Kazuaki Hirata

Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats.

In an attempt to define the role of the pineal secretory melatonin and an analogue, 6‐hydroxymelatonin (6‐OHM), in limiting oxidative stress, the present study investigated the cisplatin (CP)‐induced alteration in the renal antioxidant system and nephroprotection with the two indolamines. Melatonin (5 mg/kg), 6‐OHM (5 mg/kg), or an equal volume of saline were administered intraperitoneally (i.p.) to male Sprague–Dawley rats 30 min prior to an i.p. injection of CP (7 mg/kg). After CP treatment, the animals each received indolamine or saline every day and were sacrificed 3 or 5 days later and plasma as well as kidney were collected. Both plasma creatinine and blood urea nitrogen increased significantly following CP administration alone; these values decreased significantly with melatonin co‐treatment of CP‐treated rats. In the kidney, CP decreased the levels of GSH (reduced glutathione)/GSSG (oxidized glutathione) ratio, an index directly related to oxidative stress. When animals were treated with melatonin, the reduction in the GSH/GSSG ratio was prevented. Treatment of CP‐enhanced lipid peroxidation in the kidney was again prevented in animals treated with melatonin. The activity of the antioxidant enzyme, glutathione peroxidase (GSH‐Px), decreased as a result of CP administration, which was restored to control levels with melatonin co‐treatment. Upon histological analysis, damage to the proximal tubular cells was seen in the kidneys of CP‐treated rats; these changes were prevented by melatonin treatment. 6‐OHM has been shown to have some antioxidative capacity, however, the protective effects of 6‐OHM against CP‐induced nephrotoxicity were less than those of melatonin. The residual platinum concentration in the kidney of melatonin co‐treated rats was significantly lower than that of rats treated with CP alone. It is concluded that administration of CP imposes a severe oxidative stress to renal tissue and melatonin confers protection against the oxidative damage associated with CP. This mechanism may be reasonably attributed to its radical scavenging activity, to its GSH‐Px activating property, and/or to its regulatory activity for renal function.

Administration of Melatonin and Related Indoles Prevents Exercise-Induced Cellular Oxidative Changes in Rats

In an attempt to define the role of the pineal hormone melatonin and two analogues (5-methoxytryptamine, 5MT, and 6-hydroxymelatonin, 6HM) in limiting oxidative stress, the present study investigated the changes in glutathione, lipid peroxidation, and the activity of the antioxidant enzyme glutathione peroxidase after exercise (swimming for 60 min) with or without treatment with the indolamines mentioned. Lipid peroxidation was measured by estimating tissue levels of malondialdehyde and 4-hydroxyalkenals; the experimental animals in these studies were male Sprague-Dawley rats. In the liver, swimming exercise increased the levels of reduced glutathione (GSH) and also significantly increasing oxidized glutathione (GSSG), while decreasing the GSH/GSSG ratio, an index directly related to oxidative stress. When the animals were treated with melatonin, the concentrations of GSH and GSSG were also increased after swimming; however, no reduction in the GSH/GSSG ratio appeared. In the animals treated with 6HM the changes were the same as in those treated with melatonin. In muscle as well, the concentration of GSH and the GSH/GSSG ratio were decreased following 60 min of swimming. Pretreatment of the rats with melatonin prevented these effects. Pretreatment of the rats with both 5MT and 6HM also prevented the changes. Brain GSH/GSSG ratio was not affected by either exercise or indolamine administration. Swimming enhanced lipid peroxidation in the liver, muscle and brain; however, this was prevented in animals treated with melatonin or 6HM before swimming. Glutathione peroxidase was significantly elevated after exercise in the brain but not in the liver and muscle. It is concluded that swimming imposes a severe oxidative stress and suggests that melatonin and, to a lesser degree, 5MT and 6HM confer protection against the oxidative damage associated with swimming for 60 min. This mechanism may be reasonably attributed to their indole structure, which possibly allows these molecules to act as free-radical scavengers.

