Multiorgan Autoimmune Inflammation, Enhanced Lymphoproliferation, and Impaired Homeostasis of Reactive Oxygen Species in Mice Lacking the Antioxidant-Activated Transcription Factor Nrf2
2006; Elsevier BV; Volume: 168; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.051113
ISSN1525-2191
AutoresQiang Ma, Lori Battelli, Ann F. Hubbs,
Tópico(s)Circadian rhythm and melatonin
ResumoNuclear factor erythroid 2-related factor 2 (Nrf2) is an antioxidant-activated cap “n” collar basic leucine zipper transcription factor. To assess the function of Nrf2 in the antioxidant response, we examined mice with targeted disruption of the Nrf2 gene. Nrf2-null mice developed complex disease manifestations, with a majority exhibiting a lupus-like autoimmune syndrome characterized by multiorgan inflammatory lesions with a marked female predominance, appearance of anti-double-stranded DNA antibodies in young adulthood, intravascular deposition of immunoglobulin complexes in blood vessels, and premature death due to rapidly progressing membranoproliferative glomerular nephritis. Mechanistic analyses revealed that the null mice showed enhanced proliferative response of CD4+ T cells, altered ratios of CD4+ and CD8+ cells, and increased oxidative lesions in tissues. Analyses of antioxidant-induced gene expression showed that the knockout mice were devoid of the basal and inducible expression of certain phase 2 detoxification enzymes and antioxidant genes in hepatic and lymphoid cells in vivo. Our findings suggest that Nrf2 mediates important antioxidant functions involved in the control of peripheral lymphocyte homeostasis and autoimmune surveillance. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an antioxidant-activated cap “n” collar basic leucine zipper transcription factor. To assess the function of Nrf2 in the antioxidant response, we examined mice with targeted disruption of the Nrf2 gene. Nrf2-null mice developed complex disease manifestations, with a majority exhibiting a lupus-like autoimmune syndrome characterized by multiorgan inflammatory lesions with a marked female predominance, appearance of anti-double-stranded DNA antibodies in young adulthood, intravascular deposition of immunoglobulin complexes in blood vessels, and premature death due to rapidly progressing membranoproliferative glomerular nephritis. Mechanistic analyses revealed that the null mice showed enhanced proliferative response of CD4+ T cells, altered ratios of CD4+ and CD8+ cells, and increased oxidative lesions in tissues. Analyses of antioxidant-induced gene expression showed that the knockout mice were devoid of the basal and inducible expression of certain phase 2 detoxification enzymes and antioxidant genes in hepatic and lymphoid cells in vivo. Our findings suggest that Nrf2 mediates important antioxidant functions involved in the control of peripheral lymphocyte homeostasis and autoimmune surveillance. The nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) belongs to a subfamily of the basic leucine zipper transcription factors termed CNC bZip for cap “n” collar basic leucine zipper. The CNC bZip proteins are characterized by a highly conserved 43-amino acid module localized immediately N-terminal to the bZip DNA-binding region. The module is homologous in sequence to a region in the CNC gene, a homeotic gene involved in cephalic patterning during embryogenesis of Drosophila,1Mohler J Vani K Leung S Epstein A Segmentally restricted, cephalic expression of a leucine zipper gene during Drosophila embryogenesis.Mech Dev. 1991; 34: 3-9Crossref PubMed Scopus (108) Google Scholar, 2Chan JY Cheung MC Moi P Chan K Kan YW Chromosomal localization of the human NF-E2 family of bZIP transcription factors by fluorescence in situ hybridization.Hum Genet. 1995; 95: 265-269Crossref PubMed Scopus (58) Google Scholar hence the name CNC (for cap “n” collar). The CNC bZip family includes NF-E2, Nrf1, Nrf2, Nrf3, Bach1, and Bach2.3Chan JY Han XL Kan YW Isolation of cDNA encoding the human NF-E2 protein.Proc Natl Acad Sci USA. 1993; 90: 11366-11370Crossref PubMed Scopus (110) Google Scholar, 4Andrews NC Erdjument-Bromage H Davidson MB Tempst P Orkin SH Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein.Nature. 