Effects of calcifediol supplementation on markers of chronic kidney disease‐mineral and bone disorder in dogs with chronic kidney disease

Abstract Background Chronic kidney disease‐mineral and bone disorder (CKD‐MBD) in dogs is associated with hypovitaminosis D, increased parathyroid hormone (PTH), and increased fibroblast growth factor‐23 (FGF‐23) concentrations. Best practice for vitamin D metabolite supplementation in CKD‐MBD remains unknown. Objective To provide an extended‐release calcifediol supplement to dogs with CKD and to measure its effects on variables indicative of CKD‐MBD. Animals Ten dogs with International Renal Interest Society stages 2 and 3 CKD. Methods In a prospective study, dogs received a calcifediol supplement for 84 days. Serum 25‐hydroxyvitamin D (25[OH]D), 1,25‐dihydroxyvitamin D (1,25[OH]2D), 24,25‐dihydroxyvitamin D (24,25[OH]2D), creatinine, calcium, phosphorus, PTH, plasma FGF‐23 concentrations, and urine profiles were measured monthly during supplementation. Urine calcium to creatinine (UCa/Cr) ratios and fractional excretion of calcium, phosphorus, and sodium were determined. Results All serum vitamin D metabolite concentrations increased significantly by day 84 (P < .001): [25(OH)D (median 249.9 ng/mL; range, 149.7‐469.9 ng/mL) compared to baseline (median 50.2 ng/mL; range, 31.3‐66.0 ng/mL); 1,25(OH)2D (median 66.1 pg/mL; range, 56.9‐88.1 pg/mL) compared to baseline (median 37.3 pg/mL; range, 29.3‐56.7 pg/mL); 24,25(OH)2D (median 81.4 ng/mL; range, 22.1‐151.7 ng/mL) compared to baseline (median 15.4 ng/mL; range, 6.9‐40.6 ng/mL)]. There were no significant differences in calcium, phosphorus, PTH concentrations, UCa/Cr or fractional excretion of calcium. No dog developed ionized hypercalcemia. Plasma FGF‐23 concentrations increased by day 84 (median 1219 pg/mL; range, 229‐8824 pg/mL) compared to baseline (median 798 pg/mL; range, 103‐4.145 pg/mL) (P < .01). Conclusions and Clinical Importance Calcifediol supplementation for 84 days was well‐tolerated in dogs with IRIS stages 2 and 3 CKD. It remains to be determined how long‐term supplementation would affect CKD progression and QOL.

Conclusions and Clinical Importance: Calcifediol supplementation for 84 days was well-tolerated in dogs with IRIS stages 2 and 3 CKD. It remains to be determined how long-term supplementation would affect CKD progression and QOL.  4 These derangements correlate with International Renal Interest Society (IRIS) stage. 5,6 In people with CKD, hypovitaminosis D and increased serum PTH and FGF-23 concentrations are associated with disease progression and decreased survival. [7][8][9] In dogs with CKD, increased serum phosphorus, calcium × phosphorus product (CaPP), as well as FGF-23 concentrations are negatively correlated with survival. 10,11 In human medicine, management of CKD-MBD utilizes an integrated approach, including dietary phosphate restriction and oral phosphate binders (to reduce intestinal phosphate absorption), calcimimetics (to lower PTH by enhancing activation of the calcium-sensing receptor), and vitamin D sterols (to increase intestinal calcium absorption and to inhibit PTH genomic synthesis and secretion). 12 There is no widely accepted best practice for how to supplement vitamin D. Per the 2017 Kidney Disease Improving Global Outcomes report, vitamin D 3 (ie, cholecalciferol) and D 2 (ie, ergocalciferol) therapy remains unproven. High doses of oral vitamin D 3 or vitamin D 2 supplementation often do not reliably increase 25(OH)D concentrations in people. 13,14 Prophylactic calcitriol therapy is no longer recommended due to risk of hypercalcemia. 15 While there was not enough data to specifically comment on the use of calcifediol [25(OH)D], some recent literature suggests that calcifediol is safe and effective in treating hyperparathyroidism associated with hypovitaminosis D in people with CKD. 16,17 In veterinary medicine, there is similar emphasis on controlling oral phosphate intake and serum phosphorus concentrations, but there has been little research specifically into how to best utilize vitamin D supplementation. In healthy dogs, high doses of cholecalciferol (ie, vitamin D 3 , the precursor to 25(OH)D via hepatic metabolism) are required to affect serum 25(OH)D concentrations. 18

