Recent epidemiological studies demonstrated substantial increases in intracerebral hemorrhage and myocardial infarction in young adults abusing METH, thus our work may provide insight into such complications of METH use

VED by the IACUC at Boston University Medical Center as being consistent with humane treatment of laboratory animals and with standards set forth in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. Boston University Medical Center has had an Animal Welfare Assurance on file with the Office of Laboratory Welfare since January 1, 1986. The Animal Welfare Assurance number is A-3316-01. The Laboratory Animal Science Center at Boston University Medical Center has been accredited by the American Association for Accreditation of Laboratory Animal Care since 1971. Animals Mitofusin 2 floxed animals were originally generated by the Chan laboratory. Conditional kidney knockouts were created by breeding MFN2f/f animals with animals expressing Cre recombinase under the control of the Pax2 promoter 1Akg ). Primary cell culture As previously described. Briefly, three-week old mice were sacrificed, their kidneys removed, and cortex harvested. The cortex was minced, placed into HBSS containing collagenase IV, and incubated for 60 min at 37uC. The collagenase was neutralized by addition of fetal calf serum, washed once in red cell lysis buffer and plated into culture dishes in selection medium containing DMEM and Ham’s F12 medium and a mixture of insulin, hydrocortisone, apotransferrin and penicillin/streptomycin. Cultures were incubated for 57 days in 5% CO2 and at 37uC and the cells were characterized as proximal tubule cells. January 2012 | 221244-14-0 Volume 7 | Issue 1 | e31074 MFN2 in Renal Stress Metabolic stress ATP depletion is an established model of renal ischemia that sustains ATP content at,10% baseline values until recovery is initiated. To initiate ATP depletion, cells were washed 3 times in glucose-free DMEM followed by incubation in glucose-free DMEM containing sodium cyanide and 2deoxy-D-glucose for 13 hours. Recovery was initiated by replacing the above media with complete primary cell culture media. In vitro recombination of MFN2 floxed alleles by Cre recombinase An adenovirus expressing Cre recombinase was used to transduce MFN2f/f primary proximal tubule cells. The cells were infected with adenovirus overnight and the medium was replaced with complete primary culture media for 48 hr prior to the experiments. Recombination of the loxP sites by Cre recombinase deleted the floxed MFN2 gene creating MFN2 deficient cells in culture. Adenovirus without cre was used as control. Antibodies The MFN2 and AIF antibodies were purchased from Santa Cruz Biotechnology. The F1F0 ATPase Antibody was purchased from Invitrogen. Cytochrome c antibody was purchased from Clontech. Active-Bax antibody was purchased from Trevigen. Total Bax antibody was purchased from Cell Signaling Technologies. The appropriate secondary antibody conjugated to AlexFluor-488 for immunofluorescence or to horseradish peroxidase was used for immunoblotting. Immunoblot analysis Cell proteins were extracted with NP-40 buffer containing a protease inhibitor cocktail. Samples were ” sonicated, spun at 10,0006 g, and supernatants collected. Subcellular fractions were obtained as previously described. Briefly, cells were harvested in isotonic mitochondrial buffer containing protease inhibitor and homogenized for 40 strokes with a dounce homogenizer. Lysates were centrifuged at 5006 g for 5 min to remove unbroken cells and nuclei. “2987731 The supernatants were centrifuged at 10,0006 g for 30 min to pellet the membrane fraction. The resulting supernatant was stored as cyto