Name: nephrotoxin microinjection in zebrafish to model acute kidney injury
Uploaded: 2016-07-17T13:00:00.000+00:00
Duration: 7 min 58 s
Description: Watch this Scientific Journal Video about Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury at JoVE.com

This work was supported in part by the NIH grant DP2OD008470. Additionally, RAM was supported in part by funds provided by the University of Notre Dame Graduate School. We thank the staffs of the Department of Biological Sciences, the Center for Zebrafish Research, and the Center for Stem Cells and Regenerative Medicine at the University of Notre Dame. We especially thank the members of lab for engaging discussions about kidney biology and their helpful feedback on this work.

The procedures for working with zebrafish embryos described in this protocol were approved by the Institutional Animal Care and Use Committee at the University of Notre Dame.
1. Preparation of Solutions
<ol>
	<li>Make a 50x stock solution of E3 embryo media by mixing 73.0 g NaCl, 3.15 g KCl, 9.15 g CaCl2, and 9.95 g MgSO4 in 5 L of distilled water, and store at RT.</li>
	<li>For culturing of zebrafish embryos, dilute the 50x stock solution of E3 embryo media stock to a 1x working solution with distilled water, then add 200 &#956;l of 0.05% methylene blue to every 1 L of 1x E3 to act as a fungicide, and store at RT.</li>
	<li>Make a pigmentation blocking solution of E3 embryo media with 0.003% 1-phenyl-2-thiourea (PTU) by combining 40 ml of the 50x E3 stock with 0.06 mg PTU. Then bring the volume to a total of 2 L with distilled water.</li>
	<li>Stir the E3/PTU O/N at RT to get the PTU powder into solution. Then store the E3/PTU at RT. 
	Note: The E3/PTU has a shelf life of approximately one week, and older solutions will be less effective at blocking pigmentation development in zebrafish larvae.</li>
	<li>Make an anesthetic solution of 0.2% Tricaine (MS-222) by adding 1 g of Tricaine to 500 ml of distilled water and adjust the pH to 7.2 with 1 M Tris, pH 9.5.</li>
	<li>Prepare to make a 2% methylcellulose solution for imaging by placing a solution of 1x E3 (no methylene blue), with 1/10th volume of 0.2% Tricaine added at 4 &#176;C. 
	Note: The methylcellulose solution is a thick, clear solution that is used to gently stabilize live embryos for microscopy. After manipulations are performed the embryos can be washed in 1x E3 to dissolve the methylcellulose without injuring the animal and enabling microscopy at subsequent stages in the same specimen.</li>
	<li>When cooled to 4 &#176;C, stir the 1x E3/Tricaine solution vigorously on a stir plate and gradually add the appropriate amount of methylcellulose powder while continuing to stir vigorously. Gradually add the powder to the solution to prevent the formation of insoluble methylcellulose aggregates.</li>
	<li>After all the powder is mixed into the 2% methylcellulose/E3/tricaine solution, stir the composite solution in the 4 &#176;C cold room O/N. 
	Note: The cold temperature is best for fully dissolving the powder. If the solution is not clear after one night, stir for an additional workday and/or a second O/N.</li>
	<li>To remove air bubbles from the 2% methylcellulose/E3/tricaine solution, aliquot the mixture into 1.5 ml tubes and spin at high speed (12,000 x g) at 4 &#176;C for 30 min or up to 2 hr.</li>
	<li>Place the 2% methylcellulose/E3/Tricaine solution at 4 &#176;C for long-term storage. 
	Note: Prior to usage, aliquots should be pre-warmed to RT for working with the zebrafish specimens.</li>
</ol>
2. Preparation of Tools
<ol>
	<li>Prepare an embryo manipulator tool, used for positioning embryos for microinjection, by attaching a gel loading tip on the end of a 5&#34; straight dissecting needle, and secure with tape or superglue.</li>
	<li>Prepare microinjection needles using a needle puller, by first placing a fire polished 10 cm borosilicate glass with filament into the instrument and secure the borosilicate glass by tightening the handles.</li>
	<li>Pull the borosilicate glass to fashion fine-tapered needles. Use the following settings: heat 540, pull 245, velocity 200, and time 125.</li>
	<li>Place needles inside a Petri dish, suspending them on a strip of modeling clay to protect the needle tips, and keep the dish covered to prevent dust accumulation.</li>
	<li>To finish preparing the needle for microinjections, use a razor blade or fine forceps edge to cut the pulled end of the needle to create a sharp angular injection tip with a diameter of approximately 0.05-0.1 mm.</li>
	<li>To make the microinjection tray, prepare a 1.5% agarose/E3 solution in a 100 ml Erlenmeyer flask and dissolve the agarose by heating the E3 to a boil in a microwave then allow to cool at RT for approximately 10 min.</li>
