TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

Uric acid induces renal inflammation via activating tubular NF-κB signaling pathway

Inflammation is a pathologic feature of hyperuricemia in clinical settings. However, the underlying mechanism remains unknown. Here, infiltration of T cells and macrophages were significantly increased in hyperuricemia mice kidneys. This infiltration of inflammatory cells was accompanied by an up-regulation of TNF-α, MCP-1 and RANTES expression. Further, infiltration was largely located in tubular interstitial spaces, suggesting a role for tubular cells in hyperuricemia-induced inflammation. In cultured tubular epithelial cells (NRK-52E), uric acid, probably transported via urate transporter, induced TNF-α, MCP-1 and RANTES mRNA as well as RANTES protein expression. Culture media of NRK-52E cells incubated with uric acid showed a chemo-attractive ability to recruit macrophage. Moreover uric acid activated NF-κB signaling. The uric acid-induced up-regulation of RANTES was blocked by SN 50, a specific NF-κB inhibitor. Activation of NF-κB signaling was also observed in tubule of hyperuricemia mice. These results suggest that uric acid induces renal inflammation via activation of NF-κB signaling.     

The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes

In kidney, the ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is involved in podocyte injury and proteinuria but details of the mechanism underlying its regulation are not known. Activation of NF-κB is thought to be the predominant risk factor for kidney disease; therefore, it is postulated that UCH-L1 may be one of the NF-κB target genes. In this study, we investigated the involvement of NF-κB activation in the regulation of UCH-L1 expression and the function of murine podocytes. Stimulation of podocytes with the cytokines TNF-α and IL-1β up-regulated UCH-L1 expression rapidly at the mRNA and protein levels and the NF-κB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation. NF-κB up-regulates UCH-L1 via binding the ? 300 bp and ? 109 bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA–nuclear protein binding. In the renal biopsy from lupus nephritis patients, the expressions of NF-κB and UCH-L1 increased in immunohistochestry staining and were positively correlated. Activation of NF-κB up-regulates UCH-L1 expression following changing of other podocytes molecules, such as nephrin and snail. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1 in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes. This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB–UCH-L1 interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.     

Circulating TNF Receptors 1 and 2 Are Associated with the Severity of Renal Interstitial Fibrosis in IgA Nephropathy

The current study aimed to examine whether the levels of TNF receptors 1 and 2 (TNFR1 and TNFR2) in serum and urine were associated with other markers of kidney injury and renal histological findings, including TNFR expression, in IgA nephropathy (IgAN). The levels of the parameters of interest were measured by immunoassay in 106 biopsy-proven IgAN patients using samples obtained immediately before renal biopsy and in 34 healthy subjects. Renal histological findings were evaluated using immunohistochemistry. The levels of serum TNFRs were higher in IgAN patients than in healthy subjects. The levels of both TNFRs in serum or urine were strongly correlated with each other (r > 0.9). Serum TNFR levels were positively correlated with the urinary protein to creatinine ratio (UPCR) and four markers of tubular damage of interest (N-acetyl-β-D-glucosaminidase [NAG], β2 microglobulin [β2m], liver-type fatty acid-binding protein [L-FABP], and kidney injury molecule-1 [KIM-1]) and negatively correlated with estimated glomerular filtration rate (eGFR). Patients in the highest tertile of serum TNFR levels showed more severe renal interstitial fibrosis than did those in the lowest or second tertiles. The tubulointerstitial TNFR2-, but not TNFR1-, positive area was significantly correlated with the serum levels of TNFRs and eGFR. Stepwise multiple regression analysis revealed that elevated serum TNFR1 or TNFR2 levels were a significant determinant of renal interstitial fibrosis after adjusting for eGFR, UPCR, and other markers of tubular damage. In conclusion, elevated serum TNFR levels were significantly associated with the severity of renal interstitial fibrosis in IgAN patients. However, the source of TNFRs in serum and urine remains unclear.     

