Name: precise cellular ablation approach for modeling acute kidney injury in developing zebrafish
Uploaded: 2017-06-03T13:00:00
Description: Watch this Scientific Journal Video about Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish at JoVE.com

我们要感谢Iain Drummond博士和Vladimir Korzh博士分享肾GFP转基因株系。我们也要感谢纽特派团提供必要的资源进行这项工作。这项研究部分得到赠款的支持：K08DK082782，R03DK097443（NIH）和HSCI Pilot Grant（AV）。

本研究按照“国家卫生研究院实验动物护理与使用指南”中的建议进行。该协议由NYIT骨科医学机构动物护理和使用委员会（NYITCOM IACUC）批准。所有的手术和体内实验都是在三联麻醉下进行的，所有努力都是为了尽量减轻痛苦。 获取和维护胚胎<ol><li>通过穿过肾GFP荧光斑马鱼获得胚胎。 注：荧光标记的斑马鱼转基因株系的实例列于表1 。 </li><li>在受?...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis,+Epidemiology+And+Outcomes+of+Acute+Kidney+Injury.">Diagnosis, Epidemiology And Outcomes of Acute Kidney Injury.</a> Clin J Am Soc Nephrol. 3 (3), 844-861 (2008).</li><li>Lameire, N., Van Biesen, W., Vanholder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Kidney+Injury.">Acute Kidney Injury.</a> The Lancet. 372 (9653), 1863-1865 (2008).</li><li>Esson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+And+Treatment+of+Acute+Tubular+Necrosis.">Diagnosis And Treatment of Acute Tubular Necrosis.</a> Ann Intern Med. 137 (9), 744 (2002).</li><li>Vaidya, V. S., Ferguson, M. A., Bonventre, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Ablation+of+The+Zebrafish+Pronephros+To+Study+Renal+Epithelial+Regeneration.">Laser Ablation of The Zebrafish Pronephros To Study Renal Epithelial Regeneration.</a> J Vis Exp. (54), (2011).</li><li>Drummond, I. A., Davidson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Kidney+Development.">Zebrafish Kidney Development.</a> Methods Cell Biol. , 233-260 (2010).</li><li>Toback, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+After+Acute+Tubular+Necrosis.">Regeneration After Acute Tubular Necrosis.</a> Kidney Int. 41 (1), 226-246 (1992).</li><li>Witzgall, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+Proliferating+Cell+Nuclear+Antigen,+Vimentin,+C-Fos,+And+Clusterin+In+The+Postischemic+Kidney.+Evidence+For+A+Heterogenous+Genetic+Response+Among+Nephron+Segments,+And+A+Large+Pool+of+Mitotically+Active+And+Dedifferentiated+Cells.">Localization of Proliferating Cell Nuclear Antigen, Vimentin, C-Fos, And Clusterin In The Postischemic Kidney. Evidence For A Heterogenous Genetic Response Among Nephron Segments, And A Large Pool of Mitotically Active And Dedifferentiated Cells.</a> J Clin Invest. 93 (5), 2175-2188 (1994).</li><li>Bonventre, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiation+And+Proliferation+of+Surviving+Epithelial+Cells+In+Acute+Renal+Failure.">Dedifferentiation And Proliferation of Surviving Epithelial Cells In Acute Renal Failure.</a> J Am Soc Nephrol. 14 (90001), (2003).</li><li>Palmyre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+Epithelial+Migration+Drives+Kidney+Repair+After+Acute+Injury.">Collective Epithelial Migration Drives Kidney Repair After Acute Injury.</a> PLoS ONE. 9 (7), e101304 (2014).</li><li>Vasilyev, A., Drummond, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+Kidney+Development+In+Zebrafish.">Live Imaging Kidney Development In Zebrafish.