Visualizing Vascular Mechanisms of Salt Sensitivity
Phase: NA
Conditions: Obesity, Cardiovascular Risk Factor, Salt; Excess
Sponsor: Vanderbilt University Medical Center

Protocol Version: 5 
Protocol Date:  07/27/2020 
 
 
 
 
Visualizing Vascular Mechanisms of Salt Sensitiv
ity  
Rachelle Crescenzi , Ph.D., Research Instructor  
Department of Radiology and Radiological Sciences
 
Vanderbilt University
 Medical Center  
1161 21st Avenue South  
Medical Center North AA-1105 
Nashville, TN 37232  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol Version: 5 
Protocol Date:  07/27/2020 
 
Table of Contents:  
 
Study Schema  
1.0 
Background  
2.0 
Rationale and Specific Aims
 
3.0 
Inclusion/Exclusion Criteria
 
4.0 
Enrollment/Randomization
 
5.0 
Study Procedures  
6.0 
Risks  
7.0 
Reporting of Adverse Events or Unanticipated Problems 
involving Risk to 
Participants or Others
 
8.0 
Study Withdrawal/Discontinuation 
 
9.0 
Statistical Considerations
 
10.0 Privacy/Confidentiality Issues
 
11.0 Abbreviations  
12.0 References  
13.0 Appendix  
 
 
 

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1.0 
Backgroun d 
 
Salt sensitive blood pressure 
preferentially affects black 
Americans  
Salt sensitive blood pressure (SSBP) is a physiologic phenotype defined by a parallel change 
in blood pressure in response to dietary salt load. SSBP is fundamentally related to the body’s 
inability to clear salt, leading to elevated blood pressure and a life-long increased risk of 
cardiovascular disease. SSBP was determined to be an independent risk factor for developing 
cardiovascular disease in hypertensive patients almost two decades ago1, and more recently 
SSBP was found to portend all-cause mortality2. It is a prevalent condition that affects up to 
50% of normotensive, 75% of hypertensive3, 74% of black American adults4, and is estimated 
to be 6% more prevalent among females than males5, 6. Black Americans are preferentially 
affected by this phenotype, and represent one of the most at-risk population for cardiovascular 
diseases related to SSBP. Salt sensitivity is increased with the occurrence of metabolic 
syndrome associated with abdominal obesity7 and insulin resistance8. Current tests for SSBP 
require dietary salt loading, are time-intensive and, more importantly, provide limited 
mechanistic information on the incompletely characterized mechanisms of tissue salt storage. 
Tissue sodium homeostasis is maintained by renal and lymphatic mechanisms
 
While renal dysfunction is implicated in SSBP9, emerging evidence suggests that lymphatic 
mechanisms additionally regulate tissue salt storage and blood pressure10 and may contribute 
significantly to systemic vascular resistance11, 12. The lymphatic system is a central component 
of the body’s circulatory system, functioning in the regulation of tissue sodium homeostasis, in 
the clearance of high-density lipoproteins13 and metabolism of fatty acids7. Lymphatic 
clearance of tissue salt may represent an extra-renal system14 capable of regulating blood 
pressure15, 16 such that inhibition of this pathway is implicated in salt-sensitive hypertension17, 
18. Patients with essential hypertension, who were not evaluated for salt sensitivity, display 
elevated tissue sodium in peripheral tissues19. Similar profiles of elevated tissue sodium have 
been reported in patients with chronic kidney disease20. Additionally, impaired lymphatic 
clearance and renal dysfunction may also contribute to obesity21, 22, which is an established 
risk factor for cardiovascular disease. Our overarching hypothesis is that impaired lymphatic 
and renal vascular function is associated with elevated tissue sodium and fat storage in 
persons with SSBP.  
Magnetic resonance i maging  (MRI) tools for measurement of lymphatic and renal 
function associated with tissue sodium and 
fat deposition  
The critical limitation to interrogating lymphatic and renal clearance of sodium in vivo rests with 
a lack of noninvasive imaging technologies sensitive to sodium, lymphatic, and renal 
physiology. To address these limitations, we have recently developed a novel, noninvasive 
magnetic resonance (MR) lymphangiography technique23 and applied standardized MRI 
protocols for measuring tissue sodium and related fat deposition in adults with impaired 
lymphatic clearance. We found evidence of lymph stasis and tissue salt deposition that 

