salt-sensitivity assessmentphase was therefore successfully completed In 6 1 patients.

Patient ClassificationPatients were classified as salt sensitive when a change of

10 mm Hg or more of DBP occurred after each period of eitherlow- or high-sodium intake (i.e. , blood pressure decreasesduring the low-sodium intake period. and increases duringthe ingh-sodlum intake period) (39). Changes in blood pres-sure were evaluated with baseline blood pressure consideredto be the measurement taken at the end of the 140-mmolNaC1 diet. In contrast to our previous report (39), patients

who had an Increase in blood pressure with sodium restric-tion were not excluded from the study, and were admitted toa third group of patients, I.e. , “counterregulators.”

ReproducibilityThe assessment of reproducibility of the above procedure

was scheduled for 32 patients, who were randomly chosenamong those patients who initially participated in the study.In all cases, patients were recalled after 6 months from thelast visit of the salt-sensitivity assessment phase. The repro-duciblilty evaluation was not feasible In six patients (four forloss at follow-up, two for compliance problems during the

changes in sodium Intake), although it was successfullyperformed in the remaining ones (N = 26).

Blood Pressure and Laboratory EvaluationsThroughout the study, blood-pressure levels were mea-

sured on the last two days of each week in the Outpatient

Unit of the Institute of I Clinica Medica, after 15 mm In a

seated position in an air-conditioned room (22 to 24#{176}C),by a

standard Riva-Rocci sphygmomanometer (Zenith. Rome, It-aly) and a stethoscope placed over the brachial artery. As

recommended for nonobese adults (48), a normal-size cuffwas used to encircle the nondominant arm, which was

supported comfortably at heart level. Systolic blood pressure(SBP) was taken at Korotkoffs Phase I, and DBP was taken atKorotkoffs Phase V (48). The first measurement of blood

pressure and heart rate was excluded, and the average of thefollowing three measurements, taken at 3-mm intervals, wasconsidered. Blood pressure was always taken by the sameresearchers, who were unaware of the study purpose, design,and results.

After blood-pressure measurement, patients rested com-fortably In bed for 1 h. Blood samples for PRA, PAC, ANP,

DLS, serum Na� and K� were then withdrawn from anantecubital vein. Plasma samples were Immediately stored at

-80#{176}C.All of the assays were performed no later than 8 daysafter storage. Plasma ANP was assayed as described else-where (47). In short, ANP was extracted from plasma byAmprep c-18 columns (Amersham, Amersham, Bucking-hamshire, U.K.) activated with 4 mL methanol and 12 mLdistified water. Each column was loaded with 2.5 mL acidi-fled plasma. The eluate was collected In 1 mL ethanol, driedunder vacuum, reconstituted in 1 mL neutral phosphatebuffer, and then measured by a commercially available hu-man-ANP (99-126) RIA kit (Peninsula Laboratories, Bel-mont, CA). Synthetic human-ANP (99-126) was used as astandard. Mean recovery for ANP in this study was 80%:interassay and intra-assay variations were less than 10%.

PRA and PAC were assayed by commercially available RIAkits (Sorin Biomedica S.p.A., Vercehli, Italy). Plasma DLS wasmeasured by the method of Balzan et al. (49) with slightmodifications. In brief, plasma samples were purified asfollows: Amprep c- 1 8 columns (Amersham) were activatedwith 5 mL methanol and 20 mL distilled water. Each columnwas loaded with 2.5 mL plasma, washed with 20 mL distilledwater, and then eluted with 2 mL methanol. The eluates offour 2.5-mL aliquots of plasma were collected, dried undervacuum, and reconstituted in 1 mL ofa buffer solution (NaCl130 mmol/L, sucrose 20 mmol/L, glucose 10 mmol/L, Tris-HC1 10 mmol/L, pH 7.4). For DLS radiolmmunoassay, asolid-phase system was used (Diagnostic Products Corpora-tion, Los Angeles, CA). Digoxin (Wellcome, Pomezia, Italy)was used as a standard. The other laboratory measurementswere performed by standard laboratory methods.

