Current topicsEvidence of O-linked N-acetylglucosamine in diabetic nephropathy
Introduction
Diabetic nephropathy (DNP) significantly impairs the quality of life of patients with diabetes and contributes to the increasing financial burden associated with chronic diseases such as diabetes. However, the specific mechanisms underlying the development of DNP remain to be elucidated. Glucotoxicity is an important contributing factor in the development and progression of diabetic complications associated with several different tissues and organs. The adverse effects associated with glucotoxicity have been attributed to over-activation of the polyol pathway, increased formation of advanced glycation end products, over-activation of protein kinase C and increased flux via hexosamine biosynthesis pathway (HBP) (Brownlee 2001).
The HBP is reported to consume 2–5% of glucose taken up by the cell leading to the formation of UDP-β-N-acetylglucosamine (UDP-GlcNAc) (Buse 2006), UDP-GlcNAc is the substrate for O-GlcNAc transferase (OGT) which catalyzes the formation of a post-translational modification attaching a single N-acetyl-glucosamine to the serine and threonine residues of nuclear and cytoplasmic proteins, termed “nucleocytoplasmic O-linked beta-N-acetylglucosamine” or “O-GlcNAcylation” (O-GlcNAc) (Buse 2006). Diabetes has been shown to increase protein O-GlcNAc levels, by increasing the flux through the HBP. Several studies have shown that increased flux through the HBP leads to extracellular matrix proliferation (Buse 2006), which is one of the major criteria of the early DNP, and this process is mediated by transforming growth factor-β (TGF-β) (Kolm-Litty et al. 1998) and plasminogen activator inhibitor-1 (PAI 1) (James et al. 2000).
Sustained increases in O-GlcNAc have been associated with the pathogenesis of diseases, such as neurodegenerative diseases, cancer and diabetes (Buse 2006). There is also growing evidence, that O-GlcNAc has a pivotal role in the development of diabetic complications such as diabetic nephropathy. For example, Masson et al. (2005) demonstrated that in mesangium cell culture, increased O-GlcNAc leads to cellular hypertrophy, a characteristic of DNP. Nerlich et al. (1998) showed that Glutamine:fructose-6-phosphate amidotransferase (GFAT), which regulates glucose entry into the HBP, was present in the cells of the human kidney and GFAT expression was increased in diabetic individuals; however, the majority of studies that have examined the role of HBP in diabetic nephropathy have been carried out in cell culture experiments. Consequently, there is remarkably little known about the role of O-GlcNAc in DNP in patients with diabetes. Therefore, the goal of this study was to determine whether O-GlcNAc modification of proteins can be detected in the human kidney and if so whether diabetes is associated with an increase in O-GlcNAc levels.
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Patients
The study was approved by the Ethical Committee of the Medical Faculty of the University of Pécs, Hungary. Renal biopsy specimens were collected from patients with type 2 diabetes (without other immunopathogenic nephropathy) and previously diagnosed DNP (n = 6) and were compared to biopsies from 7 non-diabetic individuals with previously diagnosed thin basement membrane syndrome, serving as controls. The biopsies in diabetic patients were performed due to two basic reasons: 1. rapid decrease in
Results
Glomerular hypertrophy was found in most of the diabetic samples as shown in Fig. 1B. Glomerular mesangial expansion was significantly increased in diabetic patients compared to controls (20 ±0.02% vs. 28 ± 0.02%) (p < 0.05). Using the O-GlcNAc specific antibody CTD110.6, we observed O-GlcNAc modified proteins in the nuclei in both glomerular and tubular cells in the human kidney (Fig. 1, Fig. 2A). Antibody specificity was also confirmed by pre-absorption of CTD 110.6 with 20 mmol/L GlcNAc (Fig. 3).
Discussion
There is increasing evidence that O-GlcNAc plays an important role in the pathogenesis of diabetes and diabetic complications (Buse 2006), mediated primarily via an increase in HBP flux. In 1990 Marshall and co-workers showed, that glucose-induced desensitization of the glucose transport system can be prevented by inhibition of GFAT and thus concluded that GFAT is involved in the induction of insulin resistance, furthermore they showed that glucosamine is approximately 40 times more potent than
Conclusion
Nevertheless, these data provide critical support for the notion that increased HBP flux and O-GlcNAc formation contributes to DNP and provides further impetus for more mechanistic studies on the role of O-GlcNAc in mediating the adverse effects of diabetes on renal function.
Acknowledgement
We are grateful for Mary-Ann Accavitti for kindly providing CTD110.6 antibody. We also thank Tünde Faragó, Anikó Stein and Tünde Visnyei for their technical support and Lajos Markó and László Pótó for their insightful input.
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Péter Degrell and Judit Cseh contributed equally to this work.