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Involvement of Nrf2-GSH signaling in TGFβ1-stimulated epithelial-to-mesenchymal transition changes in rat renal tubular cells

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Abstract

Transforming growth factor-β1 (TGFβ1) induces epithelial-to-mesenchymal transition (EMT) in cultured renal tubular epithelial cells. This phenotypic transition has been known to be involved in the development of chronic kidney diseases by activating profibrotic gene expression. Since oxidative stress has been recognized as one of the contributors to this TGFβ1-mediated pathology, we investigated the potential involvement of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which is a key transcription factor for the regulation of multiple antioxidant genes, in TGFβ1-stimulated EMT gene changes using the rat proximal tubular epithelial cell line NRK52E. The treatment of NRK52E with TGFβ1 led to changes in EMT gene expression, including increased α-Sma and decreased E-cadherin expression. In these cells, the TGFβ1 treatment decreased the transcript level of the catalytic subunit of γ-glutamate cysteine ligase (Gclc), a glutathione (GSH) biosynthetic enzyme, and reduced the total GSH content with a concomitant decrease in Nrf2 transcription activity. Accordantly, pre-incubation with the GSH precursor N-acetylcysteine attenuated TGFβ1-stimulated EMT gene changes. The involvement of Nrf2 in EMT gene changes has been demonstrated using NRK52E cells with nrf2 knockdown or pharmacological activation. When the expression of Nrf2 was stably silenced in NRK52E cells using interfering RNA administration, Gclc expression was significantly reduced and the increase in the levels of α-Sma and fibronectin-1 by TGFβ1 was greater than those in the nonspecific RNA control group. Conversely, Nrf2 activation and subsequent Gclc increase by Nrf2-activating sulforaphane alleviated the TGFβ1-stimulated α-Sma increase and E-cadherin decrease. Collectively, these results indicate that Nrf2-GSH signaling can modulate TGFβ1-stimulated EMT gene changes and further suggest a beneficial role of Nrf2 inducers in renal pathogenesis.

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Abbreviations

TGFβ1:

Transforming growth factor-β1

EMT:

Epithelial-to-mesenchymal transition

Nrf2i:

Nrf2 knockdown NRK52E

sc:

Nonspecific scrambled RNA NRK52E

ARE:

Antioxidant-response element

ROS:

Reactive oxygen species

GSH:

Glutathione

Gclc:

Catalytic subunit of γ-glutamate cysteine ligase

Ho-1:

Heme oxygenase-1

α-Sma:

α-smooth muscle actin

Fn-1:

Fibronectin-1

Col1A1:

Collagen 1A1

Cdh1:

E-cadherin

Hprt:

Hypoxanthine–guanine phosphoribosyltransferase

SFN:

Sulforaphane

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Acknowledgments

This research was supported by the Bio and Medical Technology Development Program (NRF-2013M3A9B5075839) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning, and the Basic Science Research Program (2011-0003619) of the NRF funded by the Ministry of Education, Science and Technology.

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Authors and Affiliations

  1. College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon, Gyeonggi-do, 420-743, Republic of Korea

    In-geun Ryoo & Mi-Kyoung Kwak

  2. College of Pharmacy, Yeungnam University, Gyeongsangbuk-do, 712-749, Republic of Korea

    Dong-ha Shin

  3. Daewon Foreign Language High School, Kwangjin-gu, Seoul, 143-713, Republic of Korea

    Kyung-Shin Kang

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  1. In-geun Ryoo
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  2. Dong-ha Shin
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Correspondence to Mi-Kyoung Kwak.

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Ryoo, Ig., Shin, Dh., Kang, KS. et al. Involvement of Nrf2-GSH signaling in TGFβ1-stimulated epithelial-to-mesenchymal transition changes in rat renal tubular cells. Arch. Pharm. Res. 38, 272–281 (2015). https://doi.org/10.1007/s12272-014-0380-y

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