| Study design
Dogs were screened initially to ensure study inclusion and exclusion criteria were met. Dogs then returned within a 2-week timeframe to complete enrolment and to ensure that their serum creatinine concentrations were stable. Dogs were prescribed an extended-release 25(OH) D medication approved for use in humans with CKD (Rayaldee, OPKO Renal Health, Miami, Florida). Dosage was based on body weight to provide approximately 2.0 μg/kg/day. This dose was extrapolated from data in healthy dogs. 20 Premade capsules were available in 30 and 60-μg capsules, and dogs received either 1 or 2 capsules per day, in the evening, with food. Dogs received the supplement for 84 days and were rechecked every 28 days. After preliminary review of data from the first 4 dogs enrolled, based on the degree and rapid nature of the rise in serum 25(OH)D concentrations, calcifediol dosing was modified to be provided on a Monday-Wednesday-Friday (M-W-F) schedule.
At baseline, and for the monthly rechecks while dogs were receiving the calcifediol supplement, a complete physical examination, including body weight, body condition score and muscle condition score, as well as funduscopic examination, was performed. A blood pressure was After 84 days of calcifediol supplementation, owners were instructed to discontinue the supplement. Vitamin D metabolites, PTH, and FGF-23 concentrations were measured 28 and 56 days later. CBC and chemistry profiles were obtained 56 days after discontinuation.

| Serum and urine profiles
The serum chemistry and urine profiles, including concentrations of creatinine, total calcium (tCa), and phosphorus, were performed using a   is reported to be 10%, intra-assay coefficient of variation is reported to be 3%, and functional sensitivity is reported to be 0.3 pmol/L for this assay. Plasma FGF-23 concentrations were measured using a human-specific ELISA (Kainos FGF-23 ELISA, Japan), previously validated for measurement in dogs. 22

| Quality of life questionnaire
Owners filled out a quality of life (QOL) questionnaire at screening, baseline and every month for 3 months while receiving the calcifediol supplement (Appendix). This questionnaire was modified from 2 previouslyvalidated questionnaires for dogs with cancer 23 and cats with CKD. 24 Owners rated 14 parameters related to their dogs' (a) happiness, (b) mental status, (c) appetite and gastrointestinal health, (d) mobility and recreation, (e) general health. Each question could receive a score of 1 to 5, with lower scores corresponding to lower QOL and higher scores corresponding to higher QOL. The highest score a dog could receive was 70.

| RESULTS
A total of 11 dogs were screened for inclusion into the study.
T A B L E 1 Laboratory variables of CKD dogs at baseline, while receiving calcifediol supplementation (through day 84), and after discontinuation of calcifediol supplementation.  Note: Data are presented as medians and ranges. Normally distributed variables are noted with an asterisk (*).

| Calcium and phosphorus
Serum tCa, iCa, and phosphorus concentrations and UCa/Cr ratios were monitored during the study to evaluate for hypercalcemia, hyperphosphatemia, or increased calciuresis associated with the calcifediol supplement. The serum tCa concentrations are nearly identical to those of serum iCa concentrations, so only tCa is reported. tCa increased by 0.54% (95% CI: 0.21%, 0.88%; P = .002) at day 84. One dog appeared to respond more than all others, and, when removed, There were no observed differences in UCa/Cr over the duration of the dosing (P = .14). Fractional excretion of tCa, iCa, phosphorus, and sodium were measured and no observed differences were noted over the duration of dosing (Table 1).

| Parathyroid hormone and fibroblast growth factor-23
Serum PTH and plasma FGF-23 concentrations are listed in Table 1.
There were no observed differences in PTH concentrations at any timepoint from baseline (P = .26). For FGF-23 concentrations, differences were observed at days 84, 112, and 140 (P < .01 for all timepoints). This remained true even when 1 dog whose FGF-23 concentrations increased much more than all others was removed from the model ( Figure 4). There was no observed association between FGF-23 concentrations and FE of phosphorus (P = .64).  The optimal goal for serum 25(OH)D concentration during vitamin D supplementation in dogs is currently unknown. One study suggested that a serum concentration of 100 ng/mL is optimal, based on a plateau of serum ionized PTH concentrations in healthy dogs and dogs with hemoabdomen. 25 Despite some individual laboratories having reference ranges for normal dogs, there are no well-defined physiologic reference ranges for "normal" 25(OH)D concentrations nor for insufficiency or deficiency. In 1 study, the range of serum 25(OH)D concentrations in healthy dogs ranged from 9.5 to 249.2 ng/dL, indicating that there is tremendous variability even in a healthy dog population. 26 In people, vitamin D insufficiency is currently defined as a serum 25(OH)D concentration <30 ng/mL, and goal of vitamin D supplementation is typically to increase 25(OH)D above 30 ng/mL. 27 However, human reference ranges cannot be applied to dogs as there are significant differences in dietary intake of vitamin D, calcium and phosphorus between the species as well as baseline serum concentrations in otherwise healthy individuals.

