Yoshiyuki Ozono

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.

Thyroid function is associated with carotid intima-media thickness in euthyroid subjects.

To investigate the relationship between thyroid function and carotid intima-media thickness (CIMT) in a relatively large general population with euthyroid status we initially enrolled 1772 Japanese adults (421 men and 1351 women) who participated in a medical screening program for the general population over 40 years old. To evaluate only euthyroid subjects without vascular diseases and/or its major risk factors, 1129 were excluded and 643 participants (175 men and 468 women) were included for further analysis. Simple and multivariate linear regression analyses were performed to evaluate free thyroxine (fT4) and thyroid-stimulating hormone (TSH) levels and other existing parameters, including carotid intima-media thickness. By multivariate linear regression analysis adjusted for age and sex, free thyroxine was significantly correlated with triglycerides (beta=0.07, p=0.015), carotid intima-media thickness (beta=-0.091, p=0.049), and thyroid-stimulating hormone (beta=-0.091, p=0.003). Thyroid-stimulating hormone was significantly correlated with high-density lipoprotein cholesterol (HDL-C) (beta=-0.001, p=0.015), HbA(1c) (beta=0.038, p=0.045), carotid intima-media thickness (beta=0.27, p=0.001), and free thyroxine (beta=-0.15, p=0.003). When adjusted for confounding factors, free thyroxine was significantly correlated only with carotid intima-media thickness (beta=-0.13, p=0.043) and thyroid-stimulating hormone was significantly correlated with HDL-C (beta=-0.001, p<0.001), HbA(1c) (beta=0.04, p=0.021), and carotid intima-media thickness (beta=0.29, p=0.001). We have demonstrated that carotid intima-media thickness is independently associated with thyroid function within the normal reference range, which suggests an increased cardiovascular risk in subjects with low normal thyroid function.

High plasma osteopontin levels in patients with inflammatory bowel disease.

Background Osteopontin (OPN) plays a key role in the progression of TH1-immune-mediated disease in models of multiple sclerosis and rheumatoid arthritis. Aim To determine whether plasma OPN levels in patients with inflammatory bowel disease are associated with disease activity. Methods Plasma samples were obtained from patients with ulcerative colitis (UC, n=30), Crohns disease (CD, n=30), and healthy volunteers (controls, n=30) and enzyme immunoassay was performed. Results Plasma OPN concentrations were significantly higher in patients with Crohns disease than in controls (951.9±538.5 ng/mL and 659.0±163.7 ng/mL, respectively). OPN concentrations in patients with UC were also higher than in the controls (1149.6±791.0 and 659.0±163.7, respectively). There was a significant difference in plasma OPN level between active UC and inactive UC (2102.0±552.8 and 649.4±313.0, respectively). Moreover, a significant correlation was observed between plasma OPN concentration and disease activity, as determined by the clinical activity index in patients with UC. Conclusions Our results indicate that the plasma concentrations of OPN are elevated in patients with UC and that OPN expression is correlated with clinical activity. These results provide insight into UC pathogenesis and suggest that OPN may be a useful tool for assessing disease activity.

Tissue-specific expression of renin-angiotensin system components in IgA nephropathy

Background: The renin-angiotensin II system (RAS) has been implicated in the development of glomerulonephritis. The aims of this study were to determine (1) the expression of RAS components, angiotensin (Ang II)-forming enzymes [angiotensin-I-converting enzyme (ACE) and chymase], and Ang II receptors, and (2) the correlation between RAS expression and severity of tissue injury in IgA nephropathy (IgAN). Methods: The expression levels of ACE, chymase, and Ang II type 1 and type 2 receptor (AT1R and AT2R) mRNAs were determined by in situ hybridization in renal specimens from 18 patients with IgAN, 5 patients with non-IgA mesangial proliferative glomerulonephritis (non-IgAN) and 10 patients with nonmesangial proliferative glomerulonephritis (minimal change nephrotic syndrome, n = 5, and membranous nephropathy, n = 5). Normal portions of surgically resected kidney served as control. Results: In normal kidney, a few mesangial cells and glomerular and tubular epithelial cells weakly expressed ACE, chymase and AT1R mRNAs. In IgAN and non-IgAN samples, ACE, chymase, AT1R and AT2R mRNAs were expressed in resident glomerular cells, including mesangial cells, glomerular epithelial cells and cells of Bowman’s capsule. The glomerular expressions in IgAN were stronger than in minimal change nephrotic syndrome and membranous nephropathy. In IgAN, the expressions in glomeruli correlated with the degree of mesangial hypercellularity, whereas the expression levels were weaker at the area of mesangial expansion. IgAN with severe tubulointerstitial injury showed expression of ACE, chymase, AT1R and AT2R mRNAs in atrophic tubules and infiltrating cells and such expression correlated with the degree of tubulointerstitial damage. Conclusion: Our results suggest that renal cells can produce RAS components and that locally synthesized Ang II may be involved in tissue injury in IgAN through Ang II receptors in the kidney.

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.

Simvastatin attenuates trinitrobenzene sulfonic acid-induced colitis, but not oxazalone-induced colitis.

Purpose To determine whether simvastatin is able to inhibit inflammation in trinitrobenzene sulfonic acid (TNBS)-induced or oxazalone (OXA)-induced colitis. Results In the prophylactic protocol, simvastatin dose-dependently suppressed the decrease in body weight and inflammatory grade of TNBS-treated mice. In contrast, in the therapeutic protocol, no significant difference in body weight reduction was observed between simvastatin-treated and control mice. IFN-γ release from LP cells was significantly suppressed in mice receiving high-dose simvastatin in the prophylactic protocol. In contrast to TNBS colitis, even high-dose prophylactic simvastatin had no suppressive effects on either weight reduction or the inflammatory grade in OXA colitis. Conclusion Our results indicate that simvastatin negatively regulates inflammation in TNBS-induced colitis, but not in OXA-induced colitis. In TNBS-induced colitis, simvastatin suppressed the Th1-polarized immune response. Our findings suggest that simvastatin has potential effects as a therapeutic agent in human inflammatory bowel disease, particularly Crohn’s disease.

Intraglomerular synthesis of complement C3 and its activation products in IgA nephropathy

Background: Complement activation is thought to be pathologically important in IgA nephropathy (IgAN). Although C3 deposition in the mesangium is found in IgAN, the origin of C3 is not clear. We recently demonstrated intraglomerular C3 synthesis in the human kidney; however, the activation and pathological role of locally synthesized C3 remains unclear. Here we performed nonradioactive in situ hybridization for C3 mRNA and immunohistochemistry for C3 and its activation products, such as C3d and membrane attack complex (MAC), to determine whether locally produced C3 in glomeruli was activated in IgA nephropathy. Methods: Renal samples from 14 patients with IgAN and 5 with minimal change nephrotic syndrome (MCNS) were examined. Uninvolved portions of surgically removed kidneys with tumors served as normal controls. Results: C3 mRNA was not detected in glomeruli in control tissue and MCNS, but was strongly expressed in resident glomerular cells of IgAN, including mesangial cells, glomerular epithelial cells and the cells of Bowman’s capsule. Examination of serial sections disclosed that more than 70% of cells positive for C3 mRNA were also stained for C3 protein, C3d, and MAC. Double staining for in situ hybridization and immunohistochemistry also revealed that those C3 mRNA signals were present in intraglomerular cells positive for C3. The expression of C3 mRNA and MAC in glomeruli correlated significantly with the degree of mesangial matrix expansion. Conclusions: Our results demonstrated that locally synthesized C3 is activated in the glomeruli of IgAN and that its expression correlated with the severity of mesangial matrix expansion. These findings suggest that activation of C3 may be involved in tissue injury in IgAN through the formation of membrane attack complex.

The “Nutcracker” Phenomenon in Combination with IgA Nephropathy

The “nutcracker” phenomenon results from compression of the left renal vein between the superior mesenteric artery and the aorta. The main features of this phenomenon are non-glomerular haematuria on urinalysis and stenosis of the left renal vein with dilatation of the vein distal to the stenosis. The characteristics of two patients with the “nutcracker” phenomenon complicated by immunoglobulin A nephropathy were compared with those of 10 patients showing only the “nutcracker” phenomenon. Patients with the “nutcracker” phenomenon complicated by immunoglobulin A nephropathy showed aggravation of haematuria after upper respiratory infections, urinary red cell morphology indicating haematuria of glomerular origin, elevation of serum IgA, persistence of proteinuria, and granular casts in the urine. The coexistence of immunoglobulin A nephropathy was suspected when these characteristics were observed in patients with the “nutcracker” phenomenon. In such cases, a renal biopsy is needed for a final diagnosis.

Successful Treatment With Cyclosporine in a Caseof Hemophagocytic Syndrome Manifesting as Severe Liver Dysfunction

Forms of hemophagocytic syndrome, which affects mainly children, vary from mild to very severe and often fatal. We describe an adult patient with hemophagocytic syndrome in whom severe liver dysfunction developed. The condition continued to deteriorate despite treatment with plasma exchange, high-dose gamma globulin, and corticosteroid therapy. Treatment with cyclosporine (2.3 mg/kg/day) dramatically improved the condition and normalized liver function. Cyclosporine reduced the serum levels of ferritin, interferon-tau, interleukin-6, and soluble interleukin-2 receptor. These findings suggest that hemophagocytic syndrome accompanied with severe liver dysfunction results from hypercytokinemia, and cyclosporine is useful in preventing a fatal outcome during the acute phase.

White blood cell count and cardiovascular biomarkers of atherosclerosis.

Objective: To investigate the association with white blood cells (WBC) and atherosclerotic parameters including cardio-ankle vascular index (CAVI) and carotid intima–media thickness (CIMT) in the general population. Methods: We investigated the relationship between WBC count and metabolic syndrome components, CAVI and CIMT in 3738 Japanese study participants. Results: WBC count weakly correlated with CAVI in men (β = 0.61, p = 0.043), but not in women (β = 0.35, p = 0.17). On the other hand, WBC did not correlate with CIMT in either men or women (p = 0.41 and p = 0.71, respectively). Conclusion: WBC count was associated with lipids, blood pressure and body mass index, although the correlations with CAVI and CIMT were weak or absent.