Analysis of Calcium Balance in Chronic Kidney Disease

We hypothesize that by CKD Stage 4 patients are no longer able to maintain calcium balance
by excreting their daily intake of calcium. We also hypothesize that the retention of
calcium is not reflected in the serum concentration, as the body attempts to maintain the
serum calcium concentration within narrow limits.

We will evaluate balance at two different levels of calcium intake. The two different levels
were chosen to be at the extremes of normal calcium intake to provide insight into balance
at low normal and high normal loads.

Specific expected experimental finding:

Directional Hypotheses:

Primary Hypothesis:

Stage 4 CKD patients (eGFR 15-29 ml/min/m2) will excrete 20% of the absorbed calcium on the
high calcium (2000 mg) diet. On the low calcium (800 mg) diet, CKD stage 4 patients will
excrete 50% of the absorbed calcium. Gastrointestinal absorption will be 20% on both diets
resulting in a net positive calcium balance of 320 mg on the high calcium intake and 80 mg
on the lower calcium intake.

Stage 3 CKD patients (eGRF 30-59 ml/min/m2) will excrete 50% of the absorbed calcium on the
high calcium diet and 80% of the absorbed calcium on the low calcium diet. Gastrointestinal
absorption will be 20% on both diets resulting in a net positive calcium balance of 200 mg
on the high calcium intake and 32 mg on the lower calcium intake.

In addition, we hypothesize a linear decrease in fractional excretion of absorbed calcium as
eGFR decreases.

Secondary Hypothesis:

Stage 4 CKD patients (eGFR 15-29 ml/min/m2) will excrete 60% of their absorbed phosphorus on
the 1600 mg phosphate diet. Stage 3 CKD patients (eGRF 30-59 ml/min/m2) will be expected to
excrete 80% of the absorbed phosphorus on the 1600 mg phosphate diet. Gastrointestinal
phosphate absorption is expected to be 60% on both diets resulting in a net positive
phosphorus balance of 192 mg and 384 mg in stage 3 and stage 4 CKD respectively. In
addition, we hypothesize a decrease in fractional excretion of absorbed phosphorus as eGFR
decreases.

The goal will be to study the following groups of patients:

1. Stage 3 CKD (eGFR 30-59 ml/min/m²)

2. Stage 4 CKD (eGFR 15-29 ml/min/m²)

Estimated glomerular filtration rate (eGFR) will be calculated by the four variable
Modification of Diet in Renal Disease (MDRD) equation which includes the serum creatinine,
patients' age, gender and ethnicity.

Subjects with CKD will be recruited from the clinical practices of the nephrology division
at the University of Colorado HSC by their nephrologists who will screen them for stable CKD
based on historical laboratory data.

Following informed consent and prior to the start of the study, patients will be evaluated
to determine whether they are vitamin D replete. Vitamin D deficiency is common not only in
the elderly and chronically ill, but also in young healthy adults, especially at the end of
winter. In a study of young healthy adults, ages 18 to 29, 36% of 69 subjects were found to
be Vitamin D 25-OH deficient at the end of winter compared with 4% by the end of summer
(13). For this reason, all patients will be assessed for Vitamin D 25-OH repletion prior to
initiating the study diets.

This will be accomplished by checking serum Vitamin D 25-OH levels in all patients. Those
with Vitamin D 25-OH levels less than 30 ng/mL will receive ergocalciferol 50,000 IU per
week x 4 week. This is the KDOQI guideline recommendation for patients with CKD who are
vitamin deficient. After that time, their Vitamin D 25-OH levels will be checked again to
assess whether they are replete (i.e., serum level > 30 ng/mL). If their serum level is
again below 30 ng/mL, a second 4 week course of ergocalciferol will be given. If the
patient's serum Vitamin D 25-OH level remains < 30 ng/mL they will be excluded from further
study. Once replete, subjects will remain on 50,000 IU ergocalciferol monthly throughout the
study. This will ensure that vitamin D substrate is not a limiting factor in these patients.
In addition the following medications will be discontinued following informed consent and
for at least 1 month prior to the start of the first controlled diet, and for the duration
of the study: paricalcitol, doxercalciferol, rocaltrol, cinacalcet, calcium supplements
including calcium containing vitamins, or phosphate binders. Once patients are vitamin D
replete they will have their calcium and phosphorus determined and be excluded from the
study if their phosphorus exceeds 4.5 mg/dL or their calcium is outside the normal range.

