In vivo multiphoton imaging of mitochondrial structure and function during acute kidney injury.

Animals

All experiments were conducted in accordance with NIH Guidelines and were approved by the Institutional Animal Care and Use Committee. Adult male C57 mice (Harlan Labs, Indianapolis, IN) or Sprague–Dawley rats (Harlan Labs) were used (except for experiments in which Munich–Wistar rats were utilized to image superficial glomeruli). Mice weighing 20–20 g were anesthetized with inhaled isoflurane (2%), whereas rats weighing 250–400 g were anesthetized by intraperitoneal injection with thiobutabarbital sodium (Sigma Aldrich, St Louis, MO) at a dose of 160 mg/kg. They were then placed on a warming table to maintain a core temperature of 37°C, which was monitored throughout the experiment using a rectal thermometer. The right internal jugular vein was cannulated to allow the injection of fluorescent dyes. For ischemia–reperfusion experiments, after shaving the abdomen of the rat a midline incision was made through the skin and musculature to expose the abdominal cavity. The left renal artery was isolated through blunt dissection, and a 3.0 silk suture was placed around the vessel and passed through a length of polyethylene tubing that exited the abdominal cavity. The abdomen was then closed around the tubing. To induce ischemia, the tension on the two ends of the thread was increased until there was a visible cessation of kidney perfusion; the tension was subsequently relaxed to allow reperfusion. The left kidney was externalized via a lateral incision and imaged using methods described previously. 73 For gentamicin toxicity experiments, adult male Sprague–Dawley rats weighing 250–350 g were given a daily intraperitoneal injection of gentamicin (Sigma Aldrich) at a dose of 100 mg/kg, as per a previously established protocol. 36 Dye loading and microscope settings Images were acquired using an inverted Fluoview 1000 Olympus microscope adapted for multiphoton microscopy (Olympus Corporation, Tokyo, Japan) and either ×20 or ×60 objectives. Fluorescent dyes were administered via intravenous injection. HEt (200 µg/ml) (Invitrogen Molecular Probes, Eugene, OR), endogenous NADH, and MCB (5 mg/ml) (Invitrogen Molecular Probes) were excited at wavelengths of 720–760nm. TMRM (5 µg/ml) and rhodamine 123 (6 µg/ml) (Invitrogen Molecular Probes) were excited at wavelengths of 800–850nm. Emitted light was collected using three fixed band-pass filters: 420–460nm (blue), 495–540nm (green), and 575–630nm (red). For experiments involving inhibitors of renal transporters, verapamil (Sigma Aldrich) was used at a dose of 1 mg/kg body weight, whereas cimetidine (Sigma Aldrich) was used at a fixed dose of 8 µmoles (0.2 ml of a 40 µM solution) per animal.

Image analysis and statistics

Images were processed using Image J software (National Institutes of Health, MD, http://rsb.info.nih.gov/ij/ ). Regions of interest were drawn around tubules to obtain mean fluorescence signals. Mitochondrial signals were isolated by setting a threshold level to remove the background cytosolic fluorescence. Images were acquired within 5 min of intravenous dye injection, as some signals decayed with time. To account for movement in the z plane during ischemia experiments, images were collected in serial z-stacks and reconstructed to a signal-averaged 2D image using imaging software. For the gentamicin experiments, fields of PTs were chosen randomly using the green autofluorescence channel before obtaining NADH and TMRM signals. The results are presented as means ± s.e.m. Statistical differences among study groups were explored using oneway analysis of variance and groups were compared using the paired or unpaired t -test as appropriate; a P value of < 0.05 was considered significant.