Cloning and preliminary characterization of a calcium-binding protein closely related to nucleolin on the apical surface of inner medullary collecting duct cells

Calcium stone crystal attachment to the urinary epithelium plays an essenti al role in the development of kidney stones by allowing small crystals to b e retained in the kidney until they become macroscopic. We among others hav e described attachment of stone crystals to cultured renal epithelia (Wiess ner, 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 parti cipate in crystal attachment, apical membranes of cultured renal inner medu llary collecting duct were biotinylated, the cells were lysed with detergen t, the lysate was subjected to hydroxyapatite chromatography, and fractions were incubated with calcium oxalate monohydrate. Electrophoresis of materi al solubilized from the crystals showed several selectively adsorbed protei n bands. A 110-kDa band stained positively for biotin and for glycosides an d bound Ca-45. The amino acid sequence of this band was determined to be th at of a protein closely related to rat nucleolin (nucleolin-related protein ; NRP), NRP was cloned and sequenced and was 83% homologous with the previo usly 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 membra nes of cultured renal tubular cells and is bound to membrane skeletal eleme nts in a calcium-dependent fashion. The physiological role of NRP remains t o be determined; however, a pathophysiological role may be that of mediatin g the attachment to the renal tubular epithelium of calcium stone crystals.