Estrogen Receptor Alpha Signaling in Endothelial Cells Exacerbates Arterial Stiffening Via Upregulation of ENaC in Insulin Resistant Females
Phase: PHASE2
Conditions: Overweight and Obesity, Insulin Resistance
Sponsor: University of Missouri-Columbia

Page 1 of 12 
BIOMEDICAL RESEARCH PROTOCOL  
UNIVERSITY OF MISSOURI 
 
Project Title: Estrogen receptor alpha signaling in endothelial cells exacerbates arterial 
stiffening via upregulation of ENaC in insulin resistant females 
IRB Number: 2012990 
Version Number: 24 
Version Date:  October 30, 2023 
Principal Investigator: Camila Manrique 
Funding Source:  NHLBI 
 
Clinical Trial Phase: Phase II 
Clinicaltrials.gov Number: NCT03837626  
Study Drug/Study Device: Amiloride 
I. 
Research Objectives/Background  
 
 
 
 
 
 
 
 
 
Hypothesis: Estrogen action, through the endothelial cell (EC) estrogen receptor alpha (ERα) upregulates EC 
ENaC expression and activity exacerbating endothelial and arterial stiffening in overweight and obese, insulin 
resistant (IR) premenopausal females. The corollary to this hypothesis is that ENaC inhibition will have a 
greater impact on arterial stiffness in overweight and obese, premenopausal insulin resistant women than in 
overweight and obese IR postmenopausal women or age matched obese IR men.  
 
To test our hypothesis, we will use in vivo measures of endothelial function and arterial stiffness in a cohort of 
overweight and obese IR subjects treated with amiloride, an ENaC inhibitor, to accomplish the follow aim: 
 
To determine whether treatment with the ENaC inhibitor, amiloride, improves endothelial function and arterial 
stiffness in overweight and obese IR subjects in a randomized placebo-controlled trial examining pre and 
postmenopausal women and age-matched men.  
II. 
Drugs/Biologics/Devices 
Following screening, male and female subjects will be randomized to a doubled blinded placebo control trial. 
Subjects will start on either a 24-week treatment with oral medication, either amiloride, a max dose of 5 mg 
daily, or placebo intervention. 
Amiloride therapy: In previous clinical trials with higher doses (10 mg) amiloride was well tolerated.1 
Nevertheless, our team of investigators is well aware of the potential side effects (1-10%): dizziness, fatigue 
headache, hyperkalemia. Subjects will be closely monitored during the drug/placebo treatment intervention. 
After each safety visit, the PI will review blood pressure and heart rate readings, as well as results of renal 
function and potassium levels.  The treatment will be held in cases of hyperkalemia > 5.5 mEq/L, creatinine 
increases more than 30% above baseline, hypotension or severe dizziness. If the subject exhibits hyperkalemia 
or if creatinine increases more than 30% above baseline, the study team will repeat safety labs until they return 
to baseline. If a subject exhibits potassium > 5.5 mEq/L, creatinine increases more than 30% above baseline, 
hypotension or severe dizziness on the 5 mg dose, medication will be held for 1 week and a lower dose of 
amiloride will be started (half of a 5mg tablet). Once the dose of a half of a 5mg tablet has been started, a safety 
visit will be completed 1 week later. If no clinically significant abnormalities are documented, safety visits will 
proceed as scheduled for the 5 mg dose.  In the event of a severe adverse event the subject will be unblinded to 
facilitate medical care. 

Page 2 of 12 
Placebo therapy: The use of placebo and blinding of study team prevents bias of the investigator and study team 
as well as providing a control for the study. There are two components to the placebo, one being the cellulose 
found inside the capsule and the other being the gelatin pill capsule used for all study pills being dispensed.  
 
An Investigational New Drug (IND) is not included as part of this protocol because the study medication, 
amiloride is being used as an FDA approved dose in a population that is not anticipated to have increased risk or 
decreased acceptability of risk. Lastly, no information will be submitted to the FDA to support a labeling 
indication, a labeling change, or a change in advertising, as this is not the intent of the current study. 
 
