伊藤 光泰

Post-transplantation bone diseases negatively affect the quality of life of solid organ recipients. Secondary or tertiary hyperparathyroidism is a frequent complication in kidney transplantation (KTx) recipients. Treatment with immunosuppressive agents including glucocorticoids can lead to deterioration in bone metabolism in these patients. In the present study, we explored the effects of a three-year treatment period with oral alendronate (ALN) in long-term KTx recipients. Post-KTx recipients were recruited (n = 24, M/F = 12/12, mean age 52.0 +/- A 7.8 years) into this study. All patients were prescribed methylprednisolone (4.07 +/- A 0.86 mg/day) with various immunosuppressive agents. Before treatment with oral ALN (35 mg/week), the mean concentrations of intact parathyroid hormone (iPTH) and 25-hydroxyvitamin D were 139.2 +/- A 71.4 pg/mL and 20.8 +/- A 4.1 ng/mL, respectively. After 36 months of ALN treatment, mean iPTH levels increased slightly (+20.9 %). Treatment with ALN reduced bone-specific alkaline phosphatase (-35.4 %), serum type I collagen N-terminal telopeptide (-31.2 %) and osteocalcin (-55.6 %) levels. ALN did not increase bone mass after 24 months. Four patients with the highest baseline iPTH levels suffered a clinical osteoporotic fracture during the 36-month ALN treatment period. Higher iPTH levels with chronic kidney disease (CKD) at baseline were associated with the incidence of new clinical fractures during ALN treatment. In conclusion, anti-resorptive therapy with ALN can suppress bone turnover even when iPTH concentration is elevated in long-term KTx recipients. However, hyperparathyroidism with CKD seems to be associated with new clinical fractures during ALN treatment.

Aripiprazole is the only atypical antipsychotic drug known to cause the phosphorylation of AMP-activated protein kinase (AMPK) in PC12 cells. However, the molecular mechanisms underlying this phosphorylation in aripiprazole-treated PC12 cells have not yet been clarified. Here, using PC12 cells, we show that these cells incubated for 24 h with aripiprazole at 50 mu M and 25 mM glucose underwent a decrease in their NAD(+)/NADH ratio. Aripiprazole suppressed cytochrome c oxidase (COX) activity but enhanced the activities of pyruvate dehydrogenase (PDH), citrate synthase and Complex I. The changes in enzyme activities coincided well with those in NADH, NAD(+), and NAD(+)/NADH ratio. However, the bioenergetic peril judged by the lowered COX activity might not be accompanied by excessive occurrence of apoptotic cell death in aripiprazole-treated cells, because the mitochondrial membrane potential was not decreased, but rather increased. On the other hand, when PC12 cells were incubated for 24 h with clozapine at 50 mu M and 25 mM glucose, the NAD(+)/NADH ratio did not change. Also, the COX activity was decreased; and the PDH activity was enhanced. These results suggest that aripiprazole-treated PC12 cells responded to the bioenergetic peril more effectively than the clozapine-treated ones to return the ATP biosynthesis back toward its ordinary level. This finding might be related to the fact that aripiprazole alone causes phosphorylation of AMPK in PC12 cells.

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc (1638T/1638T) mice that express a truncated Apc lacking the C-terminal domain. The Apc (1638T/1638T) mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc (1638T/1638T) mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc (1638T/1638T) and Apc (+/+) mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc (1638T/1638T) mice were significantly different whereas the Apc (+/+) mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc (1638T/1638T) follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc (+/+) follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc (1638T/1638T) mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc (+/+) mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc (1638T/1638T) mice and suggest suppressed secretory activities of these cells.

Background: Interactions between CD40 and its ligand (CD40L) have important roles in T-cell-dependent activation of B cells, which may be related to the thyrotoxic activity of Graves' disease (GD). Soluble forms of CD40 ligand (sCD40L) are released from activated T cells and platelets, and several types of inflammatory cytokines are increased in patients with hyperthyroid GD. The aim of this study was to assess sCD40L and other cytokines as clinical indicators of disease activity or as possible markers of remission in GD. Methods: Serum levels of sCD40L, interleukin 18 (IL-18), tumor necrosis factor-alpha (TNF alpha), and TNF alpha receptors 1 and 2 (TNFR1 and TNFR2) were investigated in patients with active GD (GD-A), intractable GD (GD-IT), inactive GD (GD-IA), GD in remission (GD-R), and Hashimoto's thyroiditis (HT), and in control subjects (CON). Results: Serum concentrations of sCD40L were higher in the GD-A and GD-IT groups than in the HT and CON groups. Similarly, serum concentrations of IL-18, which induces Th1 cytokines, such as interferon-gamma, were higher in the GD-A and GD-IT groups than in all other groups. Serum levels of TNFR1 and TNFR2 were also significantly higher in the GD-A than in all other groups. The mean serum concentration of TNF alpha was higher in the GD-R compared with the GD-A and GD-IT groups, although the difference was not significant. Serum sCD40L concentrations in the GD-R group were lower than in the GD-A and GD-IT groups. Finally, the ratio of serum TNF alpha to sCD40L was higher in the GD-R group than in the GD-A and GD-IT groups. This is the first report that serum sCD40L is increased in active GD, and that the serum TNF alpha:sCD40L ratio is a marker for remission in GD. Conclusions: Our results suggest that not only thyrotoxicosis, but also the activity of the immunoreaction presenting as anti-thyrotropin receptor antibodies (TRAb) titer in GD, affects inflammatory cytokine serum profiles. Serum profiles of cytokines vary in patients with GD depending on disease activity. An elevated serum TNF alpha:sCD40L ratio indicates declining disease activity and reflects a shift from Th2 to Th1 dominance, suggesting that suppression of sCD40L or increased production of TNF alpha is required to initiate or maintain remission of GD.

