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Abstract: TH-PO881

Urinary Peptidomic Analysis Reveals Bioactive Uromodulin Peptides Associated with Early Type 1 Diabetes

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Van, Julie Anh Dung, University of Toronto, Toronto, Ontario, Canada
  • Clotet-Freixas, Sergi, University Health Network, Toronto, Ontario, Canada
  • Batruch, Ihor, Mount Sinai Hospital, Toronto, Ontario, Canada
  • Zhou, Xiaohua, University of Toronto, Toronto, Ontario, Canada
  • Mahmud, Farid H., Hospital for Sick Children, Toronto, Ontario, Canada
  • Diamandis, Eleftherios P., Mount Sinai Hospital and University Health Network, Toronto, Ontario, Canada
  • Scholey, James W., University of Toronto, Toronto, Ontario, Canada
  • Konvalinka, Ana, University of Toronto, Toronto, Ontario, Canada
Background

Diabetic nephropathy is the leading cause of kidney disease worldwide. Yet, current treatments cannot prevent the progressive nature of the disease, exposing our limited understanding of the early kidney response to chronic hyperglycemia. In this study, we compared the urinary peptidomes of youths with type 1 diabetes without evidence of nephropathy and age/sex-matched healthy controls, in order to determine early changes in protein processing in the hyperglycemic kidney.

Methods

The study population comprises two separate cohorts: a discovery cohort (N = 30) and an internal validation cohort (N = 30). Urines were normalized to creatinine and underwent 10kDa-filter centrifugation to isolate naturally occurring peptides. In the discovery phase, filtered peptides were then fractionated and then injected on a Q-Exactive mass spectrometer. For the validation phase, parallel reaction monitoring (PRM) assays were developed on a Q-Exactive HFX instrument. MaxQuant and Skyline were used for peptide identification and quantification. Proteasix was used to predict proteases responsible for generating differentially excreted peptides.

Results

A total of 6349 urinary peptides from 750 proteins were quantified. Of the 15 differentially excreted peptides (P < 0.05), five remained significant after Benjamini-Hochberg adjustment (q < 0.05). Seven of these top 15 peptides derive from the C-terminal region of uromodulin, which regulates uromodulin polymerization. Out of the twelve predicted proteases, hepsin, granzyme A, kallikrein-6, and plasminogen were shortlisted because they were detected in healthy kidney tissues (Human Protein Atlas) and were altered at the level of gene expression in diabetic nephropathy (Nephroseq v5 database). Differential excretion of six uromodulin peptides was validated by PRM. Two of the validated uromodulin peptides induced NF-κB signaling in HK-2 cells, suggesting a potential role in mediating inflammation in kidney cells.

Conclusion

Differences between youths with type 1 diabetes and healthy controls are reflected in the urinary peptidome before the development of microalbuminuria. Uromodulin peptides we discovered may play a significant role in the early injury in a diabetic kidney and may represent a therapeutic target.