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correlated with local subcutaneous adipose tissue volume24. Here, we will use noninvasive 
MRI to test whether similar lymphatic pathways are impaired in persons with SSBP, leading to 
tissue salt and fat storage, in comparison to the involvement of renal dysfunction in SSBP 
tissue profiles.  
In addition to MRI, several external biophysical measurement tools exist to measure limb 
volume and tissue water content. These instruments include a bioimpedance spectrometer 
(LDex U400, Impedimed Limited) for measuring extracellular water content in the limbs, and a 
tissue dielectric probe (MoistureMeterD Compact, Delfin Technologies, Inc.) for measuring 
water content of the superficial dermis and adipose layers. These instruments aid in clinical 
diagnosis of other lymphatic disorders (the LDex is FDA approved to diagnose unilateral 
lymphedema of the arms or legs), which leads us to hypothesize that they may provide 
information on salt handling physiology if there is a lymphatic component to the disease. 
However, few studies have applied these devices in concert with MRI. We will apply these 
devices to study body composition related to SSBP and determine their potential use in 
screening of the phenotype.  
2.0 
Rationale and Specific Aims
 
The following hypotheses and specific aims were designed in response to a recent call from 
the American Heart Association (AHA) for i) standardized biomarkers to enable the clinical 
diagnosis of SSBP and ii) improved understanding of human tissue salt storage mechanisms25.  
Hypothesis 1: Tissue sodium content is elevated in adults predisposed for SSBP; tissue 
sodium and adiposity provide biomarkers for SSBP. Aim 1: 
Black adults without 
hypertension (20 male; 20 female) with BMI range=18 to 40 kg/m2 and age range=18-55 years 
will be assessed for SSBP according to recent guidelines26, and scanned using MRI to quantify 
tissue sodium and fat fraction in the lower extremities. Multivariable analyses will be applied to 
quantify relationships between tissue sodium, fat fraction as a surrogate of BMI, age, and sex 
with SSBP status. Impact.  Unique tissue sodium and fat profiles may provide internal, 
mechanistic biomarkers of SSBP that could be used as endpoints in clinical trials of SSBP.  
Hypothesis 2: Tissue sodium content is elevated in tissues affected by impaired 
lymphatic flow in adults with SSBP. Aim 2: 
The same participants will be scanned using 
noninvasive MR lymphangiography to quantify lymphatic flow velocity in the lower extremities. 
Non-parametric tests will be applied to evaluate the association between lymphatic flow and 
tissue sodium content in adults evaluated for SSBP. Impact.  Identifying the extent of lymphatic 
dysfunction in SSBP could motivate novel treatment strategies to improve lymphatic pumping 
and tissue sodium clearance.  
Hypothesis 3: Elevated tissue sodium content is associated with reduced renal 
perfusion in adults with SSBP.  Aim 3: 
The same participants will be imaged using 
noninvasive MR spin-labeling techniques to measure kidney perfusion (ml blood/100g 

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tissue/min). Non-parametric tests will be applied to evaluate the association between kidney 
perfusion and peripheral tissue sodium content in adults evaluated for SSBP. Impact.  Imaging 
of renal cortical circulation, where sodium-filtering nephrons are located, related to tissue 
sodium stores in SSBP may have potential to portend salt sensitive hypertension. 
The overall goal  of this work is to provide noninvasive imaging tools to assess a more 
complete spectrum of mechanisms underlying SSBP, which will motivate the use of these tools 
in future clinical trials to evaluate risk of SSBP or therapies to modify tissue sodium storage.  
Study procedures will evaluate healthy and obese normotensive black adults using a set of 
already tested non-invasive MRI techniques developed at Vanderbilt University Institute of 
Imaging Science (VUIIS) and biophysical instruments available at the VUMC (IRB #103172, 
#160199). Participants will also be assessed for SSBP using current standard practices for 
measuring changes in blood pressure after periods of high salt diet and low salt diet through 
the Clinical Research Center following IRB-approved protocols developed by collaborator Dr. 
Deepak Gupta (IRB #151943). 
3.0 
Inclusion/Exclusion Criteria
 
Inclusion criteria will include identification as black race, age between 18 and 55 years, and 
BMI between 25 and 40 kg/m2. Additionally, subjects must be normotensive or pre-
hypertensive adults who are willing to adhere to study diets and who are able to provide 
informed consent.  
The following exclusion criteria will apply:  
• Prevalent cardiovascular disease or use of medications for cardiovascular disease 
• Current or prior history of hypertension or use of blood pressure lowering medications 
• Current or prior history of diabetes mellitus or use of anti-diabetic medications 
• Prevalent renal disease (eGFR < 60 ml/min/1.73m2), abnormal serum sodium or 
potassium 
• Current or prior smoker 
• Current pregnancy  
• Current steroid use 
• Contraindications to MRI 
• Persons who are deemed clinically unsuitable for an MRI by their treating physician 
and/or VUIIS MRI technologist 
• Severe claustrophobia 
• Open wounds on the top of the feet or hands or at locations of interest for measurement 
of bioimpedance and tissue dielectric.     
Also excluded are subjects incapable of giving informed written consent: 

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• Subjects who are non-English speaking 
• Subjects who cannot adhere to the experimental protocols for any reason, or have an 
inability to communicate with the researcher. 
• Subjects who have limited mental ability to give informed consent, mentally retarded, 
altered mental status, mental disability, confusion, or psychiatric disorders. 
• Prisoners 
 
