Hattori Tatsuya, Taguchi Kazumi, Chaya Ryosuke, Hamamoto Shuzo, Okada Atsushi, Yasui Takahiro
Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Urolithiasis. 2025 Mar 25;53(1):58. doi: 10.1007/s00240-025-01732-1.
In inflammation, osteopontin (OPN) acts as both a stone matrix component for calcium oxalate (CaOx) crystal formation and an inflammatory mediator. While previous studies have demonstrated the individual roles of OPN and macrophages (Mφ) in renal CaOx stone formation during inflammation, their interaction remains poorly understood. This study aimed to elucidate the role of OPN in modulating Mφ function during crystal formation, using an ex vivo model. Bone marrow-derived macrophages (BMDM) were isolated from eight-week-old male C57BL/6J wild-type and OPN knockout mice. BMDMs from OPN-positive (BMDM) and OPN-negative (BMDM) mice were co-cultured with fluorescently labeled CaOx monohydrate (COM) crystals for phagocytosis assays and analyzed using the IN Cell Analyzer 6000. We further performed real-time quantitative reverse transcription PCR and RNA sequencing to identify gene expression profiles and clarify the role of OPN in Mφ function. The assay analysis demonstrated that phagocytosis rates were significantly higher in BMDM than in BMDM. Inflammatory markers, such as IL-6, TNF, CD44, were upregulated following COM exposure, and IL-6 expression was significantly lower in BMDM than in BMDM. RNA sequencing revealed that BMDM exhibited a less pro-inflammatory and more anti-inflammatory phenotype (Csf2, Irf5, Itgax, Csf1, Cd163), resembling M2-like Mφs. Further functional analysis indicated that OPN knockdown in Mφs increased the S100 family and CREB signaling, which enhanced the M2-like phenotype shift and phagosome formation. In conclusion, OPN plays a critical role in enhancing pro-inflammatory Mφ function, potentially limiting COM phagocytosis. Modulating OPN expression in circulating Mφs may represent a therapeutic approach for kidney stone disease.
在炎症过程中，骨桥蛋白（OPN）既是草酸钙（CaOx）晶体形成的结石基质成分，又是一种炎症介质。虽然先前的研究已经证明了OPN和巨噬细胞（Mφ）在炎症期间肾CaOx结石形成中的各自作用，但它们之间的相互作用仍知之甚少。本研究旨在使用体外模型阐明OPN在晶体形成过程中调节Mφ功能的作用。从8周龄雄性C57BL/6J野生型和OPN基因敲除小鼠中分离出骨髓来源的巨噬细胞（BMDM）。将来自OPN阳性（BMDM）和OPN阴性（BMDM）小鼠的BMDM与荧光标记的一水合草酸钙（COM）晶体共培养以进行吞噬试验，并使用IN Cell Analyzer 6000进行分析。我们进一步进行了实时定量逆转录PCR和RNA测序，以鉴定基因表达谱并阐明OPN在Mφ功能中的作用。试验分析表明，BMDM中的吞噬率显著高于BMDM。COM暴露后，IL-6、TNF、CD44等炎症标志物上调，且BMDM中的IL-6表达显著低于BMDM。RNA测序显示，BMDM表现出炎症性较低和抗炎性较强的表型（Csf2、Irf5、Itgax、Csf1、Cd163），类似于M2样Mφ。进一步的功能分析表明，Mφ中OPN的敲低增加了S100家族和CREB信号传导，这增强了M2样表型转变和吞噬体形成。总之，OPN在增强促炎性Mφ功能中起关键作用，可能会限制COM的吞噬作用。调节循环Mφ中的OPN表达可能是一种治疗肾结石疾病的方法。
