Glo1-/- zebrafish mutants display differential susceptibility of vascular and renal systems

Background and aims:

The formation of microvascular complications in patients suffering from diabetes mellitus, is associated with the accumulation of advanced glycation endproducts (AGEs). AGEs are mainly produced by reactive dicarbonyl methylglyoxal (MG), which modifies proteins, lipids and DNA and therefore alters their function. The effects of elevated MG levels in vivo are poorly understood. Our aim is to analyze the impact of a knockout of the glyoxalase system, the main MG-detoxifying system, on vasculature and kidney structure and function under normoglycemic and hyperglycemic conditions in the zebrafish.

Materials and methods:

For a permanent knockout of Glo1 and Glo2 in vascular tg(fli:EGFP) and renal tg(wt1b:EGFP) zebrafish reporter lines, the CRISPR/Cas9 technology was used. Mosaic, heterozygous and homozygous mutant zebrafish embryos were phenotypically analysed using fluorescence and confocal microscopy.

Results:

Methylglyoxal alters development of the pronephros in Glo1-Glo2-CRISPR injected zebrafish embryos (crispants) resulting in a decreased length of the neck and an increased length of the glomerulus. Regarding the vasculature, Glo1-CRISPR injected zebrafish embryos show altered intersomitic blood vessels which could not be aggravated in Glo1/Glo2 double injected embryos. In order to study a permanent gene knockout of Glo1, Glo1-knockout zebrafish lines were generated. Glo1^-/- embryos are viable and show normal pronephric structures and kidney function under normoglycemic conditions.

Conclusion:

The Glyoxalase system is important in the development of vasculature and pronephros in zebrafish embryos, whereas preliminary Glo1^-/- data suggests the presence of a compensatory mechanism activated during generation of a stable mutant line.