Sitagliptin Case Study

Drugs and chemicals
Sitagliptin (Sita) was manufactured by Merck Sharp & Dohme Ltd (Pavia, Italy), dissolved in saline. Gentamicin sulfate was obtained as ampoules from Memphis Co., for Pharm. and Chem. Ind. (Cairo, Egypt). Other used chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Animals
Animal procedures and care were in accordance with the National Institute of Health guide for the care and use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by ethical guidelines (Ain Shams University, Egypt). Forty eight male Wistar rats weighing 180–220 g were used. All rats were maintained under standard conditions of temperature, about 24±1 ˚C, with 12-h light–dark cycle. Food and tap water were
Control group: received 1 ml/day saline orally; GM group: received gentamicin (100 mg/kg/day) intraperitoneally [22]; Sita+GM group: received sitagliptin (30 mg/kg/day) by oral gavage [20] simultaneously with gentamicin; Sita group: received sitagliptin alone. Treatment continued for 14 days.
Sample collection
On the 14th day, after the last gentamicin injection was applied, rats were placed in individual metabolic cages to collect 24-hour urine to quantify protein content. Blood samples were collected through retro-orbital plexus under anesthesia. Serum was separated to measure creatinine and BUN.
Then animals were euthanized and kidneys were excised. For each group, 6 rats were used for histological examinations and other 6 for biochemical investigations. One kidney was used for mitochondrial separation while the other was homogenized to prepare 10% homogenate to determine adenosine triphosphate (ATP) content. A part of the homogenate was then centrifuged at 14,000 rpm for 1 h at 4 °C and the supernatant was used to estimate oxidative stress biomarkers. Protein content in the supernatant was determined according to method described by Lowry et al. [23]. Estimation of Kidney
BUN and serum creatinine levels were evaluated by an autoanalyzer (Olympus Instruments, Tokyo, Japan).

Estimation of Oxidative Stress Biomarkers in Renal Homogenate
Malondialdehyde levels (MDA), indicator of lipid peroxidation, were measured using thiobarbituric acid reaction according to Placer et al. [24]. Catalase (CAT) activity was measured based on the decomposition of hydrogen peroxide at 240 nm according to Aebi [25]. Superoxide dismutase (SOD) activity was assessed by pyrogallol autoxidation according to Marklund [26]. Reduced glutathione (GSH) concentration was measured according to Ellman [27]. Glutathione peroxidase (GSH-Px) activity was determined in accordance to procedure described by Beutler [28].

Isolation of mitochondrial fraction from kidney tissues
Briefly, a portion of the fresh kidney tissue was homogenized in isolation buffer [250 mM Sucrose, 1 mM EGTA and 10 mM Tris (pH 7.4)]. The homogenates were centrifuged at 600 g for 10 min at 4˚C to remove cell debris. The obtained supernatant was again centrifuged at 10000 g for 10 min at 4˚C. Then the mitochondrial fraction in the pellets was obtained