Tomomi Aizawa

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

BackgroundMesangial 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.MethodsWe 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)-β, 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).ResultsWe found that activation of TLR3 signaling could induce the expression of CXCL1 in MCs. NF-κB, IRF3 and IFN-β, 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.ConclusionTLR3 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.

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‐β. 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‐β 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.

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

AbstractAimIt 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. MethodsWe 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)-β/retinoic acid-inducible gene-I (RIG-I) pathway by reverse transcriptase-polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay.ResultsWe 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-β or RIG-I decreased CCL5 expression induced by TNF-α followed by poly IC.ConclusionPretreatment with TNF-α leads marked activation of the TLR3/IFN-β/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.

Efficacy of mizoribine-tacrolimus-based induction therapy for pediatric lupus nephritis:

Background Recent advances in the management of lupus nephritis (LN) have also contributed to a favorable outcome in patients with pediatric-onset LN. Nevertheless, we believe that a more effective and less toxic treatment is needed to attain optimal control of pediatric-onset LN. Methods Seven consecutive children with biopsy-proven LN (four with class III/IV and three with class V) received multitarget induction therapy consisting of mizoribine (MZR), tacrolimus (Tac), and prednisolone (PDN). They were prospectively evaluated at three, six, and 12 months, and at the latest observation point after a mean period of 32 months. Post-treatment renal biopsy was performed in two patients with class III/IV. Results Despite gradually tapering the dose of concomitantly administered PDN, a significant improvement compared with baseline values was observed in the urinary, serological, and clinical assessment measures even at three months of treatment, and the favorable changes persisted throughout the treatment period in most of the study participants except for one. In two patients who underwent post-treatment renal biopsy, a marked histologic improvement was confirmed. No serious adverse events were observed. Conclusions Multitarget therapy may be an attractive option for the treatment of pediatric-onset LN. Further studies involving a larger number of patients 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

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.

Toll-Like Receptor 3 Signaling Contributes to Regional Neutrophil Recruitment in Cultured Human Glomerular Endothelial Cells

Background: Given the importance of neutrophil recruitment in the pathogenesis of glomerulonephritis (GN), the representative neutrophil chemoattractant C-X-C motif chemokine 1 (CXCL1)/GROα and the adhesion molecule E-selectin in glomerular endothelial cells (GECs) play a pivotal role in the development of GN. Endothelial Toll-like receptor 3 (TLR3) is thought to be involved in the inflammatory response via innate immunity. However, the role of endothelial TLR3 signaling in the expression of neutrophil chemoattractants and adhesion molecules remains to be elucidated. Thus, we aimed to examine this issue. Methods: We treated normal human GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expressions of CXCL1 and E-selectin using quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against TLR3, interferon (IFN)-β, nuclear factor (NF)-κB p65, and IFN regulatory factor (IRF) 3. We also used immunofluorescence to examine the endothelial expression of CXCL1 in biopsy specimens from patients with crescentic and non-crescentic purpura nephritis (PN). Results: We found that the activation of TLR3 induced the endothelial expression of CXCL1 and E-selectin, and that this involved TLR3, NF-κB, IRF3, and IFN-β. Intense endothelial CXCL1 expression was observed in biopsy specimens from patients with crescentic PN. Conclusion: These findings support a role for glomerular antiviral innate immunity in the pathogenesis of GN. Intervention of glomerular TLR3 signaling may therefore be a suitable therapeutic strategy for treating GN in the future.

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-β pathways (i.e., TLR3/IFN-β/retinoic acid-inducible gene-I (RIG-I)/CCL5, and TLR3/IFN-β/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-β 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.

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 β2 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.

