Abstract
Calcium stone crystal attachment to the urinary epithelium plays an essential role in the development of kidney stones by allowing small crystals to be retained in the kidney until they become macroscopic. We among others have described attachment of stone crystals to cultured renal epithelia (Wiessner, J. H., Kleinman, J. G., Blumenthal, S. S., Garancis, J. C., and Mandel, G. S. (1987) J. Urol. 138, 640-643). To isolate protein(s) that may participate in crystal attachment, apical membranes of cultured renal inner medullary collecting duct were biotinylated, the cells were lysed with detergent, the lysate was subjected to hydroxyapatite chromatography, and fractions were incubated with calcium oxalate monohydrate. Electrophoresis of material solubilized from the crystals showed several selectively adsorbed protein bands. A 110-kDa band stained positively for biotin and for glycosides and bound (45)Ca. The amino acid sequence of this band was determined to be that of a protein closely related to rat nucleolin (nucleolin-related protein; NRP). NRP was cloned and sequenced and was 83% homologous with the previously sequenced nucleolar protein nucleolin. Using temperature-induced phase partitioning with Triton X-114, NRP was associated with both the insoluble membrane skeleton pellet and the soluble aqueous phase but not the soluble detergent phase. This association with the membrane skeleton was increased in the presence of calcium. Thus, NRP is associated with the apical membranes of cultured renal tubular cells and is bound to membrane skeletal elements in a calcium-dependent fashion. The physiological role of NRP remains to be determined; however, a pathophysiological role may be that of mediating the attachment to the renal tubular epithelium of calcium stone crystals.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF151373
We have proposed that apical membrane glycoproteins in renal tubules may play a role in attachment of calcium oxalate and calcium phosphates to the epithelium, thereby providing a mechanism for retention
We describe a novel cell surface glycoprotein, NRP, closely related to nucleolin and propose that this protein plays an important role in crystal attachment in kidney stone disease
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF151373. We have proposed that apical membrane glycoproteins in renal tubules may play a role in attachment of calcium oxalate and calcium phosphates to the epithelium, thereby providing a mechanism for retention. We analyzed surface proteins from rat inner medullary collecting duct (IMCD) cells for their ability to bind ionic calcium, calcium oxalate crystals, and HA with high affinity. We describe a novel cell surface glycoprotein, NRP, closely related to nucleolin and propose that this protein plays an important role in crystal attachment in kidney stone disease. Nucleolin or a protein closely related to it has been demonstrated in association with the plasma membranes of some cells, where it functions as a receptor for lipoproteins, viruses, extracellular matrix, growth factors, and other molecules (4 –9). We examine the influence of calcium on the distribution of NRP
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