Calcium and Phosphorus Whole-Body Balance and Kinetics in Patients With Moderate Chronic Kidney Disease
Phase: NA
Conditions: Phosphorus and Calcium Disorders, Chronic Kidney Disease Mineral and Bone Disorder, Chronic Kidney Diseases
Sponsor: University of Minnesota

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Effects of Dietary Phosphorus Restriction on Whole-Body 
Calcium and Phosphorus Balance and Kinetics in Patients 
with Moderate Chronic Kidney Disease 
 
Sharon M. Moe, MD, Distinguished Professor of Medicine 
Department of Medicine/Division of Nephrology 
950 W. Walnut Street, R2-202 
Indianapolis, IN 46202 
 
 
 
 
 
 
 
 
 
 
 
 
 
Kathleen Hill Gallant, PhD, Assistant Professor 
Department of Nutrition Science 
Purdue University 
 
 
 
 
Support Provided by: 
National Institutes of Health 
 
 
 
 

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Table of Contents: 
 
Study Schema 
1.0 
Background & Rationale 
2.0 
Objective(s) 
2.1 
Primary Objective 
2.2 
Secondary Objective 
2.3 
Tertiary/Exploratory/Correlative Objectives 
3.0 
Outcome Measures 
3.1 
Primary Outcome Measures 
3.2 
Secondary Outcome Measures 
3.3 
Tertiary/ Exploratory/ Correlative Outcome Measures 
4.0 
Eligibility Criteria 
4.1 
Inclusion Criteria 
4.2 
Exclusion Criteria 
5.0 
Study Design 
6.0 
Enrollment/Randomization 
7.0 
Study Procedures 
8.0 
Study Calendar 
9.0 
Reportable Events 
10.0 Data Safety Monitoring 
11.0 Study Withdrawal/Discontinuation 
12.0 Statistical Considerations 
13.0 Data Management 
14.0 Privacy/Confidentiality Issues 
15.0 Follow-up and Record Retention 
16.0 References 
17.0 Appendix 
 

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Abbreviations 
 
1,25D 
1,25-Dihydroxyvitamin D 
25D 
25-Hydroxyvitamin D 
AE 
Adverse Event 
ANOVA 
Analysis of Variance Analysis 
Ca 
Calcium 
Ca-44/44Ca Calcium-44 stable isotope 
CKD 
Chronic Kidney Disease 
CKD-EPI 
Chronic Kidney Disease Epidemiology Collaboration 
CKD-MBD 
Chronic Kidney Disease-Mineral Bone Disorder 
CRC 
Clinical Research Center 
CTSI 
Clinical and Translational Science Institute 
DSMB 
Data Safety Monitoring Board 
eGFR 
Estimated Glomerular Filtration Rate 
FGF23 
Fibroblast Growth Factor 23 
HIPAA 
Health Insurance Portability and Accountability Act  
IRB 
Institutional Review Board 
IV 
Intravenous 
LCa/HP 
Low Calcium/High Phosphorus Diet 
LCa/LP 
Low Calcium/Low Phosphorus Diet 
NIH  
National Institutes of Health 
P 
Phosphorus 
P-33/33P 
Phosphorus-33 Radioisotope 
PEG 
Polyethylene Glycol 
PI 
Principal Investigator 
PTH 
Parathyroid Hormone 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
 
1.0 
Background & Rationale 
 
Chronic kidney disease (CKD) is a common chronic disease, affecting approximately 26 million American 
adults (1). CKD-mineral bone disorder (CKD-MBD) is a condition of disordered bone and mineral metabolism 
that contributes to morbidity and mortality in patients with CKD (2). CKD-MBD is characterized by the presence 
of all or some of the following features: laboratory abnormalities related to calcium and phosphorus homeostasis, 
bone disease, and vascular calcification. These abnormalities begin to appear early in the disease process (3), 
and virtually all patients with moderate to advanced CKD exhibit some or all features of the disorder. The clinical 
consequences of CKD-MBD are dire and include increased cardiovascular events, bone fragility fractures, and 
mortality (4-7). In fact, cardiovascular events are the leading cause of death in patients with CKD (8, 9). Thus, 
prevention and management of CKD-MBD is of critical importance to reduce morbidity and mortality in these 
patients. Calcium (Ca) and phosphorus (P) homeostasis are central to CKD-MBD development. However, lack 
of understanding on Ca and P metabolism in CKD-MBD progression precludes the development of 
effective therapies. 
 
Ca and P are intimately related in physiology and metabolism(10). They share common hormonal 
regulators that include parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25D), and fibroblast growth 
factor 23 (FGF23) that act on a common tissue axis of intestine, kidney, bone, and parathyroid glands. 
Treatments aimed at managing CKD-MBD affect the hormonal and tissue axes of both ions and have diverse 
effects on Ca and P serum levels. For example, calcitriol lowers serum P while increasing serum Ca (11, 12); 
whereas calcimimetics lower serum P while also lowering serum Ca (13). More importantly, serum or plasma 

