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1.
Free Radic Biol Med ; 89: 83-90, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26159508

ABSTRACT

Diabetes is characterized, in part, by activation of toxic oxidative and glycoxidative pathways that are triggered by persistent hyperglycemia and contribute to diabetic complications. Inhibition of these pathways may benefit diabetic patients by delaying the onset of complications. One such inhibitor, pyridoxamine (PM), had shown promise in clinical trials. However, the mechanism of PM action in vivo is not well understood. We have previously reported that hypohalous acids can cause disruption of the structure and function of renal collagen IV in experimental diabetes (K.L. Brown et al., Diabetes 64:2242-2253, 2015). In the present study, we demonstrate that PM can protect protein functionality from hypochlorous and hypobromous acid-derived damage via a rapid direct reaction with and detoxification of these hypohalous acids. We further demonstrate that PM treatment can ameliorate specific hypohalous acid-derived structural and functional damage to the renal collagen IV network in a diabetic animal model. These findings suggest a new mechanism of PM action in diabetes, namely sequestration of hypohalous acids, which may contribute to known therapeutic effects of PM in human diabetic nephropathy.


Subject(s)
Collagen Type IV/drug effects , Diabetes Mellitus, Experimental/prevention & control , Hypochlorous Acid/toxicity , Kidney/drug effects , Proteolysis/drug effects , Pyridoxamine/pharmacology , Vitamin B Complex/pharmacology , Amino Acid Sequence , Animals , Bromates/toxicity , Chromatography, Liquid , Collagen Type IV/chemistry , Collagen Type IV/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Humans , In Vitro Techniques , Kidney/pathology , Male , Molecular Sequence Data , Oxidants/toxicity , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Tandem Mass Spectrometry
2.
Diabetes ; 64(6): 2242-53, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25605804

ABSTRACT

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes. We demonstrate that hypohalous acid-derived modifications of renal tissues and extracellular matrix (ECM) proteins are significantly elevated in experimental diabetic nephropathy. Moreover, diabetic renal ECM shows diminished binding of α1ß1 integrin consistent with the modification of collagen IV by hypochlorous (HOCl) and hypobromous acids. Noncollagenous (NC1) hexamers, key connection modules of collagen IV networks, are modified via oxidation and chlorination of tryptophan and bromination of tyrosine residues. Chlorotryptophan, a relatively minor modification, has not been previously found in proteins. In the NC1 hexamers isolated from diabetic kidneys, levels of HOCl-derived oxidized and chlorinated tryptophan residues W(28) and W(192) are significantly elevated compared with nondiabetic controls. Molecular dynamics simulations predicted a more relaxed NC1 hexamer tertiary structure and diminished assembly competence in diabetes; this was confirmed using limited proteolysis and denaturation/refolding. Our results suggest that hypohalous acid-derived modifications of renal ECM, and specifically collagen IV networks, contribute to functional protein damage in diabetes.


Subject(s)
Bromates/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Animals , Hypochlorous Acid/metabolism , Integrins/metabolism , Kidney/metabolism , Kidney/pathology , Male , Mice , Mice, Knockout , Molecular Dynamics Simulation , Nitric Oxide Synthase Type III/metabolism , Rats , Rats, Sprague-Dawley
3.
Biochem Biophys Res Commun ; 456(2): 610-4, 2015 Jan 09.
Article in English | MEDLINE | ID: mdl-25499815

ABSTRACT

Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS(-/-) C57BLKS and eNOS(-/-) C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology.


Subject(s)
Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 2/complications , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/urine , Aconitic Acid/metabolism , Aconitic Acid/urine , Animals , Glycine/analogs & derivatives , Glycine/metabolism , Glycine/urine , Hippurates/metabolism , Hippurates/urine , Indican/metabolism , Indican/urine , Keto Acids/metabolism , Keto Acids/urine , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide Synthase Type III/genetics , Phenylacetates/metabolism , Phenylacetates/urine
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