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

NOTE: Ethics Statement: All experiments were conducted in accordance with Massachusetts General Hospital guidelines for animal use in research.
1. Advance Preparation
<ol>
	<li>Determine how many adult zebrafish 6-12 months old to injure. Plan to injure 10-20% more fish than will be needed. In order to minimize variability, use age matched sibling fish reared together in the same tank so that they are roughly the same size and have less genetic variability. Female fish are much easier to inj...

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The zebrafish is ideal for studying regeneration of adult organs including the adult mesonephric kidney6. Recent studies have taken advantage of molecular markers and new transgenic reporter lines to better characterize the steps that occur during nephron regeneration and what cells might be responsible7,12. Observation of urinary casts has been used for over a century to diagnose kidney disease in humans16. Here we use the presence of casts as an easy, noninvasive and early indicator of ...

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

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin sodium sulfate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">G1264</td>
			<td>TOXIC, purity varies from batch to batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic transfer pipets</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 ml Norm-Ject syringes</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">72520</td>
			<td>green plastic syringes, ordinary 1ml syringes are OK, but harder to read accurately</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30G1/2 needles</td>
			<td style="width:101px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>IRRITANT</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">16% paraformaldehyde</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td>Make 4% in 1xPBS for working solution</td>
		</tr>
	</tbody>
</table>

kidney regeneration in adult zebrafish by gentamicin induced injury

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

Gentamicin injury can be confirmed visually by the observation of renal epithelial casts in the water (Figure 1). Different dosages of gentamicin were used to injure adult wildtype TuAB zebrafish, resulting in increasing numbers of lhx1a+ cellular aggregates in the regenerating kidney (Figure 1A-D). White casts can easily be seen in the water 1 day after injury (Figure 1E-H). A low dose of gentamicin resulted in most fish having...

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

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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

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

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

Research

JoVE Journal

Developmental Biology

10.7K visualizações.  Massachusetts General Hospital. Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. 