Conditions: Chronic Kidney Disease Cardiovascular Disease
Keywords: hyperphosphatemia vascular dysfunction chronic kidney disease cardiovascular disease endothelial dysfunction
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 66
Healthy volunteers: no

Brief summary:
The proposed research is a randomized-controlled trial to determine the effectiveness of reducing serum phosphorus using a phosphate binder, lanthanum carbonate, for improving the function of arteries in adults with moderate to severe chronic kidney disease (CKD). \[COMIRB 13-0328\] Additionally, it will determine phosphorus balance among adults with CKD and whether there is a difference in phosphorus balance after three months of treatment with lanthanum carbonate. \[COMIRB 15-0384\]

Detailed description:
Chronic kidney disease (CKD) is a major health concern both in the general and Veteran populations. Indeed, the prevalence of CKD in a large Veteran population is 20%. Cardiovascular disease (CVD) is significantly increased in CKD and is an important cause of morbidity and mortality. As much as 80% of all CVD is associated with vascular dysfunction, particularly impaired endothelium-dependent dilation (EDD), measured by brachial artery flow-mediate dilation (FMD), and stiffening of the large elastic arteries, measured by aortic pulse-wave velocity (aPWV). Not surprisingly, patients with CKD demonstrate these dysfunctional vascular phenotypes. Even in early stages of CKD, there is an increase in oxidative stress resulting in structural and functional vascular changes, which, in turn, contributes to vascular dysfunction (impaired EDD and large elastic artery stiffening). In CKD, phosphorus remains within the normal range (2.5-4.5 mg/dL) until late in the disease. However, elevated serum phosphorus, even within the normal range, is associated with impaired EDD and with indirect measures of arterial stiffness. Whether lowering serum phosphorus in patients with CKD will improve EDD and arterial stiffness is unknown. This study is a randomized-controlled trial of lanthanum carbonate, a non-calcium based phosphate binder, to treat vascular dysfunction. The efficacy of phosphate binding with lanthanum carbonate for treating vascular endothelial dysfunction and large elastic artery stiffness in patients with stage IIIb and IV CKD (estimated glomerular filtration rate 15-45 mL/min/1.73m2) with baseline serum phosphorus of 2.8-5.5 mg/dL will be assessed. The study will also determine if lowering serum phosphorus with lanthanum carbonate also reduces circulating and endothelial cell markers of oxidative stress. This study could shift clinical practice guidelines by establishing a novel therapy for reducing CVD risk in CKD patients not requiring chronic hemodialysis. \[COMIRB 13-0328\]

Little is known about phosphorus balance in CKD. It is assumed that CKD patients remain in neutral phosphorus balance despite decreases in kidney function. Serum phosphorus remains in the normal range until late in CKD thus making it difficult to recognize perturbations in phosphorus balance. Indeed, among CKD patients treated with the non-calcium containing phosphate binder, sevelamer, serum phosphorus did not change after six weeks of treatment but urinary phosphate excretion, parathyroid hormone, and fibroblast growth factor-23 changed significantly, suggesting a shift in phosphorus homeostasis. However, two other studies found that patients with CKD III-IV treated with calcium-containing phosphate binders remained in neutral phosphorus balance. There are no studies evaluating the effects of non-calcium based phosphate binders on phosphorus balance among patients with CKD nor other studies examining the effect of changing phosphorus balance on vascular function.

An extension of the above-described 12-week prospective randomized, placebo-controlled double-blind trial (COMIRB 13-0328) will be conducted in a subset of subjects. A total of 24 subjects from COMIRB 13-0328 will be recruited to participate in the Phosphorus Balance sub-investigation (12 subjects treated with lanthanum carbonate and 12 subjects treated with placebo). \[15-0384\] They will consume a diet with a fixed phosphorus content (1000 +/- 50 mg) for seven days. They will then be admitted to the inpatient Center for Translational Clinical Research at the University of Colorado Denver for 48 hours to accurately collect urine and stool samples. The goal of the Phosphorus Balance sub-investigation (COMIRB 15-0384) is to determine whether lowering serum phosphorus, accomplished during the parent phosphorus lowering randomized-controlled trial (COMIRB 13-0328), affects phosphorus balance compared to those subjects treated with placebo. A key secondary goal is to determine if differences in phosphorus balance affect vascular function as measured by FMD. \[15-0384\]

To ensure adequate enrollment in the Phosphorus Balance Study (COMIRB 15-0384), an amendment was approved to recruit patients with stage IIIb and IV CKD with normal or modestly elevated serum phosphorus (2.8-5.5 mg/dL) who are not currently participating in the parent Phosphorus Lowering RCT (COMIRB 13-0328). Similar to the Phosphorus Lowering RCT, these patients will follow a low phosphorus diet and will be randomized to lanthanum carbonate or placebo for 12 weeks (run-in period) prior to beginning the current Phosphorus Balance protocol.

Arms:
arm 1: Eligible subjects who are randomly assigned to the experimental arm will receive lanthanum carbonate (1500-4500 mg/day in divided doses) titrated to serum phosphorus levels. arm 2: Eligible subjects who are randomly assigned to the placebo arm will receive placebo tablets (identical to the active lanthanum carbonate tablets) to be taken 3 times daily and titrated to serum phosphorus levels.

Interventions: intervention 1: Lanthanum carbonate intervention 2: placebo intervention 3: Ascorbic Acid intervention 4: Nitroglycerin intervention 5: Flow-mediated dilation measurement intervention 6: Aortic pulse-wave velocity intervention 7: Endothelial cell collection
Intervention descriptions: intervention 1: Non-calcium containing phosphorus binder intervention 2: Table identical to lanthanum carbonate but with no active ingredient intervention 3: Intravenous administration during measurement of flow mediated dilation. intervention 4: Drug that is administered under the tongue that relaxes blood vessels. To be administered during measurement of flow mediated dilation. intervention 5: Measurement of the blood flow in the brachial artery, an artery in the upper arm, using ultrasound. intervention 6: Measurement of the stiffness of the arteries using a transcutaneous tonometer, a small device placed over the skin over the carotid, brachial, radial and femoral arteries. intervention 7: Collection of endothelial cells from vein in arm. A flexible wire is inserted through an IV into a forearm vein to collect endothelial cells for further study. This is performed at the baseline visit and and the final study visit.

Protocol document:
Prot_SAP_000.pdf: 
COVER PAGE 
 
Title: Phosphate Lowering Trial 
NCT Number: 02209636   
Document Date: 
 
Main Protocol (Aims 1 and 2) – 8/14/2015 
 
Substudy Protocol (Aim 3) – 6/07/2017 


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 COMIRB Protocol 
 
PROTOCOL #: 
13-0328 
 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Project Title: Phosphate Lowering to Treat Vascular Dysfunction in Chronic Kidney Disease 
 
Principal Investigator:  
Anna Jovanovich, M.D. 
Version Date: 08/14/2015 
 
I. Hypotheses and Specific Aims:  
 
Risk of cardiovascular diseases (CVD) is significantly elevated in patients with chronic kidney disease (CKD); 
however, the increased cardiovascular risk in this patient population is only partially explained by traditional 
cardiovascular risk factors 1-6.  In earlier stages of CKD, serum phosphorus levels are maintained within the 
normal range by up regulating fibroblast growth factor 23 (FGF23) and a consequent increase in the fractional 
excretion of phosphorus by the kidney 7-10.  However, elevated serum phosphorus even within the normal 
range, as is the typical case in CKD patients not requiring chronic hemodialysis, has emerged within the last 
decade as a novel, independent predictor of CVD risk and associated mortality 9, 11, 12. 
 
CKD patients also exhibit vascular endothelial dysfunction and increased arterial stiffness 13, 14, two 
independent predictors of future CVD.  A modest elevation in serum phosphorus contributes to this phenotype 
of vascular dysfunction 15-18.  Recent evidence supports that an acute increase in serum phosphorus impairs 
endothelium-dependent dilation (EDD) and increases vascular oxidative stress 15.  In addition, both vascular 
endothelial dysfunction and increased oxidative stress have been identified as important predictors of mortality 
and cardiovascular events in patients with CKD 19, 20.  Thus, reducing serum phosphorus burden in CKD 
patients not requiring chronic hemodialysis may reduce cardiovascular events by improving vascular 
endothelial function and reducing large elastic artery stiffness.  However, it is currently unknown if reducing 
serum phosphorus to a low normal laboratory range (<2.8 mg/dL) with oral phosphate binders enhances 
vascular function in this population. 
Our primary goal is to establish the efficacy of phosphate binding with lanthanum carbonate, a non-absorbed, 
non-calcium-containing phosphate binder, for treating vascular endothelial dysfunction and large elastic artery 
stiffness in patients with stage IIIb or IV CKD (estimated glomerular filtration rate (eGFR) 15-45 
mL/min/1.73m2) with baseline serum phosphorus of 2.8-5.5 mg/dL.  A key secondary goal is to determine the 
integrative physiological mechanisms underlying the beneficial effects of phosphate binding with lanthanum. 
 
Hypothesis 1: 3 months of treatment with lanthanum (starting dose of 1500-3,000 mg/day according to 
baseline serum phosphorus; up-titrated monthly as needed based on serum phosphorus) vs. placebo will 
improve endothelium-dependent dilation (EDD) and reduce large elastic artery stiffness in patients with CKD.  
 
Specific Aim 1: To determine conduit artery (brachial artery flow-mediated dilation; FMDBA) EDD and large 
elastic artery stiffness (aortic pulse-wave velocity; aPWV) before and after 3 months of lanthanum or placebo. 
 
Hypothesis 2: The improvements in EDD and arterial stiffness in the lanthanum group will be associated with 
reduced oxidative stress. 
 
Specific Aim 2: To measure FMDBA and aPWV during normal vs. inhibited oxidative stress, under the 
conditions described in Specific Aim 1; to measure circulating and endothelial cell markers of oxidative stress. 
 
II. Background and Significance:  
 
Chronic Kidney Disease and Cardiovascular Diseases.  Chronic kidney disease (CKD) is a major public 
health concern that has reached epidemic proportions, affecting ~20 million individuals (~13.1% of the 
population) in the United States alone.  Patients with chronic kidney disease (CKD) are more likely to die of 
cardiovascular diseases (CVD) than to progress to end stage renal disease 9, 21-23, and the risk of 
cardiovascular mortality or a cardiovascular event is significantly increased compared to the general population 


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24-26.  While patients with CKD exhibit a high presence of traditional CVD risk factors, they can only partially 
explain the increased incidence of CVD in this population 27.  Therefore, it is imperative to identify interventions 
targeting non-traditional cardiovascular risk factors to reduce CVD incidence in patients with CKD.   
 
Vascular Function and CKD.  As much as 80% of all CVD are associated with dysfunction and disorders of 
arteries 28.Two of the greatest contributors are the development of vascular endothelial dysfunction, most 
commonly assessed as impaired endothelium-dependent dilation (EDD), and stiffening of the large elastic 
arteries (aorta and carotid arteries) 29.  Patients with CKD demonstrate impaired EDD, as assessed by brachial 
artery flow-mediated dilation (FMDBA)30-32, as well as large elastic artery stiffening, as indicated by increased 
aortic pulse wave velocity (aPWV)13, 33, 34.  Importantly, both measures are independent predictors of future 
cardiovascular events and mortality 35-38. 
 
Integrative Physiological Mechanisms of CKD and Vascular Function.  A key mechanism contributing to 
impaired EDD, even in earlier stages of CKD, is increased oxidative stress 39-41, defined as excessive 
bioavailability of reactive oxygen species (ROS) relative to antioxidant defenses.  Superoxide, a major ROS in 
arteries, reacts with nitric oxide (NO) to form another ROS, peroxynitrite.  Peroxynitrite, in turn, decreases the 
bioactivity of tetrahydrobiopterin (BH4), the essential cofactor for NO synthesis by endothelial NO synthase 
(eNOS) 42, 43.  Together, the formation of peroxynitrite and reduced BH4 decrease NO bioavailability 44, 45.  This 
reduction in NO is a key physiological mechanism in the CKD-associated impairment in EDD 46, 47. Reduced 
NO bioavailability is a common mechanism contributing to both impaired EDD and increased arterial stiffness.  
 
Phosphate, CKD and Vascular Function.  It is widely accepted that phosphorus is maintained within the 
normal range (2.5-4.5 mg/dL) as CKD develops by a compensatory up-regulation of fibroblast growth factor 23 
(FGF23) that increases fractional excretion of phosphorus, thus controlling serum phosphorus to avoid the 
potentially deleterious consequences of hyperphosphatemia on the vasculature 7-10.  However, elevated serum 
phosphorus even within the normal range, as is the typical case in early stage CKD, has emerged within the 
last decade as a novel, independent predictor of CVD risk and associated mortality 9, 11, 12.  Recent evidence 
suggests that the increase in CVD risk may be modulated by the adverse influence of phosphorus upon 
vascular function 15, 48.  
 
Phosphate and EDD.  Impaired EDD is one component of vascular dysfunction that may be modulated by 
elevations in serum phosphorus.  Following an acute high phosphate meal (1,200 mg), FMDBA is impaired in 
young adults.  This occurs despite the fact that the elevation in serum phosphorus is only slightly above the 
normal range (4.56 mg/dL) 15.  Increased serum phosphorus also impairs EDD in both healthy 15 and CKD 
rodent models 16.  In CKD patients with baseline hyperphosphatemia (>5.5 mg/dL), lowering serum 
phosphorus with the phosphate binder sevelamer improves FMDBA compared to calcium acetate49, 50. 
However, the efficacy of phosphorus lowering prior to CKD patients becoming overtly hyperphosphatemic, as 
well as the physiological mechanisms contributing to any functional improvements with phosphorus lowering, 
are currently unknown.   
 
Phosphate and Arterial Stiffness.  The available evidence suggests that serum phosphorus may also 
increase arterial stiffness.  Modest increases in serum phosphorus in healthy adults and patients with 
moderate CKD are independently associated with an increased ankle-brachial index (an indirect measure of 
arterial stiffness), even when serum phosphorus is within the normal range 17.However, it is unknown if 
lowering serum phosphorus in CKD patients reduces arterial stiffness, as well as the mechanisms involved. 
 
