Protective effects of epigallocatechin-3-gallate (EGCG) in arsenic-induced fibrogenic changes in human kidney epithelial cells

Epigallocatechin-3-gallate (EGCG) possesses antioxidant, anti-inflammatory, and epigenetic properties, but its renoprotection is yet to be fully explored. Therefore, this study aimed to determine the potential of EGCG to attenuate arsenic-induced acute kidney injury and renal fibrosis.

To investigate the cytotoxic effect of arsenic (NaAsO2) on kidney epithelial cells, caki-1 cells, a human clear cell renal cell carcinoma (ccRCC) line that have epithelial characteristics, was used. To achieve our first objective, caki-1 cells were cultured and treated with varying concentrations of arsenic for 72 hours and subsequently assessed for viability. The level of ROS production and the mRNA expression of oxidative genes were also assessed. Our study indicates that arsenic at 10ng/ml, is cytotoxic to caki-1 cells as seen by growth inhibition in MTT assay. Furthermore, arsenic induced overproduction of ROS in a concentration-dependent manner. Consistent with these findings, the level of mRNA expression of antioxidant genes (MnSOD and GPX1) was significantly increased. Therefore, this study suggests that arsenic causes acute kidney injury in acute exposure.

During long-term arsenic exposure, arsenic induced fibrogenic changes. Caki-1 cells were exposed to 10 ng/ml arsenic and allowed to reach approximately 80% confluency. They were then sub-cultured into a new flask, treated again and the cycle of treatment and subculturing continued for 7 months. These cells were subsequently assessed for viability and ROS production. The level of expression of oxidative and fibrosis marker genes were investigated at transcript level. Results show a significant increase in cell proliferation and production of ROS. mRNA expression of markers for fibrosis and ECM (fibronectin, αSMA and vimentin) were significantly increased whereas the level of E-Cadherin was reduced. There was no significant change in the expression of representative markers for antioxidation, suggesting that the prolonged increase in ROS may have overpowered the cellular antioxidant defense system. We therefore concluded that chronic exposure to arsenic leads to fibrogenic changes via oxidative stress.

To determine the renoprotective effect of EGCG, acute and long-term arsenic exposed cells were treated with EGCG. The cell viability and ROS production was determined. Additionally, the transcript level of epigenetic, fibrotic, and oxidative marker genes were determined by qRT-PCR. EGCG inhibited ROS production and partially restored the growth pattern in both acute and long-term arsenic groups, indicating its protective effects. In the long-term arsenic-exposed cells, fibrogenic and ECM markers (fibronectin, vimentin and α-SMA) were upregulated at the transcript level while E-cadherin was downregulated. However, EGCG significantly downregulated fibronectin, vimentin and α-SMA while upregulating E-cadherin, depicting its antifibrotic effect in these kidney cells via inhibition of epithelial-to-mesenchymal transition (EMT). Moreover, acute arsenic treatment led to the elevation of the mRNA expression of DNMT1, DNMT3a, DNMT3b, MBD4, HAT1, HMT1 and HDAC1. In turn, EGCG downregulated these markers which were previously upregulated by arsenic treatment. In the long-term arsenic exposure, EGCG downregulated DNMT1, MBD4, HMT1, HAT1, and HDAC1 which were previously upregulated by arsenic, while upregulating DNMT3a and DNMT3b which were previously downregulated by arsenic. Overall, the result of this study suggests that EGCG can protect these kidney epithelial cells from arsenic-induces oxidative stress and fibrogenic changes potentially through mechanisms regulated by epigenetic alterations.

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