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

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.