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

Abstract

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...

Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis

“…Thus pretreatment of COM crystals with urinary macromolecules inhibits their binding to cultured renal cells. This corroborates the results of other studies reporting that coating of the crystal surface with OPN (24), a highly acidic region of nucleolin-related protein (37), unidentified 200 (47)-and 39-kDa (19) proteins, bikunin (9), and fibronectin (38) inhibits COM crystal adhesion to cultured renal cells.…”

Acidic polyanion poly(acrylic acid) prevents calcium oxalate crystal deposition

“…In the case of intratubular nucleation, some investigators have proposed and developed evidence for a specific attachment process to renal tubular cells. [23][24][25][26][27] Alternatively, crystals may enlarge individually through growth or may aggregate with others, resulting in fixation within the kidneys on the basis of size alone. 28 In either case, whether fixation occurs at the level of the individual crystal or small crystal aggregates, further enlargement to clinically significant size appears to take place through addition of free-floating crystals or nucleation of crystals secondarily on the surface of aggregates already fixed to tissue.…”

Preconditioning of the distal tubular epithelium of the human kidney precedes nephrocalcinosis

“…The localization of renal calcifications in preterm neonates is unknown, while in transplanted kidneys nephrocalcinosis clearly presents as intratubular microcalcifications [9,10]. Recent investigation has shown involvement of hyaluronan, osteopontin, CD44, nucleolin-related-protein, annexin II, and Tamm-Horsfal-protein in the processes of tubular crystal formation and/or retention [12][13][14][15][16]. An important step in the development of nephrocalcinosis is the retention/trapping of passing crystals to the luminal surface of cells in the distal nephron.…”