Vinaiphat Arada, Aluksanasuwan Siripat, Manissorn Juthatip, Sutthimethakorn Suchitra, Thongboonkerd Visith
Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital; and Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand.
Proteomics. 2017 Aug;17(15-16). doi: 10.1002/pmic.201700192. Epub 2017 Jul 18.
We have previously identified changes in the cellular proteome of renal tubular cells induced by low-dose (100 μg/mL) and high-dose (1000 μg/mL) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals. However, the functional significance of such expression data remained unclear. In this study, we performed comparative analyses and functional investigations of four proteomic datasets to define potential mechanisms by which renal tubular cells responded to differential crystal types and doses. The data showed that high-dose induced greater changes than low-dose, whereas COM induced more changes than COD. Luciferin-luciferase ATP assay revealed increased intracellular ATP level by high-dose of both COM and COD. OxyBlot assay and Western blotting showed accumulated intracellular oxidized proteins but decreased ubiquitinated proteins by high-dose of both crystals. Flow cytometric analysis of cell death showed that high-dose of both crystals, particularly COM, significantly increased cell death. Also, crystal adhesion assay showed higher degree of cell-crystal adhesion in high-dose and COM when compared to low-dose and COD, respectively. Finally, pretreatment of epigallocatechin-3-gallate revealed a protective effect on COM/COD crystals-induced oxidative stress and cell-crystal adhesion. Collectively, these data may provide a better understanding of cellular responses of renal tubular cells to COM/COD crystals in kidney stone disease.
我们之前已经鉴定了由低剂量（100μg/mL）和高剂量（1000μg/mL）一水合草酸钙（COM）和二水合草酸钙（COD）晶体诱导的肾小管细胞的细胞蛋白质组变化。然而，这些表达数据的功能意义仍不清楚。在本研究中，我们对四个蛋白质组数据集进行了比较分析和功能研究，以确定肾小管细胞对不同晶体类型和剂量作出反应的潜在机制。数据表明，高剂量诱导的变化比低剂量更大，而COM诱导的变化比COD更多。荧光素-荧光素酶ATP测定显示，高剂量的COM和COD均使细胞内ATP水平升高。OxyBlot测定和蛋白质印迹显示，高剂量的两种晶体均使细胞内氧化蛋白积累，但泛素化蛋白减少。细胞死亡的流式细胞术分析表明，高剂量的两种晶体，尤其是COM，显著增加细胞死亡。此外，晶体粘附试验显示，与低剂量和COD相比，高剂量和COM的细胞-晶体粘附程度更高。最后，表没食子儿茶素-3-没食子酸酯预处理显示对COM/COD晶体诱导的氧化应激和细胞-晶体粘附具有保护作用。总体而言，这些数据可能有助于更好地理解肾小管细胞在肾结石疾病中对COM/COD晶体的细胞反应。
