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 2

TFEB gain and loss of function modifies the effect of Tac in HK-2 cells.

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TFEB gain and loss of function modifies the effect of Tac in HK-2 cells....
TFEB nuclear translocation in HK-2 cells infected with empty (LV-NC) or TFEB-S142A/S211A–overexpressing lentivirus (LV-TFEB Mut) was determined by (A) immunofluorescence and (B) Western blot (n = 3). Scale bar, 20 μm. (C) Cell viability and (D) KIM-1 concentration in LV-NC or LV-TFEB mutated HK-2 cells treated with vehicle or Tac (50 μM for 24 hours) were determined by CCK-8 (n = 6) and ELISA (n = 5), respectively. CCK-8, cell counting kit-8. (E) TGF-β and α-SMA protein levels in the whole cell and TFEB in the nucleus were determined by Western blot (n = 3). (F) COL1A1 mRNA level was determined by qPCR (n = 4). (G) TFEB protein in HK-2 cells stably expressing control shRNA (shNC) or TFEB shRNA (shTFEB) was determined by Western blot (n = 3). (H) Cell viability and (I) KIM-1 concentration in shNC or shTFEB HK-2 cells treated with vehicle or Tac (50 μM for 24 hours) was determined by CCK-8 (n = 6) and ELISA (n = 5), respectively. (J) TGF-β and α-SMA protein levels in whole cell and TFEB in nucleus were determined by Western blot (n = 3). (K) COL1A1 mRNA level was determined by qPCR (n = 4). Data are shown as mean ± SD and analyzed by 1-way ANOVA (C–F and H–J) or 2-tailed Student’s t tests (G). *P < 0.05, **P < 0.01, ***P < 0.001.