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levels of Ca or P do not reflect whole-body status of either mineral (14, 15). This challenges both clinical research 
on Ca and P homeostasis, as well as clinical treatment decision-making. Thus, there is a critical need for 
knowledge of whole-body Ca and P physiology that can only be obtained from formal metabolic balance and 
kinetic studies of both ions simultaneously. However, such studies are largely absent in the scientific literature. 
Thus, significant knowledge gaps exist regarding whole-body Ca and P physiology that include how dietary 
Ca and P intakes, as well as kidney disease and its treatments, influence both Ca and P whole-body retention, 
intestinal absorption, and the transfer of Ca and P to and from the various body pools, such as intestine, bone, 
blood, and soft-tissue. Dietary P restriction is a commonly prescribed therapy for patients with CKD because it 
is presumed to reduce P retention and serum P, but this is based on little evidence (16). It is important to 
determine the effectiveness of dietary P restriction on its proposed benefits, because it is a difficult diet for 
patients to follow and may lead to consequences such as dietary protein or calcium deficiency, which can have 
negative consequences for bone health and mortality risk. The goal of this project is to generate preliminary 
data, including estimates of effect sizes and variances, on the effects of dietary P restriction therapy on Ca and 
P whole-body balance and full kinetics in patients with CKD. We will use this data in a future large research grant 
proposal that will include an adequately powered study to test the hypothesis that a high P diet, in the context 
of a typical low Ca diet, leads to P retention with negative Ca balance, and that dietary P restriction modestly 
reduces P retention but does not alleviate negative Ca balance. This will provide a foundation for further studies 
examining the effects of other factors on whole-body Ca and P physiology, including common pharmaceutical 
agents (e.g., various P binders, calcitriol, and calcimimetics), biological factors (e.g., age, race, and sex), CKD 
severity, and other nutritional interventions. 
 
Additionally, preliminary and feasibility data will be collected on magnesium (Mg) absorption using stable 
oral and IV Mg isotopes (similar to the use of stable Ca isotopes). Dietary Mg and absorption is of interest in 
CKD patients as a potential inhibitor of calcification. Yet, to our knowledge, Mg absorption has not been studied 
in CKD patients (though stable Mg isotopes have been used for absorption studies in human subjects ranging 
from infants to postmenopausal women) (17-19).  
 
 
2.0 
Objectives 
 
Aim 1. Determine intestinal Ca P, and Mg fractional absorption, effect sizes and variance estimates 
in patients with CKD on a low Ca diet with high P diet versus dietary P restriction. We will give CKD 
patients oral and IV Ca P and and Mg isotopes during a controlled-feeding, 48-hour inpatient study. In this 
study, we will use two dietary conditions and a randomized 2-period, cross-over design (low Ca/high P & low 
Ca/low P). Fractional Ca, P and Mg intestinal absorption will be determined by kinetic modeling of serum and 
urine oral and IV tracer data.  
 
Aim 2. Determine Ca, P, and Mg balance and full kinetics, effect sizes and variance estimates in 
patients with CKD on a low Ca diet with high P diet versus dietary P restriction. A subset of subjects 
from Aim 1 will be studied as inpatients for an additional 12 days (5 as inpatient) after the 48-hour inpatient 
absorption testing protocol and in the same randomized 2-period cross-over design with the low Ca/high P & 
low Ca/low P diets. Total Ca, P, and Mg data will be determined from diet, urine, and fecal samples, and will be 
used to determine mineral balances. Further, we will use serum, fecal, and urine tracer data to determine full 
kinetic modeling including rates of transfer of the three ions between body pools, such as to and from bone. 
 
3.0 
Outcome Measures/Endpoints 
3.1 
Primary Outcome Measures 
1. Fractional Ca Absorption, by kinetic modeling of Ca-44 isotopic data (by ICP-MS) over 48 
hour between Days 8-10 
2. Fractional P Absorption, by kinetic modeling of P-33 isotopic data (from scintillation 
counting) over 48 hour between Days 8-10 
3. Calcium Balance, calculated from diet, urine, and fecal Ca assessed by ICP-OES, 
throughout Days 8-14 

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4. Phosphorus Balance, calculated from diet, urine, and fecal P assessed by ICP-OES, 
throughout Days 8-21(the last week is outpatient follow-up) 
5. Calcium Kinetics, by full kinetic modeling of Ca-44 isotopic data (by ICP-MS), throughout 
Days 8-21(the last week is outpatient follow-up) 
6. Phosphorus Kinetics by full kinetic modeling of P-33 isotopic data (by scintillation 
counting), throughout Days 8-21 
 
3.2 
Secondary Outcome Measures 
1. Fractional Mg Absorption, by kinetic modeling of Mg isotopic data (by ICP-MS) over 48                
hour between Day 8-10 
2. Magnesium Balance, calculated from diet, urine, and fecal P assessed by ICP-OES, 
throughout Days 8-14 
3. Magnesium Kinetics by full kinetic modeling of Mg isotopic data (by ICP-MS), throughout 
Days 8-21 (the last week is outpatient follow-up) 
4. Serum & Urine Ca by standard clinical chemistry, all time points collected  
5. Serum & Urine P by standard clinical chemistry, all time points collected  
6. Serum & Urine Mg by standard clinical chemistry, all timepoints collected 
7. Serum Fibroblast Growth Factor 23, intact, by ELISA kit, Day 1, all time points on Day 
8-10, Day 15  
8. Serum Parathyroid Hormone, intact, by ELISA kit, Day 1, all time points on Day 8-10, 
Day 15  
9. Serum 1,25-dihydroxyvitamin D, by ELISA kit, Day 1, all time points on Day 8-10, Day 
15  
 
