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

这项工作由美国国立卫生研究院资助DP2OD008470部分得到了支持。另外，RAM是部分由圣母研究生院的大学提供了资金支持。我们感谢生物科学系，中心斑马鱼研究，中心的员工干细胞与再生医学在Notre Dame大学。我们特别感谢实验室的成员参与有关肾脏生物学及其对这项工作有帮助的反馈讨论。

对于在本协议中所描述斑马鱼胚胎工作的过程被机构动物护理和使用委员会在Notre Dame大学的批准。 1.溶液的制备<ol><li>通过在RT混合73.0克氯化钠，3.15克氯化钾，9.15克CaCl 2，并在5升蒸馏水9.95克硫酸镁 ，并存储使E3胚胎介质的50倍原液。 </li><li>对于斑马鱼胚胎的培养中，稀释的E3胚胎媒体库存的50倍原液用蒸馏水1X工作液，再加入200微升的0.05％的亚甲基蓝的每1升1倍的E3作为杀菌剂，并储存在RT。 </li><li> 40毫升50×E3库存与0.06毫克PTU组合使E3胚胎媒体与0.003％1-苯基-2-硫脲（PTU）一个色素沉着封闭溶液。然后使体积到总共2L的蒸馏水。 </li><li>炒出的E3 / PTU O / N在室温得到PTU粉成溶液。然后存储E3 /在PTURT。 注意:E3 / PTU具有大约一周的保质期，和更老的解决方案将是在斑马鱼幼体阻断色素发展不太有效。 </li><li>通过加入1g三卡因到500毫升蒸馏水使0.2％三卡因（MS-222）的麻醉剂溶液并用1M Tris，pH值9.5将pH调节至7.2。 </li><li>制备通过将1×E3（无亚甲蓝）的溶液中，使用于成像的2％的甲基纤维素溶液中，用0.2％三卡因加入在4℃1/10体积。 注意:甲基纤维素溶液是用来轻轻稳定显微镜活胚胎厚，透明的溶液。操作被执行后的胚胎可以在1×E3洗涤以溶解甲基纤维素不损伤动物并在同一检体后续阶段使显微镜。 </li><li>时冷却至4℃，剧烈搅拌在搅拌盘1倍E3 /三卡因溶液逐步添加甲基适量同时继续纤维素粉末剧烈搅拌。逐渐将粉末添加到该溶液中，以防止不溶性甲基纤维素聚集体的形成。 </li><li>所有的粉末混合到2％甲基纤维素/ E3 /三卡因溶液后，搅拌在4℃冷室O / N复合材料解决方案。 注:低温是最好的粉末溶解完全。如果一个晚上后，解决的办法是不明确的，搅拌额外的工作日和/或第二O / N。 </li><li>从2％甲基纤维素/ E3 /三卡因溶液，等分试样的混合物在4℃下30分钟或高达2小时除去气泡进入以高速（12 000 XG）2.4毫升管和旋转。 </li><li>放置在4℃的2％甲基纤维素/ E3 /三卡因溶液长期储存。 注意:在此之前，用法，等份应预先温热至RT用于与斑马鱼标本工作。 </li></ol> 2.工具的制备<ol><li>准备一个胚胎操纵的工具，用于采购订单sitioning胚胎显微注射，通过连接在一个5"直解剖针的端部的凝胶加载提示，并用胶带或强力胶固定。 </li><li>首先将火准备用针管注射拔针头，抛光10厘米硼硅玻璃长丝放入仪器，并拧紧处理安全硼硅玻璃。 </li><li>拉硼硅玻璃时尚细锥形针。使用以下设置:加热540，拉245，速度200和125的时间。 </li><li>培养皿内放置针，上橡皮泥条暂停，以保护他们的针尖，并保持覆盖，防止灰尘堆积的菜。 </li><li>完成制备用于显微注射的针，用剃刀刀片或细镊子边缘切针的拔出端以创建一个尖锐的角注射针尖的直径为约0.05-0.1毫米。 </li><li>为了使显微注射托盘，准备在一个10 1.5％琼脂糖/ E3溶液0毫升锥形瓶中，并通过在微波的E3加热到沸腾，然后允许在室温冷却约10分钟，溶解琼脂糖。 </li><li>倒入冷却的琼脂糖/ E3溶液放入培养皿，使基础具有大约0.5cm的深度。当这已固化的倾具有大约0.3厘米的深度的第二层，并插入预制胚胎以及模具和允许琼脂糖在RT固化。 注意:当插入模具插入顶层琼脂/ E3层，将所述模具放入琼脂以一个角度以减少气泡的数目。 </li><li>当整个注射托盘已成立，抬预制胚胎以及塑造出缓慢而小心地使用scoopula，然后填写与E3的解决方案和存储的菜在4℃。 </li></ol> 3.胚胎制备方法<ol><li>通过将分隔成交配室设立斑马鱼交配坦克，并与系统中的水满。 </li><li>放置一个鱼分频器的S每侧UCH每个交配室包含一雌一雄鱼，并覆盖每个配合腔。 </li><li>第二天早晨，取出分隔，使产卵。 </li><li>检查鱼后约30分钟，并返回成年人水族馆后，通过传递配合水箱水通过细筛网过滤工具收集他们的胚胎。 </li><li>倒置滤网过培养皿和E3冲洗收集胚胎。 </li><li>在28.5°CO / N孵育胚胎。 </li><li>当胚胎已达到24-26体节阶段26，倒出大部分E3介质中，然后dechorionate加入100微升的50毫克/毫升的链霉蛋白酶胚胎的每个培养皿大约15温育在室温（23℃）的菜分钟。 注意:这将需要约24-25小时，从受精的时间，如果他们被收集在方式描述并在28.5℃培养O / N胚胎达到24-26体节阶段。 </li><li>装满菜E3然后轻轻漩涡撞出从胚胎绒毛膜。 </li><li>倒出E3 /链霉，用25毫升新鲜E3漂洗菜。重复冲洗步骤去除所有链霉。 </li><li>要阻止色素沉着的发展，倒出E3并用25毫升新鲜的E3 / PTU的时候胚胎已经达到24小时受精后更换。 注意:对于实验跨越数日，每日更换一次新鲜的媒体E3 / PTU解决方案，以保持干净的菜。 </li></ol> 4.肾毒素显微注射的解决方案<ol><li>关于注射的当天，制备用适当的车辆控制沿着所需肾毒素溶液。 </li><li>涡流或轻轻混匀，以确保药物（s）是/都是在溶液中，检查水柱的管和顶部的两侧。 注意:肾毒素溶液可以制备成含有荧光缀合的葡聚糖的痕量监测显微注射效率和也在subseq评估肾清除uent时间点20,28。 </li><li>通过螺纹细凝胶加载提示到针的后部装入〜2-3微升肾毒素的修整显微注射针和用一块胶带的垂直暂停针以使该溶液通过重力来填充修整针尖。 </li><li>一旦针尖是满的，固定在显微加载针。 </li><li>通过将矿物油的液滴到微米滑动测试注射体积和注射进油来评价液滴尺寸。 500复注射体积的直径为0.1毫米。 </li><li>除去从培养箱胚的培养皿，并通过加入约5mL的0.2％三卡因的对胚胎菜麻醉。 </li><li>为了确保完全麻醉，轻轻地用胚胎操纵工具的尖端碰触胚胎。缺乏运动的指示充分麻醉。 </li><li>麻醉成功后，转移胚胎到注塑模具用TRANSF呃吸管。 </li><li>机动每个胚胎成不同以及模具放置头井使得行李箱沿凹陷休息和尾巴竖起走出抑郁的最深处的区域。 </li><li>将填充进针显微和针尖位置，旁边是一个胚胎。 </li><li>轻轻地向前移动操纵杆针头插入尾容器并踩下脚踏板提供显微注射。 注:成功注入可以通过观察液体来衡量进入流通。另外，用荧光结合的葡聚糖的nephrotoxicant的共注射使研究者来验证到程序随后成功注入。 </li><li>轻轻地向后拉操纵杆，从胚胎中取出针。 </li><li>注射后，将胚胎转移到一个干净的菜和冲洗以除去三卡因，并用新鲜的E3 / PTU替换。 </li><li>孵育胚胎到所需的时间点到驴小号的形态，其可通过摄影在甲基纤维素安装介质，或处理根据需要被记录为实验分析。 注:胚胎的复苏应该评估，以确定个人的水肿，其中常见的指示肾损伤的百分比。 </li></ol>

