Nrf2 suppresses lupus nephritis through inhibition of oxidative injury and the NF-κB-mediated inflammatory response
2013; Elsevier BV; Volume: 85; Issue: 2 Linguagem: Inglês
10.1038/ki.2013.343
ISSN1523-1755
AutoresTao Jiang, Fei Tian, Hongting Zheng, Samantha A. Whitman, Yifeng Lin, Zhigang Zhang, Nong Zhang, Donna D. Zhang,
Tópico(s)Endometriosis Research and Treatment
ResumoThe generation of reactive oxygen species has a pivotal role in both acute and chronic glomerular injuries in patients with lupus nephritis. As the transcription factor Nrf2 is a major regulator of the antioxidant response and is a primary cellular defense mechanism, we sought to determine a role of Nrf2 in the progression of lupus nephritis. Pathological analyses of renal biopsies from patients with different types of lupus nephritis showed oxidative damage in the glomeruli, accompanied by an active Nrf2 antioxidant response. A murine lupus nephritis model using Nrf2+/+ and Nrf2-/- mice was established using pristine injection. In this model, Nrf2-/- mice suffered from greater renal damage and had more severe pathological alterations in the kidney. In addition, Nrf2+/+ mice showed ameliorative renal function when treated with sulforaphane, an Nrf2 inducer. Nrf2-/- mice had higher expression of transforming growth factor β1 (TGFβ1), fibronectin, and iNOS. In primary mouse mesangial cells, the nephritogenic monoclonal antibody R4A activated the nuclear factor-kappa B (NF-κB) pathway and increased the level of reactive oxygen species, iNOS, TGFβ1, and fibronectin. Knockdown of Nrf2 expression aggravated all aforementioned responses induced by R4A. Thus, these results suggest that Nrf2 improves lupus nephritis by neutralizing reactive oxygen species and by negatively regulating the NF-κB and TGFβ1 signaling pathways. The generation of reactive oxygen species has a pivotal role in both acute and chronic glomerular injuries in patients with lupus nephritis. As the transcription factor Nrf2 is a major regulator of the antioxidant response and is a primary cellular defense mechanism, we sought to determine a role of Nrf2 in the progression of lupus nephritis. Pathological analyses of renal biopsies from patients with different types of lupus nephritis showed oxidative damage in the glomeruli, accompanied by an active Nrf2 antioxidant response. A murine lupus nephritis model using Nrf2+/+ and Nrf2-/- mice was established using pristine injection. In this model, Nrf2-/- mice suffered from greater renal damage and had more severe pathological alterations in the kidney. In addition, Nrf2+/+ mice showed ameliorative renal function when treated with sulforaphane, an Nrf2 inducer. Nrf2-/- mice had higher expression of transforming growth factor β1 (TGFβ1), fibronectin, and iNOS. In primary mouse mesangial cells, the nephritogenic monoclonal antibody R4A activated the nuclear factor-kappa B (NF-κB) pathway and increased the level of reactive oxygen species, iNOS, TGFβ1, and fibronectin. Knockdown of Nrf2 expression aggravated all aforementioned responses induced by R4A. Thus, these results suggest that Nrf2 improves lupus nephritis by neutralizing reactive oxygen species and by negatively regulating the NF-κB and TGFβ1 signaling pathways. Lupus nephritis (LN) is a severe autoimmune disease associated with a high rate of morbidity and mortality. A large body of evidence indicates that production of autoantibodies and glomerular immune-complex deposition are the initial events in the pathogenesis of the disease.1.Cameron J.S. Lupus nephritis.J Am Soc Nephrol. 1999; 10: 413-424PubMed Google Scholar, 2.Couser W.G. Basic and translational concepts of immune-mediated glomerular diseases.J Am Soc Nephrol. 2012; 23: 381-399Crossref PubMed Scopus (145) Google Scholar, 3.Kaveri S.V. Mouthon L. Bayry J. Basophils and nephritis in lupus.N Engl J Med. 2010; 363: 1080-1082Crossref PubMed Scopus (26) Google Scholar, 4.Grande J.P. Experimental models of lupus nephritis.Contrib Nephrol. 2011; 169: 183-197Crossref PubMed Scopus (18) Google Scholar The deposition of the immune complex triggers a cascade of events in the inflammatory response that are accompanied by the generation of reactive oxygen species (ROS), which have a pivotal role in both acute and chronic glomerular injuries in LN patients.5.Kovacic P. Jacintho J.D. Systemic lupus erythematosus and other autoimmune diseases from endogenous and exogenous agents: unifying theme of oxidative stress.Mini Rev Med Chem. 2003; 3: 568-575Crossref PubMed Scopus (33) Google Scholar, 6.Nath K.A. Fischereder M. Hostetter T.H. The role of oxidants in progressive renal injury.Kidney Int Suppl. 