\\Integrative Physiological Mechanisms of CKD and Phosphate on Vascular Function.  No direct evidence 
is available in humans as to the physiological mechanisms mediating any improvements in vascular function 
with phosphorus lowering.  However, work in endothelial cell culture supports a role of oxidative stress and NO 
bioavailability, as administration of high phosphate increases production of superoxide 15, 51, 52 and reduces NO 
bioavailability 15, 51, 53 and eNOS activation/expression 15, 53.  In rodents, modest reductions in dietary phosphate 
decreases oxidative stress 54, 55.  Thus, reduced oxidative stress, which in turn increases NO bioavailability, 
likely contributes to any improvements in vascular function in CKD patients following phosphorus lowering.   
 
III. Preliminary Studies/Progress Report:   
 


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We have demonstrated that there is a longitudinal relation between higher serum phosphorus and all-cause 
mortality and cardiovascular events (myocardial infarction, stroke and peripheral vascular disease), and a 
cross-sectional association between elevated serum phosphorus and worsening pulse pressure (PP).  These 
data were obtained from the Homocysteine Study in Advanced Kidney Disease (HOST), which was a 
prospective, randomized, double-blind study on the effects of folate, vitamin B6, and vitamin B12 replacement 
on all-cause mortality and cardiovascular events in patients with severe CKD and elevated homocysteine 56.  
As the intervention did not have an effect on the proposed outcomes, it represented a unique opportunity to 
convert the data obtained from a randomized control trial into an observational study.  The HOST dataset 
contains detailed information on demographics, cardiovascular risk factors, prevalent CVD, circulating markers 
(including serum calcium, phosphorus and intact PTH) and usage of cardioprotective medications in all study 
participants.  In addition, an endpoints committee adjudicated all endpoints.  For the purpose of this analysis, 
we included 1,099 HOST participants.  The mean (+SD) age of the participants was 69±11 years, 98% were 
male, and 26% were black.  The mean estimated Modified Diet Renal Disease (MDRD)-GFR was 18±6 
mL/min/1.73 m2 and most participants (n=718; 65%) had an estimated MDRD-GFR of 15-29 mL/min/1.73 m2.  
The mean serum phosphorus concentration was 4.3±1.3 mg/dL; 36% and 12% had a serum phosphorus 
concentration greater than 4.5 and 5.5 mg/dL, respectively.  These descriptive data support that most of 
the patients that will be screened for the proposed study will have serum phosphorus < 5.5 mg/dL.  
 
Higher serum phosphorus levels are independently associated with CVD events and mortality in 
patients with advanced CKD: During a median follow-up of 2.9 years, 472 (42.9%) patients died from any 
cause and 237 (21.6%) had a cardiovascular event. Higher serum phosphorus levels were positively 
associated with higher risks for composite of death and cardiovascular events. Identical results were observed 
when cardiovascular events were examined separately (not shown). Compared to the lowest quartile, the 2 
highest quartiles of serum phosphorus were associated with a significantly elevated risk for the composite of 
death and cardiovascular events, after multivariate adjustment (Table 1; hazards ratios (HR)). The analysis 
was repeated using serum phosphorus levels as a continuous variable instead of quartiles.  After multivariable 
adjustment (Model 3), the HR of the composite endpoint of death and cardiovascular events increased by 1.17 
(95% CI; 1.02-1.33; p=0.02) per 1 mg/dL increase in serum phosphorus.  These results provide important 
support to our aim to evaluate the efficacy of phosphorus lowering on vascular endothelial function and arterial 
stiffness (two independent predictors of CV events and mortality) in patients with advanced CKD. 
 
 
Table 1. Association of Higher Serum Phosphorus with Risk of Cardiovascular Events & Death in the HOST Study 
Phosphorus Concentrations 
Q1 (3.5 mg/dL) 
Q2 (3.6-4.1 mg/dL) 
Q3 (4.2-4.8 mg/dL) 
Q4 (>4.9 mg/dL) 
Model 1 
1.00 
1.35(0.87- 2.09) 
1.70 (1.12- 2.57) 
1.93(1.29- 2.90) 
Model 2 
1.00 
1.36 (0.88- 2.11) 
1.79(1.18-2.73) 
2.18(1.44-3.31) 
Model 3 
1.00 
1.19 (0.76-1.86) 
1.50 (1.01-2.31) 
1.68 (1.05-2.68) 
 
Model 1: Unadjusted; Model 2: Adjusted for age, gender and race; Model 3: Adjusted for covariates in Model 2 plus smoking, 
diabetes, hypertension, prevalent CVD, body mass index, systolic and diastolic blood pressure, treatment arm, eGFR, albumin, 
calcium, 25-hydroxyvitamin D, intact PTH, LDL-cholesterol, high density lipoprotein-cholesterol, triglycerides and use of angiotensin 
converting enzyme inhibitors, angiotensin receptor blockers and β-blockers. 
 
In addition, in a cross-sectional analysis of the relation between serum phosphorus and PP in the HOST 
cohort, higher serum phosphorus levels are independently associated with a wider PP in patients with 
advanced CKD.  The mean (+SD) PP in this population was 71 (20) mmHg.  In adjusted linear regression 
models (Model 3), each 1 mg/dL increase in serum phosphorus was associated with a 2 mmHg increase in PP 
(=2.06.9; p=0.005).  These data support the notion that serum phosphorus lowering should reduce large 
elastic artery stiffness, as increased PP is an expression of arterial stiffness. 
 
IV.  Research Methods 
 
A.  Outcome Measure(s): A prospective, randomized, placebo-controlled double-blind trial of 60 patients will 
be performed to assess the effects of the phosphate binder lanthanum in stage IIIb and stage IV chronic kidney 
disease (CKD) patients with normal or modestly elevated serum phosphorus (2.8-5.5 mg/dL) on vascular 
endothelial function and arterial stiffness.  Vascular endothelial function will be assessed as endothelium-
dependent dilation via brachial artery flow-mediated dilation (FMDBA).  Arterial stiffness will be assessed as 
aortic pulse-wave velocity (aPWV) using applanation tonometry.  The primary outcomes are the differences 


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between the active and placebo groups in change in FMDBA and aPWV at week 12 compared to baseline. 
Secondary aims will determine whether phosphorus lowering improves vascular function through reduced 
oxidative stress and/or inflammation by assessing pre-/post- differences between groups for FMDBA and aPWV 
measured during normal vs. inhibited oxidative stress and by measuring circulating and endothelial cell 
markers of oxidative stress and inflammation.  Additional blood chemistries and vital signs will be monitored 
throughout the study for subject safety.  All testing will take place it the laboratory in the University Physicians 
Incorporated (UPI) Building at the University of Colorado Anschutz Medical Campus. 
 
B. Description of Population to be Enrolled: Study Design and Research Methods  
 
Subjects.  After obtaining their written informed consent, men and post-menopausal women aged 40-79 yrs 
with stage IIIb and stage IV CKD (eGFR 15-45 mL/min/1.73m2) and normal or modestly elevated serum 
phosphorus (2.8-5.5 mg/dL) will serve as subjects.  Patients will undergo screening for eGFR and phosphorus 
at the UPI Building to determine study eligibility.  Estimated GFR will be calculated using the 4-variable MDRD 
prediction equation 57. Major inclusion/exclusion criteria are presented below:  
 
Inclusion Criteria: 
1) Aged 40-79 yrs; women must be post-menopausal (serum phosphorus increases after menopause in 
women 58) 
2) CKD stage IIIb or IV (eGFR with the 4-variable MDRD prediction equation: 15-45 mL/min/1.73m2; stable 
renal function in the past 3 months) 
3) Baseline serum phosphorus 2.8-5.5 mg/dL (stable in the past 3 months); not taking phosphate binders 
4) Serum albumin concentration >3.0 g/dL 
5) Mini-mental state examination (MMSE) score of  24 (cognitive function screening); ability to provide 
informed consent 
 
Exclusion Criteria: 
 
1) Patients with advanced CKD requiring chronic dialysis 
2) Patients at high risk for gastrointestinal events or alterations in gastrointestinal anatomy(e.g., history of 
gastrointestinal surgery, colon cancer), hypomotility disorders (e.g., constipation, ileus), current bowel 
obstruction, or fecal impaction  
3) Significant co-morbid conditions that lead the investigator to conclude that life expectancy is less than1 
year 
4) Expected to undergo living related transplant in next 6 months 
5) History of severe congestive heart failure (i.e., EF < 35%) 
6) History severe liver disease 
7) Hospitalization in the past 3 months 
8) BMI >40 kg/m2 (FMD measurements can be inaccurate in severely obese patients) 
9) Taking medication(s) that interact with agents administered during experimental sessions (e.g., sildenafil 
interacts with nitroglycerin) 
10) Active vitamin D analogue use (i.e., calcitriol, paricalcitol, doxercalciferol) 
11) Alcohol dependence or abuse  
12) Current tobacco abuse 
13) Use of prednisone and other immune suppressant medications 
 
C. Study Design and Research Methods   
 
Experimental Design (Figure 1).   A 12-week randomized, placebo-controlled, double-blind design study with 
lanthanum (starting dose of 1,500-3,000 mg/day according to baseline serum phosphorus; up-titrated monthly 
as needed based on serum phosphorus) will be conducted.  After providing informed consent, subjects will 
undergo screening prior to study start.  Subjects who meet inclusion criteria will have baseline measurements 
performed as described below.  Measurements will be made under supine, overnight fasted (water only) 
conditions.  Patients will then be randomly assigned to receive either lanthanum or placebo (see dosing 
below).  Allocation to the active drug versus placebo group will be random, and a stratification process will be 
used on the basis of baseline FMDBA of <4% and ≥4%  to protect against a baseline imbalance that would not 


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be well handled by other statistical procedures.  With counseling and adherence monitoring from a nurse, 
subjects will maintain their baseline diet throughout the study, including intake of phosphate of <1,000 mg/day. 
Measurements will then be repeated after 12 weeks.  The investigators involved in the acquisition and analysis 
of key outcomes will be blinded to the treatment assignment of the subjects as well as serum phosphorus 
levels. 
 
Figure 1: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Subjects will come to the lab at the UPI Building on 9 separate occasions: 
• 
Session 1:Screening measurements, complete 3 day diet record 
• 
Session 2: Baseline measurements 
• 
Resting arterial blood pressure and blood sampling of circulating markers (see below) 
• 
Venous endothelial cell collection (for subsequent protein measurements) 
• 
FMDBA (endothelium-dependent dilation) and aortic PWV (arterial stiffness), with and without 
acute inhibition of oxidative stress via systemic i.v. infusion of ascorbic acid [vitamin C]) 
• 
Endothelium-independent dilation (EID) to sublingual nitroglycerin 
• 
Begin study treatment (lanthanum or placebo) 
• 
Sessions 3-8: Weekly/monthly check-in (see below) 
• 
Session 9: Identical to Session 2 (note: subjects will not take a dose the a.m. prior to testing session) 
• 
Subjects will also complete a second 3 day diet record 
 
Lanthanum dosing.  The starting dose of lanthanum will be determined from a sliding scale according to 
baseline serum phosphorus (Table 2).  During the first 4 weeks, the dose of the phosphate binder will be 
titrated weekly to achieve serum phosphorus less than 2.8 mg/dL (during this period serum phosphorus will be 
measured weekly).  After the initial 4 weeks, the dose of lanthanum will be titrated every four weeks until the 
end of the study to achieve serum phosphorus in the aforementioned target.  This dose will be up-titrated as 
needed based on serum phosphorus (if not <2.8 mg/dL, dose will be increased by 1 tablet [500 mg] for each 
meal).  If serum phosphorus decreases to <2.5 mg/dL, the dose of lanthanum will be down-titrated.  Tablets 
will be taken with each meal (if a subject consumes <3 meals/day, the dosing will be adjusted accordingly). 
 
Table 2: 
 
 
 
 
 
 
 
Rationale of using serum phosphorus for up-titration of lanthanum: 
(1) Although high FGF23 levels have also been associated with death and cardiovascular events in CKD, it is 
presently unclear what cutoff should be used as a treatment target. 
(2) Normalization of FGF23 with use of phosphate binders is not plausible, as factors other than serum 
phosphorus may be involved in the regulation of FGF23. 
(3) A compelling idea would be to up-titrate the lanthanum dose to lower phosphorus.  Although we considered 
this option, we preferred the up-titration on the basis of serum phosphorus as the epidemiological data on 
the relation between elevated phosphorus and outcomes is significantly stronger than for changes in 
phosphorus. To date, a relation between FePO4 and outcomes in patients with CKD has not been 
demonstrated59. 
Serum Phosphorus (mg/dL) 
Starting Dose (mg) 
2.8-4.0  
1,500 (1 tablet with each meal) 
4.0-4.5 
 3,000(2 tablets with each meal) 
4.5-5.5 
4,500(3 tablets with each meal) 
Subjects randomly assigned to Fosrenol or placebo 
 
Baseline Measurements 
Screening 
12 weeks Lanthanum 
12 weeks placebo 
 
Baseline Measurements 
 
Post Measurements 
 
Post Measurements 
Session 1 
Session 2 
Sessions 3-8  
Session 9 


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(4) Finally, phosphorus and FGF23 will be followed closely in the study to obtain better understanding of how 
phosphate binding affects these important markers of phosphate homeostasis. 
 
Those who drop-out/do not complete the study will undergo an end-of study visit which will include identical 
measurements to the ones planned for session 9.  For those completing the entire study, total participation is 
approximately 13 hours spread over 9 visits (approximately 1 week for screening + 12 weeks the research 
study). 
 
Power. An effect size of 1.2 was estimated from published data assessing the effects of sevelamer on vascular 
function in CKD patients with hyperphosphatemia 49, 50.  A sample size of 10 subjects/ group was calculated 
based on this effect size, 90% power and an alpha level of 0.05.  However, to ensure that there is adequate 
power, as patients without overt hyperphosphatemia may not respond as strongly to the intervention, studies 
will be completed on 20 subjects/group.  Assuming a dropout rate of ~30% (10 subjects/group) based on 
our experience with similar studies, I will plan to enroll up to ~30 subjects/group (~60 total subjects total).   
 
Study Sites 
Patients for the study will be recruited from the Nephrology Clinic at the UC-Denver Anschutz Medical Campus 
and from the Denver VA Medical Center Renal Clinic with access to >950 CKD patients from all over the 
Denver/Boulder metro area (at least 33% with ICD 9 code 585.3 and 585.4 as diagnosis) with over 2146 visits 
in the past year.  
 
