Abstract Body: Introduction. The renin-angiotensin system (RAS) is a crucial cardiovascular and renal function regulator. Podocytes, glomerular epithelial cells that play a pivotal role in maintaining the renal filtration barrier, are key targets for RAS peptides. Our findings showed that RAS can promote calcium (Ca²⁺) and nitric oxide (NO) signaling in podocytes. Moreover, a decrease in NO bioavailability directly correlates with glomerular filtration barrier damage, Ca²⁺ overload, and the development of salt-sensitive hypertension. We hypothesized that RAS-mediated NO production is crucial for Ca²⁺ control and cytoskeletal rearrangements in podocytes, contributing to glomerular filtration regulation.
Methods. We performed live confocal imaging on human podocytes and freshly isolated rat glomeruli to detect the production of NO (DAF-FM) and Ca²⁺ (Fluo-8). RAS peptides (Ang II, III, IV, 1-7, 1-9, and TRV120027) were used to stimulate cells, and NO and Ca²⁺ production were measured. Blockers for Ang II Type 1 (AT1R), Ang II Type 2 (AT2R), and MAS receptors were used to identify the pathways involved in NO production. The dynamic changes in podocyte cytoskeleton mediated by RAS-NO intracellular signaling were detected using rhodamine-phalloidin staining and scanning ion microscopy. The NOS isoform-specific blockers were used to determine the source of NO in podocytes. ANOVA with post-hoc was used for comparisons.
Results. Angiotensins II and III generate the most profound increases in NO levels in podocytes. Ang II, Ang III, and Ang 1-7 promote NO production by activating AT2R (n ≥ 20, p < 0.01). Interestingly, the increase in NO modulates AT1R- and β-arrestin-mediated Ca²⁺ signaling (n ≥ 20, p < 0.05), suggesting the role of the NO/AT2R axis in Ca²⁺ control in podocytes. NO production correlates with podocyte volume increase, and scanning ion microscopy experiments further confirmed that AT2R activation and corresponding NO elevation are responsible for the protrusion of podocyte's lamellipodia. Finally, we found that in podocytes, NOS1 is responsible for NO bioavailability, accounting for 69±5% of the total NO response, while the remaining NO levels are generated by NOS2 (39±5%).
Conclusion. Our data show that RAS activation promotes NO production in podocytes, mainly via the AT2R pathway. NO is crucial for Ca²⁺ control in podocytes and lamellipodia formation, suggesting the essential role of NO in glomerular filtration barrier function.