Increased level of glycoxidation product Nε-(carboxymethyl)lysine in rat serum and urine proteins with aging: Link with glycoxidative damage accumulation in kidney

doi:10.1016/S0003-9861(02)00735-X

Archives of Biochemistry and Biophysics

Volume 411, Issue 2, 15 March 2003, Pages 215-222

https://doi.org/10.1016/S0003-9861(02)00735-X Get rights and content

Abstract

Accumulation of carboxymethylated proteins (CML-proteins) is taken as a biomarker of glycoxidative stress which is thought to contribute to the age-related impairment in tissue and cell function. To investigate the occurrence and extent of glycoxidative damage with aging in rat kidney, serum and urine, we have prepared a polyclonal antibody against CML-modified bovine serum albumin. We subsequently used it for immunolocalization and in enzyme-linked immunosorbent assays to evaluate CML-protein content. In the serum, CML-protein level was 1.43±0.14 pmol CML/μg protein at 3 months and significantly increased by 50% from 10 to 27 months (1.50±0.14 pmol CML/μg protein vs 2.27±0.26 pmol CML/μg protein), albumin and transferrin being the main modified proteins. In the urine, CML-protein level was 2.50±0.14 pmol CML/μg protein at 3 months and markedly increased from 10 months (2.99±0.24 pmol CML/μg protein) to 27 months (3.76±0.25 pmol CML/μg protein), with albumin as the main excreted modified protein. Immunolocalization of CML-proteins in kidney provided evidence for an age-dependent increased accumulation in extracellular matrices. Intense staining of the glomerular basement membrane (GBM), Bowman’s capsule, and the tubular basement membrane was found. Additionally, the CML content for collagen from GBM was 195.85±28.95 pmol CML/μg OHPro at 3 months and significantly increased from 10 months (187.61±21.99 pmol CML/μg OHPro) to 27 months (334.55±62.21 pmol CML/μg OHPro). These data show that circulating CML-protein level in serum and urine and CML accumulation in nephron extracellular matrices with aging are increasing in parallel. The CML-protein measurement in serum and urine may thus be used as an index for the assessment of age-associated glycoxidative kidney damage.

Section snippets

Animals

Male Wistar rats (WAG/Rij) were born and raised in the animal care facilities of the Commissariat à l’Energie Atomique (CEA Gif-sur-Yvette, France) and fed a commercial diet (DO4; UAR, Villemoisson sur Orge, France). This strain remained lean even when fed ad libitum and did not suffer from age-associated nephropathy [21]. With age, these animals developed a slight but significant proteinuria mainly due to albumin leakage [22], [23]. Cohorts were constituted of young (3-month-old), adult

Characterization of CML antibodies

The specificity of the purified antibodies assessed by Western blot showed no reactivity with native BSA while CML–BSA was strongly labeled (Fig. 1A, left panel). Comparison of immunoreactivity curves of these polyclonal antibodies with commercial monoclonal antibody clone 6D12 revealed comparable affinity toward free antigens (Fig. 1B), indicating that our polyclonal antibodies can be used as a potent antibody for further immunochemical analysis.

CML content in circulating proteins and in GBM collagen

Aging resulted in a significant increase in

Discussion

The main question prompting this investigation was whether serum proteins undergo carboxymethylation in parallel with extracellular matrix (ECM) proteins, especially renal tissues, with aging, thereby corroborating the hypothesis that an age-associated increased glycoxidative stress contributes to the generation of advanced glycation end products. Indeed in previous work, we showed using anti-AGE polyclonal antibodies that AGEs accumulate in rat renal ECM mainly in glomerular basement membrane

Acknowledgements

The authors are highly grateful to Dr. G. Carrard for critical reading of the manuscript and helpful suggestions. The financial support of the MENRT (Institut Universitaire de France et Université Paris7), the Fondation pour la Recherche Médicale, and the European Union (QLRT “Protage” Grant QLK6-CT1999-02193) is gratefully acknowledged.