Decoy receptor 3 inhibits renal mononuclear leukocyte infiltration and apoptosis and prevents progression of IgA nephropathy in mice

The progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis, is associated with high levels of mononuclear leukocyte infiltration into the kidney. These cells consist mainly of T cells and macrophages. Our previous study showed that a decoy receptor 3 (DCR3) gene therapy can prevent the development of a mouse autoimmune glomerulonephritis model by its potent immune modulating effects (Ka SM, Sytwu HK, Chang DM, Hsieh SL, Tsai PY, Chen A. J Am Soc Nephrol 18: 2473-2485, 2007). Here, we tested the hypothesis that DCR3 might prevent the progression of IgAN, an immune complex-mediated primary glomerulonephritis, by inhibiting T cell activation, renal T cell/macrophage infiltration, and protecting the kidney from apoptosis. We used a progressive IgAN (Prg-IgAN) model in B cell-deficient mice, because the mice are characterized by a dramatic proliferation of activated T cells systemically and progressive NF-κB activation in the kidney. We treated the animals with short-term gene therapy with DCR3 plasmids by hydrodynamics-based gene delivery. When the mice were euthanized on day 21, we found that, compared with empty vector-treated (disease control) Prg-IgAN mice, DCR3 gene therapy resulted in 1) systemic inhibition of T cell activation and proliferation; 2) lower serum levels of proinflammatory cytokines; 3) improved proteinuria, renal function, and renal pathology (inhibiting the development of marked glomerular proliferation, crescent formation, glomerulosclerosis, and interstitial inflammation); 5) suppression of T cell and macrophage infiltration into the periglomerular interstitium of the kidney; and 5) a reduction in apoptotic figures in the kidney. On the basis of these findings, DCR3 might be useful therapeutically in preventing the progression of IgAN.     

Nuclear factor-κB activation in human monocytes stimulated with lipopolysaccharide is inhibited by fibroblast conditioned medium and exogenous PGE2

The nuclear factor κB (NF-κB) is thought to be crucially involved in the gene activation of several cytokines, including tumor necrosis factor α (TNF). Previously, we showed that fibroblast conditioned medium (FCM) is able to inhibit both TNF mRNA accumulation and protein release in peripheral blood-derived human monocytes (PBM) stimulated with lipopolysaccharide (LPS). In this study we have investigated the effect of FCM on the LPS-induced DNA-binding activity of NF-κB, by means of electrophoretic shift assay (EMSA). We provide evidence that FCM strongly inhibits the LPS-induced NFκB activation in PBM. Furthermore, we show that exogenous PGE₂ mimics the NFκB inhibitory effect of FCM. On the other hand, FCM produced in the presence of indomethacin does not inhibit NF-κB activation by LPS. Our results lend further support to the hypothesis that inflammatory and immune responses of monocytes/macrophages may be modulated at the molecular level by signals originating from tissue structural cells such as fibroblasts.     

Dose-dependent deleterious and salutary actions of the Nrf2 inducer dh404 in chronic kidney disease

Oxidative stress and inflammation play a central role in the progression and complications of chronic kidney disease (CKD) and are, in part, due to impairment of the Nrf2 system, which regulates the expression of antioxidant and detoxifying molecules. Natural Nrf2-inducing phytochemicals have been shown to ameliorate kidney disease in experimental animals. However, owing to adverse outcomes a clinical trial of a synthetic Nrf2 activator, bardoxolone methyl (BARD), in CKD patients was terminated. BARD activates Nrf2 via covalent modification of reactive cysteine residues in the Nrf2 repressor molecule, Keap1. In addition to Nrf2, Keap1 suppresses IKKB, the positive regulator of NF-κB. Treatment with a BARD analog, dh404, at 5–20 mg/kg/day in diabetic obese Zucker rats exacerbates, whereas its use at 2 mg/kg/day in 5/6 nephrectomized rats attenuates, CKD progression. We, therefore, hypothesized that deleterious effects of high-dose BARD are mediated by the activation of NF-κB. CKD (5/6 nephrectomized) rats were randomized to receive dh404 (2 or 10 mg/kg/day) or vehicle for 12 weeks. The vehicle-treated group exhibited glomerulosclerosis; interstitial fibrosis and inflammation; activation of NF-κB; upregulation of oxidative, inflammatory, and fibrotic pathways; and suppression of Nrf2 activity and its key target gene products. Treatment with low-dose dh404 restored Nrf2 activity and expression of its target genes, attenuated activation of NF-κB and fibrotic pathways, and reduced glomerulosclerosis, interstitial fibrosis, and inflammation. In contrast, treatment with a high dh404 dosage intensified proteinuria, renal dysfunction, and histological abnormalities; amplified upregulation of NF-κB and fibrotic pathways; and suppressed the Nrf2 system. Thus therapy with BARD analogs exerts a dose-dependent dimorphic impact on CKD progression.     

