Anat Cell Biol.  2015 Mar;48(1):66-74. 10.5115/acb.2015.48.1.66.

Cisplatin induces primary necrosis through poly(ADP-ribose) polymerase 1 activation in kidney proximal tubular cells

• Park S
• Yoon SP
• Kim J

• KMID: 2046077
• DOI: http://doi.org/10.5115/acb.2015.48.1.66

Abstract

Treatment with cisplatin for cancer therapy has a major side effect such as nephrotoxicity; however, the role of poly (ADP-ribose) polymerase 1 (PARP1) in necrosis in response to cisplatin nephrotoxicity remains to be defined. Here we report that cisplatin induces primary necrosis through PARP1 activation in kidney proximal tubular cells derived from human, pig and mouse. Treatment with high dose of cisplatin for 4 and 8 hours induced primary necrosis, as represented by the percentage of propidium iodide-positive cells and lactate dehydrogenase release. The primary necrosis was correlated with PARP1 activation during cisplatin injury. Treatment with PJ34, a potent PARP1 inhibitor, at 2 hours after injury attenuated primary necrosis after 8 hours of cisplatin injury as well as PARP1 activation. PARP1 inhibition also reduced the release of lactate dehydrogenase and high mobility group box protein 1 from kidney proximal tubular cells at 8 hours after cisplatin injury. Oxidative stress was increased by treatment with cisplatin for 8 hours as shown by 8-hydroxy-2'-deoxyguanosine and lipid hydroperoxide assays, but PARP1 inhibition at 2 hours after injury reduced the oxidative damage. These data demonstrate that cisplatin-induced PARP1 activation contributes to primary necrosis through oxidative stress in kidney proximal tubular cells, resulting in the induction of cisplatin nephrotoxicity and inflammation.

Keyword

Cisplatin; Nephrotoxicity; Necrosis; Poly(ADP-ribose) polymerase 1; Kidney proximal tubular cell

MeSH Terms

Animals
Cisplatin*
Humans
Inflammation
Kidney*
L-Lactate Dehydrogenase
Lipid Peroxides
Mice
Necrosis*
Oxidative Stress
Poly(ADP-ribose) Polymerases*
Propidium
Cisplatin
L-Lactate Dehydrogenase
Lipid Peroxides
Poly(ADP-ribose) Polymerases
Propidium

Figure

• 

  Fig. 1 Cisplatin induces necrosis in kidney proximal tubular cells. After 18 hours of starvation, HK-2, LLC-PK1, and MCT cells were treated with 10, 25, 50, 100, 200, or 400 µM of cisplatin in phosphate buffered saline (control) for 0, 2, 4, or 8 hours. (A) The percentage of propidium iodide (PI)-positive cells was assessed in 10 field (×400) per well. (B) Lactate dehydrogenase (LDH) release was measured enzymatically using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Error bars represent SD (n=3 experiments). *P<0.05, **P<0.01, ***P<0.001 versus 0 hour.

• 

  Fig. 2 Cisplatin increases poly(ADP-ribose) polymerase (PARP) activity in kidney proximal tubular cells. After 18 hours of starvation, HK-2, LLC-PK1, and MCT cells were treated with 10, 25, 50, 100, 200, or 400 µM of cisplatin in phosphate buffered saline (control) for 0, 2, 4, or 8 hours. PARP activity was measured by a universal PARP assay kit (Trevigen). Error bars represent SD (n=3 experiments). *P<0.05, **P<0.01, ***P<0.001 versus 0 hour.

• 

  Fig. 3 Correlation between the percentage of propidium iodide (PI)-positive cells and poly(ADP-ribose) polymerase (PARP) activity in kidney proximal tubular cells during cisplatin injury. After 18 hours of starvation, HK-2, LLC-PK1, and MCT cells were treated with 10, 25, 50, 100, 200, or 400 µM of cisplatin in phosphate buffered saline (control) for 0, 2, 4, or 8 hours. The Pearson correlation between the percentage of PI-positive cells and the level of PARP activity at all cisplatin-dose levels and time points shown in Figs. 1A and 2.

• 

  Fig. 4 Poly(ADP-ribose) polymerase 1 (PARP1) inhibition reduces necrosis induced by cisplatin in kidney proximal tubular cells after cisplatin injury. After 18 hours of starvation, HK-2, LLC-PK1, and MCT cells were treated with 400 µM cisplatin for 8 hours. The cells were also treated with high or low dose of PJ34 at 2 hours after treatment with cisplatin. (A) PARP activity was measured by a universal PARP assay kit. (B) Necrosis in HK-2 cells was detected by propidium iodid (PI) staining. Nuclei were counterstained with DAPI. Scale bars=50 µm. (C) The percentage of PI-positive cells was assessed in 10 fields (×400) per well. Error bars represent SD (n=3 experiments). **P<0.01 versus control. †P<0.05, ††P<0.01, †††P<0.001 versus vehicle.

• 

  Fig. 5 Poly(ADP-ribose) polymerase 1 (PARP1) inhibition reduces plasma membrane disruption and HMGB1 release induced by cisplatin in kidney proximal tubular cells. After 18 hours of starvation, HK-2 cells were treated with 400 µM cisplatin for 8 hours. The cells were also treated with high or low dose of PJ34 at 2 hours after treatment with cisplatin. After 8 hours of cisplatin injury, the cells and media were harvested. (A) Lactate dehydrogenase (LDH) release was measured enzymatically using a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. (B) The levels of released HMGB1 in cell culture media were measured using Western blot analysis. Protein bands were quantified using Lab Works analysis software (Ultra-Violet Products). Error bars represent SD (n=3 experiments). **P<0.01 versus control. †P<0.05, ††P<0.01, †††P<0.001 versus vehicle.

• 

  Fig. 6 Poly(ADP-ribose) polymerase 1 (PARP1) inhibition ameliorates ATP depletion induced by cisplatin in kidney proximal tubular cells. After 18 hours of starvation, HK-2 cells were treated with 400 µM cisplatin for 8 hours. The cells were also treated with high or low dose of PJ34 at 2 hours after treatment with cisplatin. The level of ATP was measured by an ATP assay kit (BioVision). Error bars represent SD (n=3 experiments). **P<0.01, ***P<0.001 versus control. ††P<0.01, †††P<0.001 versus vehicle.

• 

  Fig. 7 Poly(ADP-ribose) polymerase 1 (PARP1) inhibition reduces oxidative stress induced by cisplatin in kidney proximal tubular cells. After 18 hours of starvation, HK-2 cells were treated with 400 µM cisplatin for 8 hours. The cells were also treated with high or low dose of PJ34 at 2 hours after treatment with cisplatin. The levels of 8-OHdG (A) and lipid hydroperoxide (B) were measured by assay kits (Cayman). Error bars represent SD (n=3 experiments). **P<0.01 versus control. †P<0.05, ††P<0.01, †††P<0.001 versus vehicle.

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