The 24-h urinary excretion level of active kalhikrein wasmeasured on fresh urine samples by hydrolysis of the chro-mogenic trypeptide substrate H-D-V-L-R-pNA (S-2266;Kabivitrum, MOlndal, Sweden) according to the method ofAmundsen et al. (50).

Statistical Analyses

The statistical evaluation was performed by a PC OlivettiM-380XP1 (Olivetti, Ivrea, Italy). The statistical softwareSPSS (SPSS Inc., Chicago, IL) was used. To evaluate intra-group statistical significances. we used the paired t test andanalysis of variance for repeated measures. To establish thedifferences among groups, we used unpaired t test and

one-way analysis of variance followed by post hoc analysis

(Bonferroni’s test). Linear regression and correlation wereused to evaluate the relationship between two variables.Descriptive parameters were analyzed by the chi-squared

method.


446 Volume 7 ‘ Number 3 ‘ 1996

The results of each statistical test were considered signil-icant when the associated probability value (P) was <0.05.Unless otherwise stated, data are given as mean ± SD.

RESULTS

General Characteristics

The general characteristics of the study population

are given in Table 1.

When all patients were considered (N = 6 1 ; mean age,

47. 1 ± 2.9 yr), baseline SBP and DBP were 160.7 ± 5.4

mm Hg and 104.8 ± 2.9 mm Hg, respectively. Urinary

Na� excretion was 123.9 ± 14.6 mmob/24 h, whereas

urinary K� excretion was 53. 1 ± 9.5 mmol/24 h (mean

urinary Na/K ratio, 2.3 ± 0.6). PRA and PAC bevels were

0.40 ± 0.18 ng/L per s and 302.9 ± 52.3 pmob/L,

respectively. Plasma ANP and DLS levels were 2 1 .2 ±

9. 1 fmol/mL and 34.2 ± 10.5 pg/mL, respectively.

Urinary active kallikrein excretion was 1 .09 ± 0.37

U/24 h and were negatively correlated with plasma ANP

levels (r = -0.677, P < 0.0005).

Changes in dietary sodium intake induced minor

changes in SBP and DBP bevels (Table 2), although the

investigated hormones showed significant variationsduring the different diets. In particular, PRA, PAC and

urinary kabhlkrein levels significantly increased with

sodium restriction and decreased with sodium load

(Table 2). The opposite behavior was demonstrated for

circulating ANP and DLS bevels (Table 2).

At the end of the salt-sensitivity evaluation, 23

(37.7%) men from a total of6l hypertensive men, were

classified as salt sensitive, 28 (45.9%) as salt resis-

tant, and 10 ( 16.4%) as counterregulating. Their gen-

eral characteristics are given in Table 1 . As shown in

Table 1 , salt-sensitive patients had higher fasting insu-

bin (P < 0.05) and triglyceride (P < 0.05) levels than did

salt-resistant and counterregulating patients. Counter-

regulating patients were younger than salt-resistant and

salt-sensitive patients (P < 0.05) (Table 1). The batter

group showed higher baseline blood-pressure levels

than the other two groups (P < 0.02) (Table 1). A positive

history for hypertension occurred more frequently in

salt-sensitive patients than in both of the remaining

groups (P < 0.02) (Table 1).

Blood Pressure and Hormonal Data

Salt-Sensitive Group (N = 23 patients; mean age,

48. 1 ± 3. 1 yr). SBP was 163.7 ± 4.9 mm Hg at the end

of the normal-sodium diet, increased to 1 7 1 .8 ± 7.4

mm Hg (P < 0.0001) after the high-sodium diet and

decreased to 158.7 ± 8.2 mm Hg(P < 0.0001) after the

low-sodium diet (Figure 2). After the high-sodium

intake, SBP was significantly higher than after the

low-sodium intake (P < 0.000 1) (Figure 2). DBP was106.8 ± 2.7 mm Hg after the normal-sodium intake,

increased to 1 16.3 ± 5.8 mm Hg(P < 0.0001) after the

high-sodium diet, and decreased to 94.7 ± 4.5 mm Hg

(P < 0.000 1) after the bow-sodium intake period (Fig-ure 2). After the high-sodium intake period, DBP was

significantly higher than after the bow-sodium intake(P < 0.0001) (Figure 2).