The authors have nothing to disclose.

治疗药物的不同数量已与AKI 29有关。已经出现了在理解由许多单独的化合物，如氨基糖苷类庆大霉素30和广泛使用的化学治疗顺铂31,32引起的损伤显著研究进展。参与这些条件下一些病理变化，但是，仍正在进行的研究的主题。一个新兴的挑战仍然是如何理解多种药物产生不利影响的患者，高危人群尤其是那些如在重症监护设置和老人29。因此，模式，使关于药物?...

AKI是肾功能可能导致灾难性的健康后果1的突然损失。 AKI是一个显著的医疗保健问题，全世界由于约20％的住院患者中的发病率很高，在重症监护的情况下30-50％甚至更高的速率和老人，以及50-70％的1-3死亡率。不幸的是，AKI的发病率不断上升，预计未来十年进一步升级，部分原因是由于多种因素的多样性，可诱发急性肾损伤，包括手术后应激，缺血和接触到的肾毒性，如抗生素和化疗药物4。 AKI涉及肾脏内突细胞损伤，在肾，这是必要的功能单元，并且是由一个血液过滤器和排出尿进入中央集合管1的分段小管通常发生。当肾的一个显著数目是AKI过程中被损坏，直接影响包括从循环通过从死亡和垂死的细胞1由于阻塞肾单位在废弃物清除中断，并减少或废除流体流动。随着时间的推移，肾小管阻塞可导致肾整，永久地降低了肾功能1退化。在肾脏生理改变以下的AKI也涉及复杂的炎性事件，可导致慢性结疤1。 尽管有这些成果，肾有一定能力的AKI的进行重构肾小管上皮细胞5,6后接受再生。虽然出现了肾再生的分子越来越了解，该机制仍然在许多方面难以捉摸和必要继续调查7。在何种程度AKI导致永久性肾损害也仍然不明。目前的研究表明，对肾脏的再生潜能是最高以下AKI的较不严重的情况下，而更明显或反复发作导致慢性肾脏病（CKD）和终末期肾病（ESRD），需要救生移植或透析8,9-高潮。此外，个人从CKD已经患有处于收缩AKI 8,9的严重发作的风险更大。两者合计，很明显，持续的基础和临床研究是至关重要的理解，治疗和预防AKI。 研究与动物模型已经在欣赏AKI 10期间发生的本地和环境的改变的进展工具。为了扩大这种认识以及开发新的疗法，斑马鱼的动物模型已经在以各种方式11,12使用。斑马鱼肾脏的肾单位，在胚胎和成人两种，显示与哺乳动物13-16高度保护。另外，在泽肾上皮损伤brafish类似于高等脊椎动物，由此肾小管细胞的局部破坏之后是管内增殖和肾结构17-19的重建过程。在胚胎，但是，从像顺铂的肾毒性广泛肾小管损伤与杀伤力20,21相关联。相比之下，成年斑马鱼生存AKI并表现在肾脏实质性的再生能力。例如，在暴露于氨基糖苷类抗生素庆大霉素，斑马鱼再生肾小管上皮损伤，增加新的肾单位以及22-24。虽然这些庆大霉素引起的AKI的研究提供了宝贵的资料，了解来自不同的肾毒性肾功能损害仍然至关重要升值的影响，并应对不同类型的损害25。 斑马鱼胚胎，由于其大小，透明度和遗传易处理性，对肾毒素的研究很多好处&lt;SUP&gt; 25，其中，用于管理调查的分子（多个）注射20,21的方法。肾是由24小时受精后（HPF）形成，并开始约48 HPF 26,27来过滤血液。因此，胚胎肾的迅速形成和功能便于实验分析。然而，显微注射的过程中具有技术挑战，并有可能是一个陡峭的学习曲线，以掌握该技术。在这个视频文章中，我们将介绍如何执行显微注射​​，并以提高成功的注射速度提供故障排除技巧。

<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

肾脏是容易暴露在他们从血液中过滤化学物质伤害。这会导致与在肾功能和临床症状称为急性肾损伤（AKI）的发展迅速下降有关器官损伤。药剂用于治疗的医疗情况，从细菌感染到癌症，当单独给药或与其它药物组合，可以发起AKI。斑马鱼是一个有用的动物模型来研究对肾功能的化学作用在体内 ，因为它们形成由那些保守与高等脊椎动物，包括人类在内的肾功能单位的胚胎肾。此外，斑马鱼可以用于执行遗传和化学屏幕，这提供机会以阐明AKI的细胞和分子方面和开发治疗策略如肾保护分子的鉴定。在这里，我们将演示如何显微注射入斑马鱼胚胎可以用作用于肾毒素研究一个范例。

显微注射站设立包括立体显微镜，显微操纵器和压力调节器（ 图1A）。注入板的透优选此过程（ 图1B）中，以查看标本。注射针的制备涉及拉适当硼硅酸盐玻璃，然后通过制备用切割和最后背加载针的边缘。最佳地，针尖斜面，而不是在切割过程期间钝（ 图1C），这是由急剧斜着切割器械，边缘制成，例如细钳子的切口， </stro...

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

从肾毒素，其中包括药品，从抗生素对化疗药物产生的肾损伤，可导致复杂的疾病，其发病机制尚未完全清楚。这个协议演示斑马鱼如何可以用于这些条件下，它可以被应用到的肾脏保护措施的识别疾病建模。

肾毒素显微注射斑马鱼到模型急性肾损伤

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.8K 次观看.  University of Notre Dame. 从肾毒素，其中包括药品，从抗生素对化疗药物产生的肾损伤，可导致复杂的疾病，其发病机制尚未完全清楚。这个协议演示斑马鱼如何可以用于这些条件下，它可以被应用到的肾脏保护措施的识别疾病建模。 

视频: 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.

肾再生的成年斑马鱼庆大霉素性损伤

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

科研

发育生物学

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.

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 ...

生物学

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.

测试使用微量注射蚜虫病毒传播的生理障碍

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.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

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.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

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.

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 ...

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.

激光烧蚀斑马鱼Pronephros研究肾小管上皮再生

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.

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 ...

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.

成人斑马鱼中西斯铂诱导的急性肾损伤模型

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

医学

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://jove.unicartagenaproxy.elogim.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.

将肾毒素静脉显微注射到斑马鱼胚胎中:一种将肾毒性药物输送到斑马鱼胚胎血液中的技术

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