Long-term clinicopathologic observation in a case of steroid-resistant nephrotic syndrome caused by a novel Crumbs homolog 2 mutation.

Recent advances in high-throughput sequencing for clinical genetic testing have revealed novel disease-causing genes, such as Crumbs homolog 2 (CRB2) for early-onset steroid-resistant nephrotic syndrome (SRNS). We report the long-term clinicopathologic observation of a Japanese female patient with SRNS caused by a newly identified compound heterozygous mutation of CRB2 (p.Arg628Cys and p.Gly839Trp located in the 10th and 11th epidermal growth factor-like domains, respectively). She was initially examined during a mass urinary screening for 3.5-year-old children in Japan. Although she developed long-standing SRNS without any extrarenal clinical signs thereafter, her renal function was well-preserved over the next 17 years. In total, six sequential renal biopsy specimens revealed histologic alterations ranging from minor glomerular abnormalities to advanced focal segmental glomerulosclerosis (FSGS). A genetic analysis for SRNS performed at 19 years of age revealed a newly identified compound heterozygous mutation in CRB2. Glomerular CRB2 immunoreactivity in biopsy specimens from the patient was scanty, whereas intense expression was observed in those from patients with idiopathic FSGS or in controls. To our knowledge, this is the first report regarding a long-term outcome in a case of SRNS due to an identified CRB2 mutation. Although the phenotype of CRB2 mutation-related syndrome is now expanding, we believe that this case might provide a novel clinicopathologic aspect of this syndrome.

Cylindromatosis (CYLD), a Deubiquitinase, Attenuates Inflammatory Signaling Pathways by Activating Toll-Like Receptor 3 in Human Mesangial Cells.

BACKGROUND/AIMS: Cylindromatosis (CYLD), a deubiquitinase, negatively regulates nuclear factor-κB in various cells. However, its potential roles in glomerular inflammation remain unclear. Because the activation of the Toll-like receptor 3 (TLR3)/type I interferon (IFN) pathways plays a pivotal role in chronic kidney diseases (CKD), we examined the role of CYLD in the TLR3 signaling in cultured human mesangial cells (MCs). METHODS: We stimulated CYLD-silenced MCs with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of dsRNA, and studied representative TLR3/IFN-ß pathways (i.e., TLR3/IFN-ß/retinoic acid-inducible gene-I (RIG-I)/CCL5, and TLR3/IFN-ß/melanoma differentiation associated gene 5 (MDA5)/CXCL10 axes) using RT-PCR, western blotting, and ELISA. We also used immunofluorescence staining and microscopy to examine mesangial CYLD expression in biopsied specimens from patients with CKD. RESULTS: CYLD silencing resulted in an increase of poly IC-induced RIG-I and MDA5 protein levels and increased CCL5 and CXCL10 mRNA and protein expression, but unexpectedly decreased mRNA expressions of RIG-I and MDA5. Interestingly, CYLD silencing did not affect IFN-ß or the phosphorylated STAT1 (signal transducers and activator of transcription protein 1). CYLD was highly expressed in biopsied specimens from patients with proliferative lupus nephritis (LN). CONCLUSION: CYLD inhibits post-transcriptional regulation of RIG-I and MDA5 expression following TLR3 activation in MCs. CYLD may be involved in the pathogenesis of CKD, especially pathogenesis of LN.

Clarithromycin attenuates the expression of monocyte chemoattractant protein-1 by activating toll-like receptor 4 in human mesangial cells.

