Aniela Jakubowski

The Inflammatory Cytokines TWEAK and TNFα Reduce Renal Klotho Expression through NFκB

Proinflammatory cytokines contribute to renal injury, but the downstream effectors within kidney cells are not well understood. One candidate effector is Klotho, a protein expressed by renal cells that has antiaging properties; Klotho-deficient mice have an accelerated aging-like phenotype, including vascular injury and renal injury. Whether proinflammatory cytokines, such as TNF and TNF-like weak inducer of apoptosis (TWEAK), modulate Klotho is unknown. In mice, exogenous administration of TWEAK decreased expression of Klotho in the kidney. In the setting of acute kidney injury induced by folic acid, the blockade or absence of TWEAK abrogated the injury-related decrease in renal and plasma Klotho levels. TWEAK, TNFα, and siRNA-mediated knockdown of IκBα all activated NFκB and reduced Klotho expression in the MCT tubular cell line. Furthermore, inhibition of NFκB with parthenolide prevented TWEAK- or TNFα-induced downregulation of Klotho. Inhibition of histone deacetylase reversed TWEAK-induced downregulation of Klotho, and chromatin immunoprecipitation showed that TWEAK promotes RelA binding to the Klotho promoter, inducing its deacetylation. In conclusion, inflammatory cytokines, such as TWEAK and TNFα, downregulate Klotho expression through an NFκB-dependent mechanism. These results may partially explain the relationship between inflammation and diseases characterized by accelerated aging of organs, including CKD.

The Cytokine TWEAK Modulates Renal Tubulointerstitial Inflammation

TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily of cytokines. In addition to binding and activating the fibroblast growth factor-inducible 14 receptor, TWEAK may regulate apoptosis, proliferation, and inflammation; however, the role of this system in kidney injury is unknown. In vitro, it was found that TWEAK induced the sustained activation of NF-kappaB in a murine tubular epithelial cell line (MCT). NF-kappaB activation was associated with degradation of IkappaB-alpha; translocation of RelA to the nucleus; and increased mRNA and protein expression of monocyte chemoattractant protein-1, RANTES, and IL-6. Similarly, in vivo, the systemic administration of TWEAK induced renal NF-kappaB activation, chemokine and IL-6 expression, and interstitial inflammation in mice. Parthenolide, which prevents IkappaB-alpha degradation, inhibited TWEAK-induced NF-kappaB activation and prevented the aforementioned changes in vitro and in vivo. After folic acid-induced acute kidney injury, fibroblast growth factor-inducible 14 expression increased in mouse tubular epithelium. Neutralization of TWEAK decreased the expression of chemokines in tubular cells and reduced interstitial inflammation. In conclusion, TWEAK has NF-kappaB-dependent proinflammatory effects on tubular epithelial cells in vitro and in vivo. Moreover, blockade of TWEAK reduces tubular chemokine expression and macrophage infiltration, suggesting that TWEAK modulates acute kidney injury by regulating the inflammatory response.

Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells

Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte‐mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor–like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline‐deficient, ethionine‐supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor‐inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE‐induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2‐week‐CDE‐treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin–positive cells. Stimulation of an Fn14‐positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFκB) activation and dose‐dependent proliferation, which was diminished after targeting of the p50 NFκB subunit by RNA interference. Conclusion: TWEAK acts directly and stimulates LPC mitosis in an Fn14‐dependent and NFκB‐dependent fashion, and signaling via this pathway mediates the LPC response to CDE‐induced injury and regeneration. (HEPATOLOGY 2010)

Vascular Cell Adhesion Molecule (VCAM)-Ig Fusion Protein Defines Distinct Affinity States of the Very Late Antigen-4 (VLA-4) Receptor

The Very Late Antigen-4 receptor (VLA-4) (alpha 4 beta 1) is constitutively expressed on leukocytes and plays a role in cell trafficking, activation and development through its interaction with two alternative ligands, Vascular Cell Adhesion Molecule (VCAM-1) and fibronectin (FN). VLA-4-dependent cell adhesion is augmented by various stimuli, such as divalent cations, certain beta 1-specific monoclonal antibodies (mAbs) and cell activation. However, the steps of the adhesive process which they affect are currently undefined. In order to investigate whether or not these stimuli affect the primary step, VLA-4/ligand binding, we employed a recombinant VCAM-IgG fusion protein (VCAM-Ig) as a soluble ligand for VLA-4. Using this soluble ligand, we have directly demonstrated that the VLA-4 receptor can exist in at least three different affinity states on the cell surface. Two distinct high affinity states are induced on normal peripheral blood T cells, one by the anti-beta 1 mAb TS2/16, and one of 15-20 fold higher affinity by the divalent cation Mn2+. Interestingly, activation through the T cell receptor (TcR), through CD31 or by the Macrophage Inflammatory Protein-1 beta chemokine (MIP-1 beta) do not detectably increase VLA-4 affinity although they do augment VLA-4 dependent cell adhesion in vitro. Thus, VCAM-Ig binding defines high affinity VLA-4 receptors, revealing unique effects of the TS2/16 mAb and Mn2+ cations in vitro, and distinguishes VLA-4/VCAM interactions from subsequent steps in cell adhesion.

