In Vivo Ischemia-Reperfusion Protocol in Rat

All animal procedures were carried out in accordance with the French legislation for animal care and experimentation (C06–101). Adult female Wistar rats (190–240 g; 7–8 weeks old) received daily intraperitoneal injections for 3 days with 0.9% control saline or GC7 (3 mg/kg in saline solution). On day 4, rats were anesthetized with isoflurane. Using a left flank incision, the left renal artery was prepared and held with a vascular clamp for 40 minutes. The kidney was then observed for 1 minute after removal of the clamp to assess reperfusion, and the incision was sutured. Sham-operated animals were subjected to left flank incisions only. The contralateral right kidney was left untouched and served as an internal control. Twenty-four hours postsurgery, independent renal clearances of the left ischemic and right nonischemic kidneys were performed.³⁹ Urine was collected separately from each kidney, and the FEs of glucose, Na⁺, and phosphate ions were determined using the hexokinase method (Colorimetric Kit; Randox Laboratories), flame atomic absorption spectrometry, and anion exchange chromatography (Dionex AS50; AS11 column), respectively. NGAL measurements were performed using a commercial NGAL kit (Bioporto) and are expressed as urinary NGAL-to-creatinine ratios to compensate for urinary dilution/concentration.