Name: kidney regeneration in adult zebrafish by gentamicin induced injury
Uploaded: 2015-08-03T14:30:00.000+00:00
Duration: 6 min 25 s
Description: Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

NOTE: Ethics Statement: All experiments were conducted in accordance with Massachusetts General Hospital guidelines for animal use in research.
1. Advance Preparation
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>Prepare gentamicin solution. Gentamicin stock solution may be stored in -20 &#176;C and thawed for later use. For example, for an 80 mg/kg dose (80 mg gentamicin/kg of bodyweight) in a fish weighing 0.5 g, prepare 2 mg/ml of gentamicin in phosphate buffered saline as a convenient working solution. This provides a 20 &#181;l of gentamicin for intraperitoneal injection into the fish. 
	NOTE: In general, 80-120 mg/kg achieve good results, but doses as low as 40 mg/kg may be appropriate. Gentamicin may also be purchased in solution to minimize hazardous exposure to lab personnel. 
	NOTE: Gentamicin in high doses can be toxic. Wear gloves and mask when weighing powder.</li>
	<li>Prepare 100 ml of 0.016% tricaine water for anesthetizing fish. Prepare a 25x stock of tricaine (4 g/1 L) dissolved in sterile milliQ water adjust to pH 7 and store at 4 &#176;C until use. Dilute 4 ml in 100 ml of fish water for a working concentration of 0.016%. 
	NOTE: Tricaine is an anesthetic and skin irritant, wear gloves when handling.</li>
	<li>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 &#181;l gradations) with the prepared gentamicin solution and attach a 30&#189; G 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.</li>
	<li>Prepare a scale with a clean surface or weigh boat as well as paper towels for drying fish and for holding fish for injection.</li>
	<li>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.</li>
</ol>
2. Intraperitoneal Injection of Gentamicin
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Scoop up the fish again and place it in the weigh boat on the zeroed scale and weigh the fish. Round to the nearest 0.25 g, and calculate the appropriate amount of gentamicin to inject. 
	NOTE: For a 0.5 g fish use a 20 &#181;l injection at 80 mg/kg dose.</li>
	<li>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&#8217;s head pointing left, with the belly easily accessible.</li>
	<li>Hold the syringe with the needle at a 45&#176; 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. 
	NOTE: Wear gloves when handling gentamicin solution.</li>
	<li>Drop each fish into an individual container. Observe the fish and ensure its recovery from anesthesia. Keep fish at 28.5 &#176;C overnight. 
	NOTE: If the fish doesn&#8217;t revive immediately, use a plastic transfer pipet to irrigate water across its gills to revive it.</li>
	<li>Use the same syringe and needle when injecting multiple fish with the same dose of gentamicin. Dispose of the used syringe and needle into the appropriate biohazard sharps container.</li>
</ol>
3. Post Injection Observation of Injury
<ol>
	<li>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 min after gill movement stops.</li>
	<li>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.</li>
</ol>
4. Care of Recovering Fish
<ol>
	<li>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/500 ml 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.</li>
	<li>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.</li>
</ol>
5. Analysis of Injured Kidneys
<ol>
	<li>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 min until gill movement stops, then remove the head with a razor blade and open the body cavity and remove the internal organs with forceps.</li>
	<li>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 kidney15 and continue with standard in situ hybridization.
	<ol>
		<li>Briefly, wash the kidneys in PBST (phosphate buffered saline 0.5%Tween 20), permeabilized with proteinase K in PBST (10 &#181;g/ml) for 1 hr at room temperature. Post-fix with 4% paraformaldehyde, and washed again with PBST.</li>
		<li>Then pre-hybridize the kidneys overnight at 68&#160;&#176;C&#160; in hybridization buffer (50% formamide, 5x SSC, 50 &#181;g/ml heparin, 500 &#181;g/ml tRNA, 0.1% Tween20, pH 6.0).</li>
		<li>Incubate the samples with digoxigenin labeled probe in hybridization buffer, then wash for 5-10 min each with 100%, 75%, 50%, 25% hybridization buffer at 68&#160;&#176;C, then moved to room temperature and wash twice for 30 min with 2x SSC with 0.1% Tween20, and wash twice for 30 min with 0.2x SSC with 0.1% Tween20.</li>
		<li>Equilibrate the sample 3x 5 min in MAB (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) and blocked overnight at 4 &#176;C in MAB with 10% goat serum. Then incubate samples overnight in anti-digoxigenin-AP Fab 1:5,000 in MAB.</li>
		<li>Wash the sample 5x for 1 hr in MAB, and equilibrate 3x for 15 min in NTMT (0.1 M Tris pH9.5, 0.05 M MgCl2, 0.01 M NaCl, 50 &#181;l Tween20).</li>
		<li>Then treat the kidneys with NBT/BCIP to detect signal. Fix the kidneys with 4% paraformaldehyde and incubated in dimethylformamide to remove excess NBT/BCIP, bleached overnight in deionized water. Wash the kidneys and then photograph in PBST with 50% glycerol under a coverslip.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests to disclose.