1993; 362: 722-728Crossref PubMed Scopus (566) Google Scholar, 5Chan JY Han XL Kan YW Cloning of Nrf1, an NF-E2-related transcription factor, by genetic selection in yeast.Proc Natl Acad Sci USA. 1993; 90: 11371-11375Crossref PubMed Scopus (295) Google Scholar, 6Moi P Chan K Asunis I Cao A Kan YW Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region.Proc Natl Acad Sci USA. 1994; 91: 9926-9930Crossref PubMed Scopus (1226) Google Scholar, 7Itoh K Igarashi K Hayashi N Nishizawa M Yamamoto M Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins.Mol Cell Biol. 1995; 15: 4184-4193Crossref PubMed Scopus (361) Google Scholar, 8Oyake T Itoh K Motohashi H Hayashi N Hoshino H Nishizawa M Yamamoto M Igarashi K Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.Mol Cell Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (522) Google Scholar NF-E2, the founder member of the group, controls the transcriptional regulation at the hypersensitive site 2 of the human β-globin gene cluster locus.6Moi P Chan K Asunis I Cao A Kan YW Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region.Proc Natl Acad Sci USA. 1994; 91: 9926-9930Crossref PubMed Scopus (1226) Google Scholar Nrf1, Nrf2, and Nrf3 were cloned as a result of searching for NF-E2-related proteins binding to the NF-E2 DNA-binding sequence, whereas Bach1 and Bach2 were proteins distantly related to NF-E2 but capable of binding to MafK,8Oyake T Itoh K Motohashi H Hayashi N Hoshino H Nishizawa M Yamamoto M Igarashi K Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site.Mol Cell Biol. 1996; 16: 6083-6095Crossref PubMed Scopus (522) Google Scholar a member of the small Maf proteins that form heterodimers with the CNC bZip proteins for DNA binding. Whereas NF-E2 is strictly expressed in hematopoitic cells and is required for regulation of β-globin gene expression, Nrf2 is ubiquitously expressed in animal tissues, and its role in β-globin expression remains uncertain.9Chan K Lu R Chang JC Kan YW NRF2, a member of the NRE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development.Proc Natl Acad Sci USA. 1996; 93: 13943-13948Crossref PubMed Scopus (518) Google Scholar Instead, increasing evidence reveals that Nrf2 mediates the antioxidant response element (ARE)-dependent transcriptional regulation of a battery of genes. These include phase II enzymes such as NAD(P)H:quinine oxidoreductase 1 (NQO1) and glutathione S-transferase A1 (GSTA1), which detoxify endogenous and exogenous chemicals through reduction and conjugation reactions, and antioxidative enzymes and proteins such as thioredoxin, heme oxygenase 1 (HO-1), and ferritin, which participate in reactive oxygen species (ROS) metabolism or directly scavenge ROS. Thus, Nrf2 appears to function as a key regulator of the antioxidant response. Consistent with this notion, a number of genetic studies have implicated Nrf2 in a range of protective responses against chemical-induced lesions or disease states. Loss of function of Nrf2 is associated with increased sensitivity to chemical carcinogenesis such as benzo(a)pyrene-induced gastric neoplasia,10Ramos-Gomez M Kwak MK Dolan PM Itoh K Yamamoto M Talalay P Kensler TW Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.Proc Natl Acad Sci USA. 2001; 98: 3410-3415Crossref PubMed Scopus (987) Google Scholar acetaminophen-induced hepatotoxicity,11Chan K Han XD Kan YW An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen.Proc Natl Acad Sci USA. 2001; 98: 4611-4616Crossref PubMed Scopus (651) Google Scholar hyperoxic lung damage,12Cho HY Jedlicka AE Reddy SP Kensler TW Yamamoto M Zhang LY Kleeberger SR Role of NRF2 in protection against hyperoxic lung injury in mice.Am J Respir Cell Mol Biol. 2002; 26: 175-182Crossref PubMed Scopus (585) Google Scholar pulmonary injury by butylated hydroxytoluene13Chan K Kan YW Nrf2 is essential for protection against acute pulmonary injury in mice.Proc Natl Acad Sci USA. 1999; 96: 12731-12736Crossref PubMed Scopus (524) Google Scholar or diesel exhaust,14Li N Alam J Venkatesan MI Eiguren-Fernandez A Schmitz D Di Stefano E Slaughter N Killeen E Wang X Huang A Wang M Miguel AH Cho A Sioutas C Nel AE Nrf2 is a key transcription factor that regulates antioxidant defense in macrophages and epithelial cells: protecting against the proinflammatory and oxidizing effects of diesel exhaust chemicals.J Immunol. 