Urinary purine metabolite concentrations in dogs consuming the test diet were similar to baseline values for all dogs that completed the study. The urinary purine values achieved while consuming the test diet may be appropriate for managing these dogs with the SLC2A9 mutation and historical clinical urate urolithiasis. The management of canine urate urolithiasis has involved the restriction of dietary purine often by lowering dietary protein intake in order to decrease concentrations of urinary purine metabolites. In short-term trials in healthy beagle dogs, a casein-based diet formulated with 10.4% protein and 1% potassium citrate (dry matter basis) significantly decreased the urinary activity product ratios of uric acid, sodium urate, and ammonium urate as well as 24-h urinary uric acid excretion compared to a meat based diet with 31.4% protein (dry matter basis) [14]. We found a significant decrease in 24-h urinary uric acid concentrations only at the 6 and 12-month visits compared to baseline when evaluated by the CE method. No other differences were noted with regard to other purine metabolites when evaluated by either method. It is unclear at this time why only one variable was significantly different. It was likely not due to urine dilution because the urine specific gravity was not significantly different over time. Furthermore, no other purine concentrations were significantly lower. These results suggest that the test diet may be suitable alternative for managing dogs with genetic hyperuricosuria and a history of clinical urate urolithiasis.
While studies in dogs are not published regarding the possible side effects of long-term protein restriction, it has been demonstrated that protein requirements increase in older dogs secondary to increased protein turnover [15], and the impact of low protein diets in individuals with lower energy requirements might be more pronounced. We did not find any significant difference at 12 months compared to baseline with regard to body composition or most plasma amino acid concentrations. This was interesting as most dogs in this study were younger or middle aged at enrollment, and their maintenance energy requirements (MER) were relatively low with a median of just 88% (range 35–118%) of the estimated values for inactive pet dogs established by the National Research Council (MER = 95 x kgBW0.75) [16]. As such, of the 7/9 dogs eating the lower protein diet at the baseline visit, 4 were not eating protein in concentrations to meet the NRC minimal requirement (2.6 g/kgBW0.75), while 2 dogs were consuming protein in amounts that fell between the minimal requirement and the recommended allowance, and only 1 exceeded the recommended allowance (3.3 g/kgBW0.75). After consuming the test diet for 2 months, all dogs were ingesting protein in concentrations that exceeded the NRC recommended allowance per metabolic BW. By the 6 and 12 month visits, one dog’s energy requirements had decreased so that in order to maintain stable body weight the amount of test diet was reduced to the extent that the protein intake fell below the NRC minimal requirement. Regardless, we did not find any significant difference at 12 months compared to baseline with regard to body composition or most plasma amino acid concentrations for any dogs. However, our sample population was small and we did not compare essential amino acid profiles among the diets. Larger, longer- term studies may be warranted to examine the benefit, if any, of higher dietary protein concentrations.
We noted significant differences between two different methodologies for analyzing urinary uric acid. The CE method is fast and simple; however, the careful preparation of all standards is necessary for validation. Because this method is not readily available at our institution, we commonly use the colourimetric assay, which is useful for evaluating urinary uric acid trends. The primary clinical indication for determination of 24-h urinary uric acid excretion is to titrate the dosage of allopurinol [9].
Decreasing the urinary concentration of calculogenic substances by increasing urine volume is one of the cornerstones of urolithiasis prevention [17]. In the current study, mean USG was maintained <1.020 at every visit and was not significantly different from baseline, likely because we encouraged owners to continue to provide added water for their dogs. The test diet is only available as a dry formulation, but all dogs consumed the test diet readily with appropriate amounts of water added to produce target USG (<1.020). This test diet with added water appears to maintain low urinary purine metabolite excretion for dogs that require a purine-restricted diet. However, USG should be monitored periodically in dogs with clinical urolithiasis.
Aciduria is considered a risk factor for urate urolithiasis because ammonium and hydrogen ions may precipitate with uric acid [18]. In the current study, urinary pH was lower than historically recommended [18] for urate urolithiasis management at all visits and regardless of methodology. Urinary alkalinizing agents such as potassium citrate could be considered to increase the urine pH, although studies suggest supplementation in healthy dogs may have inconsistent effects on urinary pH [19].
