Identification of hyaluronan as a crystal-binding molecule 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 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.
Verkoelen
C F
CF
Erasmus Urological Stone Treatment and Research Rotterdam (Eurostarr), and Department Urology, Erasmus University Rotterdam, The Netherlands. Verkoelen@uro.fgg.eur.nl
Van Der Boom
B G
BG
Romijn
J C
JC
D000818:Animals; D002129:Calcium Oxalate / Q000737:chemistry* / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D002810:Chondroitinases and Chondroitin Lyases / Q000494:pharmacology; D003460:Crystallization; D004285:Dogs; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006820:Hyaluronic Acid / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology; D007668:Kidney / Q000166:cytology*; D007669:Kidney Calculi / Q000737:chemistry / Q000378:metabolism; D010969:Plastics; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D014945:Wound Healing / Q000502:physiology
D002250:Carbon Radioisotopes; D006025:Glycosaminoglycans; D010969:Plastics; D002129:Calcium Oxalate; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; D002810:Chondroitinases and Chondroitin Lyases