Stromal down-regulation of macrophage CD4/CCR5 expression and NF-κB activation mediates HIV-1 non-permissiveness in intestinal macrophages

Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.     

Visfatin contributes to the differentiation of monocytes into macrophages through the differential regulation of inflammatory cytokines in THP-1 cells

Visfatin is a novel multifunctional adipocytokine with inflammatory properties. Although a link between visfatin and atherosclerosis has recently been suggested, its actions in the development of atherosclerosis remain unknown. Therefore, we investigated a potential role and underlying mechanism(s) of visfatin in monocytes/macrophages differentiation, a critical early step in atherogenesis, using phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cell models. The co-incubation of PMA with visfatin-induced CD36 expression with a concomitant increase in the phagocytosis of latex beads compared with PMA alone treatment. Moreover, visfatin markedly increased interleukin (IL)-1β secretion by enhancing IL-1β mRNA stability in a short-term incubation. Visfatin also significantly elevated the secretion of IL-6 as well as IL-1β in a longer incubation period, which was partially suppressed by nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, and c-Jun-N-terminal kinase (JNK) inhibitor, SP600125. Furthermore, silencing IL-1β successfully blocked IL-6 secretion, CD36 expression, and NF-κB activation in response to visfatin. Collectively, these results suggest that visfatin enhances the IL-1β-dependent induction of IL-6 and CD36 via distinct signaling pathways mediated by JNK and NF-κB, respectively, and consequently, leading to the acceleration of monocytes/macrophages differentiation.     

NF-κ B激活在兔急性心肌梗死再灌注后无复流中的意义

目的：观察心肌核因子-κB（NF-κB）在急性心肌梗死（AMI）再灌注后无复流的活化情况,探讨NF-κB促进无复流发生发展的作用机制。方法：24只新西兰大白兔随机分为假手术组（冠状动脉只穿线不结扎）和缺血再灌注组（结扎冠状动脉2小时,再灌注1小时）,每组12只。采用凝胶阻滞迁移分析方法（EMSA）检测正常区、缺血区和无复流区心肌组织中NF-κB活性;ELISA法测定不同时点血浆中白细胞介素-6（IL-6）、超敏C反应蛋白（CRP）以及肿瘤坏死因子-α（TNF-α）的含量;光镜、电镜观察心肌组织病理变化。结果：（1）与正常区相比,缺血区和无复流区心肌组织中NF-κB活性异常升高（P〈0.01）。（2）与结扎前相比结扎后2h、再灌注后1h血浆IL-6、CRP、TNF-α水平呈进行性升高（P均〈0.05）。（3）NF-κB的活性与无复流面积、血浆IL-6、CRP以及TNF-α水平呈正相关（分别为r=0.844,P〈0.01;r=0.682,P〈0.05;r=0.687,P〈0.05;r=0.893,P〈0.01）。（4）无复流面积与血浆IL-6、CRP以及TNF-α水平呈正相关（分别为r=0.861,P〈0.01;r=0.806,P〈0.01;r=0.877,P〈0.01）。（5）光镜及电镜结果显示无复流区的心肌组织损伤较缺血区更为严重。结论：急性心肌梗死再灌注后无复流现象的发生可能与局部心肌组织中NF-κB的过度活化有关,活化的NF-κB通过促进IL-6、TNF-α等炎症因子的表达,参与无复流的发生发展过程。     

