<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT03753204</url>
  </required_header>
  <id_info>
    <org_study_id>182038</org_study_id>
    <nct_id>NCT03753204</nct_id>
  </id_info>
  <brief_title>Salt-Sensitivity and Immunity Cell Activation</brief_title>
  <official_title>Salt-Sensitivity and Immunity Cell Activation</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>Vanderbilt University Medical Center</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>Vanderbilt University Medical Center</source>
  <oversight_info>
    <has_dmc>No</has_dmc>
    <is_fda_regulated_drug>Yes</is_fda_regulated_drug>
    <is_fda_regulated_device>No</is_fda_regulated_device>
    <is_us_export>No</is_us_export>
  </oversight_info>
  <brief_summary>
    <textblock>
      Salt-sensitive hypertension affects nearly 50% of the hypertensive and 25% of the&#xD;
      normotensive population, and strong evidence indicates that reducing salt intake decreases&#xD;
      blood pressure and cardiovascular events. The precise mechanisms of how dietary salt&#xD;
      contributes to blood pressure elevation, renal injury, and cardiovascular disease remains&#xD;
      unclear. Our data indicated that monocytes exhibit salt sensitivity, and the investigators&#xD;
      hypothesize that of salt sensitivity of these and similar immune cells correlate with the&#xD;
      hypertensive response to salt intake. Currently, the research tools for diagnosing&#xD;
      salt-sensitivity are costly, time consuming and laborious. In this study the investigators&#xD;
      will identify monocyte salt-sensitivity as a marker of salt-sensitive hypertension.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      The investigators will employ the Weinberger protocol in 20 patients as previously reported.&#xD;
      They will advise participants to maintain their usual diet and salt consumption until the&#xD;
      onset of the protocol and provide them with a container and appropriate instructions to&#xD;
      collect a 24-hour urine specimen the day before admission to the hospital. Admission will&#xD;
      take place two weeks later, for washout of the effect of the medications, and on an evening,&#xD;
      for the patient to rest overnight in the hospital before onset on the protocol. During these&#xD;
      two weeks, untreated blood pressure will be monitored either by the patients at home or in&#xD;
      interim scheduled visits, to assure that it does not exceed 180/110 mmHg, in which case the&#xD;
      protocol will be discontinued for safety. On admission, volume of the baseline urine will be&#xD;
      measured, and aliquots sent to the lab or frozen at -800C for research analytes. On the&#xD;
      morning after admission (6 am) an ambulatory blood pressure monitor (SpaceLabs 90207 or&#xD;
      90210) will be set up for continuous blood pressure recording throughout the study (every 15&#xD;
      minutes from 6 am until 10 pm and every 30 minutes from 10 pm to 6 am), body weight will be&#xD;
      recorded with the patient wearing a gown (same scale as future days), blood samples will be&#xD;
      obtained for the analytes of the study described below (baseline tests), and a new 24-hour&#xD;
      collection will be started for the period of salt loading at 8 am. Salt loading will be&#xD;
      achieved by the combination of a high-salt diet (isocaloric, with 160 mEq Na and 70 mEq K),&#xD;
      which the patient will consume in its entirety, and an infusion of 2L of saline (300 mEq&#xD;
      Na+). Patients will have free access to water but their food will be limited to that provided&#xD;
      by the protocol. They will retire to bed at 10 pm. On the following morning (6 am) body&#xD;
      weight and blood samples will again be obtained, and the 24-hr collection will be completed&#xD;
      with an intentional voiding at 8 am. These laboratory data will reflect the effect of salt&#xD;
      loading. At 8 am, subjects will be subjected to salt depletion. This is accomplished by&#xD;
      administering an isocaloric diet containing 10 mEq Na and 70 mEq K and continued unlimited&#xD;
      water intake. At 8 am, 12 noon and 4 pm, subjects will be given 40 mg of furosemide orally.&#xD;
