Nrf2 mediates hypoxia-inducible HIF1α activation in kidney tubular epithelial cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF1α) transcription factors protect against ischemic acute kidney injury (AKI) by upregulating metabolic and cytoprotective gene expression. In this study, we tested the hypothesis that Nrf2 is required for HIF1α-mediated hypoxic responses using Nrf2-sufficient (wild-type) and Nrf2-deficient (Nrf2-/-) primary murine renal/kidney tubular epithelial cells (RTECs) and human immortalized tubular epithelial cells (HK2 cells) with HIF1 inhibition and activation. The HIF1 pathway inhibitor digoxin blocked hypoxia-stimulated HIF1α activation and heme oxygenase (HMOX1) expression in HK2 cells. Hypoxia-mimicking cobalt (II) chloride-stimulated HMOX1 expression was significantly lower in Nrf2-/- RTECs than in wild-type counterparts. Similarly, hypoxia-stimulated HIF1α-dependent metabolic gene expression was markedly impaired in Nrf2-/- RTECs. Nrf2 deficiency impaired hypoxia-induced HIF1α stabilization independent of increased prolyl 4-hydroxylase gene expression. We found decreased HIF1α mRNA levels in Nrf2-/- RTECs under both normoxia and hypoxia-reoxygenation conditions. In silico analysis and chromatin immunoprecipitation assays demonstrated Nrf2 binding to the HIF1α promoter in normoxia, but its binding decreased in hypoxia-exposed HK2 cells. However, Nrf2 binding at the HIF1α promoter was enriched following reoxygenation, demonstrating that Nrf2 maintains constitutive HIF1α expression. Consistent with this result, we found decreased levels of Nrf2 in hypoxia and that were restored following reoxygenation. Inhibition of mitochondrial complex I prevented hypoxia-induced Nrf2 downregulation and also increased basal Nrf2 levels. These results demonstrate a crucial role for Nrf2 in optimal HIF1α activation in hypoxia and that mitochondrial signaling downregulates Nrf2 levels in hypoxia, whereas reoxygenation restores it. Nrf2 and HIF1α interact to provide optimal metabolic and cytoprotective responses in ischemic AKI.

Overactivation of intestinal sterol response element-binding protein 2 promotes diet-induced nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver that may progress to hepatic fibrosis and nonalcoholic steatohepatitis (NASH). Mechanisms underlying NAFLD and NASH are not yet fully understood. Dietary cholesterol was recently shown to be a risk factor for the development of NASH, suggesting a role for intestinal handling of cholesterol. One important regulator of cholesterol homeostasis is the sterol response element-binding protein-2 (SREBP-2) transcription factor. We tested the hypothesis that the overactivation of intestinal SREBP-2 increases the susceptibility to diet-induced NASH. A transgenic mouse model with intestine-specific overexpression of active SREBP-2 (ISR2 mice) driven by villin promoter was used. ISR2 mice and their wild-type littermates were fed a regular chow diet or a high-fat, high-cholesterol (HFHC) diet (15% fat, 1% cholesterol) for 15 wk. Results showed that HFHC feeding to ISR2 mice caused hepatic inflammation with increased levels of proinflammatory cytokines. Histological examination demonstrated extensive fibrosis after a HFHC diet associated with a perivascular as well as pericellular collagen deposits in ISR2 mice compared with wild-type littermates. The severe hepatic inflammation and advanced fibrosis in ISR2 mice was not associated with a difference in lipid accumulation in ISR2 mice compared with wild type littermates after HFHC feeding. These data indicate that overactivation of intestinal SREBP2 promotes diet-induced hepatic inflammation with features of human NASH resulting in rapid severe fibrosis and provide a novel link between regulatory processes of intestinal cholesterol and progression of fatty liver.NEW & NOTEWORTHY The current study highlights the role of overactivation of intestinal SREBP-2 transcription factor in the progression of hepatic fibrosis associated with diet-induced NASH. Mice with intestine-specific overexpression of SREBP-2 demonstrated more inflammation and severe fibrosis in the liver in response to 15 wk of being fed a high-cholesterol, high-fat diet as compared with their wild-type littermates. These data demonstrate a novel link between intestinal regulatory processes of cholesterol metabolism and the pathogenesis of fatty liver diseases.

Myeloid Nrf2 Protects against Neonatal Oxidant-Stress-Induced Lung Inflammation and Alveolar Simplification in Mice.

