Analysis of Calcium Balance in Chronic Kidney Disease
Analysis of Calcium Balance in Chronic Kidney Disease
Subjects with normal kidney function are able to excrete in their urine the calcium and
 phosphorus absorbed via the gastrointestinal tract and are thereby are able to maintain
 calcium and phosphorus balance. Patients with chronic kidney disease often have normal blood
 levels of calcium and phosphorus but no research has been conducted to determine if they are
 able to excrete their daily intake of these minerals. If they are not able to excrete their
 daily intake, these minerals could be deposited in soft tissue and blood vessels and
 contribute to the increased risk of cardiovascular disease seen in patients with chronic
 kidney disease. This study will determine if subjects with chronic kidney disease are in
 balance primarily with regard to calcium and at what level of kidney function they are no
 longer able to maintain balance. Understanding whether patients with chronic kidney disease
 are in balance will help direct future therapeutic interventions.
We hypothesize that by Chronic Kidney Disease (CKD) late Stage 3 and 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:

 Late Stage 3/Stage 4 CKD patients (Estimated glomerular filtration rate [eGFR] 15-40
 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.

 Subjects with normal kidney function (eGRF > 60 ml/min/m2) will excrete > 90% of the absorbed
 calcium on the high calcium diet and > 90% of the absorbed calcium on the low calcium diet.
 Gastrointestinal absorption will be 20% on both diets resulting in no net positive calcium
 balance on the high calcium intake or the lower calcium intake.

 Secondary Hypothesis:

 Late Stage 3/Stage 4 CKD patients (eGFR 15-40 ml/min/m2) will excrete 60% of their absorbed
 phosphorus on the 1600 mg phosphate diet. Normal subjects(eGRF > 60 ml/min/m2) will be
 expected to excrete > 90% 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 late stage 3/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. Late Stage 3/Stage 4 CKD (eGFR 15-40 ml/min/m²)

 2. Normal subject(eGFR > 60 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 Health Sciences Center (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 dihydroxy-vitamin D (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 Kidney Disease Outcome Quality Initiative (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 General Clinical
 Research Center (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 Parathyroid hormone (PTH), and fibroblast growth factor (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
 kilocalorie (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 (15-40 ml/min/m² for late stage CKD stage 3
 stage 4, and eGFR > 60 ml/min/m² for normal subjects)

 -  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.