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The role of hypoxia-inducible factor-1 α in zinc oxide nanoparticle-induced nephrotoxicity in vitro and in vivo

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ABSTRACT

Background: Zinc oxide nanoparticles (ZnO NPs) are used in an increasing number of products, including rubber manufacture, cosmetics, pigments, food additives, medicine, chemical fibers and electronics. However, the molecular mechanisms underlying ZnO NP nephrotoxicity remain unclear. In this study, we evaluated the potential toxicity of ZnO NPs in kidney cells in vitro and in vivo.

Results: We found that ZnO NPs were apparently engulfed by the HEK-293 human embryonic kidney cells and then induced reactive oxygen species (ROS) generation. Furthermore, exposure to ZnO NPs led to a reduction in cell viability and induction of apoptosis and autophagy. Interestingly, the ROS-induced hypoxia-inducible factor-1α (HIF-1α) signaling pathway was significantly increased following ZnO NPs exposure. Additionally, connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1), which are directly regulated by HIF-1 and are involved in the pathogenesis of kidney diseases, displayed significantly increased levels following ZnO NPs exposure in HEK-293 cells. HIF-1α knockdown resulted in significantly decreased levels of autophagy and increased cytotoxicity. Therefore, our results suggest that HIF-1α may have a protective role in adaptation to the toxicity of ZnO NPs in kidney cells. In an animal study, fluorescent ZnO NPs were clearly observed in the liver, lungs, kidneys, spleen and heart. ZnO NPs caused histopathological lesions in the kidney and increase in serum creatinine and blood urea nitrogen (BUN) which indicate possible renal possible damage. Moreover, ZnO NPs enhanced the HIF-1α signaling pathway, apoptosis and autophagy in mouse kidney tissues.

Conclusions: ZnO NPs may cause nephrotoxicity, and the results demonstrate the importance of considering the toxicological hazards of ZnO NP production and application, especially for medicinal use.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0163-3) contains supplementary material, which is available to authorized users.

No MeSH data available.


Measurement of apoptosis in HEK-293 cells treated with ZnO NPs. a Annexin V/PI staining in HEK-293 cells treated with ZnO NPs. The induction of apoptosis and necrosis was determined by flow cytometric analysis of Annexin V and PI staining. b Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry. HEK-293 cells treated with different concentrations of ZnO NPs were assessed using Annexin V/PI staining. Cells were incubated with 0–25 μg/ml ZnO NPs for 24 h. *p < 0.05 versus control. The data are presented as the mean ± standard deviation of three independent experiments. c Western blotting for procaspase-3, cleaved-caspase-3 and Bax in HEK-293 cells. Cells were treated with 0–25 μg/ml ZnO NPs for 24 h
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Fig3: Measurement of apoptosis in HEK-293 cells treated with ZnO NPs. a Annexin V/PI staining in HEK-293 cells treated with ZnO NPs. The induction of apoptosis and necrosis was determined by flow cytometric analysis of Annexin V and PI staining. b Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry. HEK-293 cells treated with different concentrations of ZnO NPs were assessed using Annexin V/PI staining. Cells were incubated with 0–25 μg/ml ZnO NPs for 24 h. *p < 0.05 versus control. The data are presented as the mean ± standard deviation of three independent experiments. c Western blotting for procaspase-3, cleaved-caspase-3 and Bax in HEK-293 cells. Cells were treated with 0–25 μg/ml ZnO NPs for 24 h

Mentions: Apoptosis in HEK-293 cells was measured by flow cytometry following Annexin V and PI staining (Fig. 3a and b). We observed a substantial increase in percentage of apoptotic cells following treatment with ZnO NPs. The expression of apoptosis-related proteins (cleaved-caspase 3 and Bax) was examined by western blotting analysis (Fig. 3c). The cleaved-caspase 3 and Bax protein levels increased following ZnO NP treatment compared with the control. Furthermore, we analyzed the type II programmed cell death, autophagy, which is characterized by the presence of acidic vesicular organelles (AVOs) in the cell cytoplasm. AVO formation was detected and measured by vital staining with acridine orange (AO) [38]. We found a significantly increased amount of AO-positive cells in the ZnO NP treatment group compared to the control (Fig. 4a and b). We detected autophagosomes in ZnO NP-treated cells using a green fluorescence-labeled LC3 immunofluorescence assay (Fig. 4c). The results showed that the number of vacuoles (green dots inside cells) increased conspicuously. Meanwhile, we observed increased expression of the autophagy-related proteins LC3-II, Beclin 1 and p62 following ZnO NP treatment (Fig. 4d). Next, we performed TEM analysis to analyze the ultrastructures of the HEK-293 cells treated with ZnO NPs (Fig. 4e). In the cytoplasm of the cells treated with ZnO NPs, we observed a large number of autophagic vacuoles.Fig. 3


