Prevention of Acute Kidney Injury by Nitric Oxide in Prolonged Cardiopulmonary Bypass. A Double Blind Controlled Randomized Trial in Cardiac Surgical Patients With Endothelial Dysfunction.
Phase: PHASE3
Conditions: Acute Kidney Injury
Sponsor: Massachusetts General Hospital

 
 
 
 
 
 
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ONLINE DATA SUPPLEMENT 
PROTOCOL: “Nitric Oxide to Reduce Acute Kidney Injury in Patients with Pre-existing 
Endothelial Dysfunction Requiring Prolonged Cardiopulmonary Bypass: 
 A Randomized Clinical Trial” 
 
Steering Committee: Pankaj Arora1, Taylor B Thompson2, Thoralf M Sundt3, Joseph V. Bonventre4, 
Robert M. Kacmarek5, Warren M. Zapol6, Fumito Ichinose6, Lorenzo Berra4,6. 
 
1, Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, AL 
2, Pulmonary Division Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA 
3, Division of Cardiac Surgery, Corrigan Minehan Heart Center, Massachusetts General Hospital, Boston, 
MA 
4, Renal Unit, Department of Medicine, Brigham Women’s Hospital, Boston, MA 
5, Respiratory Care Services Department, Massachusetts General Hospital, Boston, MA 
6, Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, 
MA. 
 
 
 
Dates of study protocol to Massachusetts General Hospital Investigational Review Board: 
Approval Created: July 26, 2016 
Approved: September 01, 2016 
 
Clinicaltrials.gov Protocol Number: NCT02836899 
 
 
 
 

 
 
 
 
 
 
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Synopsis and Study Schema 
 
 
Title 
Peri-operative Nitric Oxide Therapy to Reduce Acute Kidney Injury in Cardiac 
Surgery Patients with Endothelial Dysfunction Requiring Prolonged 
Cardiopulmonary Bypass: A Randomized Clinical Trial.   
  
 
Study Objective(s)  
To determine whether Nitric Oxide (NO) administered during cardio-pulmonary 
bypass (CPB) and after prolonged CPB reduces clinically significant acute renal 
injury (AKI) (in the first 7 days after surgery) and other major short-term 
complications (at 6 weeks and 90 days) and whether the benefits are maintained 
for one year in patients undergoing prolonged CPB for cardiac surgery. 
 
Study Design  
A single academic center, prospective, randomized, double-blind controlled trial 
comparing treatment with NO versus nitrogen (N2) in adult patients undergoing 
prolonged CPB heart surgery. 
 
Study Population 
 
Main selection criteria 
Inclusion 
 
1. 
Provide written informed consent 
2. 
Age ≥ 18 years of age 
3. 
Elective cardiac or aortic surgery with estimated CPB >90 minutes 
4. 
Clinical evidence of endothelial dysfunction assessed by a specifically 
designed questionnaire (Figure 3) 
5.       Stable pre-operative renal function without evidence of a plasma   
             creatinine increase of ≥ 0.3 mg/dL within 3 months of study entry and    
             without receiving renal replacement therapy (RRT) 
 
 
Exclusion 
 
 
 
1. 
eGFR less than 30 ml/min/1.73 m2 
2. 
Emergent cardiac surgery 
3. 
Life expectancy < 1 year at the time of enrollment 
4.          Mean pulmonary artery pressure ≥ 40 mm Hg and pulmonary        
 
vascular resistance > 4 Wood Units 
5.          Left ventricular ejection fraction < 30% by echocardiography      
 
obtained within three months of enrollment 
6. 
Hemodynamic instability as defined by a systolic blood pressure < 
90mmHg 
7. 
Administration of one or more Packed Red Blood Cell (PRBC) 
transfusions in the week prior to enrollment 
8. 
X-ray contrast infusion less than 48h before surgery 
9. 
Evidence of hemolysis from any other origin: 
a. Intravascular: 
i.  Intrinsic RBC defects leading to hemolytic anemia 
(eg, enzyme deficiencies, hemoglobinopathies, 
membrane defects) 

 
 
 
 
 
 
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ii.  Extrinsic: liver disease, hypersplenism, infections 
(eg, bartonella, babesia, malaria), treatment with 
oxidizing exogenous agents (eg, dapsone, nitrites, 
aniline dyes), exposure to other hemolytic agents (eg, 
lead, snake and spider bites), lymphocyte leukemia, 
autoimmune hemolytic disorders 
b. Extravascular:  
Infection (eg, clostridial sepsis, severe malaria), 
paroxysmal cold hemoglobinuria, cold agglutinin 
disease, paroxysmal nocturnal hemoglobinuria, IV 
infusion of Rho(D) immune globulin, IV infusion of 
hypotonic solutions 
 
 
Total expected number of subjects: 250 
 
Recruiting Center  
Departments of Anesthesia and Cardiac Surgery at the Massachusetts 
General Hospital, Boston, MA 
 
Study Drug or Intervention  
Patients will be randomized into one of two groups; one group will 
receive NO during CPB and for 24-hours after the procedure, the other 
group will receive N2. 
 
Evaluation Criteria  
The occurrence of AKI and any other organ damage when evaluated by 
the SOFA score1, prolonged ventilation or mortality reduction.  
 
Primary Endpoints 
Difference in the incidence of AKI between the control group (receiving 
N2) versus the study group (receiving NO).  AKI is defined by abrupt 
(within 48h) reduction in kidney function correlated to an absolute 
increase in serum creatinine of 0.3 mg/dL or more (≥26.4 μmol/L) or a 
percentage increase in serum creatinine of 50% or more (1.5-fold from 
baseline) at any time during the first 7 days after surgery or, finally, a 
reduction in urine output with a documented oliguria of < 0.5 ml/Kg/h for 
> 6h.2 
Secondary Endpoints 
Secondary renal endpoints: Difference between the groups of AKI 
severity as reflected by the changes of serum creatinine and urinary 
output (KDIGO stages)2.  Difference between groups of incidence and 
severity (KDOQI stages 3, 4, 5)3 of Chronic Kidney Disease (CKD) at 1 
year after surgery.  New requirement for RRT following AKI during 
hospitalization and at 6 weeks, 90 days and one year after surgery.  
Incidence of Major Adverse Kidney Events (MAKE)4 at 6 weeks, and 1 
year after surgery.  
Secondary non-renal endpoints: Incidence and severity of other single 
organ dysfunction (SOFA), prolonged cardiovascular support, 
Vasoactive-Inotropic Score (VIS)72,73, duration of mechanical ventilation.  
Evaluation of ICU and hospital length of stay. 
Exploratory Endpoints 
Incidence and severity of AKI related to the presence of CKD at baseline, 
duration of CPB, duration of aortic cross-clamp, levels of free Hb, levels 

 
 
 
 
 
 
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of NO consumption, pulmonary pressure at baseline, cardiovascular risks 
associated with endothelial dysfunction, scheduled procedure and 
EuroSCORE II5, 6.  Differences between groups of delirium7, 8.  
 
Safety Endpoints 
Difference between groups of mortality at 6 weeks, 90 days and 1 year 
after surgery and stratification according to the initial EuroSCORE II.  
Differences between the two groups of transfusions with plasma and 
stored or autologous red blood cells (RBCs) and bleeding.  Incidence of 
non-fatal stroke.  Incidence of peri-operative and non-perioperative non-
fatal myocardial infarction10.  Continuous Monitoring of MetHb with a 
non-invasive co-oximetry11, 12 monitor in order to prevent 
methemoglobinemia (defined as MetHb > 5%).  Continuous monitoring 
of Nitrogen Dioxide (NO2) in inhaled breath (threshold level < 5 ppm) to 
prevent reaching dangerously high levels (goal < 5 ppm). Differences 
between groups of post-operative infections, cardiac arrhythmias and 
other non-cardiac post-operative complications.   
Statistical Considerations 
Intention to treat analysis and per protocol analysis will be carried out.  
Comparison of the proportion of patients developing AKI.  The sample 
size of this trial was calculated based on the primary endpoint: the 
reduction of AKI incidence.  In recent reports from US and European 
studies at major academic centers, AKI incidence has been reported to be 
between 50 to 60% after prolonged CPB.  In our recent trial of NO in 
cardiac surgery in Xian, China, the sample size was calculated to find a 
30% reduction in the incidence of AKI (n=212 in that trial).  We estimate 
a greater (35%) reduction in the incidence of AKI because we anticipate 
that our predominantly Caucasian population at MGH with endothelial 
dysfunction could be more assisted by the beneficial therapeutic 
properties of NO.  Thus, in the NO group, the incidence of AKI is 
expected to decrease from 55% to 35.75%.  The sample size needed to 
detect a difference, assuming an alpha error of 0.05 (two-sided test) and a 
study power of 0.8, is 114 patients per group.  In order to account for 
possible dropouts, we increased our sample size by 10%.  We will enroll 
250 patients.  
Duration of Study Period 
Study participants will be followed up for 1 year after cardiac surgery. 
 
Data Safety Monitoring Board   
o Michael Fitzsimons, MD (Site DSMB Member). Division Chief of Cardiac Anesthesia at 
Massachusetts General Hospital, Boston, MA 
o Francesco Nordio, PhD (DSMB Independent Statistician).  Lead Biostatistician, TIMI Study 
Group, Brigham Women’s Hospital, Boston, MA 
o John Prowle, MA MB BChir MSc MD FRCP FFICM (DSMB Chair).  Clinical reader in Critical 
Care Nephrology in the Critical Care and Peri-operative Medicine Research Group at William 
Harvey Research Institute, Queen Mary University of London, London, UK. 
 
