This study describes a reproducible large animal model of renal ischemia reperfusion injury, which can be used to study the pathophysiology of acute kidney injury, and explore potential therapeutic modalities. This method is highly reproducible and minimizes the impact of inflammatory response by limiting the amount of insult to the body compared to an open approach. This preclinical model mimics clinical scenarios, such as renal transplantation, renal hypoperfusion following cardiogenic shock, transcatheter procedures resulting in renal ischemia, and cardiovascular procedures with prolonged cardio-circulatory arrest times.
To begin, disinfect the right lateral area of the neck by applying betadine and then 95%ethanol three times. Drape the animal in a sterile fashion. Perform a cutdown for the catheterization of the right carotid artery and the right jugular vein.
Retract the sternocleidomastoid muscle laterally, and dissect it down to the right carotid artery and the right jugular vein. Insert a 5F angiography sheath in both the artery and the vein. Secure it with a silk 2-0 suture.
Insert a 5F angiography sheath using the Seldinger technique into the left femoral artery by puncturing the artery with a hollow needle. Insert a soft-tip guide wire through the lumen and advance it into the femoral artery. Hold the guide wire secure with a hand while removing the needle.
Pass the angiography sheath over the guide wire into the femoral artery and withdraw the guide wire by using ultrasound guidance if necessary. Intravenously administer 200 IU per kilogram sodium heparin to achieve systemic anticoagulation. To perform angiography, inject an iodinated contrast agent under fluoroscopy to identify the renal arteries.
Identify the renal arteries and manually advance the guide wire in the guiding catheter. Position the 5F JL4 guiding catheter in the left renal artery through the right carotid artery. Position the second 5F JL4 guiding catheter in the right renal artery through the left femoral artery.
Use the guide wires to direct a 5F percutaneous transluminal angioplasty, or PTA dilation catheter, in each renal artery. Position each balloon catheter in place, and connect a pressure line to each catheter. Check the presence of arterial pulse wave forms in the pressure monitor to ensure the correct positioning of the catheter.
Inflate each balloon and aim for a pressure of approximately 2.5 atmospheric pressure inside the balloon. To confirm the cessation of blood flow to the kidneys, observe the flattening of the pulse waveform at the tip of the balloon catheter. Inject iodinated contrast medium in a 1:1 dilution with saline and check for any opacification of the renal vessels.
After 60 minutes of occlusion, carefully deflate and remove the balloon catheters from the renal arteries. Perform an angiography using a 1:1 diluted contrast medium to confirm renal artery patency and the establishment of renal reperfusion. Remove the 5F angiography sheath from the left femoral artery.
Apply from pressure at the site of catheterization for 30 minutes. Reverse the effect of heparin by the administration of three milligrams per kilogram of protamine until normalization of active clotting time. To sample urine during the post-operative period, secure a tube to the Foley catheter with a silk 2-0 suture using an interrupted stitch on the skin.
For blood sampling, leave the angiography sheaths in the right carotid artery and the right jugular vein in place, and secure them with a silk 2-0 suture. administer three milligrams per kilogram of bupivacaine at the incision site to minimize pain. Continue to hydrate the animal with 0.9 sodium chloride for a total of two hours following the end of ischemia.
Close the neck incision with a silk 2-0 suture using an interrupted stitch in two layers. Place a fentanyl patch of 25 to 50 micrograms per hour on the back of the animal to minimize post-operative pain, and monitor the animal on mechanical ventilation until it awakens. Collect the final blood and urine samples and calculate the urine output.
Perform a midline laparotomy incision using a size-10 blade from the xiphoid down to the mid pelvis. Use a straight lateral retractor to retract the abdominal skin. Dissect the lateral peritoneal attachments of the abdominal wall to expose the right and left retroperitoneum.
Identify and bluntly dissect both renal arteries and veins. Ligate both renal arteries and veins with a 2-0 silk suture and perform bilateral nephrectomies to collect whole-tissue specimens for histological and metabolic analysis. Renal function was assessed by determining the urine output, eGFR, plasma creatine, and BUN.
Following 60 minutes of bilateral renal artery occlusion, urine output was significantly decreased and remained this way at six and 24 hours following reperfusion. Similarly, a significant decrease was observed in eGFR, which dropped from baseline as compared to the end of ischemia at two hours, six hours, and 24 hours of reperfusion. Plasma creatinine was significantly increased at two, six, and 24 hours of reperfusion compared to baseline.
BUN was increased as compared to baseline at six and 24 hours of reperfusion. Evident necrotic and hemorrhagic areas were unevenly distributed in both kidneys at the end of the 60 minutes of bilateral renal ischemia and the 24 hours of reperfusion. Masson's trichrome staining revealed confluent coagulative necrosis, which was located at the proximal tubules of the renal cortex.
Plastic-embedded sections were also assessed. All Masson's trichrome slides were evaluated for cell necrosis, loss of brush border, cast formation, and tubule dilation. Acute tubular necrosis, or ATN scoring, showed significant injury to the renal cortex and medulla.
When performing this protocol, check the positioning of the balloon sites on the proximity of RC, and totally occlude the flow to the kidney. Make sure it's not kinking in that water. Following this procedure, endovascular catheter flow probes can be inserted in the renal arteries to assess renal blood flow, or renal biopsies can be performed to assess renal tissue injury.
