Gene expression of TWEAK/Fn14 and IP-10/CXCR3 in glomerulus and tubulointerstitium of patients with lupus nephritis

Aim: The role of the tumour necrosis factor‐like weak inducer of apoptosis (TWEAK)/Fn14 and interferon‐inducible protein (IP‐10)/CXCR3 axis in the pathogenesis of lupus nephritis were studied. Methods: The mRNA expression of TWEAK, Fn14, IP‐10 and CXCR3 were quantified in the glomerulus and tubulointerstitium of 42 patients with lupus nephritis (LN group) and 10 healthy controls. Results: As compared to controls, LN patients had higher glomerular expression of TWEAK and Fn14, but glomerular CXCR3 expression was lower in the LN group. Similarly, the LN group had higher tubulointerstitial expression of TWEAK and Fn14, but lower tubulointerstitial expression of CXCR3, than controls. Glomerular TWEAK expression of class V nephritis was significantly higher than class IV nephritis. Glomerular expression of CXCR3 significantly correlated with proteinuria (r = ?0.532; P = 0.019), whereas tubulointerstitial CXCR3 significantly correlated with serum creatinine (r = ?0.447; P = 0.029). Conclusion: In patients with lupus nephritis, there is an increase in intra‐renal expression of TWEAK and Fn14, and a decrease in CXCR3 expression. Intra‐renal expression of CXCR3 correlates with proteinuria and renal function. Our findings suggest that the TWEAK/Fn14 and IP‐10/CXCR3 axis may contribute to the pathogenesis of lupus nephritis.     

Roles of tumour necrosis factor‐related weak inducer of apoptosis/fibroblast growth factor‐inducible 14 pathway in lupus nephritis

As one of the manifestations of patients with systemic lupus erythematosus, lupus nephritis (LN) has high morbidity and mortality. Although the explicit mechanism of LN remains to be fully elucidated, there is increasing evidence to support the notion that tumour necrosis factor‐related weak inducer of apoptosis (TWEAK), acting via its sole receptor, fibroblast growth factor‐inducible 14 (Fn14), plays a pivotal role in such pathologic process. TWEAK/Fn14 interactions occur prominently in kidneys of LN, inducing inflammatory responses, angiogenesis, mesangial proliferation, filtration barrier injuries, renal fibrosis, etc. This review will specify the important roles of TWEAK/Fn14 pathway in the pathogenesis of LN with experimental data from cellular and animal models. Additionally, the raised levels of urinary and serum soluble TWEAK correlate with renal disease activity in patients with LN. The neutralizing antibodies targeting TWEAK or other approaches inhibiting TWEAK/Fn14 signals can attenuate renal damage in the murine lupus models. Therefore, to focus on TWEAK/Fn14 signalling may be promising in both clinical evaluation and the treatment of patients with LN.     

TWEAK, a multifunctional cytokine in kidney injury

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF superfamily that activates the Fn14 receptor. TWEAK may regulate cell proliferation, cell death, cell differentiation, and inflammation. TWEAK and Fn14 are constitutively present in the kidney. Sources of TWEAK and Fn14 include intrinsic renal cells and infiltrating leukocytes. Basal Fn14 expression is low, but Fn14 is greatly upregulated during kidney injury. TWEAK contributes to kidney inflammation promoting chemokine secretion by renal cells through canonical and non-canonical NFκB activation. TWEAK also promotes tubular cell proliferation. However, TWEAK induces mesangial and tubular cell apoptosis under proinflammatory conditions. These data indicate that TWEAK is a multifunctional cytokine in the kidney, the actions of which are modulated by the cell microenvironment. Confirmation of the role of TWEAK in kidney injury came from functional studies in experimental animal models. The TWEAK/Fn14 pathway contributed to cell death and interstitial inflammation during acute kidney injury, to glomerular injury in lupus nephritis, to hyperlipidemia-associated kidney injury, and to tubular cell hyperplasia following unilateral nephrectomy. Circulating soluble TWEAK (sTWEAK) levels are a potential biomarker of adverse outcomes in chronic kidney disease and urinary sTWEAK is a potential biomarker of lupus nephritis activity. The available evidence suggests that TWEAK may provide diagnostic information and be a therapeutic target in renal injury. Its role in human kidney disease should be further explored.     

