Measurement of urinary glycosaminoglycans in dogs
Abstract Recent work in humans with protein losing nephropathies has revealed increased urine concentrations of sulfated glycosaminoglycans (GAGs). Differences exist between normal patients, those with glomerulonephritis (GN), and those with amyloidosis thus potentially allowing differentiation without a renal biopsy. Aims of this study were to validate a simple spectrophotometric assay used to measure canine urinary GAGs, establish a normal reference range, and determine optimal storage conditions. Urine GAG concentrations were measured in a limited number of dogs with glomerulonephritis or amyloidosis. Fourteen healthy dogs were placed in metabolic cages and all urine was collected for 24 hours. Serum and urine creatinine concentrations were measured at the beginning and end of the collection period. Urine collected at the beginning of the 24-hr period was centrifuged and the supernatant used to measure a spot GAG concentration and a spot glycosaminoglycan to creatinine ratio (GCR). A well mixed aliquot of the 24-hr sample was centrifuged, the supernatant used to measure the 24-hr total GAG, and stored at 4Â°C and -20Â°C for 1, 7, and 30 days. All dogs were used to determine effects of time and temperature (n=14), however, only dogs with an endogenous creatinine clearance > 2 ml/min/kg (n=10) were used to determine normal values. A standard absorption curve using a 1,9-dimethlymethylene blue dye and dilutions of chondroiton-4-sulfate was developed to estimate total GAG concentration. Repeated measures analysis of variance was used to test for effects of storage temperature and time on stability of urinary GAG. A p-value of < 0.05 was considered significant. Relationships between spot urinary GAG concentration, spot urinary GAG to creatinine ratio (GCR) and 24-hr total GAG excretion were estimated using simple linear regression. Single urine samples were collected by cystocentesis from dogs with GN or renal amyloidosis. The diagnosis was confirmed by clinical evaluation or by histologic analysis. Urine protein, creatinine and GAG concentrations were measured. There were no time or temperature effects on urine GAG concentrations for up to 1 day at 4Â°C and 30 days at -20Â°C. Mean 24-hr total GAG excretion Â± standard deviation was 1.586 Â± 0.461 mg/kg of body weight. Mean spot GAG concentration and spot GCR were 5.007 Â± 1.588 mg/dl and 0.023 Â± 0.01 respectively. Neither spot GAG concentration (R2=0.4216) nor GCR (R2= 0.0839) were adequate predictors of 24-hr total GAG. The GCRâs from dogs with renal disease were not different from normal dogs. This study established normal total urinary GAG values in dogs. Contrary to findings in humans, there was no correlation between 24-hr total sulfated GAG and spot GCR in dogs, limiting clinical utility of this test. Further work is needed to determine if either total sulfated GAG or the spot GCR can be used to differentiate causes of protein-losing nephropathies in dogs.