Anat Cell Biol.  2016 Sep;49(3):165-176. 10.5115/acb.2016.49.3.165.

Poly(ADP-ribose) polymerase 1 contributes to oxidative stress through downregulation of sirtuin 3 during cisplatin nephrotoxicity

• Yoon SP
• Kim J

• KMID: 2353341
• DOI: http://doi.org/10.5115/acb.2016.49.3.165

Abstract

Enhanced oxidative stress is a hallmark of cisplatin nephrotoxicity, and inhibition of poly(ADP-ribose) polymerase 1 (PARP1) attenuates oxidative stress during cisplatin nephrotoxicity; however, the precise mechanisms behind its action remain elusive. Here, using an in vitro model of cisplatin-induced injury to human kidney proximal tubular cells, we demonstrated that the protective effect of PARP1 inhibition on oxidative stress is associated with sirtuin 3 (SIRT3) activation. Exposure to 400 µM cisplatin for 8 hours in cells decreased activity and expression of manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase (GPX), and SIRT3, while it increased their lysine acetylation. However, treatment with 1 µM PJ34 hydrochloride, a potent PARP1 inhibitor, restored activity and/or expression in those antioxidant enzymes, decreased lysine acetylation of those enzymes, and improved SIRT3 expression and activity in the cisplatin-injured cells. Using transfection with SIRT3 double nickase plasmids, SIRT3-deficient cells given cisplatin did not show the ameliorable effect of PARP1 inhibition on lysine acetylation and activity of antioxidant enzymes, including MnSOD, catalase and GPX. Furthermore, SIRT3 deficiency in cisplatin-injured cells prevented PARP1 inhibition-induced increase in forkhead box O3a transcriptional activity, and upregulation of MnSOD and catalase. Finally, loss of SIRT3 in cisplatin-exposed cells removed the protective effect of PARP1 inhibition against oxidative stress, represented by the concentration of lipid hydroperoxide and 8-hydroxy-2'-deoxyguanosine; and necrotic cell death represented by a percentage of propidium iodide-positively stained cells. Taken together, these results indicate that PARP1 inhibition protects kidney proximal tubular cells against oxidative stress through SIRT3 activation during cisplatin nephrotoxicity.

Keyword

Poly(ADP-ribose) polymerases; Sirtuin 3; Cisplatin; Nephrotoxicity; Oxidative stress; Manganese superoxide dismutase; Catalase; Glutathione peroxidase; Lysine acetylation

MeSH Terms

Acetylation
Catalase
Cell Death
Cisplatin*
Deoxyribonuclease I
Down-Regulation*
Glutathione Peroxidase
Humans
In Vitro Techniques
Kidney
Lipid Peroxides
Lysine
Oxidative Stress*
Plasmids
Poly Adenosine Diphosphate Ribose*
Poly(ADP-ribose) Polymerases*
Propidium
Sirtuin 3*
Superoxide Dismutase
Transfection
Up-Regulation
Catalase
Cisplatin
Deoxyribonuclease I
Glutathione Peroxidase
Lipid Peroxides
Lysine
Poly Adenosine Diphosphate Ribose
Poly(ADP-ribose) Polymerases
Propidium
Sirtuin 3
Superoxide Dismutase

Figure

• 

  Fig. 1 Poly(ADP-ribose) polymerase 1 (PARP1) inhibition improves expression and activity of antioxidant enzymes in cisplatin-induced injury to kidney proximal tubule epithelial cells. (A) Manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPX) expressions were examined by western blot analysis with respective antbodies. Anti–β-actin antibody was used as a loading control. Veh, vehicle. (B) The intensities of protein bands were quantified using the Lab Works analysis software. (C) Those activities were measured as described in the "Materials and Methods" section. n=4 experiments in each group. a)P<0.05 versus control. b)P<0.05 versus vehicle.

• 

  Fig. 2 Poly(ADP-ribose) polymerase 1 inhibition reduces cisplatin-induced acetylation of manganese superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPX) in kidney proximal tubule epithelial cells. (A) Proteins in whole cell lysates were immunoprecipitated (IP) with MnSOD, copper/zinc superoxide dismutase (CuZnSOD), catalase, and GPX, respectively; and subjected to western blot with anti–acetyl lysine antibody. Veh, vehicle. (B) The acetylation levels were represented as a ratio of acetyl lysine expression to enzyme expression. The intensities of protein bands were quantified using the Lab Works analysis software. n=4 experiments in each group. a)P<0.05 versus control. b)P<0.05 versus vehicle.

