Name: evaluation of zebrafish kidney function using a fluorescent clearance assay
Uploaded: 2015-02-20T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay at JoVE.com

由Jaipreet Bharj提供的技术援助。这项工作是由欧盟第七框架计划（SYSCILIA -241955）和荷兰肾脏基金会（CP11.18）资助。

伦理学声明：动物保养，畜牧业和程序是由动物（科学程序）所有的动物实验已经进行了下符合生物授予内政大臣（PIL号七千八百九十二分之七十零）许可证法案1986年界定和控制服务管理组和生物服务伦理委员会，SGUL，伦敦，英国。所作的所有努力，以减少使用动物的数量，并以尽量减少痛苦，提高福利完善两个程序和畜牧业。 1.制备仪器，麻醉，并且荧光染料<ol><li>使?...

<ol>
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斑马鱼提供了一个宝贵的工具，人类遗传病模型，他们作为一个科学仪器体内研究使用使许多生物系统，包括肾脏的遗传击穿详细的研究。很多现在被理解如何斑马鱼肾脏发展和功能。惊人的相似，以人类nephrogenesis和同源性致病基因21已示于理解如何斑马鱼已成为基本的基因功能导致肾疾病的病理学缺陷如何。事实上，遗传操作，可以毫不费力地使用反义吗啉击倒技术或更先进的?...

人肾起着从血液过滤代谢废物和回收所需溶质以维持细胞稳态的关键作用。有一个数字，引起肾功能障碍的人类遗传疾病。最常见的遗传性肾脏病是常染色体显性多囊肾病（ADPKD）的特征在于肾脏肾小管内的流体填充囊的发展;引起cystogenesis的损害是不利的肾功能10。多囊肾具有1的发生：800 - 1：1000，占8 -的患者在末期肾衰竭（ESRF）11 10％。几个基因有牵连导致多囊肾多囊，包括-1（PKD1）和-2（PKD2），约占85％的病例分别为12,13 15％。此外，对于与PKD1基因产物-2定位于纤毛和是基本的ciliogenesis 14,15。现在有人类遗传性疾病，被称为公认的家庭在ciliopathies，影响纤毛的功能，并导致CKD 16。 越来越多的影响纤毛发育和功能的人类遗传疾病的画在这个曾经被认为是残留的细胞器全球利益。该纤毛，头发般的细胞突起，富含受体和必要的关键细胞信号事件的传导离子通道。该纤毛由一个微管系轴丝，典型地构造成9径向地布置微管双峰具有或不具有中央一对单峰的微管的。该轴丝结构定义的纤毛作用的类型和模式。 9 + 2微管排列赋予蠕动到它被用在跨越上皮表面流体的运动的纤毛。 9 + 0构造为非能动但被认为是主要的功能在细胞信号传递活动17。除了CKD，睫状功能障碍的后果是一组特征ciliopathy功能，包括，肥胖症，视网膜变性，多趾，以及认知缺损16。然而，CKD是其中最不利于患者生活质量，因此后面的适当的体内模型的发展纤毛相关CKD的主要动力。 斑马鱼是一个很好的模型，以了解人类遗传病的病因。他们的快速发展，生产大量的鸡蛋，透明的组织，和前宫内生长使得斑马鱼发育过程进行可视化和生物操纵的事件相当容易。基因可以使用的基因组编辑工具（CRISPR 18和TALENS 19）最近成功进行遗传改变，撞倒使用反义吗啉代技术20，或通过加入化合物以它们的水生环境中的药理学调节。事实上，斑马鱼提供了一个平台，承接éxperiments不在容许在其它动物模型。而斑马鱼都比较简单脊椎动物（相比人类）它们共同与人类有许多共同的功能保守的器官，基因，和信令进程。例如，斑马鱼肾是非常相似的结构和功能相比人类21,22。然而，与哺乳动物肾，通过相的连续发展，各带标记的较发达的肾（前肾，中肾和后肾），胚胎斑马鱼只开发了一个前肾，肾的最不成熟的形式。虽然百万肾单位，可以发现在形成哺乳动物肾的积木，斑马鱼胚胎只具有两个。肾小球，它允许在初始血液滤液，融合在主动脉中线只是腹侧。通过肾小球成运行尾端沿轴线经由泄殖腔退出之前熔合pronephric肾小管血液过滤器。在pronephric TUbules被大量纤毛与运动纤毛是宽容到滤液对尾3,4退出流通。这种简单的pronephric结构经过几个星期的幼虫生长在那里他们最终发展成一个更复杂的结构中肾21斑马鱼保持动态平衡。然而，斑马鱼从未开发出后肾21。尽管斑马鱼特质，斑马鱼肾单位被分段与基因表达谱等于在哺乳动物观察，从而提供了在体内模型无与伦比为nephrogenesis 3,22。 常规的患者通过一系列的血液和尿中的测试测试肾功能。通常将血液溶解的盐，尿素和肌酸酐进行分析。高水平的尿素，肌酸酐和异常盐浓度是指示问题具有肾功能。用比色试纸尿液检测的蛋白质，布卢异常水平D，脓液，细菌和现在的尿液样本中的糖。这样的测试通常需要大约30毫升尿液或5 - 加入10ml的血液。它已经难以在体内模式生物，如斑马鱼翻译这些类型的测定，以小的，主要是由于收集足够的血液或尿液进行测定的不可能性质。在这里，我们解决缺乏相应的斑马鱼肾功能检查，并描述了创新技术的研究。用荧光染料注射入血流中，我们能够通过肾脏，以监测和个别地量化随着时间的推移过滤和荧光活性的排泄从血液。该方法可用于研究疾病引起的肾损害，这是我们提供的一个例子。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">P-97 SUTTER Flaming/Brown type micropipette puller</td>
			<td style="width:136px;">Intracel</td>
			<td style="width:136px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">borosilicate standard wall capillaries</td>
			<td style="width:136px;">Harvard Apparatus</td>
			<td style="width:136px;">30-0017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glass microscope slides</td>
			<td style="width:136px;">VWR International</td>
			<td>631-0109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Epoxy Resin Glue</td>
			<td style="width:136px;">Evo-Stik</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Rhodamine B 10,000 MW labeled Dextran</td>
			<td>Life technologies&#160;</td>
			<td>D-1824</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Phenylthiourea&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene blue&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">air compressor&#160;</td>
			<td>Jun-Air</td>
			<td>OF302-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer III&#160;</td>
			<td>Parker Instruments&#160;</td>
			<td>051-0500-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">compact 3-axis control micromanipulator&#160;</td>
			<td>Marzhauser</td>
			<td>MM33&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dissecting stereo microscope</td>
			<td style="width:136px;">Nikon</td>
			<td>SMZ1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">microloader tips</td>
			<td>Eppendorf</td>
			<td align="right">5242956003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 forceps&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stage micrometer&#160;</td>
			<td>Pyser- SGI</td>
			<td>02A00404</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of zebrafish kidney function using a fluorescent clearance assay