1994; 45: S111-S115Abstract Full Text PDF PubMed Scopus (49) Google Scholar Detection of lipid oxidation, oxidative DNA damage, and protein oxidation in lupus patients provide strong evidence for the involvement of ROS in this disease.7.Evans M.D. Cooke M.S. Akil M. et al.Aberrant processing of oxidative DNA damage in systemic lupus erythematosus.Biochem Biophys Res Commun. 2000; 273: 894-898Crossref PubMed Scopus (55) Google Scholar, 8.Frostegard J. Svenungsson E. Wu R. et al.Lipid peroxidation is enhanced in patients with systemic lupus erythematosus and is associated with arterial and renal disease manifestations.Arthritis Rheum. 2005; 52: 192-200Crossref PubMed Scopus (195) Google Scholar, 9.Morgan P.E. Sturgess A.D. Hennessy A. et al.Serum protein oxidation and apolipoprotein CIII levels in people with systemic lupus erythematosus with and without nephritis.Free Radic Res. 2007; 41: 1301-1312Crossref PubMed Scopus (33) Google Scholar Moreover, several mechanisms by which ROS promotes acute and chronic tissue damage in LN have been described in detail.10.Grande J.P. Mechanisms of progression of renal damage in lupus nephritis: pathogenesis of renal scarring.Lupus. 1998; 7: 604-610Crossref PubMed Scopus (57) Google Scholar Nrf2 is a major regulator of the antioxidant response and is a primary cellular defense mechanism.11.Itoh K. Ishii T. Wakabayashi N. et al.Regulatory mechanisms of cellular response to oxidative stress.Free Radic Res. 1999; 31: 319-324Crossref PubMed Scopus (303) Google Scholar, 12.Kensler T.W. Wakabayashi N. Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway.Annu Rev Pharmacol Toxicol. 2007; 47: 89-116Crossref PubMed Scopus (2759) Google Scholar It regulates target genes encoding intracellular antioxidants, phase II detoxifying enzymes, and other effectors that promote cell survival and maintain cellular redox homeostasis.13.Zhang D.D. Mechanistic studies of the Nrf2-Keap1 signaling pathway.Drug Metab Rev. 2006; 38: 769-789Crossref PubMed Scopus (846) Google Scholar Nrf2 upregulates its target genes through an antioxidant response element in the regulatory regions of these genes in response to oxidative stress.14.Chan K. Han X.D. Kan Y.W. 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 The essential role of Nrf2 in combating oxidative stress has been clearly demonstrated by the findings that Nrf2-null (Nrf2-/-) mice have increased sensitivity to a variety of perturbations.12.Kensler T.W. Wakabayashi N. Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway.Annu Rev Pharmacol Toxicol. 2007; 47: 89-116Crossref PubMed Scopus (2759) Google Scholar, 14.Chan K. Han X.D. Kan Y.W. 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, 15.Motohashi H. Yamamoto M. Nrf2-Keap1 defines a physiologically important stress response mechanism.Trends Mol Med. 2004; 10: 549-557Abstract Full Text Full Text PDF PubMed Scopus (1378) Google Scholar Considering the important role of ROS in the pathogenesis of LN, we hypothesize that Nrf2 protects against renal injury by neutralizing ROS and thus reducing tissue damage. In addition, several studies show that some Nrf2 inducers can inhibit the activation of the NF-κB pathway,16.Ahmad R. Raina D. Meyer C. et al.Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179.J Biol Chem. 2006; 281: 35764-35769Crossref PubMed Scopus (215) Google Scholar, 17.Karuri A.R. Huang Y. Bodreddigari S. et al.3H-1,2-dithiole-3-thione targets nuclear factor kappaB to block expression of inducible nitric-oxide synthase, prevents hypotension, and improves survival in endotoxemic rats.J Pharmacol Exp Ther. 2006; 317: 61-67Crossref PubMed Scopus (25) Google Scholar, 18.Sriram N. Kalayarasan S. Sudhandiran G. Epigallocatechin-3-gallate augments antioxidant activities and inhibits inflammation during bleomycin-induced experimental pulmonary fibrosis through Nrf2-Keap1 signaling.Pulm Pharmacol Ther. 2009; 22: 221-236Crossref PubMed Scopus (142) Google Scholar a redox-sensitive transcription factor. However, the interaction between these two pathways in LN remains unclear. In this study, we investigated the role of Nrf2 in LN. As the pivotal pathway for redox homeostasis, we hypothesize that Nrf2 is essential to maintain renal function and antagonize renal damage during the progression of LN. A total of 60 human kidney biopsies were collected; 39 were from lupus patients and 12 were from healthy people. Compared with normal glomeruli, the glomeruli of LN patients showed expansion of mesangium, increased cellularity, fibrinoid necrosis, and thickening of capillary