Urinary PRM analysis of early type 1 diabetes
Van JAD, Clotet-Freixas S, Zhou J, Batruch I, Sun C, Glogauer M, Rampoldi L, Elia Y, Mahmud F, Sochett E, Diamandis EP, Scholey J, Konvalinka A. Peptidomic analysis of urine from youths with early type 1 diabetes reveals novel bioactivity of uromodulin peptides in vitro. Mol Cell Proteomics. 2019 Dec;
- Organism: Homo sapiens
- Instrument: Q Exactive HF-X
urine, peptidomics, type 1 diabetes, kidney disease
Chronic hyperglycemia is known to disrupt the proteolytic milieu, initiating compensatory and maladaptive pathways in the diabetic kidney. Such changes in intrarenal proteolysis can be captured in the urinary peptidome. We thus examined the urinary peptidomes of otherwise healthy youths with type 1 diabetes and their non-diabetic peers. This cross-sectional study included two separate cohorts for the discovery (N = 30) and initial validation (N = 30) of differential peptide excretion. Peptide bioactivity was predicted using PeptideRanker and subsequently verified in vitro. Proteasix and the Nephroseq database were used to identify putative proteases responsible for peptide generation and examine their expression in diabetic nephropathy. A total of 6550 urinary peptides were identified in the discovery analysis. Of the 15 differentially excreted peptides (P < 0.05), seven derived from a small region (589SGSVIDQSRVLNLGPITRK607) near the C-terminus of uromodulin. Excretion rates of five uromodulin peptides were validated using parallel reaction monitoring (P < 0.05). One of the validated peptides activated TLR4-dependent NFκB signalling, stimulated cytokine release, and enhanced neutrophil migration in vitro. In silico analyses identified several possible proteases such hepsin, cathepsin B, and meprin A to be responsible for generating these peptides. In summary, uromodulin processing in the kidney produces bioactive peptides, which may be differentially excreted in early diabetes.
We followed the Standard Protocol for Urine Collection and Storage, created by the Human Kidney and Urine Proteome Project (HKUPP) and the Human Proteome Organization (HUPO). Following collection, all second-morning midstream urine samples were kept at 4C until further processing. All urine samples were centrifuged at 1000g for 10 minutes to remove intact cells and debris. The supernatants were then stored at -80C. This initial processing step was completed within 3 hours of urine collection to obviate the need for urine preservatives. Samples were de-identified and randomized so that investigators were blinded to experimental groups during sample processing. When ready, frozen samples were thawed, vortexed, and centrifuged at 1000 g for 10 minutes to remove any remaining cells and debris. Sample volumes were normalized to 20 μmol of creatinine. After adjusting the pH to 8 using ammonium bicarbonate, we used Vivaspin Centrifugal Concentrators (VivaProducts) with 10-kDa cut-off membranes to isolate urinary peptides. Crude heavy-labeled peptides were ordered from JPT Peptide Technologies and were spiked into the samples just prior to Vivaspin centrifugation. We added dithiothreitol to a final concentration of 2mM to reduce protein disulfide bonds; and subsequently iodoacetamide to a final concentration of 4 mM to alkylate and prevent the re-formation of disulfide bonds. The peptides were then passed through solid-phase extraction Oasis HLB cartridges (Waters Corporation), after adjusting pH to 4 with formic acid. To remove urinary pigments, we added ethyl acetate, vortexed and centrifuged the sample, and then discarded the supernatant. The samples were then loaded onto disposable Evotips and subsequently injected in duplicate onto the Evosep One nLC system coupled to a Q Exactive HF-X hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) on a 22-minute gradient.
All participants were asked to provide a single second-morning, midstream urine sample. To be eligible for inclusion into this cross-sectional study, all participants were 19 years of age or younger at time of urine collection, free of significant comorbidity (ie., hypertension, proteinuria, renal disease, macrovascular disease, and chronic inflammatory disease), and not taking corticosteroid, anti-inflammatory, or anti-hypertensive drugs. Our study population comprises two separate cohorts: a discovery cohort (N = 30) and an internal verification cohort (N = 30). As such, our study population includes a total of 60 urine samples from 60 youths.
Created on 8/19/19, 9:11 PM