4.0 
Enrollment/Randomization
 
We will enroll 20 black adults (10 female and 10 male) who meet the inclusion and exclusion 
criteria. To analyze study hypotheses, and for comparison to normal weight individuals, we will 
share data from the study cohort of Dr. Deepak Gupta (IRB #151943).  
The following recruitment approaches will be used:  Vanderbilt CTSA (VICTR) Research 
Notification Distribution List, the linked email system which reaches Vanderbilt faculty and 
staff, as well as members of the Middle Tennessee community, and ResearchMatch, a 
national online registry maintained at Vanderbilt which allows people to self-register and 
express interest as research participants. Additional patients may be recruited from external 
sites. IRB approved flyers/advertisements of the study may be distributed in the public local to 
Vanderbilt and surrounding areas as additional means of subject recruitment for this study. 
The SUBJECT LOCATOR program is part of a toolset available through VICTR that enables 
teams to specify inclusion/exclusion criteria for a specific study.  The inclusion/exclusion 
criteria are codified for computable use and combined with data coming through VU Clinical 
Systems to proactively identify individuals who might qualify for a study.  Once a 'match' is 
made, research study personnel are alerted using confidential messaging or a secure web 
portal and they may then use the information to further review the subject's information using 
the Vanderbilt Electronic Medical Record.  If the subject is further deemed a candidate for the 
study, the study personnel will notify the subject's care provider who will then ask the subject if 
they would be interested in communicating with study personnel. 
At the screening visit, history study consent will be obtained, a medical and physical 
examination administered, and protocol eligibility determined.  If a subject meets inclusion 
criteria, without any exclusion criteria, the subject will be enrolled.  Subjects will receive serial 
low- and high-salt diets in a randomized crossover design. The randomization (using permuted 
blocks) will occur to one of two dietary sequences: (1) high-salt, washout, then low-salt diet, or 
(2) low-salt, washout, then high-salt diet. Randomization assignment will occur at screening 
and before baseline to ensure sufficient time to prepare the subjects’ provided meals.  
5.0 
Study Procedures  
Screening Visit  

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This will occur in the Vanderbilt Clinical Research Center (CRC). Subjects will arrive in a 
fasting (at least 6 hours with no food or drink) state. After informed consent has been obtained, 
inclusion/exclusion criteria will be reviewed to confirm that the subject meets study eligibility 
requirements. Subject’s medical history and concomitant medications will be discussed and 
documented. Subjects will then undergo a physical exam, including measurement of height 
and weight, and “vital signs” (blood pressure and heart rate). Subjects will have a 5ml (1/3 
tbsp) fasting blood draw in order to assess glucose, electrolyte and fluid balance, and kidney 
function, and will undergo a urine pregnancy test (if the woman is of child-bearing potential). 
Subjects will speak with study personnel who will review physical activity level and any food 
restrictions or allergies. You will be asked questions about your ability to undergo an MRI scan, 
including questions about metal implants. Study personnel will review the low- and high-salt 
dietary protocols and provide the subject with study diet instructions.  If needed, study 
personnel may then schedule an appointment for the subject to meet with a dietician for further 
review of dietary needs.  If subject agrees to adhere to study diets, then randomization (using 
permuted blocks) will occur to one of two aforementioned dietary sequences: (1) high-salt, 
washout, then low-salt diet, or (2) low-salt, washout, then high-salt diet.  
The subject will then be given instructions on 24-hour urine collections, which will be obtained 
at 4 study time points (baseline, end of diet 1, end of washout, and end of diet 2). A baseline 
study visit will then be scheduled. Additionally, all subjects will be asked to discontinue use of 
the following over-the-counter-medications (NSAIDS, decongestants, cold medicines) one 
week prior to study and to remain off these agents until the study is completed.  
Baseline  Visit   
In the 24-hours prior to the baseline visit, the subject will collect urine for quantification of urine 
sodium excretion and creatinine. For the baseline visit, the subject will arrive to the CRC in a 
fasting (at least 6 hours with no food or drink) state, return the 24-hour urine collection, and 
then be asked to lay supine. A physician or study research coordinator will review the subject’s 
medical history to assess for any changes since screening that may potentially exclude the 
subject from participating. After 1 hour and while the subject is still supine, the CRC nurse will 
record blood pressure and heart rate (average of 3 measures made 5 minutes apart) in the left 
arm using an automated cuff. The CRC nurse will also draw venous blood samples (25 ml = 1 
3/4 tablespoons) for measurements of a basic metabolic panel and plasma renin. Blood 
samples (cells, plasma and serum) may also be collected for storage at -80ºC for future 
investigations. The subject will be given another 24-hour urine collection kit, and diet 1 
materials. If the subject was randomized to begin with the low-salt diet, he or she will pick up 
the prepared low-salt meals from the CRC.  If the subject was randomized to begin with the 
high-salt diet, he or she will receive high-sodium bullion packets. 
MRI Exam 

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The subject may undergo a baseline MRI scan on the same day as the baseline visit or on a 
separate day, to occur either before beginning study diets, or at least 1 week after the diet 2 
visit. The subject will undergo the following noninvasive magnetic resonance imaging (MRI) 
scan at the Vanderbilt University Institute of Imaging Science (VUIIS), following an established 
protocol (no longer than 1.5 hours total) to quantify baseline tissue sodium content, whole-
body fat composition, lymphatic function, and kidney vascular function. No MRI methods used 
require the administration of any exogenous contrast agents, and therefore will be completely 
non-invasive. In total, noninvasive MRI protocols will be applied within a 90-minute scan 
session.  
Tissue sodium content can be visualized and quantified non-invasively in humans with MRI.27-
31  Sodium content in leg will be imaged with a detector tuned to sodium resonance (Rapid, 
Germany) and a 3.0T MRI scanner (Ingenia Elition, Philips Medical Systems, Best, The 
Netherlands). Four tubes containing aqueous solutions with 10, 20, 30, and 40 mmol/L NaCl 
will serve as calibration standards by relating intensity to a concentration in a linear trend 
analysis.  In parallel, we will quantify tissue water content using conventional MRI methods, 
including a fat-saturated inversion recovery sequence with spin density contrast.27-29  The total 
acquisition time for this protocol is 15 minutes. 
Body composition including fat/water fraction will be measured using a whole-body multi-echo 
Dixon MRI (AMRA® Body Composition Protocol32, AMRA Medical AB, Linkoping, Sweden). 
Volunteers will be positioned supine, head-first in the MRI scanner. The total acquisition time 
for this protocol is 6 minutes. 
A 16-channel torso coil will be placed over the lower extremities of the volunteer. Noninvasive 
MR lymphangiography will be performed to measure lymphatic vascular function in the legs33. 
The total acquisition time for this protocol is 11 minutes. 
With the torso coil placed over the abdomen, the kidneys will be imaged with MR 
relaxometry34, 35 and an arterial-spin-labeling sequence applied bilaterally over the kidneys and 
renal artery to measure cortical perfusion. The total acquisition time required for kidney 
imaging is 10 minutes. 
 
Biophysical Exam  
During the baseline visit and at the time of MRI, body composition will also be measured using 
bed-side, biophysical instruments: a bioimpedance spectroscopy unit (L-Dex U400) and/or a 
tissue dielectric probe (MoistureMeterD) in the arms and legs, and locations of interest on the 
body. This will require approximately 15 minutes while resting supine, and results are intended 
to determine whether accessible, bed-side devices can provide similar information as MRI. 

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Low - and High -Salt Diets  
All low-salt meals will be provided to participant by the study. Subjects will pick up the 
prepared meals, snacks, and sodium free water at the CRC at times and intervals most 
convenient to the participant. The low-salt diet will include a maximum of 600mg (26 mEq) of 
sodium per day. Subjects are encouraged to consume all food as provided and expected to 
have no substitutions or additions. The calorie needs for weight maintenance will be assessed 
at screening. The low salt meal plan contains 2500-2800 calories. This calorie level is sufficient 
for the majority of the research population. Additional low-salt snacks will be added as need 
should the subject require additional calories. The high-salt diet supplies will consist of the 
subject’s “usual” diet, supplemented with 2 bouillon broth packets each day, which will be 
provided by the study personnel. Each broth packet contains 48 mEq sodium for a total of 
approximately 100 mEq additional sodium intake per day. This method conveniently raises 
total salt intake to >200mEq/day (> 4,600 mg sodium/day) in over 95% of healthy volunteers 
obviating the need for additional prepared meals and visits.  
Subjects, while on the low- and high-salt phases of the diet, will receive calcium 
supplementation. Calcium will be supplied by 2 antacid tablets taken by mouth; each will 
provide 750 mg of calcium.  
Diet 1 visit  
The same protocol as the baseline visit (described above) will be followed, with the exception 
that the subject will not undergo MRI. Other study procedures, including return of the 24-hour 
urine collection, blood pressure, and heart rate measures, obtaining venous blood draw in the 
supine position will be identical to the baseline visit. The biophysical exam may be performed 
while subjects are laying supine. After this Diet 1 visit, the subject will enter a minimum seven-
day washout period. This will consist of the subject adhering to their “usual” diet. They will be 
given their 24-hour urine collection kit, to be completed before the “washout” visit.    
 
