The overall goal of this procedure is to induce ischemia in mice to study acute kidney injury. This is accomplished by first like eating and then removing the right kidney. The second step is to prepare and put the kidney into a Lucite cup.
Next, a hanging weight system is applied to the isolated renal artery to induce ischemia. Ultimately, results can be obtained that show attenuated or severe kidney injury following ischemia by measuring kidney function via FITC inulin clearance. This hanging weight system for inducing renal ischemia can help answer key questions in the field of acute and chronic kidney injury due to ischemia.
Demonstrating the procedure will be Alexander Barla, a medical student from University of Pennsylvania, who is in our lab to do research on kidney injury this year. And Douglas Ard, a research assistant working also in our group. While renal ischemia can be performed simultaneously in both kidneys, a simpler method of removing the right kidney, followed by ischemic treatment of the remaining left kidney is demonstrated here.
To begin, place an anesthetized animal onto a heated table in a left lateral decubitus position. Secure the lower and upper extremities to the table with tape. Clean the skin and cover the incision area with mineral oil to prevent inhalation of mouse hair and reduce allergen exposure.
Make an incision over the right flank into the skin and cut the muscle wall with a coagulation electrode to prevent bleeding. Ligate the renal pedicle, including the renal artery and renal vein with a silk suture. Use scissors to cut out the right kidney, taking care not to interfere with the adrenal vessels.
Use sutures to close the muscle wall and then the skin. Once the first wound is closed, place the animal in a right lateral decubitus position. Make an incision over the left flank and visualize the left kidney.
Carefully separate the left kidney from the connective tissues, avoiding the adrenal gland and vessels. The next step is to turn the kidney on its ventral side into a Lucite cup. To do this, hold the surrounding tissue with forceps while carefully pushing the lower pole of the kidney into the cup.
Next, use forceps to push the upper pole of the kidney into the cup. Use cotton swabs soaked with saline at 37 degrees Celsius to keep the kidney wet and warm throughout the procedure. Once the kidney is in the cup, the kidney holder is restrained ventrally.
To maintain tension on the renal vessels, fix the kidney holder so that the renal artery can be easily identified on top of the renal vein. Isolate the renal artery away from adjacent tissues and vessels with fine forceps. Carefully separate the artery from the vein lying underneath.
Next place a seven zero silk suture under the renal artery using forceps. As a guide, attach a weight of approximately one gram to each end, and place the ends of the sutures over a small pole. With the weights hanging freely, the renal artery should be occluded.
Successful occlusion is confirmed by a change of color from red to pale. The duration of ischemia will depend on the study. Here, the ischemic period is 30 minutes after ischemia, the weights are lifted, and a reperfusion time between 30 and 60 minutes is allowed.
After reperfusion, release the tension of the kidney holder and gently move the kidney out of the cup back into the animal To determine the extent of kidney injury, one hour following renal ischemia, FITC inulin clearance can be measured to assess glomerular filtration rate. The first step is to cannulate the left jugular vein for continuous infusion of sodium chloride and FITC labeled inulin. After preparing the animal as shown earlier, make an incision along the neck.
Isolate the dark blue vein from the surrounding tissue. Tie 2 4 0 sutures around the vein about one centimeter apart. Place a clamp on the vein before the proximal suture to stop blood flow.
Next, close the distal knot and place a weight on the distal end of the suture to maintain tension on the vein, use micro scissors to cut a small opening into the vein. Hold the opening with fine forceps while inserting the catheter, remove the clamp to advance the catheter further. Once in place, close the second proximal suture over the vein and the catheter to place the bladder catheter for timed urine collection.
First, cut the skin over the bladder. Next, carefully pull the bladder out through this hole. Once isolated, secure the bladder with wet tissue and place a length of suture over it.
Cut a short 1.5 centimeter piece of a catheter and heat one of the ends to smooth the edge and facilitate insertion. Place a suture around the bladder, then cut a hole between the vessels at the top of the bladder to prevent bleeding. Insert the smooth end of the catheter and close the suture over both the catheter and the bladder.
Now that the catheters are in place, maintain a continuous infusion rate of 800 microliters per hour per 25 grams of body weight. FITC inulin is added to the infusion for evaluation of whole kidney glomerular filtration rate. Allow the animal to stabilize for 20 minutes before collecting samples At four 20 minute intervals, collect the urine by placing the end of the bladder catheter into a small einor tube.
Record the weight of the tube for and after collection. To determine the amount of urine, obtain a blood sample via retroorbital venous plexus collection in the middle of each time period for measurement of FITC inulin as an alternative to measuring inulin clearance by direct cannulation, creatinine clearance and serum creatinine can be measured using a metabolic cage. After recovery from renal injury, the misa placed into metabolic cages for determination of renal function.
Parameters for 24 hours weigh each water bottle before placing into the cage. This weight will be used later to determine the amount of water consumed during the study. Weigh the water bottles and use this weight subtracted from the starting weight to determine the amount consumed Renal function can be assessed by measuring plasma and urine levels of creatinine, sodium, and potassium at different time points after ischemia.
Following this procedure, cytokine levels can be measured to reveal the extent of inflammation after acute kidney ischemia.
