Premium
Role of NAD(P)H oxidase in transforming growth factor‐β1‐induced monocyte chemoattractant protein‐1 and interleukin‐6 expression in rat renal tubular epithelial cells
Author(s) -
ZHANG HAIYAN,
JIANG ZONGPEI,
CHANG JIE,
LI XIAOYAN,
ZHU HENGMEI,
LAN HUI Y,
ZHOU SHUFENG,
YU XUEQING
Publication year - 2009
Publication title -
nephrology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 61
eISSN - 1440-1797
pISSN - 1320-5358
DOI - 10.1111/j.1440-1797.2008.01072.x
Subject(s) - nad+ kinase , nad(p)h oxidase , p22phox , microbiology and biotechnology , western blot , nadph oxidase , oxidase test , small hairpin rna , monocyte , chemistry , intracellular , reactive oxygen species , biology , apoptosis , biochemistry , enzyme , gene knockdown , immunology , gene
SUMMARY Aim: This study investigated the role of NAD(P)H oxidase in transforming growth factor‐β1 (TGF‐β1)‐induced reactive oxygen species (ROS) generation, monocyte chemoattractant protein‐1 (MCP‐1), and interleukin‐6 (IL‐6) expression in rat renal tubular epithelial NRK‐52E cells. Methods: The cells were treated with 10 ng/mL TGF‐β1, either in the presence or absence of the NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI), or short hairpin RNA (shRNA) suppressing p67phox expression. Expression of NAD(P)H oxidase subunits, MCP‐1, and IL‐6 at the mRNA levels was detected by reverse transcription polymerase chain reaction, while expression of NAD(P)H oxidase subunit p67phox protein was analyzed by western blot and MCP‐1 by enzyme‐linked immunosorbent assay. The cellular ROS generation was visualized using 2′,7′‐dichlorodihydrofluorescein diacetate by confocal microscopy. Results: Compared to control, TGF‐β1 upregulated NAD(P)H oxidase subunit p67phox mRNA by 3.59‐fold ( P < 0.01), but had no effect on p22phox, gp91phox and p47phox NAD(P)H subunits. TGF‐β1 was also able to significantly increase intracellular ROS ( P < 0.05), MCP‐1 ( P < 0.01) and IL‐6 ( P < 0.05) expression in NRK‐52E cells. Further studies showed that generation of ROS and upregulation of MCP‐1 and IL‐6 by TGF‐β1 were significantly blocked by addition of DPI or shRNA‐p67phox ( P < 0.01), suggesting that these effects were NAD(P)H oxidase‐dependent. Conclusion: TGF‐β1 differentially regulates the expression of NAD(P)H oxidase subunits and mediates MCP‐1 and IL‐6 expression in rat renal tubular cells via the NAD(P)H oxidase/p67phox‐dependent mechanism.