Expression of glomerular heparan sulphate domains in murine and human lupus nephritis.

BACKGROUND: Recently, we identified specific N- and 6-O-sulphated heparan sulphate (HS) domains on activated glomerular endothelial cells. In this study, we evaluated in lupus nephritis the expression of different HS domains on glomerular endothelium and in the glomerular basement membrane (GBM). METHODS: The expression of specific glomerular HS domains and the presence of immunoglobulins (Ig) were determined by immunofluorescence staining of kidney sections of patients with nephritis due to systemic lupus erythematosus (SLE) and MRL/lpr lupus mice. The expression/presence of glomerular HS domains and Ig was also evaluated after eluting Ig from renal sections of lupus mice using two elution methods, and in renal sections of lupus mice treated with heparinoids. RESULTS: Both MRL/lpr mice and patients with lupus nephritis showed a decreased expression of HS in the GBM. The expression of N- and 6-O-sulphated HS domains on glomerular endothelium was decreased in MRL/lpr mice, but increased in SLE patients. MRL/lpr mice had more extensive glomerular Ig deposits than SLE patients. After elution of Ig, the glomerular endothelial expression of N- and 6-O-sulphated HS domains in MRL/lpr mice was recovered and even increased above normal levels, while the expression of HS in the GBM was restored to normal levels. Treatment with heparinoids prevented Ig deposition and preserved the expression of glomerular HS domains at normal levels in lupus mice. CONCLUSION: The expression of specific HS domains on glomerular endothelium and in the GBM is changed during lupus nephritis due to masking by Ig deposits and induction of inflammatory N- and 6-O-sulphated HS domains.     

Enhanced renal leukotriene production in murine lupus: role of lipoxygenase metabolites.

To investigate the potential role of leukotrienes in murine lupus, we measured renal hemodynamics and renal leukotriene production in MRL-lpr/lpr mice at 12 and 20 weeks of age. Over this age range, these animals develop overt manifestations of autoimmune disease with nephritis similar to human SLE. In the current study, we demonstrated that glomerular filtration rate (GFR) and PAH clearance (CPAH) deteriorated with age in MRL-lpr/lpr mice, but not in MRL(-)+/+ controls. Impaired renal hemodynamic function in MRL-lpr/lpr mice was associated with enhanced ionophore-stimulated production of both leukotriene B4 (LTB4) and leukotriene C4 (LTC4) by preparations of renal cortex. There was a significant inverse correlation between GFR and in vitro production of both LTC4 and LTB4 in kidneys from MRL-lpr/lpr mice, but not in control animals. In addition, in vitro LTC4 production was correlated with the severity of renal histomorphologic abnormalities. Administration of the specific peptidoleukotriene receptor antagonist SKF104353 to 20 week old MRL-lpr/lpr mice significantly improved both GFR and CPAH, whereas this agent had no effect of renal hemodynamics in MRL(-)+/+ controls. These results suggest that renal production of LTC4 and LTB4 is increased in MRL-lpr/lpr mice with nephritis, and that enhanced production of peptidoleukotrienes causes reversible renal dysfunction. Increased leukotriene production within the kidney may therefore be important in the pathogenesis of lupus nephritis.     

Circulating CSF-1 Promotes Monocyte and Macrophage Phenotypes that Enhance Lupus Nephritis