 
III. 
Recruitment Process 
Subjects will be recruited by a number of means.   
1. A separate informatic screen will be completed to identify clinic patients from the Division of 
Endocrinology, the Department of Medicine and the Department of Family Medicine who are between 
the ages of 30 and 70, overweight or obese and prediabetic. A letter will be sent notifying them of their 
eligibility to participate in a research study designed to lower future risk of heart disease risk. 
2. Subjects may also respond to the recruitment flyer or advertisement.  For all recruitment, subjects will 
be given an email address and/or a phone number to contact. Subjects may also be given a link to 
complete an informational screening via Qualtrics with their waiver of documentation of consent.  
3. The recruitment material will be posted around University of Missouri campus, as well as other public 
places such as swimming pools, churches, and grocery stores.  We will advertise through MU Info, TV 
slides throughout the University of Missouri, other MU provided resources, local newspapers, local TV 
stations and via multiple social media sources. Additional avenues of advertisement might become 
available throughout the study, and we will utilize only the approved recruitment material information. 
We also plan to use the service called Studykik (https://studykik.com/), which is a clinical trial 
recruitment site. No data are collected using this site - it is only used to provide potential subjects with 
information about the study and provide staff contact information.  We will use Washington University 
School of Medicine StudySearch Website (an online listing of research studies).  In addition, we will use 
Washington University Research Participant Registry (RPR) -an IRB approved database of participants 
who have consented to be contacted for research studies and social media I.E. Facebook for assistance 
with recruitment. We will used Research, Innovative and Impact website for additional recruitment 
 
Subjects randomized to amiloride (5 mg)  or placebo
Side Effects (none or minimal) Creatinine within 
30% of baseline value, potassium <5.5 mEq/L, 
absense of hypotension,  no severe dizziness.
Continue with 5mg of amiloride/placebo treatment
and safety visits as scheduled.
Side Effects (clinically significant) Creatinine >30% 
of baseline value, potassium >5.5 mEq/L, 
hypotension,  or severe dizziness.
Medication held for 1 week, then treatment  
resumed at half of a 5mg tablet.  Additional saftey 
visit 1 week after new dosing starts. Safety visits 
continue as scheduled.
Severe adverse events (defined in protocol)
Medication will be stopped. 
Subjects will be unblinded to facilitate medical 
care.
Subjects are monitored for symptoms of 
hypotension, changes in vital signs, electrolytes and 
renal function.
Figure 1. Treatment Flow Chart based on management of side effects and adverse events. 

Page 3 of 12 
(https://research.missouri.edu/). We will use Research Match. We will used BuildClinical to manage 
digital marketing recruitment for this study. BuildClinical is a data-driven digital marketing company 
that helps academic researchers recruit participants for research studies more efficiently using social 
media, software, and machine learning. They will ensure that all IRB-approved guidelines and 
procedures are followed during recruitment. They use study-specific advertisements to engage 
participants on digital platforms such as Facebook, Google, WebMD, etc. Participants who click on the 
advertisements will be redirected to a study-specific landing page. On the landing page, the person can 
complete an online pre-screen questionnaire. Pre-screening data is then routed to BuildClinical's Secure 
Socket Layer (SSL) software, which encrypts all information to keep it private and HIPAA compliant. 
Their backend servers are stored in secure data centers in the USA. All currently approved 
advertisement could potentially be used with any of the above listed means of recruitment.   
IV. 
Consent Process 
One consent form will be used that describes in detail the amiloride/placebo study.   
 
Telephone/Qualtrics screening 
Subjects who contact us via phone or email to inquire about participation will answer screening questions 
via phone or Qualtrics questionnaire, with their waiver of documentation of consent. These questions are 
for preliminary screening only and are not used as study data since the data represent self-report.  The 
questions include queries about health history/habits, age, sex, height, weight, tobacco use, medications/ 
supplements, over the counter medications use, illness, chronic conditions, and family history of diabetes 
or heart disease.  The study design is described in general terms to subjects, with mention of factors most 
likely to impact subject interest in participating.  If the subject is interested in being screened for 
participation, we then make an appointment for a screening visit.  
Consenting/screening visit 
The first visit to the Clinical Translational Science Unit (CTSU) will take up to an1 hour and include a detailed 
written consent for the study.  Review of the consent form will occur with study personnel in a quiet, unhurried 
setting. Comprehension will be assessed by asking the subjects to explain the study in their own words. 
Participants will have adequate opportunity to review the informed consent and to ask any questions they may 
have about the research protocol, compensation, risks, and benefits of taking part in the study. 
After signing the consent form, medical information is obtained by the nurse or physician including DOB, 
gender, ethnic/racial category, height, body weight (history of body weight gain or loss), vital signs, waist 
circumference, and medical history is obtained using a questionnaire. A fasting blood draw is taken to test for 
biochemistries (volume no more than 25 mL).  Eligibility will be communicated with subjects following the 
screening visit and baseline appointments will be scheduled accordingly. Upon request subjects will be 
provided with a copy of the results. 
 
V. 
Inclusion/Exclusion Criteria 
Inclusion criteria:  
- 
30 to 70 years of age at randomization 
- 
Body mass index (BMI) 25.1-50 kg/m2 or waist circumference > 88 cm (> 35 in) in women and >102 
cm (>40 in) in men. 3, 4  
- 
One other characteristic of metabolic syndrome (elevated triglycerides ≥150 mg/dl; HDL cholesterol 
<40 mg/dl in men and <50 mg/dl in women; blood pressure ≥130/85 mm Hg or treatment for 
hypertension; impaired fasting glucose (≥100 mg/dl)) or fasting insulin level >10 mU/L (correlates with 
insulin resistance).  

