Name: nephrotoxin microinjection in zebrafish to model acute kidney injury
Uploaded: 2016-07-17T13:00:00
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 w...

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 hig...

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 border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Sodium Chloride</td>
			<td style="width:229px;">American Bioanalytical</td>
			<td style="width:129px;">AB01915</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB01652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>American Bioanalytical</td>
			<td>AB00366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea (PTU)</td>
			<td>Aldrich Chemistry</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate (Tricaine)</td>
			<td>Fluka Analytical</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass</td>
			<td>Sutter Instruments Co.</td>
			<td>BF100-50-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming/Brown Micropipette puller</td>
			<td>Sutter Instruments Co.</td>
			<td>Mo. P097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Diposable Petri Dishes, Sterile, Corning:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm x 15mm</td>
			<td>VWR</td>
			<td>25373-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100mm x 15mm</td>
			<td>VWR</td>
			<td>25373-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;(microinjection tray) 150mm x 15mm</td>
			<td>VWR</td>
			<td>25373-187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Temperature Incubator</td>
			<td>Fischer Scientific</td>
			<td>11 690 516DQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Dissecting Tweezer</td>
			<td>Roboz Surgical Instruments Co.</td>
			<td>RS-5010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">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 (Fig...

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 ...

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