To investigate the regulatory role and mechanism of secreted phosphoprotein 1 (SPP1) on macrophage phenotype transformation in the early stage of ischemia-reperfusion induced acute kidney injury (AKI).

In this study, male C57BL/6J mice were used to establish a bilateral renal ischemia-reperfusion injury (IRI)-induced AKI model. Renal tissue injury, CD68⁺ macrophage infiltration, and SPP1 protein expression levels were evaluated by histopathological staining, immunostaining, and Western blotting. The mice bone marrow-derived macrophages were isolated and induced, and transcriptome sequencing was performed to analyze differentially expressed genes after stimulation with recombinant SPP1 protein. Furtherly RAW264.7 cells were stimulated with recombinant SPP1 protein, and the levels of IL-1β, TNF-α, CD86, inducible nitric oxide synthase, and reactive oxygen species were detected by qPCR and flow cytometry. For the in vivo intervention experiments, mice were randomly divided into three groups (n=6 per group): a sham group, an IRI+ IgG control group (treated with 10 mg/kg isotype IgG after IRI modeling), and an IRI+ anti-SPP1 group (treated with an equal dose of SPP1 neutralizing antibody after IRI modeling). Renal injury and inflammatory indicators were compared among the groups.

Significant tubular injury accompanied by increased CD68⁺ macrophage infiltration was observed in the renal corticomedullary junction after IRI modeling (P<0.05). SPP1 expression in proximal tubular epithelial cells was also significantly upregulated (P<0.05). Transcriptome analysis revealed that stimulation with exogenous recombinant SPP1 protein induced extensive alterations in the gene expression profile of macrophages. The 3, 141 differentially expressed genes identified were mainly enriched in inflammation- and immune-related pathways. In vitro experiments with RAW264.7 cells demonstrated that SPP1 promoted macrophage polarization towards a pro-inflammatory phenotype, as evidenced by increased mRNA expression of the aforementioned pro-inflammatory factors, a higher proportion of CD86⁺ cells, and elevated reactive oxygen species levels (all P<0.05). In vivo intervention experiments indicated that the SPP1 neutralizing antibody significantly attenuated renal tubular necrosis in the IRI mice and downregulated the expression of kidney injury molecule-1 and IL-1β (all P<0.05).

In the renal IRI, SPP1 expression was upregulated, promoting macrophage polarization towards a pro-inflammatory phenotype, enhancing oxidative stress, and thereby exacerbating renal inflammatory injury.