Page 4 of 12 
 
Subjects will be excluded if there is evidence of: 
1. History of type 1 or type 2 diabetes 
2. Known cardiovascular events within the last 12 months (stroke, acute coronary event, revascularization, 
heart failure hospitalization).  
3. History of uncontrolled thyroid disease, chronic liver disease (cirrhosis) or GFR <50 ml/min. 
4. Use of potassium sparing medications (angiotensin II receptor blockers, angiotensin converting enzymes 
inhibitors or mineralocorticoid receptor blockers) or use of potassium supplements. 
5. Active cancer (This criterion does not apply to those subjects with basal cell carcinoma or stage 1 
squamous cell carcinoma of the skin) 
6. Excessive alcohol consumption (>14 drinks/week for men, >7 drinks/week for women) 
7. Current tobacco use 
8. Non controlled hypertension  
9. Participation in regular exercise > 3 days/wk per week at a moderate or vigorous intensity  
10. Pregnancy or lactation in women (or women not using contraceptives) 
11. Women who plan to become pregnant during the duration of the trial  
12. Chronic use of NSAIDs  
13. Potassium level > 5.0 mqE/L at time of screening  
14. Blood pressure at screening <110/70 
VI. 
Number of Subjects 
Men (n=72) and women (n=87) (total=159 overweight/obese subjects) with IR will be recruited for this 
study.  Postmenopausal women are those who have not menstruated for at least 6 months.5, 6 
Table 1 presents the estimated sample sizes necessary to test the response to amiloride and compare the 
responses of men and women. We hypothesize that the improvement in carotid femoral pulse wave velocity 
(cfPWV) in men will be intermediate between the two female groups. Because of the negative effect of 
estrogen-stimulated ENaC activation, we believe that amiloride will benefit premenopausal women more (a ∆, 
reduction of 1.0 m/s) than the other groups. Our sample size also allows for a greater variation in the response 
of premenopausal women due to the effects of reproductive hormones in this group7 (SD of the change of 1.2 
m/s, Table 1). By contrast, we anticipate that the response of post-menopausal women (Table 1) will be equal 
to that of the older men or could even be the lowest of all the groups, given a lesser contribution of estrogen 
signaling in the activation of ENaC in this population. In summary, the final sample sizes listed below will give 
90% power to detect each group's changes in cfPWV. 
 
VII. 
Study Procedures/Design/Treatment Plan 
Planned visits: 
Table 1.  Repeated-
measures design for 
amiloride treatment 
Baseline and 
treatment  
cfPWV 
(m/s) 
SD of 
the ∆ 
Power Alph
Calculated 
sampled 
size 
     Final recruitment  
target 2:1    
       Amiloride       
Placebo    
1.  Premenopausal 
women 
7.2 --> 6.2 
1.2 
90% 
0.05 
30 
34 
17 
2.  Young men (30-50 y) 
7.1 --> 6.6 
0.5 
90% 
0.05 
21 
24 
12 
3.  Old men (51-70 y) 
8.0 --> 7.5 
0.5 
90% 
0.05 
21 
24 
12 
4.  Postmenopausal 
women 
8.0 --> 7.5 
0.5 
90% 
0.05 
21 
24 
12 

