Oxidative Stress and Metabolism: The NF–Erythroid 2 p45–Related Factor 2:Kelch–like ECH–Associated Protein 1 System and Regulatory T Lymphocytes in Ischemic AKI

Ischemia/reperfusion causes AKI by exposing renal cells to oxidative stress and depriving them of nutrients. One part of the complex response to such stresses is activation of NF–erythroid 2 p45–related factor 2 (Nrf2).¹ Nrf2 is a member of the cap’n’collar family of genes, so-called because the prototypic members regulate embryonic development of the labral (cap) and mandibular (collar) segments of Drosophila; Nrf2 is the most studied of this family because of its importance in cancer, inflammatory and metabolic diseases, ischemia/reperfusion, drug metabolism, and aging.^2–4 It is conserved in mammals, fowl, worms, and insects. In this issue of JASN, Noel et al.⁵ report the use of genetic tools to specifically augment Nrf2 activity in CD4 lymphocytes. This resulted in increased activity of regulatory T cells (Tregs),⁶ decreased maladaptive inflammation, and decreased injury during ischemic AKI.

The goal of this editorial is to place these important findings in the context of our growing understanding that renal leukocytes (and other cells) may be regulated by oxidative stress and cellular metabolism. This regulation may be mediated by Nrf2 and other regulatory systems. In this regard, there are two fundamental considerations: first, oxidative stress triggers redox switches, and second, Nrf2 is now recognized as a regulator of cellular metabolism.

Oxidative stress is produced by mitochondria, the NOX family of enzymes, and other cellular processes during ischemic AKI and has two major effects.⁷ (1) Effects of free radicals: some work^8–11 has focused on the catastrophic free radical effects of reactive oxygen molecules, such as superoxide. They kill cells by irreversibly oxidizing and damaging macromolecules. (2) Effects of nonradical oxidants: the above free radicals are rapidly converted to nonradical oxidants, such as hydrogen peroxide. They do not themselves impair cell viability but reversibly oxidize cysteine, arginine, histidine, and other amino acids to change protein conformation and thus, function. In other words, these post-translational modifications of proteins are “reversible redox-regulated switches” that control protein functions in living cells.^7,12 These nonradical oxidants may be the major pathway of oxidative stress.

One of the best understood of these reversible switches is Kelch–like ECH–associated protein 1 (KEAP1; also called inhibitor of Nrf2). KEAP1 is important for this editorial, because it provides a mechanism by which oxidative stress, produced during ischemic AKI, would regulate Nrf2. KEAP1 targets Nrf2 for ubiquitylation and then, proteosomal degradation.² In response to such oxidative stress, critical cysteines of KEAP1 are oxidized, and KEAP1 no longer targets Nrf2 for degradation. Noel et al.⁵ increased Nrf2 activity in CD4 lymphocytes by conditionally knocking out KEAP1 in these cells.

In addition to its effect on Nrf2, KEAP1 has additional complex biology. It also contributes to activation of NF-κB and thus, survival and proinflammatory genes as well as bcl2 and thus, apoptosis.¹³ In addition, KEAP1 may bind the cytoskeleton; this binding suggests that it may be regulated by or regulate cell shape. Furthermore, KEAP1 may bind to the PGAM5 on the mitochondrial outer membrane. It may, thus, better respond to reactive oxygen species produced by mitochondria or regulate the mitochondrial contribution to intermediary metabolism.²

Although the best understood step in Nrf2 regulation is its interaction with KEAP1, Nrf2 also receives information from tyrosine kinases, mammalian target of rapamycin (mTOR), and other signaling systems that connect it to cellular metabolism and extracellular growth factors and cytokines; altogether, these signals determine which, if any, of the over 200 Nrf2–regulated genes are transcribed in particular cells under specific circumstances.^2,3 In addition to KEAP1, ubiquitylation and proteosomal degradation of Nrf2 are also regulated by β–transducin repeat–containing protein in conjunction with glycogen synthase kinase.¹⁴

The best understood genes activated by Nrf2 are those that decrease the original oxidative stress that triggered the KEAP1 redox switch. These genes code for antioxidant systems, such as glutathione peroxidase, thioredoxin, NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, etc. Such molecules would set any protein redox switches in a reduced (e.g., thiol or R–SH) position. This would be one mechanism by which Nrf2 would activate Tregs, and it would be consistent with the known increased reducing power seen in these lymphocytes.^15,16 It would also be consistent with the observations by Noel et al.⁵ and other observations that Nrf2 activation decreased autoimmunity (possibly by increasing Treg activity) and the ability of changes in the intracellular redox potential to regulate T cells.¹⁷ However, an additional mechanism is suggested by the recent literature and discussed below.