Background: Chronic elevation of cardiac troponin T (TnT) levels as measured by conventional assays is strongly associated with structural heart disease and cardiovascular events. A new high-sensitivity assay for TnT makes it possible to measure concentrations more than a factor of 5 lower than the limits of detection of conventional assays. We evaluated the utility of serum TnT as a risk marker of cardiovascular disease in 409 outpatients with type-2 diabetes mellitus (T2DM). Results: TnT was detectable (&gt 0. 002 ng/mL) in 80% of patients, and elevation in TnT levels (&gt 0. 014 ng/mL) was found in 19. 3%, suggesting a higher prevalence of structural heart diseases in T2DM patients. A history of cardiovascular disease was noted in 89 (22%) patients. Patients with diabetic microvascular complications and those with abnormal electrocardiogram including left ventricular hypertrophy had higher TnT levels. Patients with increased levels of TnT (&gt 0. 014 ng/mL) were older, had higher values of N-terminal pro-B-type natriuretic peptide (NT-proBNP), C-reactive protein, cystatin C, and urinary albumin/creatinine ratio, had lower values of hemoglobin and estimated glomerular filtration rate, and had a higher prevalence of cardiovascular disease compared with those without increased TnT levels. In stepwise logistic analysis, NT-proBNP (odds ratio 7. 40 per 10-fold increase, P &lt 0. 0001) and cystatin C (18. 0 per 1. 0 mg/L, P &lt 0. 0001) were independently associated with elevation of TnT levels. HsTnT level, cystatin C, and HDL-cholesterol were also independent risk factors for history of major cardiovascular diseases in T2DM patients. Conclusion: This new high-sensitivity TnT assay may be useful for stratifying cardiovascular risk in outpatients with T2DM. © 2011 The Japan Diabetes Society.

Inorganic phosphate (Pi) is required in many biological processes, including signaling cascades, skeletal development, tooth mineralization, and nucleic acid synthesis. Recently, we showed that Pi transport in osteoblasts, mediated by Slc20a1, a member of the type III sodium-dependent phosphate transporter family, is indispensable for osteoid mineralization in rapidly growing rat bone. In addition, we found that bone mineral density decreased slightly with dysfunction of Pi homeostasis in aged transgenic rats overexpressing mouse Slc20a1 (Slc20a1-Tg). Bone and tooth share certain common molecular features, and thus, we focused on tooth development in Slc20a1-Tg mandibular incisors in order to determine the role of Slc20a1 in tooth mineralization. Around the time of weaning, there were no significant differences in serologic parameters between wild-type and Slc20a1-Tg rats. However, histological analysis showed that Slc20a1-Tg ameloblasts formed clusters in the papillary layer during the maturation stage as early as 4 weeks of age. These pathologies became more severe with age and included the formation of cyst-like or multilayer ameloblast structures, accompanied by a chalky white appearance with abnormal attrition and fracture. Hyperphosphatemia was also observed in aging Slc20a1-Tg rats. Micro-computed tomography and electron probe microanalysis revealed impairments in enamel, such as delayed mineralization and hypomineralization. Our results suggest that enamel formation is sensitive to imbalances in Pit1-mediated cellular function as seen in bone, although these processes are under the control of systemic Pi homeostasis.

Sekiguchi S, Suzuki A, Asano S, Nishiwaki-Yasuda K, Shibata M, Nagao S, Yamamoto N, Matsuyama M, Sato Y, Yan K, Yaoita E, Itoh M. Phosphate overload induces podocyte injury via type III Na-dependent phosphate transporter. Am J Physiol Renal Physiol 300: F848-F856, 2011. First published February 9, 2011; doi:10.1152/ajprenal.00334.2010.-Uptake of Pi at the cellular membrane is essential for the maintenance of cell viability. However, phosphate overload is also stressful for cells and can result in cellular damage. In the present study, we investigated the effects of the transgenic overexpression of type III Pi transporter Pit-1 to explore the role of extracellular Pi in glomerular sclerosis during chronic renal disease. Pit-1 transgenic (TG) rats showed progressive proteinuria associated with hypoalbuminemia and dyslipidemia. Ultrastructural analysis of TG rat kidney by transmission electron microscopy showed a diffuse effacement of the foot processes of podocytes and a thickening of the glomerular basement membrane, which were progressively exhibited since 8 wk after birth. TG rats died at 32 wk of age due to cachexia. At this time, more thickening of the glomerular basement membrane and segmental sclerosis were observed in glomeruli of the TG rats. Immunohistochemical examination using anticonnexin 43 and anti-desmin antibodies suggested the progressive injury of podocytes in TG rats. TG rats showed higher Pi uptake in podocytes than wild-type rats, especially under low Pi concentration. When 8-wk-old wild-type and TG rats were fed a 0.6% normal phosphate (NP) or 1.2% phosphate (HP) diet for 12 wk, HP diet-treated TG rats showed more progressive proteinuria and higher serum creatinine levels than NP diet-treated TG rats. In conclusion, our findings suggest that overexpression of Pit-1 in rats induces phosphate-dependent podocyte injury and damage to the glomerular barrier, which result in the progression of glomerular sclerosis in the kidney.

