Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells

Abstract

Diabetes mellitus leads to a high incidence of several so-called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). Cultured HPMCs isolated from 13 different patients (mean age 38.7+/-16 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg ml(-1)), as well as to their respective low glycosylation controls. Amadori adducts, but not their respective controls, elicited a marked increase of NF-kappaB activation, as determined by electromobility shift assays and transient transfection experiments. Additionally, Amadori adducts significantly increased the production of NF-kappaB-related proinflammatory molecules, including cytokines, such as TNF-alpha, IL-1beta or IL-6, and enzymes, such as cyclooxygenase-2 and inducible nitric oxide (NO) synthase, this latter leading to the release of NO by HPMCs. The effects of Amadori adducts were mediated by different reactive oxygen and nitrosative species (e.g. superoxide anions, hydroxyl radicals, and peroxynitrite), as they were blunted by coincubation with the appropriate scavengers. Furthermore, NO generated upon exposure to Amadori adducts further stimulated NF-kappaB activation, either directly or after combination with superoxide anions to form peroxynitrite. We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF-kappaB-related proinflammatory signals in human mesothelial cells.

Figure 1

Amadori adducts activate NF-κB in HPMCs. (a) DNA-binding activity of NF-κB was assessed after 1 h treatment of HPMCs with either NHb or HHb (both at 10 nM) or gBSA and nBSA (both at 0.25 mg ml⁻¹). A representative EMSA assay is shown, n=9. (b) NF-κB-dependent transcriptional activation was assessed in cells transiently transfected with p5 × NF-κB-Luc plasmid. HPMCs were exposed for 12 h to the above-described compounds, after which luciferase activity (RLUs) was measured, n=32. (c) Expression of NF-κB1 and NF-κB2 mRNAs after HPMCs exposure to HHb or NHb (both at 10 nM) was analysed by RT–MPCR assay. A representative blot and densitometry values of the ratio mRNA NF-κB1, 2/GAPDH mRNA are shown, n=9. Results are expressed as mean±s.e.m. ^*P⩽0.05 vs basal; ^†P⩽0.05 vs the respective glycosylation control.

Figure 2

Stimulation by Amadori adducts of NF-κB-related proinflammatory gene expression. The effect of Amadori adducts on either human iNOS (a), human −327/+59 COX-2 (c), and human IL-6 (e) promoters was studied using luciferase-based reporter plasmids in transiently transfected HPMCs. Cells were treated for 12 h with HHb and NHb (both at 10 nM), gBSA, and nBSA (both at 0.25 mg ml⁻¹) or a cytokine mixture (TNF-α+IL-1β, 10 ng ml⁻¹ each), n=32. The effect of a 6 h treatment with the above-described compounds on iNOS (b), COX-2 (d), and IL-6 (open bars) and IL-6 receptor (solid bars) (f) mRNA levels was also determined by RT–MPCR assays. Representative blots are shown, n=9. DRB: 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (10 μM). Results are expressed as mean±s.e.m. ^*P⩽0.05 vs basal; ^†P⩽0.05 vs the respective glycosylation control; ^**P⩽0.05 vs HHb-treated cells.

Figure 3

Blockade of Amadori-induced stimulation of NF-κB-related proinflammatory factors by PDTC. (a) HPMCs were treated for 12 h with HHb (10 nM) or gBSA (0.25 mg ml⁻¹) either alone or in combination with PDTC (100 μM) and the luciferase-based expression of human iNOS, −327/+59 human COX-2, and human IL-6 was studied in transiently transfected HPMCs. (b) NOS and COX activities were determined after HPMCs exposure to the above-described treatments, n=32. ^*P⩽0.05 vs basal; ^**p⩽0.05 vs the correspondent Amadori (gBSA or HHb)-treated cells.

Figure 4

Participation of ROS in the proinflammatory effects induced by Amadori adducts in HPMCs. (a) Transient transfection experiments were performed to measure NF-κB-dependent transcription activity after 12 h exposure to HHb (10 nM) or gBSA (0.25 mg ml⁻¹) either alone or co-incubated with superoxide dismutase (SOD 200 U ml⁻¹), Tempol (Temp, 100 μM) or dimethylthiourea (DMTU, 1 mM), n=18. Under the same conditions, human iNOS and −327/+59 human COX-2 (b), as well as human IL-6 (d) promoters luciferase-based reporter activities were assessed by transient transfection assays, n=18. (c) A representative immunoblot for iNOS and COX-2 protein is shown after 12 h exposure of HPMCs to HHb either alone or in combination with Tempol at the above-described concentrations, n=9. ^*P⩽0.05 vs basal. ^**P⩽0.05 vs the correspondent Amadori (gBSA or HHb)-treated cells.

Figure 5

NO and peroxynitrite modulate NF-κB-dependent transcriptional activity in HPMCs. (a) HPMCs transiently transfected with pNF-κB-Luc were treated with HHb (10 nM) alone or in combination with the iNOS inhibitors 1400W (10 μM) and L-NAME (100 μM), dexamethasone (Dex, 1 μM) or PDTC (100 μM) for 12 h, after which luciferase activity was measured, n=12. (b) NF-κB-dependent transcription was further determined in HPMCs treated for the same time period with S-nitroso-N-acetylpenicillamine (SNAP, 10 μM) and sodium nitroprusside (SNP 100 μM) either alone or in the presence of HHb. HHb was also coincubated with PDTC (100 μM), n=9. (c) NF-κB-dependent transcription was determined in HPMCs treated for 8 h with HHb alone or in combination with the peroxynitrite scavengers uric acid (0.1, 1, and 10 μM) and Trolox (1, 5, and 10 μM) or PDTC (100 μM), n=9. (d) Representative immunoblot for nitrated protein expressions after exposure for 12 h to either NHb or HHb (both at 10 nM) or HHb in combination with Tempol or DMTU (100 μM and 1 mM, respectively).^*P⩽0.05 vs basal; ^**P⩽0.05 vs HHb-treated cells.