The zebrafish is ideal for studying regeneration of adult organs including the adult mesonephric kidney6. Recent studies have taken advantage of molecular markers and new transgenic reporter lines to better characterize the steps that occur during nephron regeneration and what cells might be responsible7,12. Observation of urinary casts has been used for over a century to diagnose kidney disease in humans16. Here we use the presence of casts as an easy, noninvasive and early indicator of the extent of acute kidney injury after gentamicin injection. We observe that fish that do not produce casts are either only mildly injured (and do not form new nephrons) or too injured to recover and usually die within the first 3 days. Therefore, a critical step within the protocol is the individual observation of fish after injury, and the presence of urinary casts as an indicator of proper injury levels. We also see lhx1a expression in aggregates starting at day 4 after injury. This indicates that the first 3 days after injury are a crucial period for recovery.
If a large number of fish are uninjured, or too many fish are dying during the course of the experiment, check to make sure that the intraperitoneal injections are being done properly. It may help to add a nontoxic dye such as phenol red to the gentamicin solution to visualize any leakage during injection. Marking the needle with a permanent marker or a piece of tape 2 mm from the tip can serve as a guide to prevent pushing the needle too far into the fish. Additionally, the dosage of gentamicin may need to be adjusted. Inject fish with a range of gentamicin doses and test the kidneys for expression of lhx1a mRNA either by in situ hybridization or quantitative RT-PCR.
A limitation of this technique is that it requires housing individual fish overnight before selecting injured fish for experimentation. To investigate very early events in regeneration (within the first 24 hr after injury) fish may need to undergo continuous experimental treatment, even during the individual observation period. The cost of treating individual fish with drugs, for example, may be very high. Our technique is an improvement on the existing method of acute kidney injury using gentamicin, because it allows the researcher to eliminate outliers by utilizing a noninvasive visual screening process. This robust, uniform and reproducible injury model can now be used to effectively test hypotheses about kidney regeneration in the adult zebrafish.
We observed that although regeneration occurs in both male and female zebrafish (all data shown here are from females), regeneration in response to gentamicin injection in males is much more variable. This may be due to technical reasons, because their bodies are long, thin and more muscular, while females have a much larger belly with looser, thinner skin, allowing easier injection and a larger peritoneal cavity to accommodate injection volumes without leakage. An alternative possibility is that androgens produced by males may have some effect on the regeneration process17. Nachtrab et al. found that the effect of androgens was strain dependent, which indicates that genetic background might modulate androgen levels. Slight variations in genetic background of the TuAB fish used in our study might explain the variability seen in males, but presumably androgens are not the only source of variation and other genetic loci may contribute to heterogeneity in the response to injury. We have also found that different transgenic lines of zebrafish may respond differently to gentamicin injury. For example, the Tg(Lhx1a:GFP)12 line in our hands requires 120 mg/kg for efficient induction of injury (data not shown). These observations highlight the importance of using same-sex sibling controls in injury studies to minimize experimental variability.
