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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

Published: July 17th, 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

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

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 decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

AKI is an abrupt loss of kidney function that can lead to devastating health consequences1. AKI is a significant healthcare issue worldwide due to its high incidence of approximately 20% among hospitalized patients, with even higher rates of 30-50% in critical care cases and the elderly, and mortality rates of 50-70%1-3. Unfortunately, the prevalence of AKI has been increasing and is projected to escalate further over the next decade, due in part to the diversity of factors that can induce AKI, which include post-operative stress, ischemia, and exposure to nephrotoxins such as antibiotics and chemotherapeutic drugs4.

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The procedures for working with zebrafish embryos described in this protocol were approved by the Institutional Animal Care and Use Committee at the University of Notre Dame.

1. Preparation of Solutions

  1. Make a 50x stock solution of E3 embryo media by mixing 73.0 g NaCl, 3.15 g KCl, 9.15 g CaCl2, and 9.95 g MgSO4 in 5 L of distilled water, and store at RT.
  2. For culturing of zebrafish embryos, dilute the 50x stock solution of E3 embryo media stock to a 1x working solution w.......

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A microinjection station set up includes a stereomicroscope, micromanipulator and pressure regulator (Figure 1A). Transillumination of the injection plate is preferable to view specimens during this procedure (Figure 1B). Preparation of the injection needle involves pulling the appropriate borosilicate glass, followed by preparing the edge with cutting and finally back-loading the needle. Optimally, the needle tip is beveled rather than blunt (Fig.......

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A diverse number of therapeutic agents have been associated with AKI29. There have been significant research advances in understanding the damage induced by many individual compounds, such as the aminoglycoside gentamicin30 and the widely used chemotherapeutic cisplatin31,32. Some pathological changes involved in these conditions, however, remain the subject of ongoing study. One emergent challenge remains understanding how multiple drugs adversely affect patients, especially those in hig.......

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This work was supported in part by the NIH grant DP2OD008470. Additionally, RAM was supported in part by funds provided by the University of Notre Dame Graduate School. We thank the staffs of the Department of Biological Sciences, the Center for Zebrafish Research, and the Center for Stem Cells and Regenerative Medicine at the University of Notre Dame. We especially thank the members of lab for engaging discussions about kidney biology and their helpful feedback on this work.

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Name Company Catalog Number Comments
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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