Role of NAD(P)H oxidase in transforming growth factor-beta1-induced monocyte chemoattractant protein-1 and interleukin-6 expression in rat renal tubular epithelial cells

Haiyan Zhang, Zongpei Jiang, Jie Chang, Xiaoyan Li, Hengmei Zhu, Hui Y Lan, Shu-Feng Zhou, Xueqing Yu
Nephrology 2009, 14 (3): 302-10

AIM: This study investigated the role of NAD(P)H oxidase in transforming growth factor-beta1 (TGF-beta1)-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-beta1, 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-beta1 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-beta1 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-beta1 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-beta1 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.

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