Macrophages mediate kidney disease and are prominent in a mouse model (MRL-Fas^lpr) of lupus nephritis. Colony stimulating factor-1 (CSF-1) is the primary growth factor for macrophages, and CSF-1 deficiency protects MRL-Fas^lpr mice from kidney disease and systemic illness. Whether this renoprotection derives from a reduction of macrophages and whether systemic CSF-1, as opposed to intrarenal CSF-1, promotes macrophage-dependent lupus nephritis remain unclear. Here, we found that increasing systemic CSF-1 hastened the onset of lupus nephritis in MRL-Fas^lpr mice. Using mutant MRL-Fas^lpr strains that express high, moderate, or no systemic CSF-1, we detected a much higher tempo of kidney disease in mice with the highest level of CSF-1. Furthermore, we uncovered a multistep CSF-1-dependent systemic mechanism central to lupus nephritis. CSF-1 heightened monocyte proliferation in the bone marrow (SSC^lowCD11b⁺), and these monocytes subsequently seeded the circulation. Systemic CSF-1 skewed the frequency of monocytes toward “inflammatory” (SSC^lowCD11b⁺Ly6C^high) and activated populations that homed to sites of inflammation, resulting in a more rapid accumulation of intrarenal macrophages (CD11b⁺CSF-1R⁺ or CD68⁺) that induced apoptosis of tubular epithelial cells, damaging the kidney. In humans, we found increased levels of CSF-1 in the serum, urine, and kidneys of patients with lupus compared with healthy controls. Furthermore, serum and urine CSF-1 levels correlated with lupus activity, and intrarenal CSF-1 expression correlated with the histopathology activity index of lupus nephritis. Taken together, circulating CSF-1 is a potential therapeutic target for lupus nephritis.Identifying molecules that mediate experimental lupus nephritis may uncover therapeutic targets and biomarkers. MRL-Fas^lpr mice develop a systemic autoimmune disease akin to human lupus nephritis and thus are a powerful tool to probe for molecules that regulate kidney disease in these patients.^1,2 Kidney disease in MRL-Fas^lpr mice is rapid, progressive, and predictable.³ Moreover, the time frame is sufficiently slow to tease apart the pathogenesis, and sufficiently fast to be efficient. Thus, these mice are a powerful tool to probe for therapeutic targets and biomarkers in human lupus nephritis.Macrophages (Mø) regulate kidney disease.⁴ Mø originate from pluripotent stem cells in the bone marrow that differentiate into mature monocytes (Mo), which enter the blood stream^5,6 and traffic to the kidney. Growing evidence implicates Mø as mediators of lupus nephritis because intrarenal Mø (CD68⁺, F4/80⁺) increase with advancing disease in MRL-Fas^lpr mice.⁷ Mø require the colony stimulating factor-1 (CSF-1), their principle growth factor, to differentiate, survive, and multiply.⁸ Our prior studies indicate that CSF-1 is central to lupus nephritis. Implanting cells generating CSF-1 into the kidney of MRL-Fas^lpr mice incites local Mø-rich inflammation.^9,10 Moreover, CSF-1-deficient mice (Csf1^op/op;MRL-Fas^lpr) are protected from kidney disease and systemic illness.^11,12 However, the Csf1^op/op;MRL-Fas^lpr mice are frail and have skeletal abnormalities and numerous other defects.^13–16 Thus, it is possible that the effect of deleting CSF-1 on lupus in MRL-Fas^lpr mice is, at least in part, not directly related to the reduction of Mø. Moreover, CSF-1-generating cells implanted into the kidney induce inflammation that is restricted to the area adjacent to the implant site.⁹ Thus, the systemic effect of CSF-1 during the initiation and progression of Mø-dependent lupus nephritis remains unclear.Understanding the effect of circulating and tissue CSF-1 expression is key to designing a therapeutic treatment. CSF-1 is expressed in the circulation and is upregulated in the kidney in MRL-Fas^lpr mice with lupus nephritis.^17–19 Intrarenal CSF-1 expression occurs during inflammation and expression is largely limited to tubular epithelial cells (TECs).²⁰ Moreover, the rise in circulating CSF-1 precedes intrarenal CSF-1 expression and is bimodal in MRL-Fas^lpr mice. CSF-1 is upregulated in neonates, declines to normal levels, and then progressively rises with advancing kidney disease in MRL-Fas^lpr mice.¹⁹ Moreover, an increase in CSF-1 in the circulation precedes overt kidney pathology in MRL-Fas^lpr mice.^18,19 However, it is not clear whether CSF-1 in the circulation, apart from intrarenal CSF-1, is central to the progression of lupus nephritis in MRL-Fas^lpr mice. Therefore, we propose to test the hypothesis that systemic CSF-1 hastens the progression of Mø-rich lupus nephritis. Furthermore, we hypothesize that circulating CSF-1 increases the frequency of circulating Mo (SSC^lowCD11b⁺), which are more readily recruited to the kidney and, in turn, induce injury.Finally, preclinical studies are a first step in identifying therapeutic targets and biomarkers for lupus nephritis and require validation in humans. Therefore, we propose to test the hypothesis that CSF-1 is upregulated in the circulation, urine, and kidneys of patients with active lupus nephritis.     

