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Biology

肾毒素显微注射斑马鱼到模型急性肾损伤

Published: July 17, 2016
doi:
10.3791/54241
,
1Center for Zebrafish Research, Department of Biological Sciences,University of Notre Dame, 2Center for Stem Cells and Regenerative Medicine, Department of Biological Sciences,University of Notre Dame

Summary

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。

Abstract

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。

Introduction

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<SUP> 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。

Protocol

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。 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?…

Representative Results

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…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

Sodium Chloride American Bioanalytical AB01915
Potassium Chloride American Bioanalytical AB01652
Calcium Chloride American Bioanalytical AB00366
N-Phenylthiourea (PTU) Aldrich Chemistry P7629
Ethyl 3-aminobenzoate (Tricaine) Fluka Analytical A5040
Borosilicate glass Sutter Instruments Co. BF100-50-10
Flaming/Brown Micropipette puller Sutter Instruments Co. Mo. P097
UltraPure Agarose Invitrogen 15510-027
Magnesium Sulfate Sigma-Aldrich M7506
Methylene Blue Sigma-Aldrich M9140
Falcon Diposable Petri Dishes, Sterile, Corning:
60mm x 15mm VWR 25373-085
100mm x 15mm VWR 25373-100
 (microinjection tray) 150mm x 15mm VWR 25373-187
Low Temperature Incubator Fischer Scientific 11 690 516DQ
Micro Dissecting Tweezer Roboz Surgical Instruments Co. RS-5010
Micrometer Ted Pella, Inc. 2280-24
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References

