SUSCEPTIBILITY TO KIDNEY DISEASES FROM CHRONIC EXPOSURE TO ARSENIC OR NICOTINE
Chang, Yu Wei
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Kidney diseases and cancer are two of the dominant urologic diseases worldwide. The number of the new cases of kidney diseases and cancers has increased in multiple countries including the United States over the past decade. Environmental contaminants, such as arsenic in drinking water and tobacco smoke (with nicotine as a major component), have been reported to increase the risk for these diseases. However, the mechanistic basis for environmental contaminants-induced adverse effects in kidney is poorly understood. Hence, the main objective of this research was to evaluate the molecular mechanism of arsenic or nicotine-induced chronic kidney disease (CKD) and kidney cancer by using HK-2 normal human kidney epithelial cells as an in vitro model. To simulate chronic exposure, HK-2 cells were maintained in arsenic or nicotine -containing medium for up to six months. The effects of treatments on growth, morphology, and molecular changes, as well as neoplastic transformation in HK-2 cells was periodically monitored. Various methods such as MTT assay, cell cycle analysis, gene expression by qRT-PCR, western blot, immunofluorescence, and soft agar assay were used to analyze the adverse effects. The results revealed a growth inhibition in cells with acute exposure (72 hrs) to arsenic or nicotine, whereas long-term exposure (1 to 3 months) to arsenic or nicotine increased cell proliferation in HK-2 cells. Additionally, the morphological changes related to epithelial to mesenchymal transition (EMT) and cancer stem cell-like spheres were observed in long-term arsenic or nicotine treated cells; these changes persisted during chronic exposure (6 months) with intermittent appearance and disappearance. Moreover, molecular markers revealed that long-term arsenic exposed cells acquired properties, such as increased expression of pro-fibrotic genes and extracellular matrix proteins, similar to kidney fibrosis. Such fibrotic properties were not observed in nicotine-treated cells. In order to evaluate the role of DNA methylation in arsenic-induced fibrotic properties, cells were treated with DNA demethylating agent 5-Aza-2'-Deoxycytidine (5-Aza-2’-dC). Interestingly, 5-Aza-2’-dC reversed arsenic-induced changes in morphology and molecular markers associated with fibrosis. Therefore, the results suggest that these arsenic-induced fibrotic changes were related to alteration in DNA methylation. In 1M nicotine-treated cells, in addition to the EMT, the acquisition of stem cell-like phenotype was also observed. The treatment of demethylating agent 5-Aza-2’-dC and histone deacetylase inhibitor (HDACi) abrogated the formation of stemness suggesting the role of epigenetic alteration in acquisition of stemness. To further understand the role of oxidative stress in nicotine-induced stemness, these cells were also treated with antioxidant N-Acetyl-Cysteine (NAC). The complete abrogation of stemness by NAC treatment suggests the role of oxidative stress in nicotine-induced stemness. Interestingly, the 10M nicotine-exposed cells did not initially acquire stemness. However, lowering the levels of ROS after antioxidant treatment, these cells acquired the stem-like spheres suggesting that lower levels, but not relatively higher levels of oxidative stress is causing stemness. Our data also revealed that NAC reversed the altered expression of epigenetic regulatory genes suggesting the role of oxidative stress in epigenetic modifications during nicotine-induced stem cell phenotype. Finally, the formation of colonies in soft agar revealed that chronic exposure to arsenic or nicotine induced neoplastic transformation in HK-2 cells. The analysis of marker genes and oncogenic pathways suggests that arsenic or nicotine caused neoplastic transformation through alterations in tumor suppressor and/or oncogene as well as the activation of oncogenic pathway(s). In conclusion, the findings of this study revealed for the first time that arsenic can increase the risk of kidney fibrosis, whereas chronic exposure to arsenic or nicotine can lead to kidney cancer development. More importantly, the results support the clinical significance of epigenetic therapeutics and antioxidants in inhibition of arsenic and/or nicotine-induced CKD and kidney cancer.