walls (Figure 1, compare panel A with panel B–F). Deposition of immunoglobulin within the glomeruli was seen in all classes (Figure 1). Nrf2 was barely expressed in normal glomeruli (Figure 1), whereas the expression was elevated in lupus patients (Figure 1). Consistent with these results, the Nrf2 downstream gene NAD(P)H dehydrogenase, quinone 1(NQO1) was also upregulated in the glomeruli of LN patients, confirming the activation of the Nrf2 pathway. (Figure 1, compare panel S with panel T–X). DNA damage induced by oxidative stress was measured using an antibody against 8-Oxo-dG. Positive nuclear staining was detected in the tissues from LN patients, but not in the normal kidney tissues (Figure 1, compare panel Y with panel Z–D’). To obtain a quantitative analysis of Nrf2, NQO1, and 8-Oxo-dG expression, the glomeruli from each section were analyzed by i-Solution software (Vancouver, Canada). The average expression of Nrf2, NQO1, and 8-Oxo-dG is shown as a percentage in Figure 1 (Figure 1). Compared with the normal kidney, the expression levels of Nrf2, NQO1, and 8-oxo-dG were significantly higher in the glomeruli from any class of LN (Figure 1, *P<0.05, **P<0.05). However, the expression of Nrf2, NQO1, and 8-Oxo-dG did not correlate with the severity of LN, as type III, but not type IV, which is the severest form of LN, had the highest expression of Nrf2. Taken together, these data indicate that the glomeruli of LN patients experience oxidative stress, which triggers the activation of the Nrf2-mediated antioxidant response. The expression of Nrf2 was also analyzed in other representative types of nephritis; acute proliferative glomerulonephritis, IgA nephropathy, and purpuric nephropathy demonstrated positive Nrf2 expression (Figure 1). These findings suggest that the increased activation of Nrf2 in glomeruli from different types of nephritis is a general phenomena resulting from immune-complex deposition. To further explore the role of Nrf2 in the progression of LN, a pristane-induced murine model was used. During the course of the experiment, two Nrf2-/- mice in the pristane treatment group died at the 21st and 23rd week, respectively. Nevertheless, the survival rate between Nrf2+/+ and Nrf2-/- groups showed no significant difference (Figure 2a). The spleen to body weight ratio, however, was significantly increased in the pristane-treated groups, regardless of the genotype (Figure 2b). In addition, Nrf2-/- mice had a significantly higher spleen to body weight ratio compared with Nrf2+/+ mice in both untreated and treated groups (Figure 2b, #P<0.05). Urine albumin-to-creatinine ratio (UACR), an indicator of kidney vascular permeability, was measured every 4 weeks following pristane injection to show any alterations in renal functions. Pristane markedly increased UACR in a time-dependent manner in both genotypes (Figure 2c, *P<0.05). Nrf2-/- mice, however, showed trends toward a higher UACR than Nrf2+/+ mice starting at 16 weeks, which reached its apex at the end of the study (28 weeks post pristane injection) (Figure 2c, #P<0.05). The sera from the mice were collected and used to measure autoantibody titers at a 1:50 dilution. Anti-ssDNA, anti-nRNP, and anti-Histone Igs were significantly increased after pristane treatment (Figures 2d–f). Nrf2-/- mice had higher levels of anti-ssDNA antibodies with or without treatment of pristane (Figure 2d, #P<0.05). Treatment of pristane induced the deposition of IgG and IgM within the glomeruli in both Nrf2+/+ and Nrf2-/- mice (Figures 2g and h). In addition, another lupus model was set using Nrf2+/+ mice only to study the protective role of sulforaphane on LN. Although the administration of sulforaphane did not significantly affect the body to spleen weight ratio (Supplementary Figure 1A online), it ameliorated the renal function (Supplementary Figure 1B online, #P<0.05). Taken together, these results indicate that Nrf2-/- mice suffer greater renal damage after pristane treatment and that the increased Nrf2 level can ameliorate these complications, implicating the essential role of Nrf2 in protecting against pristane-induced LN. Download .jpg (.18 MB) Help with files Supplementary Figure 1 Glomerular lesions, such as mesangial cell proliferation, mesangium expansion, and infiltration of inflammatory cells, were observed in both Nrf2+/+ and Nrf2-/- mice following pristane injection (Figure 3). One notable observation is that untreated Nrf2-/- mice displayed a similar degree of glomerular damage as the pristane-treated wild-type mice (Figure 3, compare panel B with C). Pristane treatment