The 2K1C mouse model is a reliable and reproducible method to induce renal artery stenosis. This protocol is valuable in elucidating molecular mechanisms involved in renin expression control during renal vascular hypertension. This animal model mimics unilateral renal artery stenosis in humans.
The reliability and reproducibility make it a good model to discern new therapeutic targets for treating renal vascular hypertension. This technique requires a basic knowledge of performing surgery in mice. There are two critical parts:tubing preparation, and placement of tubing in the renal artery.
Both are described in detail. Constriction of the right renal artery using precisely-cut tubing is key. This video of the procedure will show learners how to cut the tubing, then access and constrict the artery.
Begin by cutting the tubing for the stenosis procedure. Cut a 0.5-millimeter-length piece of polyurethane tubing with a sharp scalpel. Then make a cuff by removing 0.2-millimeters of the circumference with a lengthwise cut.
Record the weight of the mice, which should be between 18 and 22 grams, to perform renal artery stenosis with the polyurethane tube. The weight of the mice is critical to perform the surgery. After anesthetizing and administering the painkiller to the mouse, place it back inside the cage until it is fully unconscious.
Pinch the toe with forceps to check if the mouse is fully anesthetized and ready for the surgery. Lay the mouse on its back on a paper towel away from the surgery area and remove the hair of the lateral abdomen with an electric hair clipper following the opposite direction of hair growth. Clean the shaved area with a sterile alcohol gauze pad and apply topical povidone-iodine solution.
Open the sterilized bag containing all the surgical equipment. Working under the dissecting microscope, use sterile sharp scissors to make a small flank incision about 0.5 centimeters away from the vertebrae. Proceed along the lumbar vertebrae and make a one-inch incision.
Pull back the skin and muscle to expose the kidney. Then, clean and remove the surrounding fat with cotton swabs to isolate the renal artery. Isolate the renal nerve from the renal artery using the curved forceps.
Clean the oozed fluid near the renal artery with a cotton swab. Place two nylon sutures under the right renal artery, make loose knots, and place the cuff around the main renal artery approximately equidistant between the kidney and aorta bifurcation. Close the cuff using the nylon sutures, making three knots for each suture to avoid the probability of losing the sutures after the surgery.
Close the incision in the muscle by applying a simple continuous suture and make simple interrupted sutures to close the skin. Return the mice to their cage and leave the half cage on a circulating water heating pad for two to three hours for animals to recover from the surgery. Provide the animals with gel diet recovery food.
On the next day, administer painkiller. Weigh the mice for the next two days and consult with the veterinarian if a mouse loses more than 20%of its weight. Inspect the mice daily to assess for redness, swelling, pain, or infection.
Record the weight of each mouse. After euthanizing the animal, place it on a sterile platform in supine position. Secure and extend the limbs to limit movement and thoroughly spray the mouse with 70%ethanol.
Use scissors to make a midline incision and open the abdomen and chest area. Pull back the skin and peritoneum wall, then carefully expose the heart and puncture the right ventricle. Remove both kidneys using forceps, keeping track of which kidney was stenosed.
Remove the kidney capsules, clean them from any fat, and record the weight of each kidney separately. Cut a longitudinal section of both kidneys and fix them in 4%PFA overnight at four degrees Celsius for in situ hybridization and immunohistochemistry. Isolate the cortex of the remaining kidney and flash freeze it in liquid nitrogen to perform a Western blot.
Store the samples at minus 80 degrees Celsius until further analysis. Renal artery constriction increases renin expression in the stenosed kidney, while repressing expression in the contralateral kidney. The two-kidney one-clip model of stenosis induced renin expression and increased kidney injury.
Expression of renin and prorenin was measured using immunoblotting. The data show that renin and prorenin expression increased in the stenosed kidney compared to contralateral and sham kidneys, suggesting that the cuff was constricting the renal artery and causing changes in renal perfusion. Immunohistochemistry was performed to visualize the localization of renin expression.
The images confirmed increased expression of renin in the clipped kidney. Moreover, juxtaglomerular cell recruitment along the afferent arterial was seen in the stenosed kidney. To investigate the effect on renin mRNA expression levels, in situ hybridization was performed.
The data suggests increased renin mRNA and JG cell recruitment in the stenosed kidney. Another characteristic of renal artery stenosis is the upregulation of kidney injury markers due to changes in kidney perfusion, superoxide production, and hypertension. Therefore, N-GAL was measured with immunoblotting, showing that the acute kidney injury marker was highly upregulated in the stenosed kidney.
When attempting this protocol, remember to place the nylon sutures around the renal artery before placing the cuff. It helps expedite the procedure.
