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 4

GADD45α is regulated by TFEB in TICN.

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GADD45α is regulated by TFEB in TICN. (A) Venn diagram showing the overl...
(A) Venn diagram showing the overlap between TFEB direct target genes identified in HEK293T cells (23) and differentially expressed genes in the kidneys of wild-type or kidney-specific TFEB-overexpressed mice (24). (B) GADD45A mRNA and (C) protein levels in HK-2 cells treated with vehicle or Tac (25, 50, and 75 μM) for 24 hours were determined by qPCR (n = 4) and Western blot (n = 3), respectively. (D) Gadd45a mRNA and (E and F) protein in C57BL/6 mice injected with vehicle or Tac (1.5 mg/kg/d) for 6 weeks were determined by qPCR (n = 6), Western blot (n = 3), and immunohistochemistry (n = 6), respectively. Scale bar, 50 μm. (G) ChIP-qPCR was performed in HK-2 cells and HEK293T cells to identify the enrichment of TFEB onto the GADD45A promoter region (n = 4). IgG served as an antibody control. (H) The potential TFEB binding sites in the promoter of GADD45α and luciferase constructs were presented. (I) Luciferase reporter assay in HK-2 cells and HEK293T cells (n = 3). (J) GADD45A mRNA and (K) protein in HK-2 cells transfected with TFEB-S142A/S211A lentivirus or TFEB shRNA were measured by qPCR (n = 4) and Western blot (n = 3), respectively. Data are shown as mean ± SD and analyzed by 1-way ANOVA (B and C) or 2-tailed Student’s t tests (D, G, and I–K). *P < 0.05, **P < 0.01, ***P < 0.001.