This method can help answer key questions in the nephrology field about the role of cell surface protein trafficking in proteinuric and not proteinuric kidney disease. The main advantages of this technique are that it facilitates the study of cell surface protein trafficking in-vivo and that more than one protein can be analyzed per experiment. Though this method can provide insight into glomerular cell surface trafficking, can also be applied to other organ systems that are perfused and are accessible by isolation techniques.
Begin by confirming a lack of response to toe pinch and disinfecting the ventral side of the anesthetized mouse with 70%isopropyl. Make a median cut through the skin from the pelvis to the sternum and use tweezers to remove the skin from the abdominal fascia. Cut the skin on both sides of the abdominal midline and make a medium through the abdominal muscle layer from the bladder to the xiphoid.
Use the scissors and forceps to divide the muscles into four quadrants And use two surgical clamps to secure the upper two quadrants towards the neck of the animals. Place the animal under a dissecting microscope and use a sterile swap to move aside the visceral organs. Using fine scissors, cut the hepatic phrenic ligament.
Using fine surgical tweezers, place one suture around the hepatic mesenteric artery branial of the renal arteries. And around the aorta, proximal to the renal arteries, at the adrenal gland. Free the distal aorta from the fascia, fat and other tissue.
Then, clamp the vena cava and the aorta at the height of the bifurcation and place another suture around the aorta distal of the renal arteries. Use tweezers to tighten the proximal aortic suture, attenuating the blood flow and cut a small hole half of the diameter of the aorta into the aorta, distal to the renal arteries. Insert a 10 centimeter catheter attached to a 10 milliliter syringe containing five milliliters of ice cold PBS plus calcium and magnesium into the aorta.
And fix the catheter with the prepared ligatures. Then begin perfusing the kidneys at a two milliliter per minute flow rate. Using fine scissors to cut a hole into the renal vein at the renal arteries and tightening the suture around the hepatic and mesenteric arteries.
After the entire volume of PBS has been delivered replace the perfusate with five milliliters of ice cold PBS plus calcium and magnesium supplemented with 0.5 milligrams per milliliter biotin for surface labeling. Continue to perfuse the kidneys at a two milliliter per minute flow rate. When the entire volume has been delivered, attach a syringe containing five milliliters of ice cold PBS plus calcium and magnesium supplemented with 100 millimolar glycine to the catheter.
Quench the glomeruli with all five milliliters of the glycine solution at a two milliliter per minute flow rate. Then perfuse the kidneys with five milliliters of ice cold PBS plus calcium and magnesium supplemented with 200 microliters of magnetic beads per milliliter at a two milliliter per minute flow rate. The embolization of the glomeruli with the brown magnetic beads will be visible on the kidney surface.
When all of the beads have been delivered remove the capsule of the kidneys and harvest the kidneys at the hilum. Place the kidneys in a 10 centimeter cell culture dish containing 15 milliliters of PBS plus calcium and magnesium. Use a new double edged blade to cut the kidneys into the smallest pieces possible.
Transfer the tissue into a two milliliter tube containing one milliliter of collagenase A for 30 minutes at 37 degrees Celsius, mixing the tissue solution gently before and after the digestion with a modified 1000 microliter pipette tip. At the end of the incubation, filter the digested tissue through a 100 micron cell strainer. Use a cell scraper and ice cold PBS plus calcium and magnesium to mash the remaining tissue through the cell strainer.
Bring the final volume of the kidney single cell suspension up to 50 milliliters with fresh ice cold PBS plus calcium and magnesium and collect the kidney cells by centrifugation. Remove the supernatant then re-suspend the pellet with slightly less than 1.5 milliliters of ice cold PBS plus calcium and magnesium while vortexing and transfer the glomerular suspension into a new two milliliter tube. To wash the glomeruli, place the tube into a magnet to aggregate the glomeruli.
After one minute, use a 1000 microliter pipette to remove the supernatant. And remove the tube from the magnet. Add one milliliter of PBS plus calcium and magnesium to the tissues.
Pipette the glomeruli up and down one time and vortex the cells. Return the tube to the magnet and wash the glomeruli again as just demonstrated. Until a sample purity has reached at least 90%by light microscope analysis at a 40 to 100 X magnification.
Then collect the magnetic bead purified glomeruli by centrifugation. To achieve a greater than 95%purity, the glomeruli needed to be washed thoroughly as demonstrated. Biotin perfusion facilitates labeling of the capillary loops in biotin but not controlled perfused mouse kidneys.
Immune precipitation of the biotin fraction of glomerular extracts reveals that the glomerular transmembrane proteins nephrin and podocalyxin are immunoprecipitated with the biotin but not with the control fraction. Staining of the immunoprecipitated fraction with streptavidin further confirms the presence of biotin laded nephrin in the biotin perfused but not control perfused animals. Endothelial protein vascular endothelial cadherin and intracellular adhesion molecule two are also immunoprecipitated within the biotin fraction.
In the early phase of nephrotoxic nephritis a reduced expression of cell surface nephrin is observed in nephrotoxic nephritis animals compared to controls. Indeed densitometic analysis shows a significant reduction of biotin laded nephrin in nephrotoxic nephritis animals compared to controllers. Quantitative analysis of the total nephrin to actin ratio reveals no significant differences between the control and nephrotoxic nephritis mice.
In addition, equal amounts of podocytes are observed in nephrotoxic nephritis and control animals. In late nephrotoxic nephritis, nephrotoxic nephritic animals exhibit a recovery of nephrin expression with no significant differences in cell surface nephrin measured between control and nephrotoxic nephritic mice and an overall total nephrin reduction in nephrotoxic nephritic animals. Further, podocyte numbers are decreased compared to control animals.
While attempting this procedure, it's important to remember to keep the syringes bubble free during their connection in order to avoid air embolism of the glomeruli and to work under ice cold conditions once kidney perfusion has started. Following this procedure other methods like the isolation of glomeruli RNA and protein can be performed to answer additional questions about protein expression or interactions in healthy and diseased kidneys.
