ASPECTOS BIÓTICOS.

Evidence suggesting that Nrf2 plays a role in regulating several metabolic processes in organs including the liver, kidney and brain is mounting (figure 1.10) (Copple, 2012). Dysregulation of the Nrf2 pathway has also been implicated in the pathogenesis of diverse diseases such as neurodegeneration, cancer and chronic kidney diseases (Zhao et al., 2011b; Copple, 2012). Transgenic Nrf2 null mice have been shown to experience enhanced susceptibility to various drug-induced toxicities including those associated with acetaminophen (Chan et al., 2001; Enomoto et al., 2001), carbon tetrachloride (Xu et al., 2008) and butylated hydroxyl toluene (Chan et al., 1999). Notably, a recent study saw the opposite in which the intrahepatic shunt present in the Nrf2 KO mice may affect acetaminophen metabolism and hence reduce the sensitivity of these animals to the toxicological effects of acetaminophen (Skoko et al., 2014). These observations suggest Nrf2 as a potential therapeutic target in a variety of pathological contexts. This will be further discussed in the following sub sections.

Figure 1.11 – Role of Nrf2 in protection against disease. Selected examples of implications of Nrf2 in diseases and its therapeutic significance in various organs

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1.5.1 Liver:

Drug-induced liver toxicities are a major cause of liver failure and a key factor in drug attrition (Ostapowicz et al., 2002; Park et al., 2011). Early studies noted that Nrf2 KO mice were more susceptible to acetaminophen-induced liver injury (Chan

et al., 2001; Enomoto et al., 2001). Conversely, Keap1 KO mice show a high resistance to the drug’s toxicity (Okawa et al., 2006). Studies have shown the induction of Nrf2 and regulated genes by acetaminophen and its reactive metabolite within the non-toxic and toxic dose ranges (Goldring et al., 2004; Copple

et al., 2008a). Other compounds have also been associated with enhanced hepatotoxicity in Nrf2 KO animals, such as pentachlorophenol, carbon tetrachloride, pyrazole and arsenic (Umemura et al., 2006; Lu et al., 2008; Xu et al., 2008; Jiang et al., 2009).

Pharmacological activation of Nrf2 using the potent inducer CDDO-Im has been shown to attenuate the liver injury provoked by acetaminophen in Nrf2 WT mice (Reisman et al., 2009). In Nrf2 KO mice, the effect of CDDO-Im was lost, highlighting the involvement of Nrf2 in CDDO-Im induced protection against acetaminophen liver injury. Further to this, a similar effect was observed when sauchinone, an antioxidant lignan and inducer of Nrf2, was used to pre-treat mice prior to administration of toxic doses of APAP (Kay et al., 2011). Other small molecule inducers of Nrf2 such as oltipraz and BHA (butylated hydroxyanisole) have been shown to protect against the oxidative and electrophilic stress associated with various drug-induced liver injuries (for a review see (Klaassen et al., 2010). These findings together suggest the possibility of using Nrf2-inducing agents as an adjuvant/co-treatment to prevent drug toxicities in the liver.

1.5.2 Kidney:

Kidney toxicity is a major concern in the development of new therapeutic drugs. Therefore, an understanding of the mechanisms that underlie kidney injury will inform the development of safe and effect drug candidates. Aged female Nrf2 KO mice develop lupus-like autoimmune nephritis, suggesting that Nrf2 regulates

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homeostasis in the aging kidney (Yoh et al., 2001). In addition, Nrf2 KO mice exposed to streptozotocin show enhanced susceptibility to hyperglycemia-induced diabetic nephropathy (Yoh et al., 2008; Jiang et al., 2010b). Elucidating the role of Nrf2 in the kidney could prove useful in the management of acute kidney injury occurring as a result of disease or drug administration. The role of Nrf2 in conferring protection against kidney injury has been highlighted in animal models of renal ischaemic-reperfusion (Yoon et al., 2008; Wu et al., 2011b) and also in models in which nephrotoxicity is induced by heavy metals (Chen et al., 2009a; Molina-Jijon et al., 2011; Prabu et al., 2012), cyclosporine A (Louhelainen et al., 2006) and ochratoxin A (Cavin et al., 2007; Cavin et al., 2009). The pre-induction of Nrf2 in these models ameliorates the deleterious effects of the nephrotoxins for example, treatment of WT mice with CDDO-Im has been shown to reduce the nephrotoxic effects of cisplatin (Aleksunes et al., 2010). Furthermore, a phase II trial of CDDO- Me for the treatment of patients with lymphomas noted an enhanced glomerular filtration rate and reduced levels of creatinine and blood urea nitrogen in the CDDO-Me treatment group indicative of an improvement of renal function (Jiang et al., 2010b). These studies together highlight an important role for Nrf2 in the kidney and identify the transcription factor as a promising target for treating a number of renal diseases. For a review, see (Shelton et al., 2013)

1.5.3 Brain:

Mao et al. and Jin et al. reported several nervous system dysfunction phenotypes such as neuronal apoptosis; tissue swelling in Nrf2 KO mice (Jin et al., 2009; Mao et al., 2011). Using Nrf2 activators CDDO-ethyl amide (CDDO-EA) and CDDO- trifluoroethyl amide (CDDO-TFEA), the dysfunctional effect was found to be alleviated in Nrf2 WT mice, but not in Nrf2 KO mice, indicating that the effect is attributable to the induction of Nrf2 and its target genes. This highlights the important role of Nrf2 in the brain and nervous system. In support of this, CDDO-Me has been shown to confer protection against Huntington’s disease in a transgenic mouse, improving the behavioural phenotype and brain pathology (Stack et al.,

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2010). In addition, Cullinan et al., 2003 highlighted the role of Nrf2 in protecting cells against the deleterious effects associated with misfolded proteins as a result of stress induced in the endoplasmic reticulum (Cullinan et al., 2003). This is of importance as a number of disorders in the nervous system are associated with misfolded protein. In Parkinson’s disease models, Nrf2 has been shown to confer cytoprotection against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)(Chen

et al., 2009b; Jazwa et al., 2011). Induction of the Nrf2 pathway (either by over expressing Nrf2, silencing Keap1 or using an inducer of Nrf2 such as CDDO-Me) inhibited the progression of disease in Parkinson’s (Barone et al., 2011) and Alzheimer’s disease models (von Otter et al., 2010), suggesting the modulation of the Nrf2 pathway as a novel therapeutic strategy. For a review, see (Zhang et al., 2013).

1.5.4 Lipid Metabolism:

Recently, studies have highlighted a role for Nrf2 in regulating lipid metabolism. The proteomic profiling of Nrf2 KO mice revealed a significant upregulation of fatty-acid synthetic pathway proteins highlighting a role for Nrf2 in the regulation of fatty acid synthesis in the liver (Kitteringham et al., 2010). In keeping with this, Nrf2 KO mice fed a high fat diet show an elevated total hepatic lipids and polyunsaturated fats (Shin et al., 2009). In addition, overexpression of Nrf2 in Keap1 KO mice or the treatment of the WT mice with inducers of Nrf2 (CDDO-Im or oltipraz) depleted lipid-synthesis genes (Shin et al., 2009; Chowdhry et al., 2010). Although these findings illustrate a negative control of lipid synthesis by Nrf2, there are conflicting reports showing the opposite. For a review, see (Schneider et al., 2013). Nonetheless, these findings highlight Nrf2 as a novel therapeutic target in lipid- related disorders, such as obesity and metabolic syndrome (Yu et al., 2011; Schneider et al., 2013).

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