Monitoring Renal Blood Flow With Contrast Enhanced Ultrasound During Coronary Angiogram

What will be done in this protocol? Specific to Aim 1: To investigate the correlation
between changes in RBF after exposure to iodinated contrast agent, measured by CEU, on
occurrence of acute kidney injury (AKI) in a population of subjects at risk for CN.

Procedures: Subjects with an estimated glomerular filtration rate (eGFR), measured by the
Modification of Diet in Renal Disease (MDRD) formula, between 30 - 60 ml/min/1.73 m2 who are
scheduled for a coronary angiogram will be enrolled in this study. These subjects are
considered to be at higher risks of CN. It is important to clarify that all subjects are
scheduled for a coronary angiography for clinical reasons before enrollment in this study.
Coronary angiography is not a part of interventions of this protocol. The decision regarding
the need for coronary angiography and the strategies for its prevention are made by the
subjects' healthcare providers and not the research team.

The details of protocol will be discussed with all interventional cardiologists and
permission will be obtained to discuss study details with potential subjects. Eligible
subjects will be identified by searching the electronic medical record of individuals who
are listed in the cardiac catheterization laboratory schedule for inclusion and exclusion
criteria. Subjects who appear to qualify for study participation based on medical record
review the inclusion and exclusion criteria will be approached. The details of the study
goals, procedures and risks will be discussed with each individual and questions will be
answered. The subjects will then be asked to sign the informed consent form. They will then
be enrolled in the study.

Since undergoing an X-ray examination or exposure to iodinated contrast media are
contraindicated in pregnancy, we assume all women of child bearing age having a test to rule
out pregnancy before enrolling in the study. If this test is not done, we will contact the
subject's cardiologist and arrange for it as a part of clinical care and not a research
related procedure.

At the time of enrollment data on subjects' age, race, gender, ethnicity, history of
coronary artery disease, diabetes and congestive heart failure will be collected. Subjects
with a history of heart failure will be asked about presence of symptoms of heart failure
such as shortness of breath at rest or with activities, fatigue, palpitation and dyspnea.
This information will be used to identify the New York Heart Association (NYHA) class of
heart failure which is considered a risk factor for CN. List of medications used by subjects
will also be recorded. Vital signs will be recorded.

Blood samples for basic metabolic panel and urine samples for urinalysis will be collected
and sent to the University of Virginia lab. More blood and urine samples will be collected
for measurement of the concentrations of biomarkers of kidney injury neutrophil
gelatinase-associated lipocalin (NGAL), cystatin C, beta 2 microglobulin, epithelial growth
factor (EGF), uromodulin, osteopontin, albumin and creatinine. These samples will be sent to
the the O'Brien Center at the University of San Diego, California for analysis..

Baseline 2-dimensional kidney ultrasound images will be obtained. All study subjects will
undergo baseline contrast enhanced ultrasound imaging of the kidneys using Definity
intravenous infusion as described in details below. They will then receive intravenous
infusion of normal saline for prevention of CN. Please see "hydration protocol" below.

Immediately before transfer to the cardiac catheterization lab and after volume expansion
with i.v. normal saline, CEU imaging of the kidneys using Definity will be repeated.

Subjects will undergo coronary angiogram procedure as planned by their interventional
cardiologists. Immediately after this procedure, CEU imaging of the kidneys using Definity
will be repeated. Subjects will be monitored for a minimum of 30 minutes after completion of
Definity infusion. Blood samples for basic metabolic panel and urine samples for urinalysis
will be collected and sent to the UVA lab. The research study procedures for this visit will
end at this time. Routine cardiac catheterization lab procedures will be followed at this
time.

Information on the volume and type of ICM will be recorded. Subjects will be discharged as
scheduled by the cardiology team. One to three days (24 - 72 hours) later the subjects will
return for a follow up visit. During the follow up visit, questions will be asked to assess
for potential adverse events. Vital signs will be obtained (blood pressure and heart rate).
Blood samples for basic metabolic panel and urine samples for urinalysis will be collected
and sent to the UVA lab. More blood and urine samples will be collected for measurement of
the concentrations of biomarkers of kidney injury NGAL, cystatin C, beta 2 microglobulin,
EGF, uromodulin, osteopontin, albumin and creatinine. These samples will be sent to the
O'Brien Center at the University of San Diego in California.

The outcome of the study, AKI, will be defined as a rise of greater than 0.3 mg/dL or 25% in
the baseline serum creatinine concentration 48 hours after exposure to ICM.

Specific Aim 2: To validate the RBF data obtained by CEU by comparing it to measurements
obtained by a Doppler ultrasound probe placed inside the main renal artery.

Procedures: Up to 25 subjects with normal renal functions who are scheduled for a coronary
angiography will be studied in this part of the proposed study. Blood samples for basic
metabolic panel and urine samples for urinalysis will be collected and sent to the UVA lab.

