Identification and dissection of the Nrf2 mediated oxidative stress pathway in human renal proximal tubule toxicity.

ABSTRACT

The identification and dissection of cellular stress mechanisms is fundamental to understanding the susceptibility of the kidney to chemicals and pharmaceuticals and for the development of renal biomarkers indicative of sub lethal injury. Here, we utilised whole genome DNA microarrays in an attempt to uncover molecular mechanisms of response to nephrotoxin exposure. Human renal proximal tubular cells (HK-2) were treated for 12h and 48 h with 5 µM Cadmium (Cd), 30 µM Diquat Dibromide (Diq), and 5 µM Cyclosporine A (CsA). Nephrotoxin treatment resulted in an alteration of a total of 4608 transcripts. Ingenuity Pathways Analysis™ revealed the anti-oxidant and detoxification Nrf2 pathway as the most significantly enriched signaling pathway in the selected dataset. Activation of this transcription factor was confirmed as nuclear translocation and paralleled the temporal alterations of compound induced H(2)O(2) production. Transcriptomics, western blot and immunofluorescence showed an induction of both HO-1 and NQO1 with Cd and Diq exposure, but not with CsA treatment. Knockdown of Nrf2 by siRNA, reduced compound induced NQO1 mRNA to basal levels and attenuated toxin induced HO-1 mRNA expression. siRNA knock down of HO-1, but not NQO1, enhanced Cd induced H(2)O(2) production and Cd induced toxicity. Using an un-biased transcriptomic approach we have identified the Nrf2 pathway as the most significant signaling response in renal epithelial cells challenged with nephrotoxin. This study highlights the importance of this pathway and particularly HO-1 in renal epithelial adaptation to oxidative stress.