Poly(ADP-ribosyl)ation Regulates the Dynamic Assembly of Stress Granules and Alleviates Renal Pyroptosis in AKI

Background: Acute kidney injury (AKI), a critical clinical condition with complex mechanisms, lacks effective therapies. Stress granules (SGs), dynamic membraneless organelles, enable cells to endure various stressors. Proper and dynamic assembly is essential for SGs’ function, among which poly(ADP-ribosyl)ation (PARylation) is an important post-translational mode. However, the roles of SGs in kidney remain unclear. Pyroptosis, characterized by cellular swelling and severe inflammation, is a specific form of tubular cell death in AKI. Although both SGs and pyroptosis are triggered by cellular stress, they result in contrasting cell fate. The crosstalk between SGs and pyroptosis, as well as how it influences cell fate, has been poorly studied. Methods: Cisplatin was used to establish AKI mice model as well as in vitro experiment. PJ34, a poly(ADP-ribose) polymerase 1 inhibitor, was used to suppresses PARylation. Poly (ADP-ribose) glycohydrolase (PARG) siRNA was transfected to inhibit the dePARylation and disassembly of SGs. Immunofluorescence (IF) was utilized to detect SGs. Additionally, Western blot, immunohistochemistry, and co-immunoprecipitation were employed in this study. Results: IF staining revealed that, both in vitro and in vivo, the SGs marker protein G3BP1 formed distinct aggregates with cisplatin stimulation. The size and number of SGs peaked at 24 h and then disappeared gradually. Interestingly, interference with PARylation using PJ34 suppressed cisplatin-induced SGs assembly, whereas PARG siRNA-mediated inhibition of G3BP1 dePARylation increased SGs size. These findings suggest that PARylation regulates SGs' dynamic assembly. Notably, DEAD-box helicase 3 X-linked (DDX3X), an NLR family pyrin domain containing 3 (NLRP3) inflammasome activator, was observed to co-localize with G3BP1-labeled SGs, accompanied by reduced DDX3X-NLRP3 binding in the cytoplasm. This resulted in reduced NLRP3-associated inflammation. These data suggest that the formation of SGs leads to the sequestration of DDX3X, thereby blocking NLRP3 inflammasome activation and subsequent pyroptosis. Conclusion: SGs play a critical role in the survival of stressed tubular cells. SGs formation sequesters DDX3X, thereby regulating NLRP3 inflammasome activation and pyroptosis. Collectively, these findings reveal a potential mechanism underlying the protective role of SGs during AKI.