Immediately before undergoing coronary angiography, each subject will undergo CEU study
using Definity to measure baseline RBF. Femoral or radial access has already been obtained
to perform the coronary angiogram. A guide catheter is used to selectively engage the renal
artery and 5 cc of contrast will be injected to perform renal angiography. If the renal
artery appears angiographically normal and is without evidence of any degree of
atherosclerotic renal artery stenosis, then a Doppler flow wire (0.014") will be advanced
into the main renal artery. If the patient is not already systemically anticoagulated for
the coronary angiogram or coronary intervention, then heparin is administered (50 U/kg) to
prevent clotting on the Doppler wire. Proper positioning of the Doppler wire will be
confirmed by injecting 1-2 cc of contrast. Velocity of the blood flow within the main renal
artery will be measured directly using this Doppler wire. After completion of these
measurements, coronary angiography procedures will be performed as scheduled. Measurement of
blood velocity in the main renal artery with the Doppler probe will be repeated before
removal of the arterial catheter. CEU using Definity for measurement of RBF will be repeated
at this point. Subjects will be monitored for a minimum of 30 minutes after completion of
Definity infusion and a urine sample will be obtained and sent to the UVA lab for
urinalysis. Blood samples for basic metabolic panel will be collected and sent to the UVA
lab. The changes from baseline in the velocity of blood after exposure to ICM obtained by
Doppler probe will be compared to the values obtained by CEU. Multiple measurements (at
least three) using each method will be performed at each time point and the mean of these
measurements will be compared.

Contrast Enhanced Ultrasound Subjects will be connected to a continuous pulse oximeter and
heart monitor. The Definity vial will be placed at room temperature before being used. It
will be activated after shaking the vial using Vialmix for 45 seconds. It will be used
immediately after activation. 1.3 mL of Definity will be mixed in 30 ml of preservative free
saline. Infusion of Definity into a peripheral vein will be started at 2 ml/min and titrated
for optimal image quality (not to exceed 10 ml/min at any time). After reaching steady state
(2 -3 minutes) baseline contrast ultrasound imaging of right kidney will be performed with
low mechanical index (MI) of 0.1. High MI (1.0) impulse will be used to disrupt Definity
microbubbles within the kidney tissue. This would result in a very brief time period during
which renal cortex is free of microbubbles and appears completely anechoic (time zero). As
continuous infusion of microbubbles continues, during the wash-in period renal tissue is
replenished with microbubbles. Time-intensity curve after disruption of tissue microbubbles
will be fitted to a rising exponential function: intensity (y) = basic intensity (BI) + A
[1-exp(-beta.t) using specialized software. The slope of this curve or beta is related to
the velocity of microbubbles (or blood) that enter the tissue and the plateau of the curve
or A is related to the relative blood volume within the tissue. Since blood flow is the
movement of a certain volume of blood at a certain speed, the product of the two (A.beta)
would represent tissue blood flow. After disruption of microbubbles within the kidney tissue
with a high MI pulse, continuous imaging of the kidney with low MI (0.1) will continue for
about 15- 30 seconds to generate the time-intensity graph. The high MI pulse and
replenishment sequence will be repeated two more times. An average of the three parameters
obtained at each time period will be used for the final analysis. Infusion of Definity will
stop at this point. A urine sample will be collected for urinalysis after 30 minutes of
stopping Definity to assess for potential renal related adverse events.

Hydration Protocol:

0.9% sodium chloride (normal saline) solution will be infused intravenously at a rate of 3
mL/kg/hr for one hour. This will be followed by normal saline given at a rate of 1 ml/kg/hr
for the remainder of the time before and also during and at least for one hour after
coronary angiogram. Patients with heart failure and documented ejection fraction of < 30%
will receive normal saline infusion at a reduced rate of 1.5 ml/kg/hr for the 1st hour
followed by 0.5 mL/kg/hr after that for the same time periods as for other subjects. At any
point, if a subject experiences shortness of breath, oxygen saturation will be measured and
intravenous hydration protocol will be stopped.

Inclusion For specific aim 1 - Subjects with reduced kidney function

1. Adult (>18 years of age) men and women

2. All races and ethnicities

3. Chronic kidney disease stage 3 with an estimated GFR by MDRD formula between 30 - 59
ml/min/1.73 m2

4. Being scheduled for a coronary angiography study as part of their clinical care

For specific aim 2 - Subjects with normal kidney function

1. Adult (>18 years of age) men and women

2. All races and ethnicities

3. Being scheduled for a coronary angiography study

4. Estimated GFR greater than or equal to 60 ml/min/1.73 m2

Exclusion For specific aim 1 - Subjects with reduced kidney function

1. Estimated GFR greater than 60 or below 30 ml/min/1.73 m2

2. History of kidney transplantation

3. Known history of a right to left intracardiac shunt

4. Pregnancy or lactation

5. History of allergies to Definity®

6. History of Pulmonary Hypertension

For specific aim 2 - Subjects with normal kidney function

1. Estimated GFR less than 60 ml/min/1.73 m2

2. History of kidney transplantation

3. History of a right to left intracardiac shunt

4. Pregnancy or lactation

5. History of allergies to Definity®

6. History of recent major bleeding

7. History of allergy to heparin or heparin-induced thrombocytopenia

8. History of renal artery stenosis

9. Evidence of renal artery stenosis during the placement of Doppler wire in the renal
artery

10. History of Pulmonary Hypertension