Tissue copper – AGE interactions in the aetiopathogenesis of tissue damage in the kidney and the heart in diabetes and their experimental treatment by selective Cu(II)-chelation

Abstract

Diabetes is a disease characterised by high blood glucose levels that affects over 300 million people worldwide. Moreover, more than 3 million people die from diabetic complications of the heart and kidney each year. Pathophysiologic changes in the kidneys and hearts of diabetic patients have been shown to correlate with the enhanced formation of a class of post-translational protein modifications termed advanced glycation end-products (AGEs), which are implicated as key factors in the pathogenesis of tissue damage in diabetes and related diseases. For example, enhanced AGE formation in collagen has been linked to corresponding decreases in pepsin digestibility and acid solubility, which may well contribute to in vivo fibrosis in the kidney and heart. Transition metals such as copper can catalyse in vitro AGE formation, in part through increased oxidative or ’glycoxidative’ stress. Altered copper metabolism occurs in diabetes, wherein the Cu(II)-selective chelator triethylenetetramine (TETA) has been shown to ameliorate diabetic tissue damage in both kidney and heart. The objective of this thesis has been to investigate changes in the structure and function of the extracellular matrix in the kidneys and hearts of healthy and diabetic rats, which had received TETA or corresponding placebo treatments. It was hypothesized that this approach would add to existing understanding of the molecular mechanisms of copper-catalysed AGE formation in diabetes, and of those by which TETA can prevent or reverse organ damage. Analysed here were alterations in collagen structure, the behaviour of enzymes of collagen metabolism and the integrity of defences against oxidative/glycoxidative stress in the heart and kidneys. In addition, collagen extracts from these organs were characterised by quantitative and qualitative methods that included measurement of post-translational modification. Dysregulated collagen metabolism was detected in diabetic kidneys, wherein TETA treatment normalised protein levels without altering the transcription of corresponding genes: these protein-level effects may have been mediated at least in part through actions on collagen-degrading proteases. Collagen from diabetic kidneys displayed altered post-translational modifications whereas TETA treatment partially lowered collagen-AGE levels towards control values. Evidence for altered collagen metabolism was also detected in diabetic hearts at the transcriptional but not the protein level. By contrast, no changes in post translational modification were detected in collagen from diabetic hearts. TETA modified the transcription of some proteases in the diabetic heart but had no measurable action at the protein level. In summary, the occurrence of dysregulated collagen metabolism has here been shown to occur in the hearts and kidneys of diabetic rats, and TETA treatment partially normalized some of the observed changes. Furthermore, a previously unrecognised link between copper homeostasis, collagen-AGEs and collagen-degrading proteases in the diabetic kidney has been identified and partially characterized in this work.