2004; 173: 3467-3481PubMed Google Scholar and premature ovarian failure by the occupational ovotoxicant 4-vinyl cyclohexine diepoxide.15Hu X Roberts JR Apopa PL Kan YW Ma Q Accelerated ovarian failure induced by 4-vinyl cyclohexene diepoxide in Nrf2 null mice.Mol Cell Biol. 2006; 26: 940-954Crossref PubMed Scopus (92) Google Scholar Although Nrf2 is dispensable for life in mice, it cooperates with Nrf1 to regulate antioxidant gene expression during early embryonic development. A combined deletion of Nrf1 and Nrf2 results in marked oxidative stress and increased apoptosis in the liver leading to embryonic lethality, suggesting overlapping functions among the CNC bZip proteins and possibly compensation for each other in the defense against oxidative stress during embryonic hepatogenesis.16Leung L Kwong M Hou S Lee C Chan JY Deficiency of the Nrf1 and Nrf2 transcription factors results in early embryonic lethality and severe oxidative stress.J Biol Chem. 2003; 278: 48021-48029Crossref PubMed Scopus (243) Google Scholar The molecular mechanism of Nrf2 action is best understood for the induction of phase II genes, such as the induction of Nqo1 by phenolic antoxidants.17Nguyen T Sherratt PJ Pickett CB Regulatory mechanisms controlling gene expression mediated by the antioxidant response element.Annu Rev Pharmacol Toxicol. 2003; 43: 233-260Crossref PubMed Scopus (1069) Google Scholar, 18Ma Q Kinneer K Bi Y Chan JY Kan YW Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap ‘n’ collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction.Biochem J. 2004; 377: 205-213Crossref PubMed Scopus (175) Google Scholar In the absence of an activator, Nrf2 is a labile protein localized in the cytoplasm forming a complex with Keap1. Keap1 anchors Nrf2 in the cytoplasm by interacting with cytoskeletal proteins. Moreover, Keap1 regulates the ubiquitin-26S proteasome-mediated turnover of Nrf2 through a Cul3-based E3 ligase. Binding of tert-butylhydroquinone (tBHQ) to Keap1 stabilizes Nrf2. Activated Nrf2 translocates into the nucleus and forms a heterodimer with a Maf protein. The dimer then binds to AREs located in the enhancer regions and mediates the transcription of the genes. Variations of this Nrf2/ARE paradigm have been observed in the transcriptional regulation of a number of genes, suggesting high adaptability of Nrf2 action in a broad range of biological functions. For instance, Nrf2 can interact with activating transcription factor 4 in the induction of Ho-1,19He CH Gong P Hu B Stewart D Choi ME Choi AM Alam J Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein: implication for heme oxygenase-1 gene regulation.J Biol Chem. 2001; 276: 20858-20865Crossref PubMed Scopus (382) Google Scholar compete with Jun/Fos for binding to ARE/AP1 sites in the induction of thioredoxin by hemin,20Kim YC Masutani H Yamaguchi Y Itoh K Yamamoto M Yodoi J Hemin-induced activation of the thioredoxin gene by Nrf2: a differential regulation of the antioxidant responsive element by a switch of its binding factors.J Biol Chem. 2001; 276: 18399-18406Crossref PubMed Scopus (258) Google Scholar or cross-interact with aryl hydrocarbon receptor (AhR) signaling for induction of murine Nqo1 by AhR agonist 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD).18Ma Q Kinneer K Bi Y Chan JY Kan YW Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap ‘n’ collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction.Biochem J. 2004; 377: 205-213Crossref PubMed Scopus (175) Google Scholar Here, we report that loss of Nrf2 in mice is associated with the development of autoimmune-mediated lesions in multiple