Protocol for kidney regeneration and new nephron development in adult zebrafish by gentamicin induced injury (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Iain Drummond; Caramai Kamei; Yan Liu

Keywords

[‘gentamicin’, ‘zebrafish’]

Subjects

[‘Cell biology’, ‘Experimental organisms’]

Release Date

2016-08-01

Abstract

TBD

Procedure

Advance preparation

1. Determine how many adult zebrafish 6-12 months old to injure. Plan to injure 10-20% more fish than will be needed. In order to minimize variability, use age matched sibling fish reared together in the same tank so that they are roughly the same size and have less genetic variability. Female fish are much easier to inject compared to the male fish, due to the larger capacity of the abdomen. However, males and females give similar results.
2. When doing the experiment for the first time, plot a dose curve to determine the appropriate gentamicin dosage for a specific strain of fish. Test each new batch of gentamicin before use in experiments since the purity of gentamicin varies from batch to batch. Proper dosage should be determined by assessing expression of injury markers such as lhx1a. (See Figure 1A-D).
3. Prepare fresh gentamicin solution before each experiment. For example, for an 80mg/Kg dose (80mg gentamicin/Kg of bodyweight) in a fish weighing 0.5g, prepare 2mg/ml of gentamicin in phosphate buffered saline as a convenient working solution. This provides a 20 microliters of gentamicin for intraperitoneal injection into the fish.

NOTE: In general, 80-120mg/Kg achieve good results, but doses as low as 40mg/Kg may be appropriate. Gentamicin may also be purchased in solution to minimize hazardous exposure to lab personnel. CAUTION! Gentamicin in high doses can be toxic. Wear gloves and mask when weighing powder.

1. Prepare 100mL of 0.016% tricaine water for anesthetizing fish. Prepare a 25x stock of tricaine (4g/1L) dissolved in sterile milliQ water adjust to pH 7 and store at 4 degrees until use. Dilute 4mL in100mL of fish water for a working concentration of 0.016%. CAUTION! Tricaine is an anesthetic and skin irritant, wear gloves when handling.
2. Make a fish scoop out of a plastic transfer pipet by cutting the bulb into a scoop shape and cutting 2 slots in the bottom to drain water. Load 1ml syringes (with 10 microliter gradations) with the prepared gentamicin solution and attach a 30G1/2 needle. Remove any air bubbles. Twist the needle on the syringe to make sure that the angled tip on the needle is facing away and the syringe markings are facing forward and readable. For control injections, prepare a syringe and needle with sterile PBS.
3. Prepare a scale with a clean surface or weigh boat as well as paper towels for drying fish and for holding fish for injection.
4. Prepare individual small half-liter containers with lids for holding fish for observation overnight post injection. Small transparent plastic mating cages are useful for this purpose. These containers should NOT be white - ideally they should be clear so that the white epithelial casts shed by the fish can be observed on a black bench top. Fill each container with enough fish water for the fish to swim comfortably.

Intraperitoneal injection of gentamicin

1. Anesthetize the fish in 0.016% tricaine solution in fish water. Wait for the gill ventilation rate to slow and for the fish to no longer respond to touch.
2. Scoop up the fish using the fish scoop, moving from head toward tail in order to avoid injuring the gills or fins. Place the fish on paper towels to absorb the excess water by turning the fish out gently on its side and shake off excess water from the scoop.
3. Scoop up the fish again and place it in the weigh boat on the zeroed scale and weigh the fish. Round to the nearest 0.25g, and calculate the appropriate amount of gentamicin to inject.

NOTE: For a 0.5g fish use a 20 microliter injection at 80mg/Kg dose.

1. Scoop up the fish again and place it on a dry folded paper towel. If injecting using the right hand, hold the paper towel in the left hand and place the fish’s head pointing left, with the belly easily accessible.

2. Hold the syringe with the needle at a 45 degree angle to the skin of the belly, anterior to the cloaca. Push the needle just under the skin, then decrease the angle and slide the needle forward under the skin, avoiding the internal organs. Depress the plunger the appropriate amount, pause to make sure no liquid is coming out around the needle, then withdraw the needle. If holding the fish steady is a problem, brace elbows against torso to stabilize them. CAUTION! Wear gloves when handling gentamicin solution.

