O-glycosyltransferase activation in leukocytes contributes to early renal function impairment in type 2 diabetes

Leukocytes infiltration into renal tissue is an early feature of diabetic kidney disease. Leukocyte homing and extravasation is influenced by O-glycosylation of serine and threonine rich proteins by enzyme Core 2 β-1, 6-N-acetylglucosaminyltransferase (Core-2). We hypothesized that hyperglycemia induces activation of Core-2 O-glycosylates adhesion molecule very late antigen-4 (VLA-4), thereby increasing adhesion and extravasation of leukocytes to endothelial cells and promoting renal inflammation. We isolated peripheral blood leukocytes from age-matched healthy volunteers (CON; n = 20) and patients with a minimum known duration of 5 years of type 2 diabetes (T2D; n = 20). In total, 12 T2D had microalbuminuria. Heparinized blood was used to assess leukocyte subpopulations, and subunits of VLA-4 on lymphocytes and monocytes. Isolated leukocytes were lysed for measuring Core-2 activity and O-glycosylation on VLA-4. Core-2 activity was higher in T2D than in CON (891 ± 320 vs. 73 ± 23 pmol/h/mg protein, P < 0.01) and associated positively with urinary albumin excretion (R = 0.54, P < 0.05), HbA1c (R = 0.62, P < 0.001) and high sensitive C-reactive protein (R = 0.51, P < 0.01), but negatively with glomerular filtration rate (R =-0.47, P < 0.01). Despite unchanged total expression of VLA-4 on leukocytes, Core-2 induced O-glycosylation was higher in T2D than in CON (2.7 ± 0.5 vs. 0.5 ± 0.1 arbitrary units, P < 0.05). In vitro, we found lower adhesion capacity of leukocytes transfected with Core-2 knockdown sequence compared to untransfected cells, under normoglycemic as well as hyperglycemic conditions (n = 2). In conclusion, Core-2 induced VLA-4 O-glycosylation potentially contributes to the pathogenesis of early stage of diabetes-related kidney disease.