tissues. The lesions were characterized by disseminated inflammation, appearance of anti-double-stranded DNA (dsDNA) antibodies at young ages, vascular deposition of immunoglobulin complexes, and premature death due primarily to rapidly progressing glomerulonephritis. Surviving mice frequently died of neoplasia of lymphoid, histiocytic, or endothelial origins. Nrf2-null mice exhibited increased proliferative response of CD4+ cells and altered ratios of CD4+ and CD8+ peripheral T cells. Finally, the Nrf2-null mice were devoid of the basal and inducible expression of a number of phase II and antioxidant-responsive genes and concomitantly displayed increased oxidative lesions in multiple tissues. Together, the findings suggest interplay between Nrf2 function, ROS defense, and autoimmune surveillance in the development of autoimmune disorders. Nrf2 knockout mice (kindly provided by Dr. Y.W. Kan, University of California, San Francisco, CA) were re-derived at Jackson Laboratory to assure specific pathogen-free status.18Ma Q Kinneer K Bi Y Chan JY Kan YW Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap ‘n’ collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction.Biochem J. 2004; 377: 205-213Crossref PubMed Scopus (175) Google Scholar Nrf2−/− and Nrf2+/+ mice on a genetic background of 129SVJ were maintained at an environmentally controlled National Institute for Occupational Safety and Health animal facility, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. The mice were barrier maintained with a light/dark cycle of 12 hours at a constant temperature (22°C) in HEPA-filtered, individually ventilated microisolator cages (Thoren Caging Systems, Hazleton, PA). Irradiated food (6% fat; 7913; Harlan-Teklad, Madison, WI) and water were provided ad libitum. Bedding was sterile Beta chip from Northeastern Products Company (Warrensburg, NY). Mice were observed daily for signs of illness. Moribund mice showing severe edema or lethargy were sacrificed for histopathological examinations. Mouse breeding, morbidity, and mortality were recorded using the Colony software, version 3.0 (Locus Technology, Inc., Orland, ME). Lymph nodes (from the mandibular and axillary regions) and spleen were collected. Lymphocytes were isolated by gentle disruption of the tissues with the flat end of a syringe plunger against the bottom of a 70-μm nylon cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ), followed by centrifugation at 1200 × g for 10 minutes. Red blood cells in spleen samples were removed by lysis with ammonium chloride lysing reagent (BD Pharmingen, San Diego, CA) and repeated centrifugation and washing. CD4+, CD8+, or CD45R/B220+ cells were isolated using BD IMagnet according to manufacturer's instructions (BD Pharmingen). Briefly, an appropriate number of lymph node cells or splenocytes were suspended in 1× BD IMag buffer and blocked for nonspecific binding with anti-mouse CD16/CD32 monoclonal antibody (BD Pharmingen) on ice for 15 minutes, followed by a brief spin. Cell pellets were re-suspended in 0.5 ml of IMag buffer, mixed with 50 μl of BD IMag anti-mouse CD4, CD8a, or CD45R/B220 particles, and incubated at 4°C for 30 minutes. The cells labeled with IMag particles were placed in the BD IMagnet and were separated from unlabeled cells by magnetic force. The separation process was repeated one more time. Isolated CD4+, CD8+, or CD45R/B220+ cells were examined for purity by fluorescence-acitvated cell sorter (FACS) as described below and were used for cell proliferation and FACS analysis. For staining of cell surface antigens, ∼5 × 105 to 1 × 106 cells were blocked with anti-mouse CD16/CD32 on ice for 15 minutes. The cells were then stained with R-phycoerythrin-conjugated anti-mouse CD4, flourescein isothiocyanate (FITC)-conjugated anti-mouse CD8a, or peridinin chlorophyll-α protein-conjugated anti-mouse CD45R/B220 monoclonal antibodies (BD Pharmingen) on ice in the dark for 15 minutes. After washing, the cells were resuspended in staining buffer and analyzed using FACSCalibur (Becton Dickinson, San Jose, CA). To