Cell experiment:

Normal rat kidney proximal tubular cells (NRK-52E) are maintained in six-well 100 or 150-mm, or 150-mm plates in a humidified incubator gassed with 5% CO2 and 95% air at 37°C in DMEM containing 5% fetal calf serum (FCS). Cells are grown to 60% confluence and divided into four treatment groups: 1) untreated (Untx), 2) CS, 3) CS+Mitoquinone (MitoQ), and 4) CS+DecylTPP. Untreated cells remained at 37°C in DMEM containing 5% FCS (group 1). CS is initiated by washing cells with cold PBS twice and storing them in UW/Viaspan solution alone (4 h at 4°C) (group 2), CS+Mitoquinone (1 μM) (group 3), or CS+DecylTPP (1 μM) (group 4). In separate experiments, cells are exposed to CS plus RW by replacing UW solution alone or UW solution containing Mitoquinone or DecylTPP with DMEM containing 5% FCS overnight (18 h at 37°C)[1].

Animal experiment:

Mice[2]Male CD1 mice (30-35 g) or C57BL/6J mice (20-25 g) are used. Seven intraperitoneal injections of a supramaximal dose (50 μg/kg) of Caerulein, a CCK-8 analogue, are given on an hourly basis to induce hyperstimulation acute pancreatitis (CER-AP). Control mice receive equal volumes of PBS injection. In the Mitoquinone treatment groups, Mitoquinone at 10 mg/kg (dose 1) or 25 mg/kg (dose 2) is given at the first and third injections of Caerulein. Similarly, dTPP at 9.6 mg/kg (dose 1) or 24 mg/kg (dose 2) is given for the dTPP treatment group. Mitoquinone and dTPP are at the same molar concentration at doses 1 and 2. Mice are sacrificed at 12 h after the first Caerulein injection to collect samples. Bile acid-induced AP is achieved by retrograde infusion of TLCS into the pancreatic duct (TLCS-AP). After induction of anesthesia, TLCS applied using a mini infusion pump at a speed of 5 μL/min for 10 minutes. Successful infusion of TLCS into pancreas is demonstrated by a diffuse light blue colour (methylene blue) appearing in the pancreatic head. Control mice receive sham surgery without TLCS infusion. In the treatment groups, Mitoquinone (10 mg/kg) or dTPP (9.6 mg/kg) is given at 1 h and 3 h after TLCS infusion. Mice are sacrificed at 24 h after the TLCS infusion or sham surgery.

References:

[1]. Mitchell T, et al. The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renaltubular cells and rat kidneys. J Pharmacol Exp Ther. 2011 Mar;336(3):682-92.
[2]. Huang W, et al. Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis. Mediators Inflamm. 2015;2015:901780.