Abstract: FR-PO716
Fluorescence Resonance Energy Transfer (FRET) Based Visualization of cGMP Signaling in Glomerular Endothelial Cells and Podocytes
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1304 Glomerular Diseases: Podocyte Biology
Authors
- Rutkowski, Nelli, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, NRW, Germany
- Görlitz, Frederik, Advanced Bioimaging Center, Berkeley, California, United States
- Höhne, Martin, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, NRW, Germany
- Schermer, Bernhard, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, NRW, Germany
- Benzing, Thomas, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, NRW, Germany
- Hackl, Matthias, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, NRW, Germany
Background
Cyclic guanosine-3′,5′-monophosphate (cGMP) is an ubiquitous intracellular second messenger and is generated by the nitric oxide (NO)/soluble guanylate cyclase (sGC) and natriuretic peptide (NP)/particulate guanylate cyclase (pGC) signaling cascades. Drugs targeting this pathway have been used successfully in the treatment of vascular disease and sparked interest in translation to renal disease. In the kidney field, the literature relies on cell culture and biochemical methods, and lacks both temporal and spatial resolution to reveal the interplay and potential divergence between cell layers of a glomerulus e.g. endothelial cells and podocytes.
Methods
Acute kidney slices from mice expressing the genetically encoded fluorescent FRET-based cGMP biosensor cGi-500 exclusively in the cytosol of glomerular endothelial cells or podocytes were prepared. Binding of free cGMP leads to a decrease in FRET efficiency, as evidenced by a simultaneous change in donor and acceptor emission intensity in opposite directions. The donor/acceptor emission ratio (CFP/YFP) reflects the change in cGMP concentration over time. Agents modulating the cGMP pathway were applied with a superfusion system to record intensity measurements of a time series with confocal microscopy.
Results
Both endothelial cells and podocytes respond to stimulation with ANP (via pGC) and SNAP (NO donor, via sGC) with an increase in cytosolic cGMP concentrations. While cGMP concentrations decrease rapidly after incubation with an NO donor in the washout phase, cGMP concentrations remain high for a prolonged period after NP incubation. Simultaneous stimulation with ANP and SNAP leads to an additive response in endothelial cells, whereas cGMP concentrations in podocytes evolve in the opposite direction. Preliminary data suggest that enhanced activation of cGMP-degrading phosphodiesterases (PDE) contribute, as demonstrated by the use of the nonspecific inhibitor IBMX.
Conclusion
Levels of cGMP in glomerular endothelial cells and podocytes respond to stimulation of the NO-sGC and NP-pGC pathways. Additive stimulation leads to a further increase in cGMP levels in endothelial cells but a decrease in cGMP levels in podocytes. This is most likely due to increased PDE activity in podocytes.
Funding
- Government Support – Non-U.S.