induced Nrf2 expression in glomeruli of Nrf2+/+ mice (Figure 3, compare panel E with F). As expected, Nrf2 was not detected within glomeruli in Nrf2-/- mice (Figure 3). Furthermore, treatment with pristane increased NQO1 expression in Nrf2+/+ mice (Figure 3, compare panel I with J). Although NQO1 was not detected in the untreated Nrf2-/- mice, it was detected in some glomeruli of pristane-treated Nrf2-/- mice, indicating the possible activation of NQO1 by an Nrf2-independent pathway (Figure 3, compare panel K with L). Nrf2-/- mice were more sensitive to pristane-induced oxidative DNA damage than Nrf2+/+ mice (Figure 3, compare panel N with P). Interestingly, Nrf2-/- mice displayed higher levels of oxidative damage even in the untreated condition (Figure 3, compare panel M with O), suggesting that basal levels of Nrf2 are essential for the protection of cells from DNA damage. In addition, administration of sulforaphane ameliorated the pathological alterations in glomeruli of Nrf2+/+ mice induced by pristane (Supplementary Figure 2C–F online). Moreover, sulforaphane induced activation of Nrf2 and inhibited the deposition of fibronectin and production of 8-Oxo-dG within glomeruli (Supplementary Figure 2G–V online). The pathological alterations were assessed and scored (Figure 3R, Supplementary Figure 2B online). Although some pathology end points were not significantly protected by sulforaphane, the trend is there. All stained sections were analyzed by the i-Solution software (Figure 3, *P<0.05 vs. control; #P<0.05 vs. Nrf2 +/+ mice). Similarly, sulforaphane ameliorated the oxidative stress and deposition of extracellular matrix (Supplementary Figure 2A online). Collectively, these results demonstrate that Nrf2 is essential in protecting against both basal and pristane-induced renal injuries. Download .jpg (.33 MB) Help with files Supplementary Figure 2 The mRNA level of key factors of glomerulosclerosis, TGFβ1 and fibronectin, were measured in order to explore the role of Nrf2 in LN. Consistent with our previous report,19.Jiang T. Huang Z. Lin Y. et al.The protective role of Nrf2 in streptozotocin-induced diabetic nephropathy.Diabetes. 2010; 59: 850-860Crossref PubMed Scopus (326) Google Scholar the basal mRNA level of TGFβ1 was higher in Nrf2-/- mice than in Nrf2+/+ mice (Figure 4a, TGFβ1 panel). Similarly, the level of fibronectin was also elevated in Nrf2-/- mice (Figure 4a, FN panel). In response to pristane treatment, the mRNA levels of TGFβ1 and fibronectin were markedly increased in Nrf2-/- mice (Figure 4a, TGFβ1 and FN panel). The inflammation-associated enzyme iNOS also showed a similar expression pattern with TGFβ1 (Figure 4a, iNOS panel). Sulforaphane suppressed the mRNA level of TGFβ1, fibronectin, and iNOS after pristane treatment, but induced the transcription of NQO1 (Supplementary Figure 1C–F online). As expected, the protein level of NQO1 was increased by pristane treatment in Nrf2+/+, but not in Nrf2-/-, mice (Figure 4b), implicating the existence of oxidative stress in the murine LN model. The protein level of fibronectin was increased after treatment of pristane, and it was higher in Nrf2-/- mice than in Nrf2+/+ mice (Figures 4b and c). In addition, the protein level of Nrf2 and NQO1 was induced in Nrf2+/+ mice with administration of sulforaphane (Supplementary Figure 1G online). More importantly, the inflammation-related pathway, NF-κB, showed increased activation in Nrf2-/- mice compared with Nrf2+/+ mice after pristane treatment (Figure 4b). Taken together, these data demonstrate that Nrf2-/- mice have excessive expression of TGFβ1, fibronectin, and iNOS after pristane treatment. In addition, the increased activation of NF-κB in Nrf2-/- mice may explain the more severe renal injuries in Nrf2-/- mice and suggests that Nrf2 may negatively regulate NF-κB. To further confirm the negative effects of Nrf2 on the NF-κB and TGFβ1 pathways, an in vitro cell culture system was used. Primary mesangial cells were isolated from MRL/lpr mice that are prone to developing lupus. The monoclonal antibody R4A, an anti-dsDNA antibody that was found to be nephritogenic, was used to treat the primary mesangial cells. A non-nephritogenic MOPC-141 antibody was included as a negative control. As expected, expression of Nrf2 and NQO1 decreased significantly after Nrf2-siRNA transfection (Figures 5a and i). Interestingly, knockdown of Nrf2 increased the expression of iNOS. R4A treatment did not change the expression of Nrf2 and NQO1, but increased the expression of iNOS in cells transfected with either control-siRNA or Nrf2-siRNA (Figure 5a). The