      An additional 12-hour urine collection will be started at 8 am and ended with an intentional&#xD;
      void at 8 pm (reflecting the period of furosemide natriuresis). A new 12-hour collection will&#xD;
      start at 8 pm, to be closed with intentional voiding the next morning at 8 am (reflecting the&#xD;
      period of salt depletion). The next morning (6 am) the last recording of body weight and&#xD;
      drawing of blood samples will take place, and by 8 am the 12-hour urine collection for the&#xD;
      period of salt depletion will be completed. The patients will be given breakfast from a&#xD;
      regular hospital diet, which may be supplemented with salty food or fluids if the salt&#xD;
      depletion intervention produced dizziness or documented orthostasis. Once the patient is&#xD;
      stable and exhibits tolerance to the upright posture, he/she will be discharged home with&#xD;
      instructions to resume antihypertensive medications, unless the presence of orthostasis&#xD;
      requires a modification in the regimen, which will be advised by a physician member of the&#xD;
      research team.&#xD;
&#xD;
      Laboratory blood measurements: Tests to be conducted in the 3 daily sets of blood samples&#xD;
      will include; CBCs and routine chemistries (including serum creatinine, Na+ and K+) at the&#xD;
      central laboratory of VUMC; Plasma renin activity by radio-immunoassay; Plasma aldosterone by&#xD;
      radio-immunoassay; Plasma eicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid&#xD;
      (20-HETE) at the eicosanoid core of VUMC by liquid chromatography/tandem mass spectrometry.&#xD;
&#xD;
      Urinalysis: Tests to be conducted in the 4 urine specimens (24-hr baseline, 24-hr salt&#xD;
      loading, 12-hr furosemide natriuresis, and 12-hr salt depletion) will include; Na+, K+ and&#xD;
      creatinine concentration at the central laboratory of VUMC; Urine eicosatrienoic acids (EETs)&#xD;
      and 20-hydroxyeicosatetraenoic acid (20-HETE) at the eicosanoid core of VUMC by liquid&#xD;
      chromatography/tandem mass spectrometry.&#xD;
&#xD;
      Definition of salt sensitivity of blood pressure: Blood pressure data from the monitors will&#xD;
      be downloaded and the average of the systolic blood pressure of the day of salt loading, from&#xD;
      12 noon (end of the saline infusion) until 10 pm will be used as the BP for the salt-loading&#xD;
      period of study. The average of the systolic blood pressure of the day of salt depletion,&#xD;
      from 12 noon (second dose of furosemide) until 10 pm will be used as the BP for the&#xD;
      salt-depleted period of study. A fall in systolic BP ≥10 mm Hg from the salt-loading to the&#xD;
      salt-depletion periods will be used to classify a subject as salt sensitive.&#xD;
&#xD;
      Sodium MRI imaging: The investigators will non-invasively detect Na+ storage in tissues of&#xD;
      humans at the Vanderbilt imaging institute. They will quantify Na+ content in the skin by&#xD;
      23Na MRI with rigorous adherence to previously reported approaches.6, 37, 38. Imaging is done&#xD;
      on a Philips Achieva 3.0T MR scanner (Philips Healthcare, Cleveland OH, USA) with a 23Na&#xD;
      quadrature knee coil (Rapid Biomedical GmbH, Rimpar, Germany). The investigators use&#xD;
      calibration phantoms (aqueous solutions with increasing NaCl concentrations) as reference&#xD;
      standards and scan them together with sections through the subject's calf muscles for quality&#xD;
      control. The left lower leg (the widest part of calf region) is scanned with the skin closely&#xD;
      in contact with the hard surface of the phantom holder for 3 minutes and 52 seconds. All&#xD;
      imaging data are processed off-line with custom MATLAB (R2013a) scripts. Na+ quantification&#xD;
      is performed by comparing signal intensities between tissue and calibration phantoms on the&#xD;
      Na+ image. A linear relationship (Na+ concentration vs signal intensity) is assessed based on&#xD;
      the phantom data, and results from a linear regression are applied to tissue regions to&#xD;
      quantify Na+ content.&#xD;
&#xD;
      Immune Cell Activation Studies: The investigators will determine if salt sensitivity is&#xD;
