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

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-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.


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Cellular uptake and cytotoxicity of ZnO NPs by HEK-293 cells. a TEM analysis of ZnO NP morphology. ZnO NPs were mainly spherical in shape. The scale bar represents 100 nm. b Cell viability was measured using the MTS assay. ZnO NPs decreased the viability of HEK-293 cells in a concentration-dependent manner. The cells were treated with 0, 15, 20, 25, 30 or 35 μg/ml ZnO NPs for 24 h. All of the MTS values were normalized to the control values (no particle exposure), which were regarded as 100 % cell viability. *p < 0.05 versus control. c Uptake of ZnO NPs detected by fluorescence confocal microscopy. ZnO NPs are shown in red and DAPI (blue) is a nuclei-specific marker. The cells were treated with 20 μg/ml ZnO NPs for 8 h. d, The results of the SSC data and flow cytometry demonstrated that ZnO NPs were apparently engulfed by HEK-293 cells. e Quantification of the scatter intensity in HEK-293 cells with ZnO NP treatment. The cells were treated with ZnO NPs at 0, 15, 20, or 25 μg/ml for 24 h. The data are presented as the mean ± standard deviation of three independent experiments
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Fig1: Cellular uptake and cytotoxicity of ZnO NPs by HEK-293 cells. a TEM analysis of ZnO NP morphology. ZnO NPs were mainly spherical in shape. The scale bar represents 100 nm. b Cell viability was measured using the MTS assay. ZnO NPs decreased the viability of HEK-293 cells in a concentration-dependent manner. The cells were treated with 0, 15, 20, 25, 30 or 35 μg/ml ZnO NPs for 24 h. All of the MTS values were normalized to the control values (no particle exposure), which were regarded as 100 % cell viability. *p < 0.05 versus control. c Uptake of ZnO NPs detected by fluorescence confocal microscopy. ZnO NPs are shown in red and DAPI (blue) is a nuclei-specific marker. The cells were treated with 20 μg/ml ZnO NPs for 8 h. d, The results of the SSC data and flow cytometry demonstrated that ZnO NPs were apparently engulfed by HEK-293 cells. e Quantification of the scatter intensity in HEK-293 cells with ZnO NP treatment. The cells were treated with ZnO NPs at 0, 15, 20, or 25 μg/ml for 24 h. The data are presented as the mean ± standard deviation of three independent experiments

Mentions: To assess the physical characteristics of ZnO NPs, several parameters were measured. The detailed results of the physical characterization after suspension in cell culture medium or water are presented in Table 1. ZnO NPs had a hydrodynamic diameter of 47.8 nm in water and 70.5 nm in MEM. The zeta potential of ZnO NPs in water was positive (+36.7), which ZnO NPs in MEM had negative charges (−8.09). As previously reported, all of the NPs have negatively charged surfaces in cell culture medium due to the formation of a corona of negatively charged proteins [33]. The polydispersity index (PDI) indicates the dispersion stability and solubility of NPs in water or medium, and a PDI value lower than 0.2 is associated with a high homogeneity of the particle population [34]. The values for ZnO NPs in water and MEM were 0.128 and 0.230, respectively. Furthermore, dissolution of NPs is an important property that influences their mode of action (e.g. antimicrobial properties, toxicity, medicinal applications and environmental impact) [35]. In our case, the release of zinc ions after 24 h of incubation was low, <1 % of the initial concentrations for ZnO NPs in water and MEM. In addition, the TEM image suggests that the particles are polydispersed and are mostly spherical in shape (Fig. 1a).Table 1


The role of hypoxia-inducible factor-1 α in zinc oxide nanoparticle-induced nephrotoxicity in vitro and in vivo
Cellular uptake and cytotoxicity of ZnO NPs by HEK-293 cells. a TEM analysis of ZnO NP morphology. ZnO NPs were mainly spherical in shape. The scale bar represents 100 nm. b Cell viability was measured using the MTS assay. ZnO NPs decreased the viability of HEK-293 cells in a concentration-dependent manner. The cells were treated with 0, 15, 20, 25, 30 or 35 μg/ml ZnO NPs for 24 h. All of the MTS values were normalized to the control values (no particle exposure), which were regarded as 100 % cell viability. *p < 0.05 versus control. c Uptake of ZnO NPs detected by fluorescence confocal microscopy. ZnO NPs are shown in red and DAPI (blue) is a nuclei-specific marker. The cells were treated with 20 μg/ml ZnO NPs for 8 h. d, The results of the SSC data and flow cytometry demonstrated that ZnO NPs were apparently engulfed by HEK-293 cells. e Quantification of the scatter intensity in HEK-293 cells with ZnO NP treatment. The cells were treated with ZnO NPs at 0, 15, 20, or 25 μg/ml for 24 h. The data are presented as the mean ± standard deviation of three independent experiments
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Related In: Results  -  Collection

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Fig1: Cellular uptake and cytotoxicity of ZnO NPs by HEK-293 cells. a TEM analysis of ZnO NP morphology. ZnO NPs were mainly spherical in shape. The scale bar represents 100 nm. b Cell viability was measured using the MTS assay. ZnO NPs decreased the viability of HEK-293 cells in a concentration-dependent manner. The cells were treated with 0, 15, 20, 25, 30 or 35 μg/ml ZnO NPs for 24 h. All of the MTS values were normalized to the control values (no particle exposure), which were regarded as 100 % cell viability. *p < 0.05 versus control. c Uptake of ZnO NPs detected by fluorescence confocal microscopy. ZnO NPs are shown in red and DAPI (blue) is a nuclei-specific marker. The cells were treated with 20 μg/ml ZnO NPs for 8 h. d, The results of the SSC data and flow cytometry demonstrated that ZnO NPs were apparently engulfed by HEK-293 cells. e Quantification of the scatter intensity in HEK-293 cells with ZnO NP treatment. The cells were treated with ZnO NPs at 0, 15, 20, or 25 μg/ml for 24 h. The data are presented as the mean ± standard deviation of three independent experiments
Mentions: To assess the physical characteristics of ZnO NPs, several parameters were measured. The detailed results of the physical characterization after suspension in cell culture medium or water are presented in Table 1. ZnO NPs had a hydrodynamic diameter of 47.8 nm in water and 70.5 nm in MEM. The zeta potential of ZnO NPs in water was positive (+36.7), which ZnO NPs in MEM had negative charges (−8.09). As previously reported, all of the NPs have negatively charged surfaces in cell culture medium due to the formation of a corona of negatively charged proteins [33]. The polydispersity index (PDI) indicates the dispersion stability and solubility of NPs in water or medium, and a PDI value lower than 0.2 is associated with a high homogeneity of the particle population [34]. The values for ZnO NPs in water and MEM were 0.128 and 0.230, respectively. Furthermore, dissolution of NPs is an important property that influences their mode of action (e.g. antimicrobial properties, toxicity, medicinal applications and environmental impact) [35]. In our case, the release of zinc ions after 24 h of incubation was low, <1 % of the initial concentrations for ZnO NPs in water and MEM. In addition, the TEM image suggests that the particles are polydispersed and are mostly spherical in shape (Fig. 1a).Table 1

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.


Related in: MedlinePlus