Page 5 of 12 
Screening visit: Written consent, DOB, gender, ethnic/racial category, height, waist circumference, body weight 
(history of body weight gain or loss, vital signs, medical history and screening laboratories. Entire visit 
is expected to take up to an 1 hour.  
Visit #1: Vital signs (blood pressure and heart rate), weight, DEXA, assessment of brachial and popliteal artery 
flow-mediated dilation (FMD), cfPWV, femoral artery blood flow, and oral glucose tolerance test 
(OGTT) (amount of blood drawn not exceeding 55 mL). OGTT will be done when glucose beverage is 
available. Entire visit is expected to take up to 6 hours. Women of childbearing age will undergo a 
pregnancy test prior to the DEXA scan and excluded if pregnancy is confirmed. Subject is dispensed the 
bottle of study drug/placebo and instructed how to take them.  
Visit #2 (1 week after starting the medication +6 days/-2 days): Vital signs, safety questionnaire for side effects, 
and blood draw for safety laboratories (basic metabolic panel). Entire visit is expected to take up to 30 
minutes. 
Visit #3 (4 weeks after starting the medication +/- 6 days): Vital signs, safety questionnaire for side effects, and 
blood draw for safety laboratories (basic metabolic panel). Entire visit is expected to take up to 30 
minutes.  
Visit #4 (8 weeks after starting the medication +/- 6 days): Vital signs, safety questionnaire for side effects, and 
blood draw for safety laboratories (basic metabolic panel). Entire visit is expected to take up to 30 
minutes. If the prior safety visit labs have been within the protocol parameters this visit may be 
completed as a phone call visit to collect safety questionnaire for side effects and any changes in the 
subject’s health.   
Visit #5 (12 weeks after starting the medication +/- 6 days): Vital signs, weight, DEXA, safety questionnaire for 
side effects, and blood draw for safety laboratories (basic metabolic panel) and general biochemistries. 
Subject also will have assessment of brachial and popliteal artery FMD and cfPWV. Women of 
childbearing age will undergo a pregnancy test prior to the DEXA scan and withdrawn from study if 
pregnancy is confirmed. Subject will be dispensed a new bottle of study drug/placebo. Entire visit is 
expected to take up to 3 hours. 
Visit #6 (16 weeks after starting the medication +/- 6 days): Vital signs, safety questionnaire for side effects, and 
blood draw for safety laboratories (basic metabolic panel). Entire visit is expected to take up to 30 
minutes. If the prior safety visit labs have been within the protocol parameters this visit may be 
completed as a phone call visit to collect safety questionnaire for side effects and any changes in the 
subject’s health. 
Visit #7 (20 weeks after starting the medication +/- 6 days): Vital signs, safety questionnaire for side effects, and 
blood draw for safety laboratories (basic metabolic panel).  Entire visit is expected to take up to 30 
minutes. If the prior safety visit labs have been within the protocol parameters this visit may be 
completed as a phone call visit to collect safety questionnaire for side effects and any changes in the 
subject’s health.   
Figure 2 

Page 6 of 12 
Visit #8 (24 weeks after starting the medication +/- 6 days): Vital signs (blood pressure and heart rate), weight, 
DEXA, waist circumference, safety questionnaire for side effects, and blood draw for safety laboratories 
(basic metabolic panel, lipid panel, and hemoglobin and hematocrit). Subject will also have assessment 
of brachial and popliteal artery FMD, cfPWV, femoral artery blood flow and oral glucose tolerance test 
(OGTT) (amount of blood drawn for safety labs, other biochemistries and OGTT not to exceed 70 mL). 
OGTT will be done when glucose beverage (75g) is available in a volume within 50ml of what was used 
in the baseline visit. These will minimize variability of the measurements related to changes in the 
volume of liquid ingested.  Women of childbearing age will undergo a pregnancy test prior to the DEXA 
scan and withdrawn from study if pregnancy is confirmed. Entire visit is expected to take up to 6 hours.  
Additional Safety Visits/unscheduled visits might be scheduled in cases of down titration of amiloride dose. 
Vital signs, safety questionnaire for side effects, and blood draw for safety labs (amount not to exceed 
15mL). Unscheduled visits may also occur if safety lab specimens are compromised for any reason or 
questionable accuracy of the result. Safety labs will then be repeated (amount of blood drawn not to 
exceed 15 mL).   
 
Medication changes and adverse events collected through out subjects’ participation.   
 
Special considerations in case of unforeseeable hardships such as public health emergencies or weather-
related events.  
 
In the event of subjects not being able to complete study visits for the reasons described above the following 
remedial steps will be taken: 
- 
Visits #3,4, 6 and 7 can be completed via a phone interview during which the subject will answer 
the safety questionnaire (no changes in compensation). 
- 
In case that subject cannot complete safety visit #2, every effort will be made to complete this 
visit within 7 days of the original scheduled date. In case that the visit cannot be completed, the 
subject will be instructed to discontinue study medication and his/her participation in the study 
will be completed. Safety officer will be informed. 
- 
In case that study visit #5 cannot be completed and providing patient has completed at least 2 in -
person visits since the medication was started, study medication will be mailed by investigational 
pharmacy for subject to continue treatment. Safety officer will be informed. Compensation will 
be unchanged.  
In the event of the Clinical Translational Science Unit (CTSU) not being available or if it is considered by the 
study physician that performing studies there can increase risk of exposure to infectious agents or related 
hazards, the following alternative sites will be made available for screening, safety and study visits.  
  
• 
MU Physical Activity and Wellness (PAW)  
• 
University hospital clinic rooms 
 
  
These locations are not carpeted, and we do not anticipate an increased risk for the subjects given that medical 
supervision and nursing staff assistance will be unchanged. 
 
Detail on procedures: 
DEXA scan: to assess body composition with dual-energy X-ray absorptiometry.  
Oral glucose tolerance test: After an overnight fast, a catheter will be inserted into an antecubital vein for 
sampling of venous blood.  Additional blood will be collected every 15-30 mins over the next 120 mins after 
consuming the glucose beverage (75 grams of dextrose).    

