Curcumin Supplementation for Improving Vascular and Cognitive Function in Chronic Kidney Disease
Phase: PHASE2
Conditions: Chronic Kidney Diseases, Cognitive Decline
Sponsor: Diana Jalal

STUDY PROTOCOL 
NCT03223883 
Aug 19, 2024 
 
STUDY DESIGN 
- Study Subjects: A total of 88 individuals (age 45-74 years) with stage IIIb and IV CKD will be recruited. 
Inclusion and exclusion criteria are shown in Table 1 and discussed in the Protection of Human Subjects 
section. Estimated GFR will be calculated based on the 4- variable Modification of Diet in Renal Disease 
(MDRD) formula 1. After obtaining informed consent, individuals with estimated GFR 15-45 mL/min/1.73m2 who 
fulfill the inclusion and exclusion criteria will be included in the study. We chose an estimated GFR cut-off of 
<45 mL/min/1.73m2 (stage IIIB) for the inclusion in the study based on data by Dr. Tamura that indicate the 
prevalence of cognitive impairment is significantly increased when estimated GFR is <50 mL/min/1.73m2 2. All 
subjects will be required to have adequately controlled blood pressure (BP), as our hypothesis is that curcumin 
reduces vascular dysfunction by targeting non-traditional risk factors for CVD. Adequately controlled BP will be 
defined as BP <140/90 mmHg in accordance with the current Joint National Committee guidelines 3. However, 
if the guidelines change for older adults without DM (based on the recent evidence from Systolic Blood 
Pressure Intervention Trial (SPRINT) 4), then we would require a systolic BP <120 mmHg for those patients.  
 
 
Table 1: Inclusion and exclusion criteria 
Inclusion Criteria 
Exclusion Criteria 
Age 45-74 years old 
Consuming a diet rich in curcumin or taking curcumin 
supplements in the past 12 months 
CKD stage IIIb and IV  
(estimated GFR: 15-45 mL/min/1.73m2)  
Life expectancy <1 year 
BMI <35 kg/m2 5 
Pregnant, breastfeeding,  
or unwilling to use adequate birth control 
Able to give informed consent 
Uncontrolled hypertension 
 
Severe liver disease 
 
Severe congestive heart failure 
 
Hospitalization within the last 3 months 
 
Active infection or antibiotic therapy 
 
Immunosuppressive therapy within the last year 
We will also screen for the use of supplements that may improve vascular function including vitamins C and E 6 
and resveratrol 7. Subjects who are taking these supplements but otherwise qualify for the study will be asked 
to undergo a 6-week wash-out period prior to participating in the study. 
- Experimental Design: 88 subjects, recruited at the University of Iowa Hospitals and Clinics (UIHC) and The 
Iowa City VA Medical Center, will be randomized to receive either placebo or curcumin (2000 mg/ day) for 12 
months and stratified randomization will be employed based on age ≥ 55 yeas 8-10 and baseline DM status 11. 
Block randomization is an alternative for consideration. Study outcomes will be evaluated at baseline, 6 
months, and 12 months (except cognitive function will be evaluated at baseline and 12 months only).  
- Curcumin supplementation: Based on our preliminary data we will use a solid lipid curcumin particle 
(SLCP, Longvida, Verdure Sciences) at a dose of 2000 mg/d.  Based on our preliminary data, we believe this 
will be an effective dose to improve vascular function in patients with CKD. Longvida will provide the 
supplement in addition to identical-looking placebo. The randomization table will be generated by Dr. You and 
provided to Longvida. Once a subject is randomized, Longvida will package and ship the study drug (curcumin 
or placebo). The package will only state “study drug” in order to guarantee blinding of all the study staff and 
participants. As we show in the Preliminary Data and Protection of Human Subjects sections, curcumin was 
well-tolerated with a small number of adverse events (mainly gastrointestinal discomfort). 
- Rational for the duration of the study: In specific aim 2, we intend to evaluate whether curcumin improves 
cognitive function in patients with CKD. Although our preliminary data suggests that curcumin may improve 
cognition in as little as 12 weeks, we believe a longer duration is necessary to minimize practice effects from 
repeated cognitive testing 12,13. Thus, the study duration will be 12 months. In addition, this longer duration will 

