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Abstract: SA-PO365

Identifying the Role of Soluble Circulating Permeability Factors in Proteinuria in a Zebrafish Parabiosis Model

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Schenk, Heiko Joachim, Hannover Medical School, Hannover, Germany
  • Müller-Deile, Janina, Hannover Medical School, Hannover, Germany
  • Schroder, Patricia Ann, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, United States
  • Schiffer, Mario, Hannover Medical School, Hannover, Germany
Background

Proteinuria may be induced by an impairment in any component of the glomerular filtration barrier. To determine the role of circulating permeability factors to cause proteinuria through glomerular damage as a result from reduced specific podocyte protein expression, we developed a parabiosis-based zebrafish model to generate a common circulation between two zebrafish larvae.

Methods

Zebrafish eggs were dechorionated at the 128-cell blastula stage and moved to the desired orientation for fusion that was achieved by a micro cell transfer from one embryo to the other with a glass micropipette. We injected npnt-morpholino (MO) into the yolk of a 1-4 cell embryo of Tg(flk1-mCherry) zebrafish and performed the fusion at a 256-cell stage with control Tg(l-fabp:eGFP-DBP) zebrafish embryos. By using a FITC-labelled MO, we ruled out a MO-transfer between both parabionts. Common circulation and loss of plasma proteins were investigated and the glomeruli of both zebrafish pairs were analyzed using electron microscopy.

Results

We fused blastulae from Tg(l-fabp:eGFP-DBP) zebrafish that express a green fluorescent vitamin D binding protein in their blood plasma with blastulae of Tg(flk1-mCherry) zebrafish that express a red fluorescent endothelium. Confocal imaging of parabiotic zebrafish with these backgrounds showed a detectable green fluorescent plasma protein from the Tg(l-fabp:eGFP-DBP) zebrafish in the red fluorescent blood vessels of the Tg(flk1-mCherry) parabiotic partner at 72 hpf, indicative of a common circulation system. Fluorescence analysis of the parabiotic zebrafish larvae after npnt knockdown revealed a reduction of the eGFP-labelled protein from the common circulatory system suggestive of proteinuria. Knockdown of npnt in the injected parabiont showed podocyte effacement and altered glomerular basement membrane. Remarkably, also the control parabiotic partner developed podocyte effacement and glomerular endothelial swelling as well but had a regular glomerular basement membrane. We could not only detect proteinuria and glomerular damage caused by the knockdown of the podocyte gene npnt in injected fish, but also in the fused partner.

Conclusion

These data suggest that circulating permeability factors may be induced by proteinuria even when an induced dysregulation of a podocyte gene is the initiating cause.