Elevated O-GlcNAc modification of proteins in diabetic kidney

Increased flux through the hexosamine biosynthesis pathway promotes the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc). In our previous immunohistochemical study, we demonstrated that the extent of O-GlcNAcylation was increased in kidneys from diabetic GK rats. In the present study, to identity marker proteins that change in their extent of O-GlcNAcylation in the diabetic kidney from GK rats, we separated total kidney proteins by two-dimensional gel electrophoresis. O-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (alpha-actin, alpha-tubulin, alpha-actinin 4, myosin) and mitochondrial proteins (ATP synthase beta, pyruvate carboxylase). This is the first report to show that alpha-actinin 4, whose mutation causes familiar segmental glomerulosclerosis (FSGS), is O-GlcNAcylated. Results of immunoprecipitation and immunoblot studies, as well as in situ Proximity Ligation Assays demonstrated that the extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence studies revealed that the levels of immunoreactivity of actin, alpha-actinin 4 and myosin were markedly increased in the glomerulus of the diabetic kidney. In contrast, the level of immunoreactive alpha-tubulin was concomitantly decreased in the proximal tubule of the diabetic kidney. Immunoelectron microscopy revealed that immunolabeling of alpha-actinin 4 increased in the foot process of podocytes. These results suggest that changes in the O-GlcNAcylation of cytoskeletal proteins are closely associated with the morphological changes in the podocyte foot processes in the glomerulus and in microvilli of proximal tubules in the diabetic kidney.