Platelet activating factor receptor blockade ameliorates murine systemic lupus erythematosus

Untreated 16-week-old MRL/MpJ-lpr/lpr (lpr) mice, when compared to congenic MRL/MpJ-+/+ (+/+) mice, are characterized by a systemic lupus erythematosus syndrome, including severe glomerulonephritis, proteinuria and reduction of renal function. We hypothesized that platelet activating factor (PAF), a potent chemotactic and proinflammatory phospholipid mediator synthesized and released by circulating cells, glomerular mesangial and renal medullary interstitial cells, may play a role in the development of renal injury in lupus mice. We assessed renal PAF synthesis in lpr as well as +/+ mice and the effect of treatment with a PAF receptor blocking agent. Treatment with the PAF receptor antagonist L659,989 for four weeks, starting at 12 weeks of age, significantly reduced acute glomerular infiltration and proliferation, and prevented chronic glomerular histological changes; proteinuria and serum creatinine levels were also significantly reduced in treated mice. Renal PAF production was increased in lpr when compared to +/+ mice, and treatment with L659,989 restored renal PAF synthesis to the control levels. Our results support the hypothesis that PAF can be one of the mediators of glomerular injury characteristic of murine lupus nephritis, and indicate the possible therapeutic utility of PAF receptor antagonists in immunologic renal diseases.     

Direct targeting of fibroblast growth factor-inducible 14 protein protects against renal ischemia reperfusion injury

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is known to have pivotal roles in various inflammatory processes. The TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14), has various unique functions under physiological and pathological conditions; however, the therapeutic potential of its direct targeting remains unknown. Here, we found that Fn14 expression was highly upregulated in ischemic renal tissues and tubular epithelial cells of patient biopsies and experimental animal models of renal injury. To clarify the function of Fn14 in ischemia reperfusion injury, we coincubated renal tubular cells with ITEM-2, an anti-Fn14 blocking monoclonal antibody, and found that it inhibited the production of proinflammatory cytokines and chemokines after injury. Furthermore, Fn14 blockade downregulated the local expression of several proinflammatory mediators, reduced accumulation of neutrophils and macrophages in ischemic tissues, and inhibited tubular cell apoptosis. Importantly, Fn14 blockade attenuated the development of chronic fibrosis after ischemia reperfusion injury and significantly prolonged the survival of lethally injured mice. Thus, we conclude that Fn14 is a critical mediator in the pathogenesis of ischemia reperfusion injury.     

Retinoic acid treatment protects MRL/lpr lupus mice from the development of glomerular disease

BACKGROUND: Retinoic acid (tRA) is an active metabolite of vitamin A with potent anti-inflammatory properties. We analyzed the effects of tRA on the development of lupus nephritis in MRL/lpr mice. METHODS: MRL/lpr mice received chow supplemented with vehicle or tRA (daily 10 mg/kg) from 8 to 14 weeks until their sacrifice. MRL/wt mice served as an additional control. RESULTS: tRA-treated MRL/lpr mice showed reduced lymphoadenopathy and splenomegaly as compared to vehicle-treated controls. Treatment reduced proteinuria to almost basal levels. Plasma IgG and anti-DNA antibodies increased comparably in both vehicle and tRA-treated mice. Vehicle-treated mice showed characteristic renal lesions. In contrast tRA-treated mice showed almost normal glomerular histology with a pronounced reduction in endocapillary cell proliferation. T-cell and macrophage infiltrates were reduced after tRA treatment within glomeruli and interstitium as compared to vehicle-treated animals. In spite of this, immune complex and complement deposition were comparable in both groups. Adoptively transferred T cells from vehicle-treated to tRA-treated MRL/lpr mice did not induce renal lesions or proteinuria. These beneficial effects of tRA treatment were associated with reduced renal expression of chemokines and inflammatory cytokines. Surprisingly, renal transforming growth factor-beta (TGF-beta) mRNA levels of tRA-treated mice were elevated, possibly indicating that TGF-beta acts as an anti-inflammatory signal in this lupus model. CONCLUSION: tRA treatment reduces lymphoproliferation and glomerulonephritis in MRL/lpr mice. This occurs in spite of unaltered anti-DNA titers and glomerular immune complex deposition, and cannot be overcome by T-cell transfer from nephritic MRL/lpr mice.     