High expression of ubiquitin conjugates and NF-κB in unstable human intracranial atherosclerotic plaques

Arteries from the first segment of the middle cerebral and adjacent normal blood vessel specimens were collected from 19 patients who had died from cerebral infarction. According to morphologic examination the atherosclerotic plaque present was classified as stable or unstable. The expression of ubiquitin conjugates and nuclear factor kappa B (NF-κB) was assessed and found significantly higher in plaques than normal tissue and higher in unstable plaques than in stable plaques. Ubiquitin conjugates and NF-κB were positively correlated with each other. It was concluded that the ubiquitin proteasome system and NF-κB possibly play a role in the development of atherosclerotic plaques in intracranial arteries.     

β-catenin与NF-κB在结直肠腺癌中的表达及其相关性研究

目的：直肠腺癌组织中β-catenin和NF-κB的表达水平及二者与结直肠癌临床病理特征之间的相关性。方法：采用免疫组化SP法检测65例结直肠腺癌和20例正常结直肠组织中β-catenin与NF—κB蛋白表达水平。结果：结直肠癌和正常结直肠组织中β-catenin的阳性表达率分别为67．7％、0％,NF—κB的阳性率分别为75．4％、15．0％,差异均有统计学意义（P〈0．05）。结直肠癌组织中β-catenin与NF．κB的表达与患者的年龄、性别、肿瘤发生部位均无关（P〉0．05）,但与TNM分期和淋巴结转移显著相关（P〈0．05）;NF-κB的表达与组织学分化程度呈显著负相关（P〈0．05）,B-catenin的表达与肿瘤的远处转移显著相关（P〈0．05）。结直肠腺癌组织中p-catenin的表达与NF—κB的表达呈显著正相关（r=0．293,P=0．018）。结论：结直肠癌中β—catenin与NF—κB的表达异常上调,在结直肠癌发生发展中起重要作用,二者联合检测有助于结直肠癌的病情和预后评估。     

Aberrant CD40-Induced NF-κB Activation in Human Lupus B Lymphocytes

Auto-reactive B lymphocytes and its abnormal CD40 signaling play important roles in the pathogenesis of systemic lupus erythematosus (SLE). In this study, we analyzed CD40 expression and CD40/CD154 induced activation of NF-κB signaling pathway in B cells from SLE patients. B cells from healthy volunteers and tonsilar B cells from chronic tonsillitis were used as negative and positive controls. Results showed CD40-induced NF-κB signaling was constitutively activated in B cells from active lupus patients, including decreased CD40 in raft portion, increased phosphorylation and degradation of IκBα, phosphorylation of P65, as well as increased nuclear translocation of P65, P50, c-Rel, which could be blocked by anti-CD154. CD154 stimulation could induce further phosphorylation and degradation of IκBα, as well as phosphorylation of P65 and nuclear translocation of P65. In addition, CD40-induced kinase activities in B cells from lupus patients mimicked that of tonsil B cells, in that IKKα/β were more activated compared to normal B cells. CD40-induced NF-κB activity was blocked by both IκB phosphorylation and proteosome degradation inhibitors in both lupus and normal B cells. All together, our findings revealed that canonical NF-κB signaling is constitutively activated in active lupus and is mediated by CD154/CD40. CD40 induced NF-κB activation is different in human lupus B lymphocytes compared with normal B cells.     

Leishmania mexicana: participation of NF-kappaB in the differential production of IL-12 in dendritic cells and monocytes induced by lipophosphoglycan (LPG)

Dendritic cells (DC) and macrophages (Mφ) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mφ IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-α, and IL-10 and nuclear translocation of NF-κB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-κB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-κB in monocytes with the subsequent decrease in IL-12 production.