Bronchopulmonary dysplasia (BPD) is a chronic condition affecting preterm infants, characterized by lung alveolar simplification/hypoalveolarization and vascular remodeling. The nuclear factor erythroid 2 like 2 (Nfe2l2, or Nrf2) plays a critical role in the cytoprotective response to neonatal hyperoxia, and its global deficiency exacerbates hypoalveolarization in mice. The abnormal recruitment and activation of myeloid cells are associated with the pathogenesis of BPD. Therefore, we employed a genetic approach to investigate the role of myeloid Nrf2 in regulating hyperoxia-induced hypoalveolarization. Pups, both wild-type (Nrf2f/f) and those with a myeloid Nrf2 deletion (abbreviated as Nrf2∆/∆mye), were exposed to hyperoxia for 72 h at postnatal day 1 (Pnd1), and then sacrificed at either Pnd4 or Pnd18 following a two-week recovery period. We analyzed the hypoalveolarization, inflammation, and gene expression related to cytoprotective and inflammatory responses in the lungs of these pups. The hypoalveolarization induced by hyperoxia was significantly greater in Nrf2∆/∆mye pups compared to their Nrf2f/f counterparts (35.88% vs. 21.01%, respectively) and was accompanied by increased levels of inflammatory cells and IL-1ß activation in the lungs. Antioxidant gene expression in response to neonatal hyperoxia was lower in Nrf2∆/∆mye pups compared to their Nrf2f/f counterparts. Furthermore, Nrf2-deficient macrophages exposed to hyperoxia exhibited markedly decreased cytoprotective gene expression and increased IL-1ß levels compared to Nrf2-sufficient cells. Our findings demonstrate the crucial role of myeloid Nrf2 in mitigating hyperoxia-induced lung hypoalveolarization and inflammatory responses in neonatal mice.

T Cell Nrf2/Keap1 Gene Editing Using CRISPR/Cas9 and Experimental Kidney Ischemia-Reperfusion Injury.

Aims: T cells play pathophysiologic roles in kidney ischemia-reperfusion injury (IRI), and the nuclear factor erythroid 2-related factor 2/kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway regulates T cell responses. We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated Keap1-knockout (KO) augments Nrf2 antioxidant potential of CD4+ T cells, and that Keap1-KO CD4+ T cell immunotherapy protects from kidney IRI. Results: CD4+ T cell Keap1-KO resulted in significant increase of Nrf2 target genes NAD(P)H quinone dehydrogenase 1, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, and glutamate-cysteine ligase modifier subunit. Keap1-KO cells displayed no signs of exhaustion, and had significantly lower levels of interleukin 2 (IL2) and IL6 in normoxic conditions, but increased interferon gamma in hypoxic conditions in vitro. In vivo, adoptive transfer of Keap1-KO CD4+ T cells before IRI improved kidney function in T cell-deficient nu/nu mice compared with mice receiving unedited control CD4+ T cells. Keap1-KO CD4+ T cells isolated from recipient kidneys 24 h post IR were less activated compared with unedited CD4+ T cells, isolated from control kidneys. Innovation: Editing Nrf2/Keap1 pathway in murine T cells using CRISPR/Cas9 is an innovative and promising immunotherapy approach for kidney IRI and possibly other solid organ IRI. Conclusion: CRISPR/Cas9-mediated Keap1-KO increased Nrf2-regulated antioxidant gene expression in murine CD4+ T cells, modified responses to in vitro hypoxia and in vivo kidney IRI. Gene editing targeting the Nrf2/Keap1 pathway in T cells is a promising approach for immune-mediated kidney diseases.

Effect of time-restricted eating on sex hormone levels in premenopausal and postmenopausal females.

OBJECTIVE: Concerns have been raised regarding the impact of time-restricted eating (TRE) on sex hormones in females. This study examined how TRE affects sex steroids in premenopausal and postmenopausal females. METHODS: This is a secondary analysis of an 8-week TRE study (4- to 6-hour eating window) conducted in adults with obesity. Men and perimenopausal females were excluded. Females were classified into two groups based on menstrual status: premenopausal (n = 12) or postmenopausal (n = 11). RESULTS: After 8 weeks, body weight decreased in premenopausal females (-3% ± 2%) and postmenopausal females (-4% ± 2%) (main effect of time, p < 0.001), with no difference between groups (no group × time interaction). Circulating levels of testosterone, androstenedione, and sex hormone binding globulin (SHBG) did not change in either group (no group × time interaction). Dehydroepiandrosterone (DHEA) concentrations decreased (p < 0.05) in premenopausal (-14% ± 32%) and postmenopausal females (-13% ± 34%; main effect of time, p = 0.03), with no difference between groups. Estradiol, estrone, and progesterone were measured only in postmenopausal females, and they remained unchanged. CONCLUSIONS: In premenopausal females, androgens and SHBG remained unchanged during TRE, whereas DHEA decreased. In postmenopausal females, estrogens, progesterone, androgens, and SHBG did not change, but DHEA was reduced.

