Delayed Postnatal Growth and Anterior Pituitary Development in Growth-Retarded (grt) Female Mice.

Growth-retarded (grt) mice display primary congenital hypothyroidism due to the hyporesponsiveness of their thyroid glands to thyroid-stimulating hormone (TSH). We examined somatic growth, anterior pituitary development, and hormonal profiles in female grt mice and normal ones. Although growth in grt females was suppressed 2 weeks after birth, the measured growth parameters and organ weights gradually increased and finally reached close to the normal levels. Grt mice exhibited delayed eye and vaginal openings and remained in a state of persistent diestrus thereafter, plasma estrogen levels being lower than those in normal mice. Grt mice that received normal-donor thyroids showed accelerated growth and their body weights increased up to the sham-normal levels, indicating the importance of early thyroid hormone supplementation. In the anterior pituitary, there were fewer growth hormone (GH) and prolactin (PRL) cells in grt mice than in normal mice as examined at 12 weeks after birth, but the numbers of these cells did not differ from those in normal mice after 24 weeks. Grt mice had more TSH cells than normal mice until 48 weeks. Plasma GH levels in grt mice were lower than those in normal mice at 2 weeks, but did not differ substantially after 5 weeks. Compared with normal mice, grt mice had significantly lower plasma PRL and thyroxine levels, but notably higher TSH levels until 48 weeks. These findings indicate that thyroid hormone deficiency in grt mice causes delayed development and growth, and inappropriate development of GH, PRL and TSH cells, followed by the abnormal secretion of hormones by these pituitary cells.

Possible involvement of thyrotropin-releasing hormone receptor 3 in the release of prolactin in the metamorphosing bullfrog larvae.

In amphibians, thyrotropin (TSH), corticotropin (ACTH) and prolactin (PRL) are regarded as the major pituitary hormones involved in metamorphosis, their releasing factors being corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and thyrotropin-releasing hormone (TRH), respectively. It is also known that thyrotropes and corticotropes are equipped with CRF type-2 receptor and AVT V1b receptor, respectively. As for PRL cells, information about the type of receptor for TRH (TRHR) through which the action of TRH is mediated to induce the release of