Background: Pyometra in dogs has been associated with renal injury. Hypothesis: Examine pyometra-related nephropathy by evaluating novel renal biomarkers. Animals: Twenty-five dogs with Escherichia coli pyometra. Fourteen clinically healthy bitches of comparable age. Methods: Prospective study. Urinary biomarkers determined by immunoassays (uIgG, uCRP, uAlb, uRBP, uTXB 2 ) or colorimetric test (uNAG) with results normalized to urine creatinine concentration. Nonparametric Mann-Whitney U-test and Wilcoxon's signed-rank test used to compare healthy dogs and dogs with pyometra, and dogs with pyometra at initial and follow-up examination.Results: Urinary biomarkers (median, range) significantly increased in dogs with pyometra (uIgG/Cr: 169.7 mg/g, 4.8-1052.9; uCRP/Cr: 0.260 mg/g, 0.006-3.030; uAlb/Cr: 89.5 mg/g, 8.8-832.7; uRBP/Cr: 1.66 mg/g, 0.05-21.44; uNAG/Cr: 5.8 U/ g, 1.6-27.7; uTXB 2 /Cr: 15.3 mg/g, 3.2-139.6) compared with healthy bitches (uIgG/Cr: 3.4 mg/g, 0.6-8.9; uCRP/Cr: below detection limit; uAlb/Cr: 17.5 mg/g, 1.3-166.3; uRBP/Cr: 0.13 mg/g, 0.02-0.44; uNAG/Cr: 2.4 U/g, 1.4-7.4; uTXB 2 /Cr: 2.4 mg/g, 1.2-4.7) (P o .001). Six months after ovariohysterectomy, urinary biomarkers in pyometra group (uIgG/Cr: 4.7 mg/g, 1.5-99.8; uCRP/Cr: below detection limit; uAlb/Cr: 13.9 mg/g, 2.1-471.2; uRBP/Cr: 0.05 mg/g, 0.02-0.32; uNAG/Cr: 1.6 U/g, 0.9-3.3; uTXB 2 /Cr: 3.3 mg/g, 1.0-6.9) were significantly lower than before surgery (P o .01), and not significantly different to those of healthy dogs (P 4 .05).Conclusion and Clinical Importance: Pyometra-related renal dysfunction affects the nephron both at glomerular and proximal tubular level and is a transient process in most dogs with E. coli pyometra.