Fibronectin as a potent inhibitor of calcium oxalate urolithiasis

Purpose: Fibronectin (230 kD.) is a multifunctional alpha 2-glycoprotein di stributed throughout the extracellular matrix and body fluids. Many investi gators have demonstrated that fibronectin, because of its cell adhesive act ion, is related to biological processes such as morphogenesis, wound healin g and metastasis. Recent studies have shown that a variety of molecules, in cluding fibronectin, inhibit endocytosis of calcium oxalate crystals in vit ro. We investigated other roles of fibronectin in calcium oxalate stone for mation. Materials and Methods: Immunoblotting of the crystal surface binding substa nce obtained from pooled healthy male urine samples was used to analyze whe ther fibronectin was adsorbed onto the surface of calcium oxalate crystals. To clarify the relationship between fibronectin and calcium oxalate crysta ls, we performed 6 experiments. Experiment I was immunohistochemical examin ation of fibronectin expression in stone forming rat model kidneys, and exp eriment 2 examined the fibronectin content of stone forming rat kidney mode ls with the enzyme-linked immunosorbent assay. Experiment 3 was designed to determine fibronectin content of Madin-Darby canine kidney (MDCK) cells st imulated by addition of calcium oxalate crystals and experiment 4 identifie d the inhibitory effect of fibronectin on calcium oxalate crystal growth by the seed crystal method. For experiment 5 we used an aggregometer system t o clarify the inhibitory effect of fibronectin on calcium oxalate crystal a ggregation and experiment 6 examined the inhibitory effect of fibronectin o n the adhesion of calcium oxalate crystals to MDCK cells. Results: In the crystal surface binding substance immunoreactive bands at 2 30 kD., which correspond to the molecular weight of fibronectin, were detec ted by Western blot analysis. In stone forming rat kidneys strong expressio n of fibronectin was found on the renal tubules to which the crystals were attached. The fibronectin content of these kidneys was significantly greate r than that of kidneys without calcium oxalate crystals. The fibronectin co ntent of MDCK cells tended to increase in proportion to the concentration o f calcium oxalate crystals added to the culture medium. The growth inhibiti on assay showed that the inhibitory effect of fibronectin on calcium oxalat e crystal growth was small in relation to the quantity of fibronectin excre ted. However, fibronectin had inhibitory effects on calcium oxalate crystal aggregation and adhesion of the crystals to MDCK cells. Conclusions: Fibronectin secretion can be stimulated by calcium oxalate cry stals, and this protein, which is excreted from the tubular cells, may inhi bit calcium oxalate crystal aggregation and attachment to cells.