Background and Aims: Gentamicin is an aminoglycoside antibiotic widely used for the treatment of many infectious diseases. Its main side effect is its nephrotoxicity, which occurs in 10-25% of therapeutic courses, despite proper monitoring and hydration of patients, can lead to acute kidney injury (AKI). We have previously demonstrated that urinary damage markers like albuminuria increased as a consequence of the addition of gentamicin. In the present work we aimed at specifically studying the renal handling of albuminuria in a nephrotoxicity model, through in situ renal perfusion experiments. Methods: Male Wistar rats were administrated by a single dose of gentamicin (150 mg/kg), or not. After 5 days, rats were anesthetized and an extracorporeal circuit for kidney perfusion was set up. The renal artery, vein and ureter of the right kidney were ligated. The renal artery of the left kidney and the urinary bladder were canulated. A catheter was placed in the right carotid artery and connected directly to the renal artery. Urine was continuously collected from a catheter placed in the urinary bladder at 10 min intervals. After 1 h of renal perfusion with blood from the carotid artery, oxygenated and warm (37 1C) Krebs-dextran (40 g/L of dextran) was perfused through the renal artery at 3 mL/min, and was discarded through the renal vein. Albuminuria was measured in the different urine fractions. Results: From the second day after gentamicin administration, albumi- nuria was significantly increased, as compared to control rats, in which urinary markers were undetectable. When exogenous Krebs solution prefunding the kidney, neither gentamicin rats nor control rats excreted albuminuria. As a control of the perfusion experiments, urinary markers still appeared in the urine in gentamicin rats whose kidney was perfused with its own blood. However, albuminuria was undetectable in control rats. Conclusions: Our results support the idea that excess albumin found in the urine (albuminuria) as a consequence of treatment with gentamicin comes from the blood ultrafiltrate reaching Bowman’s capsule and not from the renal parenchyma. More interestingly, our results provide and in situ method to test the origin of urinary biomarkers in different conditions.