 
Based on the preliminary screening of our database at the University of Colorado Hospital Renal Clinic, we 
expect at least 100 eligible patients per year. The screening evaluation, all follow-up patient visits, study 
measurements, and blood draws will take place at the UPI Building. 
 
Randomization and Treatment 
Once inclusion/exclusion criteria have been met and informed consent obtained patients will undergo baseline 
measurement of primary and secondary (exploratory) outcomes. Once baseline measurements are obtained, 
patients will be randomized to either lanthanum or placebo. Randomization will occur within 4 weeks of the 
screening evaluation and will be performed by Dr. Eugene Nuccio, a statistician, by blocks using a computer-
generated procedure to ensure equal distribution. The lanthanum and placebo will be prepared and provided 
by Shire and will be identical.  There will be no cost to the PI for the study drug and placebo  
 
a) Study Drug: Lanthanum carbonate (Fosrenol) is an FDA approved non-absorbed, non-calcium-
containing phosphate binder to lower serum phosphate in patients requiring chronic dialysis. 
 
b) Rationale for lanthanum dose: This dosing is indicated in patients with end-stage renal disease 
requiring chronic dialysis.  The population being studied in this protocol will be healthier than this patient 
group.  In addition, an IND has been obtained from the FDA.  
 
c) Blinding: The investigators and patients will remain blind to the study intervention, as the lanthanum and 
placebo will be identical.  A nurse at the UPI Building lab will be responsible for administering the pills, 
which will be stored in a locked cabinet on site.  A professional research assistant will be unblinded and 
will manage the study drug records and interact directly with the nursing staff for administration of the 
study drug.  The investigators will be blinded to laboratory results, and the research assistant will monitor 
laboratory results for safety and inform the investigator if necessary.  
 
d) Drug accountability: Pill counts will be assessed weekly during the 1st month and then monthly to 
document subject adherence. 
 
e) Withdrawal from study: Withdrawal will be done at the patient’s request, IRB request, or if there is a    
medical rationale for discontinuation as determined by the principal investigator.  
 
Safety monitoring  
The patients will be instructed to call the PI or research coordinator with any subjective complaints, in addition, 
abnormal labs will be reported to the PI immediately, who will determine if the study drug needs to be 
discontinued. The following are reported adverse events of the study drugs and the monitoring plan: 
 


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a) Lanthanum.  The safety profile of lanthanum has been studied in over 5200 subjects in completed 
clinical trials. The most common adverse reactions for lanthanum are gastrointestinal events, such as nausea, 
vomiting, and abdominal pain and they generally abate over time with continued dosing.  In end-stage renal 
disease patients receiving 4-6 weeks of treatment with lanthanum the incidence of nausea was 11% (vs. 5% 
placebo), vomiting 9% (vs. 4%) and abdominal pain 5% (vs. 0%).  Lanthanum may reduce calcium absorption 
and result in hypocalcemia (~5%, same incidence as active comparator group).  The following additional 
adverse reactions have been identified in during post-approval use of lanthanum: constipation, dyspepsia, 
allergic skin reactions, hypophosphatemia, and tooth injury (incidence not known). Lanthanum may decrease 
bioavailability of cationic antacids, quinolone antibiotics or levothyroxine.   
 
There have been reports of serious cases of gastrointestinal obstruction, ileus, and fecal impaction reported in 
association with lanthanum, some requiring surgery or hospitalization.  Risk factors for gastrointestinal 
obstruction identified from post-marketing reports include alteration in gastrointestinal anatomy (e.g., history of 
gastrointestinal surgery, colon cancer), hypomotility disorders (e.g., constipation, ileus, diabetes) and 
concomitant medications (e.g., calcium channel blockers). Some cases were reported in patients with no 
history of gastrointestinal disease. 
 
Hypophosphatemia has been reported only in post-marketing surveillance.  The risk of hypophosphatemia is 
very low.  Clinically significant (causing symptoms) hypophosphatemia associated with the use of lanthanum 
carbonate has not been reported.  
 
Safety measures will include: lanthanum dose titration and close monitoring of serum phosphate (as described 
above) to avoid severe hypophosphatemia, excluding patients at high risk for gastrointestinal events or current 
bowel obstruction, ileus and fecal impaction, repeatedly emphasizing to patients the importance of chewing 
tablets completely to reduce the risk of serious adverse gastrointestinal events such as those described above.  
Patients will also be instructed to take concomitant drugs at least one hour before or four hours after 
lanthanum to avoid potential interactions, and blood levels of concomitant drugs with a narrow therapeutic 
range will be monitored if deemed appropriate by Dr. Chonchol.   
 
Patient Stopping Criteria: 
•  Withdrawal of informed consent (subject’s decision to withdraw for any reason) 
•  Any clinical adverse event (AE), laboratory abnormality or intercurrent illness which, in the 
opinion of the investigator, indicates that continued participation in the study is not in the best 
interest of the subject 
•  Loss of ability to freely provide consent through imprisonment or involuntary incarceration for 
treatment of either a psychiatric or physical (e.g., infectious disease) illness 
•  Inability to comply with protocol 
•  Use of an investigational therapy or device other than study medication 
•  Significant non-compliance with protocol (ie, procedures, assessments, medication, etc.) 
•  Hypocalcemia that does not resolve with decreased dosage of study drug 
• 
Hypophosphatemia (<2.5 mg/dL) that does not resolve with decreased dosage of study drug 
• 
Hospitalization for gastrointestinal obstruction, ileus, and fecal impaction 
•  Serious acute hypersensitivity reactions to investigational drug 
•  Any major cardiovascular event such as myocardial infarction, stroke, unstable angina, 
thromboembolic event, or a clinically significant deterioration of a baseline cardiovascular 
condition. 
 
Study Stopping Criteria: 
The study may be stopped if:  
• The data show a significantly increased risk of serious adverse effects in one of the treatment groups.   
• Interim efficacy analyses show significant treatment benefits or futility in the intervention group.  The interim 
efficacy analyses are based on pre-specified stopping boundaries for the primary endpoint of the study which 
preserve the study wide Type I error rate.    


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• It becomes clear that successful completion of the study is not feasible (e.g. there is an excess of patient 
dropout, missing data, lack of recruitment etc). 
 
 
Standard medical treatment for all subjects/concomitant medications includes: 
All subjects must be on a stable anti-hypertensive, diabetic and lipid lowering regimen for at least one 
month prior to inclusion. 
a) Control of hypertension with anti-hypertensives with goal blood pressure less than 130/80 mm/Hg.  
Medication choice includes angiotensin converting enzyme inhibitor/angiotensin receptor blocker as first line 
agent, diuretic as second line agent, calcium channel blocker as third line agent, and beta blocker as fourth line 
agent (unless patient has known CVD and needs to be on a beta blocker regardless of blood pressure). b) 
Diabetes will be treated as needed to goal HbA1C of 7.0%. c) Control of hyperlipidemia with statins to goal of 
low density lipoprotein cholesterol (LDL-C) <100 mg/dL and total cholesterol levels to less than 200 mg/dL. d) 
Smoking cessation counseling. e) Control of anemia following the latest recommendation from the National 
Kidney Foundation. f) Control of hyperphosphatemia with phosphorus binders and down titration of the 
intervention as describe above to a goal phosphorus < 4.6 mg/dL. 
 
 
Data Collection 
 
Screening Measures and Potential Confounders: 
 
Medical History, Physical Examination and Blood Chemistries.  Subjects will report fasted for assessment 
of serum phosphorus and a standard blood chemistry analysis including a metabolic panel (with 
creatinine/calcium), blood count, lipid panel and follicle stimulating hormone (women only), then undergo a 
medical history and physical examination.  
 
Outcome Measures.  Please see below under “D. Description, Risks and Justification of Procedures and 
Data Collection Tools” 
 
Data management:  
Study data will be collected and managed using REDCap (Research Electronic Data Capture).  REDCap is a 
secure, web application designed to support data capture for research studies, providing user-friendly web-
based case report forms, real-time data entry validation (e.g. for data types and range checks), audit trails and 
a de-identified data export mechanism to common statistical packages (SPSS, SAS, Stata, R/S-Plus).  The 
system was developed by a multi-institutional consortium, which includes the University of Colorado Denver 
and was initiated at Vanderbilt University.  Currently 52 institutions in 4 countries are using the software. The 
database is hosted at the University of Colorado Denver Development and Informatics Service Center (DISC), 
which will be used as a central location for data processing and management.  REDCap data collection 
projects rely on a thorough study-specific data dictionary defined in an iterative self-documenting process by all 
members of the research team with planning assistance from the DISC. The iterative development and testing 
process results in a well-planned data collection strategy for individual studies. Both REDCap and REDCap 
Survey systems provide secure, web-based applications that are flexible enough to be used for a variety of 
types of research, provide an intuitive interface for users to enter data and have real time validation rules (with 
automated data type and range checks) at the time of entry. These systems offer easy data manipulation with 
audit trails and reporting for reporting, monitoring and querying patient records, and an automated export 
mechanism to common statistical packages (SPSS, SAS, Stata, R/S-Plus). Both systems were found to meet 
or exceed all of the security audit requirements. 
 
D.   Description, Risks and Justification of Procedures and Data Collection Tools: 
 
Primary Outcomes: 
a)Conduit artery vascular endothelial dependent dilation (EDD) and endothelial independent dilation 
(EID): Conduit artery EDD, brachial artery flow-mediate dilation (FMDBA), will be determined using high-
resolution ultrasonography (GE Vivid7 Dimension) as described originally by Celermajer et al. 60 and used 
recently by the P.I. 61, 62.  ECG-gated end-diastolic ultrasound images and Doppler flow of the brachial artery 
will be acquired during baseline and FMDBA conditions.  For FMDBA, reactive hyperemia will be produced by 


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inflating a pediatric BP cuff around the forearm to 250 mmHg for 5 minutes followed by rapid deflation.  
Brachial artery endothelium-independent dilation (EID), an index of smooth muscle sensitivity to exogenous 
nitric oxide, will be determined by measuring brachial artery dilation for 10 minutes after administration of 
sublingual nitroglycerin (0.4 mg) 63, 64.  A commercially available software package (Vascular Analysis Tools 
5.8.1, Medical Imaging Applications) will be used to acquire ECG-gated brachial artery diameters.  As recently 
recommended 65, FMDBA will be determined as the mm and % ∆ from baseline diameter 63-65.  Shear rate will 
also be calculated, and if group- or condition- differences exist, FMDBA will be adjusted accordingly 65-67. 
 
Risks of this procedure include: The assessment of FMDBA is minimally-invasive.  Inflating the blood 
pressure cuff below the elbow during this procedure may cause a mild to moderate intensity “pins and needles 
or numbing” sensation that goes away as soon as the cuff is deflated.  In addition, patients will receive a dose 
of sublingual nitroglycerin to measure EID, an essential control measure without which the results cannot be 
properly interpreted.  Sublingual nitroglycerin is a medication with a rapid vasodilator effect and is routinely 
used in patients with coronary artery disease to help relieve chest pain. Side effects of sublingual nitroglycerin 
are short-lived (30-60 minutes). Approximately 60% of people who receive nitroglycerin will experience a 
headache.  The subject may experience symptoms such as lightheadedness, and in rare instances (1 in 1000) 
lowered heart rate, which could lead to fainting.  However, these risks are primarily when a person is upright, 
and subjects will be lying supine during administration of nitroglycerin.  Finally, placing the pill under the tongue 
could cause irritation to the area in the back of the mouth.  The patients will be monitored by frequently asking 
about subjective complaints, as well as by frequent heart rate and blood pressure measurements (prior to and 
every 15 minutes after taking the medication for 1 hour).  In addition, during the 10-minute period when 
brachial artery dilation to nitroglycerin is assessed (peak activity of drug), blood pressure and heart rate will be 
assessed every 2 minutes. In the rare case that blood pressure drops drastically, an intravenous catheter will 
be available for infusion of atropine.  Still, nitroglycerin has been administered safely for over a decade in the 
laboratory the PI performed her doctoral training, as well as in other laboratories and in clinical situations 
worldwide.  Nitroglycerin will not be given to any subject with a systolic blood pressure less than 100 mmHg on 
the day the measurements are performed, history of migraine headaches or previous adverse reaction to 
administration. 
 
Justification: As discussed above in the background/significance sections, patients with CKD have an 
increased risk of death due to CVD, and endothelial dysfunction plays a critical role in the development of 
CVD.  Endothelial dysfunction is evident early in the course of kidney disease. It is unknown if administration of 
an inhibitor of inflammation improves vascular endothelial function in CKD patients. FMDBA is a non-invasive 
way to assess endothelial function. The other method available to study endothelial function (EDD) would 
include acetylcholine administration via brachial artery infusion, which is more invasive than the proposed 
methodology.  In order to interpret improvements in brachial artery dilation in response to a shear stress 
(FMDBA) following the drug intervention, it is necessary to assess smooth muscle sensitivity to nitric oxide using 
EID.  If EID is unchanged in the presence of improved FMDBA, this indicates the improved dilation in response 
to a shear stimulus is due to positive adaptations at the level of the vascular endothelium (i.e. endothelium-
dependent).  
 
b) Arterial Stiffness: Aortic pulse-wave velocity (aPWV) will be determined as described in detail previously 68, 
69.  Briefly, a transcutaneous custom tonometer (Noninvasive Hemodynamics Workstation (NIHem), 
Cardiovascular Engineering Inc., Norwood, MA) will be positioned at the carotid, brachial, radial and femoral 
arteries to non-invasively assess aPWV.  The tonometer sits on the surface on the skin and detects a 
waveform associated with the pulse at these various sites.  The time delay between the R-wave of each heart 
beat (ECG) and waveforms at these various sites is calculated using the NIHem software, and by also 
measuring the distance between these landmarks, velocity can be calculated (aPWV).  Increased pulse-wave 
velocity is indicative of increased stiffness of the aorta, a large elastic artery.  In addition, the carotid artery will 
be imaged using an ultrasound (GE Vivid 7) equipped with a linear array transducer as previously described 
51,52. Change in carotid diameter across 10-15 cardiac cycles will be analyzed using image analysis software 
(Carotid Analyzer) as previously described 51,53.  Using the change in carotid pressure also provided by 
tonometry with the NIHem and the change in diameter obtained with carotid artery ultrasound imaging, carotid 
artery compliance and the ß-stiffness index will be calculated as previously described70, 71, providing an index 


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of local arterial stiffness.  The carotid images will also be assessed for measurement of carotid artery intimal-
medial thickness (IMT), another CVD risk factor.  
 
