Name: evaluation of zebrafish kidney function using a fluorescent clearance assay
Uploaded: 2015-02-20T13:00:00
Description: Watch this Scientific Journal Video about Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay at JoVE.com

Technical assistance provided by Jaipreet Bharj. This work was supported by grants from EU-FP7 (SYSCILIA -241955) and The Dutch Kidney Foundation (CP11.18).

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....

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Zebrafish offer a valuable tool to model human genetic disease, their use as a scientific instrument for&#160;in vivo&#160;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 ...

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 polycystin-1 (PKD1) and -2 (PKD2), accounting for approximately 85% and 15% of cases respectively12,13. Furthermore, the gene products for PKD1 and -2 localize to the cilium and are fundamental to ciliogenesis14,15. There is now a recognized family of human genetic disorders, known as the ciliopathies, which affect cilia function and result in CKD16.
The growing number of human genetic diseases affecting ciliary development and function is drawing global interest in this once considered vestigial organelle. The cilium, a hair-like cellular protrusion, is enriched with receptors and ion channels necessary for the transduction of key cell signaling events. The cilium consists of a microtubule-based axoneme, typically structured into nine radially arranged microtubule doublets with or without a central pair of singlet microtubules. The axonemal structure defines the type and mode of ciliary action. The 9+2 microtubule arrangement confers motility to the cilium where it is utilised in the movement of fluids across epithelial surfaces. The 9+0 configuration is non motile but is believed to mainly function in cellular signalling events17. Apart from CKD, the consequences of ciliary dysfunction are a set of characteristic ciliopathy features that include, obesity, retinal degeneration, polydactyly, and cognitive impairment16. However, CKD is amongst the most detrimental to the patient&#8217;s quality of life and therefore a major driving force behind the development of appropriate in vivo models for ciliary related CKD.
The zebrafish is an excellent model to understand the etiology of human genetic disease. Their quick development, production of large number of eggs, transparent tissue, and ex utero growth allows zebrafish developmental processes to be visualized and biological events manipulated with considerable ease. Genes can be genetically altered using the recent success of genome editing tools (CRISPR18 and TALENS19), knocked down using antisense morpholino technology20, or pharmacologically regulated by the addition of compounds to their aquatic environment. Indeed, zebrafish offer a platform to undertake experiments that are not permissive in other animal models. Whilst zebrafish are relatively simple vertebrates (compared to humans) they share many functionally conserved organs, genes, and signaling processes in common with humans. For example, the zebrafish kidney is remarkably similar in structure and function compared to humans21,22. However, unlike the mammalian kidney that develops through a succession of phases, each marked by a more developed kidney (pronephros, mesonephros, and metanephros), the embryonic zebrafish only develops a pronephros, the most immature form of a kidney. Whilst millions of nephrons can be found forming the building blocks of the mammalian kidney, the zebrafish embryo only possess two. The glomeruli, which allow for the initial blood filtrate, are fused at the midline just ventral to the aorta. Blood filters through the glomeruli into the pronephric tubules that run caudally along the axis, fusing prior to exit via the cloaca. The pronephric tubules are heavily ciliated with motile cilia that are permissive to the flow of filtrate towards the caudal exit3,4. This simple pronephric structure maintains zebrafish homeostasis through several weeks of larval growth where they eventually develop into a more complex mesonephros structure21. However, the zebrafish never develops a metanephros21. Despite the zebrafish idiosyncrasies, the zebrafish nephron is segmented with gene expression profiles equal to that observed in mammals and thus offers an unrivalled in vivo model for nephrogenesis3,22.
Routinely patients are tested for kidney function through a series of blood and urinary tests. Typically the blood is analyzed for dissolved salts, urea and creatinine. High levels of urea, creatinine and abnormal salt concentrations are indicative of problems with kidney function. Urinalysis using a colorimetric dipstick detects abnormal levels of protein, blood, pus, bacteria and sugar present in urine samples. Such tests normally require approximately 30 ml of urine or 5 - 10 ml of blood. It has been difficult to translate these types of assays to small in vivo model organisms, such as the zebrafish, mainly due to the impossible nature of collecting sufficient blood or urine to perform the assay. Here, we address the lack of appropriate zebrafish kidney function tests and describe an innovative technique for its study. By injecting a fluorescent dye into the blood stream we are able to monitor and individually quantify over time the filtration and excretion of fluorescent activity from the blood via the kidney. This method can be used to study kidney damage caused by disease, which we provide an example of.

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evaluation of zebrafish kidney function using a fluorescent clearance assay

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 cloaca2-4. 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 &#8216;dipstick&#8217;, 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 function1,6-9.

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...

Watch this Scientific Journal Video about Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay at JoVE.com

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

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.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

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