An immunohistochemical analysis to validate the rationale behind the enhanced immunogenicity of D-ribosylated low density lipo-protein

Abstract

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease in patients with diabetes and renal failure. D-ribose is one of the naturally occurring pentose monosaccharide present in all living cells and is a key component of numerous biomolecules involved in many important metabolic pathways. Formation of D-ribose derived glycated low density lipoprotein (LDL) has been previously demonstrated but no studies have been performed to assess the immune complex deposition in the kidney of rabbits immunized with glycated LDL. In this study, LDL was glycated with D-ribose, and it was further used as an immunogen for immunizing NZW female rabbits. The results showed that female rabbits immunized with D-ribose modified LDL induced antibodies as detected by direct binding and competitive ELISA. The modified LDL was found to be highly immunogenic eliciting high titer immunogen-specific antibodies, while the native forms were moderately immunogenic. The induced antibodies from modified LDL exhibited wide range of heterogeneity in recognizing various proteins and amino acids conformers. Furthermore, our histopathological results illustrated the deposits of immune complex in glomerular basement membrane in rabbits immunized with D-ribose-LDL.

Figure 1. Level of induced antibodies against D-ribose-modified LDL.

Direct binding ELISA of D-ribose antisera (□), N-LDL antisera (▴), G-LDL antisera (♦) and preimmune sera (▪). The microtiter wells were coated with D-ribose, N-LDL and G-LDL (10 µg/ml) in direct binding ELISA of D-ribose antisera, native LDL antisera and G-LDL antisera respectively. Each data represents average of three experiments. The values represent the mean ± SD.

Figure 2. Inhibition of serum antibodies against N-LDL and G-LDL binding by D-ribose (♦), N-LDL (▴) and G-LDL (▪) respectively.

The microtiter plate was coated with D-ribose, N-LDL and G-LDL (10 µg/ml) respectively. Each data represents average of three experiments. The values represent the mean ± SD.

Figure 3. Kidney section from female rabbit immunized with native LDL showing normal morphology of glomeruli (a) (Magnification-60x); whereas, Kidney section from female rabbit immunized with D-ribose-modified LDL showing larger glomerular capillary tuft with increased number of nuclei showing proliferation of endothelium and mesenglial cells (b & c) (Magnification-60x).

Scale bar 100 µm.

Figure 4. Immunofluorescence of kidney sections of rabbit immunized with native LDL (a); whereas Immunofluorescence of kidney sections of rabbit immunized with D-ribose-modified LDL showing immune complex deposition on GBM (b & c).

Scale bar 100 µm.