Stability of glomerular and tubular renal injury biomarkers in canine urine after 4 years of storage

Abstract

The use of urine biomarkers to detect renal injury at an early stage is gaining interest in both human and veterinary medicine, 3,8,13,14,24 given that identification of early renal injury allows earlier therapeutic intervention. 17 The latter is hampered by the traditionally used diagnostic markers of decreased kidney function, serum creatinine and urea, as their concentration will only increase above the upper limit of the reference interval when >75% of nephrons are lost, making them insensitive markers for the detection of earlystage renal injury. 2,6 In contrast, urine biomarkers are generally accepted to be sensitive indicators of glomerular or tubular renal injury, and additionally have the capacity to quantify as well as localize this damage. 3,17 In research settings, enzyme-linked immunosorbent assays (ELISAs) are often used to detect and quantify biomarker proteins. Immunoassays of several glomerular and tubular biomarkers of renal injury, including immunoglobulin G (IgG) and C-reactive protein (CRP), respectively, as well as retinol-binding protein (RBP; Immunology Consultants Laboratory, Portland, OR), have been previously validated in our laboratory for use in canine urine.14 Consideration of preanalytical factors, such as sample handling and storage conditions, is important in order to perform reliable biomarker research. Because of prospective sampling, shipping, and performing analyses in batches, immediate analysis of fresh urine samples is impractical in most research settings. Therefore, storage of frozen samples for long periods (several months to years) is common. However, studies evaluating the stability of urine renal injury biomarkers during long-term storage are very scarce in veterinary medicine. Even in human medicine, relatively few studies have evaluated the impact of preanalytical factors on urine proteins. 4,7 To our knowledge, only a single urine biomarker study, investigating the stability of RBP, albumin, Abstract. Urine biomarkers are sensitive indicators of early-stage renal injury, consequently, research in this area is expanding in both human and veterinary medicine. However, studies investigating the impact of preanalytical factors, such as storage conditions, on urine biomarker concentrations are largely lacking in veterinary medicine. Therefore, we evaluated the stability of several renal injury biomarkers in canine urine after storage for 4 y at −72°C. Urine samples were collected from 26 dogs: 18 dogs with babesiosis and 8 healthy dogs. Concentrations of urine immunoglobulin G (uIgG), urine C-reactive protein (uCRP), and urine retinol-binding protein (uRBP) were measured, using validated commercial immunoassays, at the start of the study and 4 y later. To investigate the effect of long-term storage, absolute and relative differences between both measurements were compared. Additionally, dogs with babesiosis were compared with the healthy controls at both time points. Storage caused significant absolute and relative decreases in concentrations of all 3 biomarkers. Signi...

Variability of serum concentrations of cystatin C and urinary retinol‐binding protein, neutrophil gelatinase‐associated lipocalin, immunoglobulin G, and C‐reactive protein in dogs

“…In accordance with the majority of other studies of markers of kidney disease in dogs that included a healthy control group, dogs in our study also had uCRP concentrations below the detection limit . CRP is a major positive acute phase protein in dogs with a large MW (110 to 144 kDa) and therefore is unable to pass through an intact glomerular barrier, which probably was the case in the healthy dogs in our study .…”

“…uRBP, uNGAL, uIgG and uCRP assays were previously validated for use with canine urine . All immunoassays were performed in two batches and were used according to manufacturer's instructions and performed as previously described . The LOD and limit of quantification (LOQ) were determined in previous studies .…”

“…The LOD and LOQ of the uRBP, uNGAL, uIgG and uCRP assays were 14.11 ng/mL and 18.93 ng/mL, 5.35 pg/mL and 9.60 pg/mL, 19.69 ng/mL and 29.72 ng/mL and 5.28 ng/mL and 7.76 ng/mL, respectively. The concentration of each urinary marker was expressed as a ratio to urinary creatinine (/c) to account for variations in urine concentration . Urinary creatinine was determined by the modified kinetic Jaffé method …”

Weight-gain induced changes in renal perfusion assessed by contrast-enhanced ultrasound precede increases in urinary protein excretion suggestive of glomerular and tubular injury and normalize after weight-loss in dogs

“…Immunoassays for sCysC, uCRP, uIgG, uNGAL and uRBP have been described in detail elsewhere [26]. The concentration of each urinary biomarker was expressed as a ratio to urinary creatinine (/c) to account for variations in urine concentration [36].…”