GADD45α is a direct target of TFEB and contributes to tacrolimus-induced chronic nephrotoxicity
Ping Gao, Xinwei Cheng, Maochang Liu, Hui Peng, Guodong Li, Tianze Shang, Jianqiao Wang, Qianyan Gao, Chenglong Zhu, Zhenpeng Qiu, Chengliang Zhang
Ping Gao, Xinwei Cheng, Maochang Liu, Hui Peng, Guodong Li, Tianze Shang, Jianqiao Wang, Qianyan Gao, Chenglong Zhu, Zhenpeng Qiu, Chengliang Zhang
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Research Article Nephrology Therapeutics

GADD45α is a direct target of TFEB and contributes to tacrolimus-induced chronic nephrotoxicity

Abstract

Tacrolimus-induced chronic nephrotoxicity (TICN) hinders long-term use of tacrolimus, but its mechanism remains unclear. Tacrolimus exerts its pharmacological effect by inhibiting calcineurin and its substrate nuclear factor of activated T cells. Whether the inhibition of other calcineurin substrates is related to TICN remains to be explored. Transcription factor EB (TFEB), a substrate of calcineurin, plays a crucial role in homeostasis. Herein, we found that tacrolimus inhibited TFEB nuclear translocation and activity in mouse kidneys and HK-2 cells. Then, TFEB gain and loss of function rescued and exacerbated, respectively, the effect of tacrolimus in HK-2 cells. Furthermore, TFEB activation by both phosphorylation site mutation and agonist rescued TICN in mice. To elucidate the mechanism of TFEB, we analyzed ChIP-Seq data. We identified growth arrest and DNA damage-inducible 45α (GADD45α) as a transcriptional target of TFEB via ChIP and dual-luciferase reporter assays. Then we revealed that GADD45α overexpression rescued DNA damage and kidney injury caused by tacrolimus or TFEB knockdown in vitro and vice versa. The protective effect of GADD45α against TICN and DNA damage was further demonstrated by overexpressing it in mice. In conclusion, the persistent inhibition of the TFEB/GADD45α pathway by tacrolimus contributes to TICN. This study identifies a specific target for intervention in TICN.

Authors

Ping Gao, Xinwei Cheng, Maochang Liu, Hui Peng, Guodong Li, Tianze Shang, Jianqiao Wang, Qianyan Gao, Chenglong Zhu, Zhenpeng Qiu, Chengliang Zhang

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Figure 1

TFEB cytosol-to-nucleus translocation is inhibited in vitro and in vivo.

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TFEB cytosol-to-nucleus translocation is inhibited in vitro and in vivo....
HK-2 cells were exposed to Tac (25, 50, and 75 μM) for 24 hours, and then (A) TFEB mRNA and (B) protein were determined by qPCR (n = 4) and Western blot (n = 3), respectively. TFEB expression levels in cytoplasm and nucleus were normalized to β-actin and H3, respectively. qPCR, quantitative PCR. (C) After being starved for 4 hours, HK-2 cells were treated with vehicle or Tac for 24 hours, then measured by immunofluorescence. Scale bar, 20 μm. Nuclear TFEB fluorescence intensity was quantified (n = 3). (D) mRNA levels of LAMP1 and CTSD in HK-2 cells were quantified by qPCR (n = 4). C57BL/6 mice were subcutaneously injected with vehicle or Tac (1.5 mg/kg/d) for 6 weeks, and then (E) Tfeb mRNA and (F) protein in kidney were determined by qPCR (n = 6) and Western blot (n = 3), respectively. (G) TFEB (shown in red) and proximal tubule marker lotus tetragonolobus lectin (LTL) (shown in green) were measured by immunofluorescence. Arrowheads indicate nuclear localized TFEB. Scale bar, 10 μm. Nuclear TFEB fluorescence intensity was quantified (n = 6). (H) mRNA levels of Lamp1 and Ctsd in mouse kidneys were quantified by qPCR (n = 6). Data are shown as mean ± SD and analyzed by 1-way ANOVA (A–D) or 2-tailed Student’s t tests (E–H). *P < 0.05, **P < 0.01, ***P < 0.001.