 

 
 
 
 
 
 
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I. BACKGROUND AND SIGNIFICANCE. 
a. Historical background and rationale behind the proposed research 
Background and significance.  Acute kidney injury (AKI) is a common serious complication of cardiac 
surgical procedures that require prolonged (>90 minutes) cardiopulmonary bypass (CPB).  The presence 
of AKI after CPB is associated with increased morbidity and mortality.  The incidence of AKI after CPB 
depends on the type of operation, the duration of CPB and patient characteristics that adversely affect 
vascular function13-15.  In a recent study from Duke University, 54% of the 4,217 adult patients who 
underwent coronary artery bypass grafting surgery developed AKI.  Patients undergoing valve surgery, or 
a combination of both procedures, have a higher rate of AKI (as high as 60-70%)16-27.  Renal replacement 
therapy (RRT) is employed to treat the most severe cases of AKI.  In a recent European trial involving 
patients without pre-existing kidney disease who underwent cardiac surgery with CPB, RRT was required 
in 16% of the participants18.  One important risk factor for the development of surgery-associated AKI is 
pre-existing renal disease.  Compared to patients without renal impairment, patients with a pre-existing 
reduced creatinine clearance have both a significantly greater incidence of AKI and mortality after cardiac 
surgery13, 22.  Hemolysis is an important risk factor for post-surgery AKI.  The increased level of plasma 
hemoglobin (Hb) at the end of CPB closely correlates with the incidence and severity of AKI28-33.  During 
hemolysis, Hb is released into the circulation in the form of oxyhemoglobin (Oxy-Hb), which depletes 
vascular nitric oxide (NO) via the dioxygenation reaction to form methemoglobin (Met-Hb)34-36.  
Endogenous NO is a potent dilator of vascular smooth muscle and NO depletion by plasma-free 
hemoglobin produces vasoconstriction, impaired tissue perfusion and inflammation37-42.  Under normal 
physiological conditions, a decrease in vascular NO bioavailability leads to increased production of NO 
by endothelial NO synthase (eNOS), the enzyme responsible for producing vascular NO.  This sensitive 
feedback mechanism is impaired by endothelial dysfunction, which occurs in patients with 
atherosclerosis, peripheral vascular disease, hypertension, obesity, and diabetes43-45.  The inability to 
increase eNOS activity by patients with endothelial dysfunction results in reduced blood vessel dilation, 
and, consequently, reduced tissue perfusion37, 38, 40, 41, 46-48.  Therapeutic exogenous administration of NO 
gas oxidizes free plasma Oxy-Hb to Met-Hb; the latter Fe+3 species are unable to deplete plasma NO33-36, 
43, 49. 
Hypothesis of the study.  We hypothesize that administration of NO during and for 24 h after prolonged 
CPB will convert free plasma Hb to metHb and prevent NO scavenging by free plasma Oxy-Hb and 
preserve kidney function, even in patients with evidence of endothelial dysfunction.  
Rationale behind the proposed research.  1) Increased plasma-free Hb, a result of hemolysis, is 
associated with AKI following cardiac surgery with CPB.  Many factors contribute to increased plasma 
Hb in cardiac surgery patients, including prolonged CPB, transfusion of autologous red blood cells 

 
 
 
 
 
 
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(RBCs) recovered using intraoperative cell salvage devices, and the transfusion of stored RBCs44, 50, 51.  
The levels of plasma Hb are correlated with the incidence and severity of AKI28-33.  Hemolysis has also 
been identified as an important contributor to AKI after cardiac surgery16, 19, 52. 
2) Plasma Hb, in the form of Oxy-Hb, reacts with vascular NO to form Met-Hb and normal endothelium 
compensates for this reaction by increasing NO production.  In the vascular system, NO is produced by 
endothelial cells and regulates blood flow to tissue by decreasing vascular muscle tone.  During 
hemolysis, however, NO reacts with circulating plasma Oxy-Hb in a rapid and irreversible dioxygenation 
reaction that produces nitrate and Met-Hb, thereby removing NO34-36, 42, 43, 49.  Under the IRB study 
protocol #2010P000961, we have shown that healthy subjects will increase plasma levels of nitrite, a 
precursor of NO, in response to an acute and limited increase in plasma consumption of NO53.  The result 
of increased NO production is to maintain intact regulation of blood perfusion.  In cases of substantial 
hemolysis, as may occur during prolonged CPB, even healthy endothelium may be unable to produce 
sufficient NO.  Exogenous administration of NO gas may then be necessary to convert plasma Oxy-Hb to 
Met-Hb and prevent the scavenging of endogenous NO. 
3) Compared to normal endothelium, dysfunctional endothelium is less capable of producing NO.  In the 
United States, cardiac surgery patients often have pre-existing endothelial dysfunction associated with 
peripheral vascular disease, hypertension, diabetes and obesity16-23, 25, 27.  No treatment has been tested to 
supplement insufficient endothelial NO production during and after hemolysis.  Endothelial function may 
be evaluated in human brachial and digital arteries by assessing vasodilation in response to stimuli (such 
as brief ischemic periods) known to enhance the activity of eNOS, thereby increasing the production of 
endothelial NO.  Peripheral arterial tonometry is a validated method to assess endothelial function38, 43, 54. 
4) Depletion of vascular NO causes vasoconstriction.  We and other investigators have observed in pre-
clinical studies and human studies that during acute hemolysis, plasma NO consumption, measured by an 
increase in plasma NO consumption, is increased and causes vasoconstriction, characterized by an acute 
increase in both systemic and pulmonary vascular resistance (IRB protocol #2010P000961, IRB 
#2011P000700, IRB #2013P000911, IRB #2013P002596).  Intravenous administration of Hb solutions to 
animals, healthy volunteers and patients is associated with a dose-dependent increase in systemic and 
pulmonary arterial pressure38-40, 44, 47, 48, 55-59. 
5) Extra-pulmonary effects of exogenous NO administration: exogenous NO prevents plasma Oxy-Hb 
from consuming vascular NO.  Inhaled NO is FDA-approved for treating pulmonary hypertension of the 
newborn and, at the Massachusetts General Hospital, is also used to treat adult pulmonary hypertension 
and right heart failure.  When NO is breathed, it diffuses rapidly across the alveolar-capillary membrane, 
activating soluble guanylate cyclase, which converts guanosine-5'-triphosphate to cyclic guanosine 
monophosphate and relaxes the smooth muscle of pulmonary vessels60.  Over the past 2 decades, the 

 
 
 
 
 
 
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extra-pulmonary effects of NO have been recognized.  Once NO enters the pulmonary circulation, NO 
may also bind to Hb to form S-nitrosoHb, or modify other circulating proteins.  In IRB protocol 
#2010P000961, we have shown in healthy volunteers receiving autologous blood transfusions that NO 
increases plasma NO metabolites such as nitrate and, in a small amount, nitrite.  In addition, breathing 
NO can oxidize plasma Oxy-Hb to Met-Hb, thereby reducing plasma NO consumption (IRB 
#2011P000700)34, 38, 42, 61-63.  
6) Measurements of markers of tubular damage may assist standard assessments in identifying and 
characterizing early renal dysfunction.  The diagnosis of AKI is based on the KDIGO (Acute Kidney 
Injury Network) criteria2, 64, 65.  Both tools rely on the detection of an elevated level of plasma creatinine, 
which by itself has a limited value in terms of diagnosing early kidney injury after cardiac surgery.  
Elevated plasma creatinine occurs several hours after the kidney is injured, precluding timely 
intervention.  A second potential limitation of using the serum creatinine level to detect renal injury is that 
creatinine is a functional marker of glomerular filtration and not of renal tubular injury, which is one of 
the key features of post-cardiac surgery AKI.  The recent introduction of several plasma and urinary 
markers of tubular damage may allow earlier detection of AKI18, 66-68.  The ability to measure early 
markers of kidney damage facilitates clinical studies evaluating the effectiveness of a treatment that may 
prevent AKI during cardiac surgery with CPB.  In this proposed trial, we will define AKI based on the 
KDIGO criteria2.  In addition, we will measure plasma and urinary renal biomarkers to help detect the 
onset of AKI and the severity of renal tubular injury.  
 
b. Previous pre-clinical or clinical studies leading up to and supporting the proposed research 
Preliminary studies at the Massachusetts General Hospital.  
The rationale and the methods for the proposed research build on our prior Mass General Brigham IRB-
approved studies: #2010P000961, IRB #2011P000700, IRB #2013P000911, IRB #2013P002596.  Several 
of these studies were published in peer-reviewed clinical journals37, 38, 47, 48, 53, 56, 57, 60. 
1) IRB #2010P000961: Hemolysis increases NO synthesis in healthy endothelium.  We enrolled 10 
healthy adults at the MGH Blood Bank to receive one unit of 3-day stored autologous leukoreduced blood 
(not hemolyzed) followed in two weeks by an infusion of one unit of 40-day stored autologous 
leukoreduced blood (with up to 
1% hemolysis).  After transfusion 
of the 40-day, but not the 3-day 
stored blood, plasma Hb and 
nitrite levels increased 
significantly (Fig. 1A, 1B).  The 

 
 
 
 
 
 
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results suggest that hemolysis, as demonstrated by increased plasma Hb, triggers a natural compensatory 
mechanism, possibly of healthy endothelium, increasing endogenous NO production by eNOS53.  
 
2) IRB #2011P000700: Hemolysis increases plasma consumption of NO and causes pulmonary 
vasoconstriction in volunteers with endothelial dysfunction.  Breathing NO inhibits further consumption 
of plasma NO and prevents vasoconstriction.  In a second study at the MGH blood bank, we enrolled 14 
overweight volunteers with endothelial dysfunction.  Each volunteer received in a random order 
autologous 3-day stored blood (not hemolyzed), 40-day stored autologous blood (containing up to 1% 
hemolyzed blood, 40-day storage), and 40-day stored autologous blood, transfused while the volunteer 
breathed 80 ppm NO.  Transfusion with 40-day stored blood caused an increase in plasma Hb (Fig. 2A), 
plasma NO consumption, and pulmonary artery pressure as estimated by transthoracic echocardiography 
(Fig. 2B).  Plasma NO consumption strongly correlated with levels of plasma Hb (Fig. 2C) and with 
increased pulmonary artery pressure (data not shown).  Breathing NO maintained plasma NO 
consumption at pre-transfusion levels despite the increasing plasma Hb levels and pulmonary arterial 
pressure (Fig. 2B).17 
 
Figure 2A, B, and C. (A) Plasma Hb increased after transfusion with 40 day-stored blood (40d) *p<0.01 
vs. baseline (=before transfusion); 40-day stored blood transfused while the recipient inhaled NO 
(40d+NO) #p<0.01 vs. baseline.  (B) Mean PAP increased during 40d challenge (p=0.009); and decreased 
during 40d+NO challenge (p=0.005).  (C) Plasma Hb concentration at baseline, 10 min and 4 hours after 
transfusion in volunteers receiving 3-day and 40-day stored blood correlates with NO consumption.  
 