The role of hypoxia-inducible factor-1 α in zinc oxide nanoparticle-induced nephrotoxicity in vitro and in vivo
Measurement of apoptosis in HEK-293 cells treated with ZnO NPs. a Annexin V/PI staining in HEK-293 cells treated with ZnO NPs. The induction of apoptosis and necrosis was determined by flow cytometric analysis of Annexin V and PI staining. b Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry. HEK-293 cells treated with different concentrations of ZnO NPs were assessed using Annexin V/PI staining. Cells were incubated with 0–25 μg/ml ZnO NPs for 24 h. *p < 0.05 versus control. The data are presented as the mean ± standard deviation of three independent experiments. c Western blotting for procaspase-3, cleaved-caspase-3 and Bax in HEK-293 cells. Cells were treated with 0–25 μg/ml ZnO NPs for 24 h
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Fig3: Measurement of apoptosis in HEK-293 cells treated with ZnO NPs. a Annexin V/PI staining in HEK-293 cells treated with ZnO NPs. The induction of apoptosis and necrosis was determined by flow cytometric analysis of Annexin V and PI staining. b Quantification of apoptotic cells with Annexin V-stained cells using flow cytometry. HEK-293 cells treated with different concentrations of ZnO NPs were assessed using Annexin V/PI staining. Cells were incubated with 0–25 μg/ml ZnO NPs for 24 h. *p < 0.05 versus control. The data are presented as the mean ± standard deviation of three independent experiments. c Western blotting for procaspase-3, cleaved-caspase-3 and Bax in HEK-293 cells. Cells were treated with 0–25 μg/ml ZnO NPs for 24 h
Mentions: Apoptosis in HEK-293 cells was measured by flow cytometry following Annexin V and PI staining (Fig. 3a and b). We observed a substantial increase in percentage of apoptotic cells following treatment with ZnO NPs. The expression of apoptosis-related proteins (cleaved-caspase 3 and Bax) was examined by western blotting analysis (Fig. 3c). The cleaved-caspase 3 and Bax protein levels increased following ZnO NP treatment compared with the control. Furthermore, we analyzed the type II programmed cell death, autophagy, which is characterized by the presence of acidic vesicular organelles (AVOs) in the cell cytoplasm. AVO formation was detected and measured by vital staining with acridine orange (AO) [38]. We found a significantly increased amount of AO-positive cells in the ZnO NP treatment group compared to the control (Fig. 4a and b). We detected autophagosomes in ZnO NP-treated cells using a green fluorescence-labeled LC3 immunofluorescence assay (Fig. 4c). The results showed that the number of vacuoles (green dots inside cells) increased conspicuously. Meanwhile, we observed increased expression of the autophagy-related proteins LC3-II, Beclin 1 and p62 following ZnO NP treatment (Fig. 4d). Next, we performed TEM analysis to analyze the ultrastructures of the HEK-293 cells treated with ZnO NPs (Fig. 4e). In the cytoplasm of the cells treated with ZnO NPs, we observed a large number of autophagic vacuoles.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Zinc oxide nanoparticles (ZnO NPs) are used in an increasing number of products, including rubber manufacture, cosmetics, pigments, food additives, medicine, chemical fibers and electronics. However, the molecular mechanisms underlying ZnO NP nephrotoxicity remain unclear. In this study, we evaluated the potential toxicity of ZnO NPs in kidney cells in vitro and in vivo.

Results: We found that ZnO NPs were apparently engulfed by the HEK-293 human embryonic kidney cells and then induced reactive oxygen species (ROS) generation. Furthermore, exposure to ZnO NPs led to a reduction in cell viability and induction of apoptosis and autophagy. Interestingly, the ROS-induced hypoxia-inducible factor-1&alpha; (HIF-1&alpha;) signaling pathway was significantly increased following ZnO NPs exposure. Additionally, connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1), which are directly regulated by HIF-1 and are involved in the pathogenesis of kidney diseases, displayed significantly increased levels following ZnO NPs exposure in HEK-293 cells. HIF-1&alpha; knockdown resulted in significantly decreased levels of autophagy and increased cytotoxicity. Therefore, our results suggest that HIF-1&alpha; may have a protective role in adaptation to the toxicity of ZnO NPs in kidney cells. In an animal study, fluorescent ZnO NPs were clearly observed in the liver, lungs, kidneys, spleen and heart. ZnO NPs caused histopathological lesions in the kidney and increase in serum creatinine and blood urea nitrogen (BUN) which indicate possible renal possible damage. Moreover, ZnO NPs enhanced the HIF-1&alpha; signaling pathway, apoptosis and autophagy in mouse kidney tissues.

Conclusions: ZnO NPs may cause nephrotoxicity, and the results demonstrate the importance of considering the toxicological hazards of ZnO NP production and application, especially for medicinal use.

Electronic supplementary material: The online version of this article (doi:10.1186/s12989-016-0163-3) contains supplementary material, which is available to authorized users.

No MeSH data available.