Renal Function Measurement in CKD Model

At weeks 5 and 12, animals were placed into metabolic cages (Tecniplast Metabolic Cage System, Buguggiate, Italy) for 24 h to measure urine creatinine and protein levels. As described previously, urine creatinine and protein levels were measured by standard laboratory methods [34 , 36 (link), 39 (link), 40 (link)]. Blood was collected from the saphenous vein at week 5 and from the abdominal aorta at week 13 to measure serum carbamide (urea) and creatinine levels to verify the development of CKD. Urea and creatinine levels in serum were quantified by kinetic UV spectrophotometric method using urease and glutamate dehydrogenase enzymes according to the Jaffe method. The reagents and the platform analyzers were from Roche Diagnostics (Hoffmann-La Roche Ltd., Basel, Switzerland) [39 (link), 40 (link)]. Creatinine clearance, an indicator of renal function, was calculated according to the standard formula (urine creatinine concentration [μM] × urine volume for 24 h [mL])/(serum creatinine concentration [μM] × 24 × 60 min). At weeks 5 and 12 or 13, urine creatinine and volume and serum creatinine concentration were measured [39 (link), 40 (link)].