Urinary Na� excretion was 1 18.7 ± 12.8 mmol/24 h

after the normal-sodium diet, 288.7 ± 10.7 mmol/24

h (P < 0.0001) after the high-sodium diet, and 25.3 ±

8.6 mmol/24 h (P < 0.0001) after the low-sodium diet.

Body weight was 77.5 ± 2.4 kg after the normal-sodium diet, increased to 79.2 ± 2.3 kg after thehigh-sodium diet (P < 0.05), and decreased to 75.7 ±

2. 1 kg at the end of the low-sodium intake period (P <

0.05 versus normal-sodium intake; P < 0.0005 versushigh-sodium intake).

At the end of the normal-sodium intake phase. PRA

bevels were 0.32 ± 0. 15 ng/L per s, PAC levels were

TABLE 1 . General characteristics of the study population (mean ± SD)

Characteristic All Salt Sensitive Salt Resistant Counterregulating

Number 61 23 28 10Age (yr) 47.1 ± 2.9 48.1 ± 3.1 47.9 ± 2.5 45.2 ± 3.2aFamily history of hypertension (Yes/No) 26/35 15/8b 9/19 2/8Systolic blood pressure (mm Hg) 160.7 ± 5.4 163.7 ± 4�9b 159.6 ± 5.3 158.7 ± 6.1Diastolic blood pressure (mm Hg) 104.8 ± 2.9 106.8 ± 2#{149}7b 104.2 ± 3.1 103.9 ± 2.9Heart rate (bpm) 73.2 ± 5.4 73.6 ± 4.5 72.8 ± 6.1 73.1 ± 5.7

Body mass Index (kg/m2) 24.8 ± 1.6 24.7 ± 1.8 25. 1 ± 1 .5 24.8 ± 1.4Serum cholesterol (mmol/L) 4.97 ± 0.14 5.01 ± 0.18 4.92 ± 0.12 4.98 ± 0.11Serum triglycerides (mmol/L) 1.42 ± 0.14 1.51 ± 0.1 1C 1.39 ± 0.15 1.35 ± 0.16Plasma glucose (mmol/L) 5.22 ± 0.38 5.25 ± 0.43 5.18 ± 0.39 5.22 ± 0.31

Plasma Insulin (pmol/L) 22.34 ± 2.81 28.31 ± 2,95c 19.78 ± 3.25 18.93 ± 2.19Blood urea nitrogen (mmol/L) 5.32 ± 1.69 5.61 ± 1.34 5.12 ± 1.83 5.22 ± 1.91Serum creatinine (�mol/L) 92.53 ± 6.61 93.57 ± 6.31 91.49 ± 7.72 92.52 ± 5.81

Creatinine clearance (mL/s) 1.69 ± 0.30 1.71 ± 0.25 1.67 ± 0.31 1.68 ± 0.35Serum sodium (mmol/L) 141.3 ± 4.7 141.7 ± 5.3 139.8 ± 4.7 142.3 ± 4.2Serum potassium (mmol/L) 4.10 ± 0.41 4.19 ± 0.38 3.98 ± 0.57 4.12 ± 0.27

a p < 0.05 Counterregulating hypertensive patients versus salt-sensitive and salt-resistant hypertensive patients.b p < 0.02 Salt-sensitIve hypertesive patients versus salt-resistant and counterregulating hypertensive patients.C p < 0.05 Salt-sensitive hypertensive patients versus salt-resistant and counterregulating hypertensive patients.

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Figure 2. Blood-pressure changes during normal (140 mmol/day), low (20 mmol/day) and high-sodium diets (320 mmol/day) in salt-sensitive (N = 23; open squares), salt-resistant(N = 28: filled squares), and counterregulating hypertensivepatients (N = 10; dIagonal-tIlled squares). Standard devia-tions were omiffed for clarity. � � < 0.0001 versus normal-sodium diet, � P < 0.0001 versus high-sodium diet, � = P <

0.01 versus normal and versus high-sodium diet. Intergroupsignificant differences are given on the lines in the figure.

278.3 ± 75.2 pmob/L, ANP levels were 28.2 ± 8.5

fmob/mL, and DLS levels were 45.2 ± 12.3 pg/mL.

