Akira Furusu

Unexpected transcriptional induction of monocyte chemoattractant protein 1 by proteasome inhibition: involvement of the c-Jun N-terminal kinase-activator protein 1 pathway.

Proteasome inhibitors, the well-known inhibitors of NF-κB, are recently considered therapeutic agents for inflammation. However, the anti-inflammatory properties of these agents have not been fully evaluated. In this report we describe a novel effect of proteasome inhibitors on the expression of monocyte chemoattractant protein 1 (MCP-1) in mesangial cells. We found that proteasome inhibitor MG132 dose-dependently induced expression of MCP-1 at the transcriptional level. The stimulatory effect was similarly observed with other proteasome inhibitors (proteasome inhibitor 1 and lactacystin) and in other cell types (NRK fibroblasts). The 5′-flanking region of the MCP-1 gene contains multiple AP-1 sites. To explore the mechanisms involved, we examined the effects of proteasome inhibition on the AP-1 pathway. Northern blot analysis showed that MG132 rapidly induced the expression of c-jun, but not c-fos. Immunoblot analysis showed that MG132 prevented degradation of c-Jun protein. Kinase assay revealed that c-Jun N-terminal kinase (JNK) was rapidly activated by MG132. Consistent with these results, a reporter assay showed that AP-1 activity was up-regulated after treatment with MG132. Curcumin, a pharmacological inhibitor of the JNK-AP-1 pathway, abrogated the induction of MCP-1 by MG132. Similarly, stable transfection with a dominant-negative mutant of c-Jun attenuated both MG132-induced activation of AP-1 and expression of MCP-1. The transcriptional activation by proteasome inhibitors was observed not only in MCP-1, but also in other AP-1-dependent genes, including stromelysin and mitogen-activated protein kinase phosphatase 1. These data revealed that proteasome inhibition triggered the expression of MCP-1 and other genes via the multistep induction of the JNK-c-Jun/AP-1 pathway.

A multicenter randomized controlled trial of tonsillectomy combined with steroid pulse therapy in patients with immunoglobulin A nephropathy

Background The study aim was, for the first time, to conduct a multicenter randomized controlled trial to evaluate the effect of tonsillectomy in patients with IgA nephropathy (IgAN). Methods Patients with biopsy-proven IgAN, proteinuria and low serum creatinine were randomly allocated to receive tonsillectomy combined with steroid pulses (Group A; n = 33) or steroid pulses alone (Group B; n = 39). The primary end points were urinary protein excretion and the disappearance of proteinuria and/or hematuria. Results During 12 months from baseline, the percentage decrease in urinary protein excretion was significantly larger in Group A than that in Group B (P < 0.05). However, the frequency of the disappearance of proteinuria, hematuria, or both (clinical remission) at 12 months was not statistically different between the groups. Logistic regression analyses revealed the assigned treatment was a significant, independent factor contributing to the disappearance of proteinuria (odds ratio 2.98, 95% CI 1.01–8.83, P = 0.049), but did not identify an independent factor in achieving the disappearance of hematuria or clinical remission. Conclusions The results indicate tonsillectomy combined with steroid pulse therapy has no beneficial effect over steroid pulses alone to attenuate hematuria and to increase the incidence of clinical remission. Although the antiproteinuric effect was significantly greater in combined therapy, the difference was marginal, and its impact on the renal functional outcome remains to be clarified.

Transcriptional Induction of Mitogen-activated Protein Kinase Phosphatase 1 by Retinoids SELECTIVE ROLES OF NUCLEAR RECEPTORS AND CONTRIBUTION TO THE ANTIAPOPTOTIC EFFECT