Risks of this procedure include: The test is minimally-invasive and the main potential discomfort is from the 
pressure/placement of the tonometer and ultrasound probe.  
 
Justification: As discussed above in the background/significance sections, patients with CKD have an 
increased risk of death due to cardiovascular disease and large artery damage plays a critical role in CVD.  It is 
unknown if lowering serum phosphorus improves arterial stiffness in CKD patients.  These measurements will 
provide us with important insight into our specific aims.   
 
Secondary (Exploratory) Outcomes: 
 
a) Oxidative Stress-Associated Suppression of EDD and Large Elastic Artery Stiffness: The influence of 
oxidative stress on FMDBA and aPWV will be determined by infusing a supraphysiological dose of ascorbic acid 
(vitamin C; American Regent Laboratories Inc.) or isovolumic saline, as described previously by the PI’s 
doctoral training laboratory and others 63, 72-74.  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 min bolus during the “drip infusion” when peak 
plasma concentrations of ascorbic acid occur (34).  The difference in FMDBA and aPWV during ascorbic acid 
vs. saline infusion will be taken as a measure of the modulation of EDD/stiffness by oxidative stress 63, 72. 
 
Risks of this procedure include: Ascorbic acid has been infused intravenously without any unfavorable side 
effects into young and older healthy subjects, as well as patients with chronic cardiovascular diseases.  
However, administration of concentrated ascorbic acid may cause irritation to the local area around the 
infusion.  In order to reduce this risk we will dilute the infusion of ascorbic acid in sterile saline. 
 
Justification: Infusing ascorbic acid is a safe, effective and established method of acutely inhibiting oxidative 
stress.  As the difference in our primary outcome measures (FMDBA and aPWV) in the presence of ascorbic 
acid vs. saline is an index of a modulation of EDD/stiffness by oxidative stress, we will be provided with 
mechanistic insight into the physiological adaptations contributing to any improvements in vascular function 
with phosphorus lowering.  Specifically, less of an improvement in EDD/stiffness with ascorbic acid post- vs. 
pre- treatment, in the presence of improved EDD/stiffness during saline infusion post- vs. pre- treatment, would 
indicate that reduced oxidative stress contributed to the improvements in vascular function with phosphorus 
lowering. 
 
b) Vascular endothelial cell protein expression: These procedures have been described in detail previously 
61, 75-77.  A trained nurse, nurse practitioner or physician will collect endothelial cells from the intima of the 
antecubital vein of the non-dominant arm using up to 3 sterile 0.021 inch J-wires briefly advanced through a 
catheter.  The distal portion of the wire will be transferred to a 50-ml conical tube containing a buffer solution.  
Cells will be recovered by centrifugation, fixed with formaldehyde, and slides will be prepared and frozen for 
later staining for the primary antibody of interest, a complementary fluorescent secondary Alexafluor 555 
antibody (Invitrogen), VE cadherin for positive identification as endothelial cells and DAPI for nuclear integrity.  
The endothelial cells count per slide averages ~55 (range 25-92, estimated total of 200-736 endothelial cells 
per subject).  More than 90% of the endothelial cells retain cellular and nuclear integrity (as determined by 
DAPI staining).  Images will be digitally captured and analyzed using Metamorph Software (Universal Imaging 
Corp).  Values will be reported as ratios of subject endothelial cell protein expression to human umbilical vein 
endothelial cell protein expression, removing the possible confound of differences in staining intensity between 
sessions.  Using quantitative immunofluorescence, protein expression of the following proteins will be 
assessed: 
• 
nitrotyrosine, as a marker of oxidative stress (Abcam) 75, 77, 78 
• 
NADPH p47phox, manganese superoxide dismutase (MnSOD; Chemicon Intl) and copper-zinc SOD 
(CuZnSOD; Upstate) 75, 77, 78 as markers of pro and anti-oxidant signaling 
• 
total eNOS (BD Biosciences) and eNOS phosphorylated at Ser 1177 (BD Biosciences) 79 
Note: It is well establish that expression of proteins in endothelial cells obtained from venous sampling reflect 
individual and group differences observed in cells obtained from brachial artery sampling 80.  


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Risks of this procedure include: The risks associated with this procedure are those that occur with the 
placement of the venous catheters including small risk of pain, phlebitis, infection and blood clots (i.e., the 
same risks associated with venous catheterization).The risk of blood clots and infection may be slightly greater 
than those of an intravenous catheter due to increased manipulation of the blood vessel when compared with 
catheterization alone.  There is also a very low likelihood that the J-shaped wire could get stuck in the vein 
and/or that the vein could be damaged. 
 
Justification: This is a novel approach to evaluate the potential mechanisms (including expression of proteins 
modulating inflammation) by which phosphorus lowering may improve vascular function in CKD patients, with 
risk analogous to venous catheterization.  These procedures were described originally by Colombo et al.54 and 
more recently by Dr. Doug Seals’ laboratory55-58. An alternative to understanding these mechanisms would be 
via obtaining arterial endothelial cells through an arterial line. This would be considerably more invasive, and in 
light of data supporting that venous endothelial cells are a good surrogate marker for arterial endothelial cells, 
the investigators feel this risk is not justified for the purposes of this proposal. 
 
c) Laboratory Analyses: The majority of the laboratory measurements will be performed at the University of 
Colorado Clinical Laboratory using standard techniques.  Samples will be collected by research nurses at the 
UPI Building laboratory facilities and sent to the clinical laboratory or core laboratory.  
• 
Serum phosphorus (Visits 1-9): Serum phosphorus (1 mL serum) will be measured using standard 
methodology for study inclusion, drug titration and to determine the degree of phosphate lowering with 
lanthanum. 
 
• 
Follicle stimulating hormone (FSH) (Visit 1): FSH will be measured in all women <57 years of age to 
confirm post-menopausal status for study inclusion (1 mL serum).  
 
• 
Comprehensive metabolic panel (Visits 1, 2, 9): A comprehensive metabolic panel (4.5 mL plasma) will 
be performed for study inclusion and monthly throughout the study using standard methodology.  
 
• 
Lipid panel (Visits 2, 9): A lipid panel (1 mL plasma) will be performed at baseline (Visit 2) and 12 
weeks after lanthanum or placebo.  
• 
Fibroblast growth factor 23 (FGF23) (Visits 2, 9): FGF23 (200 μL serum) will be measured pre- and 
post- treatment as a marker of mineral metabolism (Kainos). 
• 
Intact Parathyroid hormone (iPTH) (Visits 2, 9): iPTH (4.5 mL plasma/serum) will be measured pre- 
and post- treatment as a marker of mineral metabolism using standard methodology. 
• 
25 hydroxyvitamin D (25(OH)D) (Visits 2, 9): 25(OH)D (4.5 mL serum) will be measured pre- and post- 
treatment as a marker of mineral metabolism by the core laboratory (Chemilluminescence, DiaSorin 
Liaison). 
• 
1,25 dihydroxyvitamin D (1,25(OH)2D) (Visits 2, 9): 1,25(OH)2D (1 mL serum) will be measured pre- 
and post- treatment as a marker of mineral metabolism by the core laboratory (EIA, Immunodiagnostic 
Systems). 
 
 
• 
Interleukin-6 (IL-6) (Visits 2, 5, 9): IL-6 (1 mL plasma) will be measured pre- and post- treatment as a 
marker of inflammation by the CTRC core laboratory (ELISA; R&D Systems). 
 
• 
C-reactive protein (CRP) (Visits 2, 5, 9): CRP (1 mL plasma) will be measured pre- and post- treatment 
as a marker of inflammation by the CTRC core laboratory (Immunoturbidimetric; Beckman Coulter). 
• 
Vitamin C (VITC) (Visits 2, 9): VITC (0.5 mL plasma) will be measured before and after infusions pre- 
and post- treatment to assess whether the study doses change Vitamin C levels.  
• 
Markers of Oxidative Damage  (Visits 2, 5, 9): (1 mL serum) will be measured before and after 
infusions pre- and post- treatment to measure markers of oxidative damage, such as F-2 isoprostanes, 
by the CTRC core laboratory (ELISA, Mercodia).  
 
• 
Future studies (Visits 2, 5, 9): For subjects signing the Informed Consent Addendum, an additional 5 
mL of serum and 9 mL of plasma will be collected for future research.   
• 
Urine phosphorus (Visits 2, 9): Urine phosphorus will be measured pre- and post- treatment as a 
marker of phosphorus excretion. 
• 
Urine creatinine (Visits 2, 9): Urine creatinine will be measured pre- and post- treatment to aid in the 
calculation of phosphorus excretion. 


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Risks of this procedure include: All of the lab work will be obtained via venipuncture and the insertion of an 
18 gauge cannula. The risks that are entailed in this procedure include discomfort and pain of venipuncture, 
failed attempt resulting in more than one attempt, bleeding upon cannula removal, and rarely thrombophlebitis 
(a non infectious inflammatory state of the vein typically due to prolonged cannulation). In the protocol the 
cannulation procedure is expected to last only for a few minutes (5-10), and the cannula will be removed 
immediately after obtaining the lab tests above and the endothelial cell collection.  A separate cannula will then 
be inserted in the dorsal aspect of the hand to infuse the saline and ascorbic acid, in order to help preserve 
access in the arm.  Subjects may be at a very small risk of anemia from the blood draws required for the 
assays described above. However, only the minimum amount of blood needed will be drawn and experienced 
research nurses/nurse practitioners will be collecting the specimens.  All blood draws will be performed on the 
arm not designated for future vascular access for dialysis.  
 
Justification: Please refer to the background/significance section for a detailed discussion of the importance 
of these serum markers with endothelial dysfunction. Patients with CKD have increased oxidative stress and 
inflammation compared to the general population, and lanthanum may improve vascular function by reducing 
oxidative stress and inflammation.  These systemic blood markers will provide important insight as to the 
mechanisms by which phosphate lowering may improve vascular function.  The other laboratory tests are 
necessary for subject inclusion and safety monitoring. 
 
E. Potential Scientific Problems:  
Although subject recruitment/retention are always challenging, we should be able to meet target enrollment 
given our location in the densely populated Denver metro area and access to the UC-Denver Nephrology Clinic 
at the University Hospital overseen by board-certified nephrologist, Dr. Chonchol, and at the Denver VA 
Medical Center Renal Clinic, staffed by Dr. Jovanovich.  We expect few difficulties with the proposed 
experimental procedures and protocols as the co-investigator has employed them during her graduate training.  
In addition, there are currently three intervention protocols in patients with CKD being performed using all/the 
majority of the proposed methods (Drs. Jablonski, Jalal and Kendrick).  We recognize that alternate phosphate 
binders could have been selected; however, we chose lanthanum (a) to avoid the potential side-effect of 
hypercalcemia that may be occur with calcium-based phosphate binders 81, (b) because calcium-based binders 
have not improved EDD in hyperphosphatemic CKD patients 49, 50, and (c) to control for the beneficial effects 
that may occur with sevelamer by mechanisms other than phosphorus lowering 82-84.  We also recognize that it 
would be of interest to compare to lanthanum to other phosphate binders, but given the duration and scope of 
the project, feel it is necessary to first compare the efficacy of lanthanum vs. placebo. 
 
F. Data Analysis Plan: 
Statistical Analysis 
General: Descriptive statistics will be used to summarize continuous variables (mean, standard 
deviation, minima, median, and maxima) and categorical variables (N and %) for baseline characteristics by 
treatment group.  Summaries will be provided by treatment group (lanthanum and placebo). Means and 
medians with variability estimates based on standard deviations or interquartile ranges will be used for 
graphical displays of continuous variables and histograms of percentages will be used to display the 
distribution of categorical variables. Highly skewed variables may be transformed prior to statistical analyses. 
Significance is defined with a two-sided alpha of 0.025 for the primary outcomes and 0.05 for the secondary 
outcomes. 
Analysis Populations: The primary analysis will be intention-to-treat. The data will also be analyzed as 
per protocol. Subjects randomized to treatment who receive at least one drug dose and complete baseline 
measurements will be included in the intention-to-treat analysis, regardless of whether they adhered to the 
assigned intervention. In the per protocol analysis, only those subjects who were adherent to the protocol 
(completed all 12 weeks of drug or placebo) and completed the end of the study measurements will be 
included. Decisions to remove subjects from the per protocol analysis set will be made prior to breaking the 
treatment blind. The intention-to-treat analysis will analyze subjects as randomized; the per protocol analysis 
will analyze subjects as treated.  
 
Prior to all analyses, data checks will be conducted to verify or correct assumptions about the data 
structure and to identify potential issues with the data being collected. This process will include range checks 


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to identify impossible values (e.g., age=0) and cross-tabulations identifying relations between key variables. 
Differences between groups at baseline will be assessed by unpaired t-tests and changes in response to 
lanthanum vs. placebo by ANOVA for repeated measures.  In the case of a significant F-value, post hoc tests 
will be performed using the Newman-Keuls method.  The influence of changes in 2˚ outcomes (blood pressure, 
mechanistic variables, etc.) on changes in 1˚ outcomes in response to lanthanum will be examined by multiple 
linear regression.  Potential confounders, such as changes in other characteristics that are known to influence 
vascular function (e.g. body mass, blood pressure) will be handled using ANCOVA.   Alpha for the two primary 
endpoints will be set at 0.025 while there will be no adjustment for secondary outcomes as they will be 
considered exploratory. 
 
G. Summarize Knowledge to be gained: 
The findings from the proposed research should provide important new information regarding the potential 
benefits of phosphorus lowering upon the reduction of vascular dysfunction in patients with CKD.  It should 
also provide important insight into the mechanisms involved in the modulation of EDD by elevated phosphorus. 
Together, this information will add to the knowledge of how to prevent and treat vascular dysfunction in 
patients with CKD.  
 