analyze responses of lymphocytes to proliferative stimuli, splenocytes, lymph node cells, or isolated CD4+, CD8+, and CD45R/B220+ cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 4 mmol/L glutamine and 10% fetal bovine serum. To measure response to anti-CD3, cells (5 × 104 or 1 × 105) were seeded in a 96-well BD BioCoat T-Cell Activation Plate precoated with anti-CD3 antibodies (BD Discovery Labware, San Diego, CA) and incubated at 37°C with 5% CO2 for 48 hours. Proliferation of the cells was measured using the 3-(4,5-dimethyl thiazole-2-yl)-2,5-diphenyl tetrazolium bromid (MTT) kit from Roche Applied Science (Indianapolis, IN). MTT labeling reagent (10 μl) was added to each well, and the plate was incubated for 4 hours in a CO2 incubator, followed by adding 100 μl of a solubilization solution into each well. The plate was kept overnight in the incubator, and cell proliferation was measured at 550 nm using a plate reader (Spectra Max 340PC; Molecular Devices, Sunnyvale, CA). For B-cell proliferation assay, purified B cells were activated with goat F(ab′)2 anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA) or lipopolysaccharide (20 μg/ml; Sigma, St. Louis, MO) for 48 hours. Proliferation was measured with the MTT kit as described above. Blood samples were collected from tail veins of age-matched (5- to 7-month-old) male and female Nrf2+/+, Nrf2+/−, or Nrf2−/− mice (all in the 129SVJ background) and C57BL/6J mice (as a separate control; The Jackson Laboratories, Bar Harbor, ME) into serum tubes (Becton Dickinson). Sera were prepared and measured for mouse anti-dsDNA or anti-single-stranded DNA antibodies using enzyme-linked immunosorbent assay (ELISA) kits from Alpha Diagnostic International (San Antonio, TX) according to supplier's instructions. ELISA was measured at 450 nm using Spectra Max 340PC plate reader. Serum BUN and total protein were determined using a COBAS MIRA Plus Chemistry Analyzer (Roche Diagnostics Corp., Indianapolis, IN). Blood was collected from the abdominal vena cava, placed in a serum microtainer tube, and incubated for approximately 20 minutes. Blood was centrifuged for 15 minutes at 3000 rpm, and serum was collected and placed in a vial. The vial was then placed in the Chemistry Analyzer. BUN and total protein were determined using Reagent for BUN from Roche Diagnostics or Reagent for Total Protein from Sigma, respectively. For hematocrit determination, blood was drawn from the abdominal vena cava into standard heparinized hematocrit tubes. One end of each tube was sealed with clay tube sealer. The tubes were centrifuged on a Damon IEC MB centrifuge (Damon, Needham Heights, MA) at 13,700 × g for 5 minutes. Hematocrit was determined with a Damon microcapillary reader (Damon). Free malondialdehyde (MDA) was measured using the Bioxytech MDA-586 kit from OxisResearch (Portland, OR). Briefly, kidney, liver, and heart tissues were collected from female Nrf2−/− or Nrf2+/+ mice at 6 months of ages or as indicated in figures. The tissues were homogenized in the presence of 5 mmol/L butylated hydroxytoluene. Extracts were prepared by centrifugation at 10,000 × g for 10 minutes. MDA contents were assayed and measured spectrophotometrically according to instructions provided by the manufacturer. Nrf2+/+ and Nrf2−/− mice (2 months old) were treated with corn oil or 3-t-butyl-4-hydroxy anisol (BHA). BHA is administered by gavage on days 1 and 3 at a dose of 400 mg/kg body weight. Liver and spleen samples were collected on day 4 after BHA treatment and were stored in RNALater (Qiagen, Valencia, CA). Total RNA was prepared using a Qiagen Total RNA isolation kit (Qiagen). Northern blotting was performed as follows. RNA samples (5 μg) were fractionated in a 1% agarose-formaldehyde gel and transferred to a Nytran membrane. The blot was probed with a dioxigenin (DIG)-labeled riboprobe prepared with the DIG-labeling kit (Roche Applied Science) for mouse genes according to established procedures.21Ma Q Renzelli AJ Baldwin KT Antonini JM Superinduction of CYP1A1 gene expression: regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced degradation of Ah receptor by cycloheximide.J Biol Chem. 2000; 275: 12676-12683Crossref PubMed Scopus (83) Google Scholar Signals were visualized by chemiluminescence using a DIG RNA detection kit with CDP Star as a substrate (Roche Applied Science). Parallel blots of the same samples were probed with a DIG-labeled mouse actin probe to ensure equal loading. Results shown were repeated two times in separate experiments with consistent observations. Real-time polymerase chain reaction (PCR) was performed using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) on a Bio-Rad iCycler (Bio-Rad, Hercules, CA) following standard procedures. Briefly, single-stranded cDNA was synthesized from 1 μg of total RNA using Superscript III reverse transcriptase (Invitrogen). For PCR reach reaction, 2 μl of DNA template, 2 μl of forward and reverse primers (10 μmol/L each), 25 μl of SYBR Green PCR Master Mix, and 19 μl of water were added to a final volume of 50 μl. Thermal cycling was performed as follows: 95°C for 3 minutes as initial denaturing, followed by 45 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 60 seconds, and a final extension at 72°C for 2 minutes. Threshold cycles (CT values) were determined using the iCycler IQ software (Bio-Rad). Real-time PCR results were normalized using 1% of input as an internal control. Relative DNA amounts were calculated from the CT values for each sample by interpolating into the standard curve obtained using a series dilution of standard DNA samples run under the same conditions. Primer sets used for real-time PCR were as follows: NQO1 forward, 5′-AAC-GGGAAGATGTGGAGATG-3′, and reverse, 5′-CGCAGTAGATGCCAGTCAAA-3′; HO-1 forward, 5′-GAGCAGA-ACCAGCCTGAACTA-3′, and reverse, 5′-GGTACAAG-GAAGCCATCACCA-3′; and β-actin forward, 5′-GACC-TCTATGCCAACACAGT-3′, and reverse, 5′-ACTCATC-GTACTCCTGGTG-3′. Tissue samples were fixed in 10% neutral buffered formalin for 1 day, subsequently routinely processed, and embedded in paraffin. Sections of 5 μm thickness were stained with hematoxylin and eosin (H&E) or with PAS. Slides were interpreted by a board-certified veterinary pathologist blinded to the genotypes of individual animals. For immunofluorescent studies of immunoglobulin complex deposition, fresh kidneys or other tissues were flash frozen in liquid nitrogen and subsequently stored at −80°C. Frozen sections were prepared using a cryostat. Sections were washed with phosphate-buffered saline (PBS; Sigma) three times (10 minutes each), followed by a 5-minute wash with PBS. Nonspecific binding was blocked by sequential 10-minute baths in 5% PBS/bovine serum albumin (IgG free) and 5% pig serum (Biogenex, San Ramon, CA). Primary antibodies were incubated with tissue sections for 2 hours in the dark as follows: FITC-goat anti-mouse IgG+A+M (Zymed, South San Francisco, CA) 1:20 in PBS plus 10% goat serum; Alexa Fluor 488-goat anti-mouse IgG (Molecular Probes, Eugene, OR) 1:20 in PBS; Alexa Fluor 488-goat anti-mouse IgM (Molecular Probes) 1:20 in PBS; or fluorescein-conjugated goat IgG fraction against mouse complement C3 (ICN/Cappell, Aurora, OH) 1:400 in PBS. Sections were rinsed three times with PBS (each for 5 minutes), covered with coverslips in Gelmount (Biomeda, Foster City, CA), and examined under a fluorescent microscope. For transmission electron microscopy, fresh kidney samples were preserved in Karnovsky's fixative, postfixed in osmium tetroxide, mordanted in tannic acid, and stained en bloc in uranyl acetate. The tissues were then dehydrated in alcohol and embedded in Epon. The sections were stained with uranyl acetate and lead citrate. Kidney samples were examined ultrastructurally using a JEOL 1220 electron microscope. The Nrf2-null mice bred normally, and their offspring grew and matured similarly to Nrf2+/+ mice. However, the mice developed a range of signs of illness after 2 months of age (Figure 1, Figure 2). Manifestations of illness were diverse, including reduced body weight, subcutaneous edema, which often started at the