Tsujihata M, Miyake O, Yoshimura K, Kakimoto K I, Takahara S, Okuyama A
Department of Urology, Osaka University Medical School, Suita, Japan.
J Urol. 2000 Nov;164(5):1718-23.
Fibronectin (230 kD.) is a multifunctional alpha2-glycoprotein distributed throughout the extracellular matrix and body fluids. Many investigators have demonstrated that fibronectin, because of its cell adhesive action, is related to biological processes such as morphogenesis, wound healing and metastasis. Recent studies have shown that a variety of molecules, including fibronectin, inhibit endocytosis of calcium oxalate crystals in vitro. We investigated other roles of fibronectin in calcium oxalate stone formation.
Immunoblotting of the crystal surface binding substance obtained from pooled healthy male urine samples was used to analyze whether fibronectin was adsorbed onto the surface of calcium oxalate crystals. To clarify the relationship between fibronectin and calcium oxalate crystals, we performed 6 experiments. Experiment 1 was immunohistochemical examination of fibronectin expression in stone forming rat model kidneys, and experiment 2 examined the fibronectin content of stone forming rat kidney models with the enzyme-linked immunosorbent assay. Experiment 3 was designed to determine fibronectin content of Madin-Darby canine kidney (MDCK) cells stimulated by addition of calcium oxalate crystals and experiment 4 identified the inhibitory effect of fibronectin on calcium oxalate crystal growth by the seed crystal method. For experiment 5 we used an aggregometer system to clarify the inhibitory effect of fibronectin on calcium oxalate crystal aggregation and experiment 6 examined the inhibitory effect of fibronectin on the adhesion of calcium oxalate crystals to MDCK cells.
In the crystal surface binding substance immunoreactive bands at 230 kD., which correspond to the molecular weight of fibronectin, were detected by Western blot analysis. In stone forming rat kidneys strong expression of fibronectin was found on the renal tubules to which the crystals were attached. The fibronectin content of these kidneys was significantly greater than that of kidneys without calcium oxalate crystals. The fibronectin content of MDCK cells tended to increase in proportion to the concentration of calcium oxalate crystals added to the culture medium. The growth inhibition assay showed that the inhibitory effect of fibronectin on calcium oxalate crystal growth was small in relation to the quantity of fibronectin excreted. However, fibronectin had inhibitory effects on calcium oxalate crystal aggregation and adhesion of the crystals to MDCK cells.
Fibronectin secretion can be stimulated by calcium oxalate crystals, and this protein, which is excreted from the tubular cells, may inhibit calcium oxalate crystal aggregation and attachment to cells.
纤连蛋白（230kD）是一种多功能α2糖蛋白，分布于整个细胞外基质和体液中。许多研究人员已证明，纤连蛋白因其细胞黏附作用，与形态发生、伤口愈合和转移等生物学过程相关。最近的研究表明，包括纤连蛋白在内的多种分子在体外可抑制草酸钙晶体的内吞作用。我们研究了纤连蛋白在草酸钙结石形成中的其他作用。
对从汇集的健康男性尿液样本中获得的晶体表面结合物质进行免疫印迹分析，以检测纤连蛋白是否吸附在草酸钙晶体表面。为阐明纤连蛋白与草酸钙晶体之间的关系，我们进行了6项实验。实验1是对结石形成大鼠模型肾脏中纤连蛋白表达的免疫组织化学检查，实验2用酶联免疫吸附测定法检测结石形成大鼠肾脏模型中的纤连蛋白含量。实验3旨在确定添加草酸钙晶体刺激后的麦迪逊-达比犬肾（MDCK）细胞中的纤连蛋白含量，实验4用晶种法确定纤连蛋白对草酸钙晶体生长的抑制作用。实验5我们使用凝集仪系统阐明纤连蛋白对草酸钙晶体聚集的抑制作用，实验6检测纤连蛋白对草酸钙晶体与MDCK细胞黏附的抑制作用。
通过蛋白质印迹分析在晶体表面结合物质中检测到对应于纤连蛋白分子量的230kD免疫反应条带。在结石形成大鼠肾脏中，在附着有晶体的肾小管上发现纤连蛋白有强烈表达。这些肾脏中的纤连蛋白含量明显高于无草酸钙晶体的肾脏。MDCK细胞中的纤连蛋白含量倾向于与添加到培养基中的草酸钙晶体浓度成比例增加。生长抑制试验表明，相对于纤连蛋白的排泄量，其对草酸钙晶体生长的抑制作用较小。然而，纤连蛋白对草酸钙晶体聚集以及晶体与MDCK细胞的黏附有抑制作用。
草酸钙晶体可刺激纤连蛋白分泌，这种从肾小管细胞排泄的蛋白质可能抑制草酸钙晶体聚集和与细胞的附着。
