Expression of interferon-stimulated gene 20 (ISG20), an antiviral effector protein, in glomerular endothelial cells: possible involvement of ISG20 in lupus nephritis.

BACKGROUND: In addition to regulating the antiviral response, increased expression of Toll-like receptor 3 (TLR3) in resident renal cells plays a role in developing some forms of glomerulonephritis. TLR3 activation leads to type I interferon (IFN) production, which induces the expression of IFN-stimulated genes (ISGs). However, the role of ISG20 expression in resident renal cells remains unclear. METHODS: Cultured normal human glomerular endothelial cells (GECs) were treated with polyinosinic-polycytidylic acid (poly IC), Escherichia coli lipopolysaccharide (LPS), R848, and CpG (TLR3, TLR4, TLR7, and TLR9 agonists, respectively). The mRNA levels of ISG20, CX3CL1/fractalkine, and CXCL10/IP-10 were measured by quantitative reverse transcription-polymerase chain reaction. ISG20 protein expression was assessed by Western blotting. RNA interference was used to knockdown IFN-ß and ISG20 expression. CX3CL1 protein levels were assessed by enzyme-linked immunosorbent assay. We performed immunofluorescence to examine endothelial ISG20 expression in biopsy specimens from patients with lupus nephritis (LN). RESULTS: In GECs, the expression of ISG20 mRNA and protein was increased by polyIC, not by LPS, R848, or CpG treatment. Moreover, ISG20 knockdown prevented poly IC-induced CX3CL1 expression but had no effect on CXCL10 expression. Intense endothelial ISG20 immunoreactivity was observed in biopsy specimens obtained from patients with proliferative LN. CONCLUSION: In GECs, ISG20 was regulated via TLR3 but not via TLR4, TLR7, or TLR9 signaling. Moreover, ISG20 was involved in regulating CX3CL1 production. In addition to regulating antiviral innate immunity, ISG20 may act as a mediator of CX3CL1 production, thereby inducing glomerular inflammation, particularly in patients with LN.

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.

Effect of sera from lupus patients on the glomerular endothelial fibrinolysis system.

BACKGROUND: Dysregulation of the coagulation fibrinolysis system in resident glomerular cells is associated with the pathogenesis of lupus nephritis. However, the role of plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) in resident glomerular cells remains undetermined. METHODS: We examined the expression of PAI-1 and tPA mRNA in cultured normal human glomerular endothelial cells (GECs) treated with serum from patients with systemic lupus erythematosus (SLE) using quantitative reverse transcription polymerase chain reactions. We determined the relationship between PAI-1/tPA mRNA expression and several clinical/laboratory parameters. Serum from 16 patients (nine patients with new-onset SLE and seven patients with stable SLE) was used in the study. RESULTS: Plasminogen activator inhibitor-1 and tPA mRNA expression was significantly higher in GECs treated with serum of patients with new-onset SLE than other groups. The PAI-1 and tPA mRNA levels were also significantly correlated in GECs treated with serum from patients with SLE. Interestingly, both PAI-1 and tPA mRNA levels in GECs were inversely correlated with serum C4 level and positively correlated with SLE disease activity. CONCLUSIONS: These results suggest that serum from patients with SLE may activate the fibrinolysis system in glomerulus, which may be involved in the pathogenesis of lupus nephritis.

Glomerular endothelial expression of type I IFN-stimulated gene, DExD/H-Box helicase 60 via toll-like receptor 3 signaling: possible involvement in the pathogenesis of lupus nephritis.

BACKGROUND: Sustained type I interferon (IFN) activation via Toll-like receptor (TLR) 3, 7 and 9 signaling has been reported to play a pivotal role in the development of lupus nephritis (LN). Although type I IFN activation has been shown to induce interferon-stimulated genes (ISGs) expression in systemic lupus erythematosus, the implication of ISGs expression in intrinsic glomerular cells remains largely unknown. METHODS: We treated cultured human glomerular endothelial cells (GECs) with polyinosinic-polycytidylic acid (poly IC), R848, and CpG (TLR3, TLR7, and TLR9 agonists, respectively) and analyzed the expression of DExD/H-Box Helicase 60 (DDX60), a representative ISG, using quantitative reverse transcription-polymerase chain reaction and western blotting. Additionally, RNA interference against IFN-ß or DDX60 was performed. Furthermore, cleavage of caspase 9 and poly (ADP-ribose) polymerase (PARP), markers of cells undergoing apoptosis, was examined using western blotting. We conducted an immunofluorescence study to examine endothelial DDX60 expression in biopsy specimens from patients with LN. RESULTS: We observed that endothelial expression of DDX60 was induced by poly IC but not by R848 or CpG, and RNA interference against IFN-ß inhibited poly IC-induced DDX60 expression. DDX60 knockdown induced cleavage of caspase 9 and PARP. Intense endothelial DDX60 expression was observed in biopsy specimens from patients with diffuse proliferative LN. CONCLUSION: Glomerular endothelial DDX60 expression may prevent apoptosis, which is involved in the pathogenesis of LN. Modulating the upregulation of the regional innate immune system via TLR3 signaling may be a promising treatment target for LN.

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.

FGF23 and Fetuin-A Interaction and Mesenchymal Osteogenic Transformation.

Recently, we found a strict bone association between Fibroblast growth factor 23 (FGF23) and Fetuin-A, both involved in cardiovascular and mineral bone disorders. In this study, an uninvestigated bone marrow positivity for both was found. Though the role of exogenous FGF23 on mesenchymal cells (MSCs) was reported, no information is as yet available on the possible production of this hormone by MSCs. To further analyze these uninvestigated aspects, we studied human primary cells and mouse and human cell lines by means of immunostaining, qRT-PCR, enzyme linked immunosorbent assays, chromatin immunoprecipitation, transfection, and a streamlined approach for the FGF23⁻Fetuin-A interaction called Duolink proximity ligation assay. Mesenchymal cells produce but do not secrete FGF23 and its expression increases during osteo-differentiation. Fibroblast growth factor 23 is also involved in the regulation of Fetuin-A by binding directly to the Fetuin-A promoter and then activating its transcription. Both FGF23 overexpression and addition induced an upregulation of Fetuin-A in the absence of osteo-inducer factors. Fibroblast growth factor 23 and Fetuin-A promoter were increased by osteo-inducer factors with this effect being abolished after FGF23 silencing. In conclusion, both FGF23 and Fetuin-A are present and strictly linked to each other in MSCs with FGF23 driving Fetuin-A production. This mechanism suggests a role for these two proteins in the osteoblast differentiation.

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.

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.