4.0 
Eligibility Criteria 
4.1 
Inclusion Criteria 
• 
Men or women, ages 30-75 years old, any race or ethnicity 
• 
Moderate CKD, defined by KDIGO as eGFR category 3b (30-44 mL/min), with albuminuria 
categories A1-A3 (Figure 1) 
• 
Female subjects must be postmenopausal (>12 months since last menstrual period), surgically 
sterile, or confirmed not pregnant by pregnancy test 
• 
Must be on stable doses of medications (except those noted in exclusion criteria) for at least 4 
weeks prior to the study 
• 
Willing to discontinue nutritional supplements (e.g. vitamin D, calcium, multivitamin/minerals, or 
others) upon enrollment until completion of the study 
• 
Adequate vitamin D status defined as serum 25D > 20 ng/mL  
 
4.2 
Exclusion Criteria 
• 
Plans to initiate dialysis within 6 months 
• 
Hypercalcemia defined as serum calcium >10.5 mg/dL within past 3 months 
• 
Hyperkalemia defined as serum potassium >5.5 mg/dL within past 3 months 
• 
Hyperphosphatemia defined as serum phosphate >5.5 mg/dL within past 3 months 
• 
Intestinal disease that alters absorption or normal intestinal function including celiac disease, 
small bowel resection, or bariatric surgery 
• 
Serious, uncontrolled underlying systemic disease including diabetes, lupus, hypertension (as 
deemed by the PI). 
• 
Pregnant or breastfeeding 
• 
Taking a phosphate binder medication, calcitriol, vitamin D analogs, calcimimetics, PTH 
analogues, and other medications that may alter Ca and P metabolism within past 30 days 
• 
Vegan or vegetarian diet, lactose intolerance and/or milk allergy 
 
 
 

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Figure 1. Current CKD Nomenclature Used by KDIGO Guidelines 
 
 
 
 
5.0 
Study Design 
 
The study design is a randomized 2-period cross-over study. Figure 2 below describes the study timeline for 
participants enrolled in the study (a larger version is in attachments). All outpatient and inpatient visits will take 
place at the Indiana CTSI Clinical Research Center located in IU Health University Hospital in Indianapolis, IN. 
For Aim 1, N = 4 subjects will be randomly assigned to controlled diets of either low Ca/high P (LCa/HP) or low 
Ca/low P (LCa/LP) for a 1-week, outpatient diet equilibration period, followed by a 48-hour inpatient Ca, P, and 
Mg absorption testing protocol. After a 1-3 week washout period, subjects only participating in Aim 1 (N = 2) 
will crossover to the other diet condition for another 1-week outpatient diet equilibration period, followed by 
another 48-hour inpatients Ca, P, and Mg absorption testing protocol.  
For Aim 2, a subset of N = 2 subjects from Aim 1 will be studied as inpatients for an additional 5 days then a 
7-day outpatient follow up for full Ca, P, and Mg balance and kinetic measures, after the 48-hour inpatient 
absorption testing protocol described under Aim 1. After a 1-3 week washout, they will crossover to the other 
diet condition for another 1-week outpatient diet equilibration period, followed by another 48-hour inpatient 
Ca, P, and Mg absorption testing protocol and additional 5 days inpatient and 7-day outpatient follow-up 
period for full balance and kinetics. 
 
 
 

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Figure 2. Study Timeline (larger version in Appendix) 
 
 
 
 
 
6.0 
Enrollment/Randomization 
 
We aim to study N = 4 subjects, although seek approval enrolling up to 6 subjects due to the small sample 
size of 4 and the need for high adherence. Based on study diet and urine and fecal collection adherence of 
>90%, N = 2 patients that meet this level of adherence will be asked to continue with the full balance and 
kinetics protocol (Aim 2) on a first-enrolled basis. Thus 2 subjects will complete the absorption study (Aim 1), 
and 2 subjects will complete the full balance and kinetic studies (Aim 1 and Aim 2). It will be determined before 
a subject signs consent which consent they will sign (Consent A = Aim 1 or consent B= Aim 1 and Aim 2). 
 
Recruitment will target moderate stage CKD patients aged 30-75 years old. Subjects will be recruited from 
patients seen in CKD clinics of the IU/Eskenazi Nephrology group who are partners of the co-investigators.  
 
• 
Personnel from the IUSM Division of Nephrology will review medical charts from nephrology clinics for 
pre-screening to determine potential eligibility.  An email will be sent to the doctors of the identified 
potential subjects to obtain their permission for enrollment and the potential subjects will be contacted 
by phone or visited by study staff during clinic. 
• 
Information about the study may be discussed with interested potential subjects by research personnel 
by phone, email or in person as preferred by the subject/clinic. 
• 
If interested, potential subjects will be either 1) scheduled for a screening visit at the Indiana Clinical 
Research Center, or 2) have screening information collected at the time of recruitment if subject is 
recruited in person at a CKD clinic by study staff.  
• 
Prior to screening, subjects will be asked to provide written informed consent, and will be given the 
opportunity to have their questions about participation answered. 
• 
Following the screening visit, all data will be evaluated by the study staff to determine eligibility of the 
subject.  If eligible, participants will be enrolled in the study, and study visit dates will be scheduled.  

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Randomization Plan 
At the beginning of the study, the study biostatistician will generate a randomization list for dietary intervention 
crossover order A or B by pairs. These orders will be A=LCa/HP to LCa/LP and B=LCa/LP to LCa/HP. Especially 
due to the small sample size, attention will be made to ensuring a balance of the two orders, which will be 
accomplished by randomizing both in pairs. 
 
 
7.0 
Study Procedures 
 
All study procedures will be conducted for the purpose of research. 
 