 
 
Washout visit   
The same protocol as the baseline visit will be followed, except that subjects will not undergo 
MRI. After this washout visit, the subject will crossover to the opposite salt diet from week 1. 
They will also be given their 24-hour urine collection kit.  
Diet 2 visit  

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Subjects will undergo the same procedures as in the Diet 1 visit (see above), except a 24-hour 
urine collection kit will not be given.  
Biomarkers  
Blood and urine samples will be coded for subject confidentiality. Measurements of interest will 
be performed at Vanderbilt University. For possible future investigations, excess blood and 
urine samples may be frozen and stored in the Vanderbilt University Core lab for 
Cardiovascular Translational and Clinical Research.  
6.0 
Risks  
Venous Blood draw: This is a routine procedure that is considered standard of care in clinical 
medicine. At each visit, subjects will undergo a single venous blood draw. All blood draws will 
be performed by trained personnel using universal precautions to protect both the subject and 
personnel. The risks to subjects are minimal, but may include pain, allergic reaction, infection 
or bleeding at the needle stick site. These usually resolve without any specific medical therapy 
over the course of minutes to days.  
MRI: Magnetic Resonance Imaging is routinely performed in clinical medicine. All imaging 
scans performed in this study are non-invasive (i.e., they do not require any injection of 
exogenous contrast agents or ionizing radiation) and therefore risks to subjects are low. Power 
monitoring and gradient switching speeds at 3.0T are applied according to the general FDA 
guidelines for MRI and controlled by the scanner company software. The effects of magnetic 
fields in an MRI scanner have been extensively studied and there are no known significant 
risks with an MRI exam. Subjects may, however, be bothered by feelings of confinement or 
claustrophobia, and by the noise made by the magnet during the procedure. Subjects will be 
asked to wear earplugs or earphones while in the magnet. Subjects may not participate in this 
study if they have a pacemaker, an implanted defibrillator or certain other implanted electronic 
or metallic devices for which MRI is contraindicated. Questioning prior to enrollment will 
determine if subjects have had brain surgery for a cerebral aneurysm, or have implanted 
medical or metallic devices, shrapnel, or other metal, such as metal in the eye. If subjects 
move rapidly within or around a magnet with a field of 3.0T they may feel dizzy. We therefore 
advise them to move slowly and, once they are positioned on the table, we will move the 
person into the magnet slowly. Emergency personnel and equipment will be on hand during 
the MRI for safety. If at any time subjects feel excessive feelings of confinement or 
claustrophobia, the scan may be terminated by the subject. The total time on which the 
participant is in the MRI scanner is estimated to be no longer than 90 minutes. 
Biophysical Exam: The external measurements taken during the visit are applied using 
standard procedures and are fast to administer using non-invasive tools that do not bring on 
any discomfort to subjects, are FDA-approved or determined safe for human use, and are 
taken within a couple of minutes while subjects are lying down. Subjects who have open 

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wounds at the site of measurement, are pregnant at the time of exam, or have an implanted 
device with a battery, such as a pacemaker, will not be allowed to receive the biophysical 
exam. 
Tolerability and safety of salt diets: While there is theoretic risk of eliciting hypertensive or 
hypotensive responses with high- and low-salt diets, the research team has decades of 
experience giving low- and high-salt diets and have demonstrated the salt diets are well 
tolerated and safe. Indeed, the salt content of the “high-salt” diet is 25-55% higher than the 
“average’ salt diet for people living in middle Tennessee. The low-salt diet is also well 
tolerated, with low rates (<10%) of withdrawal, which we have accounted for in our sample size 
and power calculations. We have not observed hypovolemia or hypotension on the low-salt 
diet, even in lean healthy individuals. All subjects will be monitored for adverse effects and 
during the study visits a physician will be available to monitor the safety of the subject through 
the protocol, and between study visits, a physician will be available 24 hours a day by phone 
and/or pager to subjects.    
Private health information: This information will be collected during the course of the study. 
However, only key study personnel will have access to this information, which will be stored in 
a HIPAA compliant, password protected REDCap database. No protected health information 
will be shared with employers, insurers, or non-research personnel.  
7.0 
Reporting of Adverse Events or Unanticipated Problems Involving Risk to 
Participants or Others
 
 
Adverse events (AEs) will be reported according to IRB policies and procedures. Reporting will 
depend on adverse event severity: 
Grading of Severity 
0. No AE or within normal limits. 
1. Mild AE. 
2. Moderate AE. 
3. Severe AE resulting in inpatient hospitalization, or a persistent or significant 
disability/incapacity. 
4. Life-threatening or disabling AE. 
5. Fatal AE. 
AE’s of grade three or higher will be reported immediately to the IRB using the Report of 
Adverse Events per IRB policies and procedures.  Every six months the PI will summarize any 
AE’s to the IRB.  In conjunction with the IRB, the PI will determine if modifications to the 
protocol are warranted. 

