Fong-Ngern Kedsarin, Sueksakit Kanyarat, Thongboonkerd Visith
Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Center for Research in Complex Systems Science, Mahidol University, 6th Floor-SiMR Building, 2 Wanglang Road, Bangkoknoi, Bangkok, 10700, Thailand.
J Biol Inorg Chem. 2016 Jul;21(4):463-74. doi: 10.1007/s00775-016-1355-x. Epub 2016 Apr 26.
Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells.
一水草酸钙（COM）晶体与肾小管上皮细胞的黏附是肾结石形成过程中的关键步骤。寻找细胞顶端膜上潜在的晶体受体可能会带来预防肾结石疾病的新方法。我们之前的研究在MDCK细胞的顶端膜上鉴定出了大量晶体结合蛋白。然而，它们作为潜在晶体受体的功能作用尚未得到验证。本研究旨在探讨热休克蛋白90（HSP90）作为COM晶体受体的潜在作用。通过剥离法从极化的MDCK细胞中分离出顶端膜，并将回收的蛋白质与COM晶体一起孵育。蛋白质印迹分析证实顶端膜和晶体结合部分中存在HSP90。未经透化处理的免疫荧光染色和激光扫描共聚焦显微镜检查证实了完整细胞顶端膜上存在表面HSP90表达。晶体黏附试验表明，用特异性抗HSP90抗体阻断表面HSP90以及用小干扰RNA（siRNA）敲低HSP90，可显著降低COM晶体在MDCK细胞顶端表面的结合（分别降低约1/2和2/3）。此外，晶体内化试验显示，在含有内化COM晶体的内吞小泡膜上存在HSP90。而且，用抗HSP90抗体预处理MDCK细胞可显著降低晶体内化（降低约1/3）。综上所述，我们的数据表明，HSP90作为肾小管上皮细胞顶端膜上COM晶体的潜在受体，参与了晶体向细胞内的内吞/内化过程。