facial area and spread to the whole body gradually (Figure 2A), pale ears and toes, cutaneous ulcers, ring-shaped tail redness and necrosis (Figure 2B), eye inflammation (Figure 2A), and neurological symptoms (dysmetria, tremors, lethargy, and seizures) (Figure 2A). These signs were rarely observed in Nrf2+/+ mice with the same genetic background during the period of study (Figure 1A). Moreover, a clear female predominance of the illness was seen (Figure 1A). The morbidities of the female mice were 29, 79, and 100% at ages of 15, 25, and 55 weeks, respectively, whereas, those of the male were 4, 33.3, and 58.3% at the same ages (Figure 1A). In addition, female mice appeared to develop signs of illness earlier (10 versus 14 weeks) and more severely than their male littermates.Figure 2Gross phenotypes of Nrf2-null mice. A: Nrf2−/− mice exhibited diverse signs of illness, including facial and whole-body edema, tail necrosis (a and b, indicated by arrow), dysmetria (b), and conjunctivitis (c). B: Tails of Nrf2-null mice developed focal, multifocal, or circumferential erythematous lesions (a−d); initial tail lesions are at the distal end and progress proximally (b and c). C: Splenomegaly and enlarged lymph nodes. Splenomegaly (a) and enlarged lymph nodes (b) were seen in Nrf2−/− (right) but not Nrf2+/+ (left) genotypes. The sizes of the liver and kidney were similar from the same mice (c).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Nrf2-null mice exhibited shortened life spans compared with the wild-type control mice (Figure 1B). Similar to the morbidity, a clear gender-dependent difference in survival was observed. Female mice died earlier and at a higher rate than male mice. The survival rates of the female at ages of 15, 25, and 55 weeks were 71.4, 50, and 21.4%, respectively, substantially lower that those of the male (ie, 96, 87.5, and 58.3% at the same ages). Thus, loss of Nrf2 in mice caused a spontaneous, gender-dependent, and diverse spectrum of disease manifestations and mortality in the absence of apparent exposure to exogenous toxic chemicals. Histopathological examination of the diseased mice revealed a high prevalence of glomerulonephritis. Among the mice that died or were euthanized because of severe illness, the glomerulonephritis prevalence was 88% (31 with glomerulonephritis, 3 without glomerulonephritis, and 1 unevaluated because of extensive tissue autolysis). The mean age of Nrf2-null mice with glomerulonephritis was 9 ± 1 (SE) months with a range of 2 to 22 months. Glomerulonephritis was the primary cause of death in 69% (22 of 32) of Nrf2-null mice in this necropsy series, causing death in mice as young as 2 months of age. Glomerulonephritis was seen in both female and male mice and was the cause of death in 88% (15 of 17) of female and 47% (7 of 15) of male mice. Spontaneous death was often accompanied by subcutaneous edema and lethargy. The corresponding histopathological alterations seen in affected Nrf2-null mice were varying manifestations of membranoproliferative glomerulonephritis (Figure 3). Antemortem BUN concentration was elevated in mice with the most severe histopathological changes in glomeruli (Figure 4). In wild-type and heterozygous mice, membranoproliferative glomerulohephritis was uncommon, and signs of renal failure were not observed.Figure 4Correlation of BUN and glomerulonephritis severity. Antimortem blood urea contents (BUN) and glomerulonephritis pathology scores (reflecting severity of glomerulonephritis) of Nrf2+/+, Nrf2+/−, and Nrf2−/− mice were evaluated for correlation using the Sigma Plot software.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In most cases of glomerulonephritis, glomeruli were enlarged with mesangial cell proliferation, thickening of glomerular basement membranes, and frequent adhesions between Bowman's capsule and glomeruli. Variable fibrosis indicated chronicity, ranging from fibrosis of Bowman's capsule to complete glomerulosclerosis. In many kidneys, renal tubules and Bowman's space contained eosinophilic materi
Referência(s)