(1341) OPN Released by Renal Tubular Cells Promotes Macrophage Influx and Tubular Cell Pyroptosis in AKI

Introduction: To facilitate the discovery of novel therapeutic targets SA-AKI, understanding the molecular mechanism is vital. In our research, we analyzed bulk and single-cell transcriptomic data of various AKI models. Findings were then validated in vitro and in vivo, revealing the roles of secretory and intracellular osteopontin (OPN) in SA-AKI.

Methods: Firstly, public bulk and single-cell transcriptomic data from various murine AKI models was analyzed. Ligands and receptors analysis between injured PTCs and immune cells was carried out to identify candidate genes that were critical. Secondly, the expression of Spp1 in different models of AKI and OPN levels in patient urine and blood samples were measured. The effect of secretory OPN in mediating macrophage chemotaxis was studied in a co-culture system of primary renal tubular cells (PRTCs) and bone marrow-derived macrophages (BMDMs). The role of intracellular OPN in LPS-stimulated PRTCs was also investigated. In vitro results were finally validated using OPN neutralizing antibody in vivo.

Results: Activation of inflammatory pathways, dysfunction in metabolism and cell death were identified as common changes in AKI of different etiologies. OPN was significantly upregulated in various models. Single-cell transcriptomic analysis identified the potential role of OPN in mediating the crosstalk between injured PTCs and macrophages. Upregulation of OPN was confirmed in blood samples of AKI patients, in mouse models and LPS-stimulated PRTCs. Secretory OPN was found to facilitate macrophage immigration in vitro. Strikingly, intracellular OPN interacted with NLRP3 and regulated pyroptosis in PRTCs. OPN knock-down in PRTCs repressed the activation of NLRP3 inflammasome, cleave of Caspase-1 and GSDMD, as well as the release of IL-1β and IL-18. OPN neutralization in mouse model significantly inhibited macrophage influx and reduced tubular cell death in the kidney, alleviating SA-AKI and improving kidney function.

Conclusions: Activation of inflammatory signal pathways, dysfunction in metabolism and cell death caused by cell injuries are key mechanisms of AKI. OPN facilitates macrophage chemotaxis and immigration and tubular cell pyroptosis by interacting with NLRP3. Targeting OPN in SA-AKI leads to reduced kidney injury and improved kidney function.