Page 7 of 12 
Arterial Stiffness and Blood Pressure: The SphygmoCor XCEL device will be used to assess blood pressure 
and aortic pulse wave velocity, a marker of arterial stiffness. A blood pressure cuff will be wrapped around the 
upper arm and upper leg. They will periodically inflate to ~200 mmHg for less than 60 seconds. A pressure 
sensor (tonometer) will be placed over the skin of the neck region to obtain the pressure wave form in the 
carotid artery.   This measure will be performed in duplicate.  
 
Brachial and Popliteal artery FMD: Arterial measurements will be performed by imaging the brachial and 
popliteal artery longitudinally using high-resolution duplex ultrasonography. Brachial and popliteal artery 
vasodilatory responses to hyperemia (i.e. flow mediated dilation) will be examined by inflating a cuff, distal to 
the antecubital fossa up to 250 mmHg for 5 minutes. Before, during and after rapid release of the cuff, brachial 
artery blood flow velocity and diameter will be continuously measured. Before, during and after rapid release of 
the cuff popliteal, artery blood flow velocity and diameter will be continuously measured. This measure will be 
performed in duplicate.  
 
OGTT with leg blood flow: After an overnight fast, a catheter will be inserted into an antecubital vein for 
sampling of venous blood.  Blood will be collected every 15-30 mins over the next 120 mins after consuming 
the glucose beverage (75 grams of dextrose).   Moreover, before and during the OGTT, we will be using an 
ultrasound to measure femoral artery blood flow.   
 
VIII. Potential Risks/Adverse Events 
Phlebotomy:  The total blood withdrawn will not exceed 550 mL in any 24-week period. Hematocrit is 
measured during screening and before the interim and final visits to check that it is within safe limits.  All 
subjects will be advised to refrain from donating blood in public blood drives, during their participation in this 
project.   
These activities are associated with a small risk of phlebitis, bruising and minor pain.  Antiseptic technique will 
be used by nurses who are experts in phlebotomy and catheter placement.  If phlebitis occurs, it will be treated 
conservatively with heat and when appropriate, with antibiotics. 
Carotid-femoral PWV and femoral artery blood flow: These procedures pose no risks. 
Brachial and Popliteal artery FMD:  FMD is a measurement of conduit artery endothelial function.  There is 
risk of minor discomfort in the arm during the five minutes of cuff inflation. 
Oral Glucose Tolerance Test (OGTT): This procedure could cause possible nausea from glucose beverage.  
DEXA and anthropometrics:  Radiation exposure during DEXA is equivalent to about two percent of the 
average radiation dose from all sources (natural background radiation, consumer appliances, radon gas, medical 
tests, etc.) that a person in the United States receives each year.  Subjects who have participated in any other 
research study or medical procedure involving significant ionizing radiation exposure (e.g., multiple chest x-
rays) in the past 12 months will be excluded.  For female subjects, a urine pregnancy test is administered before 
the DEXA.  Body weight is measured to the nearest 0.1 kg and height to the nearest 0.1 cm.  
Amiloride therapy: In previous clinical trials with higher doses (10 mg) amiloride was well tolerated.1 
Nevertheless, our team of investigators is well aware of the potential side effects (1-10%): dizziness, fatigue 
headache, hyperkalemia. Subjects will be closely monitored during the drug/placebo treatment intervention. 
After each safety visit, Dr. Manrique and Dr. Bostick will review blood pressure and heart rate readings, as well 
as results of renal function and electrolyte levels.  The treatment will be discontinued in case of hyperkalemia > 
5.5 mEq/L, creatinine increases more than 30% above baseline, hypotension or severe dizziness. If the subject 
exhibits hyperkalemia or if creatinine increases more than 30% above baseline, the study team will repeat safety 
labs until they return to baseline. If a subject exhibits potassium > 5.5 mEq/L, creatinine increases more than 
30% above baseline, hypotension or severe dizziness on the 5 mg dose, medication will be held for 1 week and 
a lower dose of amiloride will be started (half of a 5mg tablet). Once the dose of half of a 5mg tablet has been 
started, a safety visit will be completed 1 week later. If no clinically significant abnormalities are documented, 
safety visits will proceed as scheduled for the 5 mg dose.  If there are clinically significant abnormalities 

Page 8 of 12 
documented after down titration of amiloride, subjects will discontinue medication and participation in the 
clinical trial is terminated. In the event of a severe unexpected adverse event the subject will be unblinded to 
facilitate medical care. 
Placebo: Pill Capsule:  There is a potential risk for an allergic reaction to the gelatin capsule.  
Placebo: Cellulose:  These are no known risks.  
 