Many zebrafish transgenic lines use a heat shock sensitive promoter as a means to induce expression of a transgene. Since zebrafish are poikilothermic and do not regulate their body temperature, they are forced to perform physiological functions at the temperature of their environment. In the context of experiments using the hsp70 promoter to express dominant transgenes to modulate the regeneration response, we unexpectedly observed that the regeneration response was accelerated by higher temperature. Therefore, determining the effect of temperature on the regeneration response in a given system becomes crucial to planning the timing of experiments (capturing the peak of the regeneration response for example) and makes comparison to regeneration at normal temperatures problematic. Other inducible systems using for instance CreERT and tamoxifen for transgene induction may be better suited to studies of adult kidney regeneration. Similarly, newer genetic methods of injury induction such as CreERT activation of toxic transgenes or inducible injury using bacterial nitroreductase/metronidazole18,19 will broaden the range of possible experiments to better understand neonephrogenesis and kidney regeneration.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. Although mammals are able to repair existing nephrons after injury using differentiated epithelial cells1-4, they seem to lack a pool of reserved stem cells5 and are unable to form new nephrons de novo. In contrast to mammals, adult fish are able to form new nephrons throughout adult life to support the growth of the fish and in response to injury6,7. The zebrafish, Danio rerio, is an invaluable model organism for the study of organ regeneration8-10 and has the potential to provide powerful insights into applications for engineering the repair of human kidneys. The Tg(Lhx1a:EGFP) transgene11 has been shown to label a pool of nephron progenitor cells in the adult zebrafish kidney12, however mechanisms controlling the response of lhx1a+ cells to injury remain unclear.
The aminoglycoside gentamicin is a widely used antibiotic with known nephrotoxic and ototoxic effects in humans13. Intraperitoneal injection of gentamicin is an established method of inducing acute kidney injury in fish6. This injury in fish mimics the loss of tubular epithelium and scarring of glomeruli that occurs in humans after gentamicin overdose14. Inducing injury in zebrafish by gentamicin injection is a convenient way of inducing a strong, synchronous regeneration response, with many new nephrons produced and simultaneously proceeding through stages of formation, proliferation and differentiation.
This protocol details our method for robust and reproducible injury in the adult zebrafish kidney by utilizing a noninvasive visual screening process to minimize outliers. We take advantage of the fact that injury with gentamicin leads to death of epithelial kidney tissue and formation of renal tubular casts, which then accumulate into masses in the mesonephric ducts and cloaca. These are passed by the fish and can be observed visually in the water. This allows us to screen for fish with strong nonlethal injury, which can then be pooled for further experimentation. Minimizing the numbers of uninjured fish or fish that die before they reach the endpoint of the experiment leads to more uniform and efficient data collection and analysis. In addition, no special devices or reagents are required, making this method cost-effective and appropriate for use in an academic or teaching setting. Using our method, here we show the increasing effects of gentamicin dose on kidney regeneration as well as the effect of increased temperature.

