髙栁 武志

Osteoporosis patients with chronic kidney disease (CKD) are becoming common in our superaging society. Renal dysfunction causes phosphorus accumulation in the circulating plasma and leads to the development of CKD-mineral bone disorder (MBD). We have previously reported that type III Pi transporter-overexpressing transgenic (Pit-1 TG) rats manifest phosphate (Pi)-dependent podocyte injury. In the present study, we explored the effect of risedronate on Pi-induced podocyte injury in vivo. Pit-1 TG rats and wild-type rats at 5 weeks old were divided into a risedronate-treated group and an untreated group. We subcutaneously administered 5 μg/kg body weight of risedronate or saline twice a week during the experimental period. Risedronate did not alter serum creatinine levels at 5, 8, and 12 weeks of age. However, electron microscopy images showed that thickening of the glomerular basement membrane was improved in the risedronate treatment group. Furthermore, immunostaining for podocyte injury markers revealed that both desmin- and connexin43-positive areas were smaller in the risedronate-treated group than in the untreated group, suggesting that bisphosphonates could rescue Pi-induced podocyte injury. In conclusion, our findings suggest that risedronate could maintain glomerular barrier function by rescuing Pi-induced podocyte injury.

We previously showed that aripiprazole increases intracellular NADPH and glucose-6-phosphate dehydrogenase mRNA in PC12 cells. Aripiprazole presumably activates a system that concurrently detoxifies reactive oxygen species and replenishes NADPH. Nrf2, a master transcriptional regulator of redox homeostasis genes, also activates the pentose phosphate pathway, including NADPH production. Therefore, our aim was to determine whether aripiprazole activates Nrf2 in PC12 cells. Aripiprazole increased mRNA expression of Nrf2-dependent genes (NAD(P)H-quinone oxidoreductase-1, Nqo1; heme oxygenase-1, HO1; and glutamate-cysteine ligase catalytic subunit) and protein expression of Nqo1 and HO1 in these cells (p < 0.05). To maintain increased Nrf2 activity, it is necessary to inhibit Nrf2 degradation; this is done by causing Nrf2 to dissociate from Keap1 or beta-TrCP. However, in aripiprazole-treated cells, the relative amount of Nrf2 anchored to Keap1 or beta-TrCP was unaffected and Nrf2 in the nuclear fraction decreased (p < 0.05). Aripiprazole did not affect phosphorylation of Nrf2 at Ser40 and decreased the relative amount of acetylated Nrf2 (p < 0.05). The increase in Nqo1 and HO1 in aripiprazole-treated cells cannot be explained by the canonical Nrf2-degrading pathways. Further experiments are needed to determine the biochemical mechanisms underlying the aripiprazole-induced increase in these enzymes.

Objectives: According to our previous work, aripiprazole exerted a protective effect on hydrogen peroxide (H2O2)-treated PC12 cells; haloperidol did not. Because aripiprazole has distinct affinities to a set of neurotransmitter receptor subtypes, this study aimed to clarify which subtype is responsible for rescuing cells from 0.25 mM H2O2 exposure.Methods: A set of compounds, which are more specific to each subset of G-protein coupled receptors, wereexamined for their ability to mimic the pharmacological effects of aripiprazole or haloperidol, including their Ki values.The viability of PC12 cells cultured with test compounds with or without H2O2 was assessed using WST-8 reagent.Results: Results from in vitro studies using PC12 cells showed that agonism at serotonin 5-HT2C-receptors based on the antagonism against 5-HT2B-receptors played a significant role in resistingH2O2-induced cell death. However, the use of a specific 5-HT2B-receptor agonist instead of a 5-HT2B-receptor antagonist completely negated the effect of a specific 5-HT2C-receptor agonist. Furthermore, unlike the dopamine D1-receptor specific antagonist, none of the agonists of dopamine D2-, D3-, and D4-receptors ameliorated the cytopathic effects of H2O2.Conclusion: Antagonism at 5-HT2B-receptors is fundamental for the protection of PC12 cells against the cytopathiceffects caused by 0.25 mM H2O2. However, the role of negatively regulated cyclic adenosine monophosphate in this phenomenon requires further investigation.