  1. Basile, D. P., Anderson, M. D., Sutton, T. A. Pathophysiology of acute kidney injury. Compr. Physiol. 2, 1303-1353 (2012).
  2. Ostermann, M. Diagnosis of acute kidney injury: kidney disease improving global outcomes criteria and beyond. Curr. Opin. Crit. Care. 20, 581-587 (2014).
  3. Fluck, R. J. Acute kidney: improving the pathway of care for patients and across healthcare. Curr. Opin. Nephrol. Hypertens. 24, 511-516 (2015).
  4. Silver, S. A., Cardinal, H., Colwell, K., Burger, D., Dickhout, J. G. Acute kidney injury: preclinical innovations, challenges, and opportunities for translation. Can. J Kidney Health Dis. 2, 30 (2015).
  5. McCampbell, K. K., Wingert, R. A. Renal stem cells: fact or science fiction?. Biochem. J. 444, 153-168 (2012).
  6. Li, Y., Wingert, R. A. Regenerative medicine for the kidney: stem cell prospects and challenges. Clin. Transl. Med. 2, 11 (2013).
  7. Romagani, P., Lasagni, L., Remuzzi, G. Renal progenitors: an evolutionary conserved strategy for kidney regeneration. Nat. Rev. Nephrol. 9, 137-146 (2013).
  8. Kline, J., Rachoin, J. S. Acute kidney injury and chronic kidney disease: it’s a two-way street. Ren. Fail. 35, 452-455 (2013).
  9. Chawla, L. S., Kimmel, P. L. Acute kidney injury and chronic kidney disease: an integrated clinical syndrome. Kidney Int. 82, 516-524 (2012).
  10. Sanz, A. B., Sanchez-Niño, M. D., Martìn-Cleary, C., Ortiz, A., Ramos, A. M. Progress in the development of animal models of acute kidney injury and its impact on drug discovery. Expert Opin. Drug Discov. 8, 879-895 (2013).
  11. McCampbell, K. K., Wingert, R. A. New tides: using zebrafish to study renal regeneration. Transl Res. 163, 109-122 (2014).
  12. McKee, R. A., Wingert, R. A. Zebrafish renal pathology: emerging models of acute kidney injury. Curr. Pathobiol. Rep. 3, 171-181 (2015).
  13. Wingert, R. A., et al. The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. PLoS Genet. 3, 1922-1938 (2007).
  14. Wingert, R. A., Davidson, A. J. The zebrafish pronephros: a model to study nephron segmentation. Kidney Int. 73, 1120-1127 (2008).
  15. Wingert, R. A., Davidson, A. J. Zebrafish nephrogenesis involves dynamic spatiotemporal expression changes in renal progenitors and essential signals from retinoic acid and irx3b. Dev Dyn. 240, 2011-2027 (2011).
  16. McCampbell, K. K., Springer, K. N., Wingert, R. A. Analysis of nephron composition and function in the adult zebrafish kidney. J. Vis. Exp. (90), e51644 (2014).
  17. Johnson, C. S., Holzemer, N. F., Wingert, R. A. Laser ablation of the zebrafish pronephros to study renal epithelial regeneration. J. Vis. Exp. (54), e2845 (2011).
  18. Palmyre, A., et al. Collective epithelial migration drives kidney repair after acute injury. PLoS One. 9, e101304 (2014).
  19. Fogelgren, B., et al. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis. Am. J. Physiol. Renal Physiol. 307, F1334-F1341 (2014).
  20. Hentschel, D. M., Park, K. M., Cilenti, L., Zervox, A. S., Drummond, I. A., Bonventre, J. V. Acute renal failure in zebrafish: a novel system to study a complex disease. Am. J. Physiol. Renal. Physiol. 288, F923-F929 (2005).
  21. Cosentino, C. C., Roman, B. L., Drummond, I. A., Hukriede, N. A. Intravenous microinjections of zebrafish larvae to study acute kidney injury. J. Vis. Exp. (42), e2079 (2010).
  22. Zhou, W., Boucher, R. C., Bollig, F., Englert, C., Hildebrandt, F. Characterization of mesonephric development and regeneration using transgenic zebrafish. Am. J. Physiol. Renal. Physiol. 299, F1040-F1047 (2010).
  23. Diep, C. Q., et al. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature. 470, 95-101 (2011).
  24. McCampbell, K. M., Springer, K. N., Wingert, R. A. Atlas of cellular dynamics during zebrafish adult kidney regeneration. Stem Cell Int. , 547636 (2015).
  25. Sharma, P., Sharma, S., Patial, V., Singh, D., Padwad, Y. S. Zebrafish (Danio rerio): a potential model for nephroprotective drug screening. Clinical Queries: Nephrol. 3, 97-105 (2014).
  26. Gerlach, G. F., Wingert, R. A. Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration. Wiley Interdiscip Rev Dev Biol. 2, 559-585 (2013).
  27. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310 (1995).
  28. Hanke, N., et al. ‘Zebrafishing’ for novel genes relevant to the glomerular filtration barrier. Biomed. Res. Int. 2013, 658270 (2013).
  29. Kane-Gill, S. L., Goldstein, S. L. Drug-induced acute kidney injury: a focus on risk assessment for prevention. Crit. Care Clin. 31, 675-684 (2015).
  30. Lopez-Novoa, J. M., Quiros, Y., Vicente, L., Morales, A. I., Lopez-Hernandez, F. J. New insights into the mechanism of aminoglycoside nephrotoxicity: an integrative point of view. Kidney Int. 79, 33-45 (2010).
  31. Ozkok, A., Edelstein, C. L. Pathophysiology of cisplatin-induced acute kidney injury. Biomed. Res. Int. 2014, 967826 (2014).
  32. Perazella, M. A., Moeckel, G. W. Nephrotoxicity from chemotherapeutic agents: clinical manifestations, pathobiology, and prevention/therapy. Semin. Nephrol. 30, 570-581 (2010).
  33. Cheng, C. N., Verdun, V. A., Wingert, R. A. Recent advances in elucidating the genetic mechanisms of nephrogenesis using zebrafish. Cells. 4, 218-233 (2015).
  34. Pickart, M. A., Klee, E. W. Zebrafish approaches enhance the translational research tackle box. Transl. Res. 163, 65-78 (2014).
  35. Cheng, C. N., Li, Y., Marra, A. N., Verdun, V., Wingert, R. A. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization. J. Vis. Exp. (89), e51604 (2014).
  36. Galloway, J. L., Wingert, R. A., Thisse, C., Thisse, B., Zon, L. I. Combinatorial regulation of novel erythroid gene expression in zebrafish. Exp. Hematol. 36, 424-432 (2008).
  37. McKee, R., Gerlach, G. F., Jou, J., Cheng, C. N., Wingert, R. A. Temporal and spatial expression of tight junction genes during zebrafish pronephros development. Gene Expr. Patterns. 16, 104-113 (2014).
  38. Li, Y., Cheng, C. N., Verdun, V. A., Wingert, R. A. Zebrafish nephrogenesis is regulated by interactions between retinoic acid, mecom, and Notch signaling. Dev. Biol. 386, 111-122 (2014).
  39. Gerlach, G. F., Wingert, R. A. Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta. Dev. Biol. 396, 183-200 (2014).
  40. Cheng, C. N., Wingert, R. A. Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in the zebrafish. Dev. Biol. 399, 100-116 (2015).
  41. Lessman, C. A. The developing zebrafish (Danio rerio): A vertebrate model for high-throughput screening of chemical libraries. Birth Defects Res. C Embryo Today. 93, 268-280 (2011).
  42. Poureetezadi, S. J., Wingert, R. A. Congenital and acute kidney disease: translational research insights from zebrafish chemical genetics. Gen. Med. 1, 112 (2013).
  43. Poureetezadi, S. J., Donahue, E. K., Wingert, R. A. A manual small molecule screen approaching high-throughput using zebrafish embryos. J. Vis. Exp. (93), e52063 (2014).
  44. Peng, H. C., Wang, Y. H., Wen, C. C., Wang, W. H., Cheng, C. C., Chen, Y. H. Nephrotoxicity assessments of acetaminophen during zebrafish embryogenesis. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 151, 480-586 (2010).
  45. Wu, T. S., Yang, J. J., Yu, F. Y., Liu, B. H. Evaluation of nephrotoxic effects of mycotoxins, citrinin and patulin, on zebrafish (Danio rerio) embryos. Food Chem. Toxicol. 50, 4398-4404 (2012).
  46. Ding, Y. J., Chen, Y. H. Developmental nephrotoxicity of aristolochic acid in a zebrafish model. Toxicol. Appl. Pharmacol. 261, 59-65 (2012).
  47. Zennaro, C., et al. Podocyte developmental defects caused by adriamycin in zebrafish embryos and larvae: a novel model of glomerular damage. PLoS One. 9, e98131 (2014).
  48. Ding, Y. J., Sun, C. Y., Wen, C. C., Chen, Y. H. Nephroprotective role of resveratrol and ursolic acid in aristolochic acid intoxicated zebrafish. Toxins. 7, 97-109 (2015).
  49. Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of zebrafish embryos to analyze gene function. J. Vis. Exp. (27), e1115 (2009).
  50. Yuan, S., Sun, Z. Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J. Vis. Exp. (27), e1113 (2009).
  51. Christou-Savina, S., Beales, P. L., Osborn, D. P. Evaluation of zebrafish kidney function using a fluorescent clearance assay. J. Vis. Exp. (96), e52540 (2015).

Tags

ZebrafishAcute Kidney InjuryNephrotoxinGentamicinCisplatinEmbryoMicroinjectionAgaroseE3MatingSpawningEmbryo CollectionIncubationSomite Stage

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McKee, R. A., Wingert, R. A. Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury. J. Vis. Exp. (113), e54241, doi:10.3791/54241 (2016).
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