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Abstract: TH-PO564

Exploring Mechanisms of Protein Influence on Calcium Oxalate Kidney Stone Formation

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Wesson, Jeffrey, VA Medical Center, Milwaukee, Wisconsin, United States
  • Berger, Garrett K., Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Eisenhauer, Jessica A., Medical College of Wisconsin - Kidney Disease Center, Milwaukee, Wisconsin, United States
  • Hoffmann, Brian R., Medical College of Wisconsin, Milwaukee, Wisconsin, United States
Background

Calcium oxalate monohydrate (COM) crystals are the primary constituent of most kidney stones, but urine proteins in stone matrix are believed to be critical to binding crystals into stones. Recent data have shown that hundreds of proteins appear in stone matrix with no explanation for inclusion of these various proteins. We have proposed a stone formation model with protein stimulated COM aggregation based on polyanion-polycation aggregation, which is supported by finding that matrix is highly enriched in strongly anionic and strongly cationic proteins. Many proteins are likely drawn to such aggregates due to their limited solubility in water. Finding similar protein enrichment in both polyarginine (pR) induced aggregates of urine proteins and COM stone matrix would support this hypothesis.

Methods

Purified proteins (PP) were obtained from random urine samples from six healthy adults by ultradiafiltration. Protein aggregation was induced by adding pR to PP solutions at each of two concentrations; 0.25 and 0.5 µg pR /µg of PP. The resulting protein aggregates were separated by centrifugation, yielding aggregate (pRB) and supernatant fractions. Samples of each fraction and the original PP mixture were lyophilized and sent to the Proteomics Core Laboratory at Mayo Clinic for analysis.

Results

SDS gel electrophoresis revealed selective inclusion of urine proteins in pRB, which was shown to mimic COM matrix protein distributions by mass spectrometry analysis. Notable differences include enrichment of albumin and uromodulin in the pRB at the 0.5 µg pR addition compared to relative exclusion from COM matrix, while at 0.25 µg pR, albumin stayed in solution likely due to its weaker anionic charge, suggesting that aggregation was "overdriven" at the 0.5 µg pR addition. Many intracellular or nuclear proteins, that were prominent in COM matrix, were not observed in pRB, likely reflecting their absence in PP.

Conclusion

Aggregates induced by pR addition to PP samples collected a protein mixture that mimicked the protein distribution observed in COM matrix, supporting our hypothesis. The apparently discordant behavior of uromodulin may simply reflect its anionic character in this overdriven model. Future experiments will need to include observations of selective protein binding to COM crystal surfaces for comparison with these data.

Funding

  • NIDDK Support