Mitochondrial Dysfunction in Chronic Kidney Disease
Phase: PHASE2
Conditions: Hemodialysis-Induced Symptom, Mitochondrial Diseases
Sponsor: Vanderbilt University Medical Center

 
11/9/17 
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Mitochondrial and Chronic Kidney Disease 
NCT 03177798 
11/09/2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 
Statistical Considerations 
11.0 
Privacy/Confidentiality Issues 
12.0 
Follow-up and Record Retention 
 
 

 
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1.0 
Background 
 
Cardiovascular complications in end stage renal disease (ESRD)  
Patients undergoing maintenance hemodialysis (MHD) have a high risk of developing 
cardiovascular events. Cardiovascular death accounts for more than 40% of overall 
mortality in these patients. Depending on the age group, the risk of cardiovascular death 
is between 5 and 100 times higher than in the general population.1, 2 Furthermore, the 
survival rate after myocardial infarction in patients on MHD is only 40% at one year.3 
Pharmacological interventions that reduce the risk of cardiovascular events in the general 
population are ineffective in patients undergoing MHD. Two different prospective, placebo 
controlled studies did not show a protective effect of statins in ESRD patients despite 
significant reduction in LDL cholesterol.4, 5 A more recent study showed that the 
combination of simvastatin/ezetimibe, while effective in CKD patients not on dialysis, did 
not reduce the occurrence of atherosclerotic events in patients on MHD.6, 7 Similarly, the 
only prospective clinical trial of an ACE inhibitor showed no protective effect on 
cardiovascular events in these patients.8 
 
Inflammation and oxidative stress: risk factors for cardiovascular events in ESRD 
The 
increased 
incidence 
of 
cardiovascular 
events 
and 
cardiovascular death in ESRD cannot 
be explained by the traditional risk 
factors, (e.g. age, male, diabetes, 
hypertension, 
hyperlipidemia, 
and 
smoking).9 Other factors have been 
implicated in the pathogenesis of 
accelerated atherosclerosis in ESRD. 
Among them oxidative stress and systemic inflammation may play a central role (Figure 
1).10, 
11 
Chronic 
inflammation 
is 
commonly 
observed 
in 
ESRD 
patients.12 Inflammatory markers, particularly interleukin 1 (IL-1), interleukin 6 (IL-6), and 
tumor necrosis factor-alpha (TNFa), are chronically elevated in patients with ESRD, even 
before initiation of MHD.13 HD procedure itself also induces an acute increase in circulating 
concentrations of inflammatory markers.14, 15 These data suggest that both, chronic uremia 
and recurrent HD contribute to inflammation.16, 17 Inflammatory cytokines also predict 
cardiovascular endpoints in patients with ESRD. For instance, interleukin 6 (IL-6) predicts 
mortality and correlates with the severity of carotid atherosclerosis.18, 19 Oxidative stress 
occurs when the reactive oxygen species (ROS) production is higher than the antioxidant 
capacity and is common in patients undergoing MHD. Markers of oxidative stress are 
increased in ESRD.20-23 For example, F2-Isoprostanes levels are increased before, during, 
and after HD procedure. Other markers of oxidative stress correlate with mortality and 
morbidity in ESRD: plasmalogen is associated with increased mortality,24 and 
malonaldehyde correlates with history of cardiovascular disease.25 Inflammation and 
oxidative stress play a role in the pathogenesis of atherosclerosis and the interaction 
between these factors is complex. Regardless of the initial event (either oxidative stress 
or inflammation), ROS are present in the atherosclerotic wall.26 In turn, ROS induce the 
activation of signaling pathways including the nuclear factor kappa β (NF-κβ) cascade. 
Activation of NF-κβ results in the expression of cytokines and cell adhesion molecules,27 
leukocyte recruitment, and more ROS production in the atherosclerotic lesion. 
 
Figure 1. Risk factors for cardiovascular risk in ESRD 

 
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Mitochondrial dysfunction contribute to atherosclerosis, oxidative stress and 
inflammation in ESRD 
The mitochondria are one of the main sources of ROS. Around 2% of oxygen is 
incompletely reduced by the mitochondria and converted into ROS. Superoxide is the 
main ROS produced by mitochondria, which is then converted by manganese superoxide 
dismutase into hydrogen peroxide (H2O2). H2O2 can then diffuse across the mitochondrial 
membranes or be decomposed by either glutathione peroxidase or catalase. ROS 
produced by the mitochondria can activate the NLPR3 inflammasome inducing the 
production of interleukins (e.g. IL-1β).28 There is a clear association between 
atherosclerosis, inflammation, oxidative stress and mitochondrial dysfunction.29, 30 
Mitochondrial DNA mutations are more common in atherosclerotic aorta, with none or little 
damage in nuclear DNA.30 Furthermore, mitochondrial DNA mutations are significantly 
more frequent in hearts from patients with coronary disease compared to age-matched 
healthy subjects.31 Using magnetic resonance spectroscopy (MRS), previous studies have 
shown that patients undergoing MHD exhibit skeletal muscle mitochondrial dysfunction 
when compared to healthy individuals.32-34 But no study has compared patients with 
different stages of CKD. It has also been suggested that mitochondrial dysfunction worsen 
years after HD initiation; 32, 35 however, this hypothesis has not been tested yet. 
Nevertheless, previous studies have shown abnormalities on mitochondrial metabolism in 
skeletal muscles biopsies36-38 and in peripheral blood mononuclear cells (PBMCs) from 
patients with CKD.39, 40 Mitochondrial dysfunction in skeletal muscle may also explain why 
patients with CKD exhibit poor exercise tolerance and rapid fatigability.41, 42 It is still 
unknown if mitochondrial dysfunction is the result of intrinsic mitochondrial defects or 
changes in mitochondrial number. These two factors can be differentiated by measuring 
at the same time mitochondrial function and mitochondrial volume density, which has 
never done in patients with CKD. Mitochondrial volume density is usually measured with 
electron microscopy. Other methods that correlate with electron microscopy are 
fluorescent dyes, mitochondrial enzymes activities and mitochondrial DNA content (or 
mitochondria DNA copy number). The latter has recently been proposed as a potential 
marker of mitochondrial dysfunction.43 Accordingly, a recent study showed an inverse 
correlation between mitochondrial DNA copy number in PBMCs and survival rate in 
patients undergoing MHD,44 suggesting that mitochondrial dysfunction may play a role in 
all-cause mortality in ESRD. 
 
