Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

Summary

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay¹ that allows quantitative analysis of zebrafish kidney function in CKD.

Abstract

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca^2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish⁵. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria ‘dipstick’, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function^1,6-9.

Introduction

The human kidney plays a crucial role in filtering metabolic waste from the blood and recovering required solutes to sustain cellular homeostasis. There are a number of human genetic diseases that cause kidney dysfunction. The most common inherited renal disease is autosomal dominant polycystic kidney disease (ADPKD) characterized by the development of fluid filled sacs within nephritic tubules; the damage caused by cystogenesis is detrimental to kidney function¹⁰. ADPKD has an occurrence of 1:800 - 1:1,000 and accounts for 8 - 10% of patients in end stage renal failure (ESRF)¹¹. Several genes have been implicated to cause ADPKD including polycys....

Protocol

Ethics Statement: Animal maintenance, husbandry, and procedures are defined and controlled by the Animals (Scientific Procedures) Act 1986. All animal experimentation has been carried out under licenses granted by the Home Secretary (PIL No. 70/7892) in compliance with Biological Services Management Group and the Biological Services Ethical Committee, SGUL, London, UK. All efforts were made to reduce the number of animals used and to refine both procedures and husbandry in order to minimize suffering and enhance welfare.

1. Preparation of Instruments, Anesthetic, and Fluorescent Dye

1. Using a micropipette puller and borosilicate s....

Results

Bardet-Biedl syndrome (BBS) is a rare heterogeneous ciliopathy that affects approximately 1:160,000 people worldwide¹⁶. Patients present with a number of associated problems including polycystic kidneys, subsequently patients frequently require kidney dialysis or transplantation²⁴. ESRF is the most common cause of death in BBS, with around 30% of patients developing CKD¹⁶. Currently, 20 non-related genes have been implicated in BBS with no published genotype-phenotype association. The BBS.......

Discussion

Zebrafish offer a valuable tool to model human genetic disease, their use as a scientific instrument for in vivo research have enabled detailed studies of the genetic breakdown of many biological systems, including the kidney. Much is now understood about how the zebrafish kidney develops and functions. The striking similarities to human nephrogenesis and homology with disease causing genes²¹ has illustrated how zebrafish have become fundamental in understanding how defects in gene function .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
P-97 SUTTER Flaming/Brown type micropipette puller	Intracel	P-97
borosilicate standard wall capillaries	Harvard Apparatus	30-0017
Glass microscope slides	VWR International	631-0109
Epoxy Resin Glue	Evo-Stik
Rhodamine B 10,000 MW labeled Dextran	Life technologies	D-1824
N-Phenylthiourea	Sigma-Aldrich	P7629
Methylene blue	Sigma-Aldrich	M9140
Ethyl 3-aminobenzoate methanesulfonate salt	Sigma-Aldrich	A5040
methylcellulose	Sigma-Aldrich	M0512
air compressor	Jun-Air	OF302-15
Picospritzer III	Parker Instruments	051-0500-900
compact 3-axis control micromanipulator	Marzhauser	MM33
Dissecting stereo microscope	Nikon	SMZ1000
microloader tips	Eppendorf	5242956003
Dumont #5 forceps	Sigma-Aldrich	F6521
stage micrometer	Pyser- SGI	02A00404