Anat Cell Biol.  2019 Sep;52(3):312-323. 10.5115/acb.18.192.

Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells

• Moon D
• Kim J

• KMID: 2459547
• DOI: http://doi.org/10.5115/acb.18.192

Abstract

Cyclosporin A (CsA) does not only exert a toxic effect on kidney parenchymal cells, but also protects them against necrotic cell death by inhibiting opening of mitochondrial permeability transition pore. However, whether CsA plays a role in hydrogen peroxide-induced kidney proximal tubular cell death is currently unclear. In the present study, treatment with CsA further increased apoptosis and necrosis in HK-2 human kidney proximal tubule epithelial cells during exposure to hydrogen peroxide. In addition, hydrogen peroxide-induced p53 activation and BH3 interacting-domain death agonist (BID) expression were higher in CsA-treated cells than those in non-treated cells, whereas hydrogen peroxide-induced activation of mitogen-activated protein kinases including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase and activation of protein kinase B were not significantly altered by treatment with CsA. In oxidant-antioxidant system, reactive oxygen species (ROS) production induced by hydrogen peroxide was further enhanced by treatment with CsA. However, expression levels of antioxidant enzymes including manganese superoxide dismutase, copper/zinc superoxide dismutase, and catalase were not altered by treatment with hydrogen peroxide or CsA. Treatment with CsA further enhanced mitochondrial membrane potential induced by exposure to hydrogen peroxide, although it did not alter endoplasmic reticulum stress based on expression of glucose-regulated protein 78 and 94. Taken together, these data suggest that CsA can aggravate hydrogen peroxide-induced cell death through p53 activation, BID expression, and ROS production.

Keyword

Cyclosporin; Cell death; Hydrogen peroxide; p53; Reactive oxygen species

MeSH Terms

Apoptosis
Catalase
Cell Death*
Cyclosporine*
Endoplasmic Reticulum Stress
Epithelial Cells*
Humans
Hydrogen Peroxide
Hydrogen*
JNK Mitogen-Activated Protein Kinases
Kidney*
Membrane Potential, Mitochondrial
Mitogen-Activated Protein Kinases
Necrosis
Permeability
Phosphotransferases
Proto-Oncogene Proteins c-akt
Reactive Oxygen Species
Superoxide Dismutase
Catalase
Cyclosporine
Hydrogen
Hydrogen Peroxide
JNK Mitogen-Activated Protein Kinases
Mitogen-Activated Protein Kinases
Phosphotransferases
Proto-Oncogene Proteins c-akt
Reactive Oxygen Species
Superoxide Dismutase

Figure

• 

  Fig. 1 Cyclosporin A (CsA) enhances hydrogen peroxide (H2O2) injury in human kidney proximal tubule epithelial cells. Human kidney proximal tubule epithelial HK-2 cells were cultured in RPMI 1640 until reaching 80% confluence. (A) HK-2 cells were treated with either CsA (1, 10, or 100 nM) or 1% dimethyl sulfoxide (vehicle) for 1 hour and then exposed to 1 mM H2O2 or distilled water (control) for 60 minutes. Cell viability was measured using MTT assay (n=9 wells from 3 experiments per condition). In box plots, whiskers represent the minimum and maximum; bases represent the interquartile range between the first and third quartiles; and midlines represent the median. (B) HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H2O2 or control for 30, 60, or 120 minutes. Cell viability was measured using MTT assay (n=9 wells from 3 experiments per condition). (C, D) HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H2O2 or control for 60 minutes. Cell death was analyzed by flow cytometry with an annexin V-FITC detection kit after treatment with FITC-conjugated annexin V and propidium iodide. The flow cytometry assay distinguishes among normal (lower left), early apoptosis (lower right), late apoptosis (upper right), and necrosis (upper left). Three experiments were performed to evaluate the cell death. In each experiment, three samples per experimental condition were included. a)P<0.05 vs. control. b)P<0.05 vs. vehicle. c)P<0.01 vs. 0 mol/l.

• 

  Fig. 2 Cyclosporin A (CsA) increases p53 activation and BH3 interacting-domain death agonist (BID) expression after H2O2 injury in human kidney proximal tubule epithelial cells. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H2O2 or control for 60 minutes (n=6 experiments per condition). (A) Phosphorylation level of p53 (p-p53, 53 kDa) and total expression level of p53 (t-p53, 53 kDa) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (B–D) Intensities of p-p53 and t-p53 protein bands were quantified using the AzureSpot software. (E) BID (22 kDa) expression was measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (F) Intensity of BID protein expression was quantified using the AzureSpot software. a)P<0.05 vs. control. b)P<0.05 vs. vehicle.

• 

  Fig. 3 Cyclosporin A (CsA) does not alter activations of mitogen-activated protein kinases and protein kinase B (AKT) after H2O2 injury in human kidney proximal tubule epithelial cells. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H2O2 or control for 60 minutes (n=6 experiments per condition). (A) Phosphorylation levels of p38 (p-p38, 43 kDa), c-Jun N-terminal kinase (p-JNK, 46 and 55 kDa), and extracellular signal-regulated kinase (p-ERK, 42 and 44 kDa), and their total expression levels (t-p38, p-JNK, and p-ERK) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (B, C, E, F, H, I) Intensities of p-p38, t-p38, p-JNK, total JNK (t-JNK), p-ERK, and total ERK (t-ERK) protein bands were quantified using the AzureSpot software. (D, G, J) Activations of p38, JNK, and ERK indicated by their respective ratio of phosphorylation level to total expression level. (K) AKT phosphorylation (p-AKT) and total expression (t-AKT) with a molecular mass of 56 to 62 kDa were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (L, M) Intensities of p-AKT and t-AKT protein bands were quantified using the AzureSpot software. (N) AKT activation based on the ratio of phosphorylation to total expression. a)P<0.05 vs. control.

• 

  Fig. 4 Cyclosporin A (CsA) enhances reactive oxygen species (ROS) production induced by H2O2 in human kidney proximal tubule epithelial cells without altering expression level of antioxidant enzymes. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H2O2 or control for 60 minutes (n=3 experiments per condition). (A) ROS production was measured using oxidative sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate. (B) Expression levels of manganese superoxide dismutase (MnSOD; 25 kDa), copper/zinc superoxide dismutase (CuZnSOD; 23 kDa), and catalase (64 kDa) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (C–E) Intensities of MnSOD, CuZnSOD, and catalase protein bands were quantified using the AzureSpot software. N.S., not significant. *P<0.05 vs. control, #P<0.05 vs. vehicle.

• 

  Fig. 5 Cyclosporin A (CsA) increases mitochondrial depolarization induced by H2O2 in human kidney proximal tubule epithelial cells. HK-2 cells were treated with either 10 nM CsA or vehicle for 60 minutes and then exposed to 1 mM H2O2 or control for 60 minutes. (A) Percentage of mitochondrial membrane potential was measured using tetramethylrhodamine ethyl ester perchlorate dye (n=9 wells per 3 experiments per condition). The mitochondrial membrane potential in the group with control plus vehicle was taken as 100%. a)P<0.05 vs. control. b)P<0.05 vs. vehicle. (B) Expression levels of 94 kDa glucose-regulated protein (GRP94) and 78 kDa glucose-regulated protein (GRP78) were measured by western blot analysis. Antibody of β-actin (43 kDa) as a loading control was used for normalization. (C, D) Intensities of GRP94 and GRP78 protein bands were quantified using the AzureSpot software (n=6 experiments per condition). N.S., not significant.

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