Screening Visit 
Following informed consent, a screening visit of approximately 60 minutes to assess eligibility will include a 
medical history, renal panel, and urine albumin/creatine ratio (UACR). Recent lab values from the medical chart 
within ~3 months may be used if available. eGFR will be calculated from serum creatinine using the CKD-EPI 
equation (20). A urine pregnancy test of human gonadotropin will be performed for subjects of childbearing 
potential. 
 
Pre-Study Washout Period and Run-In Period 
Otherwise eligible subjects who take drugs that potentially alter calcium and phosphate balance or homeostasis 
including high dose cholecalciferol or ergocalciferol (1000 U/day or 50,000 U/wk, respectively), active vitamin D 
metabolites, calcimimetics, PTH analogues in the last 30 days, will undergo a 30-day pre-study washout from 
these drugs to be eligible for the study, with their physician approval.  
 
All subjects will be given 600 IU/d of an oral vitamin D supplement (cholecalciferol) for 2-weeks prior to beginning 
the controlled feeding period and through completion of the study. This is to ensure vitamin D intake adequacy 
prior to the study, as was done for our previous balance study in CKD patients(14). 600 IU/d is the current 
recommended dietary allowance from the Institute of Medicine (18). 
 
Dietary Interventions 
The cross-over will consist of a randomly assigned order of two dietary conditions:  
1) Low Ca (500 mg/d)/high P (1500 mg/d) diet (LCa/HP)  
The LCa/HP diet is at levels within the range observed for a typical American diet poor in Ca and high in 
P (18-21) 
 
2) Low Ca (500 mg/d)/low P (800 mg/d) diet (LCa/LP)  
The LCa/LP diet represents coupling a typical poor Ca intake with the commonly prescribed dietary P 
restriction of 800 mg/d (22).  
The magnesium content will be identical in both diets.  Diets will be designed by a registered dietitian of the 
Bionutrition Center of the Indiana CTSI using nutrient analysis software, and mineral composition will be 
confirmed by chemical analysis of diet composites prior to and during the studies. The LCa/LP diet will be 
designed to contain zero-to-minimal phosphate food additives, and an equal portion of protein from animal and 
plant sources. The LCa/HP diet will be achieved in part through higher amounts of phosphate food additives, 
which is representative of a typical American diet. Diet levels will be designed to be as close to the above targets, 

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but final values may differ by approximately +/- 10%. Actual levels of the diets will be reported in the results of 
this study 
All meals will be prepared in the metabolic kitchen at the Indiana CTSI CRC, with ingredients weighed with 
precision to 0.1 g. Outpatient meals will be provided as pack-outs for the subjects to take home; Inpatient meals 
will be prepared and trayed for the subjects. Diet composites will be prepared alongside subject diets, 
homogenized, ashed, and analyzed for accurate dietary Ca, P, and Mg intakes during the balance studies. Any 
leftovers not eaten will be weighed back and chemically analyzed and considered in the balance calculations 
(Aim 2). 
A non-absorbable fecal marker, polyethelene glycol (PEG), molecular weight 3350, will be given in capsule form 
~1 g with each meal (~3 g/d), to assess % fecal PEG recovery for fecal collection compliance, as well as steady-
state by fecal Ca:PEG, P:PEG, and Mg:PEG ratios (23). This will be in the meal pack-outs or on the patient meal 
trays while inpatient. 
 
Baseline Measures 
A baseline outpatient visit of approximately 60 minutes will occur on “Day 1” of the study. This is when the 
controlled research diet will begin. Subjects will arrive to this appointment fasting at least 8 hours and will have 
blood and urine collected for baseline biochemistries. These will include a renal panel, serum 25D, and urine Ca, 
P, creatinine, and additional serum collected for additional measures such as baseline bone and mineral 
biochemistries and uremic retention solutes. eGFR will be calculated from serum creatinine using the CKD-EPI 
equation (17). Subjects will receive 3 days of meal pack-outs and will return on the 3rd or 4th day for the remaining 
meals until the inpatient stay. We will obtain meal adherence data over the outpatient period using meal 
checklists, PEG capsule pill counts, and leftover food weigh-backs. Anthropometrics (weight, and height) and 
vital signs will be taken at the baseline visit. Subjects will also be asked to bring a stool collection from home to 
the Day 1 visit, the stool sample should be collected within 3 days prior to the visit. Stool collection supplies will 
be given to the subjects at the screening visit.  
 
Ca, P, Mg Absorption Tests (Aim 1) 
Following the 7-day outpatient equilibration period on the controlled diet, participants will be asked to fast 
overnight at least 8 hours and will be admitted to the Indiana CTSI clinical research center (CRC) on the morning 
of study Day 8. Subjects will continue the controlled diet for the 2-day inpatient testing. We will collect fasting 
blood and urine, and then subjects will be given their assigned diet breakfast with a beverage containing 10 μCi 
P-33 (range 8-11 uCi) and 10 mg stable Ca-44(range ~8-11 mg), and 40-70 mg Mg-26  to drink halfway through 
their meal. Blood samples will be taken pre-dose, then 5, 15, 30, 60, 90, 120, 180, 240, 360 min, and 24h post-
dose. All urine will be collected during the test days in 2-hour intervals until 6-hours post-dose, and then 6h-24h 
post-dose with collections timed and recorded by CRC staff. These urine collections will be individually measured 
for volume and a small aliquot taken for time point measurements, and then we will combined the values of the 
intervals to give a total 24h value. On Day 9, subjects will receive the same breakfast as Day 8, at the same 
clock time, but with no isotope; at 25h post-oral dose, an IV push of 10 μCi P-33 (range 8-11 uCi) and 6 mg 
stable Ca-44 (range ~5-7 mg), and 20-30 mg stable Mg-25  in sterile saline will be given over 2 minutes. Serum 
and urine collections post-dose will follow the same time points as during Day 8 and will continue through the 
24-hour post-IV dose on Day 10, prior to discharge. The amount of isotope in the sequential blood and urine 
samples will be determined by liquid scintillation counting or inductively coupled plasma mass spectrometry for 
P-33 and Ca-44/Mg-25/Mg-26, respectively. Time points are approximate, and exact clock time of blood and 
urine collections will be recorded throughout the inpatient study and used in the kinetic analysis. Thus, only 
missed time point collections will be considered protocol deviations. 
 