Nrf2 Regulates Anti-Inflammatory A20 Deubiquitinase Induction by LPS in Macrophages in Contextual Manner.

The aberrant regulation of inflammatory gene transcription following oxidant and inflammatory stimuli can culminate in unchecked systemic inflammation leading to organ dysfunction. The Nrf2 transcription factor dampens cellular stress and controls inflammation by upregulating antioxidant gene expression and TNFα-induced Protein 3 (TNFAIP3, aka A20) deubiquitinase by controlling NF-kB signaling dampens tissue inflammation. Here, we report that Nrf2 is required for A20 induction by inflammatory stimuli LPS in monocyte/bone marrow derived macrophages (MDMΦs) but not in lung-macrophages (LDMΦs). LPS-induced A20 expression was significantly lower in Nrf2-/- MDMΦs and was not restored by antioxidant supplementation. Nrf2 deficiency markedly impaired LPS-stimulated A20 mRNA expression Nrf2-/- MDMΦs and ChIP assays showed Nrf2 enrichment at the promoter Nrf2-/- MDMΦs upon LPS stimulation, demonstrating that Nrf2 directly regulates A20 expression. Contrary to MDMΦs, LPS-stimulated A20 expression was not largely impaired in Nrf2-/- LDMΦs ex vivo and in vivo and ChIP assays showed lack of increased Nrf2 binding at the A20 promoter in LDMΦ following LPS treatment. Collectively, these results demonstrate a crucial role for Nrf2 in optimal A20 transcriptional induction in macrophages by endotoxin, and this regulation occurs in a contextual manner.

Preconditioning the immature lung with enhanced Nrf2 activity protects against oxidant-induced hypoalveolarization in mice.

Bronchopulmonary dysplasia (BPD) is a chronic disease of preterm babies with poor clinical outcomes. Nrf2 transcription factor is crucial for cytoprotective response, whereas Keap1-an endogenous inhibitor of Nrf2 signaling-dampens these protective responses. Nrf2-sufficient (wild type) newborn mice exposed to hyperoxia develop hypoalveolarization, which phenocopies human BPD, and Nrf2 deficiency worsens it. In this study, we used PND1 pups bearing bearing hypomorphic Keap1 floxed alleles (Keap1f/f) with increased levels of Nrf2 to test the hypothesis that constitutive levels of Nrf2 in the premature lung are insufficient to mitigate hyperoxia-induced hypoalveolarization. Both wildtype and Keap1f/f pups at PND1 were exposed to hyperoxia for 72 h and then allowed to recover at room air for two weeks (at PND18), sacrificed, and lung hypoalveolarization and inflammation assessed. Hyperoxia-induced lung hypoalveolarization was remarkably lower in Keap1f/f pups than in wildtype counterparts (28.9% vs 2.4%, wildtype vs Keap1f/f). Likewise, Keap1f/f pups were protected against prolonged (96 h) hyperoxia-induced hypoalveolarization. However, there were no differences in hyperoxia-induced lung inflammatory response immediately after exposure or at PND18. Lack of hypoalveolarization in Keap1f/f pups was accompanied by increased levels of expression of antioxidant genes and GSH as assessed immediately following hyperoxia. Keap1 knockdown resulted in upregulation of lung cell proliferation postnatally but had opposing effects following hyperoxia. Collectively, our study demonstrates that augmenting endogenous Nrf2 activation by targeting Keap1 may provide a physiological way to prevent hypoalveolarization associated with prematurity.

Isoquinoline Kelch-like ECH-Associated Protein 1-Nuclear Factor (Erythroid-Derived 2)-like 2 (KEAP1-NRF2) Inhibitors with High Metabolic Stability.

Pharmacological activation of NRF2 (nuclear factor erythroid 2-related factor 2) arises from blocking the interaction of NRF2 with its negative regulator, KEAP1 (Kelch-like ECH-associated protein 1). We previously reported an isoquinoline-based NRF2 activator, but this compound showed negative logD7.4 and a -2 charge at physiological pH, which may have limited its membrane permeability. In this work, we report potent, metabolically stable analogs that result from replacing a carboxymethyl group at the 4-position with a fluoroalkyl group.