Sphingomyelin Phosphodiesterase Acid-Like 3b is Essential for Toll-Like Receptor 3 Signaling in Human Podocytes.

Recent studies have revealed the importance of cell membrane stability in normal cell function. Sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), a lipid modifying enzyme that converts sphingomyelin to ceramide in the cell membrane, is expressed in macrophages and regulates Toll-like receptor (TLR) 4 signaling by altering cell membrane fluidity. SMPDL3b is also expressed in human podocytes, which are involved in the pathogenesis of several glomerular diseases such as diabetic kidney disease, focal segmental glomerulosclerosis, and idiopathic nephrotic syndrome in children; however, the role of SMPDL3b in podocyte innate immunity is unclear. As podocytes are equipped with innate immune systems including TLR3, and viral infections often exacerbate proteinuria in children with idiopathic nephrotic syndrome, we hypothesized that changes in SMPDL3b expression levels could affect anti-viral responses via TLR3 signaling in podocytes, consequently impairing normal podocyte function. To examine the role of SMPDL3b in TLR3 signaling in podocytes, we treated conditionally immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), to activate TLR3 signaling. The cells were then transfected with small interfering RNA against SMPDL3b. Poly IC activated the TLR3 pathway, whereas knockdown of SMPDL3b attenuated poly IC-induced interferon-ß/chemokine C-X-C ligand 10 expression in podocytes. To our knowledge, this is the first report demonstrating SMPDL3b involvement in podocyte innate immunity; these results suggest that SMPDL3b is essential for adequate anti-viral responses in podocytes, possibly by modulating lipid metabolism in the cell membrane.

IFIT Proteins Are Involved in CXCL10 Expression in Human Glomerular Endothelial Cells Treated with a Toll-Like Receptor 3 Agonist.

INTRODUCTION: Various viruses including a novel coronavirus (SARS-CoV-2) can infect the kidney. When viruses invade the glomeruli from the bloodstream, glomerular endothelial cells (GECs) initiate the innate immune reactions. We investigated the expression of interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) 1/2/3, antiviral molecules, in human GECs treated with a toll-like receptor (TLR) 3 agonist. Role of IFIT1/2/3 in the expression of C-X-C motif chemokine ligand 10 (CXCL10) was also examined. METHODS: Human GECs were cultured and stimulated with polyinosinic-polycytidylic acid (poly IC), a synthetic TLR3 agonist. Real-time qPCR, Western blotting, and ELISA were used to examine the expression of IFIT1/2/3, IFN-ß, and CXCL10. RNA interference against IFN-ß or IFIT1/2/3 was also performed. RESULTS: Expression of IFIT1/2/3 and CXCL10 was induced by poly IC in GECs. The inductions were inhibited by RNA interfering of IFN-ß. Knockdown of IFIT1/2/3 decreased the CXCL10 expression. Knockdown of IFIT3 decreased the expression of IFIT1 and IFIT2 proteins. CONCLUSION: IFIT1/2/3 and CXCL10 were induced by poly IC via IFN-ß in GECs. IFIT1/2/3 may increase the expression of CXCL10 which induces lymphocyte chemotaxis and may inhibit the replication of infected viruses. These molecules may play a role in GEC innate immune reactions in response to viruses.

Interleukin-6 via Toll-Like Receptor 3 Signaling Attenuates the Expression of Proinflammatory Chemokines in Human Podocytes.

BACKGROUND: Although toll-like receptor 3 (TLR3) signaling is involved in the development of certain chronic kidney diseases, the specific molecular mechanisms underlying inflammatory reactions via activation of TLR3 signaling in human podocytes remain unclear. Interleukin (IL)-6 is a pleiotropic cytokine associated with innate and adaptive immune responses; however, little is known about the implication of IL-6 via the activation of regional TLR3 signaling in the inflammatory reactions in human podocytes. METHODS: We treated immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, and assessed the expression of IL-6, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against IFN-ß and IL-6. RESULTS: We found that the activation of TLR3 induced expression of IL-6, MCP-1, CCL5, and IFN-ß in human podocytes. RNA interference experiments revealed that IFN-ß was involved in the poly IC-induced expression of IL-6, MCP-1, and CCL5. Interestingly, IL-6 knockdown markedly increased the poly IC-induced expression of MCP-1 and CCL5. Further, treatment of cells with IL-6 attenuated the expression of CCL5 and MCP-1 mRNA and proteins. CONCLUSION: IL-6 induced by TLR3 signaling negatively regulates the expression of representative TLR3 signaling-dependent proinflammatory chemokines in human podocytes.