Contribution of mitogen-activated protein kinases to NMDA-induced neurotoxicity in the rat retina

We examined the contributions of the mitogen-activated protein kinases (MAPKs) family [extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK)] to N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Detection of apoptotic cell death in the retinal ganglion cell layer (RGCL) and the inner nuclear layer (INL) by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining began 6 h after intravitreal NMDA (100 nmol) injection and continued to increase thereafter. Western blot analysis showed that phosphorylated MAPKs (p-MAPKs) were expressed in the retina following a temporal manner: maximal expression of phosphorylated ERK (p-ERK) at 1 h, maximal expression of phosphorylated p38 (p-p38) at 6 h, and beginning of phosphorylated JNK (p-JNK) significant increase at 6 h after injection. An immunohistochemical/TUNEL co-localization study showed that p-JNK- and p-p38-positive cells in the RGCL were frequently TUNEL-positive, whereas few p-ERK-positive cells were TUNEL-positive. Moreover, co-injection of inhibitors for JNK (0.2 nmol SP600125) and/or p38 (2.0 nmol SB203580) with NMDA was effective in ameliorating NMDA-induced apoptotic cell loss in the RGCL 12 h after injection, as shown by TUNEL-positive cell counts. These inhibitors also protected the inner retina as shown by morphometric studies such as cell counts in the RGCL and measurement of the IPL thickness 7 days after injection. On the other hand, an ERK inhibitor (2.0 nmol U0126) did not suppress NMDA-induced cell death in the RGCL nor thinning of the IPL. These findings suggest that JNK and p38 are proapoptotic in NMDA-induced cell death in the RGCL, but not ERK.

Amelioration of diabetic tubulointerstitial damage in liver-type fatty acid-binding protein transgenic mice

BACKGROUND Renoprotection of liver-type fatty acid binding protein (L-FABP) was demonstrated in a streptozotocin (STZ)-induced diabetic mouse model. METHODS Established human L-FABP (hL-FABP) transgenic (Tg) mice and wild-type (WT) mice were divided into two groups: diabetic mice were uninephrectomized and injected with STZ; control mice were uninephrectomized and injected with a citrate buffer alone. Although mouse L-FABP was not expressed in WT mice, hL-FABP was expressed in the proximal tubules of the diabetic Tg mice and in the control Tg mice at 8 and 14 weeks after these injections. RESULTS The expression of renal hL-FABP increased significantly in diabetic Tg mice compared to control Tg mice. A number of macrophages (F4/80) infiltrating the interstitium, the gene expressions of MCP-1, MCP-3, TGF-beta, Fas, Bax and RAGE were significantly lower in diabetic Tg kidneys compared with diabetic WT kidneys. In the diabetic Tg kidneys, the degree of the tubulointerstitial injury and the deposition of type IV collagen were significantly lower than that of diabetic WT kidneys. The expressions of catalase and glutathione peroxidase-1 were significantly lower in diabetic Tg kidneys compared with diabetic WT kidneys. CONCLUSIONS Renal L-FABP ameliorated the tubulointerstitial damage of type 1 diabetic mice.

