We would like to thank Dr. Iain Drummond and Dr. Vladimir Korzh for sharing kidney GFP transgenic lines. We would also like to thank NYITCOM for providing necessary resources to conduct this work. This study was in part supported by grants: K08DK082782, R03DK097443 (NIH), and the HSCI Pilot Grant (AV).

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the NYIT College of Osteopathic Medicine Institutional Animal Care and Use Committee (NYITCOM IACUC). All surgery and in vivo experimentation was performed under tricaine anesthesia, and all efforts were made to minimize suffering.
1. Obtaining and Maintaining Embryos
<ol>
	<li>Obtain embryos by crossing kidney GFP fluorescent zebrafish. 
	NOTE: Examples of fluorescently labeled zebrafish transgenic lines are listed in Table 1.</li>
	<li>Keep the embryos at 28.5 &#176;C in E3 solution during the 24 h post-fertilization.</li>
	<li>After 24 h, replace the medium with an E3 solution that contains 0.003% 1-phenyl-2-thiourea (PTU). 
	NOTE: PTU is used because it blocks the tyrosinase-dependent steps in melanogenesis and hence prevents pigmentation. Here, this solution is referred to as E3-PTU.</li>
	<li>Raise the fertilized embryos at 28.5 &#176;C until they are &#62;6 dpf, at which point the kidneys have matured. 
	NOTE: It is best to use larvae in the 7-9 dpf window.</li>
	<li>Using a fluorescent dissecting microscope at 40X magnification and green fluorescence emission filters, select the larvae with bright kidney GFP fluorescence.</li>
</ol>
2. Mounting the Zebrafish for Live-imaging
<ol>
	<li>If photoablating the embryos prior to 3 dpf, remove the chorion from any unhatched zebrafish before mounting the zebrafish for imaging.
	<ol>
		<li>Manually dechorinate with forceps (preferred) or use a chemical treatment with a protease mixture. 
		NOTE: Dechorination allows for the best imaging results.</li>
	</ol>
	</li>
	<li>Rinse out the chorion debris in the petri dish using E3-PTU solution and refill the dish with E3-PTU.</li>
	<li>Prepare 1-2% Low-Melting Point (LMP) agarose with 0.2 mg/mL tricaine solution to mount the zebrafish for kidney photoablation and live imaging.
	<ol>
		<li>If LMP agarose was already prepared beforehand, reheat it in the microwave to melt it.</li>
	</ol>
	</li>
	<li>Once the LMP agarose is prepared, place a 35 mm plastic petri dish on a dissecting microscope. Place a pulled glass probe nearby to properly orient the anesthetized larvae. 
	NOTE: It is important to do this step in a timely fashion and to have everything in place, because the embryos must be oriented before the agarose solidifies in about 1-3 min.</li>
	<li>Before transferring the embryo to the agarose, make sure that the agarose is cool (i.e.&#160;at about body temperature). Use a plastic or glass transfer pipette to place the embryo into the cooled-down melted agarose-tricaine solution. 
	NOTE: This is done because agarose that is too hot can be detrimental to the embryo, leading to cardiac arrest or death</li>
	<li>Using a transfer pipette, redraw the embryo in the agarose solution and position the embryo/larva in the middle of a 35 mm dish.</li>
	<li>Quickly spread the agarose containing the embryo/larva to evenly cover the bottom of the 35 mm dish; the best volume of agarose to use is ~1-1.1 mL.</li>
	<li>Use the glass probe to orient the embryo in the best possible position for photoablation and imaging. 
	NOTE: Movie 1 shows the best orientation of the embryo/larva for unilateral photoablation. This step is the most time-sensitive and should be performed quickly before the agarose starts to gel, as any attempt to reorient the fish after gelling begins is detrimental.
	<ol>
		<li>Orient the larva so that the kidney segment of interest is perpendicular to the beam path while the contralateral segment is away from the laser beam (see Movie 1). 
		NOTE: This can be achieved by turning the larva as shown in Movie 1.</li>
	</ol>
	</li>
	<li>Allow about 15 min for the agarose to solidify. Cover the petri dish to minimize evaporation. Once the agarose has solidified, the embryo or larva is ready for photoablation.</li>
</ol>
3. Laser Ablation
<ol>
	<li>Operate the confocal imaging system. 
	NOTE: When using the referenced imaging platform (see the table of materials), the recommended settings include 40X magnification, a 0.8 NA water dipping lens, and the first dichroic mirror set to allow both 488&#160;&#38; 405 nm illumination of the sample. The detection should be performed on the green channel.</li>
	<li>Position the dish containing the immobilized zebrafish on a confocal stage. 
	NOTE: This procedure is optimized for the upright configuration using a 40-60X water dipping lens. However, it should be possible to modify the procedure for an inverted microscope.</li>
	<li>In the upright configuration, position the petri dish containing the embryo on top of a microscope slide and keep it in place using modeling clay (e.g., plasticine). Position the glass slide with the petri dish onto the stage of the confocal microscope.</li>
	<li>Add 3 mL of the imaging solution E3-PTU (see step 1.3) with 0.2 mg/mL tricaine. 
	NOTE: The imaging solution can also be added immediately before placing the slide on the stage. The addition of imaging solution should be done very carefully to prevent the agarose from floating off the bottom of the dish.</li>
	<li>Switch to the water dipping lens (40&#160;or 60X). Slowly raise the stage, making sure that the lens is slightly angled to prevent air from becoming trapped in the beam path. Once the lens touches the solution at an angle, center it so that it is in the field of view of the embryos.</li>