Podocyte sphingomyelin phosphodiesterase acid-like 3b decreases among children with idiopathic nephrotic syndrome.

AIM: Sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b), a regulator of the cytoskeleton, is expressed on podocytes. Recent reports present evidence that it is directly targeted by rituximab in the treatment of intractable nephrotic syndrome. However, the implications of SMPDL-3b for treatment of paediatric-onset idiopathic nephrotic syndrome (INS) remain unclear. This study aimed to investigate the level of expression of SMPDL-3b in urine, serum, and biopsy specimens and explore its implications in treatment of patients with INS. METHODS: Levels of urinary SMPDL-3b among 31 patients (20 in remission and 11 in relapse) with INS were analysed by dot blotting. For reference of precise quantitative analysis, we examined urinary excretion of SMPDL-3b from 10 patients with INS by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in both remitted and relapsed status. The levels of serum SMPDL-3b among 20 patients (13 in remission and 7 in relapse or onset) with INS were also measured using enzyme-linked immunosorbent assay. Further, the immunoreactivity of SMPDL-3b in the biopsy specimens obtained from patients with INS was compared with those from patients with proteinuric IgA nephropathy, lupus nephritis, and non-proteinuric controls. RESULTS: Urinary excretion of SMPDL-3b in patients with INS was significantly decreased in relapse cases compared with cases of remission and other types of proteinuric glomerular disease or controls by both dot blotting and LC-MS/MS method. On the other hand, serum SMPDL-3b level in INS was not different between cases of remission and relapse. Glomerular immunoreactivity of SMPDL-3b in patient with INS in remission was almost the same level to that of control. CONCLUSION: The expression of SMPDL-3b on podocytes is specifically decreased in paediatric-onset INS and its urinary excretion level reflects such conditions.

Expression of IFN-induced transmembrane protein 1 in glomerular endothelial cells.

BACKGROUND: Glomerular endothelial cells (GECs) are directly exposed to circulating viral particles in the glomerulus. Although viral infections may trigger the development of acute kidney injury or the worsening of pre-existing chronic kidney disease, the specific molecular mechanisms underlying antiviral reactions via the activation of endothelial Toll-like receptor 3 signaling in the kidney remain to be determined. Interferon (IFN)-induced transmembrane protein 1 (IFITM1), a member of interferon-stimulated gene protein family, is involved in the prevention of viral entry into cerebral vascular endothelial cells, respiratory epithelial cells, and endometrium. However, as far as we are aware, the implication of IFITM1 associated with viral infections in GECs has not been investigated to date. METHODS: Cultured, normal human GECs were treated with polyinosinic-polycytidylic acid (poly IC), a synthesized viral double-stranded RNA, then the expression of IFITM1 was examined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. To further elucidate the poly IC-induced signaling pathway, the cells were applied to RNA interference against IFN-ß, nuclear factor-κB p65, and IFN regulatory factor 3. We also conducted an immunofluorescence study to examine endothelial IFITM1 expression in biopsy specimens from patients with chronic kidney disease. RESULTS: We found that the activation of Toll-like receptor 3 induced endothelial expression of IFITM1, and that this involved IFN regulatory factor 3 and IFN-ß, but not nuclear factor-κB. Intense endothelial IFITM1 immunoreactivity was observed in biopsy specimens from patients with lupus nephritis. CONCLUSIONS: Antiviral reaction-related endothelial expression of IFITM1 may be involved, at least in part, in the development of particularly in lupus nephritis. Further detailed studies of the implication of interferon stimulated genes, including IFITM1 in GECs are needed.