Bradykinin may play a role in oxidative stress and mitochondrial dysfunction 
Hemodialysis activates the kallikrein-kinin system and the production of bradykinin.45 
Under 
normal 
physiological 
conditions, 
bradykinin exerts its effect through B2 
receptors. Bradykinin has a dual effect over 
the cardiovascular system: it increases tPA 
release and inhibits platelet aggregation,46, 47 
but also increases oxidative stress and 
systemic inflammation.48-51 Very few studies, 
however, 
have 
studied 
the 
effect 
of 
bradykinin 
on 
oxidative 
stress 
and 
inflammation in ESRD patients. Our group 
showed 
that 
endogenous 
bradykinin 
contributes to increases in plasminogen 
activator inhibitor-1 and monocyte chemo-
attractant protein 1 following HD, consistent with a pro-inflammatory effect.52 Thus, any 
Figure 2. Relation among progressive CKD, HD, 
and activation of kallikrein-kinin system 

 
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intervention that increases bradykinin, such as ACE inhibitors, may increase the 
inflammatory response to HD. In fact, the candidate has observed that one week treatment 
with the ACE inhibitor ramipril elicits a greater pro-inflammatory response compared to 
ARB valsartan in patients undergoing MHD.53 Bradykinin also increases ROS production 
in cardiomyocytes, endothelial cells, vascular smooth muscles and renal tubular cells.51, 
54, 55 Studies in human coronary endothelial cells showed that bradykinin vasodilator 
effects depended on ROS production by NADPH oxidase.51 Other studies suggest that 
bradykinin increases the production of mitochondrial ROS.54, 55 But little is known about 
the effect of bradykinin on mitochondrial function, especially in patients undergoing MHD.  
 
Mitochondrial dysfunction in CKD.  
There is strong evidence that oxidative stress and inflammation correlate with 
cardiovascular events in ESRD patients. Mitochondrial dysfunction may be the cause of 
both oxidative stress and inflammation. Although many previous studies have studied 
mitochondrial function in patients with CKD, they have not evaluated the effect of 
progressive kidney disease and the contribution of HD on mitochondrial function. We are 
planning to study for the very first time, mitochondrial function in vivo in patients at different 
stages of CKD not yet on HD compared to patients with ESRD. This approach will allow 
us to study the role of progressive CKD on mitochondrial dysfunction. Furthermore, no 
study has simultaneously performed in vivo measurements of mitochondrial function and 
skeletal muscle biopsies in patients with ESRD. This methodology will allow us to correlate 
in vivo mitochondrial function with biochemical and ultra-structural changes in 
mitochondria. But we will also evaluate the contribution of the HD procedure on 
mitochondrial dysfunction. HD activates the kallikrein-kinin system, which increases 
bradykinin levels and may contribute to the increased oxidative stress and inflammation 
observed in these patients. Our preliminary data suggest that bradykinin contributes to 
mitochondrial dysfunction, which is also present in ESRD. Little is known about the relation 
among hemodialysis, mitochondrial function and bradykinin, however. Understanding the 
interplay among these factors would help us to direct therapeutic interventions to patients 
with ESRD. 
 
2.0 
Rationale and Specific Aims 
Approximately 500,000 people per year undergo maintenance hemodialysis (MHD) as 
renal replacement therapy for end stage renal disease (ESRD) in the United States (US). 
Patients undergoing MHD are more susceptible to cardiovascular morbidity and mortality. 
Factors such as systemic inflammation and increased oxidative stress may play a role in 
accelerated atherosclerosis in these patients. Mitochondria are the main energy 
machinery of the cell but also sources of reactive oxygen species (ROS) and oxidative 
stress. Mitochondrial dysfunction, by increasing oxidative stress and inflammation, has 
been implicated in the pathogenesis of atherosclerosis in the general population, and it 
has been described in patients on MHD. The etiology of mitochondrial dysfunction in 
ESRD is not clearly elucidated, but progressive chronic kidney disease (CKD) and MHD 
are the possible causes. While earlier studies have shown the presence of mitochondrial 
dysfunction in ESRD compared to healthy individuals, none has previously studied the 
role of progressive CKD on mitochondrial dysfunction. Several studies suggested that the 
decline in mitochondrial function occurs following initiation of MHD and that the 
hemodialysis (HD) procedure is a potential contributor to this process. HD activates the 
kallikrein-kinin system, which increases bradykinin formation. Bradykinin exerts cardio-
protective effects such as vasodilatation and tissue plasminogen activator (tPA) release, 
but also increases inflammation and oxidative stress and eventually may lead to 

 
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mitochondrial dysfunction. In fact, our preliminary data suggest that bradykinin increases 
mitochondrial superoxide production in peripheral blood mononuclear cells (PBMCs) in 
vitro, and endogenous bradykinin increases plasma isofurans during HD. The 
overarching goal of this study is to determine the role of progressive CKD and the 
activation of the kallikrein-kinin system during MHD on the development of 
mitochondrial dysfunction. In order to achieve this goal, we propose the following 
specific aims: 
 
Specific Aim 1: Test the hypothesis that mitochondrial function worsens with the 
progression of CKD and initiation of HD. We hypothesize that mitochondrial function 
gradually deteriorates with the progression of CKD. We also expect that MHD rather than 
preventing the decline worsens mitochondrial function. To test this hypothesis, we will 
measure in vivo mitochondrial function (half-time of phospho-creatinine recovery) using 
31P magnetic resonance spectroscopy (31P-MRS) in three groups of subjects: 1) patients 
with ESRD on MHD, 2) patients with CKD stages 3-5 but not on MHD and 3) control 
subjects without CKD. The subjects in each group will be matched by age, sex, race, 
diabetic status, and BMI. We will also evaluate changes in mitochondrial quantity by 
measuring markers of mitochondrial biogenesis and mitochondrial density in muscles 
biopsies. We anticipate that patients on MHD exhibit the worst mitochondrial function (i.e. 
the longest half time of phosphocreatinine recovery). 
 
Specific Aim 2: Test the hypothesis that endogenous bradykinin promotes 
mitochondrial dysfunction in patients undergoing MHD. To test this hypothesis, we 
will conduct a randomized, double-blind, placebo-controlled, 2x2 crossover study, 
comparing HOE-140, a bradykinin B2 receptor blocker, and placebo administered 1 hour 
prior to beginning of HD session and during HD. Mitochondrial function will be measured 
using 31P-MRS. We anticipate that bradykinin B2 receptor blockade with HOE-140 will 
improve mitochondrial function during HD.  
Understanding the mechanisms leading to mitochondrial dysfunction is an important step 
in terms of developing new 
strategies 
to 
prevent 
cardiovascular 
morbidity 
and mortality in ESRD. The 
candidate 
is 
in 
unique 
position to accomplish this 
project based on his training 
in mitochondrial physiology, 
clinical pharmacology and 
clinical trials. The proposed 
studies 
will 
develop 
the 
candidate 
credentials 
to 
become an independent and 
successful 
physician 
scientist.  
 
3.0 
Animal Studies and Previous Human Studies 
3A Mitochondrial DNA copy number in chronic hemodialysis 
Mitochondrial DNA (mtDNA) is susceptible to damage by oxidative stress due to the lack 
of histone protection.56 mtDNA mutations and reduced mtDNA copy number are 
Control
HTN
MHD
0
5000
10000
15000
mtDNA copy number (AU)
0.04
0.06
0.08
50000
100000
R=0.67
p=0.07
F2-Isoprostanes
mtDNA copy number (AU)
A
.
B.
p=0.03
p=0.04
Figure 3. A. Mitochondrial copy number in healthy controls, hypertensive subjects 
without history of ESRD (HTN) and chronic HD patients (n=8 in each group, 
*p<0.05 compared to chronic HD group). B. Correlation between mtDNA copy 
number and F2-Isoprostanes in ESRD patients measured 2 hours after the end of 
HD (n=8). 

 
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commonly present in cells exposed to excessive concentration of ROS.57 Using  qPCR we 
measured mtDNA copy number in white blood cells from patients on MHD, as previously 
described44 (Figure 3A). Copy number was significantly decreased in patients on MHD 
compared to healthy and hypertensive subjects, matched by age, sex, race and BMI. 
Furthermore, there was a trend toward negative correlation between mtDNA copy number 
and F2-Isoprostanes (Figure 3B), suggesting that oxidative stress may play a role in the 
observed reduction of mtDNA copy number. 
3B Markers of oxidative stress are increased in patients undergoing chronic 
hemodialysis  
F2-Isoprostanes 
and 
Isofurans, products of non-
enzymatic 
oxidation 
of 
arachidonic 
acid, 
are 
reliable 
measures 
of 
oxidative stress in plasma. 
Formation of either F2-
Isoprostanes or Isofurans 
depends on tissue oxygen 
tension. In conditions of high 
oxygen 
tension 
the 
formation of Isofurans is 
favored. This may occur due 
to 
increased 
oxygen 
delivered (breathing a high 
FIO2) or decreased oxygen 
consumption 
(as 
in 
mitochondrial dysfunction). 
Previous studies showed 
that F2-Isoprostanes are 
increased 
in 
patients 
undergoing MHD.20 We also 
found 
that 
the 
levels 
of 
Isofurans 
were 
higher 
in 
patients undergoing MHD than in patients undergoing cardiac surgery (Figure 4A). 
Furthermore, the increased ratio of Isofurans to F2-Isoprostanes suggests a preferential 
production of Isofurans in patients undergoing MHD (Figure 4B). These findings may be 
a consequence of mitochondrial dysfunction, as previously suggested.58 
3C. Role of bradykinin in oxidative stress during hemodialysis 
Our group has previously shown that, during HD, blockade of endogenous bradykinin, 
through the bradykinin B2 receptor antagonist HOE-140, decreases markers of 
inflammation.52 We also found that HOE-140 decreases the formation of Isofurans during 
HD, suggesting that endogenous bradykinin contributes to oxidative stress in patients 
undergoing HD (Figure 5A). Using isolated PBMC from healthy individuals and patients 
undergoing MHD, we found that bradykinin increased mitochondrial superoxide 
production (Figure 5B). These preliminary results suggest a role of bradykinin on 
mitochondrial ROS production. 
Isofuran ng/ml
Cardiac Surgery
Hemodialysis
0.0
0.2
0.4
0.6
0.8
p<0.001
Ratio IsoF/IsoP
Cardiac Surgery
Hemodialysis
0
1
2
3
4
5
p<0.001
A.
B.
Figure 4. Isofurans levels (A) and ratio of Isofurans (IsoF) to F2-Isoprostanes 
(IsoP) (B) in patients undergoing chronic HD compared to a cohort of patients 
that underwent cardiac surgery. 
-0.5
0.0
0.5
Vehicle
HOE-140
-60
0
30
240
Dialysis
Time (min)
Isofurans
ng/ml
p=0.032
Control
Hemodialysis
0.0
0.5
1.0
1.5
Vehicle
BK
Mitochondrial superoxide
(Arbitrary units)
p=0.04
p=0.004
p=0.008
p=0.03
A.
B.
Figure 5.  A Isofurans levels during HD in patients receiving HOE-140 or 
placebo. B. Mitochondrial superoxide levels, measured using MitoSOX™ in 
peripheral blood mononuclear cells (PBMCs) with and without bradyinin 
stimulation. 