• 

  Fig. 3 Poly(ADP-ribose) polymerase 1 inhibition restores sirtuin 3 (SIRT3) expression and activity in cisplatin-induced injury to kidney proximal tubule epithelial cells. (A) The SIRT3 expression was examined by western blot with anti-SIRT3 antibody. Anti–β-actin antibody was used as a loading control. The intensities of protein bands were quantified using the Lab Works analysis software (n=4 experiments in each group). Veh, vehicle. (B) The SIRT3 activity was measured using a SIRT3 direct fluorescent screening assay kit according to the manufacturer's instruction. (C) The total level of NAD+ was measured using a NAD+/NADH quantification colorimetric kit according to the manufacturer's instruction. n=4 experiments in each group. a)P<0.05 versus control. b)P<0.05 versus vehicle.

• 

  Fig. 4 Sirtuin 3 (SIRT3) deficiency removes the restorative effect of poly(ADP-ribose) polymerase 1 inhibition on the activities of manganese superoxide dismutase (MnSOD), catalase, and glutathione peroxidase (GPX) in cisplatin-induced injury to kidney proximal tubule epithelial cells. (A) The expression of SIRT3 was confirmed using western blot with anti-SIRT3 antibody in HK2 cells transfected with either control double nickase plasmid (WT) or SIRT3 double nickase plasmid (KO). Antibodies to green fluorescent protein (GFP) and β-actin were used as a transfection control and a loading control, respectively. The experiment was repeated at least four times. (B) After the starvation, the SIRT3 KO and WT cells were treated with cisplatin plus/minus PJ34. Proteins in whole cell lysates obtained from the cells were immunoprecipitated with MnSOD, catalase, and GPX, respectively; and subjected to western blot with the antibody to acetyl lysine. IP, immunoprecipitation. (C) The acetylation levels were represented as a ratio of acetyl lysine expression to enzyme expression. The intensities of protein bands were quantified using the Lab Works analysis software. (D) The activities of MnSOD, catalase, and GPX were measured in SIRT3 KO or WT cells treated with cisplatin plus/minus PJ34. n=4 experiments in each group. a)P<0.05 versus vehicle. b)P<0.05 versus WT.

• 

  Fig. 5 Sirtuin 3 (SIRT3) deficiency removes the restorative effect of poly(ADP-ribose) polymerase 1 inhibition on the expressions of manganese superoxide dismutase (MnSOD) and catalase in cisplatin-induced injury to kidney proximal tubule epithelial cells. The expressions of MnSOD, catalase, and glutathione peroxidase (GPX) were measured using western blot analysis in SIRT3-KO or -WT cells treated with cisplatin plus/minus PJ34. Anti–β-actin antibody was used as a loading control. n=4 experiments in each group. a)P<0.05 versus vehicle. b)P<0.05 versus WT.

• 

  Fig. 6 Sirtuin 3 (SIRT3) deficiency removes the restorative effect of poly(ADP-ribose) polymerase 1 inhibition on forkhead box O3a (FOXO3a) transcription activity in cisplatin-induced injury to kidney proximal tubule epithelial cells. Luciferase activity was measured in SIRT3-KO or -WT cells transiently transfected with a FOXO3a promoter-reporter (FHRE-Luc) and treated with cisplatin plus PJ34 or vehicle. n=4 experiments in each group. a)P<0.05 versus vehicle. b)P<0.05 versus WT.

• 

  Fig. 7 Sirtuin 3 (SIRT3) deficiency removes the protective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition on oxidative stress and necrotic cell death in cisplatin-induced injury to kidney proximal tubule epithelial cells. (A) The level of lipid hydroperoxide was measured in SIRT3-KO and -WT cells treated with cisplatin plus PJ34 or vehicle. (B) The level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured in the cells using an assay kit. (C) The mitochondrial membrane potential was measured using tetramethylrhodamine, ethyl ester assay. (D) The percentage of propidium iodide (PI)–positive cells was assessed in 10 field per well. (E) Scheme of PARP1-dependent SIRT3 pathway during cisplatin injury. FOXO3a, forkhead box O3a; MnSOD, manganese superoxide dismutase; GPX, glutathione peroxidase. n=4 experiments in each group. a)P<0.05 versus vehicle. b)P<0.05 versus WT.

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