Urinary active kalbikrein excretion resulted of 0.62 ±

Fern et al


TABLE 2. Primary clinical and endocrine changes after different sodium intake periods in a group of 61hypertensive patients (mean ± SD)#{176}

, .

CharacterIsticNormal NaCI

.

dietLow NaCI diet

.

High NaCI diet

Weight (kg) 76.8 ± 4.5 75.6 ± 33b 78.2 ± 4.1Systolic blood pressure (mm Hg) 160.7 ± 5.4 161.3 ± 85b 168.7 ± 8.9cDiastolic blood pressure (mm Hg) 104.8 ± 2�9b 104.2 ± 56b 107.7 ± 6.1Heart rate (bpm) 73.2 ± 5.1 73.5 ± 4.9 73.3 ± 4.9Plasma renin activity (ng/L per 5) 0.40 ± 0. 18 0.62 ± 0.22c d 0.26 ± 0. 15C

Plasma aldosterone (pmol/L) 302.9 ± 52.3 360.3 ± �55C. d 239.8 ± 73,6cPlasma ANP (fmol/mL) 21.2 ± 9.2 12.3 ± 49c,d 29.3 ± 7.2cPlasma DLS (pg/mL) 34.2 ± 10.5 26.3 ± 8.7c d 42.7 ± 16.6�Urinary active kallikreln (U/24 h) 1 .09 ± 0.37 1.40 ± 0.35c d 0.79 ± 0.22cUrinary Na� excretion (mmol/24 h) 123.9 ± 14.6 26.9 ± 6.3c d 291 .4 ± 8.7cUrinary K� excretion (mmol/24 h) 53.1 ± 9.5 56.7 ± 8.9 52.3 ± 74C

a During the normal-sodium diet. patients assumed 140 mmol NaCI per day; during the low-sodium diet. patients assumed 20 mmol NaCI per day;and during the high-sodium diet. patients assumed 320 mmol NaCI per day. ANP. atrial natriuretic peptide; DLS. digoxin-like substance.b p < 0.05 versus high-sodium diet period.C p < o.ooo5 versus normal-sodium diet period.d p < 0.0005 versus high-sodium diet period.

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0.31 U/24 h. The urinary excretion ofactive kalllkreinand plasma ANP bevels were inversely correlated (r =-0.615, P < 0.002). Hormonal parameter changesthat occurred with sodium restriction and sodium

load are given in Figure 3.Salt-Resistant Group (N = 28 patients; mean age,

47.9 ± 2.5 yr). With regard to blood pressure, SBPafter the normal-sodium intake was 159.6 ± 5.3 mmHg and did not change after both the high- ( 162.3 ±

9.4 mm Hg, not significant ENS]) and the low-sodium

diets (159.6 ± 9.7 mm Hg, NS) (Figure 2). DBP was104.2 ± 3. 1 mm Hg after the normal-sodium intakeand was not significantly influenced by sodium intakevariations ( 105. 1 ± 5.3 mm Hg INS] at the end of thehigh-sodium diet and 103.8 ± 5.8 mm Hg ENS] at theend of the low-sodium intake) (Figure 2). Body weightwas 76. 1 ± 3.8 kg after the normal-sodium diet, 77.2± 3.5 kg after the high-sodium diet (NS), and 75.2 ±

2.8 kg after the bow-sodium diet (NS).

The urinary Na� excretion level was 1 25.9 ± 13.7

mmol/24h after the normal-sodium diet, increased to

296. 1 ± 12.5 mmob/24 h after the high-sodium diet (P< 0.005), and decreased to 28.5 ± 7.7 mmol/24 h

NaCI DIET after the bow-sodium diet (P < 0.005). After the nor-mal-sodlum intake period, PRA bevels were 0.4 1 ±

0. 19 ng/L per s, PAC bevels were 308.9 ± 88.7 pmol/L,and ANP levels were 18.6 ± 9.3 fmol/mL. Plasma DLSbevels were 30. 1 ± 8.9 pg/mL. The urinary activekalbikrein excretion bevel was 1 .39 ± 0.44 U/24 h. The

hormonal parameter changes induced by differentsodium diets are given in Figure 3.