</a> Methods Mol Biol. , 55-70 (2012).</li><li>Korzh, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Compound+Transgenic+Zebrafish+In+Development:+A+Tale+of+Green+Fluorescent+Protein+And+Killerred+.">Visualizing Compound Transgenic Zebrafish In Development: A Tale of Green Fluorescent Protein And Killerred .</a> Zebrafish. 8 (1), 23-29 (2011).</li><li>Bollig, F., Mehringer, R., Perner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+comparative+expression+analysis+of+a+second+wt1+gene+in+zebrafish.">Identification and comparative expression analysis of a second wt1 gene in zebrafish.</a> Dev Dyn. 235 (2), 554-561 (2006).</li><li>Liu, Y., Pathak, N., Kramer-Zucker, A., Drummond, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+controls+the+differentiation+of+transporting+epithelia+and+multiciliated+cells+in+the+zebrafish+pronephros.">Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros.</a> Development. 134 (6), 1111-1122 (2007).</li><li>Diep, C. Q., Ma, D., Deo, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+adult+nephron+progenitors+capable+of+kidney+regeneration+in+zebrafish.">Identification of adult nephron progenitors capable of kidney regeneration in zebrafish.</a> Nature. 470 (7332), 95-100 (2011).</li><li>Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+mesonephric+development+and+regeneration+using+transgenic+zebra+fish.">Characterization of mesonephric development and regeneration using transgenic zebra fish.</a> Am J Physiol. 299 (5), F1040-F1047 (2010).</li><li>Fisher, S., Grice, E. A., Vinton, R. M., Bessling, S. L., McCallion, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+RET+regulatory+function+from+human+to+zebrafish+without+sequence+similarity.">Conservation of RET regulatory function from human to zebrafish without sequence similarity.</a> Science. 312 (5771), 276-279 (2006).</li><li>Seiler, C., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+labeling+of+the+zebrafish+pronephric+duct+and+tubules+using+a+promoter+from+the+enpep+gene.">Transgenic labeling of the zebrafish pronephric duct and tubules using a promoter from the enpep gene.</a> Gene Expr Patterns. 11, 118-121 (2011).</li><li>Lin, H. F., Traver, D., Zhu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thrombocyte+development+in+CD41-GFP+transgenic+zebrafish.">Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.</a> Blood. 106 (12), 3803-3810 (2005).</li><li>Choo, B. G., Kondrichin, I., Parinov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+transgenic+Enhancer+TRAP+line+database+(ZETRAP).">Zebrafish transgenic Enhancer TRAP line database (ZETRAP).</a> BMC Dev Biol. 6 (1), 5 (2006).</li><li>Parinov, S., Kondrichin, I., Korzh, V., Emelyanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tol2+transposon-mediated+enhancer+trap+to+identify+developmentally+regulated+zebrafish+genes+in+vivo.">Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.</a> Dev Dyn. 231 (2), 449-459 (2004).</li></ol>

