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. 2024 Jan;20(1):151-165.
doi: 10.1080/15548627.2023.2252265. Epub 2023 Aug 31.

Melatonin attenuates sepsis-induced acute kidney injury by promoting mitophagy through SIRT3-mediated TFAM deacetylation

Zhiya Deng  1   2 , Man He  1   2 , Hongbin Hu  1 , Wenqian Zhang  1   2 , Yaoyuan Zhang  1 , Yue Ge  1   3 , Tongtong Ma  1 , Jie Wu  1 , Lulan Li  1 , Maomao Sun  1   2 , Sheng An  1   2 , Jiaxin Li  1 , Qiaobing Huang  1   2 , Shenhai Gong  1   4 , Jiaxing Zhang  1   5 , Zhongqing Chen  1 , Zhenhua Zeng  1   2
Affiliations

Melatonin attenuates sepsis-induced acute kidney injury by promoting mitophagy through SIRT3-mediated TFAM deacetylation

Zhiya Deng et al. Autophagy. 2024 Jan.

Abstract

AKI: acute kidney injury; ATP: adenosine triphosphate; BUN: blood urea nitrogen; CLP: cecal ligation and puncture; eGFR: estimated glomerular filtration rate; H&E: hematoxylin and eosin staining; LCN2/NGAL: lipocalin 2; LPS: lipopolysaccharide; LTL: lotus tetragonolobus lectin; mKeima: mitochondria-targeted Keima; mtDNA: mitochondrial DNA; PAS: periodic acid - Schiff staining; RTECs: renal tubular epithelial cells; SAKI: sepsis-induced acute kidney injury; Scr: serum creatinine; SIRT3: sirtuin 3; TFAM: transcription factor A, mitochondrial; TMRE: tetramethylrhodamine.

Keywords: Acetylation; acute kidney injury; autophagy; critical care; post-translational modification; sepsis.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Higher plasma melatonin level resulted in shorter recovery time and less renal mitochondrial damage in patients with SAKI. (A) Flowchart of the enrolled patients. (B) the plasma melatonin level in non-SAKI patients (n = 13) and SAKI (n = 35). (C) the plasma melatonin level in SAKI recovery (n = 21) and non-recovery SAKI patients (n = 14). The data of (B and C) are presented as the median ± IQR. Statistical significance was assessed by Mann-Whitney U test and P < 0.001. (D) SAKI recovery analyses ratified by melatonin median (29.5 pg/ml) in SAKI cohort (melatonin ≥29.5 pg/ml, n = 18, melatonin <29.5 pg/ml, n = 17). Statistical significance was assessed by log-rank test and *P < 0.05. (E) the level of plasma melatonin was negatively correlated with urinary mtDNA in SAKI patients, n = 35. (F) the level of plasma melatonin did not correlate with Scr, n = 35. (G) the level of plasma melatonin did not correlate with BUN, n = 35. (H) the level of plasma melatonin did not correlate with eGFR, n = 35. Statistical significance of (E-H) was assessed by Spearman’s rank correlation analysis. We present P and r in those figures. SAKI: sepsis-induced acute kidney injury, Mel: melatonin, mtDNA: mitochondrial DNA, Scr: serum creatinine, BUN: blood urea nitrogen, eGFR: estimated glomerular filtration rate.
Figure 2.
Figure 2.
Melatonin attenuates SAKI via activating SIRT3. (A) effects of melatonin and SIRT3 inhibitor 3-TYP on the survival times in CLP-induced septic mice; n = 10; statistical significance was assessed by log-rank test and *P < 0.0167. (B) Scr level; n = 6. (C) BUN level; n = 6. (D) Pathological observation of kidney tissue (upper panel: H&E staining of kidney cortex; lower panel: PAS staining of kidney cortex; scale bar: 100 μm) and histologic scores were evaluated based on H&E and PAS staining (n = 6). (E) tubular injury was analyzed by LCN2-positive staining with RTECs marker LTL (green), injury marker LCN2 (red), and cell nuclei marker DAPI (blue) (scale bar: 100 μm); n = 6. (F) the urinary mtDNA level from mice; n = 4. (G) the mitochondrial membrane potential of HK-2 cells was measured by TMRE fluorescence; n = 10. (H) ATP levels of HK-2 cells; n = 10. (I) the mitoROS of HK-2 cells were measured by mitoSOX staining (red) with DAPI (blue) (scale bar: 40 μm), and mtROS was statistically quantified by mtSOX fluorescence (n = 6). These data are presented as the mean ± SD and *P < 0.05. Statistical significance was assessed by one-way ANOVA. CLP: cecal ligation and puncture, LPS: lipopolysaccharide, Mel: melatonin, Scr: serum creatinine, BUN: blood urea nitrogen, H&E: hematoxylin and eosin staining, PAS: periodic acid–Schiff staining, LCN2: lipocalin 2, LTL: lotus tetragonolobus lectin, mtDNA: mitochondrial DNA, TMRE: tetramethylrhodamine, ATP: adenosine triphosphate.
Figure 3.
Figure 3.
Melatonin-induced activation of SIRT3 promotes mitophagic flux. (A) mKeima was expressed in HK-2 cells and live cell was imaged by confocal microscopy (mKeima-561 nm: red; mKeima-488 nm: green; scale bar: 20 μm); 561 nm/488 nm mKeima was statistically quantified (n = 6). (B) representative western blot with densitometric analysis of protein expression of PINK1, PRKN, LC3-II and SQSTM1 in HK-2 cells; and the protein expression levels were standardized relative to the level of GAPDH; n = 6. (C) the representative ultrastructure images of mouse renal epithelial cells observed by transmission electron microscopy; and the number of autophagosomes (black arrow) or autolysosomes (red arrow) was calculated in 20 randomly selected 20,000× fields (upper panel: scale bar: 5 μm; lower panel: scale bar: 1 μm, n = 20). (D) the representative ultrastructure images of mitophagosomes in Mel+CLP group observed by transmission electron microscopy; (scale bar: 300 nm). The data are presented as the mean ± SD. Statistical significance was assessed by one-way ANOVA and *P < 0.05. CLP: cecal ligation and puncture, LPS: lipopolysaccharide, Mel: melatonin.
Figure 4.
Figure 4.
Melatonin attenuates SAKI via promoting mitophagic flux. (A) effects of melatonin and chloroquine-blocked mitophagy on the survival times in CLP-induced septic mice; n = 10; statistical significance was assessed by log-rank test and *P < 0.0167. (B) Scr level; n = 6. (C) BUN level; n = 6. (D) Pathological observation of kidney tissue (upper panel: H&E staining of kidney cortex; lower panel: PAS staining of kidney cortex; scale bar: 100 μm) and histologic scores were evaluated based on H&E and PAS staining (n = 6). (E) tubular injury was analyzed by LCN2-positive staining with RTECs marker LTL (green), injury marker LCN2 (red), and cell nuclei marker DAPI (blue) (scale bar: 100 μm); n = 6. (F) the urinary mtDNA level from mice; n = 4. (G) the mitochondrial membrane potential of HK-2 cells was measured by TMRE fluorescence; n = 10. (H) ATP levels of HK-2 cells; n = 10. (I) the mitoROS of HK-2 cells were measured by mitoSOX staining (red) with DAPI (blue) (scale bar: 40 μm), and mtROS was statistically quantified by mtSOX fluorescence (n = 6). These data are presented as the mean ± SD and *P < 0.05. Statistical significance was assessed by one-way ANOVA. CLP: cecal ligation and puncture, LPS: lipopolysaccharide, Mel: melatonin, CQ: chloroquine, Scr: serum creatinine, BUN: blood urea nitrogen, H&E: hematoxylin and eosin staining, PAS: periodic acid–Schiff staining, LCN2: lipocalin 2, LTL: lotus tetragonolobus lectin, mtDNA: mitochondrial DNA, TMRE: tetramethylrhodamine, ATP: adenosine triphosphate.
Figure 5.
Figure 5.
Melatonin-induced SIRT3 activation results in TFAM-K154 deacetylation. (A) acetylation on mitochondrial protein was determined by western blots using pan-acetylated lysine antibody. (B) the acetylation of TFAM in kidney tissue of mice and HK-2 cells were examined by immunoprecipitation and western blot. (C) the physical interaction between SIRT3 and TFAM in kidney tissue of mice and HK-2 cells were determined by co-immunoprecipitation. (D) the immunofluorescence analysis showed that SIRT3 colocalized with TFAM (SIRT3: red, TFAM: green, DAPI: blue, scale bar: 10 μm, n = 10). The data are presented as the mean ± SD and *P < 0.05. Statistical significance was assessed by one-way ANOVA. (E) sequence alignment of TFAM among multiple species was analyzed by the MEME suite. (F) the acetylation of TFAM in HK-2 cells with or without LPS-stimulation was examined by immunoprecipitation and western blot. (G) the acetylation of TFAM in HK-2 cells with or without SIRT3 overexpression was examined by immunoprecipitation and western blot. (H) the acetylation of TFAM in HK-2 cells with or without melatonin administration was examined by immunoprecipitation and western blot. Mito: mitochondria, Ac-K: acetylated lysine, LPS: lipopolysaccharide, Mel: melatonin, CLP: cecal ligation and puncture.
Figure 6.
Figure 6.
SIRT3-induced TFAM deacetylation is indispensable for melatonin to promote mitophagic flux. (A) representative western blot with densitometric analysis of protein expression of PINK1, PRKN, LC3-II and SQSTM1 in HK-2 cells with or without SIRT3 overexpression; and the protein expression levels were standardized relative to the level of GAPDH; n = 4. (B) mKeima was expressed in HK-2 cells with or without SIRT3 overexpression, and live cell was imaged by confocal microscopy (mKeima-561 nm: red; mKeima-488 nm: green; scale bar: 20 μm); 561 nm/488 nm mKeima was statistically quantified; n = 6. (C) representative western blot with densitometric analysis of protein expression of PINK1, PRKN, LC3-II and SQSTM1 in HK-2 cells with or without melatonin administration; and the protein expression levels were standardized relative to the level of GAPDH; n = 4. (D) mKeima was expressed in HK-2 cells with or without melatonin administration, and live cell was imaged by confocal microscopy (mKeima-561 nm: red; mKeima-488 nm: green; scale bar: 20 μm); 561 nm/488 nm mKeima was statistically quantified; n = 6. (E) representative western blot with densitometric analysis of PINK1, PRKN, LC3-II and SQSTM1 in HK-2 cells with or without SIRT3 knockdown; and the protein expression levels were standardized relative to the level of GAPDH; n = 4. (F) mKeima was expressed in HK-2 cells with or without SIRT3 knockdown, and live cell was imaged by confocal microscopy (mKeima-561 nm: red; mKeima-488 nm: green; scale bar: 20 μm); 561 nm/488 nm mKeima was statistically quantified; n = 6. The data are presented as the mean ± SD. Statistical significance was assessed by one-way ANOVA and *P < 0.05. LPS: lipopolysaccharide, Mel: melatonin.
Figure 7.
Figure 7.
Schematic diagram of the mechanism: Melatonin promotes SIRT3-induced deacetylation of TFAM-K154, thereby improve mitophagic flux to attenuate SAKI. SIRT3: Sirtuin 3, TFAM: transcription factor A, mitochondrial, ac: acetylation, SAKI: sepsis-induced acute kidney injury, RTECs: renal tubular epithelial cells.
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Grants and funding

This study was supported by the National Natural Science Foundation of China (grant numbers 82172175, 81871604 and 82002078), the Guangdong Basic and Applied Basic Research Foundation (grant number 2021A1515111028), the Medical Scientific Research Foundation of Guangdong Province of China (grant number A2022120), and the Innovation and entrepreneurship training program for College Students (grant number 202112121241).