All-trans-retinoic acid (t-RA) inhibits hydrogen peroxide (H2O2)-induced apoptosis by inhibiting the c-Jun N-terminal kinase (JNK)-activator protein 1 (AP-1) pathway. In this report, we examined the involvement of mitogen-activated protein kinase phosphatase 1 (MKP-1) in suppression of JNK and the antiapoptotic effect of t-RA and the roles of nuclear receptors in the regulation of MKP-1 by t-RA. We found that not only t-RA, but also a selective agonist of retinoic acid receptor (RAR), a selective agonist of retinoid X receptor (RXR), and a pan-agonist of RAR and RXR all induced MKP-1 at the transcriptional level. Activation of RAR was required for all of these triggering effects, but activation of RXR was required only for the RXR agonist-induced MKP-1 expression. Among the three RAR subtypes, RARα and RARγ, but not RARβ, mediated the t-RA-induced MKP-1 expression. The antiapoptotic effect of t-RA on H2O2-induced apoptosis in several cell types was correlated with the inducibility of MKP-1 by t-RA. Inhibition of MKP-1 by vanadate enhanced JNK phosphorylation and attenuated the antiapoptotic effect of t-RA. Furthermore, overexpression of MKP-1 inhibited H2O2-induced JNK phosphorylation and apoptosis. To our knowledge, this is the first to demonstrate that 1) MKP-1 is inducible by retinoids at the transcriptional level, 2) RXR and individual RAR subtypes have different roles in this process, and 3) the induced MKP-1 plays a significant role in mediating both JNK inhibition and the antiapoptotic effect of t-RA in oxidative stress.

Detection of nuclear factor-κB in IgA nephropathy using Southwestern histochemistry

BACKGROUND The transcription factor nuclear factor-kappaB (NF-kappaB) is involved in inflammatory and immune responses through induction of various cytokines and growth factors. The aim of this study is to examine the correlation between NF-kappaB expression and severity of tissue injury in immunoglobulin A (IgA) nephropathy and the mechanism of such correlation. METHODS The study included 43 renal tissue samples from 28 patients, including 28 samples of IgA nephropathy, 5 samples of non-IgA mesangial proliferative glomerulonephritis (non-IgA nephropathy), and 10 samples with nonproliferative glomerulonephritis (membranous nephropathy [MN] n = 5; minimal change nephrotic syndrome [MCNS]; n = 5). Tissue sections were examined by Southwestern histochemistry and immunohistochemistry for monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and intercellular cell adhesion molecule-1 (ICAM-1), which are regulated by NF-kappaB. Normal portions of surgically resected kidney with adenocarcinoma served as controls. RESULTS In normal kidney, MCNS, and MN sections, NF-kappaB expression was detected in a few mesangial cells and tubular epithelial cells. In IgA nephropathy and non-IgA nephropathy samples, NF-kappaB was expressed in mesangial, glomerular endothelial and epithelial cells, tubular epithelial cells, and infiltrating cells. Expression in both glomeruli and interstitium correlated with progression of tissue injury. In IgA nephropathy samples, MCP-1 and GM-CSF expression was increased in both glomeruli and interstitium and correlated with progression of tissue injury. Glomerular ICAM-1 expression was weaker in severe lesions, whereas interstitial expression correlated with progression of tissue injury. CONCLUSION Our results indicate that NF-kappaB is involved in the progression of tissue injury in IgA nephropathy through the induction of transcriptionally regulated genes.

Aldosterone breakthrough during therapy with angiotensin‐converting enzyme inhibitors and angiotensin II receptor blockers in proteinuric patients with immunoglobulin A nephropathy

Background:  We are investigating whether aldosterone breakthrough negatively impacts on the antiproteinuric effects of angiotensin‐converting enzyme (ACE) inhibitors and/or angiotensin II receptor blockers (ARB).

Suppression of Renal Tubulointerstitial Fibrosis by Small Interfering RNA Targeting Heat Shock Protein 47

Background/Aim: Unilateral ureteral obstruction (UUO) is a well-established model for tubulointerstitial fibrosis. During the progression of tubulointerstitial fibrosis, upregulation of collagen synthesis and subsequent accumulation of collagen were observed in the tubulointerstitial area. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and plays an essential role in regulating collagen synthesis. We designed small interfering RNA (siRNA) sequences for HSP47 mRNA to examine whether HSP47 is involved in the progression of renal tubulointerstitial fibrosis in a mouse UUO model. Methods: The HSP47 siRNA was injected once via the ureter at the time of UUO preparation. We also applied a new gene delivery system for siRNA using cationized gelatin microspheres. The kidneys were harvested 7 and 14 days after UUO. The HSP47 and type I, III, and IV collagen expression levels were analyzed by immunohistochemistry and Western blotting. Results: Seven days after UUO, the expression levels of HSP47 and type I, III, and IV collagens were markedly upregulated in obstructed kidneys or green fluorescent protein siRNA treated obstructed kidneys. HSP47 siRNA injection significantly reduced the protein expression levels and significantly diminished the accompanying interstitial fibrosis. Moreover, cationized gelatin microspheres as a delivery system enhanced and lengthened the antifibrotic effect of HSP47 siRNA. Conclusions: Our results indicate that HSP47 is a candidate target for the prevention of tubulointerstitial fibrosis and that selective blockade of the HSP47 expression by using siRNA could be a potentially useful therapeutic approach for patients with renal disease.

A histologic classification of IgA nephropathy for predicting long-term prognosis: emphasis on end-stage renal disease

BACKGROUND A multicenter case-control study on IgA nephropathy (IgAN) was conducted to develop an evidence-based clinicopathologic classification of IgAN for predicting long-term renal outcome. METHODS Two hundred and eighty-seven patients including those with isolated hematuria or very mild proteinuria were enrolled. During a median follow-up of 9.3 years after biopsy, 49 patients (17%) progressed to end stage renal disease (ESRD). The associations between pathological variables and the need for chronic dialysis was examined by multivariate logistic regression analysis separately in patients who required dialysis earlier than 5 years (Early Progressors) and those who required dialysis within 5 to 10 years (Late Progressors) after biopsy. RESULTS Independent pathological variables predicting progression to ESRD were global sclerosis, segmental sclerosis and fibrous crescents for Early Progressors, and global sclerosis and cellular/fibrocellular crescents for Late Progressors. Four histological grades, HG 1, HG 2, HG 3 and HG 4, were established corresponding to <25%, 25-49%, 50-74% and =75% of glomeruli exhibiting cellular or fibrocellular crescents, global sclerosis, segmental sclerosis or fibrous crescents. Eleven (7%) patients in HG 1, 12 (16%) in HG 2, 13 (31%) in HG 3 and 13 (68%) in HG 4 progressed to ESRD. Multivariate logistic analysis revealed that the risk of progression to ESRD was significantly higher in HG 2, 3 and 4 than in HG 1 (odds ratio, 2.4, 5.7 and 27.6 vs. 1.0). CONCLUSIONS Our evidence-based histologic classification can identify the magnitude of the risk of progression to ESRD and is useful for predicting long-term renal outcome in IgAN.

In situ hybridization studies of stromelysin and tissue inhibitor of metalloproteinase 1 in IgA nephropathy

Summary: Accumulation of the extracellular matrix (ECM) in IgA nephropathy (IgAN) is thought to cause deterioration of glomerular function. Stromelysin and tissue inhibitor of matrix proteinase 1 (TIMP1) may play an important role in the turnover of the glomerular ECM. However, the expression of these enzymes in human renal tissues remains undefined. In the present study, non‐radioactive in situ mRNA hybridization, which permitted the analysis at a cellular level, was performed to localize stromelysin and TIMP1 in renal tissue of IgAN. We also determined the percentage of cells positive for stromelysin or TIMP1 mRNA among intraglomerular cells. A total of 16 patients with IgAN were examined, including eight patients with severe histopathological changes and eight with mild changes. Three patients without glomerular disease were also studied. Stromelysin and TIMP1 mRNA were weakly expressed in the mesangium of normal kidneys and IgAN renal tissues with mild damage. However, the expression of both mRNA was significantly increased in the area of mesangial proliferation, in glomerular epithelial cells and in Bowmans capsule of advanced lesions. Several cells in the area of mesangial proliferation were double positive for stromelysin and TIMP1 mRNA, while certain cells positive for stromelysin mRNA did not express TIMP1 mRNA. In the interstitium, epithelial cells of certain tubules and some mononuclear cells were positively stained for these mRNA, especially in advanced lesions. Our results indicated that stromelysin and TIMP1 genes were expressed in glomerular resident cells, tubular epithelial cells and infiltrated mononuclear cells in IgAN, and their expression was enhanced in advanced tissue damage. the demonstration of a co‐expression and discordant expression of the genes indicates that each gene expression may be regulated in a cell type‐specific manner and that it could also be altered by cellular environmental factors.