basal level of ROS production was minimally changed after Nrf2 knockdown (Figure 5b). Conversely, when cells were treated with R4A, the ROS level was increased in cells transfected with control-siRNA and increased even more in cells transfected with Nrf2-siRNA (Figure 5b). R4A had no effect on the Nrf2 mRNA level (Figure 5c). However, the NQO1 mRNA expression was induced by R4A treatment and this response was abrogated by knocking down of Nrf2 (Figure 5d). iNOS mRNA level was markedly induced by R4A treatment (Figure 5e). Knockdown of Nrf2 increased the mRNA level of iNOS in both MOPC-141- and R4A-treated cells. These data support our in vivo findings demonstrating an increase in the iNOS mRNA level in Nrf2-/- mice. Similar to iNOS expression, the mRNA expression of TGFβ1 was negatively regulated by Nrf2 (Figure 5f). R4A increased TGFβ1's mRNA levels, which was further increased by Nrf2 knockdown (Figure 5f). Given the fact that iNOS is one of the direct target genes of the NF-κB pathway, the effect of Nrf2 on this pathway was measured. Phosphorylation of p65 and IκB was measured to detect activation of the NF-κB signaling pathway. Treatment of cells with R4A resulted in an increase in the level of phosphorylated p65 and IκB, whereas the total amount of p65 and IκB remained unchanged (Figure 5g). Reduction of Nrf2 by transfection of Nrf2-siRNA resulted in a much stronger activation of the NF-KB signaling pathway in response to R4A treatment (Figure 5g). Next, a κB reporter plasmid was transfected into cells with either overexpression or knockdown of Nrf2 to further confirm the negative correlation between Nrf2 and the NF-κB pathway. R4A activated the κB-dependent luciferase activity, which was reduced by ectopic expression of Nrf2 in a dose-dependent manner (Figure 5h), and induced by decreased expression of Nrf2 (Figure 5i). Collectively, these data indicate that Nrf2 negatively regulates the R4A-induced activation of the NF-κB and TGFβ1 signaling pathways, thus suppressing the expression of the downstream genes of these two pathways. Next, we determined whether the increased production of ROS and expression of iNOS induced by R4A in Nrf2-/- cells is mediated by the NF-κB pathway. An NF-κB p65 inhibitory peptide was used to block the R4A-triggered NF-κB pathway (Figure 6a). In the mesangial cells from MRL/lpr mice that have Nrf2 knockdown, the R4A-induced production of ROS was blocked by incubating the cells with p65 inhibitory peptide (Figure 6b, #P<0.05). Treatment with R4A significantly induced the mRNA expression of NQO1; however, there was no significant difference between the control and p65 inhibitor–treated groups (Figure 6c). Although the mRNA level of TGFβ1 markedly increased after R4A treatment, incubation with the p65 inhibitory peptide did not affect its expression (Figure 6e). However, p65 inhibition blocked the transcription of iNOS (Figure 6d, #P<0.05) and also decreased the protein level (Figure 6f). These data indicated that the NF-κB pathway is important for regulating ROS release and the expression of iNOS triggered by R4A when Nrf2 levels are low. They also suggest that increased activation of the NF-κB pathway in Nrf2-deficient mice or cells can account for the more severe renal injuries. Although the Nrf2-antioxidant response element signaling pathway has proved to be useful in combating several diseases,20.Zhang D.D. The Nrf2-Keap1-ARE signaling pathway: the regulation and dual function of Nrf2 in cancer.Antioxid Redox Signal. 2010; 13: 1623-1626Crossref PubMed Scopus (140) Google Scholar, 21.Hu R. Saw C.L. Yu R. et al.Regulation of Nrf2 signaling for cancer chemoprevention: antioxidant coupled with anti-inflammatory.Antioxid Redox Signal. 2010; 13: 1679-1698Crossref PubMed Scopus (165) Google Scholar the role of Nrf2 in LN remains unclear. Previous studies have shown that Nrf2-/- mice spontaneously develop lupus-like autoimmune nephritis at 60 weeks.22.Yoh K. Itoh K. Enomoto A. et al.Nrf2-deficient female mice develop lupus-like autoimmune nephritis.Kidney Int. 2001; 60: 1343-1353Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar These findings suggest that the loss of Nrf2 promotes the development of LN, indicating a preventive or protective role for Nrf2. Conflicting data, however, were obtained from the same group when they used a genetic Nrf2-/- lpr/lpr mouse model.23.Morito N. Yoh K. Hirayama A. et al.Nrf2 deficiency improves autoimmune nephritis caused by the fas mutation lpr.Kidney Int. 2004; 65: 1703-1713Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Nrf2-/- lpr/lpr mice lived longer and showed an improvement in the nephritis manifestation compared with Nrf2+/+ lpr/lpr mice. This study suggests that Nrf2 aggravates lupus. In addition, two other studies demonstrated that Nrf2-/- mice, with the same background that Takahashi’s group used, developed a lupus-like autoimmune disease with renal injuries.24.Li J. Stein T.D. Johnson J.A. Genetic dissection of systemic autoimmune disease in Nrf2-deficient mice.Physiol Genomics. 