Morphological changes in diabetic kidney are associated with increased O-GlcNAcylation of cytoskeletal proteins including α-actinin 4

Abstract

PurposeThe objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes.MethodsO-GlcNAcylated proteins were identified by two-dimensional gel electrophoresis, immunoblotting and peptide mass fingerprinting. The level of O-GlcNAcylation of these proteins was examined by immunoprecipitation, immunoblotting and in situ Proximity Ligation Assay (PLA).ResultsO-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase β, pyruvate carboxylase). The extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence and in situ PLA studies revealed that the levels of O-GlcNAcylation of actin, α-actinin 4 and myosin were significantly increased in the glomerulus and the proximal tubule of the diabetic kidney. Immunoelectron microscopy revealed that immunolabeling of α-actinin 4 is disturbed and increased in the foot process of podocytes of glomerulus and in the microvilli of proximal tubules.ConclusionThese 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. This is the first report to show that α-actinin 4 is O-GlcNAcylated. α-Actinin 4 will be a good marker protein to examine the relation between O-GlcNAcylation and diabetic nephropathy.

High glucose reduces megalin-mediated albumin endocytosis in renal proximal tubule cells through protein kinase B O-GlcNAcylation

“…We showed that glucose inside the LLC-PK1 cells increases O-GlcNAcylation, which is correlated with inhibition of albumin endocytosis, opening the possibility that this mechanism could be involved in albuminuria in the early stage of DN. In agreement, Akimoto et al (56) showed increased O-GlcNAcylation in specific PT cytoskeleton proteins in a T2DM animal model. Ji et al (57) suggested that abnormal O-GlcNAcylation of actin and tubulin alters their polymerization, reducing microtubule-dependent reabsorption mechanism in PT cells.…”

Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy

“…Post‐translational modifications, such as O‐linked N‐ acetyl‐b‐D‐glucosamine modification, have been described as responsible for morphologic changes at both the glomerular and tubular levels in the diabetic kidney, and these modifications involve cytoskeletal proteins (39). Cyto‐ skeletal changes, in turn, would affect the barrier to protein filtration, as suggested by both in vitro studies under HG conditions (40) and in vivo studies in human DN (41).…”

“…The involvement of ubiquitination in the organization of cytoskeletal proteins has been recently linked to the proteasome activity (37), whereas the role of Lys63 ubiquitination in the regulation of cellular adhesion and migration has been described only in tumor cells (38). Post-translational modifications, such as O-linked Nacetyl-b-D-glucosamine modification, have been described as responsible for morphologic changes at both the glomerular and tubular levels in the diabetic kidney, and these modifications involve cytoskeletal proteins (39). Cytoskeletal changes, in turn, would affect the barrier to protein filtration, as suggested by both in vitro studies under HG conditions (40) and in vivo studies in human DN (41).…”

Post-translational modification and regulation of actin

“…Although poorly understood, actin O-GlcNAcylation has been implicated in modulating cardiac [32] and skeletal [33] muscle contraction, in cardioprotection [34], and in tissue changes in diabetics [35], as has another glucose-mediated actin PTM, glycation (the non-enzymatic attachment of glucose to lysine residues). Notably, purified actin is susceptible to glycation, as is actin in diabetic patients and animal models [36–39], where glucose decreases polymerization and lowers F-actin levels [39–41].…”