Equine urinary N-acetyl-β-D-glucosaminidase assay validation and correlation with other markers of kidney injury

Abstract

Detection of equine acute kidney injury (AKI) is hindered by limited markers of early renal damage in horses. N-acetyl-β-D-glucosaminidase (NAG), a lysosomal enzyme in renal tubular epithelium released into urine during tubular insult, has shown promise for early identification of AKI in humans and other species. We validated an assay for NAG in equine urine and measured urinary NAG in 7 azotemic and 7 non-azotemic client-owned adult horses. The enzymatic NAG assay was validated using within- and between-run coefficients of variation (CVs), recovery following standard addition, and linearity of dilution. Intra- and inter-run CVs (21% and 3.2%, respectively), average recovery following standard addition (99–109%), and linearity under serial dilution ( R2 = 0.997) were satisfactory. Urine NAG index was significantly correlated with urinary fractional excretion of sodium (FENa; ρ = 0.76, p < 0.001) and plasma creatinine (ρ = 0.74, p = 0.001). Median urine NAG indices were higher in azotemic horses ( p = 0.006), in horses with increased urinary FENa ( p = 0.006), and in horses with increased urine gamma-glutamyl transferase index ( p = 0.032). Urine NAG can be measured in horses and shows positive correlation with 2 current renal biomarkers. Additional work is needed to establish normal equine reference intervals and characterize the increase of urine NAG index in horses in relation to tubular injury.

Value of urine IL-8, NGAL and KIM-1 for the early diagnosis of acute kidney injury in patients with ureteroscopic lithotripsy related urosepsis

“…Besides the urine markers in this study, urine N-acetyl-beta-glucosamynidase, tissue transglutaminase (tTG), transforming growth factor-β (TGF-β), Matrix metalloproteinases (MMPs), and so on are also the research hotspots. 17 , 18 , 19 , 20 This suggests that a single marker is not enough to monitor the occurrence and outcome of AKI accurately, and combined markers may be more effective. For example, urine IL-18 combined with KIM-1 is considered to have the best diagnostic value for AKI that already exists, and urine NGAL combined with IL-18 is the best marker for the diagnosis of early AKI, while urine Nacetyl-beta-glucosamynidase combined with KIM-1 and IL-18 is an ideal marker for the prognosis of AKI.…”

Biomarkers of Kidney Disease in Horses: A Review of the Current Literature

“…NAG is a large lysosomal protein of the proximal tubules and is not reabsorbed nor filtered in the glomeruli due to its size [ 50 ]. As such, measuring NAG in urine has been hypothesized to be useful for detection of kidney disease.…”

“…NAG has been evaluated in one study in horses, where a photometric assay was examined on equine urine using a biochemical analyser [ 50 ] ( Table 4 ). Phase 1 results are available from the study on 7 non-azotemic and 7 azotemic horses.…”

“…The intra-run coefficient of variation for Urine NAG/Urine Creatinine was 20% whereas the intrarun coefficient of variation was 3.2% with a high linearity during serial dilutions. NAG concentrations have been reported to be affected by urinary pH, with alkaline urine leading to a drop in concentrations; this may be applicable in the interpretation in equine patients, given the alkalinity of equine urine [ 50 ]. However, further work is needed to ascertain the effect of this on different assays [ 51 ].…”

An updated review: Laboratory investigation of equine renal disease

“…N‐acetyl‐beta‐D‐glucoaminidase (NAG) is a proximal tubular lysosomal enzyme and has received recent attention for diagnostic purposes in the equine patient, including validation of an assay for equine use (Bayless et al . 2019). Measurement of urinary NAG: Cr ratios may provide clinically useful information.…”