Histone deacetylase inhibitors protect against cisplatin-induced acute kidney injury by activating autophagy in proximal tubular cells

doi:10.1038/s41419-018-0374-7

. 2018 Feb 23;9(3):322.

doi: 10.1038/s41419-018-0374-7.

Histone deacetylase inhibitors protect against cisplatin-induced acute kidney injury by activating autophagy in proximal tubular cells

Jing Liu^{1

2}, Man J Livingston², Guie Dong², Chengyuan Tang¹, Yunchao Su³, Guangyu Wu³, Xiao-Ming Yin⁴, Zheng Dong^{5

6}

Affiliations

¹ Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, China.
² Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA, 30912, USA.
³ Department of Pharmacology & Toxicology, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA, 30912, USA.
⁴ Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
⁵ Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, China. zdong@csu.edu.cn.
⁶ Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA, 30912, USA. zdong@csu.edu.cn.

PMID: 29476062
PMCID: PMC5833747
DOI: 10.1038/s41419-018-0374-7

Histone deacetylase inhibitors protect against cisplatin-induced acute kidney injury by activating autophagy in proximal tubular cells

Jing Liu et al. Cell Death Dis. 2018.

. 2018 Feb 23;9(3):322.

doi: 10.1038/s41419-018-0374-7.

Authors

Jing Liu^{1

2}, Man J Livingston², Guie Dong², Chengyuan Tang¹, Yunchao Su³, Guangyu Wu³, Xiao-Ming Yin⁴, Zheng Dong^{5

6}

Affiliations

¹ Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, China.
² Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA, 30912, USA.
³ Department of Pharmacology & Toxicology, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA, 30912, USA.
⁴ Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA.
⁵ Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, China. zdong@csu.edu.cn.
⁶ Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA, 30912, USA. zdong@csu.edu.cn.

PMID: 29476062
PMCID: PMC5833747
DOI: 10.1038/s41419-018-0374-7

Abstract

Histone deacetylase inhibitors (HDACi) have therapeutic effects in models of various renal diseases including acute kidney injury (AKI); however, the underlying mechanism remains unclear. Here we demonstrate that two widely tested HDACi (suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA)) protect the kidneys in cisplatin-induced AKI by enhancing autophagy. In cultured renal proximal tubular cells, SAHA and TSA enhanced autophagy during cisplatin treatment. We further verified the protective effect of TSA against cisplatin-induced apoptosis in these cells. Notably, inhibition of autophagy by chloroquine or by autophagy gene 7 (Atg7) ablation diminished the protective effect of TSA. In mice, TSA increased autophagy in renal proximal tubules and protected against cisplatin-induced AKI. The in vivo effect of TSA was also abolished by chloroquine and by Atg7 knockout specifically from renal proximal tubules. Mechanistically, TSA stimulated AMPK and inactivated mTOR during cisplatin treatment of proximal tubule cells and kidneys in mice. Together, these results suggest that HDACi may protect kidneys by activating autophagy in proximal tubular cells.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

**Fig. 1. Autophagy is induced by TSA and SAHA in renal proximal tubular cells.**
a–c RPTC cells were incubated with at the indicated concentrations for 20 h in the absence or presence of 20 μM chloroquine. d–f RPTC cells were treated with 0.1 μM TSA for 8 h or 20 h in the absence or presence of 20 μM chloroquine. After treatment, whole-cell lysates were collected for immunoblot analysis of LC3B. β-Actin was probed as a loading control. a, d Representative immunoblot. b, c, e, f Densitometric analysis of LC3B signals. After normalization with β-actin, the protein signal of control was arbitrarily set as 1, and the signals of other conditions were normalized with control to calculate fold changes. Data are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^P < 0.05, significantly different from without chloroquine g–i RPTC cells were transiently transfected with mRFP-GFP-LC3 and then treated with 0.1 μM TSA for 20 h. After treatment, cells were fixed with 4% paraformaldehyde for fluorescence microscopy. g Representative images showing GFP-LC3 and mRFP-LC3 puncta in transfected cells (×630). Scale bar: 15 μm. h Quantitative analysis of GFP-LC3 and RFP-LC3 puncta. Data are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^P < 0.05, significantly different from GFP-LC3 puncta counting. i Analysis of autophagic flux rate. Data are expressed as mean ± SD. *P < 0.05, significantly different from the control group

