Urinary tract stone disease is a common clinical disorder that frequently leads to hospitalization. The objective of the proposed research is to gain new insights into the pathogenesis of urolithiasis through a comprehensive study of urinary protein inhibitors of calcium oxalate (CaOx) crystallization. The grant proposal has three Specific Aims.
Specific Aim #1 proposes to extend previous studies on uropontin (osteopontin found in the kidney) to evaluate the conformation of native and dephosphorylated uropontin and a series of synthetic unmodified and phosphorylated peptides which have aspartic acid-rich CaOx crystal binding domains within the protein sequence. The experimental plan proposes to use circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) for the structural studies to monitor differences in protein conformation and structure. It proposes to correlate these structural observations with parallel crystal growth inhibition studies.
Specific Aim #2 proposes a collaborative study to look for abnormalities in uropontin excretion in patients with accelerated CaOx nephrolithiasis. The co-investigator (Dr. Coe) has an extensive patient data base and can define those patients with at least 10 stone incidents, but a urinary CaOx supersaturation which does not account for the stone burden. The principal investigator will isolate proteins from patient urines, characterize the excretion patterns, and utilize the biophysical techniques described in Specific Aim #1 for structural characterization.
Specific Aim #3 proposes to isolate, purify and structurally study other urinary proteins that show crystal inhibitory properties. The applicant will use his set of immunologic reagents and monoclonal and polyclonal antibodies for the isolation and then use other molecular biological techniques to look for sequence homology with uropontin. Structural studies as described in Specific Aim #1 will potentially also be used.
Showing the most recent 10 out of 26 publications