Protection Against Risks 
Risks to loss of confidentiality are reduced by assigning all subjects a data identifier code.  Hard copies of data 
are stored in locked file cabinets, and only the PI and study coordinator have access to the locked files.  
Individual names or initials are not used in any discussions or publications of the data.  We have assembled a 
research team which includes scientists, physicians, and clinician-scientists with significant experience in 
human research and metabolic diseases to help anticipate and reduce the risks to subjects.  The specific protocol 
to minimize risk associated with each procedure is described below. 
 
Psychological stress:  The psychological stress from participation in this study is minimal.  However, some of 
the questions about food intake and physical activity may make the subjects feel uncomfortable.  Subjects will 
be told they may skip any portion of a questionnaire if they feel uncomfortable about answering the questions. 
Blood collection and IV lines:  Aseptic technique is used to collect blood using butterfly needles and, if 
necessary, through an IV.  Only the amount of blood necessary for analyses is withdrawn and the staff members 
performing venipuncture are nurses and trained phlebotomists.  Risk of bleeding is reduced by applying 
pressure at the site of puncture.  Bruising is treated with ice.  Fainting is prevented by drawing blood in the 
semi-recumbent position. 
Arterial stiffness, flow mediated dilation, and blood pressure:  When assessing carotid-femoral PWV, the 
blood pressure cuff will squeeze the arm and leg tightly; however, any discomfort will be alleviated as soon as 
the pressure in the cuff is released. 
DEXA:  Risks include a small radiation for the DEXA, equivalent to about 2% of the average radiation dose 
from all sources that a person in the U.S. receives each year.  Subjects who have participated in other research 
studies or medical procedures involving significant ionizing radiation exposure in the past 2 months will be 
excluded.  A urine pregnancy test will be performed before the DEXA. 
There are no alternative methods that will allow us to test our proposed hypotheses.  
 
Plan for Reporting Study Deviations 
We will report all noncompliance per the IRB’s noncompliance policy. 
 
Stopping Rules 
We will stop an individual study in the event of a serious adverse event.  If 4 or more subjects experience serious 
adverse events (as described below), the study will be stopped and the events will be discussed with the IRB to 
determine whether it is appropriate to continue and/or determine appropriate modifications to the protocol to 
avoid further adverse events. Adverse events will be considered unanticipated problem if it is unexpected, serious, 
and would have implications for the conduct of the study.  
All adverse events will be submitted for review according to current protocols. 
 
Definition of serious adverse event (SAE) 
An SAE is classified as any untoward medical occurrence that, at any dose, meets any of the following criteria 
(a – f): 
 
a. Results in death 
b. Is life-threatening 
The term ‘life-threatening’ in the definition refers to an event in which the subject was at risk of death at the 
time of the event, it does not refer to an event which hypothetically might have caused death if it were more 
severe. 

Page 9 of 12 
c. Requires inpatient hospitalization or prolongation of existing hospitalization 
A hospitalization or prolongation of hospitalization will not be regarded as a serious adverse event if 
at least one of the following exceptions is met: 
- 
 The admission results in a hospital stay of less than 12 hours 
- 
The admission is pre-planned 
- 
The admission is not associated with an adverse event related to the study medication or study procedures 
as determined by the study safety officer and data safety monitoring board.  
d. Results in persistent or significant disability / incapacity 
Disability means a substantial disruption of a person’s ability to conduct normal life’s 
functions. 
e. Is a congenital anomaly / birth defect 
f. Is another serious or important medical event as judged by the investigator. 
 
Breach of Confidentiality 
Subject confidentiality will be rigorously maintained.  The data collected as part of this study will be for research 
only.  It will be de-identified after collection.  Confidentiality of data will be assured by coding of unique subject 
identities and that coding will be known only to the research team, including the use of secure files, locked filing 
cabinets, and a unique subject coding system.  The original study data will be kept in locked cabinets (hard copy) 
or entered into a secure computer database password protected under a secure server space allocated for use by 
only the study team (electronic).  Furthermore, data analysis will be appropriately blinded, and any individual 
data presented in manuscripts will also be presented in an anonymous nature.  No identifying information will be 
disclosed.  Confirming with University of Missouri policy, all research records will be retained for a period of 7-
years following completion of the study. 
All protocols and techniques to be used will be approved by the Institutional Review Board (IRB) prior to 
initiation of any studies.  Each subject will give written informed consent after all questions have been answered 
by a study team member.  The consent form will also include a statement guaranteeing confidentiality.  Adverse 
event reports and annual summaries will not include subject-identifiable material.  No information will be given 
to anyone without permission from the subject.  Electronic communication with study team members will involve 
only coded, unidentifiable information.  Any unanticipated breach of confidentiality will be summarized and 
reported to the IRB within 5 working days of awareness 
 
IX. 
Anticipated Benefits 
There may be no benefit to the subjects in this study.  These data will aid in the understanding of how epithelial 
cell sodium channel (ENaC) contributes to the genesis of arterial stiffness in obese and insulin resistant 
subjects, preferentially in women. From the screening and baseline tests, all subjects will gain health 
information about themselves.  At the end of the study, results collected will be shared with the subject in face-
to-face meetings.  In summary, the risks of the treatments are minimal, as witnessed by millions of Americans 
who are treated with similar drugs each year.  The health testing offers some risk, which includes the blood 
draws.  It is possible however, that the benefits to the subjects may be substantial, and the new information on 
characteristics that increase cardiovascular risk in insulin resistance and obese women will benefit society.  