2004; 18: 261-272Crossref PubMed Scopus (130) Google Scholar, 25.Ma Q. Battelli L. Hubbs A.F. Multiorgan autoimmune inflammation, enhanced lymphoproliferation, and impaired homeostasis of reactive oxygen species in mice lacking the antioxidant-activated transcription factor Nrf2.Am J Pathol. 2006; 168: 1960-1974Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar Thus, the role of Nrf2 in the development of LN is still unclear and further studies are needed. In our study, we demonstrate that Nrf2 protects against LN by inhibiting the activation of the NF-κB pathway and deposition of extracellular matrix. First, the LN patients manifested increased oxidative stress in the kidney, consistent with the previous report by Moroni et al.26.Moroni G. Novembrino C. Quaglini S. et al.Oxidative stress and homocysteine metabolism in patients with lupus nephritis.Lupus. 2010; 19: 65-72Crossref PubMed Scopus (35) Google Scholar Despite the increased levels of Nrf2, NQO1, and 8-Oxo-dG were observed in all types of LN, they are more likely a result of compensatory mechanism and may be surpassed by the oxidative stress, and therefore they are not able to prevent renal injuries. Moreover, there is no significant correlation between the severity of LN and the expression of Nrf2, NQO1, and 8-Oxo-dG. The insufficient case number may account for this finding, as we only had access to 4 and 7 cases in Class I and Class V, respectively. It is interesting that Córdova et al.27.Cordova E. Velazquez-Cruz R. Centeno F. et al.The NRF2 gene variant -653G/A is associated with nephritis in childhood-onset systemic lupus erythematosus.Lupus. 2010; 19: 1237-1242Crossref PubMed Scopus (58) Google Scholar reported that the two single-nucleotide polymorphisms of Nrf2 are not associated with susceptibility to childhood-onset lupus, but it could contribute to the development of kidney malfunction. This contribution is quite reasonable, as modification of Nrf2 may increase oxidative stress and promote LN. Second, in a mouse pristane-induced lupus model, Nrf2-/- mice suffered more renal damages, strengthened the activation of NF-κB pathway, and had more deposition of extracellular matrix than wild-type mice. In addition, sulforaphane, an Nrf2 inducer, prevented the pristane-induced lupus model in wild-type mice. Our data confirmed the importance of antioxidant stress and the NF-κB pathway in lupus, which is consistent with the previous report by Tsai et al.28.Tsai P.Y. Ka S.M. Chang J.M. et al.Epigallocatechin-3-gallate prevents lupus nephritis development in mice via enhancing the Nrf2 antioxidant pathway and inhibiting NLRP3 inflammasome activation.Free Radic Biol Med. 2011; 51: 744-754Crossref PubMed Scopus (202) Google Scholar In contrast to the previous reports on spontaneous formation of autoimmune nephritis in Nrf2-/- mice, we did not observe autoimmune nephritis–like phenotype in the untreated group. Presumably, this may be due to difference in the length of the individual experiments, i.e., 36 weeks in our investigation versus 12 months in the previous report.24.Li J. Stein T.D. Johnson J.A. Genetic dissection of systemic autoimmune disease in Nrf2-deficient mice.Physiol Genomics. 2004; 18: 261-272Crossref PubMed Scopus (130) Google Scholar Although Ma, et al.25.Ma Q. Battelli L. Hubbs A.F. Multiorgan autoimmune inflammation, enhanced lymphoproliferation, and impaired homeostasis of reactive oxygen species in mice lacking the antioxidant-activated transcription factor Nrf2.Am J Pathol. 2006; 168: 1960-1974Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar reported that spontaneous glomerulonephritis could appear as early as at the age of 2 months, the large-scale death of Nrf2-/- mice caused by nephritis in those studies was observed at 36 weeks. As our data highlight, the protective effects of Nrf2 on the progression of LN come from several aspects, including ROS scavenging and inhibition of the NF-κB pathway and TGFβ1 pathway. The NF-κB pathway is crucial for the development of lupus.29.Okamoto T. NF-kappaB and rheumatic diseases.Endocr Metab Immune Disord Drug Targets. 