H. References: 
 
1. 
Kendrick J, Chonchol MB. Nontraditional risk factors for cardiovascular disease in patients with chronic 
kidney disease. Nature clinical practice. Nephrology. 2008;4:672-681 
2. 
Go AS, Chertow GM, Fan D, McCulloch CE, Hsu CY. Chronic kidney disease and the risks of death, 
cardiovascular events, and hospitalization. The New England journal of medicine. 2004;351:1296-1305 
3. 
McCullough PA, Jurkovitz CT, Pergola PE, McGill JB, Brown WW, Collins AJ, Chen SC, Li S, Singh A, 
Norris KC, Klag MJ, Bakris GL. Independent components of chronic kidney disease as a cardiovascular 
risk state: Results from the kidney early evaluation program (keep). Archives of internal medicine. 
2007;167:1122-1129 
4. 
Weiner DE, Tighiouart H, Amin MG, Stark PC, MacLeod B, Griffith JL, Salem DN, Levey AS, Sarnak 
MJ. Chronic kidney disease as a risk factor for cardiovascular disease and all-cause mortality: A pooled 
analysis of community-based studies. Journal of the American Society of Nephrology : JASN. 
2004;15:1307-1315 
5. 
Wanner C, Krane V, Marz W, Olschewski M, Mann JF, Ruf G, Ritz E. Atorvastatin in patients with type 
2 diabetes mellitus undergoing hemodialysis. The New England journal of medicine. 2005;353:238-248 
6. 
Zoccali C. Traditional and emerging cardiovascular and renal risk factors: An epidemiologic 
perspective. Kidney international. 2006;70:26-33 
7. 
Gutierrez O, Isakova T, Rhee E, Shah A, Holmes J, Collerone G, Juppner H, Wolf M. Fibroblast growth 
factor-23 mitigates hyperphosphatemia but accentuates calcitriol deficiency in chronic kidney disease. 
Journal of the American Society of Nephrology : JASN. 2005;16:2205-2215 
8. 
Isakova T, Wahl P, Vargas GS, Gutierrez OM, Scialla J, Xie H, Appleby D, Nessel L, Bellovich K, Chen 
J, Hamm L, Gadegbeku C, Horwitz E, Townsend RR, Anderson CA, Lash JP, Hsu CY, Leonard MB, 
Wolf M. Fibroblast growth factor 23 is elevated before parathyroid hormone and phosphate in chronic 
kidney disease. Kidney international. 2011;79:1370-1378 
9. 
Isakova T, Gutierrez OM, Wolf M. A blueprint for randomized trials targeting phosphorus metabolism in 
chronic kidney disease. Kidney international. 2009;76:705-716 
10. 
Kendrick J, Kestenbaum B, Chonchol M. Phosphate and cardiovascular disease. Adv Chronic Kidney 
Dis. 2011;18:113-119 
11. 
Kanbay M, Goldsmith D, Akcay A, Covic A. Phosphate - the silent stealthy cardiorenal culprit in all 
stages of chronic kidney disease: A systematic review. Blood Purif. 2009;27:220-230 
12. 
Kestenbaum B, Sampson JN, Rudser KD, Patterson DJ, Seliger SL, Young B, Sherrard DJ, Andress 
DL. Serum phosphate levels and mortality risk among people with chronic kidney disease. Journal of 
the American Society of Nephrology : JASN. 2005;16:520-528 
13. 
Briet M, Bozec E, Laurent S, Fassot C, London GM, Jacquot C, Froissart M, Houillier P, Boutouyrie P. 
Arterial stiffness and enlargement in mild-to-moderate chronic kidney disease. Kidney international. 
2006;69:350-357 


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14. 
Ghiadoni L, Cupisti A, Huang Y, Mattei P, Cardinal H, Favilla S, Rindi P, Barsotti G, Taddei S, Salvetti 
A. Endothelial dysfunction and oxidative stress in chronic renal failure. Journal of Nephrology. 
2004;17:512-519 
15. 
Shuto E, Taketani Y, Tanaka R, Harada N, Isshiki M, Sato M, Nashiki K, Amo K, Yamamoto H, Higashi 
Y, Nakaya Y, Takeda E. Dietary phosphorus acutely impairs endothelial function. Journal of the 
American Society of Nephrology : JASN. 2009;20:1504-1512 
16. 
Koobi P, Vehmas TI, Jolma P, Kalliovalkama J, Fan M, Niemela O, Saha H, Kahonen M, Ylitalo P, 
Rysa J, Ruskoaho H, Porsti I. High-calcium vs high-phosphate intake and small artery tone in advanced 
experimental renal insufficiency. Nephrology, dialysis, transplantation : official publication of the 
European Dialysis and Transplant Association - European Renal Association. 2006;21:2754-2761 
17. 
Kendrick J, Ix JH, Targher G, Smits G, Chonchol M. Relation of serum phosphorus levels to ankle 
brachial pressure index (from the third national health and nutrition examination survey). The American 
journal of cardiology. 2010;106:564-568 
18. 
Ix JH, De Boer IH, Peralta CA, Adeney KL, Duprez DA, Jenny NS, Siscovick DS, Kestenbaum BR. 
Serum phosphorus concentrations and arterial stiffness among individuals with normal kidney function 
to moderate kidney disease in mesa. Clinical journal of the American Society of Nephrology : CJASN. 
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1 
 
w 
COMIRB Protocol 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
 
Protocol #: 15-0834 
Project Title: Phosphorus Balance Study 
 
 
 
Principal Investigator: Anna Jovanovich, MD 
Version Date: 06/07/2017 
 
 
 
 
 
  
 
I. Hypotheses and Specific Aims:   
Cardiovascular disease (CVD) is significantly increased in chronic kidney disease (CKD) 
and is an important cause of morbidity and mortality.1-5 As much as 80% of all CVD is 
associated with vascular dysfunction,6 particularly impaired endothelium-dependent 
dilation (EDD),7 measured by brachial artery flow-mediated dilated (FMDBA).8,9 Not 
surprisingly, patients with CKD demonstrate these dysfunctional vascular phenotypes. In 
CKD, phosphorus remains within the normal range (2.5-4.5 mg/dL) until late in the 
disease.10-13 In earlier stages of CKD, serum phosphorus levels are maintained with the 
normal range by up-regulating fibroblast growth factor 23 (FGF23) and a consequent 
increase in the fractional excretion of phosphorus by the kidney.10-13 However, elevated 
serum phosphorus, even within the normal range, is associated with adverse CVD 
outcomes in CKD.13-15 Elevations in serum phosphorus have also been associated with 
impaired EDD and with indirect measures of arterial stiffness.16-19 Whether lowering serum 
phosphorus with phosphate binders in patients with CKD will change phosphorus balance 
and improve EDD and arterial stiffness is unknown.  
 
This investigation is an extension of a 12-week prospective, randomized, placebo-
controlled double-blind trial (Phosphorus Lowering RCT, COMIRB 13-0328) that assesses 
the effects of phosphorus lowering  with the phosphate binder, lanthanum carbonate, in 
stage IIIb and IV CKD with normal or modestly elevated serum phosphorus (2.8-5.5 
mg/dL) on vascular endothelial function and arterial stiffness. The primary goal of the 
current Phosphorus Balance protocol is to determine whether lowering serum phosphorus 
(accomplished during the parent Phosphorus Lowering RCT) with a low phosphorus diet 
and 12 weeks of treatment with lanthanum carbonate, a non-absorbed, non-calcium-
containing phosphate binder, affects phosphorus balance. The key secondary goal is to 
determine if differences in phosphorus balance affect vascular function as measured by 
FMDBA. 
 
To ensure adequate enrollment, we will also recruit patients with stage IIIb and IV CKD 
with normal or modestly elevated serum phosphorus (2.8-5.5 mg/dL) who are not currently 
participating in the parent Phosphorus Lowering RCT (COMIRB 13-0328). Similar to the 
Phosphorus Lowering RCT, these patients will follow a low phosphorus diet and will be 
randomized to lanthanum carbonate or placebo for 12 weeks (run-in period) prior to 
beginning the current Phosphorus Balance protocol. 
 
Hypothesis A.  Subjects with stages IIIb and IV CKD and normal or modestly elevated 
serum phosphorus (2.8-5.5 mg/dL) who are treated with 12 weeks of lanthanum 
carbonate and a low phosphorus diet will be in negative phosphorus balance compared to 
subjects treated with placebo.   


 
2 
 
Hypothesis B. 
Improvements in EDD will correlate with negative phosphorus balance.   
 
Aim A.  To determine phosphorus balance in subjects with stage IIIb or IV CKD after 
being treated with lanthanum carbonate and a low phosphorus diet or placebo and a low 
phosphorus diet for 12 weeks.   
 
Aim B. To determine whether phosphorus balance in each treatment group correlates 
with the FMDBA measurement performed at the end of the proposed Phosphorus Balance 
protocol. 
 
II. Background and Significance:  
Risk of cardiovascular diseases (CVD) is significantly elevated in patients with chronic 
kidney disease (CKD); however, the increased cardiovascular risk in this patient 
population is only partially explained by traditional cardiovascular risk factors.1-6 In earlier 
stages of CKD, serum phosphorus levels are maintained within the normal range by up-
regulating fibroblast growth factor 23 and a consequent increase in the fractional excretion 
of phosphorus by the kidney.10-13 However, elevated serum phosphorus even within the 
normal range, as is the typical case in CKD patients not requiring chronic hemodialysis, 
has emerged within the last decade as a novel, independent predictor of CVD risk and 
associated mortality.13-15 Furthermore, little is known about phosphorus balance and its 
association with outcomes in CKD. 
 
CKD patients also exhibit vascular endothelial dysfunction and increased arterial 
stiffness,20,21 two independent predictors of future CVD. A modest elevation in serum 
phosphorus contributes to this phenotype of vascular dysfunction.16-19 Recent evidence 
supports that an acute increase in serum phosphorus impairs endothelium dependent 
dilation (EDD) and increases vascular oxidative stress.16 In addition, both vascular 
endothelial dysfunction and increased oxidative stress have been identified as important 
predictors of mortality and cardiovascular events in patients with CKD.22,23 Thus, reducing 
serum phosphorus burden in CKD patients not requiring chronic hemodialysis may reduce 
cardiovascular events by improving vascular endothelial function and reducing large 
elastic artery stiffness. However, it is currently unknown if reducing serum phosphorus to 
a low normal laboratory range (<2.8 mg/dL) with oral phosphate binders induces negative 
phosphorus balance thus, potentially enhancing vascular function in this population. 
 
Little is known about phosphorus balance in CKD. It is assumed that CKD patients remain 
in neutral phosphorus balance despite decreases in kidney function. Serum phosphorus 
increases but remains within the normal range10-13 until late in CKD thus making it difficult 
to recognize perturbations of phosphorus balance in these patients. Indeed, elevated 
serum phosphorus even within the normal range is associated with worse CVD 
outcomes.12,14,15  In CKD subjects with normal serum phosphorus treated with six weeks 
of sevelamer, a non-calcium based phosphate binder, serum phosphorus did not change 
significantly (3.45 ± 0.65 mg/dL vs. 3.31 ± 0.64 mg/dL) but there was a significant 
decrease in total urinary phosphorus excretion (534 ± 137 mg/24 hours vs. 313 ± 135 
mg/24 hours, p<0.05) as well as in parathyroid hormone (PTH) (107 [79-130] pg/mL vs. 
69 [47-94] pg/mL, p 0.05) and fibroblast growth factor 23 (FGF23) (103 [62-142] pg/mL vs. 
54 [38-94] pg/mL, p<0.05), suggesting a shift in phosphorus homeostasis.24 However, two 
recent calcium balance studies performed in subjects with CKD stages III-IV, showed that 
subjects remained in neutral phosphorus balance despite administration of calcium-


 
3 
 
containing phosphate binders.25,26  There are no studies evaluating the effect of non-
calcium based phosphate binders on phosphorus balance in subjects with CKD nor other 
studies examining the effects of changing phosphorus balance on vascular function. 
Whether longer-term (12 weeks) treatment with non-calcium based phosphate binders 
induces a negative phosphorus balance in subjects with CKD is uncertain. It is also 
unknown if negative phosphorus balance improves vascular function.   
 
III. Preliminary Studies/Progress Report:   
Serum phosphorus is related to cardiovascular disease events and mortality as well 
as measures of arterial stiffness 
Presented below is the longitudinal relationship between higher serum phosphorus and 
all-cause mortality and CVD events (myocardial infarction, stroke and peripheral vascular 
disease), and a cross-sectional association of elevated serum phosphorus with worsening 
pulse pressure (PP).  These data were obtained from the Homocysteine Study in 
Advanced Kidney Disease (HOST), which was a prospective, randomized, double-blind 
study on the effects of folate, vitamin B6, and vitamin B12 replacement on all-cause 
mortality and CVD events in VA patients with severe CKD and elevated homocysteine.27 
As the intervention did not have an effect on the proposed outcomes, it represented a 
unique opportunity to convert the data obtained from a randomized control trial into an 
observational study. The HOST dataset has detailed information on demographics, CVD 
risk factors, prevalent CVD, circulating markers (including serum calcium, phosphorus, 
and intact parathyroid hormone), and usage of cardioprotective medications in all study 
participants. In addition, an endpoints committee adjudicated all endpoints. For the 
purpose of this analysis, we included 1,099 HOST participants. The mean (+SD) age of 
the participants was 69±11 years, 98% were male, and 26% were black. The mean 
estimated MDRD-GFR was 18±6 mL/min/1.73 m2 and most participants (n=718; 65%) had 
an estimated MDRD-GFR of 15-29 mL/min/1.73 m2. The mean serum phosphorus 
concentration was 4.3±1.3 mg/dL; 36% and 12% had a serum phosphorus concentration 
greater than 4.5 and 5.5 mg/dL, respectively. These descriptive data demonstrate that 
poor vascular function and CVD events are associated with high serum 
phosphorus.  
 
Higher serum phosphorus levels are independently associated with CVD 
events/mortality in patients with advanced CKD: During a median follow-up of 2.9 
years, 472 (42.9%) patients died from any cause and 237 (21.6%) had a CVD event. 
Higher serum phosphorus levels were positively associated with higher risks for the 
composite of death and CVD events. Identical results were observed when CVD events 
were examined separately (not shown). Compared to the lowest quartile, the two highest 
quartiles of serum phosphorus were associated with a significantly elevated risk for the 
composite endpoints, after multivariate adjustment (Table 1; hazards ratios (HR)). The 
analysis was repeated using serum phosphorus levels as a continuous variable instead of 
quartiles. After multivariable adjustment (Model 3), the HR of the composite endpoint 
increased by 1.17 (95% CI; 1.02-1.33; p=0.02) per 1 mg/dL increase in serum 
phosphorus. These results provide important support to my aim to evaluate whether 
changes in phosphorus lowering affect vascular endothelial function and arterial stiffness 
(two independent predictors of CVD events/mortality) in patients with advanced CKD. 
 
