Mus musculus
Protocols: Twenty-four hours after renal ischemia-reperfusion injury, sham and injured wild-type and Oxct1 knock-out mice receiving control or ketogenic diets were anesthetized with an intraperitoneal injection of ketamine/xylazine (Dechra, Innovet) and blood was collected from the right ventricle. Mice were then submitted to left heart perfusion with 3ml PBS (1ml/min) and perfused kidneys were collected in ice-cold HBSS. Kidney capsules were removed, and kidneys were cut into small pieces in a collagenase digestion buffer (2 mg/ml collagenase I [C9891; Sigma-Aldrich], 27 IU/ml DNAse I [D4527; Sigma-Aldrich], and HBSS [14025100; Thermo Fisher Scientific]). The samples were then incubated for 40 minutes at 37°C, shaking every 10 minutes, and digestion was stopped by adding ice-cold HBSS. Digested samples were filtered on a 40 mm cell strainer, immune cells were separated using CD45 magnetic microbeads and QuadroMACS separators with MS columns (Miltenyi Biotec). Endothelial cells were then enriched from the CD45- population using CD31 magnetic microbeads and QuadroMACS separators with MS columns (Miltenyi Biotec). Cell populations were resuspended in FACS buffer (0.1% BSA, 2 mM EDTA in PBS) and stained for flow cytometry analysis and sorting. Upon magnetic sorting, CD31+ fraction was incubated for 30min at 4°C with: CD45 (1/100, 561047; BD Pharmingen), CD31 (1:50, 130-111-355; Miltenyi Biotec) and viability dye (1/1000, 65-0865-14; Invitrogen). OneComp ebeads (01–1111–42; Thermo Fisher Scientific) and cooked cells (20 minutes at 60°C) were used for compensation. Viable endothelial cells (live CD45-CD31+ ECs) were FACS-sorted using a FACS Aria III (BD Biosciences), and collected in wash buffer (0.1% BSA in PBS). Cells were centrifuged for 6 minutes at 400xg, and resuspended in 350 ul of RLT buffer supplemented with DTT (20 ul of 2 M DTT in 1 ml RLT buffer). Samples were stored at -80°C until RNA extraction. To obtain a tamoxifen-inducible, EC-specific Oxct1 knock-out mouse line, Oxct1lox/lox mice (provided by P. Crawford) were intercrossed with VECadh-CreERT2 mice (provided by T. Mäkinen) generating VECadh-CreERT2;Oxct1lox/lox C57BL/6N mice. On subsequent intercrossing with double fluorescent mT/mG Cre reporter mice C57BL/6J, the resulting VECadhΔOXCT1:mTmG mice allowed determination of recombination efficiency after tamoxifen treatment. Tamoxifen (75 mg/kg) was administered for 5 consecutive days to 7- to 8-week-old male mice by intraperitoneal injection and experiments were started 1 week later. Before IRI or sham procedure, mice were then exposed to 3-weeks diet pre-treatment with control diet (V1535-000; Ssniff) or ketogenic diet (F3666; Plexx) with ad libitum access to food and water. For the renal ischemia/reperfusion injury experiment, Mice were anesthetized with isofluorane and placed on a heating pad (37°C) to maintain constant body temperature. An incision was performed on the back to expose the renal pedicles. A bilateral clamping was performed for 30min, then the clamping was released, and reperfusion was allowed. After suture of the wound, mice were allowed to recover, and Buprenorphine was administered (1mg/kg). Total RNA was extracted from freshly isolated murine RECs using RNeasy® Micro Kit (Qiagen) according to the manufacturer's instructions. RNA quality and quantity were measured with NanoDrop (Thermo Fisher Scientific). Libraries were prepared using the QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina (Lexogen) adjusting the number of cycles for amplification to the RNA input quantities according to manufacturer's instruction. Optimal cycle number was determined by using PCR Add-on kit for Illumina (Lexogen).
strain
C57Bl6/N
individual
A672
organism
Mus musculus
organism
Mus musculus
collection date
not collected
sex
male
batch
1
scientific_name
Mus musculus
common name
house mouse
organism part
kidney
developmental stage
adult
genotype
wild type genotype
isolate
not applicable
diet
chow diet
injury
ischemia reperfusion injury
cell_type
endothelial cell
age
3
geographic location (country and/or sea)
not collected
ENA-LAST-UPDATE
2023-12-22