2006; 6: 359-372Crossref PubMed Scopus (70) Google Scholar It controls the expression of several genes involved in inflammatory responses, such as iNOS, which is expressed at high levels in lupus and is correlated with disease severity.30.Aktan F. iNOS-mediated nitric oxide production and its regulation.Life Sci. 2004; 75: 639-653Crossref PubMed Scopus (979) Google Scholar, 31.Wang G. Pierangeli S.S. Papalardo E. et al.Markers of oxidative and nitrosative stress in systemic lupus erythematosus: correlation with disease activity.Arthritis Rheum. 2010; 62: 2064-2072PubMed Google Scholar In our study, the activation of the NF-κB pathway and expression of iNOS were negatively regulated by Nrf2. Therefore, Nrf2 may inhibit the development of lupus by suppressing the NF-κB-mediated inflammatory response in addition to its ROS-scavenging activity. The cross talk of these two pathways has been extensively investigated, yet conflicting results still exist. Both positive and negative regulation between Nrf2 and NF-κB has been reported.32.Wakabayashi N. Slocum S.L. Skoko J.J. et al.When NRF2 talks who's listening?.Antioxid Redox Signal. 2010; 13: 1649-1663Crossref PubMed Scopus (486) Google Scholar Certainly, chemopreventive compounds can activate Nrf2 while inhibiting the NF-κB pathway and its downstream genes.16.Ahmad R. Raina D. Meyer C. et al.Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179.J Biol Chem. 2006; 281: 35764-35769Crossref PubMed Scopus (215) Google Scholar, 18.Sriram N. Kalayarasan S. Sudhandiran G. Epigallocatechin-3-gallate augments antioxidant activities and inhibits inflammation during bleomycin-induced experimental pulmonary fibrosis through Nrf2-Keap1 signaling.Pulm Pharmacol Ther. 2009; 22: 221-236Crossref PubMed Scopus (142) Google Scholar, 33.Liu Y.C. Hsieh C.W. Wu C.C. et al.Chalcone inhibits the activation of NF-kappaB and STAT3 in endothelial cells via endogenous electrophile.Life Sci. 2007; 80: 1420-1430Crossref PubMed Scopus (90) Google Scholar On the other hand, many agents or conditions have been shown to increase the activity of both Nrf2 and NF-κB.32.Wakabayashi N. Slocum S.L. Skoko J.J. et al.When NRF2 talks who's listening?.Antioxid Redox Signal. 2010; 13: 1649-1663Crossref PubMed Scopus (486) Google Scholar In addition, Yang et al.34.Yang H. Magilnick N. Lee C. et al.Nrf1 and Nrf2 regulate rat glutamate-cysteine ligase catalytic subunit transcription indirectly via NF-kappaB and AP-1.Mol Cell Biol. 2005; 25: 5933-5946Crossref PubMed Scopus (187) Google Scholar reported significantly decreased activity of NF-κB in the liver isolated from Nrf2-/- mice and a lower NF-κB-binding activity in Nrf2-/- fibroblasts. However, Chen et al.35.Chen X.L. Dodd G. Thomas S. et al.Activation of Nrf2/ARE pathway protects endothelial cells from oxidant injury and inhibits inflammatory gene expression.Am J Physiol Heart Circ Physiol. 2006; 290: H1862-H1870Crossref PubMed Scopus (336) Google Scholar reported that overexpression of Nrf2 could repress the expression of NF-κB downstream genes without affecting the activity of NF-κB. Therefore, the existing literature regarding the mutual regulation between Nrf2 and NF-κB is inconsistent, and further study is needed to elucidate the molecular mechanism. As a minor point of this report, we also observed that the Nrf2 may negatively regulate TGFβ1 and fibronectin expression both in vivo and in vitro, which is consistent with our previous finding in a diabetic mouse model and a human mesangial cell model.19.Jiang T. Huang Z. Lin Y. et al.The protective role of Nrf2 in streptozotocin-induced diabetic nephropathy.Diabetes. 2010; 59: 850-860Crossref PubMed Scopus (326) Google Scholar Further studies are needed to dissect the detailed mechanisms by which Nrf2 suppresses NF-κB and TGFβ1. In summary, our study clearly demonstrates that Nrf2 protects renal cell from developing LN by scavenging ROS and inhibiting the NF-κB and TGFβ1 signaling pathways. Renal biopsies (n=60) were obtained from the Department of Pathology, Fudan University, from 2008 to 2011. Paraffin sections (2μM) were stained with hematoxylin and eosin. Immunoglobulin deposition was detected by immunofluorescence. Two individual pathologists diagnosed all the cases. According to the WHO classification, 39 cases were categorized into class I, II, III, IV, and V of LN, and nine cases into acute proliferative glomerulonephritis, IgA nephropathy, and purpuric nephropathy (three cases each). In addition, 12 cases of normal kidney with no lupus history were selected as the negative control (Table 1). The selected clinical characteristics of patients are shown in Supplementary