While consuming the test diet with added water (2-, 6- and 12-month visits for the 7 dogs that completed the study and 2- and 6-month visits for 2 dogs lost to follow up), most dogs only had mild echogenic sand in their bladders with the exception of the dog whose owner did not strictly adhere to feeding only the test diet to her dog, which developed cystic calculi at the 6- and 12-month visits. While other studies have not reported the presence or absence of renal mineralisation, we noted these findings were also subjectively static throughout the one- year study period in the dogs that completed the trial. Despite their predisposition due to gender and genetics, all dogs in the current study remained free of upper or lower urinary tract signs, despite variable amounts of mineralization noted periodically. All dogs were managed without medications such as urinary alkalinizing agents or xanthine oxidase inhibitors while consuming the test diet with added water. However, urinary sand was present in 5/7 dogs at study completion; it is unknown if lowering urinary urine acid excretion any further would be of benefit in these dogs. Furthermore, they remained free of any clinical signs, so further intervention was not initiated.
Urate calculi are not often radiodense, and contrast cystourethrograms or ultrasonography is considered more sensitive for detection of uroliths. Radiography was only able to detect 32% of cases with urate uroliths in one study, with many more dogs that required contrast cystourethrograms [20]. We opted to utilize only ultrasonography in this study for our subjective assessment of urinary mineralisation, as sedation is required at our institution to perform contrast imaging and would preclude other tests during the same visit, which required scheduled meal consumption. Actual urolith recurrence rates could not be determined in this study due to varied stages of disease of the dogs at study enrollment. Furthermore, while we did follow the dogs for one year, recurrence rates are variable and could extend past this time point. However, when evaluating clinical signs only 1 dog in our trial exhibited signs suggestive of lower urinary tract disease, and resolved without intervention.
Two dogs did develop uroliths (1 dog with cystoliths; poor diet compliance and one dog with cystoliths and nephroliths determined to be calcium oxalate). However, in the dog that developed calcium oxalate urolithiasis, not all lower urinary tract calculi were removed and none of the nephroliths were removed. The owner did not wish to pursue another surgery, due to lack of clinical signs and the intervention required. The role of diet and any other individual or environmental factors in the formation of calcium oxalate urolithiasis in this case is unknown. The urinary pH was significantly lower at the end of the study period when spot urine samples were evaluated, which could have contributed to calcium oxalate formation in this dog. The dog was monitored and a customised homemade diet was instituted after study completion to manage the complex urolithiasis. Based on this case, if progression occurs in a dog with known genetic hyperuricosuria, radiographs and ultrasound together are warranted in order to aid in identification of potential development of different types of calculi. Urine pH (preferably several spot evaluations or 24-h pooled samples) should also be evaluated to help with management strategies. Further, any subsequent calculi should be removed and submitted for analysis to aid in management, regardless of history. Of the 1650 calculi that have been submitted from Dalmatians to our laboratory, only 3/1650 (0.001%) contained only calcium oxalate, although 43/1650 (0.03%) had some portion of calcium oxalate mixed with urate or another mineral (unpublished data, University of California, Davis Stone Laboratory, Westropp, 2014).
The limitations of the study include the small sample size. It was difficult to acquire additional dogs that could visit our facility at the scheduled time periods over a one year time period, and we only included dogs with a history of clinical urate urolithiasis. We did this to ensure the purine-restricted test diet was clinically indicated for their disease process. Furthermore, actual urolith recurrence could not be accurately evaluated because some of the mineralisation noted could not be removed prior to study enrollment. Furthermore, the dogs were only studied over a one- year time period. Finally, dogs had variable states of disease when enrolled in the study. While small amounts of echogenic “sand” were visible in the bladder in some dogs during the trial, this sediment was not removed and analyzed. We assumed this sediment was comprised of urate; however, infrared spectroscopy would be required to confirm composition. It is also possible that catheterisations performed 24 h prior to the ultrasonographic evaluations could have inadvertently removed small amounts of tiny cystoliths, therefore underestimating the number of cystic calculi present at the time of imaging.
Finally, 24-h urine collections were obtained from samples that owners collected, and the dogs were not housed in metabolism cages nor had indwelling urinary catheters placed. However, there are well known limitations to entire collections of urine even in controlled environments, which impacts the accuracy of nitrogen balance studies.[21] Probably more importantly in the case of veterinary patients, many dogs do not consume their typical intake of food and water under the conditions of stressful confinement in a hospital setting, which is a large factor influencing the accuracy of assessments based on 24-h urine collections regardless of whether catheterization or free catch techniques are used. The significant negative correlation noted between urine creatinine and urine volume suggests owners did not miss a micturition.