Simultaneous Assessment of Glomerular Filtration and Barrier Function in Live Zebrafish

The zebrafish pronephros is a well-established model to study glomerular development, structure and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extent the available methods by simultaneously measuring the intravascular clearances of Alexa Fluor 647-conjugated 10 kDa dextran and FITC-conjugated 500 kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, the mean fluorescence intensities of both dextrans were measured in the vein of living 4 dpf zebrafish by confocal microscopy over time. We demonstrate that injected 10 kDa dextran is rapidly cleared from the circulation, becomes visible in the lumen of the pronephric tubule, quickly accumulates in tubular cells and is detectably excreted at the cloaca. By contrast, 500 kDa dextran cannot be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholinos against zMyh9 or zApo L1, both of which are known to play an important role in glomerular function. While glomerular filtration was reduced in zMyh9 morpholino injected larvae, glomerular barrier function remained intact. By contrast, in zApoL1 morpholino injected larvae, glomerular barrier function was compromised as 500 kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish.