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Kidney Week

Abstract: PO0524

Optimization of Oxalobacter formigenes-Derived Small Peptides with Therapeutic Potential for Hyperoxalemia, Hyperoxaluria, and Related Kidney Stones

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Hassan, Hatim A., The University of Chicago, Chicago, Illinois, United States
  • Alshaikh, Altayeb, The University of Chicago, Chicago, Illinois, United States
  • Zerweck, Jonathan, The University of Chicago, Chicago, Illinois, United States
Background

Most kidney stones (KS) are composed of calcium oxalate, and very small increases in urine oxalate enhance the stone risk. Besides KS, oxalate also potentially contributes to CKD progression and CKD - and ESRD-associated cardiovascular diseases, emphasizing the need for plasma and urinary oxalate lowering therapies, and enhancing the bowel’s ability to secrete oxalate may effectively do so. We previously discovered Oxalobacter-derived secreted factors stimulating oxalate transport by human intestinal Caco2-BBE (C2) cells and reducing urinary oxalate excretion in hyperoxaluric mice by inducing colonic oxalate secretion. We identified the small peptides P8 and P9 as the major secreted factors and they have significant therapeutic potential for hyperoxalemia and hyperoxaluria. Natural peptides are often not suitable therapeutics due to rapid degradation by proteolytic enzymes, and P8 & 9 peptides have multiple enzymatic cleavage sites.

Methods

Described under Results.

Results

To optimize P8 & 9 peptides and make them resistant to proteolytic degradation, there were subjected to the following structural modifications. N-terminal acetylation (P8-Ac & P9-Ac), C-terminal amidation (P8-Am & P9-Am), retroinverso (P8-RI & P9-RI), and replacing several glycine and lysine sites with PEG6 (P8-P & P9-P) & ornithine (P8-O & P9-O), respectively. All of these modified peptides stimulated oxalate transport by C2 cells similar to the native P8 & 9, except P9-RI (47.3% less functional) and P8-RI (nonfunctional). The native and modified peptides were then treated with different enzymes (trypsin, proteinase K, and colonic lavage fluid [CLF: mimics the colonic environment]) to evaluate the impact of such modifications using LC-MS and/or HPLC in an in vitro stability assay. Native and modified (P8/9-Ac, P8/9-Am, & P8/9-P) peptides were completely degraded by the above enzymes. P8-O and P9-O have improved stability (~57-80%) against trypsin, but they were fully degraded by proteinase K and CLF. Importantly, P9-RI is completely resistant to degradation by the above enzymes.

Conclusion

P9-RI is the most stable optimized peptide, but is less functional compared to native P9. Studies are ongoing to evaluate its in vivo therapeutic effects in lowering plasma and urinary oxalate levels in hyperoxalemic and hyperoxaluric mice.

Funding

  • NIDDK Support