3. Drop each fish into an individual container. Observe the fish and ensure its recovery from anesthesia. Keep fish at 28.5 degrees Celsius overnight. NOTE: If the fish doesn’t revive immediately, use a plastic transfer pipet to irrigate water across its gills to revive it.
4. The same syringe and needle may be reused when injecting multiple fish with the same dose of gentamicin. Dispose of the used syringe and needle into the appropriate biohazard sharps container.

Post injection observation of injury

1. The next day (1 day post injury), place the injected fish in its container on a dark surface. White casts of dead epithelial tissue excreted by the injured fish should be visible. (See Figure 1E-H). If there are no casts, either the fish was not injured by the gentamicin injection (either the dose was too low, or some of the gentamicin leaked during the injection process) or the fish was severely injured resulting in complete blockage of the ureters and cloaca with sloughed tissue.

NOTE: If the fish is unable to clear the casts from its body it will usually die within 2-3 days and is therefore unusable for longer assays. If no casts are visible, euthanize the fish in tricaine water by immersing for at least 10 minutes after gill movement stops.

1. If white tissue casts are visible, set the fish aside and continue checking the other fish. An appropriate dose of gentamicin will result in 80-90% of the fish being usable. Pool the injured fish and then split them into different treatment groups if desired.

Care of recovering fish

1. Keep fish in clean water and uncrowded environment. Dirty water and crowding will lead to infections and unintended death. Try to keep no more than 6 fish/500ml of fish water and change the water daily if possible. NOTE: This is a minimum volume for conservation of space or if the investigator wishes to perform experiments using expensive drug treatments.
2. Do not feed the fish until 3 days post injury. Recovering fish will not eat at first, and any food in the tank will decompose and promote bacteria growth. Starting at 3 days post injury, feed a small amount once a day.

Analysis of injured kidneys

1. For in situ hybridization for lhx1a mRNA expression, kidneys can be harvested from fish at desired timepoints. Euthanize fish in tricaine water on ice at least 10 minutes until gill movement stops, then remove the head with a razor blade and open the body cavity and remove the internal organs with forceps.
2. Leave the kidney in place (pigmented organ attached to the dorsal body wall) and fix the fish in 4% paraformaldehyde in PBS overnight. Dissect out the kidney and continue with standard in situ hybridization. Briefly, kidneys were washed in PBST (phosphate buffered saline 0.5%Tween 20), permeabilized with proteinase K in PBST (10ug/ml) for 1 hour at room temperature, postfixed with 4% paraformaldehyde, and washed again with PBST. Kidneys were then prehybridized overnight at 68 degrees Celsius in hybridization buffer (50% formamide, 5x SSC, 50ug/ml heparin, 500ug/ml tRNA, 0.1% Tween20, pH6.0). Samples were incubated overnight with digoxigenin labeled probe in hybridization buffer, then washed 5-10 minutes each with 100%, 75%, 50%, 25% hybridization buffer at 68 degrees Celsius, then moved to room temperature and washed twice for 30 minutes with 2x SSC with 0.1% Tween20, and washed twice for 30 minutes with 0.2x SSC with 0.1% Tween20. Equilibrated 3x 5min in MAB (0.1M maleic acid, 0.15M NaCl, pH 7.5) and blocked overnight at 4 degrees Celsius in MAB with 10% goat serum, incubated overnight in anti-digoxigenin-AP Fab (Roche) 1:5000 in MAB. Washed 5x 1hr in MAB, equilibrated 3x 15min in NTMT (0.1M Tris pH9.5, 0.05M MgCl2, 0.01M NaCl, 50ul Tween20). Kidneys were treated with NBT/BCIP to detect signal, fixed with 4% paraformaldehyde, incubated in dimethylformamide to remove excess NBT/BCIP, bleached overnight in H2O2, washed and photographed in PBST with 50% glycerol under a coverslip.

Ethics Statement: All experiments were conducted in accordance with Massachusetts General Hospital guidelines for animal use in research.

For further details and accompanying video please see:
Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury.

References

Kamei CN, Liu Y, Drummond IA. J Vis Exp. 2015 Aug 3;(102):e51912. doi: 10.3791/51912. PMID: 26275011

Consortium

(Re)Building a Kidney (RBK) Consortium