2018 ACVIM Forum Research Abstract Program: Seattle, Washington, June 14 - 15, 2018

gs with stage 2 (n= 9) and stage 3 (n= 12) were followed-up for 12 to 15 months or until death. The evaluation by means of serum creatinine concentrations, only two CKD dogs with stage 2 changed the classification to stage 3 during the follow-up, and also in those dogs serum SDMA was increased concomitantly; however other three CKD dogs with stage 2 had the increase only in SDMA (changing the classification to stage 3) and not in serum creatinine, showing the underestimation of the stage based on serum creatinine. In CKD dogs with stage 3, during the follow-up, only two dogs changed the classification to stage 4 based on serum creatinine concentrations, however when taking SDMA concentrations (> 45 μg/dL), 9 out of 12 changed the classification to stage 4 and among them, 6 out 9 dogs that had serum creatinine < 5.0 mg/dL were already in stage 4 since the beginning of the follow-up. Urinary protein to creatinine ratio (UP/C) measures the total amount of proteins in urine compared to urinary creatinine concentration, but it does not identify the origin of individual proteins. Qualitative evaluation of urinary proteins by immunodetection could add important information as to the nature of a renal disease as albuminuria is associated with glomerular disease, and retinol-binding protein (RBP) and vitamin D-binding protein (VDBP) with tubular damage due to the impairment of tubular reabsorption of low molecular weight proteins that are normally found in glomerular filtrate. The hypothesis of this study was that qualitative methods (e.g. western blotting) in association with quantitative methods (UP/C) may provide better assessment and evaluation of the origin of proteinuria. The aim was to study dogs with chronic kidney disease (CKD) and to determine the UP/C as well as to use immunoassay (western blotting) to detect urinary albumin, RBP and VDBP. The study group was composed of CKD dogs with stage 2 (n=9) and stage 3 (n=13) and they were followed up for 12 to 15 months or until death. Fifteen clinically healthy dogs of different breeds and age comprised the control group. Coomassie brilliant blue (Bradford method) was used for UP/C determination, and antialbumin (ab112986,1:500; Abcam, Cambridge, MA), anti-VDBP (ab95469,1:500; Abcam) and anti-RBP (ab48624, 1:250; Abcam) for the western blotting. Urinary albumin, RBP and VDBP were not detected in control dogs and UP/C was 0.15±0.02 (mean±SEM) with a min of 0.03 and a max of 0.4. Only one CKD dog with stage 2 had proteinuria based on UP/C (mean of the follow-up, min-max; 1.49, 0.76-3.45 ) also with loss of VDBP (tubular pattern of renal disease). In the remaining 8 out of 9 CKD dogs with stage 2, the mean of UP/C was normal (UP/C < 0.4) during the follow-up period, however the qualitative evaluation detected loss of tubular proteins (RBP and /or VDBP) in 2/8, loss of glomerular protein (albumin) in 1/8, loss of tubular and glomerular proteins in 2/8, and in 3/8 no level of proteins were found. Proteinuria (UP/C > 0.5) was observed in 7 out of 13 CKD dogs with stage 3, and a tubular pattern characterized by the predominance of low molecular weight proteins loss (RBP and or VDBP) was noticed in 5/7, and the loss of albumin and RBP, both in similar magnitude, in 2/7 (tubular and glomerular pattern). In non-proteinuric (mean during follow-up, UP/C < 0.5) CKD dogs with stage 3 (n=6), the immunodetection of albumin and RBP or VDBP were observed in 3/6, and in 1/6 only a slight amount of albumin loss