Efficacy of long-term multidrug therapy in a patient with focal segmental glomerulosclerosis

Given that unremitting proteinuria itself is harmful to the kidneys of patients with focal segmental glomerulosclerosis (FSGS), patients with idiopathic FSGS who do not respond to aggressive immunosuppressive treatment, including highdose steroid combined with calcineurin inhibitor (CNI), have a high risk of progression to renal failure. Therapy-related adverse events, however, including CNI-induced nephrotoxicity, are a major therapeutic dilemma in the immunosuppressive treatment of patients with refractory nephrotic syndrome (NS). Thus, the optimal treatment strategy for management of refractory FSGS, especially in pediatric patients, remains controversial. A 6-year-old Japanese boy with a 2 month history of generalized edema associated with persistent proteinuria was diagnosed as having prednisolone (PDN)-resistant NS at a regional hospital. Percutaneous renal biopsy indicated a lesion characteristic of FSGS. Despite aggressive immunosuppressive treatment consisting of methyl-PDN pulse therapy combined with cyclosporine (CsA) at a dose of 3.1 mg/kg daily (trough blood level, 113 ng/mL), heavy proteinuria persisted for the next 12 months. A second renal biopsy performed 11 months after the first showed aggravation of FSGS with possible CNI-induced nephrotoxicity (Fig. 1). The patient was transferred to Hirosaki University Hospital thereafter. Low-density lipoprotein apheresis proved only partially effective, and unremitting proteinuria of approximately 1–3 g/day persisted for the next 2 years. Therefore, we decided to try multidrug immunosuppressive therapy consisting of relatively low-dose tacrolimus (Tac) (0.08 mg/kg) once after the evening meal plus mizoribine (MZR; 6 mg/kg) daily, given once daily before breakfast, and temporarily increased the dose of PDN to 25 mg (1.0 mg/kg) daily. After obtaining written informed consent from his parents, this multidrug therapy was started. The blood level of Tac obtained at 12 h after dosing was around 5.0 ng/mL, and the peak blood level of MZR (2 h after dosing) was around 1.5 μg/mL. After the start of this regimen, the unremitting proteinuria gradually subsided and complete remission was achieved within 3 months of the start of the regimen, with no adverse effects. The concomitant PDN was reduced to 10 mg/kg (0.25 mg/kg) on alternate days during the next 12 months. The patient’s serum total protein and serum albumin also returned to within the normal range. Serum creatinine and estimated glomerular filtration rate have remained within the normal range for the patient’s age, and Tac and PDN were successfully withdrawn after 4 and 6 years, respectively. His short stature was also gradually improved (from −2.0 SD for his age at the start of multidrug therapy to −1.0 SD for his age at the latest observation). At present, 6.5 years after commencement of the multidrug therapy, complete remission has persisted on MZR monotherapy without adverse events. Recent advances in the treatment of CsA-resistant childhood FSGS have suggested that Tac may be an effective alternative to CsA, although Tac shares a potentially nephrotoxic effect with CsA. In contrast, the purine synthesis inhibitor MZR reportedly has relatively low clinical toxicity in patients with NS. In addition to its immunosuppressive effect, MZR appears to exert a beneficial renoprotective effect against CsA-induced intimal hyperplasia and perivascular inflammatory cell infiltration in rat models. The mechanisms of action of CNI and MZR are probably complementary, and therefore we hypothesize that combination therapy using relatively low-dose Tac once daily, possibly shortening the exposure to Tac and MZR, might be a beneficial and renoprotective regimen for the treatment of childhood refractory FSGS. With regard to the optimal blood levels of drugs, we think that a Tac trough level of approximately 5.0 ng/mL and an MZR peak level of approximately 1.5 μg/mL may be adequate for long-term multidrug therapy, although this remains to be determined in future studies. Although the present patient likely had CNI-induced nephrotoxicity due to aggressive CsA treatment prior to the multidrug regimen, renal function had been preserved. Given that we could not perform post-treatment renal biopsy in this patient, the actual nature of the renoprotective effect of MZR in a clinical setting remains speculative, but no previous report has documented the longterm (>5 years) efficacy and safety of a multidrug regimen in pediatric FSGS patients. Therefore, the present report might encourage further trials of long-term multidrug therapy in a larger number of FSGS patients in order to establish optimal treatment strategy.