	<li>Pour the cooled agarose/E3 solution into a Petri dish to make a foundation with a depth of approximately 0.5 cm. When this has solidified pour a second layer with a depth of approximately 0.3 cm and insert the prefabricated embryo well mold and allow the agarose to solidify at RT. 
	Note: When inserting the mold into the top agar/E3 layer, placing the mold into the agar at an angle to decrease the number of air bubbles.</li>
	<li>When the entire microinjection tray has set, lift the prefabricated embryo well mold out slowly and carefully using a scoopula, then fill the dish with E3 solution and store at 4 &#176;C.</li>
</ol>
3. Embryo Preparation
<ol>
	<li>Set up zebrafish mating tanks by placing dividers into mating chambers and fill with system water.</li>
	<li>Place one fish on each side of the divider such that each mating chamber contains one female and one male fish, and cover each mating chamber.</li>
	<li>The next morning, remove the dividers to enable spawning.</li>
	<li>Check the fish after approximately 30 min, and after returning the adults to the aquarium, collect their embryos by passing the mating tank water through a fine wire-mesh strainer tool.</li>
	<li>Invert the strainer over a Petri dish and rinse with E3 to collect the embryos.</li>
	<li>Incubate the embryos at 28.5 &#176;C O/N.</li>
	<li>When embryos have reached the 24-26 somite stage26, decant most of the E3 media and then dechorionate by adding 100 &#181;l of 50 mg/ml pronase to each dish of embryos and incubating the dish at RT (23 &#176;C) for approximately 15 min. 
	Note: It will take approximately 24-25 hr from the time of fertilization for the embryos to reach the 24-26 somite stage if they are collected in the manner described and incubated O/N at 28.5 &#176;C.</li>
	<li>Fill the dish with E3 and then swirl gently to dislodge the chorions from the embryos.</li>
	<li>Decant the E3/pronase and rinse the dish with 25 ml of fresh E3. Repeat rinse step to remove all pronase.</li>
	<li>To block the development of pigmentation, decant the E3 and replace with 25 ml of fresh E3/PTU when the embryos have reached 24 hr post fertilization. 
	Note: For experiments spanning several days, replace the E3/PTU solution with fresh media once daily to keep the dish clean.</li>
</ol>
4. Microinjection of Nephrotoxin Solution
<ol>
	<li>On the day of injection, prepare the desired nephrotoxin solution along with the appropriate vehicle control.</li>
	<li>Vortex or mix gently to insure the drug(s) is/are in solution, checking the sides of the tube and top of the water column. 
	Note: Nephrotoxin solutions can be prepared to contain trace amounts of fluorescently conjugated dextran to monitor microinjection efficiency and also assess renal clearance at subsequent time points20,28.</li>
	<li>Load a trimmed microinjection needle with ~2-3 &#181;l of the 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.</li>
	<li>Once the needle tip is full, secure the loaded needle in the micromanipulator.</li>
	<li>Test the microinjection volume by placing a drop of mineral oil onto a micrometer slide and inject into the oil to evaluate the droplet size. An injection volume of 500 pl has a diameter of 0.1 mm.</li>
	<li>Remove the dish of embryos from the incubator, and anesthetize by adding approximately 5 ml of 0.2% Tricaine to the embryo dish.</li>
	<li>To ensure complete anesthetization, gently touch embryo with the tip of the embryo manipulator tool. Lack of movement indicates sufficient anesthetization.</li>
	<li>After successful anesthetization, transfer embryos to the injection mold with a transfer pipette.</li>
	<li>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 up out of the depression.</li>
	<li>Insert the filled needle into the micromanipulator and position the needle tip next to an embryo.</li>
	<li>Gently insert the needle into the tail vessel by moving the joystick forward and depress the foot pedal to deliver the microinjection. 
	Note: Successful injection can be gauged by watching the liquid enter circulation. Further, co-injection of the nephrotoxicant with fluorescently conjugated dextran enables the researcher to verify successful injection subsequent to the procedure.</li>
	<li>Gently pull back on the joystick to remove the needle from the embryo.</li>
	<li>After injection, transfer the embryos to a clean dish and rinse to remove the Tricaine and replace with fresh E3/PTU.</li>
	<li>Incubate the embryo to the desired time point to assess morphology, which can be documented by photography in methylcellulose mounting media, or process for experimental analysis as desired. 
	Note: Recovery of embryos should be assessed to determine the percentage of individuals with edema, which commonly indicates renal injury.</li>
</ol>

The authors have nothing to disclose.

