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Background Early recognition of acute kidney injury (AKI) is hindered by current definitions and use of traditional, insensitive markers. Hypothesis/objectives Urinary (u) activity of γ-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP), and concentrations of heat-shock protein 70 (HSP70) and interleukins (ILs) -6 and -18, are predictive biomarkers for AKI and survival. Animals Nonazotemic, hospitalized dogs (n = 118) and healthy controls (n = 20). Methods A prospective observational study. Nonazotemic dogs at risk of AKI were recruited and their urinary biomarker concentrations were measured at presentation. Serum creatinine (sCr) and symmetric dimethylarginine (sSDMA) were measured daily until discharge/death. Results The overall case fatality rate was 18.6%. Fifteen dogs (12.7%) developed AKI, which was associated with death (relative risk, 3.2; 95% confidence interval [CI], 1.57-6.55). All 5 urinary biomarkers were significantly higher in hospitalized dogs compared to controls, with minimal overlap. uHSP70/uCr, uGGT/uCr, and uIL-6/uCr at presentation were higher in dogs which later developed AKI. Areas under the receiver operator characteristic curve (AUROC) (95% CI) for the 3 biomarkers as predictors of AKI were 0.67 (0.51-0.83), 0.68 (0.55-0.81), and 0.78 (0.65-0.91), respectively. When they were categorically classified as elevated/normal, each additional elevated biomarker increased the odds for AKI (OR, 2.83; 95% CI, 1.23-6.52, P = .01). Agreement between sCr and sSDMA was poor (Cohen's kappa = .071). The AUROC of SDMA at presentation for AKI prediction was 0.73 (0.51-0.95). Conclusions and clinical importance Kidney injury was common, irrespective of subsequent worsening of azotemia or death. The predictive value of individual urinary biomarkers was reduced by moderate sensitivities and specificities. SDMA showed moderate discriminatory utility for AKI prediction, and often displayed discordant results with sCr.