C F Verkoelen1, B G Van Der Boom, J C Romijn. 1. Erasmus Urological Stone Treatment and Research Rotterdam (Eurostarr), and Department Urology, Erasmus University Rotterdam, The Netherlands. Verkoelen@uro.fgg.eur.nl
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These results identify HA as binding molecule for COM crystals at the surface of migrating and proliferating MDCK cells.
BACKGROUND: The adherence of calcium oxalate crystals to the renal tubule epithelium is considered a critical event in the pathophysiology of calciumnephrolithiasis. 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These results identify HA as binding molecule for COM crystals at the surface of migrating and proliferating MDCK cells.