A diverse number of therapeutic agents have been associated with AKI29. There have been significant research advances in understanding the damage induced by many individual compounds, such as the aminoglycoside gentamicin30 and the widely used chemotherapeutic cisplatin31,32. Some pathological changes involved in these conditions, however, remain the subject of ongoing study. One emergent challenge remains understanding how multiple drugs adversely affect patients, especially those in high-risk populations such as those in critical care settings and the elderly29. Thus, models that enable further cellular based studies about the effects of drug-induced AKI serve an important role in improving detection of this condition and appreciating dynamic drug interactions.
Work with mammalian models like the mouse and rat have been integral to establish cellular changes in the kidney following AKI, but one limitation with these systems is that direct, real-time observation is limited due to the architectural complexity and internal location of the organ. Simply put, the kidney is not available for direct observation, which necessitates that individuals be sacrificed to analyze the kidney. These limitations can be countered by using zebrafish embryos, which form a simple kidney of two nephrons that have a conserved segment structure with other vertebrates, including humans33. Zebrafish development occurs ex utero and with minor pigmentation, allowing for direct observation of renal organogenesis and monitoring of responses to external cues34. Tools for molecular analysis, such as gene expression35-37, have been utilized with high resolution to determine the features of nephrogenesis38-40. Experimental manipulations for testing of small molecules through chemical genetics are well established in zebrafish and applicable to the kidney41-43. Indeed, in recent years, zebrafish have been applied to model nephrotoxicity of agents ranging from acetominophen to mycotoxins and aristolochic acid44-48. It is important to note that there are also significant limitations to AKI modeling in the zebrafish embryo, because of the very simplicity of renal structure at this stage in development. For example, zebrafish are not suited to addressing multifactorial interactions within renal tissue that is densely packed with nephrons that are surrounded by a complex stroma that contains multiple interstitial cell types, as is the case in the rodent or human metanephros. Thus, zebrafish are best suited to visualizing autonomous cellular and molecular changes in the nephron during AKI.
The method of microinjection described here, while having a steep learning curve, is very useful for studying both developmental and disease states including nephrotoxins. This technique can be used to test drugs singly or in combination. Additionally, injections can be performed during a wide range of development time points. A similar, equally viable method for performing intravenous microinjections in zebrafish larvae has been previously described by Cosentino, et al.21. One significant distinction in their methodology, compared to the methods described here, is that the embryo to be injected is immobilized utilizing a holding pipette21. The use of a holding pipette is a viable alternative to the injection mold. Researchers who seek to implement microinjection as a delivery method for nephrotoxicant agents should be aware of this alternative and may wish to compare the methods to identify which is personally preferable, as learning to manipulate the holding pipette so as not to damage the embryo will involve practice just as it requires practice to successfully maneuver and microinject using a simple injection mold with a depression cavity for the embryos.
When performing this procedure, it is critical to prepare appropriately sized injection needles, and to cut the angle on the needle tip to optimize smooth entry and exit into the embryonic circulation. During the injection procedure, it is critical to monitor the injection volume to assess consistency of the nephrotoxin delivery between samples. Periodic assessment of injection volume with a micrometer can be performed if the researcher is uncertain about changes in volume while performing an experiment. For example, due to the nature of the technique, the needle tip can partially or entirely clog with cellular debris. This complication can be counteracted by clearing the needle periodically to ensure consistency of the ejected fluid. Additionally, a vital dye like phenol red can be added to the mixture to act as a visual marker of the injection and assist in monitoring fluid dispersal49,50. Further, injections of tracer molecules can aid in visualizing specific cell populations, following the injection. For co-injection of fluorescent dextran moieties, specifically 10 kDa dextran conjugates, has a number of applications16,17. In this case, evaluation of fluorescent intensity can be performed immediately following the procedure to confirm successful microinjection with minimal leakage. The intensity can be measured using appropriate image capture photography and then reexamined at subsequent time points to measure the change in fluorescent intensity so as to measure renal clearance51. Further, reabsorption of the dextran in the proximal tubule provides a proxy for functionality of this nephron segment16,17.
Taken together, there are many ways that microinjection can be utilized to investigate AKI with the zebrafish, particularly as this model provides the opportunity to address pharmacokinetics in vivo. Thus, once mastered, microinjection of nephrotoxins into the zebrafish embryo provides a useful paradigm for renal studies.