      associated with the activation state of human monocytes. Heparinized blood samples (40 ml)&#xD;
      are obtained and PBMCs are isolated by Ficoll-gradient. Monocytes are isolated from the PBMC&#xD;
      by magnetic labeling and negative selection using the Miltenyi monocyte isolation kit and&#xD;
      analyzed using flow cytometry. They identify monocytes as CD45+/CD14+ cells and can further&#xD;
      examine intermediate (CD14+/CD16+ and non-classical monocytes (CD14-/CD16+). The CD14+/CD16+&#xD;
      cells which comprise about 10% of the circulation, are of particular interest they produce&#xD;
      increased levels of TNFalpha and promote T cell activation. These are increased in humans&#xD;
      with hypertension and in response to in vitro elevated Na+ exposure. Although subtypes of DCs&#xD;
      are relatively rare in number, the investigators will quantify them by measuring CD45+/CD1c+,&#xD;
      CD45+/CD141+, CD45+/CD209+, CD45+/CD83+ and the recently identified Axl/SICLEC6 cells. 7-AAD&#xD;
      is used to exclude dead cells. Intracellular staining with the single chain antibody D-11 to&#xD;
      detect IsoLG-protein adducts will be used. Surface expression of CD80 and CD86, which are&#xD;
      expressed on mature antigen-presenting cells allowing them to participate in T cell&#xD;
      co-stimulation will be measured. General characterization of peripheral blood mononuclear&#xD;
      cells by flow cytometry for other inflammatory cells including total leukocytes (CD45+&#xD;
      cells), B cells (CD45+/CD19+), total T cells (CD45+/CD3+ cells), and the T cell subtype&#xD;
      (CD3+/CD8+ and CD4+) cells will also be performed. These experiments will be performed in&#xD;
      freshly-isolated cells and also in monocytes exposed to normal salt or high salt for 48 hours&#xD;
      to determine if salt-sensitivity is reflected in the monocytes as shown in Fig. 2. Medium&#xD;
      from monocytes cultured for 48 hours is analyzed for cytokine release including IL-1 beta,&#xD;
      TNF-alpha, IL-6, and IL-23 using luminex. The investigators will also obtain plasma to assess&#xD;
      for cytokines including GM-CSF, IL-4, and Flt3 ligand that are responsible for conversion of&#xD;
      monocytes to DCs. To ensure reproducibility, the samples will be submitted to the Vanderbilt&#xD;
      Endocrinology and Metabolism Core Facility to perform a blinded Luminex analysis.&#xD;
      Measurements of O2·- production and activation of NADPH oxidase including phosphorylation of&#xD;
      p47phox and association of p47phox with gp91phox by immunoprecipitation and western blot both&#xD;
      at baseline and in response elevated Na+ will be done previously described.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Recruiting</overall_status>
  <start_date type="Actual">September 1, 2021</start_date>
  <completion_date type="Anticipated">September 15, 2025</completion_date>
  <primary_completion_date type="Anticipated">September 1, 2025</primary_completion_date>
  <phase>Phase 1/Phase 2</phase>
  <study_type>Interventional</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <allocation>N/A</allocation>
    <intervention_model>Single Group Assignment</intervention_model>
    <intervention_model_description>Participants will be given a high salt diet and 2 liters of saline (salt loading), followed by low salt diet and furosemide or lasix (salt depletion).</intervention_model_description>
    <primary_purpose>Basic Science</primary_purpose>
    <masking>None (Open Label)</masking>
  </study_design_info>
  <primary_outcome>
    <measure>The percent of participants with salt sensitive blood pressure</measure>
    <time_frame>Over 3 days</time_frame>
    <description>A reduction in systolic blood pressure &gt;9 mm Hg from the salt-loading to the salt-depletion periods will be used to classify a subject as salt sensitive</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Percent of participants with immune cell activation</measure>
    <time_frame>Over 3 days</time_frame>
    <description>We will collect blood before and after a salt-loading (Weinberger) protocol and assess immune cell activation.</description>
  </secondary_outcome>
  <number_of_arms>1</number_of_arms>
  <enrollment type="Anticipated">20</enrollment>