Patients will be placed on two diets in random order for 9 days each. Randomization to diets
will be accomplished using Permuted blocks stratified by CKD stage. Patients will receive
one of two diets for each 9 day period, and then will subsequently go on the second diet
after at least a one week "washout period". This wash-out period may last up to 4 weeks to
accommodate a patient's schedule or to study menstruating women during the same phase of
their cycle. Both patients and researchers will be blinded as to which diet the patients are
receiving.

The last two days of each study period, patients will be admitted to the GCRC where they
will collect two consecutive 24 hour urines for calcium, phosphorus, sodium, and creatinine,
as well as a stool sample (see below) for calcium and phosphorus. The stool analysis for
calcium and phosphorus will be used to determine the amount of the ingested calcium and
phosphorus that is absorbed from the diet (fractional absorption), while the urinary calcium
and phosphorus will be used to determine the fraction of absorbed mineral excreted in the
urine. The urine sodium will be used to ensure stable dietary intake while the urinary
creatinine will be used to ensure that the urine collections are complete and to determine
the tubular reabsorption of phosphorus. A non-absorbable stool marker (Brilliant Blue) will
be administered to patients on the evening of their admission to the GCRC and again the
second evening in order to demarcate the time period over which the stool collected was
formed. If the patient has not passed the second marker by the end of the 48 hour period,
they will be asked to collect their stools at home and then bring them back to the GCRC.

On the final day of the patients' GCRC admission, after collection of the second 24 hour
urine sample is complete, a final small urine collection will be obtained. This last urine
sample will be used to measure spot calcium and phosphorus concentrations in order to
compare these values to the known amount excreted in the two previous 24 hour collections.

Patients will also have a small saliva sample, approximately 3 mL, collected during their
GCRC admission that will be used to measure salivary phosphorus concentration. These saliva
samples will be stored at the GCRC until a laboratory has been identified to perform this
testing.

Patients will also have an overnight fasting serum calcium, phosphorus, creatinine, 1,25
vitamin D, 25 vitamin D, intact PTH, and FGF 23 levels measured at the completion of each 24
hour urine collection. In addition, patients will have a serum phosphorus determination
prior to their evening meal on both evenings of their GCRC stay. After a "wash-out" period
of at least one week patients will start the second week-long diet, at the end of which they
will be admitted to the GCRC for a two day period. Patients may schedule their second diet
period and GCRC stay within one to four weeks of the previous diet and GCRC stay at their
convenience. The diets will be prepared by the GCRC nutrition staff.

Diet #1 (high-calcium diet):

Elemental calcium 2000 mg, phosphorus 1600 mg

Diet#2 (low-calcium diet):

Elemental calcium 800 mg, phosphorus 1600 mg

The diets will have the same sodium and potassium concentrations, and will be diabetic 2000
kCal/ day diets. As the goal of this study is to focus on calcium balance, which should not
be affected by energy balance, there will be no adjustment for individuals who are in
negative or positive energy balance. During their stay in the GCRC subjects will drink only
deionized water or other beverages whose calcium and phosphorus content has been included in
the assessment of dietary calcium intake.

All study subjects will be counseled to only eat food prepared for the study protocol. They
will also be counseled to attempt to eat all of the food provided. They will be instructed
to bring in any food that is not consumed. They will also be asked to keep a diet history of
any food they eat that is not on the prepared diet. No supplemental vitamins, calcium,
vitamin D, or phosphate binders will be taken during the study period.

Inclusion Criteria:

- Men and women 18 years of age or greater and able to comply with the study protocol.

- eGFR within appropriate target ranges (30-59 ml/min/m² for CKD stage 3 and 15-29
ml/min/m² for CKD stage 4)

- Stable kidney function defined as an eGFR which remains within a 10ml/min range over
a 3 to 6 month period prior to study enrollment. This will be determined from
historical laboratory data.

- Willing to follow study protocol

- Have a level of understanding and willingness to cooperate with all visits and
procedures as described by the study personnel

- Overall stable medical condition, defined as no malignancy, metabolic condition or
gastrointestinal condition that would make it difficult to comply with the study
protocol and complete both dietary study periods

- Able to read the consent form and provide informed consent.

- Serum calcium and phosphorus within the normal range off of vitamin D analogues,
calcium supplements, and phosphate binders for 1 month.

Exclusion Criteria:

- 1. Age less than 18 years

- Are pregnant, plan on becoming pregnant during the study period, or breast-feeding,
if female.

- Patient declines participation.

- GI malabsorption, short bowel syndrome, frequent vomiting, or gastroparesis.

- Subject receiving synthetic parathyroid hormone, glucocorticoids, calcitonin or
bisphosphonates.