Effect of calcifediol supplementation on renin-angiotensin-aldosterone system mediators in dogs with chronic kidney disease

1 INTRODUCTION

The renin-angiotensin-aldosterone system (RAAS) plays a key role in maintaining fluid and electrolyte balance and in the regulation of blood pressure. However, up-regulation of RAAS mediators (eg, angiotensin II [AngII] and aldosterone) can result in negative sequelae.¹ RAAS upregulation occurs in chronic kidney disease (CKD) in multiple species and is thought to contribute to the development of hypertension and proteinuria as well as exacerbation of tubulointerstitial injury and glomerular damage because of pro-inflammatory and pro-fibrotic effects.^2-6 In turn, hypertension at the time of CKD diagnosis in dogs has been linked to decreased survival,⁷ and proteinuria has been shown to be associated with faster progression of CKD and decreased survival in dogs.⁸

Dogs with CKD have lower vitamin D metabolite concentrations than healthy dogs⁹ and hypovitaminosis D has been associated with progression of kidney disease and decreased survival in people.^{10, 11} Vitamin D receptor (VDR) knockout mice have increased plasma AngII concentrations and increased renal renin gene expression.^{12, 13} In remnant kidney model rats, supplementation with paricalcitol, a VDR agonist similar to calcitriol, decreases renal renin gene expression, and leads to lower serum creatinine concentration, hypertension, proteinuria, and glomerular and tubulointerstitial damage.¹⁴ In both normotensive and hypertensive humans, there is a significant association between vitamin D status and the RAAS, including higher concentrations of plasma renin and AngII in patients with low concentrations of vitamin D and a significant inverse association of 25(OH)D and plasma renin activity (PRA).^15-17 To our knowledge, no relationship between RAAS and vitamin D metabolism has been established in dogs.

Calcifediol, or 25-hydroxyvitamin D (25[OH]D) has been used in human medicine for the treatment of hypovitaminosis D associated with CKD and for the management of renal secondary hyperparathyroidism.^{18, 19} Calcifediol concentrations are correlated with calcitriol concentrations in dogs and significant increases in serum 25(OH)D can be achieved by providing additional substrate in the form of calcifediol.^{20, 21} Calcifediol significantly increases serum vitamin D metabolite concentrations, including 25(OH)D and 1,25-dihydroxyvitamin D (1,25[OH]₂D; calcitriol) in dogs.²² The primary aim of this study was to determine if calcifediol supplementation has an effect on concentration of circulating mediators associated with the RAAS. We hypothesize that supplementation with calcifediol will decrease serum RAAS mediators in dogs with CKD.

2 MATERIAL & METHODS

3 RESULTS

Ten dogs were initially evaluated for inclusion, with 4 dogs being excluded because of the usage of RAAS-inhibiting medications (n = 3) and incomplete data (n = 1). Six dogs with IRIS Stage 2 and 3 CKD were enrolled. Of these 6 dogs, 2 dogs received calcifediol daily and 4 received it on a Monday-Wednesday-Friday schedule. The median dose provided was 2.1 μg/kg per dose (range, 1.5-2.7 μg/kg). Median age was 6 years (range, 1-13 years). Breeds represented included German shepherd dog (n = 2), mixed breed (n = 2), Bernese mountain dog (n = 1), and golden retriever (n = 1). Median body weight was 26.7 kg (range, 15.3-40.1 kg). Median BCS was 5/9 on a 9-point scoring system (range, 5-6). Muscle condition score was normal in 3/6 dogs, with the remaining 3 dogs having mild muscle atrophy. There were 4 female spayed dogs, 1 castrated male, and 1 intact male.

Pertinent laboratory values for enrolled dogs are listed in Table 1. Dogs were classified as having IRIS Stage 2 (n = 4) or Stage 3 (n = 2) CKD, based on 2019 IRIS Staging of CKD guidelines. There was a significant increase in phosphorus concentration at month 5 compared to baseline. Otherwise, there were no significant differences in any laboratory values at any time points. Dogs were considered either non-proteinuric (n = 4) or borderline proteinuric (n = 2). Five dogs were eating a veterinary therapeutic renal diet while 1 dog was eating a commercially available complete and balanced diet. Dogs received: gabapentin (n = 3), aluminum hydroxide (n = 2), trazodone (n = 2), maropitant (n = 2), ketorolac ophthalmic solution (n = 2), amlodipine (n = 1), diphenhydramine (n = 1), fenbendazole (n = 1), fluoxetine (n = 1), flurbiprofen ophthalmic (n = 1), omeprazole (n = 1), phenylpropanolamine (n = 1), or sucralfate (n = 1).

Serum vitamin D metabolite concentrations are listed in Table 2. All vitamin D metabolites were significantly higher at month 3 compared to baseline. Serum concentrations of RAAS mediators are listed in Table 3 (median; range). Surrogate ACE activity, as calculated by the ratio of AngII to AngI, was significantly lower at month 3 (0.5; 0.4-1) compared to baseline (0.7; 0.6-1.3; P = .01; Figure 1). There was no significant difference in ACE activity between either month 3 and month 5 (0.6; 0.6-1.3; P = .06) or baseline and month 5 (P ≥ .99). No significant differences were noted at any time point for angiotensin I/II/IV/1-5/1-7, aldosterone, PRA, AA2, ACE2, nor ALT (Figures 2 and 3). AngIII values could not be assessed because of the majority (>75%) were below the limit of quantification.

4 DISCUSSION

This small study revealed that calcifediol supplementation for 3 months resulted in a significant decrease in surrogate ACE activity, as determined by the ratio of AngII to AngI. This finding could indicate a relationship between the RAAS and vitamin D metabolites in dogs with CKD.

Humans with varying degrees of hypovitaminosis D have significantly higher concentrations of AngII as compared to a healthy controls,¹⁵ and there is a significant inverse association between concentrations of 25(OH)D and 1,25(OH)₂D and both PRA and AngII.¹⁶ The mechanism of vitamin D-related suppression of the RAAS is thought to occur through its effects on renin transcription.^{12, 13} This effect is likely due, in part, to the effects of vitamin D on the cyclic AMP response element, in particular impacting its ligand-binding domain.²⁶ While renin is integral in converting angiotensinogen to AngI, no direct action of renin on the activity of the ACE has been established. The effects of therapies on the RAAS must also consider the balance between the classical (AngI, AngII, aldosterone, ACE, and angiotensin II type-1 receptor) and the ALT (Ang1-5, Ang1-7, ACE2, and Mas receptor) RAAS. The peptides, enzymes, and receptors of the ALT RAAS counteract the effects mediated by the classical RAAS and a shift toward a higher ALT RAAS activity is thought to be beneficial. No significant change was detected in this study in either the ACE2 surrogate or the ratio of ALT to classical RAAS activity, although this could be because of the low statistical power of this study and a type II error.

The results contained within this study could indicate an alternative role for vitamin D in the negative endogenous regulation of the RAAS, warranting further evaluation on a larger cohort of dogs. Given the small sample size, it is possible that these results are not reflective of the true influence of vitamin D on the RAAS.

One other significant finding noted in this study was that phosphorus changed over time. At month 5, phosphorus concentrations were significantly higher compared to baseline, with no significant difference between other timepoints. It seems most likely that this is related to progression of kidney disease over the study period. Additional markers of CKD-mineral and bone disorder (eg, parathyroid hormone, fibroblast growth factor-23) were evaluated previously in these dogs.²² While there was no significant difference in parathyroid hormone over the 5-month study, fibroblast growth factor-23 concentrations did increase over time, most likely consistent with disease progression.

There were limitations to this study. As this was a small study performed on a subset of samples from a prior study involving calcifediol supplementation,²² the study was likely underpowered to detect differences in some variables. As such, future studies with a larger cohort may detect differences that were not significant in this study. While dogs did receive different dosing regimens of calcifediol, the serum 25(OH)D concentrations at month 3 of supplementation did not differ between the dogs.

Equilibrium analysis of RAAS mediators is becoming more common in scientific research, but differences exist between circulating RAAS and equilibrium concentrations of RAAS, though these have shown good correlations.^27-29 Additionally, results for angiotensin IV, angiotensin 1-7, and aldosterone all included concentrations below the lower level of quantification; thus these concentrations were set at ½ of the lower level of quantification. Greater than 50% of the concentrations for angiotensin III were below the level of quantification, precluding final evaluation. Our assessment of ACE and PRA activity are calculated from measured angiotensin peptide concentrations, instead of being directly measured themselves. That said, the renin surrogate is strongly correlated to PRA measurement, the ACE surrogate has been shown to correlate to ACE activity measurement in functional ACEi studies, and these and related surrogates are now being used in human studies of hypertensive heart disease and heart failure.^{23-25, 30}

It is possible that some of the other medications that dogs received could have impacted results (eg, amlodipine; n = 1). Hypertensive cats with CKD that receive amlodipine have increased concentrations of PRA.³¹ Healthy research dogs receiving amlodipine had higher aldosterone concentrations.³² We did not appreciate any other obvious patterns in the results in this small group of dogs after removing the 4 dogs that received RAAS inhibitors.

In conclusion, supplementation with calcifediol in dogs with CKD resulted in a significant decrease in surrogate ACE activity, as determined by the ratio of equilibrium concentrations of AngII to AngI.

CONFLICT OF INTEREST DECLARATION

Dr. Quimby has received grant/research support from EveryCat Health Foundation, Morris Animal Foundation, Nestle Purina, Trivium Vet, and Zoetis. She has consulted for Boehringer Ingelheim, Dechra, Elanco, Gallant, Hill's, IDEXX, Nestle Purina, Royal Canin, Vetoquinol, and Zoetis. Dr. Parker has received grant/research support from Waltham Foundation, Nestle Purina, American Kennel Club, and OPKO Health, Inc. She has received speaker honoraria from Antech, Royal Canin, Nestle Purina, the American College of Veterinary Internal Medicine, and Veterinary Information Network, Inc. She has consulted for Elanco. No other authors declare a conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION

Authors declare no off-label use of antimicrobials.

INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION

This study used samples from a previous study at The Ohio State University (IACUC # 2013A00000122).

HUMAN ETHICS APPROVAL DECLARATION

Authors declare human ethics approval was not needed for this study.