Background. Despite recent progress of immunosuppressive therapy with newly developed agents, long-term pancreatic graft survival after pancreas transplantation still remains low. Therefore, precise assessment of beta-cell function after pancreas transplantation is necessary. Methods. Pancreatic beta-cell secretory activity was measured by means of the peripheral plasma fasting serum C-peptide (CPR) response to 1 mg of glucagon intravenously in 23 patients after pancreas transplantation. The utility of Delta CPR after injection was compared with other indices that reflect insulin secretion. Results. When we performed the test, 6 patients still needed insulin injection after the transplantation. Mean CPR before and after glucagon intravenously were 1.9 +/- 0.98 ng/mL and 4.6 +/- 2.29 ng/mL, respectively. Fasting serum CPR, secretory unit of islet in transplantation (SUIT) index, and Delta CPR after glucagon injection were significantly different between insulin users and nonusers. During follow-up (501 228 days), 3 patients could stop using insulin, and their increase of CPR (1.8 +/- 0.5 ng/mL) was significantly higher than that in continuous insulin users (0.3 +/- 0.3 ng/mL). Conclusion. Fasting CPR, SUIT index, and Delta CPR after glucagon injection could reflect beta-cell function for post-pancreas transplant patients, and glucagon stimulation test could give us additional information to predict insulin-free treatment.

In aripiprazole-treated PC12 cells, we previously showed that the mitochondrial membrane potential (Delta psi(m)) was rather increased in spite of lowered cytochrome c oxidase activity. To address these inconsistent results, we focused the NADPH generation by glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway (PPP), to titrate reactive oxygen species (ROS) that results in the Delta psi(m) maintenance. G6PD may be also involved in another inconsistent result of lowered intracellular lactate level in aripiprazole-treated PC12 cells, because PPP competes glucose-6-phosphate with the glycolytic pathway, resulting in the downregulation of glycolysis. Therefore, we assayed intracellular amounts of NADPH, ROS, and the activities of the enzymes generating or consuming NADPH (G6PD, NADP(+)-dependent isocitrate dehydrogenase, NADP(+)-dependent malic enzyme, glutathione reductase, and NADPH oxidase [NOX]) and estimated glycolysis in 50 mu M aripiprazole-, clozapine-, and haloperidol-treated PC12 cells. NADPH levels were enhanced only in aripiprazole-treated ones. Only haloperidol increased ROS. However, the enzyme activities did not show significant changes toward enhancing NADPH level except for the aripiprazole-induced decrease in NOX activity. Thus, the lowered NOX activity could have contributed to the aripiprazole-induced increase in the NADPH level by lowering ROS generation, resulting in maintained Delta psi(m). Although the aforementioned assumption was invalid, the ratio of fructose-1,6-bisphosphate to fructose-6-phosphate was decreased by all antipsychotics examined. Pyruvate kinase activity was enhanced only by aripiprazole. In summary, these observations indicate that aripiprazole possibly possesses the pharmacological superiority to clozapine and haloperidol in the ROS generation and the adjustment of glycolytic pathway.

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.

VEGF family members play important roles in angiogenesis and vascular permeability. VEGF-A-transgenic mice showed an increased vascularization with edema due to hyper-vascular permeability and subcutaneous hemorrhage as side effects. VEGF-A binds and activates two receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). To dissect the signals of these two receptors, we generated transgenic mice overexpressing either the VEGFR-2-specific ligand VEGF-ENZ-7 or VEGFR-1-specific ligand PIGF-II under the control of the Keratin-14 promoter. VEGF-E-mice showed a significant increase in vascularization (about 10-fold compared to control mice) in subcutaneous tissues, whereas PIGF-mice showed only a 2-3-fold increase. Interestingly, VEGF-E-mice did not show any clear edematous lesions or hemorrhagic spots on the skin. Microscopically, VEGF-E-induced capillary networks have a well organized structure with the recruitment of pericytes. These results indicate that VEGF-E is a new angiogenic agent with less side effects for clinical usage. (C) 2003 Elsevier Science (USA). All rights reserved.