ObjectivesTo document changes in urinary biomarker concentration and conventional diagnostic tests of acute kidney injury (AKI) following hypotension and fluid resuscitation in anaesthetized dogs. Study designExperimental, repeated measures, prospective study. AnimalsA group of six male adult Greyhound dogs. MethodsFollowing general anaesthesia, severe hypotension was induced by phlebotomy, maintaining mean arterial blood pressure (MAP) < 40 mmHg for 60 minutes, followed by resuscitation with intravenous gelatine solution to maintain MAP > 60 mmHg for 3 hours. Following euthanasia, renal tissue was examined by light microscopy (LM) and transmission electron microscopy (TEM). Urinary and serum concentrations of neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CysC), and gamma-glutamyl transpeptidase (GGT), serum creatinine and urine output were measured at baseline and hourly until euthanasia. Data are presented as mean and 95% confidence interval and analysed using repeated measures analysis of variance with Dunnett’s adjustment, p < 0.05. ResultsStructural damage to proximal renal tubular cells was evident on LM and TEM. Urinary biomarker concentrations were significantly elevated from baseline, peaking 2 hours after haemorrhage at 19.8 (15.1–25.9) ng mL–1 NGAL (p = 0.002), 2.54 (1.64–3.43) mg mL–1 CysC (p = 0.009) and 2043 (790–5458) U L–1 GGT (p < 0.001). Serum creatinine remained within a breed-specific reference interval in all dogs. Urinary protein–creatinine ratio (UPC) was significantly elevated in all dogs from 1 hour following haemorrhage. Conclusions and clinical relevanceUrinary NGAL, CysC and GGT concentrations, and UPC were consistently elevated within 1 hour of severe hypotension, suggesting that proximal renal tubules are damaged in the earliest stage of ischaemia-reperfusion AKI. Measurement of urinary biomarkers may allow early diagnosis of AKI in anaesthetized dogs. Urinary GGT concentration and UPC are particularly useful as they can be measured on standard biochemistry analysers.