 
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3D. Mitochondrial ultrastructure in 
patients undergoing MHD 
Mitochondrion ultra-structure was also 
evaluated in muscle biopsy from 5 
different patients undergoing MHD. 
Figure 6 shows some of representative 
electron 
micrographs 
of 
normal 
mitochondria (Figure 6A) and abnormal 
mitochondria with signs of cristae 
swelling (Figure 6B). There also were 
many double membrane formations 
compatible 
with 
auto-phagosomes, 
which are surrounding structures that 
resemble mitochondria (Figure 6C). This 
suggests that mitophagy, a particular 
type of authophagy, may be increased in 
patients undergoing HD. We also found 
many lipofuscin granules (Figure 6D), 
probably as result of oxidative damage 
of mitochondria and lysosomes. 
 
4.0 
Inclusion/Exclusion Criteria 
 
Inclusion Criteria 
1. Age 18 years or older. 
2. On thrice-weekly chronic hemodialysis for at least 6 months. 
3. Clinically stable, adequately dialyzed (single-pool Kt/V > 1.2) thrice weekly, with 
polysulphone membrane for at least 3 consecutive months prior to study. 
4. For Specific Aim 1 only: Chronic kidney disease stages 3-5 but not yet on 
hemodialysis (eGFR less than 59 mL/min per 1.73 m2), or subjects with no history 
of CKD. 
5. Negative serum pregnancy test 
Exclusion Criteria –both Aims 
1. History of functional transplant less than 6 months prior to study 
2. Use of anti-inflammatory medications other than aspirin < 325 mg/d 
3. Use of immunosuppressive drugs within 1 month prior to study 
4. Use of anticoagulant medication 
5. History of active connective tissue disease 
6. History of acute infectious disease within one month prior to study 
7. AIDS (HIV seropositivity is a exclusion criteria only for Specific Aim 1) 
8. History of myocardial infarction or cerebrovascular event within 3 months 
9. Advanced liver disease 
10. Gastrointestinal dysfunction requiring parenteral nutrition 
11. Active malignancy excluding basal cell carcinoma of the skin 
12. History of ACE inhibitor-associated cough or angioedema 
13. Ejection fraction less than 30% 
14. Predialysis potassium repeatedly higher than 6.0 mmol/L (confirmed on a repeated 
blood draw) 
Figure 6.  Representative electron micrograph of skeletal muscles 
from patients undergoing MHD. A.  Normal sub-sarcolemmal 
mitochondria. B. Mitochondria with signs of swelling. C. Double 
membrane structure compatible with auto-phagosome. D. 
Lipofuscin pigment (scale bar=500µm). 

 
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15. Anticipated live donor kidney transplant 
16. Use of vitamin E > 60 IU/d or vitamin C >500 mg/d 
17. Pregnant or breast-feeding 
18. History of poor adherence to hemodialysis or medical regimen 
19. Inability to provide consent 
 
Specific Aim 1 
20. Subjects with cardiac pacemaker, artificial heart valve, any metallic implant, 
permanent tattoo, or any retained foreign metallic bodies will be excluded from the 
magnetic resonance spectroscopy (MRS) study (Specific Aim 1). 
21. HIV infection 
22. Albumin levels lower than 3.5 mg/dL. 
 
5.0 
Enrollment/Randomization 
 
Subjects will be recruited from the Vanderbilt University Medical Center (including the 
Vanderbilt Nephrology Clinic and the Vanderbilt Outpatient Dialysis Clinic) the Nashville 
Veterans Administration (VA) Medical Center, and 10 outpatient hemodialysis units 
operated by Dialysis Clinics Inc (DCI) and DaVita Clinics. Written advertisements, which 
give the name and phone numbers of a contact Research Nurse, will be placed on bulletin 
boards in these locations. In addition, subjects may be informed of the study by their dialysis 
nurse or primary care provider. Subjects who call for more information will be given a brief 
description of the study protocol and, if interested, will be invited to set up a meeting with 
the Research Nurse. During this meeting, the Research Nurse or Investigator will describe 
the study protocol in detail.  Interested subjects will be invited to read and sign an IRB-
approved consent form and will be given a copy of that consent form.  
Subjects will be randomly assigned to treatment order using a permuted-block 
randomization algorithm (for Specific Aim 2). The Vanderbilt Investigational 
Pharmacy will be responsible for the storage, preparation, and labeling of all 
investigational agents and for maintaining accurate drug storage and dispensing logs 
at Vanderbilt. A Clinical Research Pharmacist, will devise standard operating 
procedures for the pharmacy to follow with regard to preparing, labeling, blinding, 
and dispensing study drug. 
 
 
 
6.0 
Study Procedures 
 
Specific Aim 1: Test the hypothesis that mitochondrial function worsens with the 
progression of CKD and initiation of HD  
 
Population 
Three different groups of patients will be evaluated: 1. Patients with ESRD and on MHD 
for at least 6 months, 2. Patients with CKD stages 3-5 (eGFR less than 59 mL/min per 
1.73 m2) not yet on MHD and who have visited a nephrologist at least 1 month prior to 
enrollment, and 3. Control subjects with no CKD. The subjects in each group (including 
the controls) will be matched by age, sex, race, diabetic status, and BMI. All participants 
will be adults 18-75 years of age. 
 
 

 
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General protocol 
After obtaining informed consent, the investigators will perform a physical examination. 
 
Physical Activity 
We will measure physical activity using triaxial accelerometer. For this purpose, the 
monitor of the size of a small pager is clipped on clothing over the right hip. Activity will be 
recorded during waking hours. Physical activity will be examined over a 7-day period 
requiring at least 5 days of recorded activity to be included in the analysis. The 
accelerometer will provide information about total energy expenditure and energy 
expenditure of activity. 
 
