Published papers

Background. Vascular endothelial growth factor (VEGF) is an endothelial mitogen that is constitutively expressed in normal human glomeruli, but its role in the kidney is still unclear. In this study, we examined the effects of VEGF on human mesangial cells (HMCs). Methods and Results. Reverse transcription-polymerase chain reaction analysis demonstrated the presence of VEGF receptor mRNA (flt-1 and KDR) in HMCs. The treatment of HMCs with VEGF did not cause a change in 3H-thymidine incorporation or cell numbers. In contrast, VEGF caused a dose- and time-dependent increase in collagen synthesis, with threefold to fivefold increases in both cell-associated and secreted collagen synthesis seen after treatment with 200 ng/ml VEGF. The effects of VEGF were attenuated by treatment of HMCs with the tyrosine kinase inhibitor herbimycin A or the MEK inhibitor PD 98059, but not with the protein kinase C (PKC) inhibitor chelerythrine. VEGF treatment also caused a marked increase in p42/p44 mitogen-activated protein kinase (MAPK) activity, but had no significant effect on HMC superoxide production. Finally, an increase in collagen synthesis was also seen in rat mesangial cells treated with VEGF. Conclusions. These results suggest that VEGF is not a mitogenic signal in HMCs, but may be involved in the regulation of the mesangial matrix in humans by a MAPK-dependent mechanism.

Tranilast has been reported to reduce restenosis rate after angioplasty, but its mechanism is still unclear. We investigated the effect of tranilast against platelet-derived growth factor (PDGF) in PDGF's proliferative effect and PDGF's inhibitory effect on cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells (VSMC). NO production was measured by Griess reaction. NO synthase (NOS) protein was evaluated by Western blot with monoclonal anti-rat inducible NOS antibody. A combination of interleukin-1β (IL-1β 1 ng/ml), tumor necrosis factor-α (TNF-α 2,000 U/ml), and lipopolysaccharide (100 ng/ml) significantly increased NO production and NOS protein, and tranilast significantly enhanced both in a dose-dependent manner. PDGF (100 ng/ml) significantly reduced both cytokine-induced NO production and NOS protein induction, but tranilast completely abolished these inhibitory effects. In the presence of cytokines, serum-stimulated cell proliferation was significantly inhibited by cytokine-induced NO, whereas PDGF-stimulated proliferation was not. On the other hand, tranilast not only inhibited the proliferative effect of PDGF directly, but also restored cytokine-induced NO production and its antiproliferative effect in the presence of PDGF.

The effects of tranilast on DNA synthesis and cell proliferation in cultured rat mesangial cells, treated with platelet-derived growth factor (PDGF), were investigated. Tranilast significantly inhibited PDGF-stimulated DNA synthesis and cell proliferation in a dose-dependent manner. In the absence of PDGF, it also enhanced cytokine-induced nitric oxide (NO) production. PDGF significantly inhibited cytokine-induced NO production, but tranilast completely abolished this inhibitory effect of PDGF. These results show that tranilast inhibits PDGF-induced proliferation of mesangial cells under both normal and inflammatory conditions.

Background. Intrarenal activation of the renin-angiotensin system has been suggested to play a pivotal role in the progression of various renal diseases, but the regulation of each component has not been fully clarified. We investigated the roles of nuclear factor κB (NF-κB) activation in the intrarenal renin-angiotensin system changes induced by proteinuria. Methods. We used unilaterally nephrectomized rats loaded with bovine serum albumin as a model of proteinuric renal injury. Renal NF-κB activation was inhibited by gene transfer of the truncated form of IκBα via injection of a recombinant adenovirus vector into the renal artery, as we reported previously. Results. Inhibition of renal NF-κB activation attenuated the increases in intrarenal angiotensinogen protein (2.0-fold in rats with protein overloading and saline injection to 1.3-fold in rats with protein overloading and injection of a truncated form of IκBα) and angiotensin II (1.8-fold to 1.2-fold), and angiotensinogen mRNA. The increases in angiotensin-converting enzyme (ACE) and angiotensin II receptor type 2 were unaffected by NF-κB inhibition. The expression of ACE2, an enzyme that metabolizes angiotensins I and II, was decreased by 37%, and NF-κB inhibition abolished the decrease. Immunohistochemical analysis revealed that the angiotensinogen and ACE2 expression changes occurred mainly in proximal tubule cells (i.e., the target of adenoviral gene transfer). Conclusion. These results indicate that proteinuria induces an increase in renal angiotensin II in an NF-κB-dependent manner. Induction of angiotensinogen and decrease in ACE2 levels may be involved in this NF-κB-dependent increase in angiotensin II.

The objective of the current study was to determine whether the balance of interleukin-1 and intracellular interleukin-1 receptor antagonist in chondrocytes in osteoarthritic human joints favors agonist action. Chondrocytes were isolated from cartilage specimens taken at the time of joint arthroplasty. Interleukin-1α, interleukin-1β, and intracellular interleukin-1 receptor antagonist messenger ribonucleic acids were assessed by reverse transcriptase-polymerase chain reaction, and chondrocyte lysates were analyzed by enzyme-linked immunosorbent assay for the respective proteins. Type I intracellular interleukin receptor antagonist transcripts were the only intracellular variant detected in osteoarthritis chondrocytes. In cartilage graded as advanced osteoarthritis both interleukin proteins in chondrocyte lysates decreased, correlating with decreased interleukin-1α and β messenger ribonucleic acids. Interleukin-1 receptor antagonist exceeded interleukin-1α in chondrocyte lysates by one order of magnitude except that in moderate osteoarthritis, antagonist was only two- to fourfold in excess. Interleukin-1α and interleukin-1β proteins were correlated closely in individual lysates, with interleukin-1β exceeding interleukin-1β by one order of magnitude. In moderately degenerated cartilage, intracellular antagonist may not be sufficiently abundant to block postulated intracellular functions of precursor interleukin-1α. Furthermore, if stored interleukin-1α, interleukin-1β, and interleukin receptor antagonist are released from chondrocytes, the localized antagonist would be insufficient to prevent signaling through cell surface receptors. Chondrocyte-derived interleukin-1α and interleukin-1β may locally overwhelm inhibition by interleukin receptor antagonist to promote the early degenerative changes in osteoarthritis.

Monocyte chemoattractant protein-1 (MCP-1), a member of the CC subfamily of chemokines, plays a crucial role in the progression of glomerulonephritis by recruitment of monocytes. Tranilast, a clinically used anti-allergic drug, has been demonstrated to have various anti-inflammatory and anti-proliferative effects, and recently has been reported to prevent restenosis after percutaneous transluminal coronary angioplasty. In this study, we investigated whether tranilast inhibits MCP-1 secretion in mesangial cells. Tranilast inhibited interleukin-1β-induced MCP-1 secretion and mRNA expression in a concentration-dependent manner. Luciferase assay showed that tranilast suppressed interleukin-1β-induced nuclear factor-κB (NF-κB)-dependent transcription. Interleukin-1β-induced Jun N-terminal kinase (JNK) activation was also suppressed selectively by tranilast. These results indicate that tranilast inhibits interleukin-1β-induced MCP-1 production, at least in part, by inhibiting NF-κB activity and that suppression of JNK activation might be involved in the inhibition of MCP-1 production. Tranilast may serve as a new therapeutic agent for glomerulonephritis through anti-chemokine property. © 2001 Elsevier Science B.V. All rights reserved.

Dysregulation of apoptosis is one of the likely underlying mechanisms of neointimal thickening, a disorder in which proinflammatory cytokines may influence the function of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis. One of these cytokines, tumor necrosis factor-α (TNF-α), induces 2 possibly conflicting pathways, 1 leading to the activation of nuclear factor-κB (NF-κB) and the other leading to caspase-mediated apoptosis. We investigated whether specific inhibition of NF-κB affects TNF-α-dependent apoptosis in human VSMCs. To inhibit NF-κB activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein IκBα (AdexIκBΔN) that lacks the phosphorylation sites essential for activation of NF-κB. The IκBΔN was overexpressed by adenoviral infection and was resistant to stimulus-dependent degradation. Electromobility gel shift and luciferase assays demonstrated that overexpression of IκBΔN inhibited NF-κB activation induced by TNF-α or interleukin-1β (IL-1β). In cells overexpressing IκBAN, TNF-α dramatically induced apoptosis, whereas IL-1β had no effect. The induction was suppressed by treatment with a selective inhibitor of the caspase-3 family, Z-DEVD-fmk, and the overexpression of IκBΔN induced TNF-α-mediated caspase-3 and caspase-2 activity. These results indicate that overexpression of IκBΔN induces TNF-α-dependent apoptosis by efficient and specific suppression of NF-κB and upregulation of caspase-3 and caspase-2 activity in human VSMCs. Our findings suggest that adenovirus-mediated IκBΔN gene transfer may be useful in the treatment of disorders associated with inflammatory conditions, such as the response to vascular injury and atherosclerosis.

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.

We report a case of Henoch-Schönlein purpura nephritis (HSPN) with acquired IgA deficiency due to parvovirus B19 infection. The patient was diagnosed as having Henoch-Schönlein purpura (HSP) at 6 years old, and subsequently developed macrohematuria and massive proteinuria of 7.4 g/day with decreased creatinine clearance of 70.2 ml/min/1.73 m2 and significantly elevated serum IgA level of 449 mg/dl. The first kidney biopsy yielded the diagnosis of severe HSPN. After the initiation of the immunosuppressive therapy, the patient was infected with parvovirus B19 and developed virus-associated hemophagocytic syndrome (VAHS). Thereafter, the serum level of IgA selectively decreased and remained undetectable until the present time. Repeated kidney biopsies performed over a period of 14 years revealed a remarkable histological improvement in association with stabilization of the patient's kidney function. Considering the severity of initial kidney injury, persistent acquired IgA deficiency was likely to add favorable effects to the immunosuppressive therapy in this patient with HSPN.