| Diet, medications, and supplements
One of the unique properties about this extended-release calcifediol medication in people is that gradual increases in serum con- Urine Ca/Cr ratios were measured to monitor for early signs of hypervitaminosis D as this can be an earlier indicator than hypercalcemia. 33 While there are not fully established normal reference ranges for UCa/Cr in dogs, 1 study used 0.05 as a cut-off to document hypercalciuria; this was based on the upper end of the 95% confidence interval for control dogs. 34 In our study, there was not a significant increase in median UCa/Cr with calcifediol supplementation.
One dog did have an increase from baseline to day 84, from 0.01 to 0.07. This dog received daily calcifediol supplementation and ultimately had the highest serum 25(OH)D concentration documented at 469.9 ng/dL. One dog had a higher baseline UCa/Cr of 0.07 with subsequent values of 0.12, 0.13, and 0.11 at days 28, 56, and 84, respectively. This dog was a 1-year-old large breed dog, and its age might have contributed to higher UCa/Cr ratios, as UCa/Cr ratios vary with age in children. 35 Despite a lack of significant increase in UCa/Cr, there was an increase in FE of calcium while dogs were receiving the extendedrelease calcifediol supplement. In 1 study of 17 normal dogs of various breeds, the range of FE of calcium was 0.053%-0.555%. 36 In another study of 48 Greyhound dogs, the range of FE of calcium was 0.03%-  36 Both of these studies entered tCa into the equation. 36,37 In our study, median FE of calcium, based on tCa, increased from 0.18% to 0.51% after 84 days of calcifediol treatment. Using iCa in the equation resulted in a larger calculated number since iCa is of lower magnitude than tCa by about 50%. Neither the use of tCa nor iCa is ideal for the calculation of FE of calcium, since neither fully accounts for the ultrafilterable fraction of calcium (ionized plus complexed fractions that cross the glomerulus). Use of tCa overestimates the amount of calcium available for glomerular filtration since it includes the proteinbound fraction which does not enter urine in the absence of severe proteinuria. Serum iCa underestimates how much calcium is available for filtration since it does include the complexed fraction of calcium that also crosses the glomerulus.
The significance for the increased FE of calcium in this study and possible long-term adverse effects remain unknown. No short-term adverse effects were identified. Theoretically future studies should evaluate dogs for the presence of calcium-related crystalluria and stones. It has been reported that FE of all electrolytes increase during CKD, and FE is not expected to match up with the absolute quantity for total daily excretion of electrolytes in CKD (mg or mEq). 21,38,39 Normal FE of phosphorus in dogs has been reported to range from 11%-41%, 36,37 which was similar to our results. There was no correlation between FE of phosphorus and plasma FGF-23. There were a few limitations of this study. It would have been ideal to obtain more complete data sets at days 112 and 140 (eg, serum iCa concentrations, UCa/Cr). This was a short pilot study primarily designed to determine (a) efficacy of serum 25(OH)D repletion and (b) tolerance of the calcifediol supplement. This study was unable to specifically determine the optimal dose of extended-release calcifediol supplementation. The data generated did not determine if, and to what extent, the peak of 25(OH)D concentrations would have continued increasing in dogs with either daily or 3 times weekly dosing. Dogs that received the supplement 3 times weekly had a lower peak of serum 25(OH)D than dogs that received daily supplementation, and there was no evidence of toxicity with this dose.
Sample size is an important consideration in any study, and this might have affected the results in certain aspects of this study. One of the goals of this study was to obtain an estimate of standard deviation to plan further studies in this area of research. This study consisted of 10 dogs based on both preclinical data from the drug manufacturer and budget considerations. Specifically, the relatively small study size might limit our ability to detect some true differences and associations in this study population.
Further comprehensive studies in larger cohorts are needed to confirm or refute these results based on the data and effect sizes of the variables presented here.