Counterregulating Group (N = 10 patients; meanage, 45.2 ± 3.2 yr). The behavior of these patientsmimicked that of the salt-resistant patients. Interest-ingby, these patients showed the greatest rise In PRA

after the low-sodium diet (Figure 3). Indeed, the meanpercentage increment of PRA bevebs was 72.9% Inthese patients, whereas It was 25% in salt-sensitive (P

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FIgure 3. Primary hormonal changes after normal (140 mmol/day), low (20 mmol/day), and high-sodium diets (320 mmol/day)In salt sensItive (N = 23; open squares), salt-resistant (N = 28; filled squares), and counterregulatlng hypertensive patients (N =

10; dlagonal-fllled squares). DLS, dlgoxln-Ilke substance: ANP, atrial natrluretic peptide. Standard deviations were omitted forclarity. * � < � versus normal-sodIum diet, ** = p < 0.0001 versus high-sodium diet. Intergroup significant dIfferences aregIven on the lInes In the figure.



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< 0.00 1 versus counterregulating patients) and53.6% in salt-resistant patients (P < 0.001 versus

counterregubating patients; P < 0.0 1 versus salt-sen-

sitive patients) (Figure 4). Counterregubating patientsalso showed the greatest ANP decrease (-59.5%) afterthe bow-sodium diet (P < 0.00 1 versus salt-sensitivepatients and salt-resistant patients) (Figure 4). Asexplained in the “Methods” section, counterregubatingpatients reported a rise in DBP levels after sodiumrestriction. In this context, it is interesting to note thatfive tenths of our counterregubators reported also a 10

mm Hg decrease in DBP levels when they followed ahigh-sodium diet.

Intergroup Comparison. No differences were foundbetween salt-resistant and salt-sensitive patients with

regard to baseline PRA and PAC bevels. On the con-trary, plasma ANP and DLS levels were significanflyhigher (P < 0.000 1 ) in salt-sensitive than in salt-

resistant and counterregulating patients (Figure 3).Moreover, compared with salt-resistant patients, thesalt-sensitive patients were characterized by bowervalues of urinary active kallikrein excretion (P <

0.000 1) (Figure 3). With regard to the hormonal mod-ifications after the different sodium diets, sodium-related PRA changes were clearly less evident in salt-sensitive patients than in salt-resistant patients(Figure 3). The opposite results were found for plasmaANP and DLS bevels. Indeed, although both hormonesincreased significantly with sodium load in both salt-sensitive and salt-resistant patients, the mean incre-ment was significantly higher in the first than in thesecond group (Figure 4). Surprisingly, mean activekalblkreln excretion changes during different sodium

diets were similar in all groups (Figure 4).Other comparisons, regarding the counterregulat-

ing group, have been given in the above paragraph.

Fern et al


IHIGH NoCI DIET VERSUSNORMAL NadI DIET

Figure 4. Primary hormonal variations in salt-sensitive (N = 23: open squares), salt-resistant (N = 28; filled squares), andcounterregulating hypertensive patients (N = 10; dIagonal-filled squares) when switched from a normal (140 mmol/day) to alow-sodium diet (20 mmol/day) (left panel), and from a normal to a high-sodium diet (320 mmol/day) (right panel). DLS,dlgoxln-ilke substance; ANP, atrial nafriuretic peptide; PRA, plasma renin activity; PAC, plasma aldosterone concentration; uKK,urinary active kalllkrein excretion. � � < 0.0001 , salt-sensitive patients versus salt-resistant and counterregulating patients: � P <

0.001 , counterregulatlng patients versus salt-resistant and salt-sensitive patients; � = P < 0.01 , salt-sensitive patients versussalt-resistant patients; 0 � = P < 0.01 , salt-resistant patients versus counterregulating patients; § P < 0.0001 , salt-resistant patientsversus counterregulatlng patients.

ReproducibilityReproducibility ofour procedure was assessed in 26

patients (i.e. , 42.6% ofthe entire initial population: 10salt-sensitive, 1 1 salt-resistant, and five counterregu-

bating patients). The individual response to changes indietary sodium intake was found to be different inthree patients, compared with the first study. Onesalt-sensitive patient became salt resistant. Of thepreviously counterregubating patients, one was foundto be salt resistant, whereas one previously salt-resistant patient became a counterregulating patient.Sodium-related changes in blood pressure in both thefirst and the second study were significanfly corre-bated (SBP changes after low-sodium diet: r = 0.720, P

< 0.0005; DBP changes after bow-sodium diet: r =0.704, P < 0.0005. SBP changes after high-sodium

diet: r = 0.672, P < 0.0005; DBP changes after high-sodium diet: r = 0.899, P < 0.0005).