Figure A: Study Design 
 
enhance our ability to detect a benefit on vascular stiffness (specific aim 1). Large elastic artery stiffness 
reflects structural changes of the extracellular matrix in the vessel wall 14. Based on this, the longer duration of 
study will allow the treatment to reverse these structural changes in the vasculature. The vascular 
measurements will be conducted at baseline, 6 months, and 12 months. In order to reduce practice effects, 
cognitive function will be measured at baseline and 12 months only.  
- Recruitment Clinics:  The trial will be conducted through CRU of the ICTS at UIHC.  Patients will be 
recruited from 2 clinics with access to >1500 CKD patients from all over the Iowa State area including: 
1) UIHC Renal Clinic: The renal clinics at UIHC see approximately 3600 patient visits annually. At least 
2500 patient visits are related to stage III/IV CKD. This is a similarly-sized practice to the prior proposal. 
2) Iowa City VA Medical Center: The VA renal clinics see approximately 1950 patient visits annually the 
majority of whom have underlying stage 3 and 4 CKD. 
Based on the preliminary screening of our database at the UIHC, we expect at least 100 eligible patients per 
year. Notably, Dr. Jalal has been successful in recruiting patients with CKD to clinical trials, including single 
center, completing recruitment prior to end of funding period on corresponding grant applications. Additionally, 
UIHC has been a successful site of recruitment for clinical researchers at the University of Iowa (for example 
Bradley Dixon). 
- Detailed sessions: The study design is illustrated in Figure A. 
(All study procedures will be conducted at UIHC Clinical Research Unit (CRU)) 
Session 1: Informed consent and screening questionnaire for inclusion and exclusion criteria including 
confirmation of stage IIIb/IV CKD on 2 separate occasions in the last 6 months. All women in child-bearing age 
will undergo pregnancy test at this visit. In addition, all participants will complete a 7-day physical activity recall 
(International Physical Activity Questionnaire 15). 
Session 2 (baseline measurements after overnight fast): 
• Blood pressure at rest 
• Endothelial cell sampling for endothelial markers of inflammation and oxidative stress (eNOS and eNOS 
phosphorylated at Ser 1177, NF-κB, NAD(P)H oxidase, and nitrotyrosine) 
• Blood sampling for fasting lipid profile 
• Blood sampling for hemoglobin A1C (for participants with 
DM) 
• Blood sampling for levels of curcumin and its metabolites 
• Blood sampling for basic metabolic profile and estimated 
GFR including cystatin C 
• Blood sampling for circulating markers of inflammation 
and oxidative stress (CRP, IL-6, MCP-1, and oxLDL) 
• Urine sampling (random) for albumin/creatinine ratio 
(ACR) 
• Evaluation of EDD and EID by measurements of BA-FMD 
and nitroglycerin-mediated dilation (NMD) 
• Aortic PWV 
• Assessment of vascular function in response to vitamin C 
(ascorbic acid) infusion 
• Administer NIH Toolbox Cognitive Battery 
• Start study drug after this visit 
Sessions 3 and 4 (month 1 and month 3 safety visits): 
• Pill count 
• Safety monitoring:  
o Completion of an adverse event questionnaire 
o Update medication list 
Session 5 (month 6 study visit): will include the same 
measurements as Session 2 except for the NIH Toolbox 
Cognitive Battery. In addition, it will include the following: 