Page 10 of 12 
X. 
Compensation 
 
We have completed multiple clinical studies involving human 
subjects with adherence rates of greater than 95% and subject 
retention rates of greater than 85%.8-12  We utilize a validated 
retention strategy published by Jeffrey et al.13 that has been 
successful.  With respect to subject compensation, as shown in 
Table 2, subjects will be paid $30 for screening visit, $100 for 
baseline visit, $50 for week 1, $100 for week 4, $75 for weeks 
8, 16 and 20, $150 for week 12 (interim), and $350 for week 24 
(final visit). Any additional unscheduled safety visits will be 
compensated $50. Thus, a total compensation of $1005 will be 
provided as study events are completed not including any 
additional visits. 
XI. 
Costs 
Subjects will not be charged for any procedures that are part of this research study.  The costs of the study will 
be covered by a NHLBI R01 grant. 
 
XII. 
Data Safety Monitoring Board 
Overall framework for safety monitoring and what information will be monitored: 
The drug treatment could pose risk to participants. The data safety monitoring plan (DSMP) for this trial 
focuses on close monitoring by the PI safety officer, local IRB and the Data Safety Monitoring Committee 
(DSMC). Monitoring will include review of the protocol and statistical analysis plan, enrollment, attrition, 
efficacy end-points, and adverse events. PI will submit an annual progress report to the NHLBI that will 
include: confirmation of adherence to data safety and monitoring plan, summary of any data or safety 
monitoring issues, description of any changes to the research protocol or the DSMC if applicable and 
new/continuing IRB approvals. 
Plan for data management: 
A password-protected database will be used to manage all study data.  To ensure confidentiality only subject ID 
numbers will be entered into the database. Signed informed consent forms are kept in a locked filing cabinet. 
Participants will not be individually identified in any publication. Participants’ right to privacy will be 
protected. 
Frequency of monitoring, including any plans for interim analysis and stopping rules: 
In addition to monitoring by the PI, study coordinator, and safety officer monitoring, the MU IRB monitors all 
aspects of the project on an annual basis. The DSMC will conduct scheduled reviews on a semiannual basis. 
Two members external to the PI institution are included in the DSMC. The frequency of the structured data 
review for this study can be summarized in the following table: 
 
Data type 
Frequency of review 
Subject accrual (adherence to protocol 
regarding demographics, 
inclusion/exclusion) 
At the end of each recruitment 
Table 2. Subject compensation 
Visit 
Compensation 
Screening 
 30 
Baseline test 
 100 
Week 1  
 50 
Week 4  
 100 
Week 8, 16 & 20 
  75 
Week 12 Interim visit  150 
Week 24 Final test 
 350 
Unscheduled visit 
50 
 

Page 11 of 12 
Adverse event rates (injuries)  
semi-annually 
Compliance to treatment  
semi-annually 
Out of range laboratory data 
semi-annually 
Stopping rules report regarding statistical 
power implications of dropouts and 
missing data 
semi-annually 
IRB review 
annually 
DSMC review  
semi-annually 
 
Process for Managing and Reporting Adverse Events, Serious Adverse Events and Unanticipated 
Problems: 
Unanticipated events will be reported to MU IRB, and both serious and non-serious adverse events will be 
reported to the MU School of Medicine Data and Safety Monitoring Committee. For reporting to the DSMC, 
adverse events will be categorized and classified according to Common Terminology Criteria for Adverse 
Events Scale (CTCAE v3.0). Safety reports will be sent to the safety officer.  The PI will be responsible for 
assembling the data and producing these reports, as well as assuring that all parties obtain copies of these 
reports. 
Qualifications and responsibilities of the Safety Officer:   
The safety officer for this trial will be Brian Bostick, MD-PhD. Dr. Bostick is an Assistant Professor in the 
Division of Cardiology.  In addition to practicing medicine and cardiology, he is a clinician scientist.  As 
Safety Officer, Dr. Bostick will review eligibility criteria with Dr. Manrique, and be unblinded as to treatment 
assignment. He will review all reports sent by the study coordinator to determine whether there is any corrective 
action, trigger of an ad hoc review, or stopping rule violation that should be communicated to the University of 
Missouri IRB and the Data and Safety Monitoring Committee. In addition, the safety officer may comment on 
whether the study investigator needs to report any specific out-of-range laboratory data. 
MU Data and Safety Monitoring Committee (DSMC):   
The DSMC provides education for investigators, research teams, and faculty regarding data and safety 
monitoring; reviews proposed Data & Safety Monitoring Plans; establishes Data & Safety Monitoring Boards; 
conducts independent, interim reviews of study safety and progress; and makes recommendations concerning 
the continuation of studies, including recommendations regarding the modification, suspension, or termination 
of a study.  The data safety monitoring board minutes will summarize the topics discussed and list the 
recommendations.  Minutes will be signed by the board chair. For the current study the DSMC has 3 voting 
members representing: Endocrinology, Diabetes and Metabolism, Cardiovascular Diseases and Echocardiology, 
and Biostatistics.  Additional members may be consulted ad hoc as needed and would be reflected in the 
minutes. In addition, among these members and for the purpose of this clinical trial, 2 voting external 
members have been included: Dr. George Bakris (University of Chicago) and Dr. Linda Peterson MD 
(Washington University).  
XIII. Multiple Sites 
N/A 

