Abstract: FR-PO704
Loss of Mitf Aggravates PAN Induced Proteinuria in Zebrafish
Session Information
- Glomerular: Basic/Experimental Pathology - II
November 03, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Glomerular
- 1002 Glomerular: Basic/Experimental Pathology
Authors
- Niggemann, Philipp, Hanover Medical School, Freiburg, Germany
- Schroder, Patricia Ann, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, United States
- Bolanos-Palmieri, Patricia, Hanover Medical School, Freiburg, Germany
- Müller-Deile, Janina, Hanover Medical School, Freiburg, Germany
- Schenk, Heiko Joachim, Hanover Medical School, Freiburg, Germany
- Beverly-Staggs, Laura L., Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, United States
- Teng, Beina, Hanover Medical School, Freiburg, Germany
- Haller, Hermann G., Hanover Medical School, Freiburg, Germany
- Schiffer, Mario, Hanover Medical School, Freiburg, Germany
Background
We developed a standardized proteinuria model in zebrafish using puromycin aminonucleoside (PAN) through treatment via the water. We noticed, that fish with the nacre mutation show a significantly higher susceptibility to PAN than AB fish upon exposure to the same dosage of PAN. (AB is a standard wild-type line)
Nacre is the name of a mutation yielding a truncated version of Microphthalmia-associated transcription factor (Mitf) in zebrafish. Mitf is an evolutionary conserved transcription factor that controls pigment cell fate in vertebrates. It is well known that a mutation in mitf leads to missing neural-crest-derived melanophores and results in a pigment-less phenotype, making this mutation a commonly used fish line to study organ development.
Methods
Tg(l-fabp:eGFP-DBP) zebrafish were backcrossed onto AB or nacre background and were exposed to PAN in the water at varying timepoints from 44 hours post fertilization (hpf) to 50 hpf. Loss of high molecular weight proteins from the circulation was measured at 96 hpf as a surrogate marker for proteinuria.
In addition, we performed mitf knockdown experiments in AB zebrafish using morpholinos. Moreover, cultured human podocytes were examined after silencing of MITF in vitro.
Results
Zebrafish homozygous for the nacre mutation and AB zebrafish after knockdown of mitf exhibited stronger proteinuria after PAN treatment compared to control animals.
Moreover, a treatment with PAN at 46 hpf yielded the strongest proteinuria. Treatments at later timepoints were less effective in proteinuria induction.
Silencing of MITF in human podocytes led to a disrupted cytoskeletal organization after PAN treatment. At the same time the expression of MITF downstream partners like INF2 was changed, indicating that MITF plays an important role for cytoskeleton recovery in podocytes.
Conclusion
We present the first reproducible PAN-nephrosis model in zebrafish, which could serve as a suitable setting for drug testing in zebrafish. Furthermore, we can demonstrate that a mutation in mitf leads to a higher susceptibility for disruption of the glomerular filtration barrier upon PAN treatment, which results in stronger proteinuria.