Salt-Sensitivity and Immunity Cell Activation

The investigators will employ the Weinberger protocol in 20 patients as previously reported.
They will advise participants to maintain their usual diet and salt consumption until the
onset of the protocol and provide them with a container and appropriate instructions to
collect a 24-hour urine specimen the day before admission to the hospital. Admission will
take place two weeks later, for washout of the effect of the medications, and on an evening,
for the patient to rest overnight in the hospital before onset on the protocol. During these
two weeks, untreated blood pressure will be monitored either by the patients at home or in
interim scheduled visits, to assure that it does not exceed 180/110 mmHg, in which case the
protocol will be discontinued for safety. On admission, volume of the baseline urine will be
measured, and aliquots sent to the lab or frozen at -800C for research analytes. On the
morning after admission (6 am) an ambulatory blood pressure monitor (SpaceLabs 90207 or
90210) will be set up for continuous blood pressure recording throughout the study (every 15
minutes from 6 am until 10 pm and every 30 minutes from 10 pm to 6 am), body weight will be
recorded with the patient wearing a gown (same scale as future days), blood samples will be
obtained for the analytes of the study described below (baseline tests), and a new 24-hour
collection will be started for the period of salt loading at 8 am. Salt loading will be
achieved by the combination of a high-salt diet (isocaloric, with 160 mEq Na and 70 mEq K),
which the patient will consume in its entirety, and an infusion of 2L of saline (300 mEq
Na+). Patients will have free access to water but their food will be limited to that provided
by the protocol. They will retire to bed at 10 pm. On the following morning (6 am) body
weight and blood samples will again be obtained, and the 24-hr collection will be completed
with an intentional voiding at 8 am. These laboratory data will reflect the effect of salt
loading. At 8 am, subjects will be subjected to salt depletion. This is accomplished by
administering an isocaloric diet containing 10 mEq Na and 70 mEq K and continued unlimited
water intake. At 8 am, 12 noon and 4 pm, subjects will be given 40 mg of furosemide orally.
An additional 12-hour urine collection will be started at 8 am and ended with an intentional
void at 8 pm (reflecting the period of furosemide natriuresis). A new 12-hour collection will
start at 8 pm, to be closed with intentional voiding the next morning at 8 am (reflecting the
period of salt depletion). The next morning (6 am) the last recording of body weight and
drawing of blood samples will take place, and by 8 am the 12-hour urine collection for the
period of salt depletion will be completed. The patients will be given breakfast from a
regular hospital diet, which may be supplemented with salty food or fluids if the salt
depletion intervention produced dizziness or documented orthostasis. Once the patient is
stable and exhibits tolerance to the upright posture, he/she will be discharged home with
instructions to resume antihypertensive medications, unless the presence of orthostasis
requires a modification in the regimen, which will be advised by a physician member of the
research team.

Laboratory blood measurements: Tests to be conducted in the 3 daily sets of blood samples
will include; CBCs and routine chemistries (including serum creatinine, Na+ and K+) at the
central laboratory of VUMC; Plasma renin activity by radio-immunoassay; Plasma aldosterone by
radio-immunoassay; Plasma eicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid
(20-HETE) at the eicosanoid core of VUMC by liquid chromatography/tandem mass spectrometry.

Urinalysis: Tests to be conducted in the 4 urine specimens (24-hr baseline, 24-hr salt
loading, 12-hr furosemide natriuresis, and 12-hr salt depletion) will include; Na+, K+ and
creatinine concentration at the central laboratory of VUMC; Urine eicosatrienoic acids (EETs)
and 20-hydroxyeicosatetraenoic acid (20-HETE) at the eicosanoid core of VUMC by liquid
chromatography/tandem mass spectrometry.

Definition of salt sensitivity of blood pressure: Blood pressure data from the monitors will
be downloaded and the average of the systolic blood pressure of the day of salt loading, from
12 noon (end of the saline infusion) until 10 pm will be used as the BP for the salt-loading
period of study. The average of the systolic blood pressure of the day of salt depletion,
from 12 noon (second dose of furosemide) until 10 pm will be used as the BP for the
salt-depleted period of study. A fall in systolic BP ≥10 mm Hg from the salt-loading to the
salt-depletion periods will be used to classify a subject as salt sensitive.

