Comparison of the effects of fructose and uric acid on MCP-1 production in HK-2 cells. Cells were treated with fructose and/or uric acid with or without allopurinol for 72 h. MCP-1 content in the medium was assayed by ELISA and is expressed as pg/mg of cellular protein. Data are means ± SEM; n = 3, each experiment is performed in triplicate. Statistical analysis was performed using nonparametric U test.

Fructose-induced MCP-1 production in HK-2 cells. (A) Dosage-dependent effect of fructose on MCP-1 production. Glucose content in the medium in this and other experiments unless indicated otherwise was 5 mM. (B) Difference in the effects of fructose and glucose on MCP-1 production. (C) Glucose attenuates effect of fructose on MCP-1 production. (D) d-Mannitol did not produce a significant effect on MCP-1 production, excluding the osmotic mechanism. (E) Similarity of the effects of fructose and tagatose on MCP-1 production. In all experiments, cells were treated with fructose, glucose, tagatose, and mannitol for 72 h. MCP-1 content in the medium was assayed by ELISA and is expressed as pg/mg of cellular protein content. Data are means ± SEM *P < 0.05 versus untreated cells or corresponding control, nonparametric U test and, in A, one-way ANOVA; ^#P = 0.06 versus 5 mM fructose + 5 mM glucose; n = 3, each experiment performed in triplicate.

KHK is required for fructose-induced MCP-1 production in HK-2 cells. (A) Expression of mRNA for KHK and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in HK-2 cells and human liver. Inverted images of the RT-PCR products on agarose gel are shown. (B) mRNA expression for KHK and GAPDH genes in nontransfected HK-2 cells and puromycin-resistant clones transfected with pRS-GFP and pRS-KHK. (C) Analysis of KHK silencing at the protein level. Representative blot and densitometric analysis (KHK/GAPDH ratio) are shown. (D) Functional test of KHK silencing: Effect of fructose on KHK-dependent ATP depletion. (E) Fructose-induced MCP-1 is abolished by KHK silencing. Cells were treated with fructose for 72h. MCP-1 content in the medium was assayed by ELISA and is expressed as pg/mg of cellular protein content or percentage of control (100%). *P < 0.05 versus untreated cells or corresponding control, nonparametric U test; n = 3, each experiment performed in triplicate.

Fructose-induced KHK-dependent MCP-1 production in HK-2 cells is mediated by ROS (A) Fructose-dependent MCP-1 increase is prevented by antioxidants [10 mM N-acetyl-cysteine (NAC), 25 μM Mn(II) tetrakis(1-methyl-4 to 6 pyridyl) porphyrin (MnTMPyP), 0.1 mM allopurinol, and 200 μM apocynin]. Cells were treated for 72 h. (B) ROS production by HK-2 cells in the presence of 5 mM fructose. ROS-induced fluorescence is shown on the left, and differential interference contrast (DIC) is shown on the right. Cells were treated with fructose for 2 h. (C) Dosage response of ROS production in HK-2 cells incubated with varying concentrations of fructose or d-mannitol for 2 h. (D) Time course of ROS production in HK-2 cells incubated in 5 mM fructose. The antioxidant tempol (5 mM) completely prevented an increase in the fluorescence in response to fructose, demonstrating specificity of ROS detection (data not shown). (E) ROS accumulation after 2 h of stimulation with 5 mM fructose in nontransfected HK-2 and HK-2 clones stably expressing KHK shRNA or GFP-shRNA as a control for irrelevant shRNA and transfection. (F) Effect of oxypurinol (0.1 mM) on fructose-induced ROS production. Data are expressed as arbitrary units of fluorescence and are means ± SEM (n = 3 performed in triplicate, each representing 20 to 30 cells). *P < 0.05 versus untreated cells or corresponding control; ^&P < 0.05 versus fructose-treated cells, nonparametric U test.

Fructose-induced intracellular uric acid production and XOR expression in HK-2 cells. (A) Expression of mRNA for XOR and β-actin in HK-2 cells. Inverted images of the RT-PCR products on agarose gel are shown. (B) Identification of uric acid in samples by MS/MS experiments. (C) Intracellular uric acid content in response to 1 and 5 mM fructose stimulation for 72 h. Data are means ± SEM and are expressed as nmol/mg of protein. *P < 0.05 versus untreated cells. (D) Analysis of the effect of fructose with and without allopurinol on the intracellular level of uric acid by tandem LC/MS/MS. Extracts contained the same amount of total protein. The x axis is the retention time, and the y axis is the intensity of the single-reaction monitoring peaks. The peak area is proportional to the quantity of uric acid.