OX40L Exacerbates Tubular Injury and Interstitial Inflammation in AKI-to-CKD Transition Through GSK3α

Background: The incidence of acute kidney injury (AKI) has been steadily increasing recently, with long-term consequences including chronic kidney disease (CKD) and end-stage renal disease. Inflammation is recognized as a critical driver in AKI-to-CKD, promoting interstitial fibrosis. Tumor necrosis factor ligand superfamily member 4 (TNFSF4), also known as OX40L, functions as a co-stimulatory ligand for T cells and plays a critical role in inflammation. However, the role and mechanisms of OX40L-mediated inflammation in AKI-to-CKD remain unclear. Methods: The expression of OX40L in tubular cells in patients with renal fibrosis and AKI-to-CKD animal models was evaluated using immunoblotting, real-time PCR, and immunostaining. Proximal tubular cell-specific Tnfsf4 knockout mice were generated to investigate the role of OX40L in AKI-to-CKD. In vitro, the effects of OX40L were assessed by OX40L siRNA or overexpression plasmids. Furthermore, immunoprecipitation-mass spectrometry (IP-MS) and co-inmunoprecipitation (Co-IP) was employed to identify OX40L-interacting proteins aiming to elucidate its functional role in the AKI-to-CKD transition. Results: We identified a significant upregulation of OX40L expression in tubular cells in patients with renal fibrosis and AKI-to-CKD animals. Elevated OX40L levels were positively correlated with tubular interstitial injury and negatively correlated with eGFR. Proximal tubule-specific knockout of OX40L attenuated tubular injury, interstitial fibrosis and renal inflammation in AKI-to-CKD mice induced by ischemia-reperfusion injury or repeated low-dose cisplatin. In vitro, OX40L overexpression markedly enhanced inflammation, apoptosis, and extracellular matrix (ECM) production in tubular cells exposed to TGF-β1 or chronic hypoxia. Mechanistically, IP-MS analysis and Co-IP suggested a potential interaction between OX40L and glycogen synthase kinase-3α (GSK3α). Silencing OX40L aggravated tubular inflammation, injury, and ECM accumulation, effects that were partially reversed by GSK3α knockdown. More importantly, OX40L overexpression markedly attenuates the ubiquitination of GSK3α in HEK293T. Conclusion: These results reveal that OX40L exacerbates tubular injury and interstitial inflammation in AKI-to-CKD transition through GSK3α, which may offer a novel therapeutic strategy to prevent or slow the progression of AKI-to CKD. Funding: Government Support – Non-U.S.