AKI is an abrupt loss of kidney function that can lead to devastating health consequences1. AKI is a significant healthcare issue worldwide due to its high incidence of approximately 20% among hospitalized patients, with even higher rates of 30-50% in critical care cases and the elderly, and mortality rates of 50-70%1-3. Unfortunately, the prevalence of AKI has been increasing and is projected to escalate further over the next decade, due in part to the diversity of factors that can induce AKI, which include post-operative stress, ischemia, and exposure to nephrotoxins such as antibiotics and chemotherapeutic drugs4.
AKI involves sudden cellular damage within the kidney, commonly occurring in nephrons, which are the essential functional units, and are comprised of a blood filter and a segmented tubule that drains urine into central collecting ducts1. When a significant number of nephrons are damaged during AKI, the immediate effects include an interruption in waste clearance from the circulation, and reduced or abrogated fluid flow through nephrons due to obstruction from dead and dying cells1. Over time, tubular obstruction can lead to degeneration of entire nephrons, which permanently reduces renal function1. Physiological alterations in the kidney following AKI also involve complex inflammatory events that can lead to chronic scarring1.
Despite these outcomes, nephrons have some capacity to undergo regeneration after AKI that reconstitutes the tubular epithelium5,6. While there has been an increasing molecular understanding of nephron regeneration, the mechanisms remain elusive in many regards and necessitate continued investigation7. The degree to which AKI results in permanent renal damage also remains unknown. Current research suggests the regenerative potential for the kidney is the highest following less severe cases of AKI, while more pronounced or repeated episodes lead to chronic kidney disease (CKD) and culminate in end stage renal disease (ESRD) that requires life-saving transplantation or dialysis8,9. Additionally, individuals already suffering from CKD are at an even higher risk of contracting a severe episode of AKI8,9. Taken together, it is clear that continued basic and clinical research is vital to understand, treat and prevent AKI.
Research with animal models has been instrumental in appreciating the progression of local and environmental alterations that occur during AKI10. To expand this understanding as well as develop new therapies, the zebrafish animal model has been employed in a variety of ways11,12. The nephrons of the zebrafish kidney, in both the embryo and adult, display a high degree of conservation with mammals13-16. Further, nephron epithelial injury in zebrafish resembles the process in higher vertebrates, whereby the local destruction of tubular cells is followed by intratubular proliferation and reestablishment of nephron architecture17-19. In the embryo, however, extensive tubule damage from the nephrotoxins like cisplatin is associated with lethality20,21. By comparison, zebrafish adults survive AKI and exhibit substantive regenerative capabilities in the kidney. For example, following exposure to the aminoglycoside antibiotic gentamicin, zebrafish regenerate tubule epithelial damage and grow new nephron units as well22-24. While these gentamicin-induced AKI studies have provided invaluable information, understanding renal damage from diverse nephrotoxins remains critical to appreciate the effects and response to different types of damage25.
The zebrafish embryo, due to its size, transparency, and genetic tractability, has many benefits for nephrotoxin studies25, where the method of microinjection20,21 is used to administer the molecule(s) for investigation. Nephrons are formed by 24 hr post fertilization (hpf) and begin to filter blood by approximately 48 hpf26,27. Thus, the rapid formation and function of the embryonic kidney facilitates experimental analysis. However, the process of microinjection has technical challenges and there can be a steep learning curve to mastering the technique. In this video article, we describe how to perform microinjections and provide troubleshooting tips in order to enhance the rate of successful injections.

<table><tbody><tr><td>Sodium Chloride</td><td>American Bioanalytical</td><td>AB01915</td><td></td></tr><tr><td>Potassium Chloride</td><td>American Bioanalytical</td><td>AB01652</td><td></td></tr><tr><td>Calcium Chloride</td><td>American Bioanalytical</td><td>AB00366</td><td></td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Aldrich Chemistry</td><td>P7629</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate (Tricaine)</td><td>Fluka Analytical</td><td>A5040</td><td></td></tr><tr><td>Borosilicate glass</td><td>Sutter Instruments Co.</td><td>BF100-50-10</td><td></td></tr><tr><td>Flaming/Brown Micropipette puller</td><td>Sutter Instruments Co.</td><td>Mo. P097</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>15510-027</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methylene Blue</td><td>Sigma-Aldrich</td><td>M9140</td><td></td></tr><tr><td>Falcon Diposable Petri Dishes, Sterile, Corning:</td><td></td><td></td><td></td></tr><tr><td>60 mm x 15 mm</td><td>VWR</td><td>25373-085</td><td></td></tr><tr><td>100 mm x 15 mm</td><td>VWR</td><td>25373-100</td><td></td></tr><tr><td>&amp;nbsp;(microinjection tray) 150 mm x 15 mm</td><td>VWR</td><td>25373-187</td><td></td></tr><tr><td>Low Temperature Incubator</td><td>Fischer Scientific</td><td>11 690 516DQ</td><td></td></tr><tr><td>Micro Dissecting Tweezer</td><td>Roboz Surgical Instruments Co.</td><td>RS-5010</td><td></td></tr><tr><td>Micrometer</td><td>Ted Pella, Inc.</td><td>2280-24</td><td></td></tr></tbody></table>

nephrotoxin microinjection in zebrafish to model acute kidney injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

A microinjection station set up includes a stereomicroscope, micromanipulator and pressure regulator (Figure 1A). Transillumination of the injection plate is preferable to view specimens during this procedure (Figure 1B). Preparation of the injection needle involves pulling the appropriate borosilicate glass, followed by preparing the edge with cutting and finally back-loading the needle. Optimally, the needle tip is beveled rather than blunt (Figure 1C), a cut that is made by sharply angling the edge of the cutting instrument, such as the fine forceps, during the cutting procedure (Figure 1D). This beveled edge is critical and will greatly affect the ability to gently insert the needle into the zebrafish.
On the day of injection, embryos were positioned using sturdy but pliable embryo manipulation tools (Figure 1E). For the injection, embryos were maneuvered, such that the torso rested along the side of the injection tray to provide leverage for the subsequent injection step (Figure 1F). While injecting, one should focus the plane of view on the embryonic torso to visualize the vascular destination for needle insertion and then observe the injected material enter the circulation (Figure 1G).
The transparency of the zebrafish embryo facilitates the microinjection 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 (Figure 2A). Here, embryo specimens were allowed to develop through the 72 hpf stage, then were either mock injected, microinjected with saline vehicle, or microinjected with 2.5 mg/ml gentamicin (Figure 2A). Subsequent live observation was conducted at one and two days post injection, using transillumination lighting with a stereomicroscope (Figure 2B). Compared to mock or saline injected embryos, only the gentamicin-injected individuals showed the development of edema (Figure 2B), in this case pericardial edema, suggesting an abrogation of normal kidney functionality consistent with previously published observations11,20,21.
<img alt="Figure 1" src="/files/ftp_upload/54241/54241fig1.jpg" /> 
Figure 1.&#160;Microinjection apparatus and tools. (A) Injection station set up with stereomicroscope (left), micromanipulator and needle holder (middle) and pressure regulator (right). (B) Transillumination of the injection plate. (C) Left: Blunt cut needle. Right: Beveled cut needle. (D) Fine forceps used to cut needle tips. (E) Manipulation tools used to insert and position the specimens into the injection mold. (F) Embryos arrayed in the microinjection mold. Scale bar = 0.5 mm. (G) Positioning of needle next to the embryo for injection. Scale bar = 0.15 mm. <a href="https://www.jove.com/files/ftp_upload/54241/54241fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54241/54241fig2.jpg" /> 
Figure 2.&#160;Experimental timeline and post injection edema assay. (A) Example nephrotoxin experimental timeline in zebrafish, here applied to a gentamicin study. Embryos were dechorionated and treated with PTU solution at 24 hr post fertilization (hpf). Each day, the PTU solution was decanted and replaced with fresh media as indicated by the smaller green arrows. At 72 hpf, embryos were anesthetized, transferred to an injection mold and then treated as follows: mock injected or injected either saline (vehicle control) or gentamicin. After reviving the embryos in fresh media, the embryos were observed at 1 and 2 days post injection (96 and 120 hpf, respectively) for analysis and image acquisition. (B) Top: uninjected zebrafish embryo. Middle: embryo injected at 72 hpf with saline. Bottom: embryo injected with 2.5 mg/ml gentamicin. Scale bar = 0.5 mm. <a href="https://www.jove.com/files/ftp_upload/54241/54241fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury at JoVE.com

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

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 kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

nephrotoxin-microinjection-in-zebrafish-to-model-acute-kidney-injury

Nanyang Technological University

Research

JoVE Journal

Biology

8.8K Views.  University of Notre Dame. 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.

Video: Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

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.

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 ...

Developmental Biology

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

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 ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

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 ...

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 ...

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 ...

Medicine

Intravenous Microinjection of Nephrotoxins into Zebrafish Embryo: A Technique to Deliver Nephrotoxic Agents into the Bloodstream of Zebrafish Embryo

Source: McKee, Robert A. et al. <a href="https://www.jove.com/video/54241" target="_blank">Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury.</a> J. Vis. Exp.&#160;(2016)
This video demonstrates the intravenous microinjection of nephrotoxins into the tail vein of a zebrafish embryo to study their chemical effects on renal function.

Source: McKee, Robert A. et al. Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury. J. Vis. Exp.&#160;(2016)
This video demonstrates the ...