Measurement of preprandial and postprandial urine calcium to creatinine ratios in male Miniature Schnauzers with and without urolithiasis

Abstract Background We aimed to identify a simple test for excessive calciuresis and predict calcium oxalate (CaOx) disease in Miniature Schnauzers. We investigated the impact of postprandial time on the urine calcium to creatinine ratio (UCa/Cr) in male dogs of this breed, with the goal of improving the utility of the UCa/Cr. Hypotheses (1) Significant differences will exist in preprandial and postprandial UCa/Cr between CaOx urolith‐forming and control Schnauzers. (2) The UCa/Cr will increase significantly from the first morning baseline at ≥1 postprandial time point(s) in both control and CaOx urolith‐forming dogs. (3) Biochemical abnormalities and other variables may be associated with urolith status. Animals Twenty‐four male Miniature Schnauzer dogs, consisting of 9 with (urolith formers) and 15 without (controls) CaOx uroliths. Methods Urine was collected before and 1, 2, 4, and 8 hours after feeding a standardized diet. Receiver operator characteristic curve analysis was performed to identify the UCa/Cr cutoff that most accurately differentiates dogs based on urolith status. Results Urolith formers had significantly higher mean UCa/Cr over the course of 8 hours. The postprandial change in UCa/Cr was not significant at any time point between or within groups. The cutoff UCa/Cr value of 0.06 had a specificity of 93% (95% confidence interval [CI], 80%‐100%) and a sensitivity of 56% (95% CI, 21%‐86%) for identifying CaOx urolithiasis. Conclusions and Clinical Importance Urolith‐forming male Miniature Schnauzers have excessive calciuresis, and the postprandial sampling time up to 8 hours is not critical. This simple urine measurement has potential as a marker of CaOx disease.


| INTRODUCTION
Calcium oxalate (CaOx) urolithiasis can be detected in approximately 26% of older, healthy dogs of high-risk breeds such as the Miniature Schnauzer. 1 Forty-eight percent of dogs will have recurrence within 3 years, 2 which may be a consequence of limited understanding of CaOx pathogenesis and a failure to address the underlying cause.
Identifying a marker of the disease will help better assess the risk of initial CaOx formation or recurrence.
Urine composition from dogs with CaOx urolithiasis differs from healthy dogs. Relative supersaturation 3 and urine calcium concentration 1,3,4 appear to be the most consistent differences. Of these, measurement of urinary calcium concentration, as a measure of urine calcium excretion (calciuresis), is more cost-effective and readily available. Although calciuresis traditionally has been assessed by performing 24-hour urine collection, convenience can be improved by performing a spot measurement.
To account for variations in urine water content throughout the day, urine calcium concentration can be indexed to concurrent urine creatinine concentration (UCa/Cr). In healthy children, the UCa/Cr correlates moderately well with 24-hour urinary calcium measurements in some studies. 5,6 Although this correlation has not been investigated in dogs, UCa/Cr has the potential to be a convenient alternative for estimating calciuresis in dogs.
No accepted reference range for UCa/Cr in dogs exists, but a recent study showed a significant difference in UCa/Cr between preprandial control and urocystolith-forming dogs of 3 different breeds. 1 Therefore, a goal of our study was to assess the UCa/Cr as a diagnostic tool to identify CaOx urolith disease and determine the most accurate cutoff value. Furthermore, previous data also shows that 6 CaOx urolith-forming dogs had increased postprandial calciuresis compared to their calcium excretion in preprandial state. 4 A better understanding of variation in calciuresis with regard to preprandial and postprandial states could improve the specificity of the UCa/Cr to differentiate between control and CaOx urolith-forming dogs. If postprandial urine calcium excretion differs between CaOx urolith-forming and non-CaOx-urolith forming dogs, this difference could be utilized to improve the discriminatory power of the UCa/Cr. A variety of other patient variables, including sex, breed, diet, concurrent diseases and medications, have been associated with CaOx urolith formation. 7 Any attempt to investigate the effect of postprandial calciuresis should attempt to control for these variable as much as possible.
Our specific aims were firstly to quantify differences in the firstmorning baseline and postprandial UCa/Cr between urolith-forming (as defined by nephroliths, urocystoliths, or both) and control male Miniature Schnauzers. The second aim was to compare the UCa/Cr in the postprandial period to baseline UCa/Cr within and between urolith-forming and control male Miniature Schnauzers.