  <condition>High Blood Pressure</condition>
  <condition>Salt; Excess</condition>
  <condition>Inflammation</condition>
  <arm_group>
    <arm_group_label>Weinberger protocol</arm_group_label>
    <arm_group_type>Experimental</arm_group_type>
    <description>During the Weinberger protocol, salt loading will be achieved by the combination of a high-salt diet (isocaloric, 160 mEq Na and 70 mEq K), 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. Salt depletion will be 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 or lasix orally.</description>
  </arm_group>
  <intervention>
    <intervention_type>Drug</intervention_type>
    <intervention_name>Furosemide 40 mg</intervention_name>
    <description>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.</description>
    <arm_group_label>Weinberger protocol</arm_group_label>
    <other_name>Lasix</other_name>
  </intervention>
  <eligibility>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  We will perform a pilot analysis in 20 hypertensive subjects controlled for gender&#xD;
             (50% men, 50% women), age (18-60 years),&#xD;
&#xD;
          -  New or pre-existing diagnosis of essential hypertension defined as systolic blood&#xD;
             pressure &gt;140 mmHg or &gt;90 mmHg diastolic or taking antihypertensive medications&#xD;
             regardless of current blood pressure.&#xD;
&#xD;
          -  BMI (18.5-24.9).&#xD;
&#xD;
          -  Only subjects who give informed consent will be studied.&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  Acute cardiovascular event(s) within the previous 6 months&#xD;
&#xD;
          -  Claustrophobia precluding obtaining an MRI&#xD;
&#xD;
          -  Inability to understand the nature, scope, and possible consequences of the study or&#xD;
             to participate in/comply with the protocol.&#xD;
&#xD;
          -  Current excessive alcohol or illicit drug use.&#xD;
&#xD;
          -  Blood pressure below the inclusion criteria levels after discontinuation of therapy&#xD;
&#xD;
          -  Presence of metal implants such as artificial joints.&#xD;
&#xD;
          -  Concomitant diabetes mellitus, type I or II.&#xD;
&#xD;
          -  Autoimmune disease.&#xD;
&#xD;
          -  Recent vaccination&#xD;
&#xD;
          -  Younger or older that inclusion criteria.&#xD;
&#xD;
          -  Pregnancy.&#xD;
      </textblock>
    </criteria>
    <gender>All</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>60 Years</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Annet Kirabo</last_name>
    <role>Principal Investigator</role>
    <affiliation>Vanderbilt University Medical Center</affiliation>
  </overall_official>
  <overall_contact>
    <last_name>Annet Kirabo, PhD</last_name>
    <phone>6153439033</phone>
    <email>annet.kirabo@vanderbilt.edu</email>
  </overall_contact>
  <location>
    <facility>
      <name>Vanderbilt University Medical Center</name>
      <address>
        <city>Nashville</city>
        <state>Tennessee</state>
        <zip>37232</zip>
        <country>United States</country>
      </address>
    </facility>
    <status>Recruiting</status>
    <contact>
      <last_name>Annet Kirabo</last_name>
      <phone>615-343-9033</phone>
      <email>annet.kirabo@vanderbilt.edu</email>
    </contact>
  </location>
  <location_countries>
    <country>United States</country>
  </location_countries>
  <reference>
    <citation>Weinberger MH, Miller JZ, Luft FC, Grim CE, Fineberg NS. Definitions and characteristics of sodium sensitivity and blood pressure resistance. Hypertension. 1986 Jun;8(6 Pt 2):II127-34. doi: 10.1161/01.hyp.8.6_pt_2.ii127.</citation>
    <PMID>3522418</PMID>
  </reference>
  <reference>
    <citation>Weinberger MH, Fineberg NS. Sodium and volume sensitivity of blood pressure. Age and pressure change over time. Hypertension. 1991 Jul;18(1):67-71. doi: 10.1161/01.hyp.18.1.67.</citation>
    <PMID>1860713</PMID>
  </reference>
  <verification_date>November 2022</verification_date>
  <study_first_submitted>November 20, 2018</study_first_submitted>
  <study_first_submitted_qc>November 21, 2018</study_first_submitted_qc>
  <study_first_posted type="Actual">November 26, 2018</study_first_posted>
  <last_update_submitted>November 18, 2022</last_update_submitted>