Inhibitory effect of anti-malarial agents on the expression of proinflammatory chemokines via Toll-like receptor 3 signaling in human glomerular endothelial cells.

OBJECTIVE: Although anti-malarial agents, chloroquine (CQ) and hydroxychloroquine (HCQ) are currently used for the treatment of systemic lupus erythematosus, their efficacy for lupus nephritis (LN) remains unclear. Given that upregulation of glomerular Toll-like receptor 3 (TLR3) signaling plays a pivotal role in the pathogenesis of LN, we examined whether CQ and HCQ affect the expression of the TLR3 signaling-induced representative proinflammatory chemokines, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) in cultured human glomerular endothelial cells (GECs). METHODS: We examined the effect of polyinosinic-polycytidylic acid (poly IC), an agonist of TLR3, on MCP-1, CCL5 and interferon (IFN)-ß expression in GECs. We then analyzed whether pretreatment with CQ, HCQ, or dexamethasone (DEX) inhibits poly IC-induced expression of these chemokines using real-time quantitative reverse transcriptase PCR and ELISA. Phosphorylation of signal transducers and activator of transcription protein 1 (STAT1) was examined using western blotting. RESULTS: Poly IC increased MCP-1 and CCL5 expression in a time- and concentration-dependent manner in GECs. Pretreating cells with CQ, but not DEX, attenuated poly IC-induced MCP-1 and CCL5 expression; however, HCQ pretreatment attenuated poly IC-induced CCL5, but not MCP-1. HCQ did not affect the expression of IFN-ß and phosphorylation of STAT-1. CONCLUSION: Considering that TLR3 signaling is implicated, at least in part, in LN pathogenesis, our results suggest that anti-malarial agents exert a protective effect against the development of inflammation in GECs, as postulated in LN. Interestingly, CQ is a rather powerful inhibitor compared with HCQ on TLR3 signaling-induced chemokine expression in GECs. In turn, these findings may further support the theory that the use of HCQ is safer than CQ in a clinical setting. However, further detailed studies are needed to confirm our preliminary findings.

Endothelial expression of fractalkine (CX3CL1) is induced by Toll-like receptor 3 signaling in cultured human glomerular endothelial cells.

Background: Endothelial expression of membrane-bound fractalkine/CX3CL1 (Fkn) reportedly acts as a strong mediator of inflammation. Toll-like receptor 3 (TLR3) axes are thought to play some roles in the development of chronic glomerulonephritis (CGN) including lupus nephritis (LN). However, detailed mechanism of TLR3-mediated Fkn expression in glomerular endothelial cells (GECs) remains to be elucidated.Methods: We examined the effect of polyinosinic-polycytidylic acid (poly IC) on Fkn expression in cultured human GECs. Fkn mRNA and protein levels were quantified by real-time PCR and enzyme-linked immunosorbent assay, respectively. To further elucidate the effects of poly IC on this signaling pathway, we used small-interfering RNA (siRNA) to knockdown expression of TLR3, nuclear factor (NF)-κB p65, interferon (IFN)-ß, and IFN regulatory factor 3 (IRF3). We then analyzed whether pretreatment of chloroquine or dexamethasone (DEX) inhibits poly IC-induced Fkn expression.Results: We found that poly IC-induced Fkn expression in GECs, and that this involved NF-κB, IFN-ß, and IRF3. Pretreating cells with chloroquine, but not DEX attenuated poly IC-induced Fkn expression in GECs.Conclusion: Since the activation of TLR3/NF-κB/IFN-ß/Fkn and TLR3/IRF3/Fkn axes is involved in inflammatory reactions in GECs, intervention of glomerular TLR3 signaling may be a suitable therapeutic strategy for treating CGN especially LN.

Cytosolic Sensors of Viral RNA Are Involved in the Production of Interleukin-6 via Toll-Like Receptor 3 Signaling in Human Glomerular Endothelial Cells.