	<li>Find the segment of interest using the fluorescence light source. Superficially position the branch targeted for injury to minimize light scattering (see Movie 1). 
	NOTE: The subsequent steps make use of referenced software (see the Table of Materials), but the procedure can be easily adapted to other platforms.</li>
	<li>Open the software. Navigate to &#34;View&#34;| &#34;Acquisition Control&#34;| &#34;C2plus Compact GUI&#34; and set &#34;pixel dwell time&#34; to &#34;1.9 &#181;s,&#34; &#34;frame size&#34; to &#34;512 x 512 pixels,&#34; and &#34;pinhole size&#34; to &#34;90 &#181;m.&#34; Set &#34;405 nm (or 408 nm in some systems) laser intensity&#34; to zero and &#34;488 laser intensity&#34; to &#34;low&#34; (preferably &#60;1%). Adjust the &#34;gain&#34; to obtain a good dynamic range but not to saturate the signal. 
	NOTE: These values are not critical and are used for consistency. When a 405 nm laser is used, it will result in increases in GFP fluorescence. To avoid saturation of the signal during photobleaching (step 3.11), the signal gain value should be scaled down on the green channel. The blue channel gain can be left at zero, as it is not used for sampling.</li>
	<li>Click &#34;Scan&#34; in the software interface to scan the kidney using a 488 nm laser. Find the segment of interest that is perpendicular to the beam path and that is well-visualized, with good GFP fluorescence. Once that region has been identified, draw a region of interest using an elliptical Region-of-Interest (ROI).
	<ol>
		<li>Navigate to &#34;ROI&#34; | &#34;Draw Elliptic ROI.&#34; 
		NOTE: This will be used to continuously measure average GFP intensity while the photobleaching takes place, permitting the administration of a precise dose of laser treatment.</li>
	</ol>
	</li>
	<li>Navigate to &#34;View&#34; | &#34;Acquisition controls&#34;| &#34;C2plus Scan Area&#34; and choose &#34;Band Scan Area.&#34; The rectangular mask will appear within that interface. Manually define a rectangular scan window using the cursor (i.e.&#160;drag, resize, and turn the mask) to cover the segment targeted for laser ablation. Right-click within the mask area to accept the selection. 
	NOTE: Only the selected region will be scanned.</li>
	<li>Begin the time measurement while monitoring the green channel using just the 488 nm laser to activate GFP. Ensure that the intensity of the 488 laser is relatively low (~1%). Measure the average intensity of the GFP in the region of interest by selecting &#34;Measure&#34; | &#34;Time Measurement.&#34; Use the &#34;Graph&#34; or &#34;Data&#34; tabs to monitor the average intensity values within the ROI. 
	NOTE: The rate of acquisition is defined by pixel dwell time and the size of the rectangular window set in step 3.9, but in general, it allows for the fast monitoring of the signal (~2-10 frames/s).</li>
	<li>Induce damage to the kidney cells with the violet laser (405 nm) by increasing the intensity to 100% by sliding the &#34;laser power&#34; control all the way to the right. The 488-nm laser can remain active if the intensity is low.
	<ol>
		<li>Observe a line graph using the &#34;Graph&#34; tab or the numerical values of GFP intensity using the &#34;Data&#34; tab and wait until it drops to a desired level, for example 50% of baseline (as shown in Figure 1). 
		NOTE: A 20 mW laser has been used as part of the referenced laser unit (see the Table of Materials); maximal intensity may vary between systems. Lower intensity can be compensated for by longer exposure (i.e.&#160;using percent GFP bleaching as a measure of total exposure).</li>
	</ol>
	</li>
	<li>Once the GFP intensity within the elliptical ROI (step 3.8) has dropped to a desired level, immediately decrease the intensity of the violet (405 nm) laser to &#34;0&#34; by moving the &#34;laser power&#34; slider all the way to the left in the software control panel.
	<ol>
		<li>Obtain a new baseline of GFP fluorescence. Confirm the ablation by obtaining a ratio of X/Y. 
		NOTE: See Figure 1B; Y = average intensity before the ablation and X = average intensity after the ablation, using an average of 4 consecutive samples within the ROI. The exact target ratio (50%, 60%, etc.) depends on the amount of injury that is desired for a given experiment.</li>
	</ol>
	</li>
</ol>
4. Time-lapse Microscopy with Propidium Iodide Staining
<ol>
	<li>Apply general time lapse considerations (outlined in a previous study15) to visualize propidium iodide staining as a correlate of kidney cell injury after photoablation.</li>
	<li>Pre-incubate the larvae in 30 &#181;M propidium iodide solution (1% DMSO in E3-PTU) for 3 h prior to embedding and photoablation, as described in steps 2 and 3.7, respectively. 
	NOTE: No additional propidium iodide is required in the agarose or the imaging solution.</li>
	<li>Induce segmental kidney injury, as described in steps 3.1-3.12.</li>
	<li>Obtain time lapse image stacks, as described in a previous study14, using a 488 nm laser (GFP) and a 461-nm laser (Propidium Iodide, PI). 
	NOTE: The two colors are superimposed in confocal stacks to correlate the disappearance of GFP and the appearance of propidium iodide staining.</li>
	<li>Set the parameters for obtaining the time-lapse image stacks as follows: virtual slice thickness = 4 &#181;m, z-stack interval = 2 &#181;m, pixel dwell time = 1.9 &#181;s, image dimensions = 512 x 512, and averaging = 2. 
	NOTE: These parameters allow for continuous, 10 &#160;to 15 min interval recording of up to 4 larvae and ensure that there is minimal photobleaching of the sample.</li>
	<li>Use imaging software compatible with the recording file format to visualize and analyze the data.</li>
</ol>