• Physical activity questionnaire 
• Pill count 
• Safety monitoring:  
o Completion of an adverse event questionnaire 
o Update medication list 
Session 6 (month 9 safety visit): identical to Sessions 3 and 4. 
Session 7 (month 12 study visit- end of study): identical to Session 2 including the administration of the NIH 
Toolbox Cognitive Battery in addition to the following: 
• Physical activity questionnaire 
• Pill count 
• Safety monitoring:  
o Completion of an adverse event questionnaire 
o Update medication list 
OUTCOME MEASUREMENTS 
- BA-FMD (primary outcome): BA-FMD will be measured at the CRU at UIHC by a trained research assistant 
using high-resolution ultrasonography (GE Healthcare, Inc.) as described originally by Celermajer et al. 16 and 
by our group 17-20. Electrocardiogram (ECG) gated end-diastolic ultrasound images and Doppler flow of the 
brachial artery are acquired during baseline and FMD conditions.  For FMD, reactive hyperemia is produced by 
inflating a pediatric forearm cuff around the forearm to 250 mmHg for 5 minutes followed by rapid deflation.  
Endothelial independent dilation (EID) is determined by measuring brachial artery dilation for 10 minutes after 
administration of sublingual nitroglycerin (0.4 mg). A commercially available software package (Vascular 
Analysis Tools 5.5, Medical Imaging Applications, LLC, Coralville, IA) is used to acquire and analyze ECG-
gated brachial artery diameters.  The analysis of the images will take place at Dr. Seals’s lab in Boulder. 
Brachial artery dilation is determined as mm and % change from baseline 21. Doppler blood flow velocity is 
recorded at baseline and for 2 minutes after cuff release. Shear rate will be calculated, and if group- or 
condition-differences exist, BA-FMD is adjusted accordingly.  We propose to use BA-FMD as opposed to ACh-
FBF since BA-FMD is less invasive and considering that BA-FMD has been utilized extensively in patients with 
CKD 19,22-24.  
- aPWV: Large elastic artery stiffness will be determined by aPWV as previously described 19,25-27 , utilizing the 
Non-Invasive Hemodynamic Workstation (NIHem, Cardiovascular Engineering, Norwood, MA). The subject is 
supine in a quiet, dim, climate-controlled room. Blood pressure readings are obtained after 20-minute rest. A 
custom trans-cutaneous tonometer is placed at the carotid and femoral arteries to non-invasively assess 
aPWV. The distance from the supra-sternal notch to the carotid is subtracted from the distance between the 
two recording sites. aPWV= distance divided by time between the foot of waveforms recorded at each site. 
Similar to BA-FMD, the aPWV images will be analyzed by Dr. Seals’s group. 
- Vitamin C (ascorbic acid) infusion: The influence of oxidative stress on BA-FMD and aPWV will be 
determined by infusing a supraphysiological dose of ascorbic acid (American Regent Laboratories Inc.) or 
isovolumic saline. A priming bolus of 0.06 g ascorbic acid/kg fat free mass (FFM) dissolved in 100 mL of saline 
will be infused intravenously at 5 mL/min for 20 minutes, followed immediately by a “drip-infusion” of 0.02 g/kg 
FFM dissolved in 30 mL of saline administered over 60 minutes at 0.5 mL/min. Vascular measurements will be 
made at the end of the 20-minute bolus during the “drip infusion” when peak plasma concentrations of ascorbic 
acid occur. The difference in BA-FMD and aPWV during ascorbic acid vs. saline infusion will be taken as a 
measure of the modulation of EDD/stiffness by oxidative stress. This technique has been developed and well 
established by the Seals research group 28-31, which included Dr. Gary Pierce. Dr. Pierce directs the 
Translational Vascular Physiology Lab at UIHC CRU, where several protocols that utilize vitamin C infusion are 
ongoing. In addition, Dr. Chonchol, who has successfully performed and reported this technique in several 
clinical trials 20, will remain a co-investigator for the application.  
- Systemic markers of inflammation and oxidative stress: High sensitivity CRP, IL-6, and MCP-1 will be 
measured as markers of inflammation. OxLDL will be measured as a marker of systemic oxidative stress.  
These markers will be measured on the serum or plasma by the UIHC clinical lab.  

