Supplementary Material for: Unravelling Pathophysiology of Crystalline Nephropathy in Ceftriaxone-Associated Acute Kidney Injury: A Cellular Proteomic Approach

<b><i>Background:</i></b> Previous studies showed that ceftriaxone can cause acute kidney injury (AKI) in the pediatric population. This study proposed a cellular model of crystalline nephropathy in ceftriaxone-associated AKI and explored the related pathophysiology by using a proteomic approach. <b><i>Methods:</i></b> Ceftriaxone was crystallized with calcium in artificial urine. Madin-Darby Canine Kidney (MDCK) cells, a model of distal renal tubular cell, were cultured in the absence (untreated control) or presence of ceftriaxone crystals for 48-h (<i>n</i> = 5 each). MDCK cells were harvested and subsequently analyzed by proteomic analysis. Protein bioinformatics (i.e., STRING and Reactome) was used to predict functional alterations, and subsequently validated by Western blotting and cellular studies. <i>p &lt;</i> 0.05 was considered statistically significant. <b><i>Results:</i></b> Phase-contrast microscopy showed increased intracellular vesiculation and cell enlargement as a result of ceftriaxone crystal exposure. Proteome analysis revealed a total of 20 altered proteins (14 increased, 5 decreased and 1 absent) in ceftriaxone crystal-treated MDCK cells as compared to untreated cells (<i>p</i> &lt; 0.05). Protein bioinformatics and validation studies supported heat stress response mediated by heat shock protein 70 (Hsp70) and downregulation of annexin A1 as the proposed pathophysiology of crystalline nephropathy in ceftriaxone-associated AKI, in which impaired proliferation and wound healing of crystal-induced distal tubular cells were outcomes. <b><i>Conclusions:</i></b> This study, for the first time, used the in vitro model of crystalline nephropathy to investigate the underlying pathophysiology of ceftriaxone-associated AKI, which should be investigated in vivo for potential clinical benefits in the future.

<b><i>背景:</i></b> 既往研究表明，头孢曲松（ceftriaxone）可在儿科人群中引发急性肾损伤（AKI）。本研究构建了头孢曲松相关性AKI相关结晶性肾病的细胞模型，并通过蛋白质组学方法探究其潜在病理生理机制。
<b><i>方法:</i></b> 头孢曲松在人工尿液中与钙结合形成结晶。以马-达二氏犬肾（Madin-Darby Canine Kidney, MDCK）细胞——远端肾小管细胞的经典模型——为研究对象，分为未处理对照组与头孢曲松结晶处理组，培养48小时，每组设置5个重复样本。收集MDCK细胞后进行蛋白质组学分析；采用蛋白质生物信息学工具（即STRING与Reactome数据库）预测细胞功能改变，并通过蛋白质印迹法与细胞实验对结果进行验证。以p<0.05作为具有统计学显著性差异的判定标准。
<b><i>结果:</i></b> 相差显微镜观察显示，头孢曲松结晶处理后，细胞内囊泡形成增多、细胞体积增大。蛋白质组学分析显示，与未处理对照组相比，头孢曲松结晶处理组的MDCK细胞中共检测到20个差异表达蛋白（14个上调、5个下调、1个缺失），且差异均具有统计学显著性（p<0.05）。蛋白质生物信息学分析与验证实验证实，热休克蛋白70（heat shock protein 70, Hsp70）介导的热应激反应以及膜联蛋白A1的下调，是头孢曲松相关性AKI相关结晶性肾病的核心病理生理机制，其最终表现为结晶诱导的远端肾小管细胞增殖受损与创伤修复能力下降。
<b><i>结论:</i></b> 本研究首次利用结晶性肾病体外模型探究了头孢曲松相关性AKI的潜在病理生理机制，未来可通过体内实验进一步研究，以探索其潜在临床应用价值。