G-rich DNA suppresses systemic lupus

Whereas the role of immune complexes in mediating renal cell and immune cell activation is well established, the contribution of sequence-specific immunomodulatory actions of the chromatin part remains unclear. Toll-like receptor-9 (TLR-9) mediates immunostimulatory effects of unmethylated microbial CpG-DNA. It was hypothesized that hypomethylated CpG-DNA in vertebrates may have similar effects and may contribute to disease progression in lupus nephritis. A synthetic G-rich DNA, known to block CpG-DNA effects, was used in this study. In macrophages, G-rich DNA suppressed CpG-DNA-but not LPS-induced production of CCL5 in a dose-dependent manner. Injections of G-rich DNA suppressed lymphoproliferation induced by CpG-DNA injections in mice. In MRL(lpr/lpr) mice with lupus nephritis, labeled G-rich DNA co-localized to glomerular immune complexes and was taken up into endosomes of TLR-9-positive infiltrating macrophages. Eleven-week-old MRL(lpr/lpr) mice that received injections of either saline or G-rich DNA for 13 wk revealed decreased lymphoproliferation and less autoimmune tissue injury in lungs and kidneys as compared with saline-treated controls. G-rich DNA reduced the levels of serum dsDNA-specific IgG2a as well as the renal immune complex deposits. This was consistent with the blocking effect of G-rich DNA on CpG-DNA-induced proliferation of B cells that were isolated from MRL(lpr/lpr) mice. As oligodeoxyribonucleotide 2114-treated MRL(lpr/lpr) mice were not exposed to exogenous CpG-DNA, these effects should relate to a blockade of CpG motifs in endogenous DNA. It is concluded that adjuvant activity of self-DNA contributes to the pathogenesis of lupus nephritis. Modulating the CpG-DNA-TLR-9 pathway may offer new opportunities for the understanding and treatment of lupus.     

Identification of a unique glomerular factor X activator in murine lupus nephritis.

The role of glomerular procoagulant activity (PCA) was studied in mice (MRL/lpr, NZBxWF,, and BXSB) that are known to develop lupus nephritis. In young mice (6 to 8 wk) without renal disease, there was no increase in spontaneous glomerular PCA. In contrast, older (5 to 8 mo) autoimmune mice had significant augmentation in glomerular PCA, coinciding with the histologic appearance of severe glomerulonephritis and renal fibrin deposition. The PCA was characterized as a serine protease that directly activated factor X. This factor X activator is not tissue factor because (1) expression of PCA was not dependent on factor VII; (2) a monoclonal antibody against the factor X activator inhibited glomerular PCA, but not tissue factor; (3) the molecular weight (66 kD) of the activator was different from that of tissue factor; and (4) concanavalin A inhibited tissue factor but not glomerular PCA. Immunohistochemical studies localized the factor X activator to the glomerular mesangium and capillary wall of 4- to 6-mo-old diseased MRL/lpr mice. Immunogold-labeled antibody bound to the dense deposits, macrophages, and endothelial cells of diseased glomeruli. These studies define the role of a unique glomerular factor X activator in murine lupus nephritis.     

