Abstract: FR-PO764
Identification of RNASE1 as a Novel Contributor to Cytoskeleton Remodeling in Podocytes
Session Information
- Glomerular Diseases: Mechanisms and Podocyte Biology
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Bolanos-Palmieri, Patricia, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
- Krüger, René, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
- Azeloglu, Evren U., Icahn School of Medicine at Mount Sinai, New York, New York, United States
- Schiffer, Mario, Universitatsklinikum Erlangen Medizinische Klinik 4 Nephrologie und Hypertensiologie, Erlangen, Bayern, Germany
Background
A dynamic cytoskeleton is critical to the health and function of the podocytes as it provides structural and signalling support to the cell. We have previously shown that recovering the cytoskeleton can be a viable strategy to treat podocyte dysfunction. In this project, we used a unique human peptide library to identify native bioactive peptides with the capacity to modify the podocyte cell structure and cytoskeleton.
Methods
To test the fractions derived from the hemofiltrate peptide library, protein kinase C epsilon knockout mouse podocytes (PKCε KO) were used as a model for cytoskeletal dysfunction. After iterative tests in cell culture, the bioactive fraction was selected and the candidate peptide (CP) was identified by mass spectrometry. We used a purified recombinant version of the CP to test its effect on PKCε KO cells and on dasatinib treated human podocytes. Morphological changes were quantified by imaging components of the cytoskeleton. Possible mechanistic targets were analyzed by western blot and transcriptomics.
Results
Using PKCε KO podocytes we systematically tested the hemofiltrate and were able to identify one fraction capable of influencing cell morphology. Further analysis showed that RNASE1 could be a mediator of the observed structural changes. To explore this, additional tests were performed using recombinant RNASE1 which showed that exposing the PKCε KO podocytes to RNASE1 lead to an increase in cell spreading and number of focal adhesions resulting in improved cell attachment and cell morphology that more closely resemble that of wild type podocytes. This result was further confirmed using other injury models like human podocytes exposed to dasatinib. Preliminary data looking into the mechanisms involved in cytoskeleton rearrangement in response to RNASE1 suggest that there may be differential tyrosine kinase signalling in podocytes treated with RNASE1. Additionally, RNA sequencing identified 18 differentially regulated genes as candidates for further analysis.
Conclusion
This systematic approach led to the identification of a novel candidate protein able to positively alter podocyte morphology. By modifying the structural changes that result from injury, RNASE1 could provide new insights into the molecular mechanisms that result in podocyte dysfunction and proteinuria as well as open new avenues for treatment.
Funding
- Private Foundation Support