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Serious and unexpected adverse events will be reported to our IRB within ten working days. All 
our adverse events will be reported as required at the time of continuing review. Protocol 
deviations and violations will be reported to our IRB within ten working days. 
8.0 
Study Withdrawal/Discontinuation
 
Subjects may withdraw from the study at any time and should notify study personnel if they 
wish to withdraw from the study. Subjects may request their biological samples to be destroyed 
at any time. However, any data or biological samples that have already been used for research 
cannot be destroyed. Subjects may be discontinued from the study at the discretion of the 
investigators’ (possible reasons listed below). Subjects will receive financial compensation for 
the visits that they complete.  
Possible reasons for withdrawal/discontinuation from study include, but are not limited to:  
• Noncompliance with treatment or procedures 
• Decision by participant/participant withdraws consent 
• Lost to follow-up 
• Starting a medication that would exclude the subject (see exclusion criteria) 
• Development of a significant medical condition specified in the exclusion criteria 
• In female subjects, becoming pregnant during study 
• Significant adverse event deemed by investigator to preclude continued participation 
 
9.0 
Statistical Considerations
 
Subjects who are overweight or obese (n=20) will be recruited through this IRB. To analyze 
study hypotheses, and for comparison to normal weight individuals, we will share data from the 
study cohort of Dr. Deepak Gupta (IRB #151943). This will include identical data acquired from 
at least n=20 subjects who are normal weight (18 to <25 kg/m2) and who meet identical 
inclusion/exclusion criteria.  
Statistical analysis procedures for Aim 1: 
Statistical analyses will be performed by key 
study personnel and members of the Center for Quantitative Sciences at the VUMC. The 
primary statistical objective of Aim 1 is to test the hypothesis that adults who are salt sensitive 
have higher baseline tissue sodium relative to adults who are not salt sensitive. To test this, we 
will divide participants into those who are salt sensitive (SS, ≥5 mmHg change in mean arterial 
pressure ∆MAP following high salt diet compared to low salt diet) or not salt sensitive (NSS, <5 
mmHg ∆MAP), according to a review of methods and recommendations from Kurtz et al.26. 
The metric ∆MAP represents a continuous variable of SSBP. A Mann Whitney U test will be 
applied to determine whether tissue sodium content is significantly different in SS and NSS 
groups. Based on a prior study of tissue sodium storage in adults who are hypertensive (n=10) 

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or normotensive (n=12)19 we anticipate a similar difference in tissue sodium of 6±4 (mean ± 
standard deviation, mmol/L) between SS and NSS adults. Our study is designed to provide at 
least 80% power to detect differences in tissue sodium with a two-sided significance of p<0.05. 
 Our secondary statistical objective of Aim 1 is to test the hypothesis that tissue sodium content 
and potential risk factors for SSBP, including biological sex, age, and tissue fat/water fraction as 
a sensitive surrogate of BMI, are related to ∆MAP as a continuous variable. We will apply 
multivariable regression analysis using tissue sodium, fat/water fraction, biological sex, and age 
as independent variables, and ∆MAP as the dependent variable among all participants. With a 
sample size of 40 , we should be able to evaluate the four predictors of ∆MAP (each with one 
degree of freedom) without over-fitting the model. 
Statistical analysis procedures for Aim 2: 
The primary statistical objective of Aim 2 is to test 
the hypothesis that tissue sodium content has an inverse relationship with lymphatic flow 
velocity, which depends on a person’s salt sensitivity. To test this hypothesis, we will apply 
multivariable regression analysis using tissue sodium as the dependent variable, and 
lymphatic flow velocity, ∆MAP, biological sex, and age as independent variables among all 
participants. 
Statistical analysis procedures for Aim 3: 
The primary statistical objective of Aim 3 is to test 
the hypothesis that elevated tissue sodium content is associated with reduced renal perfusion, 
which depends on a person’s salt sensitivity. We will apply similar statistical analyses as Aim 2 
to evaluate the relationships between renal cortical perfusion and peripheral tissue sodium 
content in adults evaluated for salt sensitivity.  
10.0 Privacy/Confidentiality Issues
 
 
Strict confidentiality will be maintained to the fullest extent by the research team, including 
keeping all data in a secure, locked cabinet with limited access and/or password protected 
databases, i.e. REDCap.  All specimens will be coded after they are obtained and the code key 
kept in a locked cabinet.  Blood samples will be coded anonymously to remain confidential and 
identifiers will be kept in a separate, secure, locked location.   Samples may be shared with 
third parties outside of Vanderbilt for future testing but will remain anonymous to the recipient.  
If samples are accidentally lost, subjects will not be re-contacted to provide a substitute 
sample.  The investigators will not be obligated to keep contact information to re-contact the 
subject.  Subjects may contact the principal investigator at any time to request that samples be 
destroyed.  
The data files generated by the MRI scanner and L-Dex software are coded using a project 
name and unique number generated sequentially.  Only the investigators will have the key to 
the subject codes. Image analysis will use the coded files. The safety surveys described above 
include questions about sensitive health issues. This information will remain with the signed 

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consent form in a secured area near the scanner. These records will remain in the 
investigators’ possession for ten years following the termination of the study and will then be 
destroyed. 
 
 
 
 
11.0 Abbreviations  
23NA: sodium 
AHA: American Heart Association 
BMI: body-mass index 
MAP, ∆MAP: mean arterial pressure, or change in mean arterial pressure following high salt 
diet compared to low salt diet 
CRC: Clinical Research Center 
eGFR: estimated Glomerular Filtration Rate 
FDA: food and drug administration 
MRI: magnetic resonance imaging 
RAAS: renin-angiotensin-aldosterone system 
REDCap: Research Electronic Data Capture 
SSBP: salt sensitive blood pressure 
VICTR: Vanderbilt Institute for Clinical and Translational Research 
VUIIS: Vanderbilt University Institute of Imaging Science 
 
12.0 References  
1. Morimoto A, Uzu T, Fujii T, Nishimura M, Kuroda S, Nakamura S, Inenaga T and Kimura G. 
Sodium sensitivity and cardiovascular events in patients with essential hypertension. Lancet. 
1997;350:1734-7. 
2. Weinberger MH, Fineberg NS, Fineberg SE and Weinberger M. Salt sensitivity, pulse 
pressure, and death in normal and hypertensive humans. Hypertension. 2001;37:429-32. 
3. Oh YS, Appel LJ, Galis ZS, Hafler DA, He J, Hernandez AL, Joe B, Karumanchi SA, Maric-
Bilkan C, Mattson D, Mehta NN, Randolph G, Ryan M, Sandberg K, Titze J, Tolunay E, Toney 
GM and Harrison DG. National Heart, Lung, and Blood Institute Working Group Report on Salt 
in Human Health and Sickness: Building on the Current Scientific Evidence. Hypertension. 
2016;68:281-8. 
4. Svetkey LP, McKeown SP and Wilson AF. Heritability of salt sensitivity in black Americans. 
Hypertension. 1996;28:854-8. 

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5. Bursztyn M and Ben-Dov IZ. Sex differences in salt-sensitivity risk approximated from 
ambulatory blood pressure monitoring and mortality. J Hypertens. 2013;31:900-5. 
6. He J, Gu D, Chen J, Jaquish CE, Rao DC, Hixson JE, Chen JC, Duan X, Huang JF, Chen 
CS, Kelly TN, Bazzano LA, Whelton PK and GenSalt Collaborative Research G. Gender 
difference in blood pressure responses to dietary sodium intervention in the GenSalt study. J 
Hypertens. 2009;27:48-54. 
7. Chen J, Gu D, Huang J, Rao DC, Jaquish CE, Hixson JE, Chen CS, Chen J, Lu F, Hu D, 
Rice T, Kelly TN, Hamm LL, Whelton PK, He J and GenSalt Collaborative Research G. 
Metabolic syndrome and salt sensitivity of blood pressure in non-diabetic people in China: a 
dietary intervention study. Lancet. 2009;373:829-35. 
8. Sharma AM, Ruland K, Spies KP and Distler A. Salt sensitivity in young normotensive 
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9. Hall JE. Renal Dysfunction, Rather Than Nonrenal Vascular Dysfunction, Mediates Salt-
Induced Hypertension. Circulation. 2016;133:894-906. 
10.Machnik A, Neuhofer W, Jantsch J, Dahlmann A, Tammela T, Machura K, Park J-K, Beck 
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Hilgers KF, Alitalo K, Eckardt K-U, Luft FC, Kerjaschki D and Titze J. Macrophages regulate 
salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-
dependent buffering mechanism. Nature medicine. 2009;15:545-52. 
11.Titze J and Luft FC. Speculations on salt and the genesis of arterial hypertension. Kidney 
Int. 2017;91:1324-1335. 
12.Laffer CL, Scott RC, 3rd, Titze JM, Luft FC and Elijovich F. Hemodynamics and Salt-and-
Water Balance Link Sodium Storage and Vascular Dysfunction in Salt-Sensitive Subjects. 
Hypertension. 2016;68:195-203. 
13.Randolph GJ and Miller NE. Lymphatic transport of high-density lipoproteins and 
chylomicrons. J Clin Invest. 2014;124:929-35. 
14.Hofmeister LH, Perisic S and Titze J. Tissue sodium storage: evidence for kidney-like 
extrarenal countercurrent systems? Pflugers Arch. 2015;467:551-8. 
15.Wiig H, Schroder A, Neuhofer W, Jantsch J, Kopp C, Karlsen TV, Boschmann M, Goss J, 
Bry M, Rakova N, Dahlmann A, Brenner S, Tenstad O, Nurmi H, Mervaala E, Wagner H, Beck 
FX, Muller DN, Kerjaschki D, Luft FC, Harrison DG, Alitalo K and Titze J. Immune cells control 
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16.Johnson RS, Titze J and Weller R. Cutaneous control of blood pressure. Curr Opin Nephrol 
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17.Titze J and Machnik A. Sodium sensing in the interstitium and relationship to hypertension. 
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18.Machnik A, Dahlmann A, Kopp C, Goss J, Wagner H, van Rooijen N, Eckardt KU, Muller 
DN, Park JK, Luft FC, Kerjaschki D and Titze J. Mononuclear phagocyte system depletion 
blocks interstitial tonicity-responsive enhancer binding protein/vascular endothelial growth 
factor C expression and induces salt-sensitive hypertension in rats. Hypertension. 
2010;55:755-61. 