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The authors declare that they have no competing financial interests.

It should be noted that the total laser power varies between systems. However, using percent GFP photobleaching allows for a readout of total energy delivered to the fluorescent kidney, independent of the variation in laser power and compensated for by the length of exposure. Keep in mind, however, that the response of different tissues to this method of photoablation varies. Even during kidney maturation, it is significantly more difficult to obtain a 50% reduction in GFP fluorescence in younger embryos than in mature l...

Acute Kidney Injury (AKI)1,2, which also can be referred to as acute renal failure, is broadly defined as a sudden impairment in kidney function3. While the level of understanding of this condition has been enhanced remarkably over the years, morbidity and mortality rates have remained high1,2. The current treatment for this condition is mostly supportive, as results from multiple clinical trials of drug therapy have been negative4,5. The kidney is unique in that it has the ability to repair itself. Therefore, supportive therapy after an early diagnosis of AKI is the best way to limit morbidity6. However, it is difficult to detect AKI early, and the mortality rate is a staggering 50-80% for those who require dialysis5. With the ability of the kidneys to repair themselves and the lack of treatment options for this condition, it is important to develop methods to enhance this nephron regeneration process.
There have been many different models used for AKI research that includes different agents of injury and animal models. In terms of agents of kidney damage, the aminoglycoside antibiotic gentamicin has been used as a nephrotoxic agent that leads to AKI7,8. However, several groups have found that gentamicin treatment is lethal to the zebrafish embryo9. It causes tubular damage that is too serious for embryo recovery, making the study of regeneration difficult without some type of intervention. Mammalian models, like the mouse and rat, are also considered valuable, but they face many limitations during the study of AKI. Perhaps the main disadvantage of rodent models is the difficulty in visualizing the rodent kidney and thus determining the precise spatiotemporal processes leading to epithelial death and repair.
Johnson et al. have reported a laser ablation-based technique to induce acute kidney injury in embryonic and larval zebrafish9. They used pulsed laser ablation to damage the kidney after an intramuscular injection with dextran conjugates. The fluorescence from dextran conjugates allows for the visualization of damage and regeneration in the tubule epithelium9. This model overcomes the two limitations mentioned above, but it does not allow for graded levels of injury and is difficult to carry out on large, arbitrary cell groups.
The new laser ablation-based zebrafish model of AKI described here addresses all of the above limitations. The pronephric kidney in larval zebrafish is a mature, functioning organ that contains segments similar to the mammalian nephron, including a glomerulus, proximal and distal tubules, and a collecting duct10. Zebrafish larvae are also optically transparent, making it feasible to observe the kidney through fluorescence techniques. Thus, zebrafish are a valuable in vivo model of AKI, and the larval pronephric kidney (5-12 days-post-fertilization (dpf)) can be used to study the cellular and molecular processes involved in kidney injury and repair.
This paper presents a method through which specific Green Fluorescent Protein (GFP)-expressing nephron segments can be photoablated using a low-energy (compared to a pulsed-laser system) violet laser light (405 nm). The GFP fluorescence allows for the targeting of a group of cells, making the changes that occur visible through the observation of GFP photobleaching. In addition, GFP (by absorbing violet light) serves as an energy sink to potentiate the injury in GFP-expressing kidney cells. Time-lapse microscopy can then be used to study the repair process. Studies have found cell proliferation, cell migration, and cell metaplasia11,12,13 to all be potential processes that may play an important role in kidney repair. However, the relative importance of these processes and the details of their interplay have been difficult to uncover due to the limitations of existing models of AKI. Using this novel approach, it was possible to show that cell migration plays a central role in kidney repair after acute injury14.