**Fig. 2. TSA enhances autophagy during cisplatin treatment of renal proximal tubular cells.**
a–c RPTC cells were untreated or treated for 20 h with cisplatin (20 μM), TSA (0.1 μM), or cisplatin+TSA in the absence or presence of 20 μM chloroquine. The cells were then collected for immunoblot analysis of LC3B. β-Actin was used as a loading control. a Representative immunoblots. b, c Densitometric analysis of LC3B signals. Data are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^#P < 0.05, significantly different from the cisplatin-only group. ^P < 0.05, significantly different from the corresponding group without chloroquine. d–f RPTC cells were transiently transfected with mRFP-GFP-LC3 and then treated as indicated. After treatment, cells were fixed with 4% paraformaldehyde for fluorescence microscopy. d Representative images showing GFP-LC3 and mRFP-LC3 puncta in transfected cells (×630). Scale bar: 15 μm. e Quantitative analysis of GFP-LC3 and RFP-LC3 puncta. Data are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^#P < 0.05, significantly different from the cisplatin-only group. ^P < 0.05, significantly different from GFP-LC3 puncta counting. f Analysis of autophagic flux rate. Data are expressed as mean ± SD. *P < 0.05, significantly different from control group. ^#P < 0.05, significantly different from the cisplatin-only group

**Fig. 3. Inhibition of autophagy by chloroquine eliminates the protective effects of TSA in cisplatin-treated RPTC cells.**
RPTC cells were untreated or treated with cisplatin (20 μM) and cisplatin+TSA (0.1 μM) in the absence or presence of 20 μM chloroquine for 20 h. a Representative images of cellular and nuclear morphology (×100). Scale bar: 400 μm. b, d Quantification of apoptosis percentage. c, e Caspase activity measured by enzymatic assays using DEVD-AFC as substrates. Data in b–e are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^#P < 0.05, significantly different from the cisplatin-only group

**Fig. 4. Autophagy is impaired in *Atg7*-KO mouse kidney proximal tubular cells.**
Wild-type (*Atg7* WT) and *Atg7*-knockout (*Atg7* KO) MPTC cells were transiently transfected with mRFP-GFP-LC3 and then untreated or treated with cisplatin (20 μM), TSA (0.1 μM), or cisplatin+TSA for 20 h. a Representative images showing GFP-LC3 and mRFP-LC3 puncta (×630). Scale bar: 15 μm. b, c Quantitative analysis of GFP-LC3 puncta and RFP-LC3 puncta. d Analysis of autophagic flux rate. Data in b, c, d are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^#P < 0.05, significantly different from the cisplatin-only group. ^*P <* 0.05, significantly different from the corresponding group in *Atg7* WT cells. e, f *Atg7* WT and *Atg7* KO MPTC cells were treated as described above without transfection. Whole-cell lysates were collected after treatment for immunoblot analysis of LC3B and ATG7. β-Actin was used as a loading control. e Representative immunoblots. f Densitometric analysis of LC3B signals. Data are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^#P < 0.05, significantly different from the cisplatin-only group. ^*P <* 0.05, significantly different from the corresponding group in *Atg7* WT cells

**Fig. 5. The cytoprotective effect of TSA is abrogated in *Atg7*-KO mouse kidney proximal tubular cells.**
*Atg7* WT and *Atg7* KO MPTC cells were untreated or treated with cisplatin (20 μM), TSA (0.1 μM), or cisplatin+TSA for 20 h. After treatment, cells were collected for morphological and biochemical analysis. a Representative images of cellular and nuclear morphology of apoptosis (×200). Scale bar: 200 μm. b Quantification of apoptosis percentage. c Efficiency of apoptosis inhibition by TSA (*Atg7*-WT vs *Atg7*-KO). d Caspase activity measured by enzymatic assays. e Efficiency of TSA reducing caspase activation (cisplatin only vs cisplatin+TSA). Data in b–e are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^#P < 0.05, significantly different from the cisplatin-only group. ^*P <* 0.05, significantly different from the corresponding group in *Atg7* WT cells

**Fig. 6. TSA enhances renal tubular autophagy during cisplatin treatment in mice.**
C57BL/6 mice were injected with saline or a single dose of cisplatin (30 mg/kg, i.p.) in the absence (−) or presence (+) of TSA (1 mg/kg, i.p., daily injection) (n = 7 for saline control, n = 18 for cisplatin-only, n = 20 for cisplatin+TSA). a, b Kidneys were collected at the indicated time points for immunoblot analysis of LC3B. β-Actin was used as a loading control. a Representative immunoblots. b Densitometric analysis of LC3B signals. Data are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^*P <* 0.05, significantly different from the cisplatin-only group. c, d Kidneys were collected 3 days after treatment for immunohistochemical staining of LC3B. c Representative images of LC3B staining (×400). Scale bar: 15 μm. d Quantitative analysis of punctate LC3B staining. Data are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#P < 0.05, significantly different from the cisplatin-only group. e–g CAG-RFP-GFP-LC3 mice were injected with saline or a single dose of cisplatin (30 mg/kg, i.p.) only or cisplatin+TSA (1 mg/kg, i.p. daily) (n = 3 for saline control, n = 3 for cisplatin-only, n = 3 for cisplatin+TSA). Kidneys were collected 3 days after treatment for fluorescence microscopy. e Representative images showing GFP-LC3 and RFP-LC3 puncta in renal tubules of CAG-RFP-GFP-LC3 mice (×630). Scale bar: 15 μm. f Quantitative analysis of GFP-LC3 puncta and RFP-LC3 puncta. Data are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#P < 0.05, significantly different from the cisplatin-only group. ^*P <* 0.05, significantly different from GFP-LC3 puncta counting. g Analysis of autophagic flux rate. Data are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#P < 0.05, significantly different from the cisplatin-only group

**Fig. 7. Chloroquine abolishes the protective effects of TSA in cisplatin-induced AKI in mice.**
C57BL/6 mice were divided into five groups for the following treatment: (1) saline (n = 8), (2) a single dose of cisplatin (30 mg/kg, i.p.) only (n = 12), (3) cisplatin+TSA (1 mg/kg, i.p., daily) (n = 15), (4) cisplatin+chloroquine (60 mg/kg, i.p., daily) (n = 6), and (5) cisplatin+TSA+chloroquine (n = 7). Blood samples and kidneys were collected 3 days after treatment for biochemical and histological analyses. a Serum creatinine. b Representative images of kidney H–E staining (×200). Scale bar: 100 μm. c Pathological score of tubular damage. Data in a and c are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#P < 0.05, significantly different from the cisplatin-only group. ^&P< 0.05, significantly different from the cisplatin+TSA group. d Representative immunoblots of cleaved caspase 3. e Densitometric analysis of cleaved caspase 3 signals. Data are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#*P <* 0.05, significantly different from the cisplatin-only group. ^&*P <* 0.05, significantly different from the cisplatin with the TSA group

**Fig. 8. Autophagy is defective in renal proximal tubules of PT-*Atg7* KO mice.**
Wild-type (PT-*Atg7* WT) and PT-*Atg7* KO mice were injected with saline or a single dose of cisplatin (30 mg/kg, i.p.) in the absence (−) or presence (+) of TSA (1 mg/kg, i.p., daily injection) (n = 5–6 for each condition). Kidneys were collected 3 days after treatment for immunoblot analysis and immunohistochemical staining of LC3B. a Representative immunoblots. β-Actin was loading control. b Densitometric analysis of LC3B signals. c Representative images of LC3B immunohistochemical staining (×400). Scale bar: 15 μm. d Quantitative analysis of punctate LC3B staining. Data in b and d are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#P < 0.05, significantly different from the cisplatin-only group. ^P < 0.05, significantly different from the corresponding groups of PT-*Atg7* WT mice

**Fig. 9. The renoprotective effect of TSA in cisplatin-induced AKI is lost in PT-*Atg7* KO mice.**
PT-*Atg7* WT and PT-*Atg7* KO mice were injected with saline or a single dose of cisplatin (30 mg/kg, i.p.) in the absence (−) or presence (+) of TSA (1 mg/kg, i.p., daily injection). Three days after treatment, blood samples and kidneys were collected for biochemical and histological analyses. a Serum creatinine. b Efficiency of TSA reducing serum creatinine (cisplatin only vs cisplatin+TSA). c BUN. d Efficiency of TSA reducing BUN (cisplatin only vs cisplatin+TSA). Data in a–d are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#P < 0.05, significantly different from the cisplatin-only group. ^*P <* 0.05, significantly different from the corresponding group of PT-*Atg7* WT mice. e Representative images of kidney H–E staining (×200). Scale bar: 100 μm. f Pathological score of tubular damage. Data are expressed as mean ± SD. ^#P < 0.05, significantly different from the cisplatinonly group. ^P < 0.05, significantly different from the PT-Atg7-WT group. g Representative immunoblots. h Densitometric analysis of cleaved caspase 3 signals. Data are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#*P <* 0.05, significantly different from the cisplatin-only group. ^*P <* 0.05, significantly different from the PT-Atg7-WT group

**Fig. 10. TSA activates AMPK and inactivates mTOR during cisplatin treatment of RPTC cells and C57Bl/6 mice.**
a–c RPTC cells were untreated or treated with cisplatin (20 μM) only, TSA (0.1 μM) only, or cisplatin+TSA for 20 h. After treatment, cells were collected for immunoblot analysis of p-AMPK, AMPK, p-P70S6K, and P70S6K. β-Actin was used as a loading control. a Representative immunoblots. b, c Densitometric analysis of p-AMPK and p-P70S6K signals after normalization with AMPK and P70S6K, respectively. Data are expressed as mean ± SD. *P < 0.05, significantly different from the control group. ^#P < 0.05, significantly different from the cisplatin-only group. d–f C57BL/6 mice were injected with saline or a single dose of cisplatin (30 mg/kg, i.p.) in the absence (−) or presence (+) of TSA (1 mg/kg, i.p., daily injection). Kidneys were collected 3 days after treatment for immunoblot analysis of p-AMPK, AMPK, p-P70S6K, and P70S6K. β-Actin was used as a loading control. d Representative immunoblots. e, f Densitometric analysis of p-AMPK and p-P70S6K signals after normalization with total AMPK and P70S6K, respectively. Data are expressed as mean ± SD. *P < 0.05, significantly different from the saline group. ^#P < 0.05, significantly different from the cisplatin-only group

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References

1. Seto E, Yoshida M. Erasers of histone acetylation: the histone deacetylase enzymes. Cold Spring Harb. Perspect. Biol. 2014;6:a018713. doi: 10.1101/cshperspect.a018713. - DOI - PMC - PubMed
1. Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discov. 2014;13:673–691. doi: 10.1038/nrd4360. - DOI - PubMed
1. Li Y, Seto E. HDACs and HDAC inhibitors in cancer development and therapy. Cold Spring Harb. Perspect. Med. 2016;6:pii: a026831. doi: 10.1101/cshperspect.a026831. - DOI - PMC - PubMed
1. Pang M, et al. Inhibition of histone deacetylase activity attenuates renal fibroblast activation and interstitial fibrosis in obstructive nephropathy. Am. J. Physiol. Ren. Physiol. 2009;297:F996–F1005. doi: 10.1152/ajprenal.00282.2009. - DOI - PMC - PubMed
1. Liu N, et al. Blocking the class I histone deacetylase ameliorates renal fibrosis and inhibits renal fibroblast activation via modulating TGF-beta and EGFR signaling. PLoS ONE. 2013;8:e54001. doi: 10.1371/journal.pone.0054001. - DOI - PMC - PubMed

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[1] Seto E, Yoshida M. Erasers of histone acetylation: the histone deacetylase enzymes. Cold Spring Harb. Perspect. Biol. 2014;6:a018713. doi: 10.1101/cshperspect.a018713. - DOI - PMC - PubMed

[2] Seto E, Yoshida M. Erasers of histone acetylation: the histone deacetylase enzymes. Cold Spring Harb. Perspect. Biol. 2014;6:a018713. doi: 10.1101/cshperspect.a018713. - DOI - PMC - PubMed

[3] Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discov. 2014;13:673–691. doi: 10.1038/nrd4360. - DOI - PubMed

[4] Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders. Nat. Rev. Drug Discov. 2014;13:673–691. doi: 10.1038/nrd4360. - DOI - PubMed

[5] Li Y, Seto E. HDACs and HDAC inhibitors in cancer development and therapy. Cold Spring Harb. Perspect. Med. 2016;6:pii: a026831. doi: 10.1101/cshperspect.a026831. - DOI - PMC - PubMed

[6] Li Y, Seto E. HDACs and HDAC inhibitors in cancer development and therapy. Cold Spring Harb. Perspect. Med. 2016;6:pii: a026831. doi: 10.1101/cshperspect.a026831. - DOI - PMC - PubMed

[7] Pang M, et al. Inhibition of histone deacetylase activity attenuates renal fibroblast activation and interstitial fibrosis in obstructive nephropathy. Am. J. Physiol. Ren. Physiol. 2009;297:F996–F1005. doi: 10.1152/ajprenal.00282.2009. - DOI - PMC - PubMed

[8] Pang M, et al. Inhibition of histone deacetylase activity attenuates renal fibroblast activation and interstitial fibrosis in obstructive nephropathy. Am. J. Physiol. Ren. Physiol. 2009;297:F996–F1005. doi: 10.1152/ajprenal.00282.2009. - DOI - PMC - PubMed

[9] Liu N, et al. Blocking the class I histone deacetylase ameliorates renal fibrosis and inhibits renal fibroblast activation via modulating TGF-beta and EGFR signaling. PLoS ONE. 2013;8:e54001. doi: 10.1371/journal.pone.0054001. - DOI - PMC - PubMed

[10] Liu N, et al. Blocking the class I histone deacetylase ameliorates renal fibrosis and inhibits renal fibroblast activation via modulating TGF-beta and EGFR signaling. PLoS ONE. 2013;8:e54001. doi: 10.1371/journal.pone.0054001. - DOI - PMC - PubMed

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Histone deacetylase inhibitors protect against cisplatin-induced acute kidney injury by activating autophagy in proximal tubular cells

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Histone deacetylase inhibitors protect against cisplatin-induced acute kidney injury by activating autophagy in proximal tubular cells

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Conflict of interest statement

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