The Effect of alpha-MSH on Tumor Necrosis Factor-alpha and NFkappaB Activation in Cultured Human Proximal Tubular Cells during Simulated Ischemia

Lee, So Young; Kim, Sang Wook; Jo, Sang Kyung; Kwon, Young Joo; Cha, Dae Ryong; Cho, Won Yong; Kim, Hyung Kyu

Abstract

BACKGROUND
Tumor necrosis factor a (TNF), potent proinflammatory cytokine, may be related with ischemia/reperfusion injury induced tubular cell inflammation and apoptosis. We examined TNF and its major nuclear transcriptional factor, NFkappaB activation in cultured human tubular cells in hypoxic condition and the effect of alpha-MSH, potent antiinflammatory agent, which have been reported to reduce renal I/R injury in rats. METHODS: Hypoxic culture condition was produced by oxidative pathway inhibitor (Antimycin 2 mM) and glycolytic pathway inhibitor (deoxy-D- glucose 2 mM and 10 mM) for 1 hour and re-oxygenation was performed by placing the cells in normal medium. The expression of TNF mRNA was studied by RT-PCR and NFkappaB DNA binding activity was analysed by Electomobility shift assays (EMSA) and cellular apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labelling (TUNEL) method and DNA laddering. These data were compared between the alpha-MSH and the vehicle-treated groups. RESULTS: Measured ATP level was 49% of control by luciferase-based assay kit. I/R injury caused an increase in TNF mRNA and NFkappaB activation and was accompanied by morphological evidence of apoptosis. alpha-MSH significantly reduced the degree of apoptosis, as well as TNF mRNA and NFkappaB activity (TNF/L19 mRNA ratio, vehicle/alpha-MSH: 105.15 +/- 16.5/18.75 +/- 0.85, p<0.05) (NFkappaB activity, vehicle/alpha-MSH: 5624/4803 densitometric index (DI), p<0.05). CONCLUSION: These findings suggest that alpha-MSH can decrease cellular apoptosis in hypoxic tubular cells and this protective effect of alpha-MSH may be related, in partially, with supression of TNF and NFkappaB activity.