<table><tbody><tr><td>Petri Dishes, 35 x 10 mm</td><td>Genesee Scientific</td><td>32-103</td><td>Procedural Usage: Step 2.4, 2.7</td></tr><tr><td>Petri Dishes, 100 x 15 mm</td><td>Midwest Scientific</td><td>910</td><td>Procedural Usage: Step 1</td></tr><tr><td>Dechorination forceps - Electron Microscopy Sciences Dumont Tweezers 5 Dumostar</td><td>Fischer Scientific</td><td>50-241-57</td><td>Procedural Usage: Step 2.1.1</td></tr><tr><td>Plastic Transfer Pipet</td><td>Globe Scientific</td><td>135030</td><td>Procedural Usage: Step 2.5, 3.6</td></tr><tr><td>Tricaine</td><td>Sigma Aldrich</td><td>A5040-25G</td><td>Procedural Usage: Step 2.3, 3.4</td></tr><tr><td>Agarose</td><td>Fischer Scientific</td><td>BP165-25</td><td>Procedural Usage: Step 2.3</td></tr><tr><td>Pulled glass probe (manufactured manually from glass capillary tubes)</td><td>Fischer Scientific</td><td>21-1640-2C</td><td>Procedural Usage: Step 2.4</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1270</td><td>Procedural Usage: Step 1.5</td></tr><tr><td>SOLA Light Engine</td><td>Lumencor</td><td>SOLA SM-5-LCR-SB</td><td>Procedural Usage: Step 1.5</td></tr><tr><td>Eclipse C2 Plus Confocal Microscope System</td><td>Nikon</td><td></td><td>Procedural Usage: Step 3</td></tr><tr><td>1x E3 Solution</td><td></td><td></td><td>Recipe used to generate: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt; , 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt; Procedural Step Usage: 1.2, 1.3, 2.2, 2.3</td></tr><tr><td>PTU</td><td>Sigma</td><td>P7629-10G</td><td>Procedural Step Usage: 1.3, 2.2, 3.4, 4.2</td></tr><tr><td>NIS Elements Software</td><td>Nikon</td><td>C2+</td><td>Procedural Usage: Step 3</td></tr><tr><td>Laser Unit</td><td>Agilent</td><td>MLC 400</td><td>Procedural Step 3.11</td></tr><tr><td>Propidium Iodide (PI)</td><td>Sigma Aldrich</td><td>P4170-100MG</td><td>Procedural Step Usage: 4.2</td></tr></tbody></table>

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.

Please note that this protocol was successfully used with a number of kidney GFP transgenic lines, including ET(krt8:EGFP)sqet11-9, ET(krt8:EGFP)sqet33-d10, and Tg(atp1a1a.4:GFP). The example results shown here were obtained using the ET(krt8:EGFP)sqet11-9 line.
Figure 1 shows the example photoablation protocol. The average GFP intensity is monitored inside the region of interest (<strong class="xfi...

Watch this Scientific Journal Video about Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish at JoVE.com

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

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 ...

precise-cellular-ablation-approach-for-modeling-acute-kidney-injury

Research

JoVE Journal

Developmental Biology

6.6K Views.  NYITCOM.  Dit werk presenteert een nieuw in vivo model van segmentale nierbeschadiging met behulp van transplantale zebravis van GFP van de nieren. Het model zorgt voor de inductie van de gerichte ablatie van nierepitheelcellen om de cellulaire mechanismen van nephron letsel en reparatie te tonen. 

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

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 ...

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

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

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 ...

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

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.

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 ...

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 ...

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

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