肾再生的成年斑马鱼庆大霉素性损伤
Summary
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.
Abstract
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.
Introduction
肾脏是流体平衡，血压调节和过滤废物从体内必需的。虽然哺乳动物可以利用受伤分化的上皮细胞后1-4修复肾单位存在，他们似乎缺乏保留的干细胞5的游泳池，并不能形成新的肾单位从头 。在对比哺乳动物，成鱼能够形成整个成年生活新肾支持鱼的生长，并且响应于损伤6,7。斑马鱼， 斑马鱼 ，是一种宝贵的模式生物器官再生8-10的研究，并有可能提供强大的洞察到应用程序中来改造人类的肾脏的修复。 的Tg（Lhx1a：EGFP）转基因11已经显示然而标记肾祖细胞的池，在成年斑马鱼肾12，控制lhx1a的响应+细胞损伤ř机制emain不清楚。
氨基糖甙类的庆大霉素是一种广泛使用抗生素与人类已知的13和肾耳毒性作用。腹腔注射庆大霉素是一家成立于鱼6诱发急性肾损伤的方法。这种损伤的鱼模仿的肾小管上皮细胞，而发生在人类过量服用庆大霉素后14肾小球疤痕的损失。斑马鱼中的庆大霉素注射诱导损伤是引起强烈的，同步再生反应，产生了许多新的肾单位，同时继续通过形成，增殖和分化阶段的便捷方式。
该协议通过利用无创可视化的筛选过程中尽量减少离群点细节我们的方法可再生的损伤的成年斑马鱼肾脏。我们注意到，与庆大霉素损伤导致的肾上皮组织和格式死亡的事实优势离子肾小管演员，然后积聚成群众中肾管和泄殖腔。这些是由鱼传递，并且可以在水目视观察。这使我们能够对鱼类具有较强的非致命伤害，然后可以为汇集进一步的实验筛选。最小化的未受伤的鱼或鱼，才到达实验的终点导致更均匀和高效的数据收集和分析该模具的数量。此外，没有任何特殊的设备或试剂要求，使得这种方法具有成本效益，并适用于在学术或教学环境中使用。使用我们的方法，在这里，我们表明肾再生庆大霉素剂量的增加效果以及增加温度的作用。
Protocol
Representative Results
Discussion
斑马鱼是理想的学习成人器官再生包括成人中肾肾脏6。最近的研究已经利用分子标记技术和转基因新记者行更好地表征在肾再生发生的步骤，哪些细胞可能是负责7,12。尿管型的观察已使用了一个多世纪来诊断肾脏疾病在人类16。在这里，我们使用管型的存在，作为庆大霉素注射后一种简单，非侵入性的和早期指标的急性肾损伤的程度。我们观察到的鱼，不产生强制类型转…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| gentamicin sodium sulfate | Sigma | G1264 | TOXIC, purity varies from batch to batch |
| plastic transfer pipets | Fisher | 13-711-7M | |
| 1 ml Norm-Ject syringes | Electron Microscopy Sciences | 72520 | green plastic syringes, ordinary 1ml syringes are OK, but harder to read accurately |
| 30G1/2 needles | Becton Dickinson | 305106 | |
| ethyl 3-aminobenzoaate methanesulfonate salt (tricaine) | Sigma | A5040 | IRRITANT |
| 16% paraformaldehyde | Electron Microscopy Sciences | 15710 | Make 4% in 1xPBS for working solution |
References
- Humphreys, B. D., et al. Intrinsic epithelial cells repair the kidney after injury. Cell Stem Cell. 2, 284-291 (2008).
- Humphreys, B. D., et al. Repair of injured proximal tubule does not involve specialized progenitors. Proc Natl Acad Sci U S A. 108, 9226-9231 (2011).
- Guo, J. K., Cantley, L. G. Cellular maintenance and repair of the kidney. Annu Rev Physiol. 72, 357-376 (2010).
- Cirio, M. C., de Groh, E. D., de Caestecker, M. P., Davidson, A. J., Hukriede, N. A. Kidney regeneration: common themes from the embryo to the adult. Pediatr Nephrol. , (2013).
- Little, M. H., Bertram, J. F. Is there such a thing as a renal stem cell. J Am Soc Nephrol. 20, 2112-2117 (2009).
- Reimschuessel, R. A fish model of renal regeneration and development. ILAR J. 42, 285-291 (2001).
- 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).
- Poss, K. D., Wilson, L. G., Keating, M. T. Heart regeneration in zebrafish. Science. 298, 2188-2190 (2002).
- Goessling, W., et al. APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development. Dev Biol. 320, 161-174 (2008).
- Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., Parsons, M. J. Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase. Mech Dev. 124, 218-229 (2007).
- Swanhart, L. M., et al. Characterization of an lhx1a transgenic reporter in zebrafish. Int J Dev Biol. 54, 731-736 (2010).
- Diep, C. Q., et al. Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature. 470, 95-100 (2011).
- 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 (2011).
- Hentschel, D. M., et al. Acute renal failure in zebrafish: a novel system to study a complex disease. Am J Physiol Renal Physiol. 288, F923-F929 (2005).
- Gerlach, G. F., Schrader, L. N., Wingert, R. A. Dissection of the adult zebrafish kidney. J Vis Exp. , (2011).
- Fogazzi, G. B., Cameron, J. S. Urinary microscopy from the seventeenth century to the present day. Kidney Int. 50, 1058-1068 (1996).
- Nachtrab, G., Czerwinski, M., Poss, K. D. Sexually dimorphic fin regeneration in zebrafish controlled by androgen/GSK3 signaling. Curr Biol. 21, 1912-1917 (2011).
- Zhou, W., Hildebrandt, F. Inducible podocyte injury and proteinuria in transgenic zebrafish. J Am Soc Nephrol. 23, 1039-1047 (2012).
- Huang, J., et al. A zebrafish model of conditional targeted podocyte ablation and regeneration. Kidney Int. 83, 1193-1200 (2013).
