Medical Proteomics Unit and Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Department of Immunology and Immunology Graduate Program, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Proteomics Clin Appl. 2008 Jul;2(7-8):1099-109. doi: 10.1002/prca.200780136. Epub 2008 Jun 18.
Adhesion of calcium oxalate (CaOx) crystals to renal tubular cells is a critical event that triggers a cascade of responses, leading to the development of kidney stones. However, the molecular mechanisms of these cellular responses remain largely unknown. We performed gel-based, differential proteomics study to examine cellular responses (as determined by altered protein expression) in Madin-Darby Canine Kidney (MDCK) cells during CaOx monohydrate (COM) crystal adhesion. Approximately 3-million MDCK cells were inoculated in each culture flask and maintained for 24 h. A total of 10 semiconfluent flasks were then divided into two groups (n = 5 per group) and the culture medium was replaced by either COM-containing (with 100 μg/mL COM crystals) or COM-free medium. The cells were grown further for 48 h. Crystal adhesion on the cell surface was clearly demonstrated using phase-contrast and scanning electron microscopy. Cell death assay using annexin V/propidium iodide double staining showed that all these samples had comparable % cell death. Cellular proteins were then extracted, resolved with 2-DE, and visualized by SYPRO Ruby staining (n = 5 gels per group). Quantitative intensity analysis revealed significantly increased abundance of 15 protein spots, whereas the other 5 were decreased. These altered proteins were then identified by quadrupole TOF (Q-TOF) MS and/or MS/MS analyses, including transcription/translation regulators, signal transduction proteins, metabolic enzymes, nuclear membrane proteins, carrier protein, cellular structural protein, chaperones, and proteins involved in biosynthesis, enzyme activation, and growth regulation. These data may lead to better understanding of the cellular responses in distal renal tubular cells during COM crystal adhesion.
草酸钙（CaOx）晶体与肾小管细胞的黏附是触发一系列反应的关键事件，导致肾结石的形成。然而，这些细胞反应的分子机制在很大程度上仍然未知。我们进行了基于凝胶的差异蛋白质组学研究，以检查 Madin-Darby 犬肾（MDCK）细胞在 CaOx 一水合物（COM）晶体黏附过程中的细胞反应（通过改变蛋白质表达来确定）。每个培养瓶接种约 300 万个 MDCK 细胞，并维持 24 小时。然后将总共 10 个半汇合的培养瓶分为两组（每组 n = 5），并用含有（100μg/mL COM 晶体）或无 COM 的培养基替换培养基。细胞进一步生长 48 小时。使用相差和扫描电子显微镜清楚地显示了晶体在细胞表面的黏附。使用膜联蛋白 V/碘化丙啶双重染色的细胞死亡测定表明，所有这些样品的细胞死亡率相当。然后提取细胞蛋白，用 2-DE 分离，并通过 SYPRO Ruby 染色可视化（每组 n = 5 个凝胶）。定量强度分析显示 15 个蛋白质斑点的丰度显著增加，而其他 5 个斑点的丰度降低。然后通过四极杆飞行时间（Q-TOF）MS 和/或 MS/MS 分析鉴定这些改变的蛋白质，包括转录/翻译调节剂、信号转导蛋白、代谢酶、核膜蛋白、载体蛋白、细胞结构蛋白、伴侣蛋白以及参与生物合成、酶激活和生长调节的蛋白质。这些数据可能有助于更好地理解 COM 晶体黏附过程中远端肾小管细胞的细胞反应。