Melatonin Receptors in the Spinal Cord

The pineal hormone, melatonin, plays an important role in the regulation of diurnal and seasonal rhythms in animals. In addition to the well established actions on the brain, the possibility of a direct melatonin action on the spinal cord has to be considered. In our laboratory, we have obtained data suggesting that melatonin receptors are present in the spinal cords of birds and mammals. Using radioreceptor binding and quantitative autoradiography assays with 2-[125I]iodomelatonin as the specific melatonin agonist, melatonin binding sites have been demonstrated in the rabbit and chicken spinal cords. These sites are saturable, reversible, specific, guanosine nucleotide-sensitive, of picomolar affinity and femtomolar density. The linearity of Scatchard plots of saturation data and the unity of Hill coefficients indicate that a single class of melatonin binding sites is present in the spinal cord membranes studied. The picomolar affinity of these sites is in line with the circulating levels of melatonin in these animals suggesting that these sites are physiologically relevant. Autoradiography studies in the rabbit spinal cord show that melatonin binding sites are localized in the central gray substance (lamina X). In the chicken spinal cord, these binding sites are localized in dorsal gray horns (laminae I-V) and lamina X. As lamina X and laminae I-II have similar functions, melatonin may have comparable roles in the chicken and rabbit spinal cords. Moreover, in the chicken spinal cord, the density of 2-[125I]iodomelatonin binding in the lumbar segment was significantly higher than those of the cervical and thoracic segments. The densities of these binding sites changed with environmental manipulations. When chickens were adapted to a 12L/12D photoperiod and sacrificed at mid-light and mid-dark, there was a significant diurnal variation in the density (maximum number of binding sites; Bmax) of melatonin binding sites in the spinal cord. After constant light treatment or pinealectomy, the Bmax of melatonin receptors in the chicken spinal cord increased significantly in the subjective mid-dark period. Moreover, there was an age-related decrease in the 2-[125I]iodomelatonin binding to the chicken spinal cord. Our results suggest that melatonin receptors in the chicken spinal cord are regulated by environmental lighting and change with development. These receptors may play an important role in the chronobiology of spinal cord function. The biological responses of melatonin on spinal cords have also been demonstrated in vitro. Melatonin decreased the forskolin-stimulated cAMP production in the chicken spinal cord explant. Preincubation with pertussis toxin blocked the melatonin effect. Our results suggest that melatonin receptors in the chicken spinal cord are linked to the adenylate cyclase via a pertussis toxin-sensitive G protein and that melatonin binding sites in spinal cords are melatonin receptors with biological functions. These receptors may be involved in the regulation of spinal cord functions related to sensory transmission, visceral reflexes and autonomic activities.

Circadian Rhythm of Melatonin Release From the Photoreceptive Pineal Organ of a Teleost, Ayu (Plecoglossus altivelis) in Flow-Thorough Culture

In the present study, we tested whether the pineal organ of ayu (Plecoglossus altivelis), an osmerid teleost close relative of salmonids, harbours a circadian oscillator regulating rhythmic melatonin release using flow‐through culture. The pineal organ maintained under light/dark cycles released melatonin in a rhythmic fashion with high levels during the dark phase. A circadian rhythm of melatonin release persisted in constant darkness for at least four cycles. Characteristics of the circadian rhythm (free‐running period, phase and amplitude) exhibited small variations among cultures when the data was normalized, indicating that this system is sufficient for the analysis of the circadian rhythm both at qualitative and quantitative levels. Six‐hour extension of the light phase from the normal onset time of the dark phase or exposure to constant light for 36 or 48 h before transfer to constant darkness significantly inhibited melatonin release. Phase shifts in the circadian rhythm of melatonin release were also observed. Thus, the ayu pineal organ contains all the three essential components of the circadian system (a circadian clock, the photoreceptor responsible for photic entrainment of the clock, and melatonin generating system as an output pathway). This system should provide a useful model for analysing the physiological and molecular basis of the vertebrate circadian system. In addition, further comparative studies using salmonids and related species including ayu will provide some insight into the evolution of the roles of the pineal organ in the vertebrate circadian system.

Pit‐1 gene expression in human pituitary adenomas using the reverse transcription polymerase chain reaction method

OBJECTIVE Previous studies of Pit‐1 expression in human pituitary tumours have produced conflicting results. We have studied expression of Pit‐1 mRNA in human pituitary adenomas, as well as in normal human and rat pituitaries, and results were compared with clinical, histological, and immunohistochemical features. In addition, expression of GH, PRL, and TSH‐β mRNA was also studied and compared with Pit‐1 gene expression.

Urinary liver type fatty acid binding protein in diabetic nephropathy.

