Association between vitamin D metabolites, vitamin D binding protein, and proteinuria in dogs

Abstract Background Proteinuria has been associated with progression of renal disease and increased morbidity and mortality in dogs and people. In people, proteinuria also has been associated with hypovitaminosis D. Little is known about the relationship between vitamin D metabolism and proteinuria in dogs. Objectives To further elucidate vitamin D status in dogs with protein‐losing nephropathy (PLN) and minimal to no azotemia. We hypothesized that vitamin D metabolites would be lower in dogs with PLN compared to healthy dogs. Animals Twenty‐three client‐owned adult dogs with PLN and 10 healthy control dogs. Methods Serum 25‐hydroxyvitamin D (25[OH]D), 1,25‐dihydroxyvitamin D (1,25[OH]2D), 24,25‐dihydroxyvitamin D (24,25[OH]2D), serum vitamin D binding protein (VDBP), and urine 25(OH)D concentrations were measured. Results Compared to healthy dogs, dogs with PLN had lower concentrations of all vitamin D metabolites (P < .01). Correlations (rho; 95% confidence interval [CI]) in dogs with PLN are reported. Serum 25(OH)D and 24,25(OH)2D concentrations were positively correlated with albumin (r = 0.47; 0.07‐0.74), and 24,25(OH)2D was negatively correlated with urine protein‐to‐creatinine ratio (UPC; r = −0.54; −0.78 to −0.16). Urine 25(OH)D‐to‐creatinine ratio was negatively correlated with serum albumin concentration (r = −0.77; −0.91 to −0.50) and positively correlated with UPC (r = 0.79; 0.53‐0.91). Serum VDBP concentration was positively correlated with serum albumin concentration (r = 0.53; 0.05‐0.81). Conclusions and Clinical Importance Dogs with PLN have decreased serum concentrations of vitamin D metabolites. Urine 25(OH)D‐to‐creatinine ratio and UPC are correlated in PLN dogs. Future studies are needed to assess additional management strategies for dogs with PLN.


| INTRODUCTION
Chronic kidney disease (CKD) is commonly diagnosed in dogs, with a prevalence of up to 25% of dogs presented to veterinary teaching hospitals. [1][2][3] Major consequences of CKD in dogs include development of proteinuria and hypovitaminosis D. 4,5 In people, proteinuria and hypovitaminosis D, characterized by decreased serum 25-hydroxyvitamin D (25[OH]D) concentrations, are associated with progression of kidney disease and decreased survival, 6,7 and vitamin D status is inversely associated with magnitude of proteinuria. [8][9][10][11] Proteinuria in people is an independent predictor of vitamin D status in the absence of other causes of vitamin D dysregulation associated with kidney disease. 12,13 Similarly in dogs, proteinuria has been shown to be associated with progression of kidney disease and increased morbidity and death. 14 An association between serum vitamin D concentration and proteinuria also has been identified in dogs. In a study of 19 dogs with azotemic CKD, serum 25(OH)D concentrations were inversely related to urine protein:creatinine (UPC) ratios. 15 However, because of the complex pathophysiology of CKD-mineral and bone disorder (CKD-MBD), it was not possible to determine whether an independent association between proteinuria and vitamin D status existed separately from other CKD variables (eg, advanced azotemia). As a result, the association between proteinuria and vitamin D status in dogs with minimal to no azotemia remains unknown.
Our primary aim was to evaluate the relationship between vitamin D metabolites ( Dogs were excluded from the study if they were <1 year of age, receiving medications known to affect proteinuria (eg, corticosteroids), had a serum creatinine concentration ≥1.4 mg/dL) or had clinical signs, physical examination findings, or abdominal imaging abnormalities consistent with concurrent diseases that would affect proteinuria. Dogs enrolled as controls were deemed healthy on the basis of a normal physical examination, CBC, serum biochemistry profile, and urinalysis. All owners signed a consent form before dogs were enrolled. 4,16

| Study design
Each dog had a complete physical examination performed, including body weight, body condition score (BCS), and muscle condition score (MCS). 17 All BCS (using the 9-point scoring system) and MCS scores were assigned by 1 author (Valerie J. Parker). Blood pressure was measured using a Doppler ultrasonographic device and appropriately sized cuffs based on dog size. Blood was collected by jugular venipuncture for CBC and serum biochemistry. Urine was collected by cystocentesis for urinalysis, urine culture, and UPC. Additional serum and urine was stored at −80 C for analysis of vitamin D metabolites and vitamin D binding protein (VDBP). Information regarding medications, diets, and dietary supplements was recorded.