Bone morphogenic protein (BMP)-7 is expressed in the adult kidney and reverses chronic renal injury when given exogenously. Here, we report that a histone deacetylase inhibitor, trichostatin A (TSA), attenuates chronic renal injury, in part, by augmenting the expression of BMP-7 in kidney side population (SP) cells. We induced accelerated nephrotoxic serum nephritis (NTN) in C57BL/6 mice and treated them with TSA for 3 weeks. Compared with vehicle-treated NTN mice, treatment with TSA prevented the progression of proteinuria, glomerulosclerosis, interstitial fibrosis, and loss of kidney SP cells. Basal gene expression of renoprotective factors such as BMP-7, vascular endothelial growth factor, and hepatocyte growth factor was significantly higher in kidney SP cells as compared with non-SP cells. Treatment with TSA significantly upregulated the expression of BMP-7 in SP cells but not in non-SP cells. Moreover, initiation of treatment with TSA after 3 weeks of NTN (for 3 weeks, until 6 weeks) partially but significantly reversed renal dysfunction. Our results indicate an important role of SP cells in the kidney as one of the possible generator cells of BMP-7 and TSA as a stimulator of the cells in reversing chronic renal disease.

We have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-I and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1-/- compared to wild-type mice. The leukocyte integrin αMβ2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte-neutrophil elastase, known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium and Weibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet. © 2007 Schattauer GmbH, Stuttgart.

MRL/MpJ-Tnfrsf6lpr (MRL-Faslpr) mice develop a spontaneous T cell-dependent autoimmune disease that shares features with human lupus, including fatal nephritis, systemic pathology, and autoantibodies (autoAb). The inducible co-stimulator (ICOS) is upregulated on activated T cells and modulates T cell-mediated responses. To investigate whether ICOS has an essential role in regulating autoimmune lupus nephritis and the systemic illness in MRL-Faslpr mice, ICOS null (-/-) MRL Faslpr and ICOS intact (+/+) MRL-Faslpr strains (wild-type [WT]) were generated and compared. It was determined that in ICOS-/- MRL-Faslpr as compared with the WT strain, (1) there is a significant reduction in circulating IgG and double-stranded DNA autoantibody isotype titers, and (2) there is an amplification of the frequency of intrarenal T cells generating IFN-γ and TNF-α in ICOS-/- versus WT mice. Of note, eliminating ICOS in the MRL-Faslpr strain does not alter renal pathology or function. Despite the reduction in circulating IgG and autoantibody isotypes (G1, G2a, and G2b), the amount of these IgG isotypes depositing in kidneys is similar. Furthermore, the systemic illness (skin, salivary and lacrimal glands, lungs, lymphadenopathy, and splenomegaly) is equivalent in ICOS-/- MRL-Fas lpr and WT mice. These findings highlight the danger of relying on individual parameters, such as quantitative serum Ig levels and T cell functions, as prognostic indicators of lupus. Copyright © 2006 by the American Society of Nephrology.

© Copyright 2018. All rights reserved. Objective. The aim was to elucidate the prognosis and risk factors associated with relapse during longterm remission maintenance therapy for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Methods. Patients with newly diagnosed AAV (n = 156) were registered in the Remission Induction Therapy in Japanese patients with ANCA-associated Vasculitides (RemIT-JAV) study, and among them, 83 patients who achieved remission were enrolled and followed up for 24 additional months in our nationwide, prospective cohort study (Co-RemIT-JAV; registration number UMIN 000006373). Patterns of maintenance therapy, effectiveness, and safety were evaluated from months 25 to 48 after the RemIT-JAV. The primary outcome measure was the rate of relapse. Secondary outcome measures included overall and renal survival, risk factors associated with relapse, and incidence rates of serious infections. Results. The patients comprised 35 men and 48 women aged 65.3 ± 12.6 years. Between months 25 and 48, the survival rate was 95% (79/83). Causes of death included 1 thyroid cancer, 1 infection, and 2 unknown reasons. Four patients had developed endstage renal disease (ESRD) by Month 24; 1 developed ESRD beyond Month 25. The relapse rate was 24% (20/83) from months 25 to 48. Multivariable analysis revealed that oral prednisolone ≤ 2.5 mg/day at Month 24 was a significant risk factor for relapse between months 25 and 48 (HR = 3.1, 95% CI 1.1-8.5). Conclusion. One-quarter of patients with AAV relapsed during maintenance therapy, and relapse was associated with the dose of oral prednisolone 24 months after the initiation of remission induction therapy in Japan.