Prior to any study procedure, subjects will report to the VUIIS for a training session for the 
31P magnetic resonance spectroscopy (31P-MRS) as described below. Mitochondrial 
function will be evaluated using 31P-MRS. Endurance will be also evaluated using the six-
minute walk test as explained below. The subjects will report to the Vanderbilt University 
Institute of Imaging Science (VUIIS) in the morning on one of the non-dialysis days. During 
this day we will perform the six-minute walk test and a training session for the 31P-MRS. 
Two days later, subjects will be asked to return to the General Clinical Research Center 
(CRC) at Vanderbilt in the morning under fasting conditions for blood sampling, 31P-MRS, 
and skeletal muscle biopsy of the vastus lateralis (see below).  
 
31P Magnetic Resonance Spectroscopy 
Mitochondria function can be evaluated using 
non-invasive techniques such as 31P-MRS, which 
evaluates the concentration of PCr and other 
phosphate-energy carrier molecules in limb 
muscles. Dr. Bruce Damon and Dr. Theodore 
Towse, faculty members of the VUIIS, have 
successfully implemented this technique that will 
be used to evaluate in vivo mitochondrial 
function. Each subject will lie  prone for 
approximately 30 minutes with a coil positioned 
over the belly of the vastus lateralis muscle. The 
position of the coil will be confirmed using scout 
localizer images and a reference positioned 
within the center of the coil (Figure 7A). After 
basal measurements, subjects will be asked to 
perform cycles of knee extension and rest. 
Measurements will be recorded during the 
exercise protocol and for additional 15 minutes 
(recovery period). Spectra will be used to 
calculate concentrations of inorganic phosphate 
(Pi), PCr, adenosine triphosphate (ATP). The 
primary end-point will be the half-time of PCr 
recovery 
(time 
to 
achieve 
half 
of 
basal 
concentration during the recovery period). A 
spectra obtained by the investigators is shown in 
Figure 7B. This method is reliable and reproducible and has been considered the “gold 
standard” to measure mitochondrial function.59, 60 Subjects will report for an initial 
familiarization session (a non-dialysis day). During this session, subjects will be 
familiarized with the exercise protocol. The subjects will be asked to return 2 days later to 
Figure 7 - A. MRI of the thigh. The white dot above the 
thigh marks the center of the 31P MRS coil and aids in 
positioning the coil over the vastus lateralis (*). B. 31P 
MRS spectra acquired at rest form the vastus lateralis 
of a healthy 37 y.o. female. 

 
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11 
perform the 31P-MRS. During the 2 days prior to testing, we will ask that subjects 1) 
perform no moderate or heavy intensity exercise within 1 day of testing; 2) not consume 
alcohol within the 1 day prior to testing; 3) not consume caffeine or use tobacco within 6 
hours prior to testing; and 4) do not eat a meal 3-6 hours before testing. 
 
Six-minute walk test 
This test will be performed as previously described.61, 62 Briefly, patients will be instructed 
to walk during 6 minutes on a 20 meter corridor. The distance in meters will be recorded. 
Patients will not be encouraged to perform the test, but they will be able to judge the time 
remaining from the large clock in the corridor. In this specific aim the test will be performed 
in a non-dialysis day. Dr Ikizler (co-mentor of the PI) has standardized the test in his lab 
and shown that physical activity is lower on dialysis days.63 
 
Muscle biopsies 
Biopsies will be obtained from the vastus lateralis skeletal muscle. Patients receiving 
anticoagulant medication will be excluded. Aspirin will be discontinued for 14 days before 
the procedure. This procedure will be performed under aseptic conditions and local 
lidocaine anesthesia in the CRC at Vanderbilt, by percutaneous needle biopsy. After 
removing subcutaneous fat, biopsies will be divided in 3 pieces, one will be immersed in 
fixatives for electron microscopy; the other 2 pieces will be immediately frozen in liquid 
nitrogen and stored at -80 ºC for DNA and RNA isolation and for future studies (e.g. 
mitochondrial proteomics). The PI has already completed 5 biopsies in patients 
undergoing chronic HD. 
 
Transmission electron microscopy to assess mitochondrial mass and morphology 
Muscle biopsies will be dissected and cut into small pieces and fixed in glutaraldehyde 
(4%) and paraformaldehyde (2%) for 2 
hours at room temperature. Samples 
will 
be 
prepared 
for 
electron 
microscopy as previously described.64 
Briefly, samples will be post-fixed in 
osmium tetraoxide (1%) for 1 hour, 
dehydrated and embedded for further 
sectioning. 
Thin 
(80 
nm) 
fiber 
transverse sections will be stained with 
uranyl acetate and lead citrate and 
examined with a transmission electron microscope. Mitochondrial volume density (% of 
muscle fiber volume occupied by mitochondria) will be determined using a standard point 
counting method.65 Representative electron micrographs from patients with ESRD and 
obtained by the PI are shown in Figure 6. 
 
Markers of mitochondrial biogenesis and mitophagy 
Markers will be measured using quantitative RT-PCR in skeletal muscles biopsies. For 
this purpose, muscle tissue will be homogenized and RNA isolated using RNeasy® 
Fibrous Tissue Mini Kit RNA (Qiagen, Chatsworth, CA), according to the manufacturer’s 
protocol. Mitochondrial biogenic factors will include PPAR gamma coactivator 1-alpha 
(PGC-1α), nuclear respiratory factors (NRF) 1 and 2, and mitochondrial transcription factor 
A (TFAM). Among the mitophagy markers we will evaluate autophagy gene 8 (ATG-8 or 
LC-3), 5 (ATG-5) and 7 (ATG-7), BCL-2/adenovirus interacting protein 3 (BNIP-3) and 
BNIP-3 like protein (BNIP-3L). After performing RNA reverse transcription, PCR analyses 
will be performed in triplicate, using SYBR Green and 18S rRNA as the calibrator 
Table 1. Variables measured in Specific Aim 1 
Variable 
Specimen 
Primary end-point: 
 
• 
Half time of PCr recovery  
(31P-MRS) 
Non-invasive 
Secondary end points: 
 
• 
Six-minute walk test 
Non-invasive 
• 
Mitochondrial volume density 
Muscle biopsy 
• 
Mitochondrial biogenic factors 
Muscle biopsy 
• 
Mitophagy factors 
Muscle biopsy 
• 
Lactate-to-pyruvate ratio 
Plasma 

 
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12 
housekeeping gene. Fluorescence signal will be detected using ABI PRISM 7500 
Sequence Detection System.  
 
Lactate-to-pyruvate ratio 
Lactate-to-pyruvate ratio in blood samples has been used as another marker of 
mitochondrial function. Blood samples will be obtained and mixed with 8% perchloric acid 
(3 to 1 volume to volume ratio). Lactate will be measured using a colorimetric method 
(Sigma-Aldrich). 
Pyruvate 
will 
be 
measured 
using 
high 
performance 
liquid 
chromatography (HPLC). Table 1 summarizes the variables that will be measured in 
Specific Aim 1. 
 
Specific Aim 2: Test the hypothesis that endogenous bradykinin promotes 
mitochondrial dysfunction in patients undergoing chronic HD. 
Population 
Adult (18 years or older) patients undergoing MHD or will be considered for the study. 
Inclusion and exclusion criteria will be the same as specified in section 4B and outlined in 
detail in Protection of Human Subjects section. During the screening period we will test 
each dialysis subject for Hepatitis B if it has not been done within 1 month. This is done 
as standard of care for the dialysis procedure.  
 
 
General protocol 
In a randomized placebo-controlled 2X2 crossover study, subjects will receive either HOE-
140 or placebo in the first study day. Prior to the first study day, subjects will report to the 
VUIIS for a training session for the 31P-MRS. The first study day will occur at least 2 days 
but not more than 2 weeks after the training session. Participants will report to the CRC 
for the study day. HOE-140 (IND # 49,049) or placebo will be infused for 30 minutes prior 
to the initiation of dialysis at a rate of 100µg/kg/h and at 50 µg/kg/h for the duration of 
hemodialysis. We have previously found that this dose of HOE-140 blocks the vasodilator 
effect of bradykinin.66 The study procedures will be performed in the CRC. Participants will 
be asked to perform the six-minute walk test, as described in Specific Aim 1. Subjects will 
then undergo HD in the CRC. Serial blood samples will be collected from the arterial tubing 
of HD 30 minutes before the beginning of HD, at the beginning of HD, 30 minutes and 1 
hour after the initiation of HD, and at the end of HD. The patient will receive the same 
dialysis treatment prescription as their standard-of-care treatment. The investigator has 
experience performing this protocol.53 At the end of HD, patients will remain in the CRC 
for 2 hours; the last blood samples will be collected and subject will be asked to perform 
the six-minute walk test again. The subject will be then transported to the VUIIS to evaluate 
mitochondrial function by 31P MRS and finally discharged home. 
One week later, the participants will return to the CRC for the second study day. The 
procedures will be the same as on the first study day, receiving the other study medication. 
 
Markers of oxidative stress and inflammation 
Table 2. Variables measured in Specific Aim 2 
Variable 
Pre-
HD 
Post-
HOE 
30 min 
HD 
60-min 
HD 
End 
HD 
120 min 
post HD 
Primary endpoint: 
 
 
 
 
 
 
• 
Half time of PCr recovery 
(31P-MRS) 
 
 
 
 
 
X 
Secondary endpoints: 
 
 
 
 
 
 
• 
Six-minute walk test 
X 
 
 
 
 
X 
• 
F2-Isoprostanes 
X 
X 
X 
X 
X 
X 

 
11/9/17 
13 
In 
the 
blood 
samples 
we 
will 
measure circulating 
markers of mitochondrial function (lactate-to-pyruvate ratio), oxidative stress (F2-
Isoprostanes and Isofurans) and inflammation. Table 2 summarizes the variables that will 
be measured in Specific Aim 2. Blood will be centrifuged immediately after collection and 
stored at -80ºC degrees until processing. F2-Isoprostanes and F2-Isofuranes are products 
of lipid peroxidation and markers of oxidative stress. Both will be measured in plasma 
using negative ion gas-chromatography mass spectroscopy as previously described.67 
Serum IL-1β, IL-6, IL-8, IL-10 and C-reactive protein will be measured using Luminex 
immunoassay technology. Monocyte chemo-attractant protein 1 concentrations will be 
measured by ELISA using commercially available kits (Linco Research, St Charles MO). 
 
7.0 
Risks 
 
Potential risks 
1. Insertion of venous catheters may cause bleeding, bruising or infection.  
2. Frequent blood draws can lead to anemia. 
3. HOE 140/Icatibant is a drug approved by the FDA for acute attacks of hereditary 
angioedema. We plan to use HOE-140 for an off-label use. We have held an IND for HOE 
140 (#49,049) since 1995 and have had extensive experience with its safe administration.  
HOE 140 may cause increased blood pressure or heart rate, though we have not observed 
this in prior studies. In addition, we have not seen an effect of HOE 140 on blood pressure 
or heart rate during hemodialysis in a previous study. While HOE 140 has a short half-life 
and there have been no apparent adverse effects to date in studies in subjects undergoing 
cardiopulmonary bypass or hemodialysis, blocking bradykinin could promote ischemia by 
decreasing fibrinolysis and increasing blood pressure.  In Specific Aim 2, we will monitor 
subjects for ischemia throughout the study. 
4. ACE inhibitors can cause birth defects, cough, angioedema, hyperkalemia, or 
hypotension. 
5. AT1 receptor blockers can cause hyperkalemia or hypotension. 
6. The collection and storage of DNA for genotyping creates the risk of release of 
information that could link subjects to stored samples and genotyping results. 
7. MRS does not use ionizing radiation; therefore there are no harmful side effects with 
temporary exposure. But some situations limit the use of MRS, such as in subjects using 
pacemaker or similar metal device, where magnetic field may lead to malfunction. 
Prosthetic metal device or any other metal pieces (e.g aneurysm clips, copper intrauterine 
device) may heat up during the study causing skin or tissue irritation. Skin irritation may 
also result in subjects with tattoos or medication patches. 
8. Muscle biopsy may cause muscle soreness, bruising, infection, a hematoma, or a slight 
chance of bleeding at the incision site. Soreness at the biopsy site may last 48 hours. 
Adequacy of protection against risks 
Recruitment and informed consent 
Written advertisements approved by the Vanderbilt University Medical Center IRB will be 
placed on bulletin boards in the dialysis centers. The advertisement will the name and phone 
numbers of a contact Research Nurse and Investigators. In addition, subjects may be 
• 
Isofurans 
X 
X 
X 
X 
X 
X 
• 
Inflammatory cytokines 
X 
X 
X 
X 
X 
X 
• 
Lactate-to-pyruvate ratio 
X 
X 
X 
X 
X 
X 

 
11/9/17 
14 
informed of the study by their dialysis nurse, primary care provider, or nephrologist. Subjects 
who call for more information will be given a brief description of the study protocol and, if 
interested, will be invited to set up a meeting with the Research Nurse. During this meeting, 
the Research Nurse or Investigator will describe the study protocol in detail. Interested 
subjects will be invited to read and sign an IRB-approved consent form and will be given a 
copy of that consent form to take home. 
Protection against risk 
In Specific Aim 1 to minimize risk of pain during the biopsy, lidocaine/prilocaine (5%) cream 
will be applied to the site of biopsy 1 hour prior to the procedure. The area will be also 
anesthetized with lidocaine (1%). We will make sure that the subject only feels pressure, but 
not pain, prior to starting the biopsy and during the procedure. To minimize the pain after 
the procedure, patients will be asked to take two acetaminophen tablets (325 mg) 
immediately after the procedure.  
To avoid the risk of infection during the biopsy, the site of incision will be clean with povidone-
iodine swabs followed by alcohol swabs (70% isopropyl alcohol), and the procedure will be 
performed under aseptic conditions. 
At the end of biopsy to avoid the risk of swelling, a bag of ice will be place over the biopsy 
area wrapped with an elastic band to keep it in place. To avoid the risk of bleeding, pressure 
will be applied to the biopsy area for 10 minutes. Thin adhesive strips will be placed over 
the skin to close the small incision. 
After the biopsy procedure, subjects will be discharged home with instruction of contacting 
the investigators immediately if they experience intense pain, signs of inflammation, 
bleeding or any kind of drainage from the biopsy site. 
To avoid any possible risk derived from the MRS study, patients will be inquired about the 
use of any metal device, tattoos, or the presence of any retained metal foreign body. 
In Specific Aim 2, blood pressure and heart rate will be measured continuously throughout 
dialysis. We will obtain an electrocardiogram before and after the study. 
Subjects will also be discontinued from the study if they develop low blood pressure as 
defined by symptoms of hypotension or a systolic blood pressure <95 mmHg.  
 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to 
Participants or Others 
 
The principal investigator will be responsible for ensuring the data integrity and the safety 
of all the study participants. All the adverse events will be recorded and reported to the 
IRB. Study progress report will be prepared every six months to describe: the screening 
and recruitment process, demographic of the participants, status and occurrence of 
adverse events (AE), drug toxicities, treatment adherence, and any incident of non-
compliance with the protocol. These reports will be prepared by the principal investigator 
and reviewed with his mentor. The reports will be presented to the Data and Safety Monitor 
Board (DSMB). AE will be classified using a 0-5 scale (0-no AE, 1-mild AE and no 
treatment required, 2-moderate AE that respond to treatment, 3-severe AE that require 
hospitalization or limit daily activities, 4-disalbling or life-threatening AE, 5-death). Any AE 
greater than or equal to 3 will be reported to the IRB and DSMB within 10 business days 
of the PI’s notification of the event. All other AEs will be reported at the time of the annual 
report to the IRB. Protocol changes will be reported to the IRB and will not be implemented 
until approved.  

 
11/9/17 
15 
The DSMB will objectively review the treatment results as they relate to human safety and 
data quality. Drs. Italo Biaggioni, Kerri Cavanaugh and Aihua Bian have agreed to be 
members of the DSMB. Dr Biaggioni, who will serve as the DSMB chair, is Professor of 
Medicine and Pharmacology and Associate Director of the Vanderbilt CRC. Dr. 
Cavanaugh is Assistant Professor in the Division of Nephrology who focuses on patient 
education and awareness regarding chronic kidney disease. Ms. Bian is a biostatistician 
with experience as a consultant with the Department of Medicine. A statement from the 
members of the DSMB is included. None of the members of the DSMB have any conflict 
of interest, whether financial or intellectual with the investigator or the study. The DSMB 
will regularly meet twice a year; in case of an unanticipated or serious adverse event, an 
extraordinary meeting will be scheduled. 
 
9.0 
Study Withdrawal/Discontinuation 
 
Blood pressure and heart rate will be measured continuously throughout dialysis.  We will 
obtain an electrocardiogram before and after the study.Blood pressure will be measured 
during the study. If at any time during the washout period, the systolic blood pressure 
exceeds 160 mmHg or the diastolic blood pressure exceeds 100 mmHg, the subject’s 
usual anti-hypertensive medications will be maximized. If at any time the systolic blood 
pressure exceeds 189 mmHg or DBP exceeds 114 mmHg, the subject will be discontinued 
from the study and his or her antihypertensive medications will be adjusted appropriately.  
Subjects will also be discontinued from the study if they develop low blood pressure as 
defined by symptoms of hypotension or a systolic blood pressure <95 mmHg.  
Serum electrolytes will be measured prior to each study day. For a serum potassium from 
5.5-5.9 mmol/L, dietary restriction will be emphasized and the potassium content of the 
dialysate will be adjusted to decrease serum potassium. A predialysis serum potassium of 
6.0-6.4 mmol/L, if confirmed and not corrected with adjustment of dialysate potassium 
concentration, will result in discontinuation of the study day. The subject may be 
rescheduled to complete the study at a later date. A confirmed predialysis serum potassium 
of 6.5 mmol/L or greater will result in immediate discontinuation.  All potassium 
measurements will be obtained prior to dialysis, which is the most effective treatment for 
hyperkalemia.  
 
10.0 
Statistical Considerations 
Statistical Analysis 
a. Sample size 
• 
Specific Aim 1: We plan to enroll 3 groups: patients with ESRD on MHD (group 1), 
patients with CKD stages 3-5 but not on MHD (group 2), and control subjects without 
CKD (group 3). In previous studies,32, 35 the half-time of PCr recovery in HD patients 
following exercise was 1.1 minutes (m) and the standard deviation was 0.35 m. Normal 
values are 0.6 m with a standard deviation of 0.24 m.32 Assuming the same coefficient of 
variation, we anticipate the mean±SD of the half-time of PCr recovery would be 
1.1±0.35m, 0.85±0.27m, and 0.6±0.19m, for groups 1, 2 and 3, respectively. Using a 2-
sample t-test, we will need 26 subjects per group to have 80% power to detect the 
difference between groups 1 and 2, and 15 subjects per group to detect the difference 
between groups 2 and 3 with a type I error rate of 0.05. For matching purposes, we plan 
to study 26 subjects in each group. The subjects will be matched by age, race, gender, 
BMI, diabetic status.  

 
11/9/17 
16 
• 
Specific Aim 2: The primary end-point is the half-time of PCr recovery (in minutes) 
measured by 31P-MRS. Samples sizes were calculated using data from previous 
studies.32, 35 A sample size of 11 will have 80% power to detect a difference in means (of 
the half time of PCr recovery) of 0.33 minutes (the placebo treatment mean of 1.1±0.35 
minutes and the HOE-140 treatment mean of 0.77±0.35 minutes, a 30% reduction), 
assuming a correlation coefficient of 0.5 between the two measures, and that results to a 
standard deviation of differences of 0.35, using a paired t-test with a 0.05 two-sided 
significance level. 
 
b Data analysis plan. 
Standard graphing and screening techniques will be used to detect outliers and to ensure 
data accuracy. The distribution of continuous endpoints will be examined for normality. In 
the case of non-normally distributed data, proper data transformations will be performed 
or non-parametric tests will be used. ANOVA or Kruskal-Wallis tests will be used to 
compare the three groups and contrasts for specific two-group comparisons will be 
implemented. Two-group comparisons will also be made using two-sample T-tests or 
Wilcoxon rank sum tests. We will use individual matching; each triplet will be matched on 
race, gender, BMI and etiology of chronic kidney disease to ensure similar distribution 
among the 3 groups on these variables. We will also assess the comparability among the 
three groups. ANCOVA analysis adjusting for these factors along with two more additional 
factors will also be conducted. Specific Aim 2 is a 2x2 crossover study with repeated 
measurements during and after HD. For this aim we will use linear mixed effects models 
with a random subject effect and with the treatment (HOE-140 versus vehicle) and the 
time trend (before, during and after HD) as fixed effects. Carry over effect of HOE-140 
treatment will be tested using the T-test approach proposed by Jones and Kenward.68 
Besides the evaluation of the treatment effect and the time trend using the mixed effect 
models, direct treatment effect will be estimated as within-subject mean difference along 
with their 95% confidence intervals. Based on our previous studies, we anticipate a drop-
out rate of 10% or less. Subjects who drop out will be replaced. Specific inferences on 
effects of interest will be made by reporting a point estimate along with a 95% confidence 
interval and the p-value. Hypotheses will be tested at the level of α=0.05. SAS® release 
9.3 (Cary, North Carolina) and the open source statistical package R (version 2.12)69 will 
be used for the analysis with the assistance of Dr. Chang Yu from the Department of 
Biostatistics at Vanderbilt University Medical Center.  
 
 
11.0 
Privacy/Confidentiality Issues 
There are many safeguards in place to prevent the release of information from this study.  
All research samples are bar coded with the subject’s unique identifier. Data sets used for 
analysis  only contain this identifier. The key to the code is protected. Only study team 
members have access to information that identifies subjects participating in the study. The 
results of tests run on research samples will not be recorded in any subject’s medical record 
and neither the subject nor his or her doctor will be told of the results. Access to the 
Vanderbilt computer network is protected at the level of firewalls, TCP wrappers and 
university assigned user IDs.  Data are secured with encryption algorithms and the network 
is maintained by the Medical Center’s Network Computer Service.   
 
12.0 
Follow-up and Record Retention 
 

 
11/9/17 
17 
We plan to enroll all subjects within the first 3.5 years of the grant. We will enroll 78 
subjects in Aim 1 and 11 subjects in Aim 2. Aim 2 is a crossover study, so patients will 
come twice to the Vanderbilt CRC for each study day.  We will utilize trained research 
nurses to assist with muscle biopsies, hemodialysis, and blood processing and 
sampling.  
Research records will be maintained for at least seven (7) years from the date the 
research is closed with the Vanderbilt University IRB. All research records will be 
accessible for inspection and copying by authorized representatives of the IRB, federal 
regulatory agency representatives, and the department or agency supporting the 
research.  
All Health Insurance Portability and Accountability Act (HIPAA) related documentation 
will be maintained for at least seven (7) years from the date of the last use or disclosure 
of the Protected Health Information (PHI). 
 
 
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