Identification of Hyaluronan as Crystal Binding Molecule at the Surface of Migrating and Proliferating MDCK Cells

Abstract. Crystals that form in tubular fluid must be retained in the kidney to become stones. Nucleolin-related protein (NRP) is found on the surface of inner medullary collecting duct (IMCD) cells in culture (cIMCD) and selectively adsorbs to calcium oxalate (CaOx). We proposed that NRP mediates attachment to the renal tubular epithelium of Ca stone crystals through an electrostatic interaction with a highly acidic region (acidic fragment [AF]) similar to those of other proteins that have been reported to affect urinary crystal formation. The current studies demonstrate that nucleolin is expressed on both apical and basolateral cell surfaces of cIMCD, reaching a peak in the late stages of mitosis and gradually declining to undetectable levels with maturation of the polarized epithelium. Scraping areas of mature monolayers stimulated the cells surrounding the defects to migrate and proliferate so as to repair them, and these areas demonstrate surface NRP expression and enhanced attachment of CaOx monohydrate crystals. Surface expression of the NRP AF was produced by cloning the NRP AF into a display vector. Transfected cIMCD demonstrating copious surface expression of AF enhanced CaOx attachment 6.7-fold compared with control cIMCD, whereas cells transfected with a vector without the AF did not differ from control. AF was also cloned into a replication-deficient adenovirus and expressed in 293 cells, resulting in AF secretion into the nutrient medium. This medium inhibited CaOx attachment to cIMCD, compared with conditioned medium from cells infected with wild-type virus. These results demonstrate that surface-bound AF can mediate CaOx attachment and that secreted AF can inhibit attachment. These results support the notion that surface-associated NRP could mediate attachment of CaOx to the renal tubule epithelium, thereby causing retention of crystals that might eventually become kidney stones.Several groups of investigators have suggested that an important early event in the formation of kidney stones is the attachment of calcium oxalate (CaOx) crystals to the apical surface of renal tubular epithelium (1-3). Well-polarized, undisturbed tubular epithelial cells in culture demonstrate little capacity for crystal attachment (4 -6). This resistance to attachment may be due to the confinement of molecules with crystal-binding properties to domains other than the apical membrane. When tight junctions between cells are disrupted in the course of cellular proliferation or migration or when cells are exposed to a number of different agents, apical membranes seem to express constituents with affinity for CaOx.Nucleolin-related protein (NRP) is a surface-associated protein of cultured inner medullary collecting duct (IMCD) cells that selectively adsorbs to CaOx and binds to membrane skeletal elements in a Ca-dependent manner (7). NRP has a highly acidic region composed of 62% Asp and Glu residues (135KNGKNAKKEDSDEEDDDDSEEEDEEDEDEEDEFE PTVMKAAAAAPAS-DEEEDDEEDDDEDDDEEDEDED-EDDSEEEAMETTPAKGKKAPVKAVPVKAKSTAEE-DEEEDDEDEEDDEE...

Section: Discussionmentioning

confidence: 99%

An Acidic Peptide Sequence of Nucleolin-Related Protein Can Mediate the Attachment of Calcium Oxalate to Renal Tubule Cells

Сорокина¹,

Wesson²,

Kleinman³

2004

“…Experiments in the scrape-wounding model suggest that cell-surface crystal binding proteins are present on the surface of regenerating and migrating cells ( Figure 4B). Candidate molecules include hyaluronan, which has been demonstrated on the surface of migrating renal cells in association with crystals (17), and heparan sulfate proteoglycan, because the protein was immunohistochemically localized to regions of ethylene glycol-treated rat kidneys that contained crystalline deposits (32). Because proliferating cells can be susceptible to crystal binding for a period of days, PGE 2 could play an especially important role by protecting cells during this critical period.…”

