Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 with distilled water, then add 200 μl of 0.05% methylene blue to every 1 L of 1x E3 to act as a fungicide, and store at RT.
3. Make a pigme....

Results

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.......

Discussion

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 cisplatin^31,32. 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.......

Materials

List of materials used in this article

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:
60 mm x 15 mm	VWR	25373-085
100 mm x 15 mm	VWR	25373-100
(microinjection tray) 150 mm x 15 mm	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