Hydrodynamic Renal Pelvis Injection: A Technique to Directly Deliver DNA into Kidney Cells of Murine Model

To deliver plasmid DNA directly into the mouse kidney cells, begin by placing an anesthetized mouse in a lateral position. Prep the mouse by removing its dorsal fur from the side. Locate an appropriate incision site in the left flank, below the ribcage.

Make a small slit in the skin and underlying muscle layers. Next, apply gentle pressure on the abdominal cavity to expose the kidney through the incision site. Partially remove the overlaying perinephric fat tissue to visualize the renal pelvis.

Now, take a syringe containing plasmid DNA in a hydrodynamic delivery buffer. Insert the needle directly into the renal pelvis - a triangular, dilated portion of the ureter in the kidney. Inject the plasmid solution rapidly such that the DNA directly reaches the renal parenchyma - the functional part of the kidney that filters blood.

The quick delivery of large fluid volume through the renal pelvis builds a high pressure, creating transient pores in the plasma membrane of renal parenchymal cells. This facilitates the entry of plasmids into the cells. Eventually, these pores close, entrapping the plasmids within the cells.

Finally, retract the needle and return the kidney to the abdominal cavity. Suture the skin and muscle layers at the incision site.

Wear sterile surgical gloves and starting from the side of incision, swab the animal with a new chlorhexidine/alcohol swab three times in a circular motion. Now, locate the incision site. Pinch the skin with tweezers, and using scissors, cut the skin layer approximately 1 centimeter from the spine below the rib cage. Make another center-cut below in the muscle layer. Locate the kidney, and without touching it, gently expose it from the abdominal cavity by applying a steady gentle pressure on the abdomen with the fingers.

Exposing the kidney is sometimes difficult. Apply gentle pressure to the abdomen and spine to avoid damaging the spleen. If organs other than the kidney come out, gently return them to the body cavity using closed forceps if necessary.

Then, gently separate the kidney from the surrounding fat just enough to visualize the renal pelvis, which can be seen as a small white dot. After visualizing the renal pelvis, use closed forceps to push down on the right side of the kidney so that the renal pelvis remains in view. Then, grasp the loaded insulin syringe in the right hand by holding it parallel to the working surface and insert the needle carefully into the renal pelvis.

Once the needle is inserted, inject the solution quickly within 3 seconds. Let the needle remain in place for approximately 10 seconds to prevent backflow of the solution. Then, remove the needle slowly and carefully and return the organ to the abdominal cavity by stretching the incision site gently using closed forceps. Now, suture the muscle layer with 5-0 absorbable sutures and place 2 to 4 independent knots. Similarly, suture the skin layer with 5-0 or 6-0 non-absorbable nylon sutures and place 2 to 4 independent knots.