Crossline-like Structure Accumulates as Fluorescent Advanced Glycation End Products in Renal Tissues of Rats with Diabetic Nephropathy.

Although formation of characteristic fluorescence (Exmax/Emmax:370/440nm) in renal tissues has been known to be enhanced in diabetic model rats and the natural fluorophores have been thought as advanced glycation end products (AGEs), these structures remain unclear to this date. Since we had already isolated Crossline derivatives as the main fluorescent AGE-products formed in a sugar-amine-model system in vitro, we tried to determine whether crossline-like structure can be found in renal tissues of rats with diabetic nephropathy. Specific polyclonal antiserum against crossline-structure and its affinity chromatography-purified antibody were prepared. The purified antibody did not recognize native rat serum albumin (RSA), but it recognized RSA modified by prolonged incubation with high concentration of glucose (AGE-RSA) as well as the crossline-hapten. The immunoreactivity of AGE-RSA was completely blocked by co-incubation with crossline-derivative. These results confirm immunochemically that in vitro-prepared AGE-proteins contain crossline-like structure. Similarly, crossline-like structure in AGE-proteins in vivo was examined immunohistochemically. In streptozotocin-induced diabetic rats, both crossline-like immunoreactivity and fluorescence (Ex/Em:370/440 nm) of renal tissues increased, and the levels rose further with progression of diabetic nephropathy. Crossline accumulation was observed in specific renal tissues such as glomeruli (basement membrane and mesangium), tubules, and Bowman's capsules. Although the contribution of crossline-like substance(s) to total fluoroescence in diabetic renal tissues has not as yet been quantitatively determined, our results provide evidence that crossline accumulation occurs in renal tissues of rats in association with the initiation and/or progression of diabetic nephropathy.