Rapid Depletion of Renal Macrophages Using Human CD59/Intermedilysin Cell Ablation Tool

We use intermedilysin mediated oblation of the human CD59 expressing cell in mice to study renal microphage regeneration, focusing on chemokine-driven monocyte recoupment and niche establishment of immune surveillance post injury. Recent development in our field reveal mechanism of renal macrophages regeneration, highlighting a specific chemokine that recruit macrophages and how immune surveillance is reestablished following acute kidney injury. We combine tamoxifen inducible cryogenetics, intermedilysin ablation, multi-parameter flow cytometry, and intravital microscopy to ablate, track, and visualize kidney macrophages in real time.

Key challenges are avoiding systemic inflammation, targeting only resident macrophages, and measuring rapid regeneration without disturbing the delicate renal mIcroenvironment. We showed monocytes restore 88%of kidney macrophages within seven days after ablation, a processes critically dependent on the CX3 CR1, CX3 CL1 signaling axis. To begin, preheat corn oil to 42 degrees Celsius.

Dissolve 100 milligrams of tamoxifen in five milliliters of preheated corn oil to prepare a stock solution with a concentration of 20 milligrams per milliliter. Administer tamoxifen intraperitoneally to 10 to 12-week-old male mice at a dose of 100 micrograms per gram body weight. After 15 days, inject a single intravenous dose of intermedilysin into the mice at a dose of 120 nanograms per gram body weight.

Monitor and confirm resident macrophage ablation and subsequent regeneration by performing flow cytometry at one, three, and seven days, post intermedilysin administration. For kidney collection, use fine forceps and scissors to make a horizontal incision in the lower abdominal region of a euthanized mouse. Extend the incision vertically along the midline up to just below the rib cage or sternum.

Gently retract the skin and muscle to uncover the thoracic cavity. Cut the right atrium open to allow blood to drain from the circulation. Insert a 21 to 23-gauge needle into the left ventricle.

Begin perfusion with 10 to 20 milliliters of cold PBS to flush out blood. Continue perfusion until the kidneys appear pale, indicating effective blood clearance. Locate the kidneys against the dorsal body wall, and gently separate them from surrounding tissues using forceps and scissors.

Excise the kidney capsules and immediately place the kidneys in cold PBS to prevent degradation. Prepare 100 milliliters of digestion enzyme cocktail by mixing calcium and magnesium-free HBSS, collagenase IV, and DNase I.Prepare the enzyme solution fresh, and keep it on ice until use. Using sterile scissors, mince the kidneys into tiny fragments in a Petri dish containing five milliliters of HBSS.

Transfer the minced kidney tissue into 15-milliliter tubes containing 10 milliliters of the prepared enzyme solution. Now incubate the tubes at 37 degrees Celsius for 30 minutes with gentle agitation to dissociate the tissue. Gently triturate the tissue using a pipette or syringe plunger to further break up cell clumps.

Remove debris by passing the cell suspension through a 40-micrometer cell strainer. Centrifuge the cell suspension at 700 G for 10 minutes at 18 degrees Celsius. Next, carefully decant the buffer.

Resuspend the pellet in five milliliters of lysis buffer before incubating at room temperature for five minutes to lyse red blood cells. Add PBS to wash the cells and remove the lysis buffer. Maintain the resulting single-cell suspension on ice until further use.

For density gradient centrifugation, first resuspend the cell pellet in 10 milliliters of 30%density gradient solution. Carefully layer this over three milliliters of 70%density gradient solution in a centrifuge tube. Centrifuge the gradient at 500 G for 30 minutes without applying a break.

After centrifugation, carefully collect the interface layer between the 30%and 70%solutions. Wash the collected cells twice with 10 milliliters of PBS, and centrifuge each wash at 500 G for 10 minutes. Resuspend the final cell pellet in one milliliter of FACS buffer in a 1.5-milliliter tube.

Count the cells using a hemocytometer under a bright-field microscope, and adjust the cell concentration to two times 10 to the power of six cells per milliliter. Pellet the cells in 1.5-milliliter tubes by centrifuging at 650 G for five minutes, and resuspend the cell pellet in one milliliter of FACS buffer. Add anti CD 16 or 32 antibody at one to 200 dilution to block nonspecific FC receptor binding.

After incubating the cells for 15 minutes at four degrees Celsius, stain the cells with aqua live/dead dye and a preconjugated antibody cocktail, including CD45 E 450, CD11 B PE SI7, HCD59 PE, and F4/80 BV 605. Add the antibody cocktail directly to the cell suspension. Incubate the sample for 30 minutes at four degrees Celsius in the dark to protect fluorophores.

Then wash the cells twice with FACS buffer. Fix the cells in 1%paraformaldehyde for 30 minutes on ice. Then wash the cells twice with FACS buffer to remove residual fixative.

Acquire stained and fixed cells using a flow cytometer. Analyze the data using the software. Identify kidney and other tissue resident macrophages and microglia using the markers CD45, CD11B, and F4/80.

Perform initial gating to select live cells using forward and side scatter profiles. Eliminate doublets using forward scatter area versus height gating. Exclude dead cells using the viability dye.

Enrich immune cells by gating on CD45 positive populations. Define kidney resident macrophages as CD11b+F4/80hi cells within CD45 positive gate as shown in the gating strategy. Define microglia populations as CD11b+CD45 intermediate cells, and perform final analysis.

A single prominent protein band was observed near 54 kilodalton in the SDS page gel, confirming the purity and expected molecular weight of recombinant intermedilysin. Purified intermedilysin exhibited strong hemolytic activity in a dose-dependent manner, achieving 50%red blood cell lysis at 7.24 nanogram per milliliter, and nearly complete lysis at 650 nanogram per milliliter. Flow cytometry analysis confirmed successful and selective depletion of renal macrophages in inducible human CD59 compound cre-R mice one day after intermedilysin administration, with no depletion in control mice.

Renal macrophages began repopulating by day three, and recovered to approximately 88%of their original population by day seven. Only 5%of newly regenerated renal macrophages expressed human CD59 by day seven, indicating that repopulation primarily occurred via monocyte recruitment rather than in situ proliferation.