Evaluation of zebrafish kidney function using a fluorescent clearance assay

J Vis Exp. 2015 Feb 20;(96):e52540. doi: 10.3791/52540.

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. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Disease Models, Animal*
  • Female
  • Fluorescent Dyes / pharmacokinetics*
  • Kidney / metabolism*
  • Male
  • Renal Insufficiency, Chronic / blood
  • Renal Insufficiency, Chronic / metabolism*
  • Renal Insufficiency, Chronic / urine
  • Zebrafish

Substances

  • Fluorescent Dyes