探讨分泌型磷酸蛋白-1(secreted phosphoprotein 1, SPP1)对缺血再灌注急性肾损伤(acute kidney injury, AKI)早期巨噬细胞表型转化的调控作用及其机制。

本研究采用雄性C57BL/6J小鼠建立双侧肾缺血再灌注损伤(ischemia-reperfusion injury，IRI)诱导的AKI模型。通过组织病理染色、免疫染色及Western印迹法评估肾组织损伤、CD68^＋巨噬细胞浸润及SPP1蛋白表达水平。分离并诱导小鼠骨髓来源巨噬细胞，利用转录组测序分析其在SPP1重组蛋白刺激后差异表达基因。进一步使用SPP1重组蛋白刺激RAW264.7细胞，通过qPCR和流式细胞术检测IL-1β、TNF-α、CD86、诱导型一氧化氮合酶及活性氧水平。在体内干预实验中，将小鼠随机分为假手术组、IRI＋IgG对照组(IRI建模后给予10 mg/kg的同型IgG)和IRI＋抗-SPP1组(IRI建模后给予等量的SPP1中和抗体)，每组各6只，比较各组肾损伤与炎症指标。

IRI建模后肾皮髓交界区可见显著肾小管损伤伴CD68^＋巨噬细胞浸润增多(P<0.05)，近端肾小管上皮细胞中SPP1表达亦明显上调(P<0.05)。转录组分析表明，外源性SPP1重组蛋白刺激后巨噬细胞基因表达谱发生广泛改变，鉴定出的3，141个差异表达基因主要富集于炎症和免疫相关通路。RAW264.7细胞实验显示，SPP1促进巨噬细胞向促炎表型转化，表现为上述促炎因子mRNA表达升高、CD86^＋细胞比例增加和活性氧水平上升(P均<0.05)。体内干预实验表明，SPP1中和抗体可显著减轻IRI小鼠的肾小管坏死，并下调肾损伤分子-1及IL-1β表达(P均<0.05)。

在肾脏IRI中SPP1表达上调，并促进巨噬细胞向促炎表型转化，增强其氧化应激，加剧肾脏炎症损伤。

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.