| Vitamin D metabolites and VDBP analysis
Serum 25(OH)D and 1,25(OH) 2 D concentrations were measured by radioimmunoassay (RIA) and 24,25(OH) 2 D concentration was measured by liquid chromatography-mass spectrometry (LC-MS). Urine 25(OH)D concentration was measured by RIA and serum VDBP concentration was measured by ELISA. As has been reported elsewhere, urine 25(OH)D concentration was assessed relative to urine creatinine concentration. 18 All vitamin D measurements were performed by a Vitamin D External Quality Assessment Scheme (DEQUAS)-certified laboratory (Heartland Assays, Inc, Ames, Iowa).

| Data analysis
Statistical analysis was performed using R 3. 6

| RESULTS
Twenty-three dogs with PLN and 10 control dogs were included.
Median age for dogs with PLN was 9.9 years (range, 3.1-13.5 years).
Median age for control dogs was 4.3 years (range, 1.4-10.3 years). Dogs with proteinuria were significantly older than control dogs (P < .001).
Breeds represented among dogs with PLN were Yorkshire Terrier  Table 1. Median UPC of the PLN cohort was 4.8 (range, 1.7-27.5). Median blood pressure was 160 mm Hg (range, 120-240 mm Hg). None of the control dogs was proteinuric. One dog had an increased systolic blood pressure of 180 mm Hg, but no specific underlying etiology was identified to account for this dog's hypertension.
To explore the association of the combination of age, body weight, UPC, serum albumin concentration, and serum creatinine concentration on the vitamin D metabolites, we used multivariable linear regression models. As seen in As seen in Table 5, an association was found between serum albumin

| DISCUSSION
We found that proteinuria in minimally to non-azotemic dogs is associated with decreased serum concentrations of vitamin D metabolites    [19][20][21][22] Activation of the VDR suppresses renin, has antifibrotic effects, and decreases glomerulosclerosis. [22][23][24] In people, several studies have demonstrated similar associations between vitamin D metabolites and proteinuria or albuminuria. [25][26][27][28] One study identified patients with focal segmental glomerulosclerosis, a disorder associated with proteinuria, to be at risk of vitamin D deficiency. 29 Another study in children with nephrotic syndrome and nor-  loss of VDBP with anti-proteinuric treatment. 44 In rats, urinary VDBP was shown to potentially be a biomarker for tubulointerstitial disease. 49 A recent study evaluated the use of urinary VDBP as a biomarker of renal tubular injury in dogs. 50 In that study, no urinary VDBP was detected in healthy dog urine, a but CKD dogs did have VDBP urinary loss that increased with stage of CKD, even without proteinuria.
In our study, serum VDBP concentration was not significantly dif- it is simply a reflection of general protein loss, including VDBP and its complexed vitamin D metabolites, rather than a causal link between albumin loss and hypovitaminosis D. Low vitamin D status also could affect albumin by its effects on inflammation. 57,58 Thus, the lack of vitamin D's immunomodulation actually may result in hypoalbuminemia, independent of urinary loss of albumin. 59 Although malnutrition often is cited as a predisposing factor to hypoalbuminemia, only 1 dog was reported to be underconditioned and 10 dogs were noted to have variable degrees of muscle atrophy, with most (n = 7) having only mild atrophy.
Our study had a few limitations. The control dog cohort included fewer dogs than the PLN cohort, and the dogs were not age-matched.
No difference in serum 25(OH)D concentrations by age however was found in a study of 320 dogs. 35 Regardless, it is difficult to assess what effects age and body weight may have had on the variables evaluated. The number of PLN dogs also was low. This could have led to a decrease in statistical power, potentially explaining some of the borderline significant results (eg, correlation between 25(OH)D and UPC).
Significant intraindividual variation can occur in day-to-day UPC in proteinuric dogs, 60 especially depending on methods of analysis (single, averaged, or 3-day pooled samples). 61 Although no studies to our