Universität zu Köln

Rutkowski, Nelli ORCID: 0000-0003-3672-4310 (2024). FRET-based visualization of cGMP dynamics in glomerular endothelial cells and podocytes. PhD thesis, Universität zu Köln.

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Abstract

Podocytes play a critical role in maintaining the integrity of the glomerular filtration barrier, a trilaminar structure that prevents the loss of macromolecules into the urine. These cells extend prominent cellular protrusions, termed foot processes, which contain an actin-based contractile system as a distinctive cellular structure. Podocytes can adapt to renal hemodynamic changes by modifying the foot process actin cytoskeleton, while they are exposed to significant physical forces resulting from pressure and fluid flow across the filtration slit. However, due to their limited capacity for self-renewal, podocyte loss is a hallmark in the progression of glomerular kidney diseases. Vasoactive factors, including angiotensin II (ANGII), are synthesized by glomerular cells upon stimulation and influence glomerular filtration. Among these factors, the role of the second messenger cyclic guanosine 5' monophosphate (cGMP) in podocytes and glomerular endothelial cells (GECs) remains elusive. Additionally, the presumed protective role of restored cGMP levels in various cardiovascular and renal diseases has sparked interest in exploring novel therapeutic strategies. This work aimed to evidence cGMP signaling in podocytes and GECs, and to elucidate cell-specific differences in cGMP dynamics. Using a stepwise approach, we analyzed isolated glomeruli and acute kidney slices from transgenic mice expressing the FRET-based ratiometric cGMP sensor (cGi500) either in podocytes or in endothelial cells. We stimulated both cGMP-synthesizing guanylyl cyclase systems (soluble (sGC) or particulate/membrane-bound (pGC)) independently or simultaneously. Both cell types responded with an increase in intracellular cGMP, while showing differences in both the intensity and duration of the response. Podocytes of cGi500 biosensor mice responded with a prolonged pGC-mediated cGMP signaling, emphasizing a predominant role for this pathway. GECs of cGi500 biosensor mice answered with a transient cGMP response after stimulation. Co-stimulation in podocytes did not result in the expected additive effect on cGMP levels, as observed in GECs. Moreover, increased cGMP levels were triggered by stimuli inhibiting cGMP degradation, cGMP export, or indirectly through calcium influx-related mechanisms in podocytes. Cell-specific differences in cGMP degradation were evident, with podocytes exhibiting comparatively lower baseline activity of the tested phosphodiesterase (PDE) isoforms. Notably, the most active isoforms, PDE3 and PDE5, had a preference towards the pGC/cGMP signaling pathway. On the contrary, in GECs, the basal activity of PDE2a was the highest of all isoforms tested, and had an equivalent impact on both cGMP pathways. In summary, our data provided unprecedented insights into cell-specific dynamics of cGMP signaling in glomerular cells. These findings contribute to a deeper understanding of cGMP signaling in renal physiology and reveal potential targets for therapeutic interventions.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Rutkowski, Nelli nelli.rutkowski@uk-koeln.de orcid.org/0000-0003-3672-4310 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-746130
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	CECAD - Cluster of Excellence Cellular Stress Responses in Aging-Associated Diseases
Subjects:	Life sciences Medical sciences Medicine
Uncontrolled Keywords:	Keywords Language cGMP signaling English FRET English Kidney English
Date of oral exam:	28 May 2024
Referee:	Name Academic Title Benzing, Thomas Prof. Dr. Kloppenburg, Peter Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/74613