TNF-like weak inducer of apoptosis (TWEAK) induces inflammatory and proliferative effects in human kidney cells.

Members of the TNF-ligand and receptor superfamilies are important in the pathogenesis of lupus nephritis, a major cause of mortality and morbidity in SLE. TWEAK, a member of the TNF-ligand superfamily, is markedly increased in urine from patients with active lupus nephritis, and urinary TWEAK levels significantly correlate with renal disease activity. To support a possible role of TWEAK in the pathogenesis of lupus nephritis and other inflammatory nephritides, we examined the effects of TWEAK in human kidney mesangial cells, podocytes and tubular cells, following our demonstration of the presence of the TWEAK receptor Fn14 on these cells. We found that TWEAK induces human kidney cells to express multiple inflammatory mediators, including RANTES, MCP-1, IP-10, MIP-1alpha, ICAM-1, and VCAM-1. Cytokine production is mediated through NF-kappaB activation, and is inhibited by anti-TWEAK monoclonal antibodies. TWEAK stimulated chemokines induced migration of human PBMC, particularly monocytes/macrophages. Furthermore, we found that TWEAK promotes kidney infiltration of inflammatory cells, and stimulates proliferation of kidney cells in vitro and in vivo. Thus, TWEAK may play an important pathogenic role in the development of glomerulonephritis by promoting a local inflammatory environment and inducing kidney cell proliferation. Blocking TWEAK/Fn14 interactions may be a promising therapeutic target in immune-mediated renal diseases.

A Novel Role for Tumor Necrosis Factor–Like Weak Inducer of Apoptosis (TWEAK) in the Development of Cardiac Dysfunction and Failure

Background— Tumor necrosis factor–like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, is a multifunctional cytokine known to regulate cellular functions in contexts of injury and disease through its receptor, fibroblast growth factor–inducible molecule 14 (Fn14). Although many of the processes and downstream signals regulated by the TWEAK/Fn14 pathway have been implicated in the development of cardiac dysfunction, the role of TWEAK in the cardiovascular system is completely unknown. Methods and Results— Herein, we demonstrate that mouse and human cardiomyocytes express the TWEAK receptor Fn14. Furthermore, we determine that elevated circulating levels of TWEAK, induced via transgenic or adenoviral-mediated gene expression in mice, result in dilated cardiomyopathy with subsequent severe cardiac dysfunction. This phenotype was mediated exclusively by the Fn14 receptor, independent of tumor necrosis factor-α, and was associated with cardiomyocyte elongation and cardiac fibrosis but not cardiomyocyte apoptosis. Moreover, we find that circulating TWEAK levels were differentially upregulated in patients with idiopathic dilated cardiomyopathy compared with other forms of heart disease and normal control subjects. Conclusions— Our data suggest that TWEAK/Fn14 may be important in regulating myocardial structural remodeling and function and may play a role in the pathogenesis of dilated cardiomyopathy.

TWEAK Activates the Non-Canonical NFκB Pathway in Murine Renal Tubular Cells: Modulation of CCL21

TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFκB activation suggestive of engagement of the non-canonical NFκB pathway. We now explore TWEAK-induced activation of NFκB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFα activated different DNA-binding NFκB complexes. TWEAK-induced sustained NFκB activation was associated with NFκB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFα used as control), induced a delayed increase in CCL21a mRNA (3.5±1.22-fold over control) and CCL21 protein (2.5±0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFκB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFα. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFκB2 and RelB translocation and CCL21a mRNA (1.5±0.3-fold over control) and CCL21 protein (1.6±0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2±0.9 vs 1.3±0.6-fold over healthy control) or deficiency of TWEAK (2±0.9 vs 0.8±0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1±1.4 vs 1.8±1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFκB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.