<table><tbody><tr><td>gentamicin sodium sulfate</td><td>Sigma</td><td>G1264</td><td>TOXIC, purity varies from batch to batch</td></tr><tr><td>plastic transfer pipets</td><td>Fisher</td><td>13-711-7M</td><td></td></tr><tr><td>1 ml Norm-Ject syringes</td><td>Electron Microscopy Sciences</td><td>72520</td><td>green plastic syringes, ordinary 1 ml syringes are OK, but harder to read accurately</td></tr><tr><td>30 G1/2 needles</td><td>Becton Dickinson</td><td>305106</td><td></td></tr><tr><td>ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td><td>Sigma</td><td>A5040</td><td>IRRITANT</td></tr><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Make 4% in 1x PBS for working solution</td></tr></tbody></table>

kidney regeneration in adult zebrafish by gentamicin induced injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Gentamicin injury can be confirmed visually by the observation of renal epithelial casts in the water (Figure 1). Different dosages of gentamicin were used to injure adult wildtype TuAB zebrafish, resulting in increasing numbers of lhx1a+ cellular aggregates in the regenerating kidney (Figure 1A-D). White casts can easily be seen in the water 1 day after injury (Figure 1E-H). A low dose of gentamicin resulted in most fish having no casts, while higher doses resulted in over 75% of fish having casts (Figure 1I). In all groups, selecting for fish with casts resulted in a higher average injury level (Figure 1J).
After visual confirmation of injury, fish can be pooled and then split into control and treatment groups for experiments (Figure 2). Wildtype TuAB adult zebrafish were injured with an 80 mg/kg dose of gentamicin. One day after injury, fish with casts were selected, pooled and then split into heat shock and no heat shock groups. Heat shock fish were exposed to 37 &#176;C for 16 hr overnight and an 8 hr rest period at 28.5&#160;&#176;C each day. At 4 days and 7 days post injury fish were fixed for whole mount in situ hybridization and injury level was assessed by density of lhx1a+ aggregates. Heat shock alone does not produce an injury response (Figure 2A, D), but heat shock accelerates the response in injured fish compared to no heat shock controls (Figure 2B, C, G). By 7 days post injury there was no longer a significant difference between injured groups (Figure E, F, H).
<img alt="Figure 1" src="/files/ftp_upload/51912/51912fig1highres.jpg" /> 
Figure 1: Regeneration in response to acute kidney injury by gentamicin is dose dependent. (A-D) lhx1a mRNA expression marks cellular aggregates, which are new nephron progenitors. (A) 7 days after control i.p. injection, wildtype TuAB adult zebrafish normally have 0-2 lhx1a+ cellular aggregates in the whole kidney. (B-D) 7 days after gentamicin injection fish display many lhx1a+ cellular aggregates, indicating robust neonephrogenesis.This response is dose dependent, with higher concentrations of gentamicin resulting in more lhx1a+ clusters. (E-H) 1 day after injection of gentamicin, fish excrete white casts consisting of dead epithelial tissue from the kidneys. (E) A collection of casts from a total of 12 fish showing the variety of shapes and sizes. Note the difference between white casts indicating injury compared to reddish-orange digested brine shrimp. (F-H) Examples of casts collected from individual fish 1 day after injury by allowing the water in the tank to settle, pipetting the sediment out and observing under a microscope. (F) Excrement with no casts, indicating no injury from a fish treated with the 40 mg/kg dose. (G) Medium sized casts indicating injury from a fish treated with the 60 mg/kg dose. (H) Larger casts with additional sloughed tissue indicating injury from a fish treated with the 80 mg/kg dose. (I) The percentage of injured fish with casts is increased at 60 mg/kg and 80 mg/kg. (J) After 7 days, injected fish were processed for whole mount in situ hybridization to assess injury. lhx1a+ aggregates were counted in a single 5x picture taken at the widest point of each kidney and kidney area was measured using ImageJ. Average number of aggregates/mm2 is shown for each dose. In each case, fish with casts had a higher average than the total group at each dose. p &#60;0.5 for 40 mg/kg compared to 80 mg/kg. Scale bar in A-D = 0.2 mm, E = 1 mm, F-H = 2mm.
<img alt="Figure 2" src="/files/ftp_upload/51912/51912fig2highres.jpg" /> 
Figure 2: Temperature dependent regeneration. 24 hr after gentamicin injection, injured wildtype TuAB adult zebrafish with casts were pooled and then split into a heat shock group and a no heat shock group. Fish were maintained at 37 &#176;C for 16 hr overnight each night afterward with an 8 hr resting period at 28.5&#160;&#176;C during each day. (A, D) Control uninjured fish did not express lhx1a after heat shock. (B, C) Heat shock accelerates the regeneration response, with more clusters already appearing at 4 days after injury compared to injured fish without heat shock (p &#60;0.5, Student&#8217;s t-test). (E, F) After 7 days, there is no significant difference between injured fish treated with heat shock and no heat shock. (G, H) Quantification of injury by counting lhx1a+ aggregates per mm2. Each point represents an individual fish. Number of fish and average response are indicated above each group. Scale bar = 0.2 mm.

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.8K Views.  Massachusetts General Hospital. Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. 

Video: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Neuroscience

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Biology

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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