Merlin C Thomas1, Mark E Cooper1 , George Jerums2,
Danielle Alberti Memorial Centre for Diabetes Complications, Baker Heart Research Institute, Melbourne 8008, Victoria, AUSTRALIA;
2University of Melbourne, Director of Endocrinology, Endocrine unit, Austin and Repatriation Medical Centre (Austin Campus), West Heidelberg, Victoria, AUSTRALIA
Prolonged hyperglycemia and oxidative stress in diabetes result in the production and accumulation of advanced glycation end products (AGEs) . AGEs are formed via the Maillard or 'browning' reaction between reducing sugars and amine residues on proteins, lipids or nucleic acids. Under normal circumstances, this reaction is slow, meaning that AGE-modification predominantly occurs in long-lived molecules such as collagen and lens proteins . The degree of AGE-modification therefore represents one mechanism to judge the 'age' of a molecule allowing the recognition of senescent targets for excretion or catabolism . In addition, as molecular turnover is reduced with increasing chronological age , the amount and variety of AGE-modified tissue increases, contributing to many of the changes recognised as signs of ageing (such as cataracts and stiffness). In diabetes, prolonged hyperglycemia and oxidative stress hasten the formation of AGEs , meaning not only that long-lived proteins become more heavily modified but also that shorter-lived molecules such as apolipoproteins become targets for de novo advanced glycation [1,2]. In addition, the intracellular formation of AGEs from reactive carbonyl intermediates may occur at a much faster rate than glucose-derived AGE formation that occurs outside the cell. These intracellular AGEs potentially represent an important source of glycation products, as AGE levels may be increased after only days of hyperglycemia, well before similar changes can be demonstrated in vitro.
AGEs have been shown to have a wide range of chemical, cellular and tissue effects implicated in the development and progression of diabetic nephropathy
Table 1. Effects of AGEs that potentially contribute to diabetic nephropathy
Intermolecular and intramolecular cross-linking [22,33-36] Disruption of cell-matrix and matrix-matrix interactions [38-40] Increased ECM synthesis [43-48] Tubuloepithelial-mesenchymal transdifferentiation  Reduced metalloprotease activity [52,53]
Induction of oxidative stress:
Increased generation of reactive oxygen species  Activation of NADPH oxidase  Mitochondrial dysfunction [1,60] Depletion of endogenous anti-oxidants 
Induction of cytokines and growth factors including: TGFßl, CTGF, VEGF, IGF-1, PDGF TNFa, IL-1B, IL-6 [1,2,19,22]
Activation of cellular pathways: PKC-MAPK pathway  Tyrosine kinase pathway  NFkB [19-22]
Other effects :
Altered expression of nephrin [70-73] Disruption of vaso-relaxation 
The accumulation of AGEs in diabetes is closely linked to the presence and duration of hyperglycemia. For example, the DCCT demonstrated a close association between hyperglycemia and AGEs levels, with significant reductions in skin AGE concentrations after intensive glycaemic intervention . However, AGEs may be considered downstream mediators of renal injury in diabetes. This is illustrated by studies showing that inhibition of advanced glycation is able to attenuate renal injury, without influencing glycaemic control . In addition, administration of exogenous AGEs in non-diabetic animals is able to generate lesions similar to those seen in diabetic nephropathy
. In the DCCT study, AGE levels were a better predictor for the development of complications of diabetes than glycated haemoglobin, with over a third of the variance in complications in that study attributable to differences in AGE indices. Notably, the influence of AGEs was even greater in the intensive control cohort, suggesting that while glycaemic control is important, it is not sufficient to prevent complications . Taken together, these studies suggest that AGEs represent a potent common mechanism by which hyperglycemia and oxidative stress may induce renal injury in patients with diabetes.
AGEs are a chemically heterogeneous group of compounds, many of which are poorly defined or remain to be identified . While AGE-modification usually commences at one amino-group, some of the best-characterised AGEs, such a pentosidine and pyrroline, form intermolecular cross-links between modified proteins. These cross-links can result in significant changes in protein structure and function, such as increased stiffness and resistance to proteolytic digestion. In addition, many of these compounds have intrinsic fluorescence, meaning that tissue fluorescence may be used as a marker for the presence of AGE modifications. For example, tissue fluorescence has been shown to increase with ageing . With the development of diabetes, there is a marked increase in tissue fluorescence in the kidney , the retina  and other sites of diabetic microvascular disease . Renal and hepatic impairment are also associated with increased tissue fluorescence, reflecting the role of these organs in the catabolism and excretion of AGEs .
Other AGEs, such as carboxymethyllysine (CML), are neither cross-links nor fluorescent. However, CML constitutes the main epitope for recognition by most commercially available antibodies used for the detection and quantification of AGEs. In clinical studies, Makita et al have reported increased serum CML-AGE levels in diabetic patients . Notably, the greatest increase in CML-AGE staining in diabetes appears to occur in the diabetic glomerulus , particularly in the expanded mesangial matrix and nodular lesions of diabetic nephropathy .
The molecular identity of the AGEs that most contribute to the development of diabetic complications including nephropathy has not been clearly determined. In recent studies, Miura et al found that fluorescent AGEs better correlated with complications in patients with type I diabetes than levels of pentosidine or non-fluorescent AGEs like CML . However, in a study of patients with type II diabetes, Beisswenger et al reported that non-fluorescent CML-AGE levels were better associated with the presence of complications including retinopathy and nephropathy . It is possible that both these measured AGEs may be a marker for the presence of (unmeasured) reactive AGE intermediates . In addition, if the majority of AGE-mediated injury occurs via activation of multi-ligand AGE receptors (see below), it is possible that the accumulation of AGEs via a variety of pathways and in a variety of molecular forms may be similarly pathogenic.
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