Nephrolithiasis, the formation of kidney stones, is a common condition seen in up to 12 percent of adults during their lifetime. As the mechanisms by which stones form are poorly understood, new knowledge is required to identify susceptible patients for early treatment and to formulate new therapeutic strategies to prevent the appearance of single and/or recurrent stones. How nascent crystals that nucleate in tubular fluid are retained in the nephron and form calculi is not known. My studies during the past 8 years demonstrate that calcium oxalate monohydrate (COM) crystals bind within seconds to anionic, sialic acid- containing glycoproteins on the apical surface of cultured monkey kidney epithelial cells (BSC-1), employed to model the tubule, suggesting one mechanism whereby crystals could be retained in the kidney in vivo. Preliminary studies have identified constitutive release of a protein by BSC-1 cells that blocks adhesion of COM crystals to the apical cell surface; it has been named the Crystal Adhesion Inhibitor, or CAI. A novel method employing COM crystal affinity chromatography was used to purify CAI. Evidence provided in this revised application demonstrates that CAI is a constituent of normal human urine. Biochemical characterization identifies it as a sialic acid-containing glycoprotein. Microsequencing of the amino terminus and 9 fragments generated by lys-C and asp-N protease cleavage reveal that CAI is novel. Two monospecific antibodies against synthetic peptides prepared using amino acid sequence information each recognize the factor on Western blots of partially- purified normal human urine, renal cell conditioned medium, and total kidney cell protein. The goal of this revised research plan is to define the potential role of CAI in human nephrolithiasis.
New Specific Aims are to: 1) Utilize 2 monospecific antibodies prepared against CAI to characterize and quantitate it in the urine of normal and stone- forming individuals; 2) Study inhibition of COM, hydroxyapatite, and uric acid crystal adhesion to renal cells by CAI isolated from conditioned medium and the urine of normal and stone-forming subjects; 3) Study inhibition of COM crystal growth by CAI isolated from conditioned medium and the urine of normal and stone-forming subjects, 4) Study inhibition of COM crystal aggregation by CAI isolated from conditioned medium and the urine of normal and stone-forming individuals, 5) Utilize the monospecific antibodies prepared against CAI to isolate affinity-purified protein and study its physical-chemical properties; 6) Study the cell biology of CAI. Achieving these specific aims will provide new knowledge about mechanisms that mediate stone formation, and provide a rational basis for design of novel strategies to treat and/or prevent this disease.
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