 
Table 1. Association of Higher Serum Phosphorus with Risk of Cardiovascular Events & Death in the 
HOST Study 
Phosphorus Concentrations 
Q1  
Q2  
Q3  
Q4  


 
4 
 
(3.5 mg/dL) 
(3.6-4.1 mg/dL) 
(4.2-4.8 mg/dL) 
(>4.9 mg/dL) 
Model 1 
1.00 
1.35(0.87- 2.09) 
1.70 (1.12- 2.57) 
1.93(1.29- 2.90) 
Model 2 
1.00 
1.36 (0.88- 2.11) 
1.79(1.18-2.73) 
2.18(1.44-3.31) 
Model 3 
1.00 
1.19 (0.76-1.86) 
1.50 (1.01-2.31) 
1.68 (1.05-2.68) 
Model 1: Unadjusted; Model 2: Adjusted for age, gender and race; Model 3: Adjusted for covariates in Model 
2 plus smoking, diabetes, hypertension, prevalent CVD, body mass index, systolic and diastolic blood 
pressure, treatment arm, eGFR, albumin, calcium, 25-hydroxyvitamin D, intact PTH, LDL-cholesterol, high 
density lipoprotein-cholesterol, triglycerides and use of angiotensin converting enzyme inhibitors, angiotensin 
receptor blockers and β-blockers. 
 
 
In addition, we performed a cross-sectional analysis of the relationship between serum 
phosphorus and pulse pressure (PP) in the HOST cohort. The results confirm that higher 
serum phosphorus levels are independently associated with a wider PP in patients with 
advanced CKD. The mean (+SD) PP in this population was 71 (20) mmHg. In adjusted 
linear regression models (Model 3), each 1 mg/dL increase in serum phosphorus was 
associated with a 2 mmHg increase in PP (=2.06.9; p=0.005). These data support the 
notion that serum phosphorus lowering should reduce large elastic artery stiffness, as 
increased PP is an expression of arterial stiffness. 
 
Feasibility of lanthanum carbonate treatment to lead to a sufficient separation in 
mean serum phosphorus between groups 
Dr. Geoff Block, Clinical Assistant Professor in the Division of Renal Diseases and 
Hypertension at the University of Colorado Denver, recently examined the effects of 
phosphate binders on parameters of mineral metabolism among patients with moderate to 
advanced CKD. A total of 148 patients (eGFR 20-45 ml/min/1.73m2) were randomly 
assigned to calcium acetate (n=30), lanthanum carbonate (n=30), sevelamer (n=30), or 
placebo (n=58). The primary endpoint was change in mean serum phosphorus from 
baseline to the average measured at months 3, 6, and 9.  Serum phosphorus decreased 
from a baseline mean of 4.2 mg/dL (for both active and placebo arms) to 3.9 mg/dL in the 
active therapy arm and 4.1 mg/dL in the placebo arm (p=0.03). These results were 
published in a peer-reviewed journal.28 Differences in serum phosphorus between the 
placebo and the lanthanum carbonate arms were further analyzed at 3 months. Serum 
phosphorus decreased from a baseline of 4.2 mg/dL (for both the lanthanum carbonate 
and placebo arms) to 3.7 mg/dL in the lanthanum carbonate arm and to 4.1 mg/dL in the 
placebo arm (p=0.04). The mean dose of lanthanum carbonate was 2.7 g/day. An 
increase in serum phosphorus of 0.3-0.5 mg/dL has been associated with a significant 
increase in relative risk of all-cause mortality and CVD events in various epidemiologic 
studies of patients with CKD. These data suggest that the treatment with lanthanum 
carbonate will lead to a sufficient and significant separation in the mean serum 
phosphorus between groups. 
 
There is a significant difference in FMDBA and aPWV between healthy individuals 
and those with CKD 
FMDBA is a well-established intermediate endpoint and validated means with which to 
examine CVD in subjects with and without CKD. In Figure 1 we demonstrate that among 8 
men studied by our group with stage III-IV CKD (aged 63±4 years) compared to 8 healthy 
age-matched control subjects (aged 60±3 years), there was a significant difference in 
FMDBA (*p<0.05). These preliminary data are additional experimental evidence supporting 
the testing of therapeutic interventions that improve FMDBA in patients with stage III-IV 
CKD. 


 
5 
 
 
 
 
 
IV. Research Methods 
A. Outcome Measure(s):  The proposed investigation is an extension of a 12-week 
prospective, randomized, placebo-controlled double-blind trial (Phosphorus Lowering 
RCT) of 60 subjects that assesses the effects of the phosphate binder, lanthanum 
carbonate, and a low phosphorus diet in stage IIIb and IV CKD with normal or modestly 
elevated serum phosphorus (2.8-5.5 mg/dL) on vascular endothelial function and arterial 
stiffness. Vascular endothelial function is assessed as endothelium-dependent dilation via 
FMDBA. Subjects with stage IIIb and IV CKD with normal or modestly elevated serum 
phosphorus (2.8-5.5 mg/dL) who are not participating in the Phosphorus Lowering RCT 
will also be enrolled and will be treated with either lanthanum carbonate or placebo and a 
low phosphorus diet for a 12-week run-in period prior to initiating the current Phosphorus 
Balance protocol in order to mimic the parent Phosphorus Lowering RCT. After 
completion of 12 weeks of either lanthanum carbonate and a low phosphorus diet or 
placebo and a low phosphorus diet, 24 research subjects (12 treated with lanthanum 
carbonate and 12 treated with placebo) will participate in the proposed Phosphorus 
Balance Study. The primary outcome is the difference in phosphorus balance between the 
active and placebo groups. Serum phosphorus is not expected to change during the 
current Phosphorus Balance protocol as subjects will remain on the same dose of active 
drug or placebo that they were on during either the parent Phosphorus Lowering RCT or 
the 12-week lanthanum carbonate or placebo run-in period. The dose of active drug or 
placebo was titrated during the first month of the parent Phosphorus Lowering RCT or the 
12-week run-in period and is expected to be stable during months 2 and 3 of both the 
parent RCT and 12-week run-in period as well as during the current Phosphorus Balance 
protocol. Secondary outcomes include whether there is a difference in FMDBA between the 
active and placebo groups and whether this correlates with a difference in phosphorus 
balance between the active and placebo groups.  
   
B. Description of Population to be Enrolled:   
Subjects. A group of 24 subjects who have completed the 12-week parent Phosphorus 
Lowering RCT or the 12-week run-in period described above will provide written informed 
consent and serve as subjects the for the current Phosphorus Balance protocol. Major 
inclusion/exclusion criteria are presented below: 
 
Inclusion Criteria: 
Figure 1 


 
6 
 
1. Completed prospective, randomized, placebo-controlled double-blind trial, 
Phosphorus Lowering to Treat Vascular Dysfunction in Chronic Kidney Disease 
(COMIRB #13-0328) or willing to participate in 12-week run-in period 
2. Aged 40-79 years; women must be post-menopausal (serum phosphorus 
increases after menopause in women58) 
3. CKD stage IIIb or IV (eGFR by 4-variable MDRD prediction equation: 15-45 
mL/min/1.73m2); stable renal function in the past 3 months) 
4. Mini-mental state examination (MMSE) score ≥24 (cognitive function screening); 
ability to provide informed consent 
 
Exclusion Criteria: 
1. Subjects with advanced CKD requiring chronic dialysis 
2. Subjects at high risk for gastrointestinal events or alteration in gastrointestinal 
anatomy (e.g., history of gastrointestinal surgery, colon cancer), hypomotility 
disorders (e.q., constipation, ileus), current bowel obstruction, or fecal impaction 
3. Significant co-morbid conditions that lead the investigator to conclude that life 
expectancy is less than 1 year 
4. Expected to undergo living-related kidney transplant in the next 6 months 
5. History of severe congestive heart failure (i.e., EF < 35%) 
6. History of severe liver disease (i.e., elevated bilirubin, AST, ALT, or evidence of 
liver cirrhosis on biopsy or imaging) 
7. Hospitalization in the past 3 months 
8. BMI ≥40 kg/m2 (FMD measurement can be inaccurate in severely obese subjects) 
9. Active vitamin D analogue use (i.e., calcitriol, paricalcitol, doxercalciferol) 
10. Alcohol dependence or abuse (i.e., as documented in subject’s chart per PCP or 
mental health provider or if stipulated by subject) 
11. Current tobacco abuse (i.e., current and habitual use of any tobacco product) 
12. Use of prednisone and other immune suppressant medications (i.e., current and 
ongoing use of prednisone or other immune suppressant medication) 
 
A Standard Operating Procedure is in place that prohibits the use of nitroglycerin (NTG) in 
subjects with systolic blood pressure <100 mmHg and with migraine headache. 
Additionally, it is part of the Standard Operating Procedure that subjects are educated 
regarding drugs that interact with lanthanum carbonate (levothyroxine, quinolone 
antibiotics, and other cationic medications) and are instructed in correct timing of 
lanthanum carbonate administration with respect to drugs that interact with it.  
 
C. Study Design and Research Methods   
Experimental Design. After completing the 12-week Phosphorus Lowering RCT (Figure 
1) or 12-week run-in period (Figure 2), a group of 24 research subjects will complete an 
additional 9-day Phosphorus Balance study. Subjects will provide informed consent for the 
proposed Balance study. Of note, all research subjects will have an FMDBA measurement 
at the beginning and end of the parent RCT or the 12-week run-in period. Subjects will 
continue taking their current dose of active drug (lanthanum carbonate) or placebo from 
the parent RCT or the 12-week run-in period and will begin a CTRC-prepared fixed low 
phosphorus diet (daily breakfast, lunch, dinner, and a snack) and will be instructed to 
consume only deionized water, which will be provided. They will also take polyethylene 
glycol 1 g with each meal (totaling 3g/day) as a non-absorbable stool marker. After 7 
days, subjects will be admitted to the CTRC for a 48-hour admission. 
 


 
7 
 
For subjects recruited from the parent Phosphorus Lowering RCT: 
• Session 1: Recruitment and Consent 
o All subjects currently participating in the parent Phosphorus Lowering RCT 
will be eligible to participate in the current Phosphorus Balance protocol 
• Session 2: Beginning of proposed Phosphorus Balance study 
o Blood sampling to monitor serum phosphorus concentration, serum 
creatinine for measurement of kidney function, and evaluation of markers of 
mineral metabolism  
o Receive CTRC-prepared fixed low phosphorus diet (daily breakfast, lunch, 
dinner, and a snack) including deionized water 
▪ 
Mifflin St. Joeur equation and activity factor of 1.4 will be used to 
determine caloric needs. Based on their caloric needs, subjects will 
be assigned to one of four kilocalorie diet categories: 2000-2300, 
2300-2600, 2600-2900, 2900-3200. The subjects will then select their 
meals from four different pre-set menus.  
▪ 
Macronutrients: 15% protein/ 30% fat/ 55% CHO ± 2% for each 
macro 
▪ 
The diets will contain the same proportion of plant and meat/dairy 
proteins to control for the different rates of absorption of phosphorus 
from plant sources versus phosphorus from meat/dairy sources 
▪ 
Micronutrients controlled: phosphorus < 1000±50 mg/day and calcium 
< 800±50 mg/day 
▪ 
Each of the 4 kilocalorie group diets will be frozen and sent  for exact 
measurement of phosphorus and calcium content. Subjects will be 
required to pick-up food in 3-day increments to prevent spoilage 
o Continue current dose of lanthanum or placebo 
o Receive polyethylene glycol 1 g PO TID with meals as a stool marker 
o Submit 1 stool specimen prior to initiating polyethylene glycol to be used as a 
comparison 
• Session 3: End of Phosphorus Balance Study 
o 48-hour admission to CTRC 
▪ 
Blood sampling to monitor serum phosphorus concentration, serum 
creatinine for measurement of kidney function, and evaluation of 
markers of mineral metabolism  
▪ 
24-hour urine and stool collection for phosphorus and calcium 
measurement (2 will be collected) 
▪ 
Subjects will continue 1 g of polyethylene glycol with each meal as a 
non-absorbable stool marker during the CTRC inpatient admission. 
▪ 
Continue CTRC-prepared fixed low phosphorus diet 
▪ 
Continue current dose of lanthanum or placebo 
o Final FMDBA measurement to be performed at the UPI Vascular Lab 
 
 
 
 
 
 
 
 
 
End Phosphorus Lowering RCT 
Begin Phosphorus Balance Study 
Figure 1 


 
8 
 
      
 
 
 
 
 
 
 
 
 
 
 
 
 
 
For subjects not enrolled in the parent Phosphorus Lowering RCT: 
• Session 1: Consent and screening measurements 
• Session 2: Baseline measurements 
o Resting arterial blood pressure  
o Blood sampling to monitor serum phosphorus concentration, serum 
creatinine for measurement of kidney function, and evaluation of markers of 
mineral metabolism  
o FMDBA (endothelium-dependent dilation) 
o Endothelium-independent dilation (EID) to sublingual nitroglycerin 
o Randomization to either lanthanum carbonate or placebo 
• Session 3-5: Biweekly/monthly check-in (see below) 
• Session 6: Beginning of proposed Phosphorus Balance study (same as Session 2 for 
those recruited from parent Phosphorus Lowering RCT) 
o Resting arterial blood pressure  
o Blood sampling to monitor serum phosphorus concentration, serum 
creatinine for measurement of kidney function, and evaluation of markers of 
mineral metabolism  
o FMDBA (endothelium-dependent dilation) and endothelium-independent 
dilation (EID) to sublingual nitroglycerin 
o Receive CTRC-prepared fixed low phosphorus diet (daily breakfast, lunch, 
dinner, and a snack) including deionized water 
▪ 
Mifflin St. Joeur equation and activity factor of 1.4 will be used to 
determine caloric needs. Based on their caloric needs, subjects will 
be assigned to one of four kilocalorie diet categories: 2000-2300, 
2300-2600, 2600-2900, 2900-3200. The subjects will then select their 
meals from four different pre-set menus.  
▪ 
Macronutrients: 15% protein/ 30% fat/ 55% CHO ± 2% for each 
macro 
▪ 
The diets will contain the same proportion of plant and meat/dairy 
proteins to control for the different rates of absorption of phosphorus 
from plant sources versus phosphorus from meat/dairy sources 
▪ 
Micronutrients controlled: phosphorus < 1000±50 mg/day and calcium 
Lanthanum carbonate 
Placebo 
Begin fixed phosphorus diet 
Begin fixed phosphorus diet 
48-hour CTRC admission 
Final FMDBA 
48-hour CTRC admission 
Final FMDBA 
Session 2 
Session 3 
7-day equilibration 


 
9 
 
< 800±50 mg/day 
▪ 
Each of the 4 kilocalorie group diets will be frozen and sent  for exact 
measurement of phosphorus and calcium content. Subjects will be 
required to pick-up food in 3-day increments to prevent spoilage 
o Continue current dose of lanthanum or placebo 
o Receive polyethylene glycol 1 g PO TID with meals as a stool marker 
o Submit 1 stool specimen prior to initiating polyethylene glycol to be used as a 
comparison 
• Session 7: End of Phosphorus Balance Study (same as Session 3 for those recruited 
from parent Phosphorus Lowering RCT) 
o 48-hour admission to CTRC 
▪ 
Blood sampling to monitor serum phosphorus concentration, serum 
creatinine for measurement of kidney function, and evaluation of 
markers of mineral metabolism  
▪ 
24-hour urine and stool collection for phosphorus and calcium 
measurement (2 will be collected) 
▪ 
Subjects will continue 1 g of polyethylene glycol with each meal as a 
non-absorbable stool marker during the CTRC inpatient admission. 
▪ 
Continue CTRC-prepared fixed low phosphorus diet 
▪ 
Continue current dose of lanthanum or placebo 
o Final FMDBA measurement to be performed at the UPI Vascular Lab 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Lanthanum dosing. The starting dose of lanthanum will be determined from a sliding 
scale according to baseline serum phosphorus (Table 1).  The dose of the phosphate 
binder will be titrated at week 2, week 4, and week 8 (Sessions 3-5) to achieve serum 
phosphorus less than 2.8 mg/dL. This dose will be up-titrated as needed based on serum 
phosphorus (if not <2.8 mg/dL, dose will be increased by 1 tablet [500 mg] for each meal).  
If serum phosphorus decreases to <2.5 mg/dL, the dose of lanthanum will be down-
Subjects randomly assigned to lanthanum carbonate or placebo 
 
Baseline Measurements 
Screening 
12 weeks Lanthanum* 
12 weeks placebo* 
 
Baseline Measurements 
Pre Balance FMD & begin fixed 
phosphorus diet 
Pre Balance FMD & begin fixed 
phosphorus diet 
Session 1 
Session 2 
Sessions 3-5  
Session 6 
7-day equilibration 
Session 7 
48-hour CTRC admission 
Final FMDBA 
48-hour CTRC admission 
Final FMDBA 
Figure 2 


 
10 
 
titrated. Tablets will be taken with each meal (if a subject consumes <3 meals/day, the 
dosing will be adjusted accordingly). 
 
Table 1: 
 
 
 
 
 
For all subjects: 
Phosphorus Balance. A simplified definition of balance will be used:  
 
Balance = oral intake – urine output – stool output.  
 
Subjects will be admitted to the CTRC to ensure adequate collection of 24-hour urine and 
stool collections; two 24-hour collections will be obtained during each CTRC admission. 
Stool phosphorus will be collected and measured according to previous protocols used at 
the CTRC.25,26 The urine and stool collected during the 48-hour admissions to the CTRC 
will be averaged to give the best estimate of phosphorus balance for each subject. While 
calcium balance is not a study end-point, we are collecting urine and stool calcium as 
these data may be important in the interpretation of phosphorus balance. Polyethylene 
glycol (PEG, 3 g/day), a non-absorbable marker, will be given with meals during the 9-day 
study to assess steady state and will be measured in the stool using a turbidimetric 
assay.26 
 
FMDBA. Measurements will be made at the UPI Vascular Lab.   
 
Lanthanum dosing. Subjects will continue taking the same dose of lanthanum carbonate 
or placebo as they are taking at the end of the phosphorus lowering RCT or during the 12-
week run-in phase. There will be no dose titration unless the serum phosphorus falls 
below 2.5 mg/dL (the lower limit of normal), in which case the dose of lanthanum 
carbonate will be decreased by 1500 mg daily (equivalent to 1 tablet with each of three 
meals daily) at a time until the serum phosphorus concentration is above 2.5 mg/dL. 
Tablets will be taken with each meal. 
 
Study Sites. Subjects will be recruited from the active prospective, randomized, placebo-
controlled double-blind trial, Phosphorus Lowering to Treat Vascular Dysfunction in 
Chronic Kidney Disease (COMIRB #13-0328). Subjects for the parent Phosphorus 
Lowering RCT were recruited from the Nephrology Clinic at the University of Colorado 
Denver Anschutz Medical Campus and from the Denver VA Medical Center Renal Clinic 
with access to >950 CKD patients from all over the Denver/Boulder metro area (at least 
33% with ICD 9 code 585.3 and 585.4 as diagnosis) with over 2146 visits in the past year. 
Subjects not recruited from the parent Phosphorus Lowering RCT will be recruiting from 
the Nephrology Clinic at the University of Colorado Denver Anschutz Medical Campus 
and from the Denver VA Medical Center Renal Clinic.  
 
The recruitment and screening as well as FMDBA measurements for the proposed 
Phosphorus Balance protocol will take place at the UPI Vascular lab, while the 48-hour 
admission for stool and urine collections as well as blood sampling will take place at the 
CTRC. 
Serum Phosphorus (mg/dL) 
Starting Dose (mg) 
2.8-4.0  
1,500 (1 tablet with each meal) 
4.1-4.5 
 3,000 (2 tablets with each meal) 
4.6-5.5 
4,500 (3 tablets with each meal) 


 
11 
 
 
Randomization and Treatment. Once inclusion/exclusion criteria have been met and 
informed consent obtained, subjects will remain in the treatment group to which they were 
originally randomized in the Phosphorus Lowering RCT. Subjects not recruited from the 
parent Phosphorus Lowering RCT will be randomized to either lanthanum carbonate or 
placebo. Randomization will be performed by Dr. Zhiying You, a statistician, by blocks 
using a computer-generated procedure to ensure equal distribution. The lanthanum 
carbonate and placebo are prepared and provided by Shire and are identical. There is no 
cost to the PI for the study drug or placebo.  
 
a) Study Drug: Lanthanum carbonate (Fosrenol) is an FDA approved non-absorbed, 
non-calcium-containing phosphate binder to lower serum phosphate in patients 
requiring chronic dialysis.  
 
b) Rationale for lanthanum dose: This dosing is indicated in patients with end-stage 
renal disease requiring chronic dialysis. The population being studied in this protocol 
will be healthier than this patient group. In addition, an IND exemption has been 
obtained from the FDA.  
 
c) Blinding: The investigators and subjects will remain blind to the study intervention, 
as the lanthanum carbonate and placebo are identical. A nurse at the UPI Vascular 
lab will be responsible for administering the pills, which will be stored in a locked 
cabinet on site. A professional research assistant will be unblinded and will manage 
the study drug records and interact directly with the nursing staff for administration of 
the study drug. The investigators will be blinded to laboratory results, and the 
research assistant will monitor laboratory results for safety and inform the 
investigator if necessary.  
 
d) Drug accountability: Pill counts will be assessed weekly at each CTRC admission. 
 
e) Withdrawal from study: Withdrawal will be done at the patient’s request, IRB 
request, or if there is a medical rationale for discontinuation as determined by the 
principal investigator.  
 
Safety monitoring. The subjects will be instructed to call the PI or research coordinator 
with any subjective complaints, in addition, abnormal labs will be reported to the PI 
immediately, who will determine if the study drug needs to be discontinued. The following 
are reported adverse events of the study drug and the monitoring plan: 
 
a) Lanthanum carbonate. The safety profile of lanthanum carbonate has been studied 
in over 5200 subjects in completed clinical trials. The most common adverse reactions for 
lanthanum carbonate are gastrointestinal events, such as nausea, vomiting, and 
abdominal pain and they generally abate over time with continued dosing. In end-stage 
renal disease patients receiving 4-6 weeks of treatment with lanthanum carbonate the 
incidence of nausea was 11% (vs. 5% placebo), vomiting 9% (vs. 4%), and abdominal 
pain 5% (vs. 0%). Lanthanum carbonate may reduce calcium absorption and result in 
hypocalcemia (~5%, same incidence as active comparator group). The following 
additional adverse reactions have been identified during post-approval use of lanthanum 
carbonate: constipation, dyspepsia, allergic skin reactions, hypophosphatemia, and tooth 


 
12 
 
injury (incidence not known). Lanthanum carbonate may decrease bioavailability of 
cationic antacids, quinolone antibiotics, or levothyroxine.   
 
There have been reports of serious cases of gastrointestinal obstruction, ileus, and fecal 
impaction reported in association with lanthanum carbonate, some requiring surgery or 
hospitalization. Risk factors for gastrointestinal obstruction identified from post-marketing 
reports include alteration in gastrointestinal anatomy (e.g., history of gastrointestinal 
surgery, colon cancer), hypomotility disorders (e.g., constipation, ileus, diabetes), and 
concomitant medications (e.g., calcium channel blockers). Some cases were reported in 
patients with no history of gastrointestinal disease. 
 
Hypophosphatemia has been reported only in post-marketing surveillance. The risk of 
hypophosphatemia is very low. Clinically significant (causing symptoms) 
hypophosphatemia associated with the use of lanthanum carbonate has not been 
reported.  
 
Safety measures will include: lanthanum carbonate dose titration and close monitoring of 
serum phosphate (as described above) to avoid severe hypophosphatemia, excluding 
patients at high risk for gastrointestinal events or current bowel obstruction, ileus and fecal 
impaction, repeatedly emphasizing to subjects the importance of chewing tablets 
completely to reduce the risk of serious adverse gastrointestinal events such as those 
described above. Per standard operating procedure, subjects will also be instructed to 
take concomitant drugs at least one hour before or four hours after lanthanum carbonate 
to avoid potential interactions, and blood levels of concomitant drugs with a narrow 
therapeutic range will be monitored if deemed appropriate by Dr. Jovanovich.   
 
Patient Stopping Criteria: 
• 
Withdrawal of informed consent (subject’s decision to withdraw for any 
reason) 
• 
Any clinical adverse event (AE), laboratory abnormality, or intercurrent 
illness which, in the opinion of the investigator, indicates that continued 
participation in the study is not in the best interest of the subject 
• 
Loss of ability to freely provide consent through imprisonment or 
involuntary incarceration for treatment of either a psychiatric or physical 
(e.g., infectious disease) illness 
• 
Inability to comply with protocol 
• 
Use of an investigational therapy or device other than study medication 
• 
Significant non-compliance with protocol (i.e., procedures, assessments, 
medication, etc.) 
• 
Hypocalcemia (<8.6 mg/dL) that does not resolve with decreased dosage 
of study drug 
• 
Hypophosphatemia (<2.5 mg/dL) that does not resolve with decreased 
dosage of study drug 
• 
Hospitalization for gastrointestinal obstruction, ileus, or fecal impaction 
• 
Serious acute hypersensitivity reactions to investigational drug 
• 
Any major cardiovascular event such as myocardial infarction, stroke, 
unstable angina, thromboembolic event, or a clinically significant 
deterioration of a baseline cardiovascular condition. 
 


 
13 
 
Study Stopping Criteria: 
The study may be stopped if:  
• The data show a significantly increased risk of serious adverse effects in one of the 
treatment groups.   
• It becomes clear that successful completion of the study is not feasible (e.g. there is an 
excess of patient dropout, missing data, lack of recruitment, etc.). 
 
Standard medical treatment for all subjects/concomitant medications includes: 
All subjects must be on a stable anti-hypertensive, diabetic, and lipid lowering 
regimens for at least one month prior to inclusion. 
a) Control of hypertension with anti-hypertensives with goal blood pressure less than 
130/80 mm/Hg. Medication choice includes angiotensin-converting enzyme 
inhibitor/angiotensin receptor blocker as first line agent, diuretic as second line agent, 
calcium-channel blocker as third line agent, and beta-blocker as fourth line agent (unless 
patient has known CVD and needs to be on a beta-blocker regardless of blood pressure). 
b) Diabetes will be treated as needed to goal HbA1C of 7.0%. c) Control of hyperlipidemia 
with statins to goal of low-density lipoprotein cholesterol (<100 mg/dL and total cholesterol 
levels to less than 200 mg/dL. d) Smoking cessation counseling. e) Control of anemia 
following the latest recommendation from the National Kidney Foundation.  
 
Data Collection 
Medical History, Physical Examination, and Blood Chemistries. Subjects recruited for 
the phosphorus lowering RCT reported fasted for assessment of serum phosphorus and a 
standard blood chemistry analysis including a metabolic panel (with creatinine/calcium), 
lipid panel, and follicle stimulating hormone (women only if not post-menopausal), then 
underwent a medical history and physical examination. Subjects continuing in the 
proposed phosphorus balance study will undergo another physical examination and fasted 
assessment of serum phosphorus and a standard metabolic panel including creatinine 
and calcium.  
 
Data management:  
Study data will be collected and managed using REDCap (Research Electronic Data 
Capture).  REDCap is a secure, web application designed to support data capture for 
research studies, providing user-friendly web-based case report forms, real-time data 
entry validation (e.g. for data types and range checks), audit trails, and a de-identified 
data export mechanism to common statistical packages (SPSS, SAS, Stata, R/S-Plus). 
The system was developed by a multi-institutional consortium, which included the 
University of Colorado Denver and was initiated at Vanderbilt University. Currently 52 
institutions in 4 countries are using the software. The database is hosted at the University 
of Colorado Denver Development and Informatics Service Center (DISC), which will be 
used as a central location for data processing and management. REDCap data collection 
projects rely on a thorough study-specific data dictionary defined in an iterative self-
documenting process by all members of the research team with planning assistance from 
the DISC. The iterative development and testing process results in a well-planned data 
collection strategy for individual studies. Both REDCap and REDCap Survey systems 
provide secure, web-based applications that are flexible enough to be used for a variety of 
types of research, provide an intuitive interface for users to enter data and have real time 
validation rules (with automated data type and range checks) at the time of entry. These 
systems offer easy data manipulation with audit trails and reporting for reporting, 


 
14 
 
monitoring, and querying patient records, and an automated export mechanism to 
common statistical packages (SPSS, SAS, Stata, R/S-Plus). Both systems were found to 
meet or exceed all of the security audit requirements. 
 