Deterioration of diabetic nephropathy (DN) is largely determined by the degree of tubulointerstitial changes rather than the extent of histological changes in the glomeruli. Therefore, a tubular marker that accurately reflects tubulointerstitial damage may be an excellent biomarker for early detection or prediction of DN. Liver-type fatty-acid binding protein (L-FABP) is a 14 kDa small molecule that is expressed in the cytoplasm of human proximal tubules. In vivo experimental studies revealed that renal L-FABP gene expression was up-regulated by various stresses that cause tubulointerstitial damage, such as massive proteinuria, hyperglycemia, hypertension, ischemia and toxins, and that urinary excretion of L-FABP was increased. Recent clinical studies of patients with type 1 or type 2 diabetes demonstrated that urinary excretion of L-FABP derived from proximal tubules is a suitable biomarker for predicting and monitoring deterioration of renal function in DN. Moreover, therapeutic interventions with renoprotective effects reduced urinary L-FABP concentrations. Therefore, urinary L-FABP measured using the Human L-FABP ELISA Kit developed by CMIC Co., Ltd. (Tokyo, Japan) was confirmed as a newly established tubular biomarker by the Ministry of Health, Labour and Welfare in Japan in 2010. This review article summarizes the clinical significance of urinary L-FABP in DN.

Pro-apoptotic role of c-Jun in NMDA-induced neurotoxicity in the rat retina

We examined the role of c‐Jun on N‐methyl‐D‐aspartate (NMDA)‐induced neurotoxicity in the rat retina. An increase in c‐Jun mRNA, c‐Jun protein and phosphorylated c‐Jun (p‐c‐Jun) levels in the retina was detected 3 hr after intravitreal injection of NMDA (20 nmol). These levels peaked after 12 hr, and then returned to their control levels by 24 hr. c‐Jun and p‐c‐Jun immunoreactivities were observed in the retinal ganglion cell layer (RGCL), especially in retinal ganglion cells (RGCs), and in the inner nuclear layer (INL) 12 hr after NMDA injection, and terminal deoxynucleotidyl transferase‐mediated nick‐end labeling (TUNEL)‐positive cells were immunopositive for c‐Jun and p‐c‐Jun. A c‐Jun antisense oligodeoxynucleotide (AS ODN), which was simultaneously injected with NMDA, penetrated the cells in the RGCL and the INL, suppressed the NMDA‐induced increase in c‐Jun and p‐c‐Jun protein levels and reduced the number of TUNEL‐positive cells in the RGCL 12 hr after the injection. The protective effect of c‐Jun AS ODN on the NMDA‐treated retina was also shown by the RGCL cell count and measurement of the IPL thickness, as well as by quantitative real‐time PCR analysis of Thy‐1 mRNA 7 days after the injection. These results suggest that c‐Jun synthesis and phosphorylation participate in NMDA‐induced neuronal cell death.

Proteasome inhibitors which induce neurite outgrowth from PC12h cells cause different subcellular accumulations of multi-ubiquitin chains.

The effects of two proteasome inhibitors on neurite outgrowth from PC12h cells were investigated in terms of the mean length of the neurites and the frequency of occurrence of cells with long neurites. Benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and benzyloxycarbonyl-isoleucyl-t-butyl-glutamyl-leucinal (PSI) caused a significant elongation of PC12h cell neurites. Since ZLLLal is known to inhibit both calpain and proteasome activity, we examined the effects of benzyloxycarbonyl-leucyl-leucinal (ZLLal) which inhibits calpain activity to the same degree as ZLLLal, but which inhibits proteasome activity only weakly. ZLLal did not induce the significant elongation of neurites at any of the concentrations we studied. These results show that the inhibition of proteasome activity causes neurite elongation. We also quantified subcellular levels of multi-ubiquitin chains and free ubiquitin after treatments with PSI, ZLLLal and ZLLal. Treatment with ZLLal had no effects on levels of water- and urea-soluble multi-ubiquitin chains or of free ubiquitin either in the nucleus or in the cytoplasm. PSI and ZLLLal induced a large accumulation of water- and urea-soluble multi-ubiquitin chains and free ubiquitin in the nucleus. Similarly, PSI and ZLLLal increased cytoplasmic levels of urea-soluble multi-ubiquitin chains. On the contrary, PSI and ZLLLal had no effect on levels of water-soluble multi-ubiquitin chains or free ubiquitin in the cytoplasm. This is the first study to demonstrate subcellular differences in the accumulation of multi-ubiquitin chains and free ubiquitin during the neurite elongation induced by proteasome inhibitors.