Robust Rat and Mouse Models of Bilateral Renal Ischemia Reperfusion Injury

Materials and Methods

Rodents and animal care. Eight-week-old Male adult Lewis Rats weighing approximately 180-240g (Charles River Laboratories, Bristol, UK) and Male adult C57BL/6 mice weighing >15 g (Charles River Laboratories) were purchased and maintained in an animal house with free access to water and pelleted food. All animal experiments were conducted according to the United Kingdom Use of Animals (Scientific Procedures) Act 1986, under license P6B0CD326. The animals were given a seven-day period of acclimatization to their new surroundings and were housed and handled according to the local institutional policies and procedures licensed by the Home Office. Ethical approval for all the protocols within the License was provided by the Animal Welfare and Ethical Review Body under the Establishment License held by Cardiff University. The ARRIVE guidelines were followed for all experiments involving use of animals.

Surgical techniques. See Table I, for further details. The animals underwent thorough health checks before transfer to individual cages 24 h prior to the intended surgical procedure. Analgesia was provided to individual animals 24 h prior to any surgical intervention until the time of tissue retrieval in the form of 200 mcg of buprenorphine (Sansoz, Camberley, UK, PC 05050650092042) dissolved in 500 ml of drinking water. Anesthesia was provided in the induction chamber using a combination of 5% isoflurane in oxygen at a rate of 2-3 ml/min. The animal was placed in a supine position, immobilized with plain surgical tape. Maintenance anesthesia was provided via tube mask covering the face by approximately 2% isoflurane delivered with approximately 1-2 ml/min oxygen. For rats, 0.3-0.4 ml of blood was taken from tail vein once the animal was fully anesthetized.

The abdomen was prepared by the removal of excess fur from the abdomen with hair trimmers. Under aseptic conditions, a midline laparotomy was performed. Once safe access to peritoneal cavity was achieved, bowels are moved to the right side gently with sterile cotton buds. The left kidney renal vascular pedicle was identified, dissected out (with fine forceps with curved tips) and clamped under direct vision with a microvascular clamp. The bowels were then moved to the left and the same process was done for the right kidney renal vascular pedicle. Care was taken to avoid any damage to surrounding structures. If IPC was utilized, this can be performed according to our previous methods (5). The kidneys were visually assessed for macroscopic ischemic injury which is evidenced by change in color from a normal pink to purplish brown. Each clamp was removed after exactly the specified time of ischemia for each kidney according to the experiment (e.g., 20 min for mice and 45 min for rats). The kidneys were visually assessed again for return of color to pink. Rectus sheath and skin were closed with absorbable (4/0 vicryl) continuous sutures. Sham controls underwent the same operation without the renal pedicle clamping. At the completion of the procedure, anesthesia was disconnected, and the animal was observed closely until full recovery.

At the end of assigned time point for culling (1 to 28 days), animals were again anesthetized using combination of isoflurane and oxygen, positioned on the operating board, and maintenance anesthesia was delivered as mentioned above. Midline wound was opened followed by opening of the pleura to gain access to the chest cavity. Total blood circulating volume was aspirated from the heart by direct puncture to cause exsanguination (and confirmation of permanent cessation of the circulation was made by surgical excision of the heart. Each kidney was carefully removed using dissecting scissors and placed in a receptacle for tissue processing and storage. Anesthesia was disconnected and the animal carcass was then packed to be disposed of as per the animal unit (Joint Biological Services Unit, Cardiff University) guidelines and procedures.

Histology. Kidney tissue was embedded in paraffin and sectioned for Hematoxylin and eosin (H&E) staining. Scoring was carried out in a blinded fashion using the Endothelial, Glomerular, Tubular, and Interstitial (EGTI) scoring system, developed specifically for animal research on kidney tissue in the context of injury (5, 6).

Renal function. Serum creatinine was measured in blood samples of rats taken pre-operatively (at 0 h) and in samples from rats and mice at time of retrieval using the Jaffe reaction.

RT-qPCR. Whole kidney tissue was stored in RNA later solution immediately at time of retrieval and frozen at −80°C. Total RNA was extracted using TRIzol reagent (Cat. No. 15596018; Life Technologies, Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to manufacturer’s instructions. RNA quality was assessed using the Agilent 2100 Bioanalyzer with RNA 6000 Nano chips (Santa Clara, CA, USA) and quantified prior to RT-qPCR. cDNA was generated using a High-Capacity Reverse Transcription Kit (Cat. No. 4368814; Life Technologies) with random primers. RT-qPCR was performed on a ViiA7 Fast Real-Time PCR System (Life Technologies). Primer sequences: NGAL (forward: 5’-GGGCTGTCCGATGAACTGAA-3’; reverse: 5’-CATTGGTCGGTGGGAACAGA-3’) and KIM-1 (forward: 5’CGGCTAACCAGAGTGACTTGT-3’; reverse: 5’-TACAGAGCCTGGAAGAAGCAG-3’) were quantified using POWER SYBR GREEN PCR Master Mix (Cat. No. 4368706; Life Technologies) with gene-specific primers, normalized to GAPDH (forward: 5’-CCTCTGACTTCAACAGCGACAC- 3’; reverse: 5’-TGTCATACCAGGAAATGAGCTTGA-3’), as previously described (5, 6).

Statistical analysis. Statistical analyses were performed using GraphPad Prism v10 software (San Diego, CA, USA). Data are expressed as mean±SD or median (and range) and assessed for statistical significance using unpaired Student’s t-test or Mann-Whitney U-test.