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Abstract: PO1702

Possible Role of the Cytosolic RNase Inhibitor in Maintaining the Integrity of the Glomerular Filtration Barrier

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Bolanos-Palmieri, Patricia, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
  • Tiefenböck, Franz, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
  • Schiffer, Mario, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
Background

The ribonuclease inhibitor (RI) is a cytoplasmic protein encoded by the RNH1 gene. The RI forms a tight non-covalent bond with members of the RNaseA superfamily and therefore it’s considered that its main function is to act as a sentinel for dysregulated RNases. The ratio of RI to substrate has also been shown to vary according to the proliferative and metabolic status of the cell suggesting that the balance in the protein-protein interactions between the RI and its substrates plays a role in maintaining cell homeostasis by impacting processes such as protein synthesis and various signaling pathways. However, the entirety of the biological roles fulfilled by the RI are yet to be described.

Methods

Immunofluorescence staining of human kidney tissue and protein detection by western blot from samples of immortalized podocytes were carried out to confirm the presence of the RI in glomerular cells. As an initial injury model, podocytes were treated with PAN to determine if changes in RI expression occur as a response. RNH1 inhibition was performed in a transgenic zebrafish line that allows for the detection of proteinuria via a GFP-tagged protein in the circulation. At 96hpf the severity of the edema phenotype and the fluorescence levels were recorded.

Results

Our preliminary data shows that the RI is present in human podocytes both in vitro and in vivo, as its expression domain coincides with glomerular cells labelled with synaptopodin. Additionally, the RI is present in cultured podocytes both before and after the temperature shift used to induce quiescence, and it appears to increase after differentiation. There is also expression modulation of RNH1 in response to PAN treatment of podocytes. In our zebrafish model, RNH1 knockdown resulted in up to a 40% increase in mild to severe edema and over 30% decrease in fluorescence suggesting that a reduction in RI might compromise the filtration barrier enough to lead to proteinuria.

Conclusion

Taken together our initial data show a promising avenue for research where the balance between members of the RNaseA superfamily and their cytoplasmic inhibitor may represent a powerful mechanism that allows the cells to modulate the proteome in response to stimuli. An imbalance in this relationship might ultimately affect the integrity of the cells in the kidney.

Funding

  • Government Support – Non-U.S.