- Endothelial cell sampling and cell protein expression: This technique performed and described by our 
group of investigators 20,30,32-34 including Dr. Jalal (PI). Briefly, J-wires are advanced into a brachial artery and/or 
an antecubital vein and withdrawn, and cells are recovered by washing and centrifugation. Collected cells are 
fixed with 3.7% formaldehyde and plated on slides. After blocking nonspecific binding sites with 5% donkey 
serum (Jackson Immunoresearch), cells are incubated with the primary antibody. Then, cells are incubated 
with CY3-conjugated secondary antibodies (Research Diagnostics). Slides are systematically scanned to 
identify endothelial cells (positive VE-Cadherin), and nuclear integrity is confirmed using 4',6'-diamidino-2-
phenylindole hydrochloride staining. Once endothelial cells with intact nuclei are identified, images are captured 
and then analyzed using Metamorph Software (Universal Imaging, Downingtown, PA) to quantify the intensity 
of CY3 staining (i.e., average pixel intensity). Values for each sample are reported as ratios of endothelial cell 
protein expression/human umbilical vein endothelial cell (HUVEC) to account for any variation in the staining 
procedure. Technicians are blinded to subject identity during the staining and analysis procedures. The 
following proteins will be evaluated: eNOS (BD Biosciences) and eNOS phosphorylated at Ser 1177 (BD 
Biosciences) as an indicator of eNOS activity 20,35, NF-κB (Santa Cruz) as a marker of pro-inflammatory 
signaling 32,36, and NAD(P)H oxidase (Upstate) 30,33,34 and nitrotyrosine (Abcam) 30,33,34 as markers of oxidative 
stress. 
- Cognitive function assessment: The NIH Toolbox for the Assessment of Neurological and Behavioral 
Function was initiated by the NIH Blueprint for Neuroscience Research to develop state-of-the-art 
measurement tools to for the collection of cognitive data in large cohort studies 37. The NIH Cognitive Toolbox 
is a validated multidimensional assessment of cognitive function domains, with minimal floor and ceiling 
effects, that enables us to evaluate (consistently) cognitive changes in response to our intervention 13. The 
cognitive battery of the NIH Toolbox will be administered to our patients by a trained research assistant 
(blinded to subject randomization) at the CRU UIHC in a quiet and private exam room over approximately 45 
minutes. The following table describes the test, subdomain, and tasks that will be applied: 
    Table 2: NIH Toolbox Cognition Battery Tests 
Subdomain 
Test name and Task description 
Executive function and 
attention 
Indicate direction of central arrow when flanking arrows are pointing in the same or 
opposite direction. (6 minutes) 
Executive function category 
switching 
Alternate sorting according to shape or color. (7 minutes) 
Working memory 
Repeat the order of items (2-8) according to size in 1 category then repeat the order of 
items according to size in 2 categories of items. (7 minutes)   
Processing speed 
Identify items images (side by side) as “same” or “not the same”. (2 minutes) 
Episodic memory 
Place pictures of individuals performing different tasks in the order they are shown. (9 
minutes) 
Written word pronunciation 
Read printed words aloud. (6 minutes) 
Auditory and word-visual 
picture matching 
Match a single word to one of four pictures. (4 minutes) 
- Curcumin Levels: These will be measured to evaluate the bioavailability of curcumin and its metabolites in 
CKD. The measurements will be conducted by Dr. Jelena Klawitter on a fee-for-service basis in the iC42 
Laboratory in the UC-Denver Anschutz Medical Campus according to the method described by Cao et al. 38. 
Curcumin (Acros Organics, Morris Plains, NJ), curcumin O-glucuronide and curcumin O-sulfate (Cell Mosaic, 
Worcester, MA), demethoxycurcumin and bisdemethoxy-curcumin (Sigma Aldrich), and tetrahydrocurcumin 
(ChromaDex, Irvine, CA) will be used in to perform liquid chromatography utilizing high-end mass spectrometry 
systems. The internal standard (IS) hesperetin will be purchased from 4C Pharma Scientific Inc. (ON, Canada). 
Heparin-treated human plasma, collected from the study participants and stored at -80 °C, will be used.  
- Clinical labs: These include basic metabolic profile (and estimated GFR), cystatin C, ACR, Hemoglobin A1C, 
and lipid profile will be measured by the UIHC clinical lab.  
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