DISCUSSION

In a previous report (39), we found that a lowurinary excretion of active kalbikrein was highly pre-dictive of a salt-sensitive behavior of blood pressure,and combined with the highest prevalence of hyper-tensive relatives. We interpreted the reduced kal-likrein excretion as the consequence of an impairedcapability of the kidney to modulate the enzyme pro-

duction during changes in sodium intake.The study presented here aimed to verify this hy-

pothesis, and to analyze the relationships amongkaffikrein and other sodium-regulating hormones.

Furthermore, because the Influence of sodium intake

on blood pressure (5 1 ,52), and the concept of saltsensitivity itself (45) are still debated, we planned to

also study the reproducibility of our procedure of

evaluating blood-pressure sensitivity to changes insodium intake.

When one considers the entire study population,blood-pressure levels were not significantly Influencedby sodium (Table 2). However, the arbitrary definition

of a cut-off value of salt sensitivity as an increment ofDBP of at beast 10 mm Hg with the high-sodium dietand of an equal decrement with the low-sodium dietIdentified 37.7% of hypertensive patients (N = 23) assalt sensitive and 45.9% (N = 28) as salt resistant.

Therefore, this study confirmed that the bbood-pres-sure response to salt restriction is markedly heteroge-neous ( 1-4,6, 15, 1 7,43,44). Furthermore, It supportsprevious studies done in hypertensive patients(3,6,39,41 ,42) and normotensive patients (18,42), in-dicating that severe salt restriction may have adversepressor effects in some individuals, because a low-

sodium diet increased DBP levels In 1 0 of 6 1 patients(16.4%).

The reexamination of 26 patients indicated a veryhigh reproducibility of our procedure of evaluatingblood-pressure response to changes in dietary sodiumintake. Indeed, only 3 of 26 patients changed theirclass, compared with the first study. Therefore, this

study further demonstrates that the pressor responseto changes in sodium intake is not a result of casualvariations of blood pressure, at beast within a short


450 Volume 7 - Number 3 ‘ 1996

period of time. Furthermore, only one patient changedfrom the salt-sensitive to the salt-resistant class,

whereas the remaining two patients switched from the

salt-resistant to the counterregubating group (N = 1)

or vice versa (N = 1 ), thus confirming the inefficacy of

dietary sodium restriction in their management. Anexcellent study by Overback et a!. ( 18), in complete

agreement with our findings, demonstrated in normo-tensive patients that the blood-pressure response to

changes in dietary sodium intake, as assessed by a

protocol similar to the current one, was highly repro-

ducible In more than 90% of the subjects. Indeed,reexamination of 3 1 normotensive patients revealed

different blood-pressure responses to changes in so-dium Intake only in three subjects (18).

On the other hand, patient selection allowed us toexclude any confounding factors rebated to bodyweight (10-12), disturbances of the carbohydrate me-tabohism ( 1 3), and age (3, 1 5, 1 7). With regard to theinfluence of sex, salt sensitivity has been reported tobe more (53) or less marked (54) in women than inmen, although Weinberger et a!. (3) were not able toobserve any difference at all. However, because sev-eral of the hormones that are involved in sodiumhomeostasis are strongly influenced by the menstrualcycle (46), and because urine collection is not possibleIn menstruating women, we decided to select onlyhypertensive men for our study.

Another problem we tried to bypass with the current

protocol Is rebated to the sequence. Indeed, the use ofthe same order in the diet sequence may result in anorder effect, limiting the efficacy of the study (3,5,6).Furthermore, each diet must be followed for a periodof time that is long enough to achieve sodium balance(55,56), and must be identical for all patients (6,57).Therefore, we designed a study in which a short periodof “normal” (I.e. , intermediate) sodium intake pre-ceded the randomization to a 2-wk period on either ahigh- or low-sodium intake. The use of capsules con-taming different amounts of sodium allowed us tomaintain the study in absolute double-blind condi-

tlons, whereas repeated measurements of 24-h un-

nary sodium excretion levels assured the perfect ad-herence to each diet.