  <last_update_submitted_qc>November 18, 2022</last_update_submitted_qc>
  <last_update_posted type="Actual">November 22, 2022</last_update_posted>
  <responsible_party>
    <responsible_party_type>Principal Investigator</responsible_party_type>
    <investigator_affiliation>Vanderbilt University Medical Center</investigator_affiliation>
    <investigator_full_name>Annet Kirabo</investigator_full_name>
    <investigator_title>Assistant Professor</investigator_title>
  </responsible_party>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Hypertension</mesh_term>
    <mesh_term>Inflammation</mesh_term>
  </condition_browse>
  <intervention_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Furosemide</mesh_term>
  </intervention_browse>
  <patient_data>
    <sharing_ipd>Undecided</sharing_ipd>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT03753204
org study id: 182038
nct id: NCT03753204
lead sponsor: 
has dmc: No
is fda regulated drug: Yes
is fda regulated device: No
is us export: No
Salt-sensitive hypertension affects nearly 50% of the hypertensive and 25% of the
      normotensive population, and strong evidence indicates that reducing salt intake decreases
      blood pressure and cardiovascular events. The precise mechanisms of how dietary salt
      contributes to blood pressure elevation, renal injury, and cardiovascular disease remains
      unclear. Our data indicated that monocytes exhibit salt sensitivity, and the investigators
      hypothesize that of salt sensitivity of these and similar immune cells correlate with the
      hypertensive response to salt intake. Currently, the research tools for diagnosing
      salt-sensitivity are costly, time consuming and laborious. In this study the investigators
      will identify monocyte salt-sensitivity as a marker of salt-sensitive hypertension.
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.
allocation: N/A
intervention model: Single Group Assignment
intervention model description: Participants will be given a high salt diet and 2 liters of saline (salt loading), followed by low salt diet and furosemide or lasix (salt depletion).
primary purpose: Basic Science
masking: None (Open Label)
measure: The percent of participants with salt sensitive blood pressure
time frame: Over 3 days
description: A reduction in systolic blood pressure >9 mm Hg from the salt-loading to the salt-depletion periods will be used to classify a subject as salt sensitive
measure: Percent of participants with immune cell activation
time frame: Over 3 days
description: We will collect blood before and after a salt-loading (Weinberger) protocol and assess immune cell activation.
arm group label: Weinberger protocol
arm group type: Experimental
description: During the Weinberger protocol, salt loading will be achieved by the combination of a high-salt diet (isocaloric, 160 mEq Na and 70 mEq K), 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. Salt depletion will be 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 or lasix orally.
intervention type: Drug
intervention name: Furosemide 40 mg
description: 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.
arm group label: Weinberger protocol
other name: Lasix
criteria: 
gender: All
minimum age: 18 Years
maximum age: 60 Years
healthy volunteers: No
last name: Annet Kirabo
role: Principal Investigator
affiliation: Vanderbilt University Medical Center
last name: Annet Kirabo, PhD
phone: 6153439033
email: annet.kirabo@vanderbilt.edu
facility: 
status: Recruiting
contact: 
country: United States
citation: Weinberger MH, Miller JZ, Luft FC, Grim CE, Fineberg NS. Definitions and characteristics of sodium sensitivity and blood pressure resistance. Hypertension. 1986 Jun;8(6 Pt 2):II127-34. doi: 10.1161/01.hyp.8.6_pt_2.ii127.
PMID: 3522418
citation: Weinberger MH, Fineberg NS. Sodium and volume sensitivity of blood pressure. Age and pressure change over time. Hypertension. 1991 Jul;18(1):67-71. doi: 10.1161/01.hyp.18.1.67.
PMID: 1860713
responsible party type: Principal Investigator
investigator affiliation: Vanderbilt University Medical Center
investigator full name: Annet Kirabo
investigator title: Assistant Professor
mesh term: Hypertension
mesh term: Inflammation
mesh term: Furosemide
sharing ipd: Undecided