Table 1 online. Permission to use the fixed tissue sections for research purposes was obtained and approved by the Ethics Committee from Fudan University, China.Table 1Selected renal biopsies from LN patientsClassificationCase (N)PatientsLupus nephritis39 Class I4 Class II9 Class III9 Class IV10 Class V7Normal kidney12Total51 Open table in a new tab Download .doc (.06 MB) Help with doc files Supplementary Information Nrf2-/- mice were originally generated in Dr Kan's laboratory (University of California, San Francisco, CA). Female mice were intraperitoneally injected once with 0.5ml of pristane (Sigma, St Louis, MO), phosphate-buffered saline at 8 weeks of age, and pristine plus sulforaphane (12.5mg/kg, Santa Cruz, Dallas, TX) every other day. The mice were euthanized 28 weeks after injection, and kidneys and spleens were isolated and weighed. Kidneys were fixed for histology analysis and frozen for protein and RNA extraction. Female MRL-lpr/lpr (MRL/lpr) mice were purchased from the Institute of Zoology, Chinese Academy of Science for isolation of primary mesangial cells. Freshly voided spot urine samples and blood samples were collected. Enzyme-linked immunosorbent assay kits were used to measure urine albumin and creatinine levels (albumin: Bethyl Laboratories, Houston, TX; creatinine: Exocell, Philadelphia, PA). The UACR ratio is expressed as micrograms of albumin to milligrams of creatinine. Enzyme-linked immunosorbent assay kits were used to measure the autoantibody titers (Alpha Diagnostic, San Antonio, TX). The deposition of antibodies was measured by immunofluorescence. Both renal biopsies and kidney tissues from the animal model were analyzed. Furthermore, the stained results were analyzed by the image quantification software (IMT i-Solution, Vancouver, Canada). The information of antibodies used in this study and the methods of image quantitation are described in the Supplementary Materials online. The protocol for detection of 8-Oxo-dG was described previously.19.Jiang T. Huang Z. Lin Y. et al.The protective role of Nrf2 in streptozotocin-induced diabetic nephropathy.Diabetes. 2010; 59: 850-860Crossref PubMed Scopus (326) Google Scholar Total RNA from kidney tissues or cells was extracted using Trizol (Life Technologies, Grand Island, NY). Equal amounts of RNA (2μg) were reverse-transcribed into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN). Taqman probes and primers, and the methods used in this study, are described in Supplementary Material online. The protocol was followed as described by Dr Putterman’s group.36.Qing X. Zavadil J. Crosby M.B. et al.Nephritogenic anti-DNA antibodies regulate gene expression in MRL/lpr mouse glomerular mesangial cells.Arthritis Rheum. 2006; 54: 2198-2210Crossref PubMed Scopus (66) Google Scholar The detailed methods are described in Supplementary Material online. The nephritogenic antibody R4A was a gift from Dr Betty Diamond,37.Katz J.B. Limpanasithikul W. Diamond B. Mutational analysis of an autoantibody: differential binding and pathogenicity.J Exp Med. 1994; 180: 925-932Crossref PubMed Scopus (121) Google Scholar and the control antibody MOPC-141 was purchased from Sigma. The dichlorofluorescein for ROS detection was purchased from Sigma. The validated small interfering RNA was from Qiagen. The dual luciferase assay system is a product of Promega (Promega, Madison, WI). The NF-κB p65 inhibitory peptide was purchased from IMGENEX (IMGENEX, San Diego, CA). The detailed protocols are described in Supplementary Material online. Results are expressed as mean±s.d. Unpaired Student’s t-tests were used to compare the means of two groups. One-way analysis of variance (Tukey test) was applied to compare the means of three or more groups. The Wilcoxon (Gehan) statistical test was used to analyze the survival rate. We thank Dr Betty Diamond for providing the R4A monoclonal antibody. This work was supported by the following grants: Science and Technology Commision of Shanghai Municipality (11ZR1402400 to TJ), National Institute of Environmental Health (ES015010 to DDZ), Nation Cancer Institute (CA154377 to DDZ), and National Natural Science Foundation of China (81228023 to DDZ and HZ). Table S1. Selected clinical characteristics of patients in the study group. Table S2. Primers and probes used for mice and humans in qRT-PCR amplification. Figure S1. Inhibited UACR and expression of TGFb1, iNOS, and FN after administration of sulforaphane. Figure S2. Renal damage induced by pristane treatment is ameliorated by sulforaphane. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki
Referência(s)