With regard to the results obtained, we confirmedthat a low active kalblkrein excretion level during anintermediate sodium intake is linked with salt sensi-

tivity of blood pressure. This association does notImply a causal relationship between kallikreln excre-tion and salt sensitivity. Similarly. It must be empha-sized that it does not prove that an impaired kal-blkreln-kinin system function determines the salt

sensitivity of blood pressure. According to this proto-

cob, we failed to demonstrate an altered kalbikreinresponse to changes in dietary sodium intake In salt-sensitive patients. Indeed, although bow active kal-hikrein excretion levels always resulted, sodium-re-bated changes in urinary excretion were similar in thesalt-sensitive patients, compared with both salt-resis-

tant and counterregulating patients.

This kalbikrein behavior is in complete agreement

with recent findings obtained in normotensive pa-

tients (37,38), demonstrating identical decreases inkailikrein excretion after a high-sodium diet in sub-

jects with either a bow or a high-enzyme excretion bevelat baseline. Interestingly, normotensive patients who

had a bow kallikreln excretion were markedly salt

sensitive, whereas the remaining subjects were salt

resistant (37,38). On the other hand, we have recentlydemonstrated a marked salt sensitivity of blood pres-sure in the presence of a normal kalbikrein response to

changes In dietary sodium chloride Intake in low-

renin hypertensive patients with a reduced kalbikreinexcretion (36).

In this context, experimental studies have already

demonstrated that kalbikrein production is regulatedby potassium rather than by sodium intake (58). In

humans, Berry et at. (59) were abbe to classify 80% of

an Utah population into groups with a high or bow-

kallikrein genotype. In particular, a bow-kablikreinexcretion bevel was combined with the highest preva-bence of hypertensive relatives, segregating as a Men-debian recessive trait (59). Enzyme excretion was de-pendent on a major gene with strong environmentalmodulation by dietary potassium In heterozygotic but

not in homozygotic subjects (60). Therefore, a reducedkallikrein excretion bevel, as webb as an increased

prevalence of familial hypertension. resulted from thepatients’ being homozygous for the bow-kalbikrein gene

or from being heterozygous and having a bow potas-

slum intake (60). In agreement with this concept, twin

studies showed that urinary potassium more than

urinary sodium correlated with urinary kalllkrein andblood-pressure levels (6 1 ). Moreover. dietary potas-sium supplementation significantly increased kal-

bikreln excretion, and reduced blood-pressure levels in

essential hypertensive patients (62). In the study pre-

sented here, potassium intake did not change duringthe assessment of salt sensitivity. As a consequence,the bow kallikrein excretion we found in salt-sensitivehypertensive patients as well as the normality of theenzyme response to changes in sodium intake should

simply reflect the casual allebic distribution and theconsequent presence of both homozygotic and het-erozygotic subjects for the bow-kallikrein gene. Never-theless, a genetic influence on kalblkrein excretiondoes not imply a causal role for kalbikrein in determin-ing a progressive sensitization of susceptible individ-

uals to an “inappropriate” sodium intake. Further-

more, we cannot exclude that the observed kalllknelnbehavior has been influenced by other factors, such asRBF (63). Indeed, unfortunately, we have not mea-sured this parameter in our patients.

With regard to the other fmdings of this study, mean

PRA levels were lower in salt-sensitive than in salt-

resistant patients. Furthermore, PRA bevels increased

slightly with bow sodium intake, confirming previous

data that showed a marked salt sensitivity of bloodpressure in patients who have a PRA unresponsive to

Fern et al


low sodium intake ( 19,3 1 ,33,43,64-66), furosemideadministration (3), and upright posture (47,65).

Counterregubating hypertensive patients showed amarked overstimulation of the renin system after so-

dium restriction. Even more interestingly, the samepatients showed the most evident reduction of plasmaANP with the bow-sodium diet. Because ANP caninhibit renin release in man (67,68), It seems intnigu-ing to speculate that a marked decrease of ANT mayhave favored PRA overstimubation. This latter phe-nomenon could be the primary contributor to theparadoxical behavior of blood pressure that wasshown by counterregulating patients.

