Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo
Sydney Tang, Joseph C.K. Leung, Katsushige Abe, Kwok Wah Chan, Loretta Y.Y. Chan, Tak Mao Chan, Kar Neng Lai
Sydney Tang, Joseph C.K. Leung, Katsushige Abe, Kwok Wah Chan, Loretta Y.Y. Chan, Tak Mao Chan, Kar Neng Lai
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Article Nephrology

Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo

Abstract

Renal tubulointerstitial injury is characterized by inflammatory cell infiltrate; however, the stimuli for leukocyte recruitment are not fully understood. IL-8 is a potent chemokine produced by proximal tubular epithelial cells (PTECs). Whether nephrotic proteins stimulate tubular IL-8 expression remains unknown. Acute exposure of human PTECs to albumin induced IL-8 gene and protein expression time- and dose-dependently. Apical albumin predominantly stimulated basolateral IL-8 secretion. Electrophoretic mobility shift assay demonstrated nuclear translocation of NF-κB, and the p65/p50 subunits were activated. NF-κB activation and IL-8 secretion were attenuated by the NF-κB inhibitors pyrrolidine dithiocarbamate and cell-permeable peptide. Albumin upregulated intracellular reactive oxygen species (ROS) generation, while exogenous H2O2 stimulated NF-κB translocation and IL-8 secretion. Albumin-induced ROS generation, NF-κB activation, and IL-8 secretion were endocytosis- and PKC-dependent as these downstream events were abrogated by the PI3K inhibitors LY294002 and wortmannin, and the PKC inhibitors GF109203X and staurosporin, respectively. In vivo, IL-8 mRNA expression was localized by in situ hybridization to the proximal tubules in nephrotic kidney tissues. The intensity of IL-8 immunostaining was higher in nephrotic than non-nephrotic subjects. In conclusion, albumin is a strong stimulus for tubular IL-8 expression, which occurs via NF-κB–dependent pathways through PKC activation and ROS generation.

Authors

Sydney Tang, Joseph C.K. Leung, Katsushige Abe, Kwok Wah Chan, Loretta Y.Y. Chan, Tak Mao Chan, Kar Neng Lai

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Effect of PI3K and PKC inhibition on albumin-induced intracellular ROS g...
Effect of PI3K and PKC inhibition on albumin-induced intracellular ROS generation and IL-8 secretion in PTECs. (a) ROS generation. PTECs were exposed for 30 min to medium alone (control), HSA (10 mg/ml), LY294002 (100 μM, added 1 h before the addition of HSA), wortmannin (500 nM, added 1 h before the addition of HSA), GF109203X (5 μM, added 1 h before the addition of HSA), or staurosporin (100 nM, added 1 h before the addition of HSA). Intracellular ROS formation was detected by flow cytometry and expressed as percentage of MFI of control cells incubated with culture medium alone. Results are means ± SD of triplicate experiments. *P < 0.0001 versus medium control, †P = 0.001 versus cells treated with HSA alone. (b) IL-8 protein secretion. PTECs were treated for 24 h with medium alone (control), HSA (10 mg/ml), LY294002 (100 μM, added 1 h before the addition of HSA), wortmannin (500 nM, added 1 h before the addition of HSA), GF109203X (5 μM, added 1 h before the addition of HSA), or staurosporin (100 nM, added 1 h before the addition of HSA). At the end of incubation, IL-8 level was measured in supernatants using ELISA, while cell number was counted. Results are means ± SD of triplicate experiments. *P < 0.0001 versus medium control, †P < 0.0001 versus cells treated with HSA alone.