 
 

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Ca, P, Mg Balance and Kinetics (Aim 2) 
For the full Ca, P, Mg balance and kinetics in Aim 2, a subset of N = 2 subjects will remain inpatient for an 
additional 5 days following the 48-hour absorption testing protocol described under Aim 1. During this time, all 
urine and fecal samples will be collected. Urine will be collected in time, 24-hour pools, as well as two 2-hour 
fasting urine collections taken on the morning of Day 15, aliquoted for fasting biochemistries, and then data 
combined with the appropriate 24-hour collection. Urine Ca, P, Mg and creatinine will be measured in each 24-
hour collection or 2-hour fasting collection. Blood samples will be taken each morning, Day 10-15, to use for full 
Ca, P, and Mg kinetic modeling. Subjects will be discharged the morning of Day 15, after the 2-hour fasting urine 
and fasting blood collection; at this point they will discontinue study diet and PEG, but continue taking vitamin 
D at home for the remainder of Day 15 through Day 21. Fasting blood biochemistries will be measured in samples 
collected during a return outpatient visit on Day 21. In addition to isotope analysis, total serum Ca, P, Mg and 
creatinine will be measured by standard clinical chemistries. All fecal samples will be collected during the 
inpatient periods, then homogenized, ashed, and analyzed for Ca-44, P-33, Mg-25, Mg-26, total Ca, total P, total 
Mg, and PEG recovery.  
 
Other Biochemistries 
Additional biochemistries will be measured in serum and urine samples collected during these studies. These will 
include FGF23, PTH, 1,25D at the end of the study and creatinine clearances. The sequential and timed serum 
and urine samples will allow for dynamic assessment of these biochemical outcomes. Data on additional minerals 
Na, K, and Mg will be obtained from the ICP analysis of urine, diet, and feces, and may be used in secondary 
analyses. Additional analyses on leftover serum, urine, or feces may include other mineral metabolism 
biochemistries (e.g. 25-hydoxyvitamin D or bone turnover biomarkers), metabolites considered to be gut-derived 
uremic retention solutes (e.g. TMAO). The fecal microbiome may be analyzed in secondary analyses.  
 
8.0 
Study Calendar 
 
See Appendix B. “Study Calendar of Assessments” 
 
9.0 
Reportable Events 
 
Collection of AEs will begin at the time of consent.  
 
Prompt reportable AEs are assessed by the principal investigator (PI) as (1) unexpected, (2) related or possibly 
related to study participation, AND (3) suggests that the research places subject(s) or others at greater risk of 
harm than was previously known.  
These will be submitted to the IRB within 5 business days of the investigators becoming aware of the 
event. 
 
SAEs are any severe SE including any event leading to hospitalization, life-threatening condition, disability or 
incapacitation, death, or other significant hazard. 
These will be reported to the IRB within 48 hours of the investigators becoming aware of the event, 
and in writing within 5 business days. 
 
AEs not requiring prompt reporting will be those that are not SAEs as described above, are anticipated (i.e. 
described in the consent form), and do not occur any more frequently or with greater severity than expected.  
 
These will be recorded and reported to the IRB at continuing review. 
 
 
 
 

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10.0 Data Safety Monitoring 
 
The Data and Safety Monitoring Plan includes establishment of a DSMB. Members of the DSMB will include 
experts experienced in clinical research and patients with CKD and will consist of a Chairperson and one 
additional member. Please see details in the DSMP attachment. 
 
Potential Risks 
Risk of Phlebotomy and IV infusion:  
    The potential risks of phlebotomy are slight, but some possible risks include: excessive bleeding, fainting or 
feeling lightheaded, bruising, and infection (which is a slight risk any time the skin is broken). During each of 
the absorption test days, approximately 80 mL (11 time points of 7 mL each, approximately 5 tablespoons) of 
blood will be drawn over 24 hours. For comparison, the average whole blood donation is 1 pint, or 473 mL 
(about 32 tablespoons). Therefore, the total amount of blood drawn over the course of this study for subjects 
studied only under Aim 1 (absorption tests only) is 348 mL (174 mL in each crossover period (screening visit [7 
mL], baseline visit [7 mL], plus the absorption test protocol [~160 mL, 22 time points of 7 mL each] which will 
be over ~48 hours, which is less than that drawn in a single blood donation. For subjects in Aim 2 (full balance 
and kinetics), they will have an additional 42 mL (6 time points of 7 mL each) drawn over the 12 days (5 days 
additional inpatient and final blood draw on Day 21 outpatient) after the 48-hour absorption testing period in 
each session, totaling approximately 432 mL drawn over the entire study (approximately 2 months). This is a 
cumulative volume of blood from sequential draws of 7 mL each, to yield approximately 3 mL of serum at each 
time point, which is needed for analysis of P-33, Ca-44, Mg-25, Mg-26 and other biochemistries of mineral 
metabolism.  
The Ca-44, P-33 and Mg-25   will be administered by IV in a sterile saline “push” infusion of 1-3 mL. Potential 
risks are rare, but could include infection at the infusion site, sepsis, or air embolism. Steps to minimize these 
risks are below. 
 