In agreement with previous findings ( 18,65,69), theANP increment after the high-sodium diet was morepronounced in salt-sensitive than in salt-resistanthypertensive patients. In this regard, some previousreports (65,70,71) have also demonstrated that theANP response to sodium load is a function of the reninstatus, being more evident in hypertensive patientswith a low renin activity than in those with a highrenin activity. Therefore, because atnial secretion ofANP is primarily a result of volume expansion

(70,72,73), we think our data support the hypothesisby Overback et a!. ( 18), who suggested that an tin-paired ability of the kidney to excrete a sodium loadmight induce a compensatory increase in ANP releasefrom the atnlal myocytes. In a similar way, differencesin extracelbular fluid volume between salt-resistant

and salt-sensitive patients may easily explain theinverse relationship between the kalhikrein and ANPconcentrations that exist in salt-sensitive hyperten-

sive patients, as well as their tendency to displaysuppressed PRA bevels.

In keeping with this interpretation, ANP behaviorwas paralleled by that of plasma DLS. Indeed, on anormal sodium-intake diet, plasma DLS bevels werehigher in salt-sensitive than in salt-resistant patients.Moreover, the increase of this substance with thehigh-sodium diet was particularly evident in the firstgroup, supporting the hypothesis that an impairedability to modulate a sodium load may induce thesecretion of an endogenous substance(s) cross-react-ing with digoxin antibodies (74,75). This finding couldbe particularly interesting because of the suggestednatriuretic action of this substance(s) (47,76). How-

ever, the effective robe of DLS as a natniuretic agent isnot undisputed (76). Therefore, we can state thatsalt-sensitive patients showed the highest sodium-rebated increment of a circulating substance(s) cross-reacting with digoxin antibodies. On the contrary. anyspeculative hypothesis that is based on the renalactions of DLS seems absolutely arbitrary.

Another interesting finding of this study Is rebated to

the higher triglyceride and fasting insulin levels wefound in salt-sensitive patients, compared with salt-resistant and counterregulating patients. In this con-text, Williams et at. (77) have already suggested that

punctiform mutations may explain the segregating sin-gle-gene effects for low urinary kalllkrein excretion, high

sensitivity to environmental “hypertensinogenic” agents,

and fasting hyperinsulinemla and hyperdyslipldemla.

According to this hypothesis, Sharma et a!. (78) have

recently described a hyperinsubinemic response to oral

glucose load in salt-sensitive normotensive patients.Furthermore, we have recently described (65,7 1 ) thepresence offasting hypeninsulinemia in a patient subsetdefined as “non-modulators” because of abnormal an-

giotensin H-mediated controb of PAC release and therenal blood supply. abnormal sodium handling, andsalt-sensitive hypertension (19,31,33).

In conclusion, we presented herewith a randomized.double-blind, cross-over study that confirmed a marked

salt sensitivity of blood pressure in hypertensive pa-

tients who have a reduced excretion of active kalllkrein.Compared with salt-resistant hypertensive patients,salt-sensitive hypertensive patients excreted less activekabllkrein when they ingested either 20 mmol, 140mmol, or 320 mmol NaCl per day. However, the kal-likrein response to different sodium diets was similar inboth groups, suggesting that the enzyme production Isreduced, although its sodium-related regulation Is nor-mal in patients who have a deleterious blood pressuresensitivity to high-sodium intake.

Sodium restriction induced a marked increase ofblood pressure in 10 of 6 1 patients ( 1 6.4%). A blood-pressure increment after sodium restriction waslinked with an overstimubation of the renin system

and suppression of ANP release, suggesting that bothof these hormonal changes may have determined theadverse pressor effects.

Finally, we confirmed that salt sensitivity Is not a

result of casual variations of blood pressure, becauseIdentical pressor responses to changes in dietary so-dium intake were found in 23 of 26 patients whoreceived again both the low and the high-sodium diets6 months after the end of the first study.

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Active Kallikrein Response to Changes in Sodium-Chloride Intake in Essential Hypertensive Patients1

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