| Case selection
The study was advertised to clients of the Veterinary Teaching Hospital (VTH) at the Virginia-Maryland College of Veterinary Medicine and to the local community. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Virginia Tech. Inclusion criteria were adult (>24 months of age), male (either neutered or intact), Miniature Schnauzer dogs. Dogs with diseases that alter calcium excretion (eg, hypercalcemia, hyperparathyroidism, renal disease, diabetes mellitus, osteolytic disease, granulomatous disease, and hyperadrenocorticism) were excluded. To identify concurrent disease, the medical history was reviewed, in addition to performing a physical examination, serum biochemistry profile, urinalysis, abdominal radiography, and urinary tract ultrasonography. Body condition score (1-9) was recorded. A diet history was collected for each subject. Dogs were excluded if they were not consuming a complete and balanced commercial adult maintenance dog food, substantiated by Association of American Feed Control Officials nutrient profiles or feeding test, as the main portion of their diet. Furthermore, dogs were excluded if they were consuming a commercial prescription veterinary urinary dog food because such a diet would be expected to alter urine constituents. Current diets were reviewed by a board-certified veterinary nutritionist. Dogs currently receiving medications that are known to alter calcium excretion (glucocorticoids, furosemide, thiazide diuretics, levothyroxine, theophylline, or potassium citrate) also were excluded. Dogs were prospectively enrolled with the informed owner consent.
Dogs were classified into 2 groups based on client history, medical records review, and results of abdominal radiography and urinary tract ultrasonography (kidneys, ureters, bladder) performed by the investigators. The groups were (1) urolith former dogs with previously removed or currently present CaOx uroliths anywhere in their urinary system or (2) breed-matched controls with no history or current presence of uroliths.
If urolithiasis was identified by either diagnostic imaging or the medical history, the following criteria then were applied to determine if it was reasonable to conclude that uroliths were composed of CaOx.
The criteria were either confirmatory CaOx urolith analysis or neutral or acidic urine with the lack of evidence of bacterial urinary tract infection, hepatic disease, or crystalluria of any type other than CaOx.
Dogs concluded to have other types of uroliths were excluded from the study.

| Study procedures
Dogs were presented to the VTH the day before sample collection.
The daily energy requirement (DER) was calculated using the equation 1.6 × 70 × BW 0.75 , where BW is the current body weight in kilograms.
All dogs were fed a standardized diet (Science Diet Adult 1-6 years Chicken and Barley Entrée Canned Dog Food; Hills Pet Nutrition, Inc, Topeka, Kansas) to meet the DER, divided into evening and morning meals, 12 hours apart. Dogs had to eat at least half of each prescribed meal to remain in the study. Dogs were walked 3-5 hours after the evening meal to ensure they urinated. The next morning, an initial urine sample was collected before feeding (first-morning or baseline).
After the baseline sample was collected, dogs were fed their morning meal. Urine was collected at 4 postprandial time points (referred to as the 1-, 2-, 4-, and 8-hour postprandial samples).
Urine was collected by voiding whenever possible, but if dogs did not voluntarily urinate, a sample was collected by aseptic urinary catheterization or cystocentesis, whichever was least stressful for individual dogs.

| Laboratory measurements
Initial preprandial blood and urine samples were submitted immediately upon collection to the VITALS Laboratory of the Virginia Maryland College of Veterinary Medicine for a serum biochemistry profile and urinalysis. If the urine dipstick detected ≤2+ proteinuria, then a sulfosalicylic acid precipitation (SSA) test was performed. If this test was also positive, then a urine protein to creatinine ratio was calculated. If the serum total calcium concentration was outside the laboratory reference range, then a serum ionized calcium concentration was determined.
The baseline and postprandial urine samples were submitted immediately for calcium and creatinine measurements. Urine calcium and creatinine concentrations were measured by spectroscopy on all 5 samples using a Beckman Coulter AU 480 analyzer, using the calcium-sensitive Arsenazo dye and the modified Jaffe procedures, respectively.

| Statistical analysis
The primary outcome was UCa/Cr, whereas the primary exposure of interest was urolith status (urolith former versus control). A power analysis using the analysis of variance (ANOVA) of PASS 8  Normal probability plots showed that UCa/Cr, ΔUCa/Cr, %ΔUCa/ Cr, age, and serum cholestrerol concentration followed a normal distribution, whereas body condition score and serum triglyceride concentration were skewed. Normally distributed variables were summarized as means (standard deviation) and skewed variables as medians (range).
Categorical variables were summarized as contingency tables. Associations between urolith status and potential confounders were assessed using 2-sample t-tests (age and cholesterol), Wilcoxon rank sum test (body condition score and triglycerides) and Fisher's exact test (presence of proteinuria, presence of hypercalciuria). A mixed-model ANOVA was used to assess the impact of urolith status (urolith former versus control) and time (first morning versus 1-, 2-, 4-, 8-hour postprandial) on UCa/Cr, ΔUCa/Cr, and %ΔUCa/Cr (separately). Each model also specified dog identification as a random effect, and residuals were inspected to verify that the errors were normally distributed with a mean of zero and a constant variance.   Table 1.
A significant postprandial effect on UCa/Cr was not identified in either group. The ΔUCa/Cr at 1, 2, 4, and 8 hours postprandial were not significantly different from zero, and the ΔUCa/Cr was not significantly different between the groups. The %ΔUCa/Cr also were not significantly different from zero or between urolith-forming and control groups (Figures 2 and 3).

| DISCUSSION
We confirmed an association of UCa/Cr with CaOx urolith status in male Miniature Schnauzers, which provides further evidence that abnormal calciuresis is a mechanism underlying CaOx urolithiasis in this population.
Significant increases in postprandial UCa/Cr were not identified, which is contrary to previous observations of postprandial calciuresis measured by the 24-hour urine collection. 4 Our study does not necessarily exclude postprandial calciuresis, but rather we conclude that the

CONFLICT OF INTEREST DECLARATION
Authors declare no 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
Approved by the Virginia Tech IACUC, application number 16-059.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.