Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Summary

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Abstract

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 adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to "dial" the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

Introduction

Acute Kidney Injury (AKI)^1,2, which also can be referred to as acute renal failure, is broadly defined as a sudden impairment in kidney function³. While the level of understanding of this condition has been enhanced remarkably over the years, morbidity and mortality rates have remained high^1,2. The current treatment for this condition is mostly supportive, as results from multiple clinical trials of drug therapy have been negative^4,5. The kidney is unique in that it has ....

Protocol

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the NYIT College of Osteopathic Medicine Institutional Animal Care and Use Committee (NYITCOM IACUC). All surgery and in vivo experimentation was performed under tricaine anesthesia, and all efforts were made to minimize suffering.

1. Obtaining and Maintaining Embryos

1. Obtain embryos by crossing kidney GFP fluorescent zebrafish.
   NOTE: Examples of fluorescently labeled zebrafish transgenic lines are listed in Table 1

Results

Please note that this protocol was successfully used with a number of kidney GFP transgenic lines, including ET(krt8:EGFP)sqet11-9, ET(krt8:EGFP)sqet33-d10, and Tg(atp1a1a.4:GFP). The example results shown here were obtained using the ET(krt8:EGFP)sqet11-9 line.

Figure 1 shows the example photoablation protocol. The average GFP intensity is monitored inside the region of interest (

Discussion

It should be noted that the total laser power varies between systems. However, using percent GFP photobleaching allows for a readout of total energy delivered to the fluorescent kidney, independent of the variation in laser power and compensated for by the length of exposure. Keep in mind, however, that the response of different tissues to this method of photoablation varies. Even during kidney maturation, it is significantly more difficult to obtain a 50% reduction in GFP fluorescence in younger embryos than in mature l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Petri Dishes, 35 x 10 mm	Genesee Scientific	32-103	Procedural Usage: Step 2.4, 2.7
Petri Dishes, 100 x 15 mm	Midwest Scientific	910	Procedural Usage: Step 1
Dechorination forceps - Electron Microscopy Sciences Dumont Tweezers 5 Dumostar	Fischer Scientific	50-241-57	Procedural Usage: Step 2.1.1
Plastic Transfer Pipet	Globe Scientific	135030	Procedural Usage: Step 2.5, 3.6
Tricaine	Sigma Aldrich	A5040-25G	Procedural Usage: Step 2.3, 3.4
Agarose	Fischer Scientific	BP165-25	Procedural Usage: Step 2.3
Pulled glass probe (manufactured manually from glass capillary tubes)	Fischer Scientific	21-1640-2C	Procedural Usage: Step 2.4
Stereomicroscope	Nikon	SMZ1270	Procedural Usage: Step 1.5
SOLA Light Engine	Lumencor	SOLA SM-5-LCR-SB	Procedural Usage: Step 1.5
Eclipse C2 Plus Confocal Microscope System	Nikon		Procedural Usage: Step 3
1x E3 Solution			Recipe used to generate: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 , 0.33 mM MgSO4 Procedural Step Usage: 1.2, 1.3, 2.2, 2.3
PTU	Sigma	P7629-10G	Procedural Step Usage: 1.3, 2.2, 3.4, 4.2
NIS Elements Software	Nikon	C2+	Procedural Usage: Step 3
Laser Unit	Agilent	MLC 400	Procedural Step 3.11
Propidium Iodide (PI)	Sigma Aldrich	P4170-100MG	Procedural Step Usage: 4.2