RIPK3 exhibits a U-shaped dose-response in AKI-to-CKD progression: Optimal therapeutic window and the TGF-β1-HMGB1 feedback loop

Acute kidney injury (AKI) survivors are at risk of progression to chronic kidney disease (CKD), but stage-specific roles of RIPK3 and optimal intervention timing remain unclear. Using a bilateral renal ischemia-reperfusion injury model in Ripk3+/+, Ripk3+/-, and Ripk3-/- mice together with time-window pharmacological inhibition, we profiled RIPK3 activity across 28 days and examined upstream regulation. RIPK3 and phosphorylated RIPK3 increased from 6 h after injury and remained elevated through day 28. Unexpectedly, RIPK3 ± mice showed the mildest injury and fibrosis, whereas Ripk3-/- mice were not superior to wild-type controls, indicating a U-shaped dose-response. Intervention timing was critical: the RIPK3 Inhibitor GSK872 initiated on day 7 improved renal function, histology, and 90-day survival more than treatment starting on day 0 or day 14, coinciding with a shift from peak repair-factor expression to sustained inflammatory cytokine elevation. Mechanistically, TGF-β1 induced RIPK3 transcription via SMAD3, while RIPK3-associated necroptotic injury promoted HMGB1 release that enhanced TGF-β1/SMAD3 signaling, forming a positive feedback loop; subacute SB431542 treatment reduced fibrosis. These findings support stage- and dose-aware RIPK3 modulation as a strategy to limit AKI-to-CKD progression.