Chiangjong Wararat, Sinchaikul Supachok, Chen Shui-Tein, Thongboonkerd Visith
Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, 12th Fl. Adulyadej Vikrom Bldg., 2 Prannok Rd., Bangkoknoi, Bangkok 10700, Thailand.
Mol Biosyst. 2011 Jun;7(6):1917-25. doi: 10.1039/c1mb05052d. Epub 2011 Apr 14.
Calcium oxalate dihydrate (COD) crystals can adhere onto the apical surface of renal tubular epithelial cells. This process is associated with crystal growth and aggregation, resulting in kidney stone formation. Glycoproteins have been thought to play roles in response to crystal adhesion. However, components of the glycoproteome that are involved in this cellular response remain largely unknown. Our present study therefore aimed to identify altered glycoproteins upon COD crystal adhesion onto tubular epithelial cells representing distal nephron, the initiating site of kidney stone formation. Madin-Darby Canine Kidney (MDCK) cells were maintained in culture medium with or without COD crystals for 48 h (n = 5 flasks per group). Cellular proteins were extracted, resolved by 2-DE and visualized by SYPRO Ruby total protein stain, whereas glycoproteins were detected by Pro-Q Emerald glycoprotein dye. Spot matching and quantitative intensity analysis revealed 16 differentially expressed glycoprotein spots, whose corresponding total protein levels were not changed by COD crystal adhesion. These altered glycoproteins were successfully identified by Q-TOF MS and/or MS/MS analyses, and potential glycosylation sites were identified by the GlycoMod tool. For example, glycoforms of three proteasome subunits (which have a major role in regulating cell-cell dissociation) were up-regulated, whereas a glycoform of actin-related protein 3 (ARP3) (which plays an important role in cellular integrity) was down-regulated. These coordinated changes implicate that COD crystal adhesion induced cell dissociation and declined cellular integrity in the distal nephron. Our findings provide some novel insights into the pathogenic mechanisms of kidney stone disease at the molecular level, particularly cell-crystal interactions.
二水合草酸钙（COD）晶体能够黏附在肾小管上皮细胞的顶端表面。这一过程与晶体生长和聚集相关，会导致肾结石的形成。一直以来，人们认为糖蛋白在应对晶体黏附方面发挥着作用。然而，参与这种细胞反应的糖蛋白质组的成分在很大程度上仍不清楚。因此，我们目前的研究旨在确定COD晶体黏附到代表远端肾单位（肾结石形成的起始部位）的肾小管上皮细胞上后发生改变的糖蛋白。将犬肾上皮细胞（MDCK）在含有或不含有COD晶体的培养基中培养48小时（每组5个培养瓶）。提取细胞蛋白，通过双向电泳进行分离，并用SYPRO Ruby总蛋白染色进行可视化，而糖蛋白则用Pro-Q Emerald糖蛋白染料进行检测。斑点匹配和定量强度分析揭示了16个差异表达的糖蛋白斑点，其相应的总蛋白水平并未因COD晶体黏附而改变。通过Q-TOF质谱和/或串联质谱分析成功鉴定了这些改变的糖蛋白，并通过GlycoMod工具鉴定了潜在的糖基化位点。例如，三种蛋白酶体亚基（在调节细胞间解离中起主要作用）的糖型上调，而肌动蛋白相关蛋白3（ARP3）（在细胞完整性中起重要作用）的一种糖型下调。这些协同变化表明，COD晶体黏附诱导了远端肾单位中的细胞解离并降低了细胞完整性。我们的研究结果在分子水平上，特别是在细胞-晶体相互作用方面，为肾结石疾病的致病机制提供了一些新的见解。