Recent work reveals that Nrf2 also regulates cellular metabolism.^2,18 Not only does this have implications for diabetes mellitus and metabolic syndrome, but it suggests another way for Nrf2 to regulate immunity. During an immune response, such as occurs during ischemic AKI, CD4 lymphocytes must profoundly change their metabolism, because they are transformed from resting cells with minimal energy needs to activated cells with high energy needs to support rapid proliferation and the production of large amounts of cytokines. In meeting these needs, aggressive CD8, Th1, Th2, and Th17 lymphocytes and natural Tregs (Treg− CD4+ and CD25+ Foxp3+) have different metabolic signatures. In contrast to the former, Tregs have high rates of lipid oxidation, low levels of the Glut1 transporter that facilitates entry of glucose into these cells, and low rates of fatty acid synthesis. A working hypothesis is that these distinct metabolic pathways not only supply energy but also, dictate the function of T cells. In other words, therapy to force T cells down one metabolic pathway or another may direct T lymphocyte differentiation—for example, increase effector T cells in cancer or increase Tregs in transplantation or autoimmune disease. Activation of AMP kinase by metformin inhibits mTOR, increases mitochondrial oxidative pathways, including fatty acid oxidation, and increases Tregs; conversely, decreasing fatty acid oxidation with Etomoxir decreased Treg generation.^19,20 Insofar as Nrf2 activation increases fatty acid oxidation,² it may also increase Tregs through this mechanism.

In addition, Nrf2 also regulates metabolism independently and as part of the mTOR metabolic response to nutrient deprivation by lymphocytes and other cells.² mTORC1 phosphorylates p62/SQSTM1 and increases Nrf2 activity by decreasing its binding to KEAP1.²¹ Under conditions of sufficient nutrients, mTOR coordinates the increased metabolism that supports proliferation and differentiation of effector T lymphocytes and other cells. Under conditions of nutrient deprivation, mTOR is inhibited, and in some model systems, Tregs instead of effector T cells are activated. Such activation of Tregs by nutrient deprivation is analogous to the effect of rapamycin and other mTOR inhibitors that are used to prevent transplant rejection.^22–25 In a model of tolerated skin transplants, nutrient levels in the transplant are low; this inhibits mTOR, and Treg activity is increased.²⁶ Nutrient deprivation of local lymphocytes may also inhibit mTOR and increase Tregs in the vicinity of cancers. In this case, the cancer deprives the local microenvironment of nutrients. A novel approach to cancer therapy is to change the metabolism of cancers, so that the nutrient microenvironment favors T effectors rather than Tregs.^27,28 Consistent with the above hypothesis is the observation that targeted mutation of mTOR, which is common to both the mTORC1 and mTORC2 pathways, directs T lymphocytes toward a Treg phenotype.²⁵ However, the effects of individually mutating specific components of either the mTORC1 and/or mTORC2 pathways are complex.²⁹

To conclude, Noel et al.⁵ showed that conditional targeted mutation of KEAP1, the inhibitor of Nrf2, in CD4 lymphocytes decreased inflammation and injury during ischemic AKI. Increased Nrf2 increased Tregs in the injured kidney. If we assume that there has been no fundamental change in T cell embryonic development caused by this mutation, then these observations illustrate an important point: oxidative stress, which occurs during ischemic AKI, may regulate cells by triggering redox switches. The best understood such redox switch is KEAP1, which has a function that is changed by oxidation of critical cysteines; the ability of KEAP1 to target Nrf2 for proteolysis is abrogated by oxidation of these cysteines. Differentiation of T cells into effector versus regulatory pathways may be dictated by the intracellular redox potential, and this may be set to a large degree by Nrf2-regulated genes. Furthermore, Nrf2 in concert with mTOR and AMP kinase may determine which metabolic pathways are executed when T lymphocytes are activated. These specific metabolic pathways may dictate the differentiation pathways taken by these lymphocytes (Figure 1).

Figure 1.

Regulation of T lymphocytes by oxidative stress and metabolism. AMPK, AMP kinase.

Disclosures

None.