Risk of Radiation Exposure:  
The risk of radiation exposure from P-33 (10 μCi in each oral and IV dose = 40 μCi total over the course of the 
study, effective dose of 35.6 mrem) is present for this study but is similar to some other common exposures. 
For example, the average radiation dose from a single round-trip cross-country flight in a commercial airplane 
across the United States is 4 mrem, from a single chest x-ray is 8 mrem, and from a single mammogram is 30 
mrem (SC Dept of Health). People in the United States receive an average of 620 mrem of radiation per year 
from natural and man-made radiation sources (Source: Purdue REM website; NCRP Report #160).  
Ca-44 Mg-25, and Mg-26 are stable isotopes so does not carry radioactivity risk. 
 
Risk of Diet Intervention:  
The controlled LCa/HP diet is based on a typical US diet that is low in Ca and is high in P due to its ubiquity in 
foods (37, 38). The LCa/LP diet changes the dietary P content to the level recommended when dietary P 
restrictions are prescribed. Thus, the controlled LCa/HP diet is at a level that would not be atypical in average, 
healthy American, and the LCa/LP diet reflects what would be commonly consumed by a CKD patient on a P 
restricted diet. Diets will be designed to meet the estimated energy needs of each subject to maintain current 
weight.  
Potential risks of the controlled diet are feelings of hunger or being overly full on the research diet. A potential 
risk for food borne illness is present as is the case with any food consumption.  
For patients with diabetes, there is risk of potential hypoglycemia with the study diet due to potential differences 
in amount and pattern of food consumed on study compared with at home. 
 

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Risk of Loss of Confidentiality: 
As with all research studies, there is a risk of loss of confidentiality. Steps to minimize this risk are described in 
the following section.  
 
Protections against Risks 
Risk of Phlebotomy and IV Infusions:  
To minimize risk from venipuncture and IV infusions, only trained personnel on the Indiana CTSI CRC will draw 
blood, place IVs, and push the IV infusions, using sterile supplies and technique. The IV infusion will be Ca-44, 
P-33, and Mg-25 in sterile saline. A sterile procedure for preparing these doses has been approved by the Indiana 
University Radioactive Drug Research Committee (RDRC) that we have used in prior studies. We send dose 
samples for each batch made to an independent certified lab for sterility and endotoxin testing.  
 
Risk of Radiation Exposure:  
Oral and IV doses of P-33 will be analyzed by liquid scintillation to confirm the appropriate radiation activity is 
given to subjects. The closest amount to 10 uCi that can be accurately measured without going over the limit of 
11 uCi per dose will be given and recorded (e.g. 1.4356 mL may be needed for exactly 10 uCi, so 1.4 mL will be 
accurately measured, administered, and recorded). The radiotracer tests will be performed at the Indiana Clinical 
Research Center under medical supervision. The protocol will be approved by the Indiana University Radionuclide 
Radiation Safety Committee (RRSC) and the Radioactive Drug Research Committee (RDRC). These committees 
have approved similar P-33 use in our prior human studies. 
 
Risk of Diet Intervention: 
If a subject reports hunger or being overly full on the research diet, the diet will be modified accordingly. 
Research kitchen staff are trained in safe food handling, and subjects will be given explicit instructions on how 
to keep their pack-out meals safe to consume (including refrigeration and reheating instruction). 
For diabetic subjects, we will collect usual home diet information (4-day food record), and the study physicians 
and dietitian will adjust the study diet and diabetes medications accordingly for the study. Diabetic subjects will 
be asked to perform fingersticks at home for blood glucose monitoring, and report to the study coordinator any 
blood sugar < 100 mg/dL. The study coordinator will alert the MD.  
The study physician orders will be amended to include a specific order to the MD (first Dr. Moorthi, then Dr. 
Moe) for any BS < 100 mg/dL. 
 
Risk of Loss of Confidentiality: 
See 14.0 Privacy/Confidentiality Issues below 
 
 
11.0 Study Withdrawal/Discontinuation 
 
Diet Adherence Assessment 
Patients will be assessed for adherence to the controlled diet on Day 3 of the controlled diet outpatient period. 
Subject who do not meet a minimum 90% adherence will be withdrawn from the study. This will be determined 
by evaluating meal check lists and returned uneaten foods. During the 48-hour absorption testing as inpatients, 
subjects must show at least 90% adherence with the study diet and complete urine and fecal collections to be 
eligible to continue the additional 12-days (5 as inpatient) for full balance and kinetic measures. This is to ensure 
high quality balance data for these preliminary studies.  

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Participants may withdraw themselves from the study at any time by informing the study staff. The study staff 
may ask the participant if they are willing to talk about their reason for wanting to withdrawal from the study 
and if they are willing to discuss potential accommodations that might be possible that would make continuing 
the study possible, if appropriate. The study staff will explicitly let the participants know that they are not 
obligated to give a reason or discuss ways they might change their mind.  
 
For participants who do not complete the entire study, data for the completed portions will be used for 
secondary/exploratory analyses, when statistically appropriate, unless the subject withdraws their consent for 
use of their data. 
 