Sodium MRI imaging: The investigators will non-invasively detect Na+ storage in tissues of
humans at the Vanderbilt imaging institute. They will quantify Na+ content in the skin by
23Na MRI with rigorous adherence to previously reported approaches.6, 37, 38. Imaging is done
on a Philips Achieva 3.0T MR scanner (Philips Healthcare, Cleveland OH, USA) with a 23Na
quadrature knee coil (Rapid Biomedical GmbH, Rimpar, Germany). The investigators use
calibration phantoms (aqueous solutions with increasing NaCl concentrations) as reference
standards and scan them together with sections through the subject's calf muscles for quality
control. The left lower leg (the widest part of calf region) is scanned with the skin closely
in contact with the hard surface of the phantom holder for 3 minutes and 52 seconds. All
imaging data are processed off-line with custom MATLAB (R2013a) scripts. Na+ quantification
is performed by comparing signal intensities between tissue and calibration phantoms on the
Na+ image. A linear relationship (Na+ concentration vs signal intensity) is assessed based on
the phantom data, and results from a linear regression are applied to tissue regions to
quantify Na+ content.

Immune Cell Activation Studies: The investigators will determine if salt sensitivity is
associated with the activation state of human monocytes. Heparinized blood samples (40 ml)
are obtained and PBMCs are isolated by Ficoll-gradient. Monocytes are isolated from the PBMC
by magnetic labeling and negative selection using the Miltenyi monocyte isolation kit and
analyzed using flow cytometry. They identify monocytes as CD45+/CD14+ cells and can further
examine intermediate (CD14+/CD16+ and non-classical monocytes (CD14-/CD16+). The CD14+/CD16+
cells which comprise about 10% of the circulation, are of particular interest they produce
increased levels of TNFalpha and promote T cell activation. These are increased in humans
with hypertension and in response to in vitro elevated Na+ exposure. Although subtypes of DCs
are relatively rare in number, the investigators will quantify them by measuring CD45+/CD1c+,
CD45+/CD141+, CD45+/CD209+, CD45+/CD83+ and the recently identified Axl/SICLEC6 cells. 7-AAD
is used to exclude dead cells. Intracellular staining with the single chain antibody D-11 to
detect IsoLG-protein adducts will be used. Surface expression of CD80 and CD86, which are
expressed on mature antigen-presenting cells allowing them to participate in T cell
co-stimulation will be measured. General characterization of peripheral blood mononuclear
cells by flow cytometry for other inflammatory cells including total leukocytes (CD45+
cells), B cells (CD45+/CD19+), total T cells (CD45+/CD3+ cells), and the T cell subtype
(CD3+/CD8+ and CD4+) cells will also be performed. These experiments will be performed in
freshly-isolated cells and also in monocytes exposed to normal salt or high salt for 48 hours
to determine if salt-sensitivity is reflected in the monocytes as shown in Fig. 2. Medium
from monocytes cultured for 48 hours is analyzed for cytokine release including IL-1 beta,
TNF-alpha, IL-6, and IL-23 using luminex. The investigators will also obtain plasma to assess
for cytokines including GM-CSF, IL-4, and Flt3 ligand that are responsible for conversion of
monocytes to DCs. To ensure reproducibility, the samples will be submitted to the Vanderbilt
Endocrinology and Metabolism Core Facility to perform a blinded Luminex analysis.
Measurements of O2·- production and activation of NADPH oxidase including phosphorylation of
p47phox and association of p47phox with gp91phox by immunoprecipitation and western blot both
at baseline and in response elevated Na+ will be done previously described.

Inclusion Criteria:

- We will perform a pilot analysis in 20 hypertensive subjects controlled for gender
(50% men, 50% women), age (18-60 years),

- New or pre-existing diagnosis of essential hypertension defined as systolic blood
pressure >140 mmHg or >90 mmHg diastolic or taking antihypertensive medications
regardless of current blood pressure.

- BMI (18.5-24.9).

- Only subjects who give informed consent will be studied.

Exclusion Criteria:

- Acute cardiovascular event(s) within the previous 6 months

- Claustrophobia precluding obtaining an MRI

- Inability to understand the nature, scope, and possible consequences of the study or
to participate in/comply with the protocol.

- Current excessive alcohol or illicit drug use.

- Blood pressure below the inclusion criteria levels after discontinuation of therapy

- Presence of metal implants such as artificial joints.

- Concomitant diabetes mellitus, type I or II.

- Autoimmune disease.

- Recent vaccination

- Younger or older that inclusion criteria.

- Pregnancy.