Pediatric Urology Fall Congress, Sept 9-11 2016, Fairmont The Queen Elizabeth
 Montréal, Canada



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Identification of Putative Urinary Biomarkers for Pediatric Stone Disease by Combining Molecular, Biochemical, and Proteomic Approaches
Joel F. Koenig, MD, Scott R. Manson, PhD, Qiusha Guo, PhD, Katelynn H. Moore, BS, Zev Leopold, BS, Paul F. Austin, MD.
Washington University School of Medicine, Saint Louis, MO, USA.

BACKGROUND:
Approximately 50% of patients with urinary stone disease (USD) experience at least one recurrence. Increasing our limited knowledge of crystal-protein and crystal-epithelial interactions could significantly improve our ability to predict recurrence and guide treatment decisions in USD. Standard proteomic approaches have made little progress since these techniques are unable to provide information about functional relevance. In this study, we (1) demonstrate the molecular importance of human urinary proteins in regulating COM adhesion and growth and (2) combine the biochemical purification of COM-binding proteins with targeted proteomics to discover possible biomarkers for USD.
METHODS:
Stone formation was modeled in vitro by assessing the adhesion of fluorescently-labelled COM crystals (COM-FITC) to confluent monolayers of IMCD3 epithelial cells in the presence or absence of human urinary proteins. Urine samples were collected from pediatric (mean age = 11.0) and adult (mean age = 64.3) patients either with or without a history of USD (n = 7-10 / group). Parallel analyses were performed on each sample including: (1) affinity chromatography using control and COM-loaded columns to purify binding proteins, (2) protein quantification to measure the total levels of COM-binding proteins, (3) HPLC-MS for high-throughput identification of COM-binding proteins, and (4) immunoblotting and ELISA to perform protein-specific analyses and validate results.
RESULTS:
While incubation of IMCD cells with COM-FITC crystals resulted in rapid binding with high affinity, the addition of human urinary proteins inhibited COM-FITC binding by 76.2%. Furthermore, urinary proteins inhibited the growth of COM crystals in artificial urine by 53.5%. The mean urinary concentration of COM-binding proteins is 72.1 μg/mL in children and 104.9 μg/mL in adults. Only 11.9% and 16.5% of urinary proteins bind to COM with high affinity in children and adults, respectively. Affinity chromatography was highly specific for purifying COM-binding proteins, as <1% of urinary proteins bound to the control column. HPLC-MS identified known stone-associated proteins including uromodulin and osteopontin along with 45 novel urinary COM-binding proteins including apolipoprotein D, pro-EGF, heparan sulfate, and pro-thrombin. The binding specificities of representative proteins were successfully validated by immunologic methods, as evidenced by 80.8% binding of uromodulin but only 0.1% binding of albumin to the COM column. Protein-specific analyses have begun to reveal associations with age and disease status, but larger study populations will be required to define these relationships. [All results are p < 0.05]
CONCLUSIONS:
This study demonstrates that urinary proteins bind to COM crystals and inhibit their adhesion and growth. There is a >2.5-fold range of variability of the total levels of COM-binding proteins in human urine, raising the possibility that these variations are an important determinant of susceptibility to USD. Additionally, differences in the levels of COM-binding proteins in adults and children may begin to explain differences in UDS, but further detailed studies are needed. Targeted proteomics generated a catalog of 47 COM-binding proteins that will serve as an important resource for identifying novel genetic causes, diagnostic biomarkers, and therapeutic targets in USD.
FUNDING SOURCES: NIH, NIDDK, SLCH Foundation, Midwest Stone Institute, and Urology Care Foundation.


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