Murine Bilateral Renal Lymphadenectomy

We study trafficking of immune cells from the renal lymph node to the kidney and of kidney antigen to the draining lymph node. Our surgical approach allows for the unique advantage that we can selectively disrupt the renal lymph nodes while leaving all other lymph nodes intact in a well-tolerated long-term survival model. Now that this model has been developed, we can explore the contributions of renal lymph node and immune responses and how the removal of these node contributes to immune-related kidney injury.

To begin, place the anesthetized animal on the operating table and apply ointment. Use tape to secure the animal's left arm in a superior direction and the tail in an inferior direction to create a relaxed but elongated posture. Shave the fur and disinfect the surgical area three times.

After confirming the depth of anesthesia using a toe pinch, make a one-centimeter oblique incision through the skin just below the spleen, approximately one centimeter inferior to the last rib. Using blunt dissection, separate the skin from the abdominal and chest wall and identify the spleen within the abdomen. Then, make a one-centimeter incision through the abdominal wall musculature just inferior to the spleen to enter the peritoneal cavity.

Identify the left kidney as a smooth organ located along the posterior abdominal wall just beneath the spleen. Using two angled forceps, bluntly dissect behind the left kidney to access the retroperitoneum and gently reflect the left kidney in an anteromedial direction. Locate the adipose tissue pad surrounding the left renal lymph node, which lies posterior to the kidney and superior to the renal vessels.

Identify and avoid the adrenal gland located superiorly, the aorta posteriorly, and the cisterna chyli anteromedially. Using fine-tipped forceps, bluntly dissect around the lymph node capsule to separate it from adjacent vessels while providing gentle counter tension with angled forceps. Use fine-tipped straight forceps to retract the lymph node superiorly.

Then use fine-point scissors to sharply cut the posteromedial attachments. Examine the area to confirm hemostasis. Next, return the kidney to its normal anatomical position.

Close the abdominal wall and then the skin using 6-0 monofilament polypropylene suture in a running stitch. Apply sterile surgical glue to seal the wound. Optionally, for improved identification of the renal lymph nodes, inject 10 to 20 microliters of sterile, filtered 5%Evans blue dye in PBS into both hind foot pads before the renal lymphadenectomy.

Within five minutes of injection, the dye reaches the renal lymph nodes via drainage along the iliac lymphatic tract. Reposition the animal into the left lateral decubitus position with the left side facing down. Use tape to secure the animal's right arm in a superior direction and the tail in an inferior direction to maintain a relaxed but elongated posture.

Now, make a one-centimeter oblique skin incision just beneath the last rib, which will be positioned slightly higher than on the left side. Using blunt dissection, separate the skin from the abdominal and chest wall. Then make a one-centimeter incision through the abdominal wall musculature to access the peritoneal cavity.

Identify the right kidney, which is a smooth organ located along the posterior abdominal wall and just beneath the liver. Using two angled forceps, bluntly dissect behind the right kidney to gain entry to the retroperitoneal space and reflect the right kidney in an anteromedial direction. Identify the adipose tissue pad surrounding the right renal lymph node, which lies posterior to the kidney and superior to the renal vessels.

Identify and avoid the adrenal gland located superior to the lymph node. Now, using fine-tipped forceps, bluntly dissect around the capsule of the lymph node to separate it from nearby vessels while applying gentle counter traction with angled forceps. Then, with fine-tipped straight forceps, pull the lymph nodes superiorly and sharply cut the posteromedial attachments with fine-point scissors.

Inspect the area to ensure hemostasis. Finally, return the right kidney to its anatomical position. Close the abdominal wall and skin in two layers using 6-0 monofilament polypropylene suture in a running pattern and apply sterile surgical glue over the incision site.

Flow cytometry confirmed successful lymph node extraction in all five left and five right renal lymphadenectomy samples, each showing over 50%CD45-positive live cells, compared to less than 2%in visceral adipose controls. Quantification of CD45-positive cells showed a highly significant difference between both left and right renal lymphadenectomy samples compared to visceral adipose tissue with no significant difference between the left and right sides.