Data Safety and Monitoring: 
The Department of Veterans Affairs Clinical Science Research and Development, Central 
Data Monitoring Committees (DMC) is acting as the DSMB for the parent RCT (13-0328). 
Since this is a VA-funded study, it is expected that the Central VA DMC will act as DSMB 
for the current protocol as well. 
 
D.   Description, Risks and Justification of Procedures and Data Collection Tools: 
 
a) Conduit artery vascular endothelial dependent dilation (EDD) and endothelial 
independent dilation (EID): Conduit artery EDD, brachial artery flow-mediate dilation 
(FMDBA), will be determined using high-resolution ultrasonography (GE Vivid7 Dimension) 
as described originally by Celermajer et al.29 and used recently by the co-mentor.31,32 
ECG-gated end-diastolic ultrasound images and Doppler flow of the brachial artery will be 
acquired during baseline and FMDBA conditions. For FMDBA, reactive hyperemia will be 
produced by inflating a pediatric BP cuff around the forearm to 250 mmHg for 5 minutes 
followed by rapid deflation. Brachial artery endothelium-independent dilation (EID), an 
index of smooth muscle sensitivity to exogenous nitric oxide, will be determined by 
measuring brachial artery dilation for 10 minutes after administration of sublingual 
nitroglycerin (0.4 mg).32,33 A commercially available software package (Vascular Analysis 
Tools 5.8.1, Medical Imaging Applications) will be used to acquire ECG-gated brachial 
artery diameters. As recently recommended,34 FMDBA will be determined as the mm and 
% ∆ from baseline diameter.32-34 Shear rate will also be calculated, and if group- or 
condition- differences exist, FMDBA will be adjusted accordingly.34-36 
 
Risks of this procedure include: The assessment of FMDBA is minimally invasive. 
Inflating the blood pressure cuff below the elbow during this procedure may cause a mild 
to moderate intensity “pins and needles or numbing” sensation that goes away as soon as 
the cuff is deflated. In addition, patients will receive a dose of sublingual nitroglycerin to 
measure EID, an essential control measure without which the results cannot be properly 
interpreted. Sublingual nitroglycerin is a medication with a rapid vasodilator effect and is 
routinely used in patients with coronary artery disease to help relieve chest pain. Side 
effects of sublingual nitroglycerin are short-lived (30-60 minutes). Approximately 60% of 
people who receive nitroglycerin will experience a headache. The subject may experience 
symptoms such as lightheadedness, and in rare instances (1 in 1000) lowered heart rate, 
which could lead to fainting. However, these risks are primarily when a person is upright, 
and subjects will be lying supine during administration of nitroglycerin. Finally, placing the 
pill under the tongue could cause irritation to the area in the back of the mouth. The 
subjects will be monitored by frequently asking about subjective complaints, as well as by 
frequent heart rate and blood pressure measurements (prior to and every 15 minutes after 
taking the medication for 1 hour). In addition, during the 10-minute period when brachial 
artery dilation to nitroglycerin is assessed (peak activity of drug), blood pressure and heart 
rate will be assessed every 2 minutes. In the rare case that blood pressure drops 
drastically, an intravenous catheter will be available for infusion of normal saline. Still, 
nitroglycerin has been administered safely for over a decade in the laboratory the co-
mentor performed her doctoral training, as well as in other laboratories and in clinical 
situations worldwide. Per standard operating procedure, nitroglycerin will not be given to 


 
15 
 
any subject with a systolic blood pressure less than 100 mmHg on the day the 
measurements are performed, history of migraine headaches, or previous adverse 
reaction to administration. 
 
Justification: As discussed above, patients with CKD have an increased risk of death 
due to CVD, and endothelial dysfunction plays a critical role in the development of CVD. 
Endothelial dysfunction is evident early in the course of kidney disease. It is unknown if 
lowering phosphorus improves vascular endothelial function in CKD patients. FMDBA is a 
non-invasive way to assess endothelial function. The other method available to study 
endothelial function (EDD) would include acetylcholine administration via brachial artery 
infusion, which is more invasive than the proposed methodology. In order to interpret 
improvements in brachial artery dilation in response to a shear stress (FMDBA) following 
the drug intervention, it is necessary to assess smooth muscle sensitivity to nitric oxide 
using EID. If EID is unchanged in the presence of improved FMDBA, this indicates the 
improved dilation in response to a shear stimulus is due to positive adaptations at the 
level of the vascular endothelium (i.e. endothelium-dependent).  
 
b) Laboratory Analyses: The majority of the laboratory measurements will be performed 
at the University of Colorado Clinical Laboratory using standard techniques. Samples will 
be collected by research nurses at the CTRC and sent to the clinical laboratory or core 
laboratory. Serum to measure phosphorus and the comprehensive metabolic panel will be 
immediately sent to the University of Colorado Clinical Laboratory for analysis. Urine will 
also be sent to the University of Colorado Clinical Laboratory for analysis of phosphorus, 
calcium, and creatitine to avoid degradation and inaccurate measurement of these 
substances, which may occur with prolonged storage. The rest of the serum, and feces 
will be collected and immediately processed for storage in a -80° C freezer until the end of 
the study when they will be batched and analyzed. The stored serum will be used to 
measure markers of mineral metabolism, including fibroblast growth factor 23 (FGF23), 
intact parathyroid hormone (iPTH), 25 hydroxyvitamin D (25(OH)D), 1,25 dihydroxyvitamin 
D (1,25(OH)2D), and the stored stool will be used to measure phosphorus.  
 
For subjects recruited from the parent Phosphorus Lowering RCT: 
• 
Serum phosphorus (Visits 2,3): Serum phosphorus (1 mL serum) will be 
measured using standard methodology for safety monitoring of serum phosphorus 
levels during treatment with active and placebo drugs and also to inform 
interpretation of data at the study end. 
 
• 
Comprehensive metabolic panel (Visits 2,3): A comprehensive metabolic panel 
(4.5 mL plasma) will be performed at the beginning and end of the study using 
standard methodology.  
 
• 
Fibroblast growth factor 23 (FGF23) (Visits 2,3): FGF23 (200 μL serum) will be 
measured at the beginning and end of the study as a marker of mineral 
metabolism. 
 
• 
Intact Parathyroid hormone (iPTH) (Visits 2,3): iPTH (4.5 mL plasma/serum) will 
be measured at the beginning and end of the study as a marker of mineral 
metabolism using standard methodology. 
 


 
16 
 
• 
25 hydroxyvitamin D (25(OH)D) (Visits 2,3): 25(OH)D (4.5 mL serum) will be 
measured at the beginning and end of the study as a marker of mineral 
metabolism. 
 
• 
1,25 dihydroxyvitamin D (1,25(OH)2D) (Visits 2,3): 1,25(OH)2D (1 mL serum) will 
be measured at the beginning and end of the study as a marker of mineral 
metabolism. 
 
• 
Stool phosphorus (Visit 3): Two 24-hour stool collections will be collected during 
each of the CTRC admissions. Stool samples will be collected, frozen, and sent for 
measurement of phosphorus content.  
 
• 
Stool calcium (Visit 3): Two 24-hour stool collections will be collected during each 
of the CTRC admissions. Stool samples will be collected, frozen, and sent for 
measurement of calcium content. 
 
• 
Stool polyethylene glycol (Visit 1-2): One stool sample will be collected (this can 
be completed at the subjects’ home) and submitted for measurement of 
polyethylene glycol as a baseline marker prior to initiating the balance study. 
Content of polyethylene glycol will also be measured in the stool collected during 
the 48-hour CTRC admission as a stool marker. 
 
• 
Urine phosphorus (Visits 3): Two 24-hour urine collections will be collected during 
each of the CTRC admissions. Urine phosphorus will be measured using standard 
technique.  
 
• 
Urine calcium (Visit 3): Two 24-hour urine collections will be collectd during each 
of the CTRC admissions. Urine calcium will be measured using standard 
technique. 
 
• 
Urine creatinine (Visit 3): Two 24-hour urine collections will be collected during 
each of the CTRC admission. Urine creatinine will be measured using standard 
technique and will be used to interpret and inform 24-hour urine phosphorus 
measurements. 
 
For subjects not enrolled in the parent Phosphorus Lowering RCT: 
• 
Serum phosphorus (Visits 1-7) 
• 
Comprehensive metabolic panel (Visits 1-7) 
• 
Fibroblast growth factor 23 (FGF23) (Visits 2,6,7) 
• 
Intact Parathyroid hormone (iPTH) (Visits 2,6,7) 
• 
25 hydroxyvitamin D (25(OH)D) (Visits 2,6,7) 
• 
1,25 dihydroxyvitamin D (1,25(OH)2D) (Visits 2,6,7) 
• 
Stool phosphorus (Visits 6,7)  
• 
Stool calcium (Visits 6,7) 
• 
Stool polyethylene glycol (Visits 6,7) 
• 
Urine phosphorus (Visits 2,6,7) 
• 
Urine calcium (Visit 6,7) 
• 
Urine creatinine (Visit 2,6,7) 


 
17 
 
 
 
 
Risks of this procedure include: All of the lab work will be obtained via venipuncture 
and the insertion of an 18-gauge cannula. The risks that are entailed in this procedure 
include discomfort and pain of venipuncture, failed attempt resulting in more than one 
attempt, bleeding upon cannula removal, and rarely thrombophlebitis (a non-infectious 
inflammatory state of the vein typically due to prolonged cannulation). A cannula will be 
inserted in the dorsal aspect of the hand to infuse the saline in order to help preserve 
access in the arm. Subjects may be at a very small risk of anemia from the blood draws 
required for the assays described above. However, only the minimum amount of blood 
needed will be drawn and experienced research nurses/nurse practitioners will be 
collecting the specimens. All blood draws will be performed on the arm not designated for 
future vascular access for dialysis.  
 
Justification: Considered markers of mineral metabolism, FGF23, iPTH, 25(OH)D, and 
1,25(OH)2D, are hormones that regulate the absorption of phosphorus from the gut and its 
excretion in the kidney. In earlier stages of CKD, serum phosphorus levels are maintained 
with the normal range by up-regulating FGF23 and a consequent increase in the fractional 
excretion of phosphorus by the kidney. FGF23 also regulates 1-α-hydroxylase and thus 
the conversion of nutritional vitamin D (25(OH)D) to its active form, 1,25(OH)2D, which in 
turn affects calcium and iPTH. Markers of mineral metabolism become dysregulated in 
CKD.10-13 To better understand and interpret changes in phosphorus and phosphorus  
balance, especially in subjects with CKD, it is necessary to know the changes that occur 
in the levels of these markers of mineral metabolism. 
 
    
E.   Potential Scientific Problems:   
Although subject recruitment/retention are always challenging, we should be able to meet 
target enrollment given that we will be recruiting from an active randomized controlled trial 
and we are expanding the protocol to enroll subjects in a parallel run-in phase should we 
be unable to achieve adequate enrollment solely from the parent RCT. We expect few 
difficulties with the proposed experimental procedures and protocols. Dr. Jablonski, co-
investigator, has employed the measurement of FMDBA during her graduate training, and 
numerous protocols measuring FMDBA are successfully underway at the UPI Vascular 
Laboratory. Furthermore, a calcium balance study utilizing a similar protocol was 
successfully conducted in healthy and CKD subjects at the CTRC by investigators 
associated with our group. We recognize that alternative phosphate binders could have 
been selected; however, we chose lanthanum carbonate a) because calcium-containing 
phosphate binders have been used in other balance studies and may confound results; b) 
to avoid the potential for hypercalcemia as observed with calcium-containing phosphate 
binders; and c) because calcium-containing binders have not improved EDD in 
hyperphosphatemic CKD subjects;37 and d) to control for the beneficial effects that may 
occur with sevelamer by mechanisms other than phosphorus lowering.38-40 We also 
recognize that it would be of interest to compare lanthanum compare with other 
phosphate binders, but given the duration and scope of the project, feel it is necessary to 
first compare lanthanum carbonate with placebo. Finally, some common cardiovascular 
disease drugs, particularly statin therapy, have established vascular effects. To control for 
statin use, subjects on statin therapy at the beginning of the parent study will remain on 
the same dose during the parent study and during the current protocol. Subjects not on 
statin therapy will be asked not to initiate statin therapy until the end of both the parent 


 
18 
 
and current protocols. If there is unequal distribution of statin use in the active and 
placebo group, we will control for this using statistical modeling. 
 
F.   Data Analysis Plan:   
We hypothesize that subjects treated with lanthanum carbonate will be in negative 
phosphorus balance compared to subjects treated with placebo. In addition, we expect 
that improvements in EDD will correlate with a negative phosphorus balance. An N = 12 
from each group will be entered into the balance study (total N = 24). We will compare 
phosphorus balance between the two groups using an Independent Samples T-test. Data 
published previously26 demonstrated that phosphorus balance was achieved on oral 
calcium carbonate vs. placebo (phosphorous balance of 153 vs. 95 mg/d with a SD = 82); 
however, the sample size of 8 in a crossover design was not enough to conclude 
equivalence. We propose to use N = 12 per group, which will give a two group 0.050 two-
sided t-test 81.3% power with a standard deviation of 43. The results of the current 
protocol will be used to aid in the interpretation of the parent Phosphorus Lowering RCT. 
Pearson correlation coefficients will be used to determine the correlation between 
phosphorus balance and FMDBA at the end of the 9-day diet intervention (i.e., the end of 
the current protocol) in each treatment group.  
 
G.  Summarize Knowledge to be Gained:   
The findings from the proposed research should provide important new information 
regarding phosphorus balance in CKD and its effect on vascular function in this population 
of patients subject to a high degree of CVD morbidity and mortality. No phosphorus 
balance study in CKD patients treated with a non-calcium-containing phosphate binder, 
such as lanthanum carbonate, has ever been conducted. Moreover, the length of time 
subjects will be treated with active or placebo drug prior to balance measurements and the 
numerous measurements of stool and fecal phosphorus exceeds that of other studies. 
Together, this information will provide important insight into phosphorus metabolism and 
vascular function in CKD. 
 
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