Section: Discussionmentioning

confidence: 99%

“…It was reported recently that COM crystals avidly adhere to renal cells that are migrating in to repair a scrape wound made in a monolayer (16), and hyaluronic acid has been proposed to mediate this phenomenon (17). In addition, studies in stoneforming humans (18,19) and oxalate-loaded rats (20) have demonstrated that renal cellular injury occurs.…”

mentioning

confidence: 99%

Modulation of Proliferating Renal Epithelial Cell Affinity for Calcium Oxalate Monohydrate Crystals

Farell¹,

Huang

Kim

et al. 2004

Abstract. Adhesion of urinary crystals to distal tubular cells could be a critical event that triggers a cascade of responses ending in kidney stone formation. Monolayer cultures of distal nephronderived MDCKI cells were used as a model to study crystal-cell interactions. COM crystal adhesion reached a peak 2 d after plating and progressively fell thereafter. The decline in crystal binding was accelerated by prostaglandin E 2 (PGE 2 ) supplementation and delayed by blockade of PG production. Crystals avidly adhered to cells that migrated in to repair a scrape wound made in the monolayer and after a transient hypoglycemic insult. Exposure of MDCKI cells to uric acid crystals and soluble uric acid was also associated with increased crystal adhesion. Treatment of physically or hypoglycemically injured cells with trypsin or neuraminidase reduced crystal binding to baseline levels, suggesting that increased exposure of cell surface glycoproteins mediated the effect, whereas PGE 2 treatment blunted crystal binding to regenerating cells. Furthermore, when cells were grown in the presence of synthetic D-mannosamine analogues that can modify the conformation of cell surface sialoglycoconjugates, crystal binding to proliferating cells was decreased, whereas blockade of N-

“…In addition, it appears mathematically unlikely that nucleation and growth of an individual crystal could produce a particle large enough to occlude a nephron lumen (4). Therefore, our laboratory (5)(6)(7)(8)(9)(10)(11) and others (12)(13)(14)(15)(16)(17) have investigated the mechanism(s) whereby urinary crystals can interact with renal epithelial cells; once adherent to a cell, smaller particles could be retained and serve as a nidus for renal stone formation.…”

mentioning

confidence: 99%

“…More recently, renal cell surface nucleolin-related protein (NRP) (18) and hyaluronan (17) have been identified as potential crystal-binding molecules. Hyaluronan appears to be present on the surface of cultured cells that are proliferating in response to injury (17), and anionic phospholipids may also mediate COM crystal adhesion to injured and possibly apoptotic cells (19,20).…”

mentioning

confidence: 99%

Annexin II Is Present on Renal Epithelial Cells and Binds Calcium Oxalate Monohydrate Crystals

Kumar¹,

Farell²,

Deganello³

et al. 2003

Abstract. Attachment of newly formed crystals to renal epithelial cells appears to be a critical step in the development of kidney stones. The current study was undertaken to identify potential calcium oxalate monohydrate (COM) crystal-binding proteins on the surface of renal tubular cells. Apical membranes were prepared from confluent monolayers of renal epithelial cells (MDCKI line), and COM crystal affinity was used to isolate crystal-binding proteins that were then subjected to electrophoresis and electroblotting. Microsequencing of the most prominent COM crystal-binding protein (M r of 37 kD) identified it as annexin II (Ax-II). When exposed proteins on the surface of intact monolayers were biotinylated and then isolated using streptavidin agarose beads, Ax-II was detected, suggesting that at least a portion is exposed on the apical cell surface. Ax-II was not completely extracted by 0.1 M Na 2 CO 3 , suggesting that at least a portion of cellular Ax-II is an intrinsic membrane-bound protein. Using confocal immunofluorescence microscopy, Ax-II was visualized together with Caveolin-1 (Cav-1) on the apical membrane of intact MDCKI cells. Cells pretreated with a monoclonal anti-Ax-II antibody bound significantly fewer COM crystals, whereas anti-LDL receptor antibody did not decrease COM binding, further suggesting a functional role for Ax-II during adhesion of crystals to intact cells. These results suggest that Ax-II avidly binds COM crystals and is present on the apical surface of MDCKI cells.