BACKGROUND: Signaling pathways induced by the activation of renal toll-like receptor 4 (TLR4) play a pivotal role in chronic kidney disease (CKD). Some recent studies suggested that clarithromycin (CAM), a 14-membered ring macrolide, exerts renoprotective effects by suppressing proinflammatory chemokines. However, its beneficial effects on signaling pathways through renal TLR4 activation are unknown. METHODS: Cultured human mesangial cells (MCs) were treated with lipopolysaccharide (LPS). Expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleukin-8 (IL-8/CXCL8) was analyzed by quantitative RT-PCR and enzyme-linked immunosorbent assay. Signaling pathways affected by CAM were determined by examining the activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) by performing western blotting. RESULTS: CAM inhibited both the mRNA and protein expression of MCP-1 without cell injury but did not affect those expressions of IL-8 in LPS-stimulated MCs. Interestingly, CAM decreased p38 MAPK activation by inhibiting phosphorylation but did not affect NF-κB activation. CONCLUSION: Our results indicated that CAM exerted renoprotective effects by suppression of p38 MAPK activity and by decreasing the expression of MCP-1 in LPS-stimulated MCs. Given the implication of TLR4 signaling in CKD, CAM may be a potential treatment of choice for CKD.

Urinary excretion of sphingomyelinase phosphodiesterase acid-like 3b in children with intractable nephrotic syndrome.

Rituximab (RTX), a specific antibody to human CD20, has been successfully used to treat intractable nephrotic syndrome (NS). Recent studies have suggested a direct effect of RTX on podocytes by targeting sphingomyelinase phosphodiesterase acid-like 3b (SMPDL-3b). Thus, we examined the urinary excretion of SMPDL-3b as well as its immunoreactivity in biopsy specimens from children with intractable NS. Urine samples from six patients (five with minimal-change NS and one with focal segmental glomerulosclerosis) and from four healthy adults were examined. Glomerular immunoreactivity and urinary excretion of SMPDL3b in proteinuric NS patients decreased compared with controls. Interestingly, urine samples obtained from the same patients at the remission stage after RTX treatment showed an increase in urinary SMPDL-3b excretion compared with the proteinuric stage. Urinary excretion level of SMPDL-3b could thus be used to predict the clinical efficacy of RTX treatment in NS patients.

Chloroquine attenuates TLR3/IFN-ß signaling in cultured normal human mesangial cells: A possible protective effect against renal damage in lupus nephritis.

BACKGROUND: Chloroquine has been reported to protect against renal damage in lupus nephritis (LN); however, its detailed mechanism in glomerular inflammation remains unclear. Upregulation of the type-I interferon (IFN) system plays a pivotal role in LN pathogenesis, therefore, we examined whether chloroquine inhibits toll-like receptor 3 (TLR3)/IFN-ß signaling in cultured normal human mesangial cells (MCs). METHODS: We examined chloroquine effect on the representative TLR3/IFN-ß-signaling axis, TLR3/IFN-ß/retinoic acid-inducible gene-I (RIG-I)/CCL5 in MCs treated with polyinosinic-polycytidylic acid (poly IC), a synthetic viral dsRNA analog and analyzed the expression of these molecules using reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). Furthermore, we subjected MCs to RNA interference against NF-κB p65. RESULTS: Pretreatment of cells with chloroquine attenuated IFN-ß, RIG-I and CCL5 expression and phosphorylation of STAT1 induced by poly IC, but not IFN-ß-induced phosphorylation of STAT1 and RIG-I expression induced by IFN-ß. Knockdown of p65 inhibited the poly IC-induced IFN-ß expression, and chloroquine pretreatment decreased the nuclear poly IC-induced translocation of NF-κB p65 in MCs. CONCLUSION: These results suggest that chloroquine attenuates mesangial TLR3 signaling in the early phase of NF-κB activation. Considering that TLRs/type-I IFNs signaling is implicated in LN pathogenesis, our results may further support regional renoprotective effects of chloroquine in treating LN.

Monitoring of Epstein-Barr virus load and killer T cells in patients with juvenile idiopathic arthritis treated with methotrexate or tocilizumab.