BACKGROUND/AIMS: Dysregulation of interleukin-6 (IL-6) production in residual renal cells may play a pivotal role in the development of glomerulonephritis (GN). Given that Toll-like receptor 3 (TLR3) signaling has been implicated in the pathogenesis of some forms of GN, we examined activated TLR3-mediated IL-6 signaling in cultured normal human glomerular endothelial cells (GECs). METHODS: We treated GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of IL-6 and the cytosolic viral RNA sensors retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) using reverse transcription quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assays. To further elucidate the effects of poly IC on this signaling pathway, we subjected the cells to small interfering RNA (siRNA) against TLR3, interferon (IFN)-ß, RIG-I, and MDA5. RESULTS: We found that poly IC induced the expression of RIG-I, MDA5 and IL-6 via TLR3/IFN-ß signaling in GECs. siRNA experiments revealed that both MDA5 and RIG-I were involved in the poly IC-induced expression of IL-6, with MDA5 being upstream of RIG-I. CONCLUSION: Interestingly, cytosolic sensors of viral RNA were found to be involved in IL-6 production via TLR3 signaling in GECs. Regional activation of TLR3/IFN-ß/ MDA5/RIG-I/IL-6 axis due to viral and "pseudoviral" infections is involved in innate immunity and inflammatory reactions in GECs. We believe this signaling pathway also plays a pivotal role in the development of some forms of GN.

Chloroquine attenuates TLR3-mediated plasminogen activator inhibitor-1 expression in cultured human glomerular endothelial cells.

BACKGROUND: Chloroquine, an antimalarial agent, has been reported to prevent the risk of thrombosis and decrease renal damage in patients with systemic lupus erythematosus (SLE); however, its detailed mechanisms remain unclear. Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis and is involved in fibrin deposition in glomeruli. Since upregulation of glomerular Toll-like receptor 3 (TLR3) signaling reportedly plays a pivotal role in the pathogenesis of lupus nephritis (LN), we examined whether chloroquine affects TLR3-mediated expression of PAI-1 in cultured human glomerular endothelial cells (GECs). METHODS: We examined the effect of polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, on PAI-1 and tissue plasminogen activator (t-PA) expression in GECs. Then, we analyzed whether pretreatment of chloroquine or dexamethasone inhibits poly IC-induced expression of these proteins using reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Poly IC increased PAI-1 expression in a time- and concentration-dependent manner, but did not affect t-PA expression in GECs. RNA interference against TLR3 inhibited poly IC-induced PAI-1 expression. Interestingly, pretreating cells with chloroquine, and also hydroxychloroquine, but not dexamethasone, attenuated poly IC-induced PAI-1 expression in GECs. CONCLUSION: Considering that TLR3 signaling is implicated in LN pathogenesis, our results suggest that chloroquine exert postulated renoprotective effects by inhibiting PAI-1 expression.

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.

BDNF repairs podocyte damage by microRNA-mediated increase of actin polymerization.

Idiopathic focal segmental glomerulosclerosis (FSGS) is a progressive and proteinuric kidney disease that starts with podocyte injury. Podocytes cover the external side of the glomerular capillary by a complex web of primary and secondary ramifications. Similar to dendritic spines of neuronal cells, podocyte processes rely on a dynamic actin-based cytoskeletal architecture to maintain shape and function. Brain-derived neurotrophic factor (BDNF) is a pleiotropic neurotrophin that binds to the tropomyosin-related kinase B receptor (TrkB) and has crucial roles in neuron maturation, survival, and activity. In neuronal cultures, exogenously added BDNF increases the number and size of dendritic spines. In animal models, BDNF administration is beneficial in both central and peripheral nervous system disorders. Here we show that BDNF has a TrkB-dependent trophic activity on podocyte cell processes; by affecting microRNA-134 and microRNA-132 signalling, BDNF up-regulates Limk1 translation and phosphorylation, and increases cofilin phosphorylation, which results in actin polymerization. Importantly, BDNF effectively repairs podocyte damage in vitro, and contrasts proteinuria and glomerular lesions in in vivo models of FSGS, opening a potential new perspective to the treatment of podocyte disorders.

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.