Protocol Version: 5 
Protocol Date:  07/27/2020 
19.Kopp C, Linz P, Dahlmann A, Hammon M, Jantsch J, Müller DN, Schmieder RE, Cavallaro 
A, Eckardt KU, Uder M, Luft FC and Titze J. 23Na magnetic resonance imaging-determined 
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B, Wabel P, Hammon M, Cavallaro A, Eckardt KU, Kotanko P, Levin NW, Johannes B, Uder 
M, Luft FC, Muller DN and Titze JM. Magnetic resonance-determined sodium removal from 
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Donahue MJ and Titze J. Tissue sodium content is elevated in the skin and subcutaneous 
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and Titze J. Seeing the sodium in a patient with hypernatremia. Kidney international. 
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28.Kopp C, Linz P, Wachsmuth L, Dahlmann A, Horbach T, Schofl C, Renz W, Santoro D, 
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and Titze J. (23)Na magnetic resonance imaging of tissue sodium. Hypertension. 2012;59:167-
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A, Eckardt KU, Uder M, Luft FC and Titze J. 23Na magnetic resonance imaging-determined 
tissue sodium in healthy subjects and hypertensive patients. Hypertension. 2013;61:635-40. 
30.Dahlmann A, Dorfelt K, Eicher F, Linz P, Kopp C, Mossinger I, Horn S, Buschges-Seraphin 
B, Wabel P, Hammon M, Cavallaro A, Eckardt KU, Kotanko P, Levin NW, Johannes B, Uder 
M, Luft FC, Muller DN and Titze JM. Magnetic resonance-determined sodium removal from 
tissue stores in hemodialysis patients. Kidney international. 2014. 
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L. Feasibility of MR-Based Body Composition Analysis in Large Scale Population Studies. 
PLoS One. 2016;11:e0163332. 

Protocol Version: 5 
Protocol Date:  07/27/2020 
33.Crescenzi R, Donahue PMC, Hartley KG, Desai AA, Scott AO, Braxton V, Mahany H, Lants 
SK and Donahue MJ. Lymphedema evaluation using noninvasive 3T MR lymphangiography. J 
Magn Reson Imaging. 2017;46:1349-1360. 
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preparation pulses. Magn Reson Imaging. 2014;32:813-8. 
35.Wang P, Zhu H, Kang H and Gore JC. R1rho dispersion and sodium imaging in human calf 
muscle. Magn Reson Imaging. 2017;42:139-143. 
 
 

Protocol Version: 5 
Protocol Date:  07/27/2020 
13.0  Appendix :  
 
Protocol Overview and Schedule of Events
 
Procedures and data 
acquired  
Screening 
Visit  
Baseline 
Visit  
Diet 1 
Visit  
Washout 
Visit  
Diet 2 
Visit  
MRI visit  
Participant information and 
informed consent obtained 
X 
 
 
 
 
 
Inclusion/exclusion criteria met 
X 
 
 
 
 
 
Medical history  
X 
 
 
 
 
 
Urine pregnancy test (females) 
X 
 
 
 
 
 
Physical examination  
X 
 
 
 
 
 
Current medications  
X 
X 
X 
X 
X 
 
Vital signs, height, and weight  
X 
X 
X 
X 
X 
X 
Heart rate and blood pressure 
(3 measurements, 5 minutes 
apart) 
 
X 
X 
X 
X 
X 
Biophysical examination 
 
X 
X 
X 
X 
X 
Dietary protocol followed (for 7 
days prior to visit) 
 
 
X 
 
X 
 
Clinical laboratory samplea 
collected 
Xb 
Xb 
Xb 
Xb 
Xb 
 
24-hour urine collected 
 
Xc 
Xc 
Xc 
Xc 
 
MRI scan 
 
 
 
 
 
Xd  
Adverse events assessed 
X 
X 
X 
X 
X 
 
Compensation offered 
 
X 
X 
X 
X 
 
a. Blood samples (cells, plasma and serum) will be collected for storage at -80ºC for possible future 
investigations  
b. Blood collection on study visit days will be fasting venous draw  
c. Subjects will bring their collection jug to the clinic where it will be processed by study staff for analysis.  
(Subjects will be provided with urine collection and storage containers and instructed to start their urine 
collection on the day prior to their clinic visit, 24 hours before they arrive to the clinic). 
d. The baseline MRI scan may occur on the same day as the baseline visit or on a separate day, to occur either 
before beginning study diets, or at least 1 week after the diet 2 visit.  Subjects will be compensated for the 
MRI visit separately.