OBJECTIVES: Methotrexate (MTX) is used for the treatment of polyarticular juvenile idiopathic arthritis (JIA), and an anti-interleukin-6 receptor monoclonal antibody (tocilizumab: TCZ) is also used and added for the treatment of intractable JIA. It has been reported that MTX might induce Epstein-Barr virus (EBV)-associated lymphoma, but the discussion about the effect of MTX and/or TCZ against reactivation of EBV in pediatric patients has been incomplete. METHODS: The EBV loads in four polyarticular JIA and three systemic arthritis JIA patients treated with MTX and/or TCZ, and the percentage of EBV-specific killer T cells (EBV-CTLs) in some patients were prospectively monitored. RESULTS: No patients had EBV-associated symptoms during the observation period. EBV loads in all patients were not significantly increased, and the levels of EBV loads were the same as EBV-seropositive healthy children following the administration of MTX and/or TCZ. EBV-CTLs were detectable during the observation period, but some patients had slightly low levels of EBV-CTLs. CONCLUSION: Treatment with MTX and/or TCZ did not severely affect EBV load and prevent induction of EBV-CTLs in JIA patients.

Interferon (IFN)-Induced Protein 35 (IFI35), a Type I Interferon-Dependent Transcript, Upregulates Inflammatory Signaling Pathways by Activating Toll-Like Receptor 3 in Human Mesangial Cells.

BACKGROUND/AIMS: Activation of Toll-like receptor 3 (TLR3) signaling followed by type I interferon (IFN) expression is crucial in antiviral and "pseudoviral" immune reactions in renal mesangial cells (MCs). These reactions are probably involved in the pathogenesis of chronic kidney disease (CKD). However, the role of IFN-induced 35-kDa protein 35 (IFI35), a type I IFN-dependent transcript, in glomerular inflammation is unclear. Here, we aimed to investigate the expression and the role of IFI35 in IFN-ß/retinoic acid-inducible gene-I (RIG-I)/CCL5 and IFN-ß/melanoma differentiation-associated gene 5 (MDA5)/CXCL10 axes in MCs. METHODS: We treated human MCs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, then analysed the IFI35 expression by reverse transcription-polymerase chain reaction and western blotting. To examine the regulation of IFI35 expression, we subjected MCs to RNA interference (siRNA) against IFN-ß, RIG-I, and MDA5. RESULTS: Activation of TLR3 by poly IC induces the IFI35 expression in MCs. siRNA against IFN-ß inhibited poly IC-induced IFI35 expression. Knockdown of IFI35 resulted in a decrease of poly IC-induced RIG-I and MDA5 protein as well as decreased CCL5 and CXCL10 mRNA and protein expression. However, it did not affect the expression of none of phosphorylated signal transducers or activator of transcription (STAT) 1 protein, or RIG-I and MDA5 in mRNA levels. CONCLUSION: Regional expression of IFI35 and its dysregulation may be involved in the pathogenesis of glomerular inflammation in CKD.

Does Dent disease remain an underrecognized cause for young boys with focal glomerulosclerosis?

Focal glomerulosclerosis (FGS) is a histologic entity that causes significant proteinuria in children. Although its etiology varies, recent reports indicated that some young male patients with FGS had underlying Dent disease. We describe the case of a 14-year-old Japanese boy who presented with persistent non-nephrotic range proteinuria, hematuria, and renal insufficiency. The patient was initially diagnosed as having FGS associated with scattered tubulointerstitial fibrosis. Although he had neither nephrocalcinosis nor family history of renal disease including urolithiasis, increased excretion of urinary ß2 microglobulin was noted. Genetic analysis for Dent disease indicated a mutation (c.726 + 1G > A) in Chloride Channel, Voltage-Sensitive 5 (CLCN5). Given a recent hypothesis that Dent disease may be underrecognized in children with FGS, a careful diagnostic evaluation for possible underlying Dent disease should be considered in young boys who present with persistent albuminuria associated with high-grade low-molecular-weight proteinuria.

Alteration in the podoplanin-ezrin-cytoskeleton linkage is an important initiation event of the podocyte injury in puromycin aminonucleoside nephropathy, a mimic of minimal change nephrotic syndrome.

Podoplanin was identified as a protein associated with the transformation of arborized foot processes of glomerular epithelial cells (podocytes) to flat feet. However, the function of podoplanin in the podocyte is not yet fully clarified. In this study, we analyzed the molecular nature of podoplanin, and its expression in rat nephrotic models and patients with minimal change nephrotic syndrome (MCNS). We demonstrated here that podoplanin has two forms: one contains abundant sialic acid and the other a lesser amount of sialic acid. Podoplanin bound ezrin to interact with the cytoskeleton. The silencing of podoplanin in cultured podocytes caused a change in the cell shape and the distribution of ezrin and actin. The expression of podoplanin was clearly reduced before the onset of proteinuria in puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, and the decrease in the expression of podoplanin became more evident at the proteinuric stage. Podoplanin was detected in normal urine samples, and the amount of urinary podoplanin markedly increased on day 1 of PAN nephropathy. Urinary ezrin was also detected. The amount of the phosphorylated ezrin was reduced, while the amount of the podoplanin-interacting ezrin increased. The podoplanin expression was reduced in a patient with active-phase MCNS. It is conceivable that the alteration of the podoplanin-ezrin-cytoskeleton linkage is an important event of the podocyte injury in MCNS.

Toll-like receptor 3 signaling contributes to the expression of a neutrophil chemoattractant, CXCL1 in human mesangial cells.

BACKGROUND: Mesangial proinflammatory chemokine/cytokine expressions via innate immunity play a pivotal role in the pathogenesis of glomerulonephritis. CXCL1/GROα is a strong neutrophil chemoattractant cytokine and reportedly plays an important role in regional inflammatory reactions. However, detailed signaling of mesangial CXCL1 expression induced by viral or "pseudoviral" immunity remains to be determined. METHODS: We treated normal human mesangial cells (MCs) in culture with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of CXCL1 by reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR and enzyme-linked immunosorbent assay. To elucidate the poly IC-induced signaling pathway for CXCL1 expression, we subjected the cells to RNA interference against Toll-like receptor (TLR) 3, retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), interferon (IFN)-ß, nuclear factor (NF)-κB p65 and IFN regulatory factor (IRF) 3. We also conducted an immunofluorescence study to examine mesangial CXCL1 expression in biopsy specimens from patients with lupus nephritis (LN) and IgA nephropathy (IgAN). RESULTS: We found that activation of TLR3 signaling could induce the expression of CXCL1 in MCs. NF-κB, IRF3 and IFN-ß, but neither RIG-I nor MDA5, were found to be involved in mesangial CXCL1 expression in this setting. Induction of CXCL1 by poly IC was inhibited by pretreatment of cells with dexamethasone. Intense glomerular CXCL1 expression was observed in biopsy specimens from patients with LN, whereas only a trace staining occurred in specimens from patients with IgAN. CONCLUSION: TLR3 signaling also contributes to the CXCL1 expression in MCs. These observations further support the implication of viral and "pseudoviral" immunity in the pathogenesis of inflammatory renal diseases, especially in LN.

Tumor necrosis factor-α synergistically enhances polyinosinic-polycytidylic acid-induced toll-like receptor 3 signaling in cultured normal human mesangial cells: possible involvement in the pathogenesis of lupus nephritis.

AIM: It has been reported that tumor necrosis factor (TNF)-α plays dual controversial roles, beneficial or detrimental, in the pathogenesis of murine lupus nephritis (LN). However, its precise role in the development of human LN remains to be determined. METHODS: We examine the effect of pretreatment with TNF-α on the toll-like receptor 3 (TLR3) signaling induced by polyinosinic-polycytidylic acid (poly IC), a synthetic analog of viral dsRNA that makes "pseudoviral" infection in cultured normal human mesangial cells, and analyzed the expression of CC chemokine ligand 5 (CCL5) via TLR3/interferon (IFN)-ß/retinoic acid-inducible gene-I (RIG-I) pathway by reverse transcriptase-polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay. RESULTS: We found synergistic effect of TNF-α, even at low level, on the expression of CCL5 induced by poly IC in a concentration-dependent manner, in comparison with that by poly IC alone. Knockdown of either IFN-ß or RIG-I decreased CCL5 expression induced by TNF-α followed by poly IC. CONCLUSION: Pretreatment with TNF-α leads marked activation of the TLR3/IFN-ß/RIG-I/CCL5 axis induced by "pseudoviral" infection. Since chronic local activation of proinflammatory cytokines including TNF-α in resident renal cells may exist in patients with active lupus, synergistic effect of TNF-α and "pseudoviral" infection is possibly involved in the development of LN.

Expressions of mRNA for innate immunity-associated functional molecules in urinary sediment in immunoglobulin A nephropathy.

AIM: It has been reported that the innate immune system plays a pivotal role in the pathogenesis of immunoglobulin A nephropathy (IgAN). To explore non-invasive monitoring of disease activity in children with IgAN, we examined whether expressions of mRNA for innate immunity-associated functional molecules: CC ligand chemokine 5 (CCL5), fractalkine/CX3CL1, interferon-γ-induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), retinoic acid-inducible gene-I (RIG-I), and toll-like receptor 3 (TLR3) in urinary sediment from patients with IgAN correlate with histologic parameters. METHODS: Twenty consecutive children with IgAN and four children with thin basement membrane disease (serving as a non-inflammatory control) were enrolled in this pilot study. Urinary mRNA expressions of target genes were examined real-time quantitative polymerase chain reaction. RESULTS: The expressions of CCL5, fractalkine and RIG-I were significantly increased in IgAN (all P < 0.05). Although no significant correlation was observed between mRNA expressions of these three molecules and clinical parameters, such as levels of urinary protein excretion, degrees of occult blood in urine and serum albumin, the expression of fractalkine was significantly correlated with the histological activity index (P = 0.022) and the chronicity index (P = 0.005). Furthermore, intense glomerular immune activity of fractalkine was observed in biopsy specimens in patients with moderately to severe proliferative IgAN. CONCLUSION: Regional expression of fractalkine may be involved in the pathogenesis of childhood IgAN. Although our present findings remain preliminary, measurement of mRNA expression of fractalkine in urinary sediment could be used as a non-invasive method for predicting histologic severity in IgAN in children. Further studies of this issue are needed.

Severe intrinsic acute kidney injury associated with therapeutic doses of acetaminophen.

Acetaminophen is a commonly used medication to manage fever and pain in children and the drug is generally considered to be safe when used at appropriate therapeutic dosages. Recently, we encountered the case of a 3-year-old Japanese girl who suffered from severe intrinsic acute kidney injury (AKI) after therapeutic doses of acetaminophen for a fever due to viral infection. Renal biopsy indicated severe acute tubular necrosis with a significant striped interstitial fibrosis and mild interstitial inflammation. Unfortunately, she developed chronic kidney disease thereafter. This is the youngest case of biopsy-proven severe intrinsic AKI associated with therapeutic doses of acetaminophen. Acetaminophen, even if administered at therapeutic dosages, may be dangerous in selected children, especially with possible pre-existing volume depletion.

Mizoribine selectively attenuates monocyte chemoattractant protein-1 production in cultured human glomerular mesangial cell: a possible benefit of its use in the treatment of lupus nephritis.

AIM: Mizoribine (MZR) is a selective inhibitor of the inosine monophosphate dehydrogenase - a key enzyme in the de novo pathway of guanine nucleotides - that was developed in Japan. Besides its immunosuppressive effects, MZR has recently been reported to suppress the progression of histologic chronicity via suppression of macrophage infiltration of the interstitium in selected patients with lupus nephritis. METHODS: We examine the direct effect of MZR in human mesangial cells on the expression of functional molecules including monocyte chemoattractants in cultured human mesangial cells (MCs) treated with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of viral dsRNA, that makes 'pseudoviral' infection, and analyzed the expression of target molecules by reverse transcriptase-polymerase chain reaction and Western blotting. Thereafter, the effect of MZR on the expressions was examined. RESULTS: Pretreatment of cells with MZR partially, but significantly, attenuates the expression of monocyte chemoattractant protein (MCP)-1 mRNA and protein, whereas the poly IC-induced expressions for the other functional molecules, such as CCL5, fractalkine and IL-8 were not influenced by MZR treatment. On the other hand, pretreatment of cells with tacrolimus did not suppress the expression of MCP-1 mRNA. CONCLUSION: Mizoribine itself selectively attenuated the expression of MCP-1 both mRNA and protein levels in MCs treated with poly IC; that is, a possible model of 'pseudoviral' infection, which may be involved in the pathogenesis of lupus nephritis.

Interaction between interferon-stimulated gene 56 and melanoma differentiation-associated gene 5 in Toll-like receptor 3 signaling in normal human mesangial cells.

BACKGROUND/AIMS: Toll-like receptor 3 (TLR3) is a pathogen recognition receptor against viral double-stranded RNA. TLR3 signaling is important in antiviral responses, but inappropriate TLR3 signaling may be related with inflammatory renal diseases. Interferon (IFN)-stimulated gene 56 (ISG56) is an IFN-inducible gene that encodes a multifunctional protein with 6 tetratricopeptide motifs and is thought to be involved in antiviral reactions, but the role of ISG56 in TLR3 signaling in mesangial cells is not known well. METHODS: Normal human mesangial cells were cultured and treated with a synthetic TLR3 ligand polyinosinic-polycytidylic acid, and the expression of ISG56 was analyzed using real-time RT-PCR and Western blot analyses. Using an RNA-interfering technique, involvement of TLR3, IFN-ß, melanoma differentiation-associated gene 5 (MDA5) or retinoic acid-inducible gene-I (RIG-I) in ISG56 expression, and of ISG56 in the expression of MDA5, RIG-I, CXCL10 and CCL5 was examined. RESULTS: Treatment of cells with polyinosinic-polycytidylic acid induced ISG56. ISG56 induction was inhibited by knockdown of TLR3 or IFN-ß, and knockdown of ISG56 resulted in the decreased expression of MDA5, RIG-I, CXCL10 and CCL5. RNA interference against MDA5 decreased ISG56 expression. CONCLUSION: ISG56 was induced by TLR3 signaling via newly synthesized IFN-ß. ISG56 is involved in the expression of MDA5, RIG-I, CXCL10 and CCL5, and ISG56 and MDA5 may constitute a positive-feedback loop. ISG56 may play a role in immune and inflammatory reactions induced by TLR3 signaling in human mesangial cells.

Glomerular expression of fractalkine is induced by polyinosinic-polycytidylic acid in human mesangial cells: possible involvement of fractalkine after viral infection.

BACKGROUND: Viral infections often trigger the onset or worsening of glomerular diseases, but the details of this mechanism are unclear. Fractalkine/CX3CL1 (Fkn) is a chemokine that induces the chemotaxis and activation of cells expressing its receptor, CX3CR1. To examine the involvement of glomerular Fkn expression in the development of glomerulonephritis after viral infection, we conducted experimental studies using human mesangial cells (MCs) in culture. METHODS: We examined the effect of polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, on Fkn expression in MCs to investigate the involvement of Fkn in the antiviral reaction of MCs. Fkn mRNA and protein were analyzed using real-time PCR and enzyme-linked immunosorbent assay. Also, an immunofluorescent study to examine mesangial Fkn expression in biopsy specimens obtained from patients with glomerulonephritis was conducted. RESULTS: Poly IC-induced Fkn expression in MCs in both a time- and dose-dependent manner, and RNA interference (RNAi) against Toll-like receptor 3 (TLR3) or interferon regulatory factor 3 (IRF3) inhibited poly IC-induced Fkn expression. Significant glomerular Fkn expression was observed in biopsy specimens from patients with immunoglobulin A nephropathy and purpura nephritis, with increasing severity of glomerular inflammation. CONCLUSION: The TLR3/IRF3/Fkn signaling pathway may, at least in part, mediate immune and inflammatory responses against viral infection in MCs.

Glomerular expression of myxovirus resistance protein 1 in human mesangial cells: possible activation of innate immunity in the pathogenesis of lupus nephritis.

Since viral infections activate type I interferon (IFN) pathways and cause subsequent release of IFN-dependent proinflammatory chemokines and cytokines, the innate immune system plays an important role in the pathogenesis of lupus nephritis (LN). It has been reported that human myxovirus resistance protein 1 (Mx1), a type I IFN-dependent transcript, acts against a wide range of RNA viruses. Although the expression of Mx1 in biopsy specimens obtained from patients with dermatomyositis and cutaneous lupus has been described, the expression of Mx1 in human mesangial cells (MCs) has remained largely unknown. We treated normal human MCs in culture with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of Mx1 by reverse transcription-polymerase chain reaction and western blotting. To elucidate the poly IC-signalling pathway, we subjected the cells to RNA interference against IFN-ß. We also conducted an immunofluorescence study to examine mesangial Mx1 expression in biopsy specimens from patients with LN. Poly IC-induced Mx1 expression in MCs are shown both time- and dose-dependently, and RNA interference against IFN-ß inhibited poly IC-induced Mx1 expression. Intense glomerular Mx1 expression was observed in biopsy specimens from patients with LN, whereas negative staining occurred in specimens from patients with IgA nephropathy or purpura nephritis. These preliminary observations support, at least in part, the theory of innate immune system activation in the pathogenesis of LN.

A young girl with refractory intestinal Behçet's disease: a case report and review of literatures on pediatric cases who received an anti-tumor necrosis factor agent.

Behçet's disease (BD) is a chronic, relapsing, multisystem inflammatory disorder classified as vasculitis and characterized by recurrent oral and genital ulcerations, uveitis, and protean clinical signs of skin, central nervous system, musculoskeletal, and gastrointestinal involvements. Among the protean manifestations due to BD, intestinal BD is often intractable, but effective treatment for intestinal BD has not been fully established. Tumor necrosis factor (TNF)-α plays a central role in inflammation in BD patients; however, there are very few reports on the successful treatment of pediatric-onset cases of intestinal BD using anti-TNF-α agents. We report the case of a 6-year-old Japanese girl with refractory intestinal BD who was successfully treated with multidrug therapy including etanercept (ETN). Also, we performed a review of literatures on pediatric cases who received an anti-TNF-α agent. To our knowledge, this is the youngest patient with intestinal BD who was successfully treated using ETN. Although further studies are needed to determine the efficacy and safety of anti-TNF-α agents in the treatment for pediatric-onset BD, we believe that even in very young patients with refractory BD, an anti-TNF-α agent may be beneficial.

Urinary fractalkine and monocyte chemoattractant protein-1 as possible predictors of disease activity of childhood glomerulonephritis.

Renal biopsy is the gold standard for confirmation of disease severity and diagnosis of glomerulonephritis (GN), but its procedure is invasive with a risk of complications. Thus, a non-invasive monitoring method is desirable especially in pediatric patients. Fractalkine and monocyte chemoattractant protein-1 (MCP-1) are proinflammatory chemokines, and both have been reported to be involved in the pathogenesis of immunocomplex-mediated GN. Recently, it has been reported that urinary fractalkine and MCP-1 may serve as possible predictors of disease activity of adult patients with GN. We, therefore, examined whether urinary levels of fractalkine and MCP-1 correlate with clinical and histologic parameters. Twenty-six consecutive children with various types of GN were enrolled in this study, including lupus nephritis, IgA nephropathy, membranous nephropathy, acute GN, and thin basement membrane disease (served as a non-inflammatory control). Urinary fractalkine and MCP-1 concentrations in the first morning urine samples obtained at the time of renal biopsy were measured by enzyme-linked immunosorbent assay, and standardized for urinary creatinine concentrations. Urinary fractalkine concentration differed significantly among the disease categories. Urinary concentrations of fractalkine and MCP-1 showed a significant positive correlation with the degree of occult blood in urine and a significant inverse correlation with the estimated glomerular filtration rate. Furthermore, the urinary MCP-1 concentration was significantly correlated with histological chronicity indices in patients with lupus nephritis and IgA nephropathy. Measurement of urinary fractalkine and MCP-1 concentrations may be useful as a non-invasive method for predicting the disease activity of GN in children.