3) IRB #2013P000911: Prolonged CPB for cardiac surgery increases hemolysis and plasma NO 
consumption.  In a pilot study, we 
enrolled 50 MGH cardiac surgery 
patients who were undergoing 
cardiac surgery with CPB.  We 
reported that plasma Hb (Fig. 3A) 
and NO consumption (Fig. 3B) 
increased in patients undergoing prolonged CPB (n=25 patients with over 140 minutes of CPB) and did 

 
 
 
 
 
 
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not increase in patients with shorter periods of CPB (n=25, less than 140 minutes of CPB ).  Duration of 
CPB was the primary determinant of hemolysis in cardiac surgery at MGH as opposed to receiving 
transfused blood from a cell saver device or from the blood bank.37 
 
4) IRB #2013P002596: Administration of NO prevents acute kidney injury during prolonged CPB (over 
90 minutes) in patients with no evidence of endothelial dysfunction: preliminary results of a double-blind 
randomized controlled trial.  In a single-center, randomized, double-blind controlled trial, we compared 
treatment with 80 ppm NO (NO group) versus control (Nitrogen, “N2” group).  In a collaborative study 
performed at Xijing Hospital in Xi’an, China, we enrolled 217 adults with normal pre-op kidney function 
without evidence of diseases associated with endothelial dysfunction.  The 217 patients underwent 
elective multiple valve replacement surgery with an estimated CPB time > 90 minutes.  Nitric oxide gas 
or N2 was administered via the oxygenator during CPB and by inhalation for 24 hours post-operatively.  
The aim of the study was to determine whether NO reduces AKI (primary outcome).  AKI was defined as 
either an increase in serum creatinine by 50% within 7 days after surgery or an increase in serum 
creatinine by 0.3 mg/dl within 48 hours after surgery.  No adverse events were associated with the 
administration of 80 ppm NO for 24 hours occurred.  Blood Met-Hb remained below 10%.  NO gas 
treatment decreased the incidence of AKI from 63% to 50% (p=0.04, primary endpoint achieved), as 
shown in Table 1.  These results are presently being written as a manuscript for submission. 
 
 
 
 
 
 
 
 
 
Plasma Hb concentration before and at the end of CPB increased to the same level in the two treatment 
groups (Fig. 4A), indicating that the extent of hemolysis was similar.  However, the level of consumption 
of NO by plasma, in patients with high plasma Hb (>100 μM) after CPB, was 10 times greater in the 
group that received N2 than in the group that received NO, as shown in (Fig. 4B).  Thus, NO 
administration markedly reduces NO consumption by Oxy-Hb. 

 
 
 
 
 
 
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We propose to replicate the trial design we conducted in China, to investigate whether the administration 
of NO prevents AKI also in an MGH population with endothelial dysfunction. 
 
c. Potential benefits to patients and/or society 
Benefits to patients: The proposed therapeutic trial builds on our recent randomized clinical trial in 217 
Chinese patients with valvular cardiac disease caused by rheumatic fever (IRB #2013P002596); these 
young patients (mean age 48 years old) did not have a history of diabetes, hypertension or obesity that are 
often associated with endothelial dysfunction.  This study demonstrates that administration of 80 parts per 
million (ppm) of NO for up to 24 hours during and after cardiac surgery with prolonged CPB reduced the 
incidence of AKI.  In other studies, in animals, we reported that endothelial dysfunction exacerbates the 
incidence of AKI and other organ injury after transfusion of hemolyzed blood61.  This proposed single-
center randomized trial at MGH will evaluate 250 patients with impaired endothelial function undergoing 
cardiac surgery with prolonged CPB.  During bypass, we will administer NO via the bypass machine 
oxygenator, and, once disconnected from CPB, we will add NO for up to 24 hours to the inspiratory 
circuit of the ventilator to determine whether this therapy decreases the incidence of AKI in these 
patients. 
Benefits to society: AKI and renal replacement therapy are some of the most expensive healthcare costs.  
One year of hemodialysis can cost up to $72,000, while a year of peritoneal dialysis costs approximately 
$53,000, according to the U.S. Renal Data System.  The cost of acute renal replacement therapy in the 
intensive care unit in the United States is about $3,629.80/day.  Nitric oxide supplementation during and 
after CPB heart surgery might reduce costs associated with dialysis and improve the overall wellness of 
post-cardiac surgical patients.  
From a scientific point of view, the results of our study will yield a new understanding of the mechanism 
by which exogenous NO maintains the homeostasis of vascular NO and will investigate whether 

 
 
 
 
 
 
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exogenous NO prevents AKI following hemolysis of prolonged CPB in patients with endothelial 
dysfunction.  In previous animal and human studies, we showed that acute and persistent depletion of 
vascular NO results from the combination of two overlapping conditions: acute depletion of vascular NO 
by hemolysis and pre-existing (chronic) endothelial dysfunction, which renders the endothelium unable to 
synthesize sufficient quantities of endogenous NO.  Our hypothesis with respect to the plasma 
bioavailability of NO is that a similar pathophysiological mechanism occurs in post-cardiac surgery 
patients who have pre-existing endothelial dysfunction.  Endogenous vascular NO is rapidly depleted 
from circulating plasma by the conversion of plasma Oxy-Hb (Hb-Fe2+) into Met-Hb (Hb-Fe3+), and the 
impaired endothelium is unable to supplement endogenous NO production.  Our study will test whether 
the administration of NO gas prevents the depletion of vascular NO by converting circulating Oxy-Hb to 
Met-Hb in patients with endothelial dysfunction. 
 
 

 
 
 
 
 
 
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II. SPECIFIC AIMS (Research Objectives) 
 
a. Specify objectives and hypotheses to be tested in the research project 
 
Hypotheses of the proposed trial: Administration of exogenous NO will (I) decrease the incidence and 
severity of AKI in patients with endothelial dysfunction undergoing prolonged CPB; (II) convert free 
plasma circulating Oxy-Hb to Met-Hb (but not intracellular oxy-Hb) thereby preventing depletion of 
endothelial NO by inhibiting the ability of plasma free hemoglobin to scavenge vascular NO.  
 
Study design: We will test these hypotheses in a randomized, double-blind clinical trial at the 
Massachusetts General Hospital of cardiac surgery patients with endothelial dysfunction who require 
prolonged CPB.  Two hundred and fifty patients will be randomized to receive either supplemental NO 
(NO group, n=125) or nitrogen (N2, Control group, n=125) during and after cardiac surgery for up to 24 
hours post-surgery.  NO will be administered via the oxygenator during CPB and by inhalation when 
mechanical ventilation is resumed.  
 
Aims of the study: The primary endpoint of this therapeutic trial is to determine whether there is a 
difference in AKI incidence between the control group (receiving N2) versus the study group (receiving 
NO).  AKI is defined by KDIGO criteria as an abrupt (within 48h) reduction in kidney function correlated 
to an absolute increase in serum creatinine of 0.3 mg/dL or more (≥26.4 μmol/L) or a percentage increase 
in serum creatinine of 50% or more (1.5-fold from baseline) at any time during the first 7 days after 
surgery or, finally, a reduction in urine output with a documented oliguria of < 0.5 ml/Kg/h for >6h2.  
Secondary renal endpoints include:  
1. Differences in AKI severity between the two groups using the following KDIGO stages2: 
 
Stage 1: Serum creatinine increase ≥26.5 μmol/l (≥0.3 mg/dl) or increase to 1.5–2.0-fold from 
baseline or urinary output <0.5 ml/kg/h for 6 h 
 
Stage 2: Serum creatinine increase >2.0–3.0-fold from baseline or urinary output <0.5 
ml/kg/h for 12 h 
 
Stage 3: Serum creatinine increase >3.0-fold from baseline or serum creatinine ≥354 μmol/l 
(≥4.0 mg/dl) with an acute increase of at least 44 μmol/l (0.5 mg/dl) or urinary output <0.3 
ml/kg/h for 24 h or anuria for 12 h or need for RRT 
2. Incidence and severity (KDOQI stages 3, 4, 5)3 of Chronic Kidney disease (CKD) at 1 year after 
surgery.  CKD is defined as a reduction in glomerular filtration rate (GFR) of less than 60 
mL/min/1.73 m2 for at least 3 months.  CKD GFR will be measured using the Chronic Kidney 
Disease Epidemiology Collaboration (CKD-EPI) equation69.  

 
 
 
 
 
 
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3. New requirement for RRT following AKI during hospitalization and at 6 weeks, 90 days, and 1 
year after surgery.  
4. Incidence of major Adverse kidney events4, a composite outcome of death, new RRT, and 
worsening renal function (defined as a 25% or greater decline in eGFR).  Since a decline of 25% 
in eGFR may not be clinically relevant, we will also evaluate a decline of 50% in eGFR to assess 
for worsened renal function as part of the composite outcome at 6 weeks, and 1 year after 
surgery. 
Secondary non-renal endpoints include: 
1. Incidence of other single organ dysfunction assessed using SOFA score during ICU stay1 
2. Incidence of prolonged cardiovascular support defined as the need for vasopressors and inotropic 
agents, a balloon pump, or a ventricular-assist device for more than 48 hours after cardiac 
surgery. 
3. Difference between groups of maximum hourly Vasoactive-inotropic score (VIS) 72,73 in the first 
7 days after surgery and duration of vasopressors and or inotropic agents support.  VIS is 
calculated as Dopamine dose (mcg/kg/min) + Dobutamine dose (mcg/kg/min) + 100 x 
Epinephrine dose (mcg/kg/min) + 10 x Milrinone dose (mcg/kg/min) + 10,000 x Vasopressin 
dose (units/kg/min) + 100 x Norepinephrine dose (mcg/kg/min) + 10 x Phenilephrine dose 
(mcg/kg/min). 
4. Difference in the duration of mechanical ventilation. 
5. Difference in ICU and hospital length of stay between groups 
 
Exploratory endpoints include: 
1. Incidence and severity of AKI related to the presence of CKD at baseline, duration of CPB, 
duration of aortic cross-clamp, levels of free Hb, levels of NO consumption, pulmonary pressure 
at baseline, cardiovascular risks associated with endothelial dysfunction, scheduled procedure and 
EuroSCORE II. 
 
Safety endpoints include:  
1. Difference of mortality at 6 weeks, 90 days and 1 year after surgery between the two groups and 
stratification according to the initial EuroSCORE II. 
2. Continuous Monitoring of MetHb with a non-invasive co-oximetry monitor in order to prevent 
methemoglobinemia (defined as MetHb > 5%)12.  
3. Continuous inhaled monitoring of Nitrogen Dioxide (NO2) (threshold level < 5 ppm) in order to 
prevent dangerously high levels of NO2. 

 
 
 
 
 
 
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4. Incidence of non-fatal stroke during hospitalization and at 6 weeks after surgery. 
5. Incidence of peri-operative and non-perioperative non-fatal myocardial infarction, as defined by 
the third universal definition of MI released in 2012 by the ESC/ACCF/AHA/WHF10. 
6. Incidence of post-operative bleeding calculated as the sum of blood loss through thoracic drains 
from the moment of closure of the chest over a period of 24 hours. 
7. Differences between the two groups of transfusions with plasma and stored or autologous red 
blood cells (RBCs) recovered using intraoperative cell salvage devices.  
8. Post-operative infections (e.g., pneumonia, wound infection, endocarditis, central line infection, 
urinary tract infection, sepsis).  
9. Cardiac arrhythmias and other non-cardiac post-operative complications (e.g., hepatobiliary 
disorders, pneumothorax, pleural effusion, vascular disorders).  
 
 
 

 
 
 
 
 
 
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III. 
SUBJECT SELECTION 
 
a. Inclusion and Exclusion criteria 
Inclusion criteria: 
1. Provide written informed consent 
2. Age ≥ 18 years of age 
3. Elective cardiac or aortic surgery with estimated CPB>90 minutes 
4. Clinical evidence of endothelial dysfunction assessed by a specifically 
designed questionnaire (Figure 3)  
5. Stable pre-operative renal function without evidence of a plasma creatinine 
increase of ≥ 0.3 mg/dL within 3 months of study entry and without 
receiving renal replacement therapy (RRT) 
 
Exclusion criteria: 
1. 
eGFR less than 30 ml/min/1.73 m2 
2. 
Emergent cardiac surgery 
3. 
Life expectancy < 1 year at the time of enrollment 
4. 
Mean pulmonary artery pressure ≥ 40 mm Hg and pulmonary vascular resistance > 4 Wood 
Units  
5. 
Left ventricular ejection fraction < 30% by echocardiography obtained within three months of 
enrollment 
6. 
Hemodynamic instability as defined by a systolic blood pressure <90 mmHg 
7. 
Administration of one or more Packed Red Blood Cell transfusion in the week prior to 
enrollment 
8. 
X-ray contrast infusion less than 48h before surgery 
9. 
Evidence of intravascular or extravascular hemolysis from any other origin: 
i. 
Intravascular: Intrinsic RBC defects leading to hemolytic anemia (eg, enzyme 
deficiencies, hemoglobinopathies, membrane defects).  Extrinsic: liver disease, 
hypersplenism, infections (eg, bartonella, babesia, malaria), treatment with oxidizing 
exogenous agents (eg, dapsone, nitrites, aniline dyes), exposure to other hemolytic 
agents (eg, lead, snake and spider bites), lymphocyte leukemia, autoimmune 
hemolytic disorders 

 
 
 
 
 
 
17
ii. 
Extravascular: Infection (e.g., clostridial sepsis, severe malaria), paroxysmal cold 
hemoglobinuria, cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, iv 
infusion of Rho (D) immune globulin, iv infusion of hypotonic solutions 
 
b. Source of subjects and recruitment methods 
The study screening and recruitment will occur at the Massachusetts General Hospital (MGH) Heart 
Center.  Each year at MGH, approximately 700 adult patients undergo cardiac surgery, with CPB lasting 
longer than 90 minutes.  Endothelial function before cardiac surgery is not routinely assessed at our 
institution.  However, we expect that the great majority of cardiac surgery patients are affected by pre-
operative endothelial dysfunction because they have one or more risk factors, including hyperlipidemia, 
hypertension, atherosclerosis, diabetes, obesity and peripheral vascular diseases.  Adult patients will be 
consented at either the outpatient pre-operative visit or in the in-patient cardiac services.  Pre-operative 
systemic endothelial function and baseline kidney function will be evaluated before surgery.  Only 
patients with stable kidney function and evidence of endothelial dysfunction will be enrolled.  Reasons for 
excluding patients from the study will be noted.  Stratification based on kidney function, evaluated by 
measurement of estimated glomerular filtration rate (eGFR)65 calculated from the serum creatinine at the 
time of enrollment, will be performed as a post-hoc analysis at the end of the study. 
  
 
 

 
 
 
 
 
 
18
IV. 
SUBJECT ENROLLMENT 
 
a. Methods of enrollment, including procedures for patient registration 
If the patient is eligible, a clinician/member of the treatment team will initially introduce the study to the 
patient and verbally obtain the patient’s permission to be contacted by licensed physicians part of the 
study staff.  We will recruit patients from two pools of cardiac patients for the enrollment and consent 
process: outpatient or admitted patients (“inpatient”).  Cardiac surgeons have been presented with the 
objectives of the study and all have opted to be a part of this study.  At MGH, cardiac surgeons require at 
least two visits prior to surgery, one for the initial consult and the other for a pre-admission testing area 
(PATA) visit closer to the anticipated surgery date.  Only licensed physicians part of the study staff will 
approach the subject for consent during their PATA visits to participate in the trial.  As for the inpatient 
pool, we shall screen the EPIC patient database system daily for cardiac surgical patients admitted to 
MGH awaiting non-emergent cardiac surgery.  Should these patients meet the inclusion criteria, we will 
approach the patient at least a day prior to their cardiac procedure for consent.  Consent will be obtained 
exclusively from the patient and not from a surrogate because cardiac surgery is usually a scheduled 
procedure.  We believe it is best to obtain consent directly from the patient when he/she is fully awake.  
After consent is obtained, the patient will be randomized to the study group.  A de-identified code will be 
assigned to the patient and registered on a dedicated enrollment log.  After consent is obtained, the patient 
will be randomized to the study group.  A de-identified code will be assigned to the patient and registered 
on a dedicated enrollment log.  
 
b. Procedures for obtaining informed consent (including timing of consent process) 
Patients will be screened in the “Cardiac surgery pre-operative clinic”, MGH Cox Building floor 6 (before 
surgery) or in the inpatient cardiac service wards at MGH.  If the patient is eligible, a clinician/member of 
the treatment team will initially introduce the study to the patient and verbally obtain the patient’s 
permission to be contacted by licensed physicians part of the study staff.  Only licensed physicians part of 
the study staff will approach the subject for consent to participate in the trial.  Consent will be obtained 
exclusively from the patient, and not from a surrogate, because cardiac surgery is usually a scheduled 
procedure.  We believe it is best to obtain consent directly from the patient when he/she is fully awake.  
After consent is obtained, the patient will be randomized to the study group.  A de-identified code will be 
assigned to the patient and registered on a dedicated enrollment log.  The signature of the consent form by 
the patient will be required prior to the initiation of any study procedures. 

 
 
 
 
 
 
19
Personal Medical Information (PMI) will be accessed by the investigator(s) only for study purposes.  
Patients who choose not to participate in this study will receive standard care according to the procedures 
of the ICU, without any repercussions. 
 
c. Treatment assignment and randomization (if applicable) 
To ensure a robust and unbiased approach, randomization should account for demographic characteristics 
of the patients (i.e.: age, sex) and baseline estimated glomerular filtration rate (eGFR)69.  Only patients 
who are cared for by experienced anesthesiologists and cardiac surgeons (more than 5 years of staff 
experience) will participate in this study.  The patients will be randomized to receive either NO (study 
drug) or N2 (placebo) alone.  In order to account for an elevated baseline pulmonary artery hypertension 
(PAH), patients with pre-operative PAH will be allocated equally to the two groups during the stratified 
randomization process.  Given that most cardiac surgical patients do not get pre-operative right heart 
catheterization, and echocardiography is a poor tool for PAH, an experienced cardiac anesthesiologist will 
obtain PAH diagnostic parameters from a pulmonary artery catheter placed after anesthesia induction on 
the day of surgery.  We selected a threshold of mean pulmonary arterial pressure ≥30 mm Hg to allocate 
patients to a PAH group versus low or non-PAH group.  The random allocation sequence will be created 
using a computerized random generation program (RedCap).  The randomization will be in blocks of 10 
patients. 
 Patients will be randomly allocated to one of the test gas study groups (80 ppm NO in N2) or the 
placebo group (N2).  The intervention will consist of administering the test gas via the CPB machine (into 
the sweep gas of the CPB oxygenator) and, after CPB via the inspiratory limb of the anesthetic or 
ventilator circuit and thereafter via the mechanical ventilator in the Intensive Care Unit (ICU).  Test gas 
administration will commence at the onset of CPB and last for 24 hours.  When patients are extubated 
they will breathe test gas via a facemask or nasal prongs.  At the end of 24 hours, inhaled NO (iNO) will 
be weaned and discontinued while carefully monitoring hemodynamics for a period of 2-4 hours.  Local 
guidelines for NO gas discontinuation will be followed.  Using Inovent (Mallinckrodt, Ikaria Inc, N.J., 
USA) or commercially available tanks of Nitric Oxide (Airgas Inc, Radnor Township, Pennsylvania) or 
volumetrically-calibrated flowmeters, pure N2 (placebo) or 800 ppm NO gas in N2 will be mixed with 
pure O2 or air to obtain the desired concentration of O2 and, in the NO treatment group, a final 
concentration of 80 ppm NO.  Nitric oxide, NO2, and O2 levels will be continuously monitored.  Patients 
in the placebo (N2) group will receive nitrogen test gas during the same 24-hour period.  The inspired 
oxygen levels will be maintained at the usual levels required for routine post-operative care.  No changes 
to the usual and customary standards of care for any intraoperative or post-operative treatment will be 
made during the study period. 

 
 
 
 
 
 
20
 
d. Blinding procedure for Nitric Oxide and Nitrogen delivery 
A double-blind study will be performed to avoid potential patient and investigator bias.  The participants 
and those analyzing data and assessing the outcomes will be blinded to group assignment.  The test gas 
tank and the gas delivery system in the operating room (OR) and at the bedside will be masked to keep 
the investigators and clinicians treating the patient blinded.  The respiratory therapist in the ICU, the PI 
and a study staff will be unblinded and will prepare the appropriate test gas tanks and NO/N2 meters.  
Blood Met-Hb levels and NO/NO2 concentrations are safety concerns that the respiratory therapist and 
study staff will monitor and regulate (NO2 levels will be maintained below 5 ppm, and Met-Hb below 
5%, if necessary by reducing NO concentration to 40 ppm or less according to MGH guidelines NO 
therapy).  

 
 
 
 
 
 
21
V. 
STUDY PROCEDURES 
 
a. Study visits and parameters to be measured  (e.g., laboratory tests, x-rays, and other 
testing) 
Screening visit: Screening will be in the “Cardiac surgery pre-operative clinic”, MGH Cox building floor 
6 (before surgery) or in the inpatient cardiac service wards.  Subjects will be screened if they require 
prolonged CPB (>90 minutes on CPB, i.e., valve replacement ± CABG) and if the primary cardiac 
surgeon of the patient agrees to enroll the patient in the study. 
Screening consists of:  
 
Review the operating room list of subjects booked for cardiac surgical procedures requiring the 
use of cardiopulmonary bypass with an estimated time > 90 minutes. 
 
Review in the Electronic Medical Chart the other inclusion/exclusion criteria including serum 
creatinine levels obtained as part of standard laboratory pre-surgical tests. 
 
Administration of a brief questionnaire to determine endothelial function with the subject verbal 
assent on the day of pre-surgical evaluation.  The questionnaire evaluates medical history, 
integrating clinical and laboratory data of screened patients (Figure 5) and it is used as a tool to 
detect patients with endothelial dysfunction. 
 
Review the research protocol: this is a protected time for the subject to ask questions and become 
familiar with all aspects of the study protocol. 
 
Consent form: The subject will be asked to sign the consent form.  The consent form will be 
obtained prior to the initiation of any study procedures.  Only patients who are eligible, as 
determined after screening and assessment of endothelial function, will be asked to sign the 
consent form.  
Should a patient be deemed ineligible and thus excluded from the study for whatever reason, including 
declining to participate, their reasons for exclusion will be noted.  Reasons for exclusion could include but 
are not limited to the inclusion or exclusion criteria. 
 
Pre-operative anesthesia management will be performed as per standardized protocols and includes the 
implementation of the KDIGO guidelines to reduce the risk of AKI, such as avoidance of nephrotoxic 
agents, discontinuation of ACEi and ARBs for the first 48 h after surgery, close monitoring of serum 
creatinine and urine output, avoidance of hyperglycemia for the first 72 h after surgery, avoidance of 
radiocontrast agents, close hemodynamic monitoring by using pulmonary artery catheter with an 
optimization of the volume status and hemodynamic parameters. 

 
 
 
 
 
 
22
 
 
     Figure 5.  Screening questionnaire to assess endothelial dysfunction. 
 
Randomization.  Patients will be randomly allocated to one of the test gas study groups (inhaled 80 parts 
per million (ppm) nitric oxide in nitrogen) or the placebo group (inhaled N2), as described previously in 
VI.c. “Treatment assignment and randomization”. 
 
Data collection.  During the hospital stay, we will prospectively collect from the patient’s chart: hospital 
stay, ICU stay, duration of ventilation, amount of blood loss through chest tubes (ml), cardiac and non-
cardiac post-operative complications, transfusions with plasma (units) and stored (units) or autologous 
(ml) red blood cells (RBCs), hospital course and mortality at 6 weeks, 90 days and 1 year after surgery 
stratified using EuroSCORE II. 
We will also assess organ injury from the patient’s chart and laboratory data, we will calculate a SOFA 
score1 and record it: 
Non-fatal stroke: assessment for non-fatal stroke by the NIH Stroke Scale at baseline before surgery and 
at 6 weeks after surgery. 
Peri-operative and non peri-operative non-fatal myocardial infarction: as defined by the third universal 
definition of MI released in 2012 by the ESC/ACCF/AHA/WHF (Definitions and Study Outcomes)10.  
 
CABG and/or valve repair/replacement and age > 40 year old for males and > 50 year old for 
females and 1 out of 8 of the following criteria: 
 
 
 
Previous coronary artery bypass graft or PTCA (+ stent) 
  Yes___ No___ 
History or presence of intermittent claudication, critical limb ischemia, or peripheral vascular disease 
with the Exception of vasculitis.  
  Yes___ No___ 
History of transient ischemic attack and/or ischemic stroke 
Yes___ No___ 
Diagnosis of diabetes (IDDM or NIDDM) requiring oral hypoglycemic agents or insulin  
Yes___ No___ 
Hypercholesterolemia (total cholesterol > 200 mg/dl or LDL > 160 mg/dl) treated with statins, ion-
exchange resins or other oral agents  
Yes___ No___ 
BMI > 40 
Yes___ No___ 
Hypertension (SBP 140 ≥ mmHg) treated with antihypertensive drugs  
Yes___ No___ 
Active smoking ≥ 10 pack - years 
Yes___ No___ 

 
 
 
 
 
 
23
We will also collect the total time (hours:minutes) of intubation of each patient, post-operative infections, 
cardiac arrhythmias and non-cardiac complications, prolonged cardiovascular support as defined as the 
need for vasopressors, inotropic agents, balloon pump, or ventricular-assist device for more than 48 hours 
after cardiac surgery, maximum daily vasoactive-inotropic score (VIS) 72,73 and duration of vasopressors 
and or inotropic agents support.  VIS is calculated as Dopamine dose (mcg/kg/min) + Dobutamine dose 
(mcg/kg/min) + 100 x Epinephrine dose (mcg/kg/min) + 10 x Milrinone dose (mcg/kg/min) + 10,000 x 
Vasopressin dose (units/kg/min) + 100 x Norepinephrine dose (mcg/kg/min) + 10 x Phenilephrine dose 
(mcg/kg/min). 
Renal failure will be defined according to the KDIGO criteria2 and Renal Replacement therapy will be 
initiated according to the subject’s treating physician’s decision and consulting nephrologist’s indication 
at Massachusetts General Hospital. 
 
Blood samples collection.  
Table.  Summary of all Measurements and Blood Drawn for the Entire Study. 
 
1 
2 
3 
4 
5 
6 
7 
8 
9 
Baseline 
After 
CPB 
POD: 
day1 
POD: 
day2 
POD: 
day3 
POD: 
day4 
POD: 
day5 
POD: 
day6 
POD: 6 
weeks 
Plasma Hb, 
mg/dL 
✔ 
✔ 
✔ 
✔ 
 
 
 
 
 
NO consumption 
NO metabolites 
✔ 
✔ 
✔ 
✔ 
 
 
 
 
 
Plasma creatinine 
 
✔ 
✔ 
✔ 
✔ 
✔ 
✔ 
✔ 
✔ 
Total Blood 
Drawn 
6 ml 
9 ml 
9 ml 
9 ml 
3 ml 
3 ml 
3 ml 
3 ml 
3 ml 
 
POD: post-operative day.  The total amount of blood that will be collected for the purpose of this trial is: 
6 ml + 9 ml + 9 ml + 9 ml + 3 ml + 3 ml + 3 ml + 3 ml + 3 ml = 48 ml of blood.  
 
 

 
 
 
 
 
 
24
Plasma samples will be collected on the day of surgery before starting the procedure, at the end of surgery 
and once a day until day 7 after the cardiac procedure or until discharge if before day 7 (for a maximum 
of 41 mL of blood over a 7-day time period).  An additional 4 ml of blood at 6 weeks after surgery will be 
collected, for a total of 48 mL of blood drawn for this study.  In the first two days after surgery, we will 
measure plasma creatinine, plasma-free Hb concentration, and plasma NO consumption.  From day 3 to 
day 7, we will measure daily plasma creatinine.  At 6 weeks after surgery, we will measure creatinine.  
Urinary output will be recorded daily from the day of surgery to day 7.  
 
b. Drugs to be used (dose, method, schedule of administration, dose modifications, 
toxicities), include Toxicity Grading Scale (if applicable)  
The intervention will consist of giving the test gas via both the CPB machine, after CPB via the anesthetic 
circuit, and thereafter via the mechanical ventilator.  Test gas administration will commence at the onset 
of CPB and last for 24 hours.  When patients are extubated, they will breathe test gas via a facemask or 
nasal prongs.  At the end of 24 hours, NO will be weaned and discontinued while carefully monitoring 
hemodynamics for a period of 2-4 hours.  Standard MGH guidelines for NO discontinuation will be 
adopted.  Using an Inovent (Mallinckrodt, Ikaria Inc, N.J., USA) or commercially available tanks of 
Nitric Oxide (Airgas Inc, Radnor Township, Pennsylvania) or volumetrically-calibrated flowmeters, pure 
nitrogen (placebo) or 800 ppm NO gas in N2 is mixed with pure O2 or air to obtain a final concentration of 
80 ppm NO.  During CPB, the test gas is delivered through the extracorporeal oxygenator, after CPB the 
NO is delivered through the inspiratory limb of the anesthetic or ventilator circuit.  NO, NO2, and O2 
levels are monitored by ozone-chemiluminescence technology (Sievers Nitric Oxide Analyzer, NOA 
280i, GE).  Subjects in the placebo group will receive nitrogen test gas during the same 24-hour period.  
When patients are extubated, they will breathe test gas via a face mask or nasal prongs.  The inspired 
oxygen levels will be maintained at the usual levels required for routine post-operative care.  The test gas 
tank in the OR and at the bedside will be covered and blinded from the investigators and clinicians 
treating the patient.  Only the respiratory therapist, the PI, a study staff, and the person changing the tanks 
and monitoring the respiratory levels of NO/NO2 and methemoglobin will know the treatment group of 
the patient.  No changes to the usual and customary standards of care for any intraoperative or post-
operative treatments will be made during the study period.  Toxicity and safety profile of Nitric Oxide 
administration is detailed in chapter VII.b. “Drug site effects and toxicity”. 
 
 

 
 
 
 
 
 
25
VI. 
BIOSTATISTICAL ANALYSIS  
 
a. Specific data variables being collected for the study  
Pre-operative collection of patient data from the MGH electronic medical chart (EPIC) will include:  
 
Patient demographics  
 
Past and current medical and surgery history and therapy 
 
Co-morbidities  
 
Pertinent cardiopulmonary test results: i.e., echocardiography, cardiac catheterization, pulmonary 
function tests, and chest imaging.  
 
Prospective collection of patient data will include:  
 
EuroSCORE II  
 
ICU and length of hospital stay 
 
Duration of mechanical ventilation 
 
Blood loss through chest tubes (ml) 
 
Units of plasma and stored RBCs transfused and volume (ml) of autologous RBCs transfused.  
 
Hospital course 
 
Mortality 
 
Organ injury (SOFA score)1 
 
Non-fatal stroke, as defined by the NIH Stroke Scale at baseline before surgery and at 6 weeks 
after surgery 
 
Delirium (CAM-ICU score)7 
 
Peri-operative and non-perioperative non-fatal myocardial infarction (MI) at 28, 90 days and 1 
year after surgery.  MI is defined by the third universal definition of MI released in 2012 by the 
ESC/ACCF/AHA/WHF10 
 
Prolonged cardiovascular support defined as the need for vasopressors and inotropic agents, a 
balloon pump, or a ventricular-assist device for more than 48 hours. 
 
Maximum hourly Vasoactive-inotropic score (VIS) 72,73 and duration of vasopressors and or 
inotropic agents support.  VIS is calculated as Dopamine dose (mcg/kg/min) + Dobutamine dose 
(mcg/kg/min) + 100 x Epinephrine dose (mcg/kg/min) + 10 x Milrinone dose (mcg/kg/min) + 
10,000 x Vasopressin dose (units/kg/min) + 100 x Norepinephrine dose (mcg/kg/min) + 10 x 
Phenilephrine dose (mcg/kg/min). 
 
Post-operative infections, cardiac arrhythmias and non-cardiac complications.  

 
 
 
 
 
 
26
 
Renal failure requiring dialysis at any time up to 1 year after surgery  
 
Acute renal injury, defined according to the KDIGO criteria2. 
 
 
b. Study endpoints 
The primary endpoint of this therapeutic trial is to determine whether there is a difference in AKI 
incidence between the control group (receiving N2) versus the study group (receiving NO).  AKI is 
defined by AKIN criteria as an abrupt (within 48h) reduction in kidney function correlated to an absolute 
increase in serum creatinine of 0.3 mg/dL or more (≥26.4 μmol/L) or a percentage increase in serum 
creatinine of 50% or more (1.5-fold from baseline) at any time during the first 7 days after surgery or, 
finally, a reduction in urine output with a documented oliguria of < 0.5 ml/Kg/h for >6h2.  
Secondary renal endpoints include differences in the treatment groups of AKI severity as reflected by 
the changes of serum creatinine and urinary output (KDIGO stages)2; incidence and severity (stage 3, 4, 
5) of Chronic Kidney disease (CKD) at 1 year after surgery3; new requirement for RRT following AKI 
during hospitalization and at 6 weeks, 90 days and one year after surgery; incidence of Major adverse 
Kidney events (MAKE) at 6 weeks, and 1 year after surgery4; Secondary non-renal endpoints include 
incidence and severity of other single organ dysfunction (SOFA)1, prolonged cardiovascular support, 
vasoactive-inotropic score (VIS) 72,73, duration of mechanical ventilation; evaluation of ICU and hospital 
length of stay. 
Exploratory endpoints include incidence and severity of AKI related to the presence of CKD at baseline, 
duration of CPB, duration of aortic cross-clamp, levels of free Hb, levels of NO consumption, pulmonary 
pressure at baseline, cardiovascular risks associated with endothelial dysfunction, scheduled procedure 
and EuroSCORE II at 28 days 6 weeks, 90 days and 1 year after surgery9.  
Finally, safety endopoints include: mortality at 6 weeks, 90 days and 1 year after surgery stratified 
according to the initial EuroSCORE II; incidence of non fatal stroke; incidence of peri-operative and non-
perioperative non-fatal myocardial infarction10; incidence of post-operative bleeding and differences 
between the two groups of transfusions with plasma and stored or autologous red blood cells (RBCs) 
recovered using intraoperative cell salvage devices; Post-operative infections (e.g., pneumonia, wound 
infection, endocarditis, central line infection, urinary tract infection, sepsis); Cardiac arrhythmias and 
other non-cardiac post-operative complications (e.g., hepatobiliary disorders, pneumothorax, pleural 
effusion, vascular disorders); continuous monitoring of MetHb with a non invasive co-oximetry12 monitor 
in order to prevent methemoglobinemia (defined as MetHb > 5%) and continuous monitoring of Nitrogen 
Dioxide (NO2) in inhaled breath (threshold level < 5 ppm) in order to prevent reaching dangerously high 
levels (goal <5 ppm).  

 
 
 
 
 
 
27
 
c. Statistical methods 
The primary aim of this study is to determine whether supplementation with NO decreases the incidence 
of AKI in patients with endothelial dysfunction.  We expect to enroll a total of 250 cardiac-surgery 
patients requiring more than 90 minutes of CPB.  Patients will be randomized as previously described in 
the “subject enrollment” section.  The two arms will be compared for homogeneity of baseline 
characteristics (age, weight, sex, and eGFR)69, intraoperative course (cardiac-surgeon/anesthesiologist, 
CPB time, amount of blood transfusions) and post-operative data (post-operative transfusions, adverse 
events directly related to surgery procedures).  To examine differences between the two groups, we will 
use an unpaired Student’s t-test for continuous variables with a normal distribution, a Mann-Whitney U 
test for continuous variables that are not normally distributed and a Fisher’s exact test for dichotomous 
variables.  Continuous variables will be described as mean±SD when normally distributed, median (IQR) 
when not normally distributed, or count (%) if a dichotomous variable.  The change in the laboratory 
markers over time and between the two groups will be tested with a repeated-measures ANOVA.  The 
incidence of AKI and post-operative adverse events after prolonged CPB will be described in terms of 
relative risk with significance level and confidence intervals for the two groups. 
 
d. Power analysis 
The sample size of this trial was calculated based on the primary endpoint: the reduction of AKI 
incidence.  Despite not having tracked the incidence, AKI is very common in our (MGH) patients after 
open-heart surgery, and it is reasonable to assume that AKI incidence is similar to those recent reports 
from US and European studies at major academic centers CPB (AKI incidence has been reported to be 
between 50 to 60%).  In our prior trial on NO in cardiac surgery, the sample size was calculated to find a 
30% reduction in the incidence of AKI (n=212 in that trial).  We estimate a greater (35%) reduction in the 
incidence of AKI because we anticipate that this population with endothelial dysfunction will be more 
affected by the beneficial therapeutic properties of NO.  Thus, in the NO group, the incidence of AKI is 
expected to decrease from 55% to 35.75%.  The sample size needed to detect a difference, assuming an 
alpha error of 0.05 (two-sided test) and a study power of 0.8, is 114 patients per group.  In order to 
account for possible dropouts, we increased our sample size by 10%.  We will enroll 250 patients, 125 in 
the NO group and 125 patients in the control (N2) group.  
 
 
VII. 
RISKS AND DISCOMFORTS  
 

 
 
 
 
 
 
28
a. Procedure-related risks for this study: vein punctures and NO treatment 
IV punctures.  Risks related to IV punctures include hematoma formation and phlebitis.  To minimize 
risks and discomfort, we will obtain blood from arterial lines, peripheral intravenous catheters, or central 
venous catheters when present.  
 
 
b. Drug side effects and toxicities:  
Nitric Oxide (NO).  
Risks of breathing NO at 80ppm for 24 hours are low.  The theoretical risks of NO breathing include the 
following: pulmonary edema (especially in CREST), methemoglobinemia, hypoxia, and hypotension.  In 
the study in neonates breathing NO for up to 4 days, study subjects had hematuria (6%), hyperglycemia 
(7%), sepsis (2%), infection (3%), stridor (3%) and hypotension (10%).  They all occurred in the placebo 
group as well and none of these findings was significantly higher in the NO group.  In a small study of 
healthy volunteers, Frostell et al reported that there were no adverse clinical events in the group inhaling 
NO.  Of note, the average baseline methemoglobin level prior to NO administration was 0.61% and this 
increased to 0.77% after 10 minutes of inhalation of NO at 80ppm.  This was a statistically significant, 
although clinically irrelevant, increase in methemoglobin levels64.  In our recent study (IRB 
#2013P002596), there were no side effects or drug toxicity associated with the use of NO at 80 ppm for 
up to 24 hours.  
 
