Glyceraldehyde-Derived Pyridinium Evokes Renal Tubular Cell Damage via RAGE Interaction

Abstract

Glyceraldehyde-derived advanced glycation end products (glycer-AGEs) contribute to proximal tubulopathy in diabetes. However, what glycer-AGE structure could evoke tubular cell damage remains unknown. We first examined if deleterious effects of glycer-AGEs on reactive oxygen species (ROS) generation in proximal tubular cells were blocked by DNA-aptamer that could bind to glyceraldehyde-derived pyridinium (GLAP) (GLAP-aptamer), and then investigated whether and how GLAP caused proximal tubular cell injury. GLAP-aptamer and AGE-aptamer raised against glycer-AGEs were prepared using a systemic evolution of ligands by exponential enrichment. The binding affinity of GLAP-aptamer to glycer-AGEs was measured with a bio-layer interferometry. ROS generation was evaluated using fluorescent probes. Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GLAP-aptamer bound to glycer-AGEs with a dissociation constant of 7.7 × 10^-5 M. GLAP-aptamer, glycer-AGE-aptamer, or antibodies directed against receptor for glycer-AGEs (RAGE) completely prevented glycer-AGE- or GLAP-induced increase in ROS generation, MCP-1, PAI-1, or RAGE gene expression in tubular cells. Our present results suggest that GLAP is one of the structurally distinct glycer-AGEs, which may mediate oxidative stress and inflammatory reactions in glycer-AGE-exposed tubular cells. Blockade of the interaction of GLAP-RAGE by GLAP-aptamer may be a therapeutic target for proximal tubulopathy in diabetic nephropathy.

Keywords: GLAP; RAGE; diabetic nephropathy; proximal tubular cells.

Figure 1

(a) Effects of RAGE-Ab, AGE-aptamer (AGE-apt), or GLAP-aptamer (GLAP-apt), on reactive oxygen species (ROS) generation in glycer-AGE-exposed tubular cells. Tubular cells were treated with 100 μg/mL glycer-AGEs or 100 μg/mL non-glycated bovine serum albumin (BSA) in the presence or absence of 5 μg/mL RAGE-Ab, 10 nM AGE-apt, or 10 nM GLAP-apt for 1 h. ROS generation was evaluated by CellRox oxidative stress reagents. N = 6–12 per group. # and ##, p < 0.05 and p < 0.01 compared to the values with 100 μg/mL glycer-AGEs. (b) The interaction of GLAP-aptamer to immobilized glycer-AGEs was analyzed by bio-layer interferometry. N = 4 per group.

Figure 2

Effects of glyceraldehyde-derived pyridinium (GLAP) or GLAP-aptamer (GLAP-apt) on ROS generation (a,b), MCP-1 (c), PAI-1 (d), and RAGE mRNA levels (e) in proximal tubular cells. Tubular cells were treated with the indicated concentrations of GLAP in the presence or absence of 5 μg/mL RAGE-Ab, 10 nM AGE-apatmer (AGE-apt), or 10nM GLAP-apt for 1 h (a,b) or for 4 h (c–e). ROS generation was evaluated by CellRox oxidative stress reagents. N = 6 per group (c–e). Total RNAs were transcribed and amplified by real-time PCR. Data were normalized by the intensity of 18S rRNA mRNA-derived signals and then related to the control values. (c,d) N = 3 per group. (e) N = 7 per group. **, p < 0.01 compared to the control values. # and ##, p < 0.05 and p < 0.01 compared to the values with 10 μg/mL GLAP alone, respectively.