NOVEL INHIBITOR OF CRYSTAL ADHESION TO RENAL CELLS

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date5/1/994/30/06

ASJC

  • Medicine(all)