Page 12 of 12 
XIV. References/Appendices 
 
1. 
Brown MJ, Williams B, Morant SV, Webb DJ, Caulfield MJ, Cruickshank JK, et al. Effect of amiloride, 
or amiloride plus hydrochlorothiazide, versus hydrochlorothiazide on glucose tolerance and blood 
pressure (pathway-3): A parallel-group, double-blind randomised phase 4 trial. The lancet. Diabetes & 
endocrinology. 2016;4:136-147 
2. 
Brunner EJ, Shipley MJ, Ahmadi-Abhari S, Tabak AG, McEniery CM, Wilkinson IB, et al. Adiposity, 
obesity, and arterial aging: Longitudinal study of aortic stiffness in the whitehall ii cohort. Hypertension. 
2015;66:294-300 
3. 
Canepa M, AlGhatrif M, Pestelli G, Kankaria R, Makrogiannis S, Strait JB, et al. Impact of central 
obesity on the estimation of carotid-femoral pulse wave velocity. American journal of hypertension. 
2014;27:1209-1217 
4. 
Grundy SM, Brewer HB, Cleeman JI, Smith SC, Lenfant C. Definition of metabolic syndrome. Report 
of the National Heart, Lung, and Blood Institute/American Heart Association Conference on Scientific 
Issues Related to Definition. 2004;109:433-438 
5. 
The Women's Health Initiative Steering C. Effects of conjugated equine estrogen in postmenopausal 
women with hysterectomy: The women's health initiative randomized controlled trial. Jama. 
2004;291:1701-1712 
6. 
Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, et al. Risks and 
benefits of estrogen plus progestin in healthy postmenopausal women: Principal results from the 
women's health initiative randomized controlled trial. Jama. 2002;288:321-333 
7. 
Syed-Abdul MM, Hu Q, Jacome-Sosa M, Padilla J, Manrique-Acevedo C, Heimowitz C, et al. Effect of 
carbohydrate restriction-induced weight loss on aortic pulse wave velocity in overweight men and 
women. Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et 
metabolisme. 2018 
8. 
Morishima T, Restaino RM, Walsh LK, Kanaley JA, Padilla J. Prior exercise and standing as strategies 
to circumvent sitting-induced leg endothelial dysfunction. Clinical science (London, England : 1979). 
2017;131:1045-1053 
9. 
Walsh LK, Restaino RM, Neuringer M, Manrique C, Padilla J. Administration of tauroursodeoxycholic 
acid prevents endothelial dysfunction caused by an oral glucose load. Clinical science (London, England 
: 1979). 2016;130:1881-1888 
10. 
Jacome-Sosa M, Hu Q, Manrique C, Parks E. Slower rate of fat absorption at dinner versus lunch is 
associated with lower concentrations of triacylglycerols (tg) the following morning. The FASEB 
Journal. 2015;29:744.747 
11. 
Abdul MMS, Hu Q, Padilla J, Manrique CM, Heimowitz C, Le N, et al. Sex-dependent effects of a low 
carbohydrate diet-induced weight loss on aortic stiffness. The FASEB Journal. 2017;31:1015.1019-
1015.1019 
12. 
Parks EJ, Rutledge JC, Davis PA, Hyson DA, Schneeman BO, Kappagoda CT. Predictors of plasma 
triglyceride elevation in patients participating in a coronary atherosclerosis treatment program. Journal 
of cardiopulmonary rehabilitation. 2001;21:73-79 
13. 
Jeffery RW, Wing RR, Sherwood NE, Tate DF. Physical activity and weight loss: Does prescribing 
higher physical activity goals improve outcome? The American journal of clinical nutrition. 
2003;78:684-689