斑马鱼的胚胎提供了一个听话的模型来研究器官和模拟人类遗传性疾病。尽管其相对简单，斑马鱼肾开发并在几乎相同的方式作为人类的功能。在人类肾脏的结构的主要区别是数以百万计的肾相比，仅具有两个斑马鱼的存在。然而，简化这种复杂的系统进入基本功能单元已资助我们如何肾脏开发和运营的理解。在斑马鱼，位于肾小球中线负责初始血液过滤成发散熔合到彼此的泄殖腔之前两侧向下胚轴运行两个pronephric肾小管。在pronephric肾小管是人口稠密通过运动纤毛有利于滤液的运动沿着分割肾小管，使各种溶质，最后才通过泄殖腔2-4退出交流。负责CKD，INC许多基因泸那些与ciliogenesis，已经研究了在斑马鱼5。然而，一个主要的平局背面已在遗传操作之后评估斑马鱼肾功能的困难。传统的检测来衡量肾功能不全的人证明非平移，以斑马鱼，主要是由于他们的水生环境和小尺寸。例如，它不是物理上能够提取血液从对于尿素，肌酐含量分析胚胎上演鱼，因为它们太小。此外，斑马鱼不产生足够的尿液用于测试在一个简单的蛋白尿&#39;试纸&#39;，这是经常在初始病人检查执行。我们描述了一种荧光测定法，利用了斑马鱼的光学透明度，定量监测荧光染料的间隙，随着时间的推移，从脉管并通过肾脏，得到读出肾功能1,6-9的。

巴比二氏综合征（BBS）是一种罕见的异类ciliopathy影响约为1：全球16 16万人。患者目前有许多相关的问题，包括多囊性肾脏，随后患者经常需要肾透析或移植24。 ESRF是死亡BBS的最常见的原因，大约有30％的患者发展CKD 16。目前，20个非相关的基因有牵连的BBS，没有发表基因型 - 表型的关联。在BBS蛋白质共享纤毛和基体，随着患者的特性特征，推断ciliopathy诊断中常见蛋白质定...

Watch this Scientific Journal Video about Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay at JoVE.com

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

斑马鱼是一种流行的工具来模拟慢性肾脏病（CKD）。然而，它们的小尺寸使得它不可能用传统方法评估肾功能。我们描述一个荧光染料肾清除率法1，允许在CKD斑马鱼肾功能定量分析。

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Research

JoVE Journal

Biology

Transplantation of Whole Kidney Marrow in Adult Zebrafish

在成年斑马鱼的全肾骨髓移植

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

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Using the optokinetic response to study visual function of zebrafish

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

斑马鱼幼虫的研究急性肾损伤的静脉Microinjections

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

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

直接进入成年斑马鱼的肾细胞移植

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

Evaluation of Mammary Gland Development and Function in Mouse Models

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Dissection of the Adult Zebrafish Kidney

成年斑马鱼肾脏的解剖

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

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

肾单位的组成与功能的成年斑马鱼肾脏分析

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