应当注意，总体激光功率在系统之间变化。然而，使用百分比的GFP光漂白允许读出传递到荧光肾的总能量，而与激光功率的变化无关，并且由曝光长度补偿。但是，请记住，不同组织对这种光消融方法的反应有所不同。即使在肾成熟期间，在成年幼虫中获得GFP荧光在年轻胚胎中降低50％明显更难。在将该协议修改和适应不同应用时，应考虑到光消解的组织反应性的这些差异。因此，监测GFP荧光的?...

急性肾损伤（AKI） 1,2 ，也可称为急性肾功能衰竭，广泛定义为肾功能的突发性损害3 。虽然多年来对这种状况的了解程度有了显着提高，但发病率和死亡率仍然高达1,2 。目前对这种病症的治疗主要是支持性的，因为药物治疗的多个临床试验的结果为阴性4,5 。肾脏是独一无二的，因为它具有修复自身的能力。因此，AKI早期诊断后的支持治疗是限制发病率的最佳途径6 。然而，很难早期检测AKI，对于需要透析的患者，死亡率为惊人的50-80％ 5。随着肾脏修复自身的能力和缺乏这种病症的治疗方案，重要的是开发增强这种肾单位再生过程的方法。 AKI研究中已经有许多不同的模型，包括不同的伤害和动物模型。就肾脏损伤剂而言，氨基糖苷类抗生素庆大霉素已被用作导致AKI 7,8的肾毒性药物。然而，有几组发现庆大霉素治疗对斑马鱼胚胎是致命的9 。它造成管状损伤对于胚胎恢复来说太严重，使得再生的研究难以进行某种干预。哺乳动物模型，如小鼠和老鼠，也被认为是有价值的，但在AKI研究期间它们面临许多限制。啮齿动物模型的主要缺点也许是visua的困难使啮齿动物肾脏化，从而确定导致上皮细胞死亡和修复的精确时空过程。 约翰逊已经报道了基于激光消融的技术在胚胎和幼虫斑马鱼中诱导急性肾损伤9 。在用葡聚糖缀合物肌内注射后，他们使用脉冲激光消融来损伤肾脏。来自葡聚糖共轭物的荧光允许在小管上皮9中的损伤和再生的可视化。该模式克服了上述两个限制，但不允许分级伤害，难以在大型，任意细胞群上进行。 这里描述的新的基于激光消融的AKI斑马鱼模型解决了上述所有限制。幼虫斑马鱼中的肾肾是一种成熟的功能性器官，其中包含与哺乳动物ne相似的节段包括肾小球，近端和远端小管以及收集管10 。斑马鱼幼虫也是光学透明的，使得通过荧光技术观察肾脏是可行的。因此，斑马鱼是AKI的有价值的体内模型，幼虫肾肾（5-12天 - 受精后（dpf））可用于研究肾损伤和修复中涉及的细胞和分子过程。 本文提出了一种通过使用低能量（与脉冲激光系统）紫激光（405nm）相比较来表达特定的表达绿色荧光蛋白（GFP）表达的肾单位片段的方法。 GFP荧光允许靶向一组细胞，通过观察GFP光漂白使得发生可见的变化。此外，GFP（通过吸收紫光）作为能量吸收器来增强GFP表达肾细胞中的损伤。延时微型然后复制可以用来研究修复过程。研究发现细胞增殖，细胞迁移和细胞化生11,12,13都是可能在肾脏修复中发挥重要作用的潜在过程。然而，由于AKI现有模型的局限性，这些过程的相对重要性及其相互作用的细节难以揭示。使用这种新颖的方法，可以显示细胞迁移在急性损伤后的肾脏修复中起核心作用14 。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Petri Dishes, 35 x 10mm</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 15mm</td>
			<td>Midwest Scientific</td>
			<td>910</td>
			<td>Procedural Usage: Step 1</td>
		</tr>
		<tr>
			<td>De-chorination 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 2 , 0.33 mM MgSO 4 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, and 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</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

急性肾损伤（AKI）是一种常见的死亡率高的疾病。具有肾脏的修复能力，可以在支持治疗后恢复足够的肾功能。然而，需要更好地了解如何在细胞水平上发生肾单位细胞死亡和修复以最小化细胞死亡并增强再生过程。斑马鱼背景是一个很好的模型系统来完成这个目标，因为它包含类似于哺乳动物肾单位的解剖段。以前，用于研究鱼类肾损伤的最常用模型是药物庆大霉素模型。然而，该模型不允许精确的时空控制损伤，因此难以研究涉及肾脏修复的细胞和分子过程。为了克服这个限制，这项工作提出了一种方法，与庆大霉素方法相反，特定的绿色荧光蛋白（GFP）-ex可以使用紫外激光（405nm）照射按压肾单位片段。这种AKI的新型模型提供了其他上皮损伤方法缺乏的许多优点。其主要优点是在健壮的体内动物模型中“拨”伤害水平和精确的时空控制能力。这种新方法有可能显着提高对肾损伤和修复机制的理解水平。

请注意，该协议已成功应用于许多肾转基因品系，包括ET（krt8：EGFP）sqet11-9，ET（krt8：EGFP）sqet33-d10和Tg（atp1a1a.4：GFP）。这里显示的示例结果是使用ET（krt8：EGFP）sqet11-9线获得的。 图1显示了实例的photoablation协议。在感兴趣的区域内监测平均GFP强度（ 图1A和电影1 ）。...

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

这项工作提出了使用肾GFP转基因斑马鱼的节段性肾损伤的新的体内模型。该模型允许诱导肾上皮细胞的靶向消融以显示肾单位损伤和修复的细胞机制。

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 ...

Research

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Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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

Mechanical Vessel Injury in Zebrafish Embryos

在斑马鱼胚胎的机械血管损伤

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

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

手术消融分析在研究涡虫再生眼

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

斑马鱼急性酒精性肝损伤的组织学分析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...