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Stable isotopic dilution analysis LC-MS/MS provides a robust reference method for measurement of protein oxidation and glycation adducts. Optimised pre-analytic processing of samples and LC-MS/MS analysis procedures are required to achieve this.
Quantitative measurement of protein oxidation and glycation adducts provides information on level of exposure to potentially damaging protein modifications, protein inactivation in ageing and disease, metabolic control, protein turnover, renal function and other aspects of body function. Reliable and clinically assessable analysis is required for translation of measurement to clinical diagnostic use. Stable isotopic dilution analysis LC-MS/MS provides a “gold standard” approach and reference methodology to which other higher throughput methods such as immunoassay and indirect methods are preferably corroborated by researchers and those commercialising diagnostic kits and reagents. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
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This would be in concordance with previous reports describing the accumulation of AGEs such as CML, pentosidine, and pyrraline in the expanded mesangial areas of patients with diabetic nephropathy and IgA nephropathy (mesangial proliferative glomerulonephritis)[24],[25]. Furthermore, AGEs have been described in the mesangium of aging rat[26],[27]. Significant GA-pyridine accumulation was indeed found in diabetic nephropathy, Wegener's granulomatosis, mesangial proliferative glomerulonephritis, and focal glomerulosclerosis.
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Advanced glycation end products (AGEs) contribute to diabetic and atherosclerotic end-organ damage, but the mechanisms of AGE-formation and AGE-induced damage are unclear. Glycolaldehyde (GA) is a Maillard-reaction intermediate and can be formed by reaction of L-serine with the myeloperoxidase-system. GA reacts with proteins to form AGEs, such as GA-pyridine, which is specific for protein modification by GA. GA-pyridine accumulates in human atherosclerotic lesions. As atherosclerosis and progressive glomerulosclerosis share many similarities, we hypothesized that GA-pyridine accumulates in renal diseases, especially those with prominent mesangial involvement.
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Non-enzymatic glycoxydation and lipoxidation of proteins continues to stimulate great interest in gerontology as both markers and promoters of aging. The first aim of the study was to determine the age-related changes in levels of N^ɛ-(carboxymethyl)lysine (CML) and 4-hydroxy-2-nonenal (HNE) present on proteins of the cardiovascular system of Fischer 344 rats and identify the particular polypeptides being modified. The second objective was to evaluate whether pharmacological administration of aminoguanidine (1 g/L in the drinking water) could reverse protein glycoxidation and lipoxidation. CML content in serum, aorta, and heart proteins from 28-month-old rats was double of that found in 4-month-old animals. AG administration to old rats for 3 months from the age of 25 months lowered CML content by 15 (P = .2275), 44 (P < .0001), and 28% (P = .0072) in serum, aorta, and heart, respectively. Serum albumin, transferrin and immunoglobulins were most prominently adducted by both CML and HNE. While the extent of albumin and transferrin modification was comparable between age groups, CML and HNE bound to immunoglobulins increased in the sera of old rats as a result of the accumulation of immunoglobulin heavy and light chains. AG treatment prevented immunoglobulin accumulation in serum, suggesting a beneficial action on renal filtration. Lipoxidation of heart mitochondrial proteins was prevalent over glycoxidation, either as CML or pentosidine. Although AG prevented HNE-induced inactivation of the α-ketoglutarate dehydrogenase complex in vitro, it had no effect in rat hearts, suggesting AG could not reach the mitochondrial matrix.

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Increased level of glycoxidation product Nε-(carboxymethyl)lysine in rat serum and urine proteins with aging: Link with glycoxidative damage accumulation in kidney

Abstract

Section snippets

Animals

Characterization of CML antibodies

CML content in circulating proteins and in GBM collagen

Discussion

Acknowledgements

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Life Sci.

Kidney Int.

Kidney Int.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Free Radicals Biol. Med.

Mech. Ageing Dev.

J. Inorg. Biochem.

Z. Ernahrungswiss.

Proc. Natl. Acad. Sci. USA

J. Lab. Clin. Med.

Drugs Aging

Biochemistry

Biochem. J.

Biochem. J.

Diabetes

Increased level of glycoxidation product N^ε-(carboxymethyl)lysine in rat serum and urine proteins with aging: Link with glycoxidative damage accumulation in kidney