Tumor Necrosis Factor–Like Weak Inducer of Apoptosis (TWEAK) Enhances Vascular and Renal Damage Induced by Hyperlipidemic Diet in ApoE-Knockout Mice

Objective—Tumor necrosis factor–like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of cytokines. TWEAK binds and activates the Fn14 receptor, and may regulate apoptosis, inflammation, and angiogenesis, in different pathological conditions. We have evaluated the effect of exogenous TWEAK administration as well as the role of endogenous TWEAK on proinflammatory cytokine expression and vascular and renal injury severity in hyperlipidemic ApoE-knockout mice. Methods and Results—ApoE−/− mice were fed with hyperlipidemic diet for 4 to 10 weeks, then randomized and treated with saline (controls), TWEAK (10 &mgr;g/kg/d), anti-TWEAK neutralizing mAb (1000 &mgr;g/kg/d), TWEAK plus anti-TWEAK antibody (10 &mgr;g TWEAK +1000 &mgr;g anti-TWEAK/kg/d), or nonspecific IgG (1000 &mgr;g/kg/d) daily for 9 days. In ApoE−/− mice, exogenous TWEAK administration in ApoE−/− mice induced activation of NF-&kgr;B, a key transcription factor implicated in the regulation of the inflammatory response, in vascular and renal lesions. Furthermore, TWEAK treatment increased chemokine expression (RANTES and MCP-1), as well as macrophage infiltration in atherosclerotic plaques and renal lesions. These effects were associated with exacerbation of vascular and renal damage. Conversely, treatment of ApoE−/− mice with an anti-TWEAK blocking mAb decreased NF-&kgr;B activation, proinflammatory cytokine expression, macrophage infiltration, and vascular and renal injury severity, indicating a pathological role for endogenous TWEAK. Finally, in murine vascular smooth muscle cells or tubular cells, either ox-LDL or TWEAK treatment increased expression and secretion of both RANTES and MCP-1. Furthermore, ox-LDL and TWEAK synergized for induction of MCP-1 and RANTES expression and secretion. Conclusion—Our results suggest that TWEAK exacerbates the inflammatory response associated with a high lipid–rich diet. TWEAK may be a novel therapeutic target to prevent vascular and renal damage associated with hyperlipidemia.

Tweak induces proliferation in renal tubular epithelium: a role in uninephrectomy induced renal hyperplasia

The tumour necrosis factor (TNF) family member TWEAK activates the Fn14 receptor and has pro‐apoptotic, proliferative and pro‐inflammatory actions that depend on the cell type and the microenvironment. We explored the proliferative actions of TWEAK on cultured tubular cells and in vivo on renal tubules. Additionally, we studied the role of TWEAK in compensatory proliferation following unilateral nephrectomy and in an inflammatory model of acute kidney injury (AKI) induced by a folic acid overdose. TWEAK increased the proliferation, cell number and cyclin D1 expression of cultured tubular cells, in vitro. Exposure to serum increased TWEAK and Fn14 expression and the proliferative response to TWEAK. TWEAK activated the mitogen‐activated protein kinases ERK and p38, the phosphatidyl‐inositol 3‐kinase (PI3K)/Akt pathway and NF‐κB. TWEAK‐induced proliferation was prevented by inhibitors of these protein kinases and by the NF‐κB inhibitor parthenolide. TWEAK‐induced tubular cell proliferation as assessed by PCNA and cyclin D1 expression in the kidneys of adult healthy mice in vivo. By contrast, TWEAK knock‐out mice displayed lower tubular cell proliferation in the remnant kidney following unilateral nephrectomy, a non‐inflammatory model. This is consistent with TWEAK‐induced proliferation on cultured tubular cells in the absence of inflammatory cytokines. Consistent with our previously published data, in the presence of inflammatory cytokines TWEAK promoted apoptosis, not proliferation, of cultured tubular cells. In this regard, TWEAK knock‐out mice with AKI displayed less tubular apoptosis and proliferation, as well as improved renal function. In conclusion, TWEAK actions in tubular cells are context dependent. In a non‐inflammatory milieu TWEAK induces proliferation of tubular epithelium. This may be relevant for compensatory renal hyperplasia following nephrectomy.

Protection Against Adoptive Transfer of Autoimmune Diabetes Mediated Through Very Late Antigen-4 Integrin

The very late antigen-4 (VLA-4) integrin expressed on the surface of lymphocytes and macrophages can regulate their migration to inflammatory sites as well as control cellular activation. The role of VLA-4 in the establishment of autoimmune diabetes is not easily predicted given the multiplicity of adhesion pathways and their differential use by various cell types. The contribution of VLA-4 to insulin-dependent diabetes mellitus was investigated by administration of VLA-4-specific monoclonal antibodies (MoAb) in an adoptive transfer model of disease in NOD mice. This study shows that VLA-4-specific MoAbs profoundly inhibit the development of diabetes with protection sustained by repeated MoAb exposure. Insulitis was completely inhibited during treatment and progressed to a severe degree once MoAb treatment was suspended, yet ∼40% of treated recipients failed to become diabetic during 1–2 months post-treatment. Although we cannot rule out depletion of a relatively minor subpopulation of cells by prolonged anti-VLA-4 MoAb exposure, this inhibition of diabetes onset by treatment with MoAbs to VLA-4 supports a dependence on VLA-4 for cellular functions leading to diabetes and demonstrates that a significant disease modifying effect can be mediated by targeting the VLA-4 integrin.