12.0 Statistical Considerations 
 
This is a pilot study to determine effect sizes and variance estimates in fractional Ca, P, and Mg 
intestinal absorption (Aim 1) and Ca, P, and Mg balance (Aim 2) in patients with moderate CKD on a 
low Ca/High P (LCa/HP) diet vs. low Ca/low P (LCa/LP) diet. These data will be used for power 
calculations to determine an adequate sample size for a subsequent clinical trial to be proposed in an 
R01.  
 
Four participants will be enrolled in this randomized cross-over study, 50% female, and we aim for at 
least ¼ black/African American and at least ¼ white race. All N = 4 will be included in Aim 1 with the 
primary outcomes of fractional Ca, P, and Mg absorption from a 48-hour testing periods. A subset of 
N = 2 subjects will be studied under Aim 2, with a week of Ca, P, and Mg balance and full kinetic 
model (12 days (5 as inpatient) beyond the initial 48-hour inpatient absorption test protocol).  
 
At the beginning of the study, the study biostatistician (Dr. McCabe) will generate a randomization list 
for dietary intervention crossover order A or B by pairs. These orders will be [LCa/HP to LCa/LP] or 
[LCa/LP to LCa/HP]. Especially due to the small sample size, attention will be made to ensuring a 
balance of the two orders, which will be accomplished by randomizing both in pairs. 
 
Based on our experience, we anticipate only a 15% attrition rate, which is only 0-1 enrolled subjects 
needing to be replaced. However, because of the small sample size of 4 and the need for high 
adherence due to the preliminary nature of these studies, we seek approval enrolling up to 6 subjects 
to complete our planned n = 4. 
 
Statistical Analysis:  A repeated measures analysis of variance (ANOVA) for cross-over designs will 
be sued to compare the LCa/HP diet with the LCa/LP diet on the primary outcomes: 
Aim 1:  
 
 
 
 
Aim 2:  
Fractional Ca Absorption   
 
Ca Balance 
Fractional P Absorption  
 
 
P Balance 
 
Data will be examined for outliers and distributional model assumptions will be checked.  Remedial 
measures will be taken as needed. 
 
Power Calculations:  The sample size required for the future R01 proposal will be based on the 
mean differences observed in the primary outcomes between the LCa/HP and LCa/LP diets, as well 
as the within- and among-patient variance estimates. We will determine sample sizes needed to 
detect a range of mean differences between diets with 80% to 90% power for a cross-over study.  
 
13.0 Statistical Data Management 
 

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Primary data will be collected via paper, in-person and phone interviews, direct data capture from 
measurement instrument and stored electronically in REDCap, OnCore, and on the IU Dept of Medicine 
Department Server, and a Box REED (secure, HIPAA compliant) folder assigned to Dr. Hill Gallant via Purdue.  
The electronic storage location will be backed up automatically every day.  Other data sources include outside 
lab data that will be stored in separate electronic files and merged with the primary data as needed.  Quality 
assurance steps will include: 1) built in range checks and 2) testing of RedCap database by study team prior to 
moving to production mode. The following quality control methods will be used:  1) single entry with random 
checks of accuracy; and 2) extraction and cleaning of data that will be used for analysis every 6 months.  
 
14.0 Privacy/Confidentiality Issues 
 
All records associated with each subject’s participation in the study will be managed using confidentiality 
standards applicable to medical records, i.e. records will only be seen by IRB-approved individuals working on 
the study and records will be kept in a secure area. To minimize the risk of loss of confidentiality, any hard 
copies of all laboratory, demographic, and health-care related information obtained, will be kept will be kept in 
secure locked file cabinets in the Nephrology research group offices of Dr. Moe in University Hospital, and in 
the Department of Nutrition Science at Purdue University in the office or locked lab spaces of Dr. Hill Gallant. 
Electronic data will be stored on a Purdue Box REED (secure, HIPAA compliant) folder or IU Dept of Medicine 
Department Network Drive, accessible only to those with IRB approval. RedCap data management (through 
the Indiana CTSI) will be used for this study. All personally identifiable and medical data will be kept 
confidential, in accordance with federal regulations. Only the Hill Gallant Lab at Purdue University will maintain 
the subject ID code.  Codes will be stored in an electronic file separate from subject files. All specimens will be 
stored with coded de-identified labels. Specimens to be analyzed by other laboratories will be delivered with 
coded de-identified labels. All the specimens and data collected that contain protected identifiable information 
will be collected specifically for research purposes. The research staff is trained in privacy regulations. Only 
IRB-approved research staff will have access to the code key. No identifiable data are transmitted via unsecure 
Internet. FileLocker, Box REED, RedCap, or encrypted email will be used. The de-identified coded data will be 
used for statistical analysis. If publications result from this study, no reference will be made to data that can 
be linked to an individual participant. 
The likelihood of a loss of confidentiality is deemed to be low due to these protections put in place. Every effort 
will be made to maintain the highest level of confidentiality. 
 
 
15.0 Follow-up and Record Retention 
 
The study duration for subjects is up to ~2 months (for those in Aim 2). We anticipate the study will be 
completed within 12 months of enrolling the 1st subject.  
 
Records will be retained while the study is active and for 3 years following the closure of the study. De-
identified coded data and samples will be kept for future research purposes, but the key code will be 
destroyed as well as other identifiable information. Electronic identifiable data will be permanently scrubbed, 
and any hard copy identifiable will be shredded. 
 
16.0 References 
 
1. 
Centers for Disease Control and Prevention. United States. Chronic Kidney Disease Surveillance 
System. Atlanta. U.S. Department of Health and Human Services. Available at http://www.cdc.gov/ckdCenters 
for Disease Control and Prevention. Available at http://www.cdc.gov/ckd. 2011. 