Viral double-stranded RNA aggravates lupus nephritis through Toll-like receptor 3 on glomerular mesangial cells and antigen-presenting cells

How viral infections trigger autoimmunity is poorly understood. A role for Toll-like receptor 3 (TLR3) was hypothesized in this context as viral double-stranded RNA (dsRNA) activates dendritic cells to secrete type I interferons and cytokines that are known to be associated with the disease activity in systemic lupus erythematosus (SLE). Immunostaining of nephritic kidney sections of autoimmune MRL(lpr/lpr) mice revealed TLR3 expression in infiltrating antigen-presenting cells as well as in glomerular mesangial cells. TLR3-positive cultured mesangial cells that were exposed to synthetic polyinosinic-cytidylic acid (pI:C) RNA in vitro produced CCL2 and IL-6. pI:C RNA activated macrophages and dendritic cells, both isolated from MRL(lpr/lpr) mice, to secrete multiple proinflammatory factors. In vivo, a single injection of pI:C RNA increased serum IL-12p70, IL-6, and IFN-alpha levels. A course of 50 microg of pI:C RNA given every other day from weeks 16 to 18 of age aggravated lupus nephritis in pI:C-treated MRL(lpr/lpr) mice. Serum DNA autoantibody levels were unaltered upon systemic exposure to pI:C RNA in MRL(lpr/lpr) mice, as pI:C RNA, in contrast to CpG-DNA, failed to induce B cell activation. It therefore was concluded that viral dsRNA triggers disease activity of lupus nephritis by mechanisms that are different from those of bacterial DNA. In contrast to CpG-DNA/TLR9 interaction, pI:C RNA/TLR3-mediated disease activity is B cell independent, but activated intrinsic renal cells, e.g., glomerular mesangial cells, to produce cytokines and chemokines, factors that can aggravate autoimmune tissue injury, e.g., lupus nephritis.     

Ligands to nucleic acid-specific toll-like receptors and the onset of lupus nephritis

Lupus nephritis develops from a combination of genetic and environmental factors such as microbial infection. A role for microbial nucleic acids (e.g., via nucleic acid-specific Toll-like receptors [TLR]) was hypothesized, in this context, because microbial nucleic acids can trigger multiple aspects of autoimmunity in vitro and in vivo. Eight-week-old MRL(lpr/lpr) and MRL wild-type mice received an injection of pI:C RNA (ligand to TLR-3), imiquimod (ligand to TLR-7), or CpG-DNA (ligand to TLR-9) on alternate days for 2 wk. Only CpG-DNA triggered the onset of lupus nephritis in MRL(lpr/lpr) mice, as defined by diffuse proliferative glomerulonephritis associated with glomerular IgG and complement C3 deposition, proteinuria, and glomerular macrophage infiltrates. None of the compounds caused DNA autoantibody production or glomerulonephritis in MRL wild-type mice. The role of CpG-DNA to trigger lupus nephritis in MRL(lpr/lpr) mice was found to relate to its potent immunostimulatory effects at multiple levels: B cell IL12p40 production, B cell proliferation, double-stranded DNA autoantibody secretion, and dendritic cell IFN-alpha production. The induction of lupus nephritis by CpG-DNA is motif specific and could be prevented by co-injection of inhibitory DNA. In summary, among the ligands tested, CpG-DNA triggers lupus nephritis in genetically predisposed hosts. These data support the concept that systemic lupus erythematosus is triggered by pathogens that release CG-rich DNA.     

Thromboxane receptor blockade reduces renal injury in murine lupus nephritis.

To investigate the role of thromboxane A2 (TxA2) in murine lupus, we assessed the effects of the specific thromboxane receptor antagonist GR32191 on immune complex glomerulonephritis in MRL-lpr/lpr mice. Forty mg/kg/day GR32191 was given by twice daily subcutaneous injection for eight weeks beginning at 12 weeks of age. This dose completely blocked the renal vasoconstriction produced by the thromboxane agonist U46619. After eight weeks of treatment, both glomerular filtration rate (GFR) (8.9 +/- 0.6 vs. 6.8 +/- 1.1 ml/min/kg; P less than 0.05) and PAH clearance (CPAH) (37.4 +/- 2.5 vs. 29.9 +/- 3.3 ml/min/kg; P less than 0.05) were significantly higher in mice given GR32191 compared to vehicle treated animals. Administration of GR32191 also reduced proteinuria from 18.1 +/- 11.6 to 3.7 +/- 1.3 mg/24 hours (P less than 0.05). In GR32191 treated MRL-lpr/lpr mice, renal hemodynamic function and proteinuria were not significantly different from congenic MRL-+/+ controls. Thromboxane receptor blockade had striking affects on renal histomorphology reducing both hyaline thrombi in glomeruli (P = 0.022) and interstitial inflammation (P = 0.006). Glomerular crescents and severity of vasculitis also tended to be reduced in mice receiving the thromboxane receptor antagonist. The overall histopathologic score in mice given GR32191 was significantly lower than vehicle treated animals (4.7 +/- 0.5 vs. 8.4 +/- 1.5; P = 0.016). These effects of GR32191 were associated with decreased excretion of thromboxane B2 (TxB2) in urine (292 +/- 37 vs. 747 +/- 155 pg/24 hr; P less than 0.005) as well as a modest reduction in glomerular deposits of IgG (semiquantitative score 2.6 +/- 0.2 vs. 3.5 +/- 0.2; P less than 0.02). Thus, chronic thromboxane receptor blockade markedly altered the course of renal disease in MRL-lpr/lpr mice, suggesting that TxA2 is an important mediator of renal dysfunction and injury in this murine model of lupus nephritis.     

Cytokine cooperation in renal tubular cell injury: the role of TWEAK

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) is a member of the TNF superfamily. TWEAK activates the Fn14 receptor, and may regulate apoptosis, proliferation, and inflammation, processes that play a significant role in pathological conditions. However, there is little information on the function and regulation of this system in the kidney. Therefore, TWEAK and Fn14 expression were studied in cultured murine tubular epithelial MCT cells and in mice in vivo. The effect of TWEAK on cell death was determined. We found that TWEAK and Fn14 expression was increased in experimental acute renal failure induced by folic acid. Cultured tubular cells express both TWEAK and the Fn14 receptor. TWEAK did not induce cell death in non-stimulated tubular cells. However, in cells costimulated with TNFalpha/interferon-gamma, TWEAK induced apoptosis through the activation of the Fn14 receptor. Apoptosis was associated with activation of caspase-8, caspase-9, and caspase-3, Bid cleavage, and evidence of mitochondrial injury. There was no evidence of endoplasmic reticulum stress. A pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp prevented TWEAK-induced apoptosis, but it sensitized cells to necrosis via generation of reactive oxygen species. In conclusion, cooperation between inflammatory cytokines results in tubular cell death. TWEAK and Fn14 may play a role in renal tubular cell injury.     

Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice

MRL/MpJ-Fas(lpr)/J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8+ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.     

Toll-like receptor-7 modulates immune complex glomerulonephritis

Viral infections may trigger immune complex glomerulonephritis via Toll-like receptors (TLR), as certain TLR trigger immunity upon recognition of viral nucleic acids. On the basis of previous findings regarding viral double-stranded RNA and TLR3 in experimental lupus erythematosus, a similar role for TLR7 that recognizes viral single-stranded RNA was hypothesized. Immunostaining of kidney sections of nephritic MRLlpr/lpr mice revealed TLR7 expression in infiltrating ER-HR3-positive macrophages and few CD11c-positive dendritic cells but not in glomerular mesangial cells as observed for TLR3. This finding was consistent with the distribution pattern of intravenously injected single-stranded RNA in nephritic MRLlpr/lpr mice. TLR7 ligation activated monocytes and dendritic cells, both isolated from MRLlpr/lpr mice, to secrete IFN-alpha, IL-12p70, IL-6, and CCL2. In vivo, a single injection of the TLR7 ligand imiquimod increased serum levels of IL-12p70, IFN-alpha, and IL-6. A course of 25 microg of imiquimod given every other day from week 16 to 18 of age aggravated lupus nephritis in MRLlpr/lpr mice. This was associated with increased glomerular immune complex deposits as well as interstitial expression of CCL2 in imiquimod-treated MRLlpr/lpr mice. Different types of viral nucleic acids seem to modulate systemic autoimmunity through specific interactions with their respective TLR. Different TLR expression profiles on immune cell subsets and nonimmune parenchymal cell types determine the molecular mechanisms involved in viral infection-associated exacerbation of lupus nephritis and possibly other types of immune complex glomerulonephritis.     

Glomerular Autoimmune Multicomponents of Human Lupus Nephritis In Vivo: α-Enolase and Annexin AI

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti–α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti–α-enolase/low anti-annexin AI IgG2 and patients with low anti–α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti–α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum.