Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.
The overall goal of this protocol is to use zebrafish to observe the effect of nephrotoxins such as gentamicin or cisplatin to model acute kidney injury. The main advantage of this technique is that the zebrafish model enables real time imaging of cell damage and regeneration after acute kidney injury. To begin this procedure, prepare an embryo manipulation tool for positioning the embryos for micro injection by attaching a gel loading tip on the end of a five inch straight dissecting needle and secure it with tape or super glue.
Next, prepare some fine tapered micro injection needles by pulling the fire polished borosilicate glasses with filament using a pipette puller. Then, use a razor blade or fine forceps edge to cut the needle tips to create sharp angular injection tips. Subsequently, place the needles on a strip of modeling clay in a petri dish to protect the needle tips.
To make the micro injection tray, prepare 1.5%agarose E3 soluation in a 100 milliliter Erlenmeyer flask by dissolving the agarose with E3 in the microwave. Then, allow the mixture to cool at room temperature for approximately ten minutes. Pour the cooled agarose E3 solution into a petri dish to make a foundation with a depth of approximately 0.5 centimeters.
After it has solidified, pour a second layer of agarose E3 solution. Then, insert the pre-fabricated embryo well mold into the agar, at an angle to avoid any air bubbles, and allow the agarose to solidify at room temperature. After the entire micro injection tray has set, lift the pre-fabricated embryo well mold out slowly and carefully, then fill the dish with E3 solution and store at four degrees celsius.
In this procedure, set up the zebrafish mating tanks by placing dividers into the mating chambers and fill with system water. Next, place one fish on each side of the divider, such that each mating chamber contains one female and one male fish, and cover both mating chambers. The next morning, remove the dividers to enable spawning.
After approximately 30 minutes, check for any embryos at the bottom of the tank. Then, return the fish to the aquarium and collect their embryos by passing the mating tank water through a fine wire mesh strainer tool. Next, invert the strainer over a petri dish and rinse it with E3 to collect the embryos.
Subsequently, incubate the embryos at 28.5 degrees celsius overnight. When the embryos reach 24 to 26 somite stage, decant most of the E3 media. Then, dechorionate them by adding E3 media, then 100 microliters of 50 milligrams per milliliter pronase to each dish of embryos, then incubating the embryos at 28 degrees celsius for 15 minutes.
After 15 minutes, fill the dish with E3 and then swirl gently to dislodge the chorions from the embryos. Then, decant the E3 pronase and rinse the dish with 25 milliliters of fresh E3.To block the development of pigmentation, replace E3 with 25 milliliters of fresh E3 PTU 24 hours post fertilization. For experiments spanning several days, replace the E3 PTU solution with fresh media once a day to keep the dish clean.
On the day of injection, prepare the desired nephrotoxin solution with the appropriate vehicle control. Vortex or mix it gently. Afterward, check the sides of the tube and top of the water column to ensure the drugs are dissolved in solution.
Load a trimmed micro injection needle with two to three microliters of nephrotoxin by threading a fine gel loading tip into the back of the needle, and suspend the needle vertically with a piece of tape to allow the solution to fill the trimmed needle tip by gravity. Once the needle tip is full, secure the loaded needle into the micro manipulator. Test the micro injection volume by placing a drop of mineral oil onto a micrometer slide and inject into the oil to evaluate the droplet size.
Next, remove the dish of embryos from the incubator and anesthetize them by adding five milliliters of 0.2%tricaine to the embryo dish. Subsequently, transfer the embryos to the injection mold with a transfer pipette. Maneuver each embryo into a different well of the mold, placing the head in the deepest area of the well such that the trunk rests along the depression and the tail sticks out a bit.
Now, insert the filled needle into the micro manipulator and position the needle tip next to an embryo. Gently insert the needle into the tail vein for micro injection. The most critical step of the procedure is the micro injection.
To ensure success, the needle must be cut at a sharp angle, it must be moved carefully into position next to the embryo. If the injection is successful you should see the liquid entering the circulation. Co-injection of the nephrotoxicant with fluorescently conjugated dextrin would enable the researcher to verify successful injection after the procedure.
Afterward, gently remove the needle from the embryo. Transfer the embryo to a clean dish and rinse to remove the tricaine with fresh E3 PTU. Then, incubate the embryo to the desired time point to assess morphology, which can be documented by photography in methyl cellulose mounting media.
Or process for experimental analysis as desired. Note that recovery of embryos should be assessed every 12 to 24 hours after the injection to determine the the percentage of individuals with edema, which commonly indicates renal injury. The transparency of the zebrafish embryo facilitates the micro injection procedure and can be further enhanced by PTU chemical treatment at 24 hpf, to lessen the development of pigmentation during a typical experimental time course.
Here, embryo specimens were allowed to develop through the 72 hpf stage then were either mock injected, micro injected with saline vehicle, or micro injected with 2.5 milligrams per milliliter gentamicin. Subsequent live observation was conducted at one and two days post injection using transillumination lighting with a stereo microscope. Compared to mock or saline injected embryos, only the gentamicin injected individuals showed the development of edema.
In this case, pericardial edema suggesting an abrogation of normal kidney functionality consistent with previously published observations. Following this procedure, other methods such as whole mount C2 hybridization can be performed in order to access changes in gene expression after nephrotoxin exposure.