MAP kinase-dependent, NF-κB-independent regulation of inhibitor of apoptosis protein genes by TNF-α

The inhibitor of apoptosis protein (IAP) family of molecules regulates apoptotic processes triggered by various stimuli. However, the mechanisms involved in the regulation of the IAP genes are not fully understood. In this report, we examined roles of nuclear factor‐κB (NF‐κB) and mitogen‐activated protein (MAP) kinases in tumor necrosis factor‐α (TNF‐α)‐induced expression of IAP genes. In human endothelial cells, TNF‐α induced c‐IAP1 and c‐IAP2, but not XIAP and TIAP/Survivin, at the transcriptional level. Inactivation of NF‐κB by overexpression of a super‐repressor mutant of IκBα did not affect the induction of IAPs by TNF‐α. In contrast, extracellular signal‐regulated kinase, p38 MAP kinase, and c‐Jun N‐terminal kinase were activated after stimulation with TNF‐α, and inhibition of each kinase by PD098059, SB203580, curcumin, or SP600125 substantially attenuated the TNF‐α‐induced c‐IAP1 and c‐IAP2 expression. To examine whether the MAP kinases‐mediated induction of IAPs contributes to survival of TNF‐α‐exposed cells, cells were pretreated with MAP kinase inhibitors and stimulated with TNF‐α. Treatment with kinase inhibitors alone did not induce apoptosis but enhanced markedly TNF‐α‐triggered apoptosis. Furthermore, overexpression of either c‐IAP1 or c‐IAP2 diminished the apoptosis‐promoting effects of MAP kinase inhibitors. These data indicated that TNF‐α induced expression of c‐IAP1 and c‐IAP2 via MAP kinases, but not via NF‐κB, and that MAP kinases participated in the inhibition of apoptosis by induction of c‐IAPs in TNF‐α‐stimulated endothelial cells. J. Cell. Physiol. 210: 703–710, 2007.

Imaging mass spectrometry analysis reveals an altered lipid distribution pattern in the tubular areas of hyper-IgA murine kidneys.

Immunoglobulin A (IgA) nephropathy is the most common glomerular disease worldwide. To investigate the pathogenesis of this renal disease, we used animal models that spontaneously develop mesangioproliferative lesions with IgA deposition, which closely resemble the disease in humans. We analyzed the molecular distribution of lipids in hyper-IgA (HIGA) murine kidneys using matrix-assisted laser desorption/ionization-quadrupole ion trap-time of flight (MALDI-QIT-TOF)-based imaging mass spectrometry (IMS), which supplies both spatial distribution of the detected molecules and allows identification of their structures by their molecular mass signature. For both HIGA and control (Balb/c) mice, we found two phosphatidylcholines, PC(16:0/22:6) and PC(18:2/22:6), primarily located in the cortex area and two triacylglycerols, TAG(16:0/18:2/18:1) and TAG(18:1/18:2/18:1), primarily located in the hilum area. However, several other molecules were specifically seen in the HIGA kidneys, particularly in the tubular areas. Two HIGA-specific molecules were O-phosphatidylcholines, PC(O-16:0/22:6) and PC(O-18:1/22:6). Interestingly, common phosphatidylcholines and these HIGA-specific ones possess 22:6 lipid side chains, suggesting that these molecules have a novel, unidentified renal function. Although the primary structure of the HIGA-specific molecules corresponding to m/z 854.6, 856.6, 880.6, and 882.6 remained undetermined, they shared similar fragmentation patterns, indicating their relatedness. We also showed that all the HIGA-specific molecules were derived from urine, and that artificial urinary stagnation-due to unilateral urethral obstruction-caused HIGA-specific distribution of lipids in the tubular area.