title: The physiological, pharmacological and toxicological roles of Nrf2 in the kidney
Creators: name: Shelton, Luke
Publisher: name: University of Liverpool Repository
Year: 2015

description: Nrf2 is a transcription factor that, under conditions of chemical stress, is able to evade its cytosolic repression and translocate to the nucleus to initiate the transcription of a battery of cytoprotective genes, such as those involved in the detoxication of xenobiotics. Nrf2 has previously been shown to afford protection against chronic and acute renal injury, yet, relatively little is known about the mechanism by which Nrf2 affords this protection, and the extent of its transcriptional roles in the kidney. This thesis seeks to further our understanding of the physiological, pharmacological and toxicological roles of Nrf2 in the kidney. Using an iTRAQ-based proteomic approach to quantify protein expression levels in the kidneys of Nrf2+/+ and Nrf2-/- mice, acutely treated with vehicle or the potent Nrf2 inducer CDDO-Me (3 mg/kg), we demonstrated that 189 proteins were differentially expressed in the Nrf2-/- mouse kidney, compared to Nrf2+/+, and 42 proteins were differentially expressed in the CDDO-Me treated Nrf2+/+ mouse kidney, compared to vehicle. The key finding was that the kidneys of Nrf2-/- mice are deficient in proteins that mediate cellular redox balance, the metabolism of a range of xenobiotics, and the regulation of core metabolic processes, including energy metabolism and the synthesis and recycling of amino acids. Functional demonstration of a reduction in energy metabolism was demonstrated by assessing total NADPH and GSH, of which Nrf2-/- mouse kidneys had 35% and 30% less than their Nrf2+/+ counterparts, respectively. A single acute dose of CDDO-Me failed to augment the expression of proteins, other than Nqo1, that were shown to be regulated by Nrf2 at the basal level in the mouse kidney, however qPCR analysis of these kidneys revealed that CDDO-Me has an effect at the transcriptional level which has not fully translated within the timeframe of this study. In summary, we have provided evidence that Nrf2 regulates the expression of an array of proteins that contribute to cell defence and the maintenance of homeostasis in the kidney, supporting current interest in Nrf2 as a novel therapeutic target in a number of renal diseases. MicroRNAs are a recently discovered RNA-regulatory element that show promise in their use as biomarkers of physiological and pathological events. In order to provide insight into the microRNAs under Nrf2 control in the kidney, we performed an unbiased microRNA array analysis on kidney homogenates from Nrf2+/+ and Nrf2-/- mice, treated with vehicle or CDDO-Me, and then validated several promising microRNA candidates using targeted qPCR analysis. Of particular note are miR-466h-3p, the expression of which was significantly increased in the CDDO-Me treated Nrf2+/+ mouse kidney and decreased in the Nrf2-/- mouse kidney, compared to their respective controls, and miR-28c and 144, which were both significantly decreased in the CDDO-Me treated Nrf2+/+ mouse kidney, and increased in the Nrf2-/- mouse kidney. This novel analysis represents the first step in characterising the renal Nrf2 microRNA-ome, which could reveal novel mechanisms of Nrf2 function and markers of its activity that could translate to the clinic. Recent interest in the use of CDDO-Me as a therapeutic intervention for late-stage chronic kidney disease has culminated in a phase III clinical trial (BEACON), which was subsequently terminated due to unforeseen adverse cardiac events, of which the cause has yet to be identified. In order to determine whether the drive to produce more potent Nrf2 inducers has inadvertently led to the generation of inherently more toxic compounds, the relationship between potency towards Nrf2 and toxicity was evaluated for CDDO-Me and related triterpenoids, and other classes of Nrf2 inducer. Using a rat H4IIE-ARE8L luciferase reporter cell line to determine in vitro therapeutic indices, it was discovered that within the compounds tested an increase in potency toward Nrf2 of four magnitudes results was associated with an increase in toxicity of only two magnitudes, resulting in a relative increase in in vitro safety. This data indicates that it is possible to generate potent Nrf2-inducers that are not inherently toxic, and suggests that therapeutic targeting of Nrf2 continues to hold promise as a novel treatment for a range of diseases. In summary, by using a proteomic approach we have identified an array of renal Nrf2-regulated proteins that contribute to various cytoprotective and metabolic processes in the kidney, supporting current interest in the therapeutic targeting of Nrf2 as treatment for renal disease. Additionally, the microRNAs under Nrf2 regulation in the kidney have also been identified, and represent the first step in fully characterising the Nrf2 microRNA-ome. Finally, it was shown that the drive to produce more potent Nrf2 inducers has not led to the generation of inherently more toxic compounds; indeed an increase in potency is associated with a relative increase in in vitro safety, suggesting that the targeting of Nrf2 is still a promising therapeutic route. Overall, the work presented in this thesis has furthered our understanding of the physiological, pharmacological and toxicological roles of Nrf2 in the kidney.