Verkoelen C F, Van Der Boom B G, Romijn J C
Erasmus Urological Stone Treatment and Research Rotterdam (Eurostarr), and Department Urology, Erasmus University Rotterdam, The Netherlands.
Kidney Int. 2000 Sep;58(3):1045-54. doi: 10.1046/j.1523-1755.2000.00262.x.
The adherence of calcium oxalate crystals to the renal tubule epithelium is considered a critical event in the pathophysiology of calcium nephrolithiasis. Calcium oxalate monohydrate (COM) crystals cannot adhere to the surface of a functional Madin-Darby canine kidney (MDCK) monolayer, but they bind avidly to the surface of proliferating and migrating cells.
To identify crystal-binding molecules (CBMs) at the surface of crystal-attracting cells, we applied metabolic labeling protocols in combination with differential enzymatic digestion and gel filtration, which was compared with [14C]COM crystal binding and confirmed by confocal microscopy.
The indication that hyaluronan [hyaluronic acid (HA)] might act as a CBM in subconfluent cultures came from studies with glycosaminoglycan (GAG)-degrading enzymes. Subsequently, metabolic-labeling studies revealed that hyaluronidase cleaved significantly more radiolabeled glycoconjugates from crystal-attracting cells than from cells without affinity for crystals. During wound repair, crystal binding could be prevented by pretreating the healing cultures with hyaluronate lyase, an enzyme that specifically hydrolyzes HA. Binding to immobilized HA provided evidence that COM crystals physically can become associated with this polysaccharide. Finally, confocal microscopy demonstrated that fluorescently labeled HA binding protein (HABP) adhered to the surface of proliferating cells in subconfluent cultures as well as to cells involved in closing a wound, but not to cells in confluent monolayers.
These results identify HA as binding molecule for COM crystals at the surface of migrating and proliferating MDCK cells.
草酸钙晶体与肾小管上皮的黏附被认为是钙肾结石病病理生理学中的一个关键事件。一水合草酸钙（COM）晶体不能黏附于功能性的Madin-Darby犬肾（MDCK）单层细胞表面，但它们能强烈结合于增殖和迁移细胞的表面。
为了鉴定晶体吸引细胞表面的晶体结合分子（CBMs），我们应用了代谢标记方案，并结合差异酶消化和凝胶过滤，将其与[14C]COM晶体结合进行比较，并通过共聚焦显微镜进行确认。
关于透明质酸[透明质酸（HA）]可能在亚汇合培养物中作为CBM起作用的迹象来自对糖胺聚糖（GAG）降解酶的研究。随后，代谢标记研究表明，透明质酸酶从晶体吸引细胞中切割出的放射性标记糖缀合物比从对晶体无亲和力的细胞中切割出的要多得多。在伤口修复过程中，通过用透明质酸裂解酶预处理愈合培养物可以防止晶体结合，透明质酸裂解酶是一种特异性水解HA的酶。与固定化HA的结合提供了证据，表明COM晶体在物理上可以与这种多糖结合。最后，共聚焦显微镜显示，荧光标记的HA结合蛋白（HABP）黏附于亚汇合培养物中增殖细胞的表面以及参与闭合伤口的细胞表面，但不黏附于汇合单层细胞的表面。
这些结果确定HA为迁移和增殖的MDCK细胞表面COM晶体的结合分子。
