Hyperglycemia induces Toll like receptor 4 expression and activity in mouse mesangial cells: relevance to diabetic nephropathy

Abstract

Diabetes is a proinflammatory state. The pattern recognition receptors, Toll-like receptors (TLRs), are increased in diabetic patients and have been suggested to play a role in diabetic nephropathy (DN). Progression of DN involves altered mesangial cell (MC) function with an expansion of the mesangial matrix. There is a paucity of data examining the role of TLR and its expression in MC. We hypothesize the expression of TLRs in the mesangium might be an important factor contributing to mesangium expansion and nephropathy. Thus we evaluated the effect of high glucose on TLR2 and TLR4 expression in mouse mesangial cells (MMC) in vitro. Exposure of MMC to 25 mM glucose for 24 h resulted in increased TLR4 mRNA and cell surface receptor expression compared with 5.5 mM glucose (P < 0.05). Interestingly, we were not able to detect expression of TLR2 in MMC. Furthermore, expression of a TLR4 downstream signaling cascade including myeloid differentiation factor 88 (MyD88), interferon regulatory factor 3 (IRF3), and Toll interleukin receptor domain containing adaptor inducing interferon-β (TRIF)-related adaptor molecule (TRAM) were significantly increased in cells exposed to 25 mM glucose (P < 0.05). There was also a significant increase in NF-κB activation along with increased secretion of inflammatory cytokines IL-6 and monocyte chemotactic protein-1. Levels of transforming growth factor-β were also significantly increased in the presence of 25 mM glucose (P < 0.05). Collectively, these data suggest that hyperglycemia activates TLR4 expression and activity in MC and could contribute to DN.

Fig. 1.

A: Hyperglycemia induces Toll like receptor 4 (TLR4) mRNA expression in mouse mesangial cells (MMC). Representative RT-PCR gel shows significantly increased TLR4 mRNA expression on exposing MMC to 25 mM glucose compared with 5.5 mM at 24 h. Densitometric values are normalized to GAPDH and are expressed as means ± SD. *P < 0.05 compared with 5.5 mM glucose or mannitol control. B: surface expression of TLR4 in MMC. TLR 4 surface expression was assessed in the MMC after glucose challenge by flow cytometry at 24 h. Values are expressed as MFI/10,000 events (means ± SD). *P < 0.05 compared with 5.5 mM glucose. #P < 0.05 compared with mannitol control.

Fig. 2.

TLR4 signaling pathways in MMC. Western blotting was performed for different adaptor proteins in the cell lysates after exposure of MMC to glucose challenge for 24 h. Densitometric ratios represent adaptor protein-to-β-actin ratios. MyD88, myeloid differentiation factor 88; IRF3, interferon regulatory factor 3; TRAM, Toll interleukin receptor domain-containing, adaptor protein-inducing interferon (TRIF)-related adaptor molecule. *P < 0.05 compared with 5.5 mM glucose.

Fig. 3.

NF-κB (p65) activation in MMC. ELISA was performed to study activation of transcription factor NF-κB (P65) in the nuclear extracts from mesangial cells at 24 h. *P < 0.05 compared with 5.5 mM glucose or mannitol control.

Fig. 4.

Hyperglycemia-induced circulating expression of proinflammatory cytokines and transforming growth factor (TGF)-β by MMC at 24 h. ELISA was performed to estimate the levels of released cytokines/mg protein in the supernatant. IL-6, monocyte chemotactic protein-1 (MCP-1), AND TNF-α are expressed as pg/mg cell protein. TGF-β is expressed as ng/mg cell protein. *P < 0.05 vs. 5.5 mM glucose.