Crystal Retention Capacity of Cells in the Human Nephron: Involvement of CD44 and Its Ligands Hyaluronic Acid and Osteopontin in the Transition of a Crystal Binding- into a Nonadherent Epithelium ANJA VERHULST,* MARINO ASSELMAN, † VEERLE P. PERSY,* MARIEKE S.J . SCHEPERS, † MARK F. HELBERT,* CARL F. VERKOELEN, † and MARC E. DE BROE* *Department of Nephrology-Hypertension, University of Antwerp, Antwerp, Belgium; and † Department of Experimental Urology, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands. Abstract. Nephrolithiasis requires formation of crystals fol- lowed by their retention and accumulation in the kidney. Crys- tal retention can be caused by the association of crystals with the epithelial cells lining the renal tubules. The present study investigated the interaction between calcium oxalate monohy- drate (COM) crystals and primary cultures of human proximal (PTC) and distal tubular/collecting duct cells (DTC). Both PTC and DTC were susceptible to crystal binding during the first days post-seeding (4.9  0.8 g COM/cm 2 ), but DTC lost this affinity when the cultures developed into confluent monolayers with functional tight junctions (0.05  0.02 g COM/cm 2 ). Confocal microscopy demonstrated the expression of the trans- membrane receptor protein CD44 and its ligands osteopontin (OPN) and hyaluronic acid (HA) at the apical membrane of proliferating tubular cells; at confluence, CD44 was expressed at the basolateral membrane and OPN and HA were no longer detectable. In addition, a particle exclusion technique revealed that proliferating cells were surrounded by HA-rich pericellular matrices or “cell coats” extending several microns from the cell surface. Disintegration of these coats with hyaluronidase sig- nificantly decreased the cell surface affinity for crystals. Fur- thermore, CD44, OPN, and HA were also expressed in vivo at the luminal side of tubular cells in damaged kidneys. These results suggest (1) that the intact distal tubular epithelium of the human kidney does not bind crystals, and (2) that crystal retention in the human kidney may depend on the expression of CD44-, OPN-, and-HA rich cell coats by damaged distal tu- bular epithelium. debroe@uia.ua.ac.be Kidney stone development requires the formation of crystals in the tubular fluid followed by their retention and accumulation in the kidney. Whereas crystal formation predominantly depends on the composition of the tubular fluid, crystal retention might de- pend on the composition of the renal tubular epithelial cell surface (1– 4). Per day, the human kidney forms about 1.5 L of urine from 150 to 180 L of ultrafiltrate. As a result of this concentration process, tubular fluid often becomes supersaturated with calcium salts, leading to the spontaneous nucleation of crystals. Providing distal tubules, collecting ducts, ureters, bladder, and the urethra with a nonadherent surface might be a natural defense mechanism against crystal retention, which is hampered when the anti-adher- ence properties are compromised. Damage to epithelial cells lining the renal tubules may play a crucial role in the disturbance of this defense mechanism. In rats, the deposition of crystals in the kidneys is higher when their crystal-inducing diet is combined with nephrotoxic agents (5,6). Likewise, crystals adhere to damaged bladder urothe- lium, but not to the healthy tissue (7). The urine of recurrent stone-formers contains enhanced levels of renal tubular cell- derived enzymes (8) and cytokines (9), indicating that the renal tissue is injured in these patients. In cell culture, it became evident that proliferation (1), scrape damage (2), or modifica- tion of cell membrane properties (10) showed increased crystal binding when compared with intact monolayers. The glycos- aminoglycan hyaluronic acid (hyaluronan; HA) was identified as one of the major cell surface crystal binding molecules in these cultures (3,11). Hyaluronic acid is a high–molecular mass polysaccharide (ranging from 1 to 10 million D) consisting of multiple repeat- ing disaccharides of glucuronic acid (GlcA) and N-acetylglu- cosamine (GlcNAc). HA performs several important biologic functions in vertebrates. In connective tissues, it mainly serves as a structural component; during embryonic development and repair processes, it provides hydrated matrices through which cells can move (12,13). HA also plays a role in the communi- cation of the cell interior with the environment through its major cell surface receptor, CD44 (14,15). The transmembrane Received June 24, 2002. Accepted August 30, 2002. Correspondence to Dr. Marc E. De Broe, University of Antwerp, Department of Nephrology-Hypertension, p/a University Hospital Antwerp, Wilrijkstraat 10, B-2650 Edegem/Antwerpen, Belgium. Phone: +32-3-821-3421; Fax: +32-3-829-0100; E-mail: debroe@uia.ua.ac.be 1046-6673/1312-0107 Journal of the American Society of Nephrology Copyright © 2002 by the American Society of Nephrology DOI: 10.1097/01.ASN.0000038686.17715.42 J Am Soc Nephrol 13: 107–115, 2003