Glycosylation on Urinary Exosome for Biomarker Discovery

IgA Nephropathy (IgAN) and thin basement membrane nephropathy (TBMN) have identical hematuria symptom but different seriousness in renal function. In case of TBMN, renal function is usually normal but IgAN is the most common cause of end-stage renal failure. In general, the biopsy of the kidney is performed to distinguish between IgAN and TBMN. There are a lot of needs for clinicians to have a sensitive and specific biomarker for differentiation IgAN and TBMN. Exosomes which are small cell-derived vesicles have been suggested as potential biomarkers in diagnosis of disease because these are secreted from original cell with intracellular fluids and apical membrane. Urinary exosomes, 40-100nm vesicles secreted to urine by renal cells, may provide non-invasive diagnosis manner of kidney disease. Glycosylation is highly sensitive to the biological environment changes and considerably affected by disease states. Glycan is known as playing key roles in cancer metastasis and intracellular recognition. Therefore, the examination of glycosylation change in urinary exosome may be a new way for potential biomarkers to differentiate TBMN and IgAN. In this study, we have analyzed 18 individuals exosomes secreted into urine from renal epithelial cells. Samples were composed of three different groups, mainly TBMN, IgAN patients, and healthy volunteers. Briefly, N-glycans from urinary exosomes were released by PNGase F digestion and then enriched by solid phase extraction using a porous graphitized carbon cartridge. N-glycans were eluted with three different solution (10% ACN, 20% ACN, and 40% ACN with 0.05% TFA in H2O) based on the glycan size and polarity. Enriched glycans were analyzed for qualitative and quantitative profiling using high resolution MALDI-TOF/TOF mass spectrometry and performed tandem MS to obtain extensive structure information. We also separated isomer-specific glycans by nano-LC chip/Q-TOF mass spectrometry. We found that high mannose glycans are high in abundance in normal group. Complex, non-sialylated fucosylated tri-, and tetraantennary glycans are significantly different between normal and patients, which suggest the potential biomarker.