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Isakova T, Wahl P, Vargas GS, Gutierrez OM, Scialla J, Xie H, Appleby D, Nessel L, Bellovich K, 
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Foley RN, Parfrey PS, Sarnak MJ. Clinical epidemiology of cardiovascular disease in chronic renal 
disease. American journal of kidney diseases : the official journal of the National Kidney Foundation. 
1998;32(5 Suppl 3):S112-9. Epub 1998/11/20. PubMed PMID: 9820470. 
8. 
United States Renal Data System. 2017 USRDS annual data report: Epidemiology of kidney disease in 
the United States. National Institues of Health, National Institute of Diabetes and Digestive and Kidney 
Diseases, Bethesda, MD, 2017. Availabe at: https://www.usrds.org/2017/view/Default.aspx. 
9. 
Gargiulo R, Suhail F, Lerma EV. Cardiovascular disease and chronic kidney disease. Dis Mon. 
2015;61(9):403-13. doi: 10.1016/j.disamonth.2015.07.005. PubMed PMID: 26328516. 
10. 
Taylor JG, Bushinsky DA. Calcium and phosphorus homeostasis. Blood Purif. 2009;27(4):387-94. doi: 
10.1159/000209740. PubMed PMID: 19299893. 
11. 
Sprague SM, Llach F, Amdahl M, Taccetta C, Batlle D. Paricalcitol versus calcitriol in the treatment of 
secondary hyperparathyroidism. Kidney international. 2003;63(4):1483-90. doi: 10.1046/j.1523-
1755.2003.00878.x. PubMed PMID: 12631365. 
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Ong LM, Narayanan P, Goh HK, Manocha AB, Ghazali A, Omar M, Mohamad S, Goh BL, Shah S, 
Seman MR, Vaithilingam I, Ghazalli R, Rahmat K, Shaariah W, Ching CH, Oral Paricalcitol in ESG. 
Randomized controlled trial to compare the efficacy and safety of oral paricalcitol with oral calcitriol in dialysis 
patients with secondary hyperparathyroidism. Nephrology (Carlton). 2013;18(3):194-200. doi: 
10.1111/nep.12029. PubMed PMID: 23311404. 
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Investigators ET, Chertow GM, Block GA, Correa-Rotter R, Drueke TB, Floege J, Goodman WG, 
Herzog CA, Kubo Y, London GM, Mahaffey KW, Mix TC, Moe SM, Trotman ML, Wheeler DC, Parfrey PS. 
Effect of cinacalcet on cardiovascular disease in patients undergoing dialysis. N Engl J Med. 
2012;367(26):2482-94. doi: 10.1056/NEJMoa1205624. PubMed PMID: 23121374. 
14. 
Hill KM, Martin BR, Wastney ME, McCabe GP, Moe SM, Weaver CM, Peacock M. Oral calcium 
carbonate affects calcium but not phosphorus balance in stage 3-4 chronic kidney disease. Kidney international. 
2013;83(5):959-66. doi: 10.1038/ki.2012.403. PubMed PMID: 23254903. 
15. 
Spiegel DM, Brady K. Calcium balance in normal individuals and in patients with chronic kidney 
disease on low- and high-calcium diets. Kidney international. 2012;81(11):1116-22. Epub 2012/02/03. doi: 
10.1038/ki.2011.490 
ki2011490 [pii]. PubMed PMID: 22297674; PMCID: 3352985. 
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17. 
Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF, 3rd, Feldman HI, Kusek JW, Eggers P, Van 
Lente F, Greene T, Coresh J, Ckd EPI. A new equation to estimate glomerular filtration rate. Annals of internal 
medicine. 2009;150(9):604-12. PubMed PMID: 19414839; PMCID: PMC2763564. 
18. 
Ross AC, Taylor CL, Yaktine AL, Del Valle HB, editors. Institute of Medicine. Dietary Reference 
Intakes for Calcium and Vitamin D: National Academy Press; 2011. 
19. 
Mangano KM, Walsh SJ, Insogna KL, Kenny AM, Kerstetter JE. Calcium intake in the United States 
from dietary and supplemental sources across adult age groups: new estimates from the National Health and 
Nutrition Examination Survey 2003-2006. Journal of the American Dietetic Association. 2011;111(5):687-95. 
doi: 10.1016/j.jada.2011.02.014. PubMed PMID: 21515114; PMCID: PMC3896234. 
20. 
Chang AR, Lazo M, Appel LJ, Gutierrez OM, Grams ME. High dietary phosphorus intake is associated 
with all-cause mortality: results from NHANES III. The American journal of clinical nutrition. 2014;99(2):320-
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21. 
Institute of Medicine. DRI: Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, 
and fluoride. Washington, DC: National Academy Press; 1997. 
22. 
Academy of Nutrition and Dietetics Evidence Analysis Library. "Chronic Kidney Disease (CKD): 
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23. 
Wilkinson R. Polyethylene glycol 4000 as a continuously administered non-absorbable faecal marker for 
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17.0 Appendix 
 
A. Figure 2 – Study Timeline – larger version 
B. Study Calendar of Assessments 
C. DSMP (submitted to NIH) 
D. Email regarding RDRC review and approval of protocol 
E. Study Description Visual Aid for use with subjects for Aim 1 (2 day absorption test only) 
F. Study Description Visual Aid for use with subjects for Aim 2 (14 day balance and kinetics)