Aldolase, a glycolytic enzyme, consists of 3 immunologically different subunits as the tetrameric form and may correlate with cell carcinogenesis. In the present study, we investigated the role of aldolase isozymes in the thyroid tissue of various thyroid disorders using immunohistochemical staining and highly sensitive enzyme immunoassay for quantitative analysis of the isozymes. Aldolase A had low tissue specificity but correlated with tumor proliferation. The relation to tumor proliferation, however, was found only in a few cases of the thyroid tumors. Aldolase B was barely detectable in these thyroid tissues. Aldolase C, however, had high activities suggesting the high thyroid tissue specificity, particularly for parafollicular cells. It was considered to be a useful marker of tumor cell differentiation. The analysis of these isozymes may be helpful for the diagnosis of medullary thyroid carcinoma and neuroendocrine tumors.

We conducted an observational study in order to assess the prevalence of hypovitaminosis D and its seasonal changes, in the Tokai area (N35.3 E137.0), in 197 normal subjects in Japan. The mean serum 25-hydroxyvitamin D (25-OHD) level measured by direct radioimmunoassay (RIA) was lowest at the end of winter, and highest at the end of summer (15.1 +/- 7.1 ng/ml in March; 21.5 +/- 5.5 ng/ml in June; 31.6 +/- 5.6 ng/ml in September; 23.1 +/- 5.3 ng/ml in December; mean +/- SD). The prevalence of hypovitaminosis D ( < 20 ng/ml) was 86.7%, 33.4%, 1.0%, and 26.0% in March, June, September, and December, respectively. Mean plasma intact parathyroid hormone (iPTH) concentration was lowest at the end of summer and highest at the end of winter (28.2 +/- 9.3 pg/ml in March; 21.7 +/- 7.0 pg/ml in June; 19.8 +/- 6.9 pg/ml in September; and 25.7 +/- 9.2 pg/ml in December; mean +/- SD). Serum 25-OHD was inversely associated with iPTH ( coefficient, - 0.223; r = 0.251; P < 0.001). Serum 25-OHD levels were higher in men than in women. The serum 25-OHD level was positively associated with age, body weight, and body mass index, but not with body fat content. These results suggest a high prevalence of hypovitaminosis D associated with elevation of iPTH in Japan, in winter, even in a sunny area.

The possible role of abnormal T cell-dependent B-cell activation in Graves' disease was investigated by comparing lymphocyte subset distribution and the production of soluble CD8 (sCD8), sCD23, IL-10 and IL-12 by peripheral blood cells (PBMC) and thyroid-infiltrating lymphocytes (TL) in vitro. In TL, the percentage of CD8(+) cells was slightly higher and the sCD8 concentration was significantly higher than in PBMC. The ratio CD23(+) cells to CD20(+) cells (activated B/pan B cells) was increased in TL compared to PBMC from Graves' or normal controls, although the percentage of CD20(+) cells was decreased. Compared to PBMC in Graves' disease, the relative ratio of IL-10 to IL-12 release (IL-10/IL-12) by unstimulated TL was increased, despite a lack of significant difference between PBMC and TL in mean values for either IL-10 or IL-12 secretion. Incubating PBMC with a combination of anti-CD40 monoclonal antibodies and interleukin-4 (IL-4) resulted in B cell activation, reflected in an increase in the sCD23 level in both controls and Graves' patients, but especially prominent in the latter. Stimulation with anti-CD40 antibody and IL-4 also decreased the percentage of CD8+ cells in PBMC but not TL from both Graves' disease and normal controls, and the percentage of CD8(+) cells in TL was higher than PBMC after the stimulation. The sCD23 concentration in TL was decreased compared to PBMC both in patients with Graves' disease and normal controls. However, in contrast to the increased responses observed in Graves' PBMC or normal controls after stimulation, sCD23 levels remained the same in stimulated TL from Graves' patients. This combination of B cell stimulants increased production of IL-10 in PBMC but not in TL obtained from patients with Graves' disease, and the increased IL-10/IL-12 ratio declined to a value no different from that in PBMC group after stimulation. Thus, T cell-dependent B-cell activation via a CD40 pathway may cause a shift in the Th-1/Th-2 balance to Th-2 dominance in Graves' disease, while increased CD8(+) cells in TL may suppress sCD23 production and IL-10-producing Th2 cells. (C) 2002 Elsevier Science Ltd. All rights reserved.