The inhalation of NO will be administered and monitored by a trained RRT.  There will be dedicated 
therapists who also administer NO as part of their clinical responsibilities.  They will be familiarized with 
the technical aspects of this protocol and supervised by one of the investigators (Dr. Lorenzo Berra), who 
has significant experience with NO administration both clinically and as part of investigational protocols.  
In addition, NO breathing is a treatment that is very common in C-ICU at Blake 8.  All personnel (MDs, 
RTs, and RNs) are familiar with this medication.  On average 5 to 10 patients are treated each month with 
NO breathing or similar pulmonary vasodilator in C-ICU at MGH.  All study subjects receiving study gas 
(NO or N2) will have continuous pulse oximetry monitoring, an RRT, and a licensed physician will be 
available for their care.  The system for the inhalation of NO continuously monitors the NO, nitrogen 
dioxide (NO2) and oxygen concentration of the inhaled gas and is stopped immediately if the 
concentration of NO increases beyond 80 ppm (clinically accepted upper limit).  The respiratory therapist 
will continuously monitor the oxygen saturation via non-invasive oximetry during the procedure.  
Methemoglobin levels will be assessed using a non-invasive continuous co-oximetry monitor.  If the 

 
 
 
 
 
 
29
methemoglobin level rises more than 5%, NO will be halved to 40 ppm and closely monitored until a 
reduction occurs.  If methemoglobin persists at > 5%, NO will be halved until methemoglobin is < 5%. 
Any study subject who experiences a side effect that is felt to be related to the study drug will not be 
allowed to continue in the protocol. 
 
c. Psychosocial risks 
We do not anticipate psychosocial risks to the study subjects from participation in this protocol.  Each 
patient will personally sign the consent form before surgery, we will not ask for a surrogate’s permission 
to enter a patient into the study.  The consent form will be obtained prior to the initiation of any study 
procedures, including those done for screening. 
Strict confidentiality will be maintained by the research team at all times, including keeping all data in a 
secure, locked cabinet with limited access.  All specimens will be coded after they are obtained and the 
code key will be kept in a locked cabinet.  
 
d. Radiation risks 
Not applicable. 
 
 

 
 
 
 
 
 
30
VIII. Potential benefits to participating individuals and to society 
 
Potential benefits to participating individuals and to society have been described in chapter I.c. 
In summary:  
a. Benefits to participating individuals: subjects breathing nitric oxide might benefit from a decreased 
occurrence of AKI after surgery and a faster recovery of the cardio-pulmonary function after CPB.  
b. Benefits to society: There is no drug available that has been shown to decrease AKI incidence after 
prolonged CPB.  If the administration of NO in this trial showed clinical benefits in patients with 
endothelial dysfunction, other drugs/treatments could be studied to target the NO pathway for the 
prevention of AKI and other organ injury after CPB.  
 
 
 

 
 
 
 
 
 
31
IX. 
MONITORING AND QUALITY ASSURANCE 
 
a. Independent monitoring of source data 
Informed consent forms, case report forms, and data will be reviewed by the principal investigator 
following enrollment of every 5 subjects.  
 
b. Safety monitoring (e.g., Data Safety Monitoring Board, etc.) 
An interim analysis by a Data Safety Management Board (DSMB) comprised of 3 members is planned at 
reaching 50% of the study population (125 patients enrolled) to ensure the safety of the study subjects.  
The safety data that will be reviewed includes: patient-, nurse-, research-assistant, or investigator-reported 
adverse events such as reason for early termination of the protocol or adverse reaction to NO.  Other data 
to be reviewed include appropriate handling and processing of blood samples and maintenance of patient 
confidentiality throughout the study.  The study will only be stopped if the interim analysis detects a 
significant increase in mortality, AKI, need for RRT, MI, post-operative hemorrhage, other significant 
morbidity in the NO test gas group. 
Members of the Data Safety Management Board (DSMB) consist of an anesthesiologist with clinical 
expertise in gas therapeutics, a cardiologist and a nephrologist.  Names and contacts of the DSMB 
members are:  
 
o 
Michael Fitzsimons, MD (Site DSMB Member).  Director Cardiac Anesthesia, 
Anesthesia Department, Massachusetts General Hospital, Boston, MA 
 
o 
Francesco Nordio, PhD (DSMB Independent Statistician).  Lead Biostatistician, TIMI 
Study Group, Brigham Women’s Hospital, Boston, MA 
 
o 
John Prowle, MA MB BChir MSc MD FRCP FFICM (DSMB Chair).  Clinical reader in 
Critical Care Nephrology in the Critical Care and Peri-operative Medicine Research Group at 
William Harvey Research Institute, Queen Mary University of London, London, UK.  
 
 
c. Outcomes monitoring 
Blood and urine samples will be collected until day 7 after CPB or until discharge from the hospital if this 
occurs before day 7 after CPB.  Follow-up will continue at 30 days, 90 days, and 1 year after surgery as 
described in chapter II.  “Specific aims” and V.  “Study procedure”. 

 
 
 
 
 
 
32
 
d. Adverse event reporting guidelines 
In accordance with PHRC policy on Adverse Event Reporting and Unanticipated Problems Involving 
Risks to Subjects, the Principal Investigator (Lorenzo Berra, MD) will report adverse events or other 
unanticipated problems to the DSMB and to the PHRC within 5 working days/7 calendar days of the date 
the investigator first becomes aware of the problems.  Mild or moderate adverse events will be presented 
in progress reports at continuing reviews.  
 
 
Stopping rules.  The review and decision regarding altering or stopping the protocol will be performed 
by the principal investigator together with the DSMB.  Mild or moderate adverse events will be presented 
in progress reports at continuing reviews.  Protocol exit criteria will be: 
a. Acute worsening hypotension: decrease in mean blood pressure of > 20 mmHg not attributable to 
other causes, such as: hypovolemia, hemorrhage, sepsis, and acute heart failure. 
b. Sudden worsening of hypoxemia: decrease of SatO2 < 80% at 100 FiO2 not attributable to other 
causes, such as: pulmonary edema, ARDS, pulmonary embolism. 
c. An increase in NO2 levels > 5 ppm from baseline. 
Or any life-threatening symptom potentially attributed to NO administration by the physician investigator. 
 
 
 

 
 
 
 
 
 
33
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38
Supplementary Methods 
 
Nitric Oxide Delivery 
 
Commercially available tanks of medical-grade NO and N2 gas that were indistinguishable in 
appearance from each other were used in the trial.  These gases were administered from the start 
of CPB and continued for a period of 24 hours.  During CPB using the Stockert S5 heart-lung 
machine (LivaNova, Mirandola, Italy), the gases were administered through the sweep gas of the 
CPB oxygenator.  Volumetrically calibrated flowmeters were used to administer pure N2 in the 
control arm and a mixture of NO in N2 in the NO arm.  O2 was then added to reach the desired O2 
concentration.  Once CPB was completed and ventilation was initiated, the same gasses were 
administered through the inspiratory limb of the ventilator.  A medical gas blender was used to 
regulate the NO concentration.  After extubation, the test gas was delivered by a high-flow nasal 
cannula.  During the delivery of NO, the concentrations of NO and NO2 were assessed using an 
inline NO/NO2 sensor (Alphasense, Great Notley, Essex, UK).  

 
 
 
 
 
 
39
Supplementary Table 1:  Surgical Features of the Study Population 
 
Total 
(N=250) 
NO 
(N=125) 
Control 
(N=125) 
SMD 
Type of Surgery 
Valve Surgery Only, n (%) 
112 (44.8%) 
55 (44.0%) 
57 (45.6%) 
0.03 
CABG Only, n (%) 
49 (19.6%) 
25 (20.0%) 
24 (19.2%) 
0.02 
Combined CABG and Valve Surgery, n (%) 
61 (24.4%) 
29 (23.2%) 
32 (25.6%) 
0.06 
Complicates AVR, n (%) 
6 (2.4%) 
3 (2.4%) 
3 (2.4%) 
<0.001 
Bentall Procedure, n (%) 
17 (6.8%) 
12 (9.6%) 
5 (4.0%) 
0.22 
Other, n (%) 
14 (5.6%) 
7 (5.6%) 
7 (5.6%) 
<0.001 
MAZE, n (%) 
40 (16.0%) 
23 (18.4%) 
17 (13.6%) 
0.13 
Septal Defect Repair, n (%) 
8 (3.2%) 
6 (4.8%) 
2 (1.6%) 
0.18 
Ascending Aorta Aneurysm Repair, n (%) 
29 (11.6%) 
16 (12.8%) 
13 (10.4%) 
0.08 
LAAA, n (%) 
45 (18.0%) 
27 (21.6%) 
18 (14.4%) 
0.19 
CABG: Number of Distal Anastomoses 
3 (2-4) 
3 (2-4) 
3 (2-4) 
0.128 
CBP Time, min 
152 (129-187) 
151 (127-187) 
153 (129-185) 
0.014 
Aortic Clamp Time, min 
111 (86-142.5) 
109 (85-143) 
113 (90-142) 
0.023 
Valve Replacement 
Mitral, n (%) 
67 (26.8%) 
32 (25.6%) 
35 (28.0%) 
0.05 
Tricuspid, n (%) 
5 (2.0%) 
1 (0.8%) 
4 (3.2%) 
0.17 
Aortic, n (%) 
88 (35.2%) 
45 (36.0%) 
43 (34.4%) 
0.03 
Pulmonary, n (%) 
2 (0.8%) 
1 (0.8%) 
1 (0.8%) 
<0.001 
Valve Repair 
Mitral, n (%) 
76 (30.4%) 
39 (31.2%) 
37 (29.6%) 
0.04 
Tricuspid, n (%) 
19 (7.6%) 
9 (7.2%) 
10 (8.0%) 
0.03 
Aortic, n (%) 
33 (13.2%) 
19 (15.2%) 
14 (11.2%) 
0.12 
Valvular Defect 
Aortic Valve Regurgitation, n (%) 
44 (17.6%) 
20 (16.0%) 
24 (19.2%) 
0.08 
Aortic Valve Stenosis, n (%) 
58 (23.2%) 
28 (22.4%) 
30 (24.0%) 
0.04 
Mitral Valve Regurgitation, n (%) 
97 (77.6%) 
48 (38.4%) 
49 (39.2%) 
0.02 

 
 
 
 
 
 
40
Mitral Valve Stenosis, n (%) 
9 (3.6%) 
5 (4.0%) 
4 (3.2%) 
0.04 
Pulmonic Valve Regurgitation, n (%) 
2 (0.8%) 
1 (0.8%) 
1 (0.8%) 
<0.001 
Pulmonic Valve Stenosis, n (%) 
1 (0.4%) 
1 (0.8%) 
0 (0%) 
0.13 
Tricuspid Valve Regurgitation, n (%) 
17 (6.8%) 
7 (5.6%) 
10 (8.0%) 
0.1 
Data are expressed as counts and percentages (%)  
Abbreviations: AVR, Aortic Valve Replacement; CABP, Coronary artery bypass graft surgery; CPB, 
Cardiopulmonary bypass; LAAA, Left atrial appendage aneurysms; NO, Nitric Oxide; SMD, Standardized 
Mean Difference.  

 
 
 
 
 
 41
Supplementary Table 2: Subgroup Analysis Among Patient with High Mean Pulmonary Artery Pressure (30-40 mm Hg) 
 
Total 
(N=52) 
NO 
(N=26) 
Control 
(N=26) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
34 (65.4%) 
16 (61.5%) 
18 (69.2%) 
0.71 (0.23-2.24) 
0.74 (0.22-2.48) 
Acute Kidney Injury Severity  
No Acute Kidney Injury, n (%) 
18 (34.6%) 
10 (38.5%) 
8 (30.8%) 
Reference 
Reference 
Stage 1, n (%) 
25 (48.1%) 
14 (53.9%) 
11 (42.3%) 
1.02 (0.30-3.45) 
0.98 (0.28-3.52) 
Stage 2, n (%) 
6 (11.5%) 
2 (7.7%) 
4 (15.4%) 
0.40 (0.06-2.77) 
0.20 (0.01-2.94) 
Stage 3, n (%) 
3 (5.6%) 
0 (0.0%) 
3 (11.5%) 
NA 
NA 
  Data are expressed as counts and percentages (%)  

 
 
 
 
 
 42
Supplementary Table 3: Subgroup Analysis Among Patient with Low Mean Pulmonary Artery Pressure (<30 mm Hg) 
 
Total 
(N=198) 
NO 
(N=99) 
Control 
(N=99) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
75 (37.9%) 
39 (39.4%) 
36 (36.4%) 
1.14 (0.64-2.02) 
1.10 (0.60-2.00) 
Acute Kidney Injury Severity  
No Acute Kidney Injury, n (%) 
123 (62.1%) 
60 (60.6%) 
63 (63.6%) 
Reference 
Reference 
Stage 1, n (%) 
54 (27.3%) 
27 (27.3%) 
27 (27.3%) 
1.05 (0.55-2.00) 
1.05 (0.54-2.05) 
Stage 2, n (%) 
15 (7.6%) 
9 (9.1%) 
6 (6.1%) 
1.58 (0.53-4.69) 
1.41 (0.45-4.36) 
Stage 3, n (%) 
6 (3.0%) 
3 (3.0%) 
3 (3.0%) 
1.05 (0.20-5.41) 
1.40 (0.24-8.15) 
Data are expressed as counts and percentages (%)  
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 43
Supplementary Table 4: Subgroup Analysis Among Patient with High Duration of Aortic Clamping (≥111 Minutes) 
 
Total 
(N=125) 
NO 
(N=59) 
Control 
(N=66) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
62 (49.6%) 
31 (52.5%) 
31 (47.0%) 
1.25 (0.62-2.53) 
1.30 (0.63-2.69) 
Acute Kidney Injury Severity  
No Acute Kidney Injury, n (%) 
63 (50.4%) 
28 (47.5%) 
35 (53.0%) 
Reference 
Reference 
Stage 1, n (%) 
43 (34.4%) 
23 (39.0%) 
20 (30.3%) 
1.44 (0.66-3.13) 
1.47 (0.65-3.35) 
Stage 2, n (%) 
12 (9.6%) 
5 (8.5%) 
7 (10.6%) 
0.89 (0.26-3.12) 
0.93 (0.25-3.53) 
Stage 3, n (%) 
7 (5.6%) 
3 (5.1%) 
4 (6.1%) 
0.94 (0.19-4.54) 
0.97 (0.19-4.94) 
Data are expressed as counts and percentages (%)  
 

 
 
 
 
 
 44
Supplementary Table 5: Subgroup Analysis Among Patient with Low Duration of Aortic Clamping (<111 Minutes) 
 
Total 
(N=121) 
NO 
(N=63) 
Control 
(N=58) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
45 (37.2%) 
23 (36.5%) 
22 (37.9%) 
0.94 (0.45-1.96) 
0.82 (0.37-1.81) 
Acute Kidney Injury Severity  
No Acute Kidney Injury, n (%) 
76 (62.8%) 
40 (63.5%) 
36 (62.1%) 
Reference 
Reference 
Stage 1, n (%) 
34 (28.1%) 
17 (27.0%) 
17 (29.3%) 
0.90 (0.40-2.02) 
0.83 (0.35-1.97) 
Stage 2, n (%) 
9 (7.4%) 
6 (9.5%) 
3 (5.2%) 
1.80 (0.42-7.73) 
1.22 (0.25-5.91) 
Stage 3, n (%) 
2 (1.7%) 
0 (0.0%) 
2 (3.5%) 
NA 
NA 
Data are expressed as counts and percentages (%)  
 
 
 

 
 
 
 
 
 45
Supplementary Table 6: Subgroup Analysis Among Patient with High Duration of Cardiopulmonary Bypass (≥152 Minutes) 
 
Total 
(N=126) 
NO 
(N=61) 
Control 
(N=65) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
60 (47.6%) 
31 (50.8%) 
29 (44.6%) 
1.28 (0.64-2.59) 
1.36 (0.66-2.81) 
Acute Kidney Injury Severity  
No Acute Kidney Injury, n (%) 
66 (52.4%) 
30 (49.2%) 
36 (55.4%) 
Reference 
Reference 
Stage 1, n (%) 
42 (33.3%) 
22 (36.1%) 
20 (30.8%) 
1.32 (0.61-2.87) 
1.38 (0.62-3.08) 
Stage 2, n (%) 
12 (9.5%) 
6 (9.8%) 
6 (9.2%) 
1.20 (0.35-4.11) 
1.44 (0.38-5.51) 
Stage 3, n (%) 
6 (4.8%) 
3 (4.9%) 
3 (4.6%) 
1.20 (0.23-6.39) 
1.40 (0.24-8.23) 
Data are expressed as counts and percentages (%)  
 
 

 
 
 
 
 
 46
Supplementary Table 7: Subgroup Analysis Among Patient with Low Duration of Cardiopulmonary Bypass (<152 Minutes) 
 
Total 
(N=122) 
NO 
(N=62) 
Control 
(N=60) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
48 (39.3%) 
23 (37.1%) 
25 (41.7%) 
0.83 (0.40-1.71) 
0.68 (0.31-1.50) 
Acute Kidney Injury Severity  
No Acute Kidney Injury, n (%) 
74 (60.7%) 
39 (62.9%) 
35 (58.3%) 
Reference 
Reference 
Stage 1, n (%) 
36 (29.5%) 
18 (29.0%) 
18 (30.0%) 
0.90 (0.40-1.99) 
0.73 (0.30-1.78) 
Stage 2, n (%) 
9 (7.4%) 
5 (8.1%) 
4 (6.7%) 
1.22 (0.28-4.51) 
1.13 (0.26-4.93) 
Stage 3, n (%) 
3 (2.5%) 
0 (0.0%) 
3 (5.0%) 
NA 
NA 
Data are expressed as counts and percentages (%)  

 
 
 
 
 
 47
Supplementary Table 8: Subgroup Analysis Among Patient with High EuroScore II (≥1.96) 
 
Total 
(N=122) 
NO 
(N=71) 
Control 
(N=51) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
65 (53.3%) 
40 (56.3%) 
25 (49.0%) 
1.34 (0.65-2.76) 
1.67 (0.77-3.61) 
Acute Kidney Injury Severity  
No Acute Kidney Injury, n (%) 
57 (46.7%) 
31 (43.7%) 
26 (50.9%) 
Reference 
Reference 
Stage 1, n (%) 
46 (37.7%) 
28 (39.4%) 
18 (35.3%) 
1.31 (0.59-2.87) 
1.61 (0.69-3.76) 
Stage 2, n (%) 
14 (11.5%) 
9 (12.7%) 
5 (9.8%) 
1.51 (0.45-5.07) 
2.03 (0.56-7.35) 
Stage 3, n (%) 
5 (4.1%) 
3 (4.2%) 
2 (3.9%) 
1.26 (0.20-8.11) 
1.71 (0.25-11.98) 
Data are expressed as counts and percentages (%)  

 
 
 
 
 
 48
Supplementary Table 9: Subgroup Analysis Among Patient with Low EuroScore II (<1.96) 
 
Total 
(N=120) 
NO 
(N=49) 
Control 
(N=71) 
Unadjusted Odds Ratio 
(95% CI) 
Adjusted Odds Ratio 
(95% CI) 
Acute Kidney Injury, n (%) 
41 (34.2%) 
13 (26.5%) 
28 (39.4%) 
0.56 (0.25-1.23) 
0.55 (0.25-1.23) 
Acute Kidney Injury Severity  
No Acute Kidney Injury 
79 (65.8%) 
36 (73.5%) 
43 (60.6%) 
Reference 
Reference 
Stage 1, n (%) 
30 (25.0%) 
11 (22.5%) 
19 (26.8%) 
0.69 (0.29-1.64) 
0.69 (0.29-1.65) 
Stage 2, n (%) 
7 (5.8%) 
2 (4.1%) 
5 (7.0%) 
0.48 (0.09-2.61) 
0.55 (0.10-3.13) 
Stage 3, n (%) 
4 (3.3%) 
0 (0.0%) 
4 (5.6%) 
NA 
NA 
Data are expressed as counts and percentages (%)