The overall goal of this protocol is to analyze the isolated kidney function in an ex vivo perfused mouse kidney. Excluding the influence of blood pressure, hormones, or the autonomic nervous system. This method can help answer key questions of Nefrology.
Giving insides into isolated organ function and serving as an elegant tool to study acute kidney injury. The main advantage of the IPK is that organ function is studied in the absence of systemic influences. After preparing the mouse for surgery, using scissors, perform a medium laparotomy from the pubic crest to the sternum.
First open the skin, and then the abdominal muscles. Next, remove the intestines and place them on the left side of the mouse, lateral from the abdomen. Now, free the bladder from connective tissue and expose both ureters and the urethra.
For these procedures, place all the ligatures using 5-0 sutures. The first ligature to place is around the left ureter. Close this tightly with a double surgical knot.
Next, place a ligature around the urethra using the same technique. Then, place a lasso ligature around the whole bladder, but leave it untied. With the first three sutures in place, use eye scissors to make a one millimeter long incision into the bladder.
Into this incision, place a cannula made of two centimeters of PE-50 tubing. Now close the ligature that lassos the bladder, securing the cannula. With the bladder cannulated, now cut the left ureter and urethra distal from the ligatures.
The bladder should now be only attached to the right ureter and should move freely. Next, remove the connective tissues and fat surrounding the abdominal aorta. Then place ligatures around the midpoint of the exposed aorta and just below the diaphragm between the superior mesenteric artery and the celiac trunk.
Keep each of these loose as well. Next, place a ligature around the superior mesenteric artery. Now, place a third ligature around the aorta directly below the right renal artery and above the left renal artery.
This is ligature number seven. Finally, put a ligature around the caudal vein package. Now place a clamp between ligature number seven and the branching of the left renal artery.
Then, make a small incision in the aorta, caudal to ligature seven, taking care not to cut the dorsal wall and stretch the opening with a vessel dilator. Into the opening, insert a two centimeter pulled PE-50 cannula, pushing the tip just into the clamp. Then, open the clamp.
Next, push the tip of the needle cranially until it reaches the junction of the right kidney artery and the aorta. Now close ligature seven, and then close the ligature at the midpoint of the abdominal aorta. Proceed by opening the chest with scissors by dissecting the diaphragm.
Then, with a single cut, separate the aorta, vena cava, heart, and vegetative nerves. Now, start pressure control of the perfusion pump and maintain the mean pressure between 80 and 100 millimeters of mercury. Now, close the three remaining untied ligatures around the aorta and caudal vein package.
Using scissors, free the right kidney from the surrounding connective tissue and release it from the adipose capsule. Cut the aorta proximally to the ligature between the superior mesenteric artery and the celiac trunk. Next, cut the superior mesenteric artery distally to the ligature around the superior mesenteric artery.
Now, remove the kidney supporting vessel bundle, taking care not to cut into the vessels themselves. Then, cut the liver at the connection to the kidney, but leave a small part of the liver attached to it, so that the vena cava is kept open by it. The next step is to remove the kidney bundle from the mouse.
Now, leave the kidney in the chamber and remove the mouse. At the connection between the liver and kidney, place a lasso ligature. Then, cannulate the vena cava with a venous line and close the lasso ligature.
The kidney should now sit stably in the box. Venous outflow through the venous line should begin immediately. Urine outflow will begin after approximately three minutes.
Peristalsis of the ureter should begin immediately and remain stable for at least one hour. Close the box. The preparation is now complete and should remain viable for at least an hour.
After using the described method on C57 black 6 mice, the tissue viability of four kidneys was tested after an hour of continuous perfusion. Pressure was maintained at a continuous 100 millimeters of mercury. Perfusate flow and vascular resistance remained steady in all four kidneys.
The glomerular filtration rate was assessed using FITC inulin clearance intended to increase over time but not significantly. Fractional excretion of sodium and potassium was assessed after 55 minutes of perfusion. These parameters increased compared to the in vivo situation.
After 55 minutes, urinals molality did not rise above the osmolality of the venous outflow. The kidneys were then fixed and their ultrastructure was assessed with TEM. Glomeruli in S1 segments of proximal tubules appeared largely unaltered.
Some, but not all S2, S3 segments of the proximal tubules in the thick ascending limbs that were not close to vascular bundles showed signs of organ damage. Upon closer inspection, distal convoluted tubules and collecting ducts did not show signs of necrosis. Overall, the kidneys mirrored what is reported for rat isolated perfused kidneys.
Following this procedure, analysis methods like imaging or facts followed by protein or RNA analysis can be performed on the tissue to obtain further insights.
