Yamaguchi Satoshi, Wiessner John, Hasegawa Andrew, Hung Linda, Mandel Gretchen, Mandel Neil
Department of Urology, Asahikawa Medical College, Asahikawa, Japan.
Int J Urol. 2002 Sep;9(9):501-8. doi: 10.1046/j.1442-2042.2002.00510.x.
The interaction between kidney urothelium and crystals is a critical event in the growth of renal calculi. When studying calcium oxalate monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells in culture, we observed that crystals also attached to areas on the coverslips devoid of cells. This phenomenon could be the result of substances produced by the cells that adhere to the glass and subsequently bind COM crystals. We investigated the characteristics of this COM binding substance.
Media was collected from cultures of MDCK cells (conditioned media) and proteins were separated by high performance liquid chromatography. The molecular weights and purity of isolated proteins were determined by polyacrylamide gel electrophoresis. The conditioned media and each separated fraction were applied to glass and to MDCK cells and COM-binding ability determined using 14C-labeled crystals. The binding of radio-labelled calcium oxalate dihydrate, brushite, uric acid, and apatite to coverslips were also studied.
Fourteen times more COM bound to coverslips incubated with conditioned media than those with control media. The molecular weight of the protein bound to the glass was determined to be 200 kDa. The COM crystals binding to this protein was 1.5 micro g/ng. Other crystals bound to a lesser extent. The incubation of cells with this protein inhibited COM binding by 39%.
The MDCK cells produce a 200-kDa protein that has a high binding affinity for COM crystals. This protein binds to glass and is responsible for crystal binding to areas devoid of cells. This protein also has an inhibitory effect on COM binding to MDCK cells in culture.
肾尿路上皮与晶体之间的相互作用是肾结石形成过程中的关键事件。在研究一水草酸钙（COM）晶体与培养的马-达二氏犬肾（MDCK）细胞的结合时，我们观察到晶体也附着在盖玻片上无细胞的区域。这种现象可能是细胞产生的物质附着在玻璃上并随后结合COM晶体的结果。我们研究了这种COM结合物质的特性。
从MDCK细胞培养物中收集培养基（条件培养基），并通过高效液相色谱法分离蛋白质。通过聚丙烯酰胺凝胶电泳测定分离蛋白质的分子量和纯度。将条件培养基和每个分离的组分应用于玻璃和MDCK细胞，并使用14C标记的晶体测定COM结合能力。还研究了放射性标记的二水草酸钙、透钙磷石、尿酸和磷灰石与盖玻片的结合。
与条件培养基孵育的盖玻片上结合的COM比与对照培养基孵育的盖玻片上多14倍。与玻璃结合的蛋白质的分子量测定为200 kDa。与该蛋白质结合的COM晶体为1.5μg/ng。其他晶体的结合程度较小。用这种蛋白质孵育细胞可使COM结合减少39%。
MDCK细胞产生一种对COM晶体具有高结合亲和力的200 kDa蛋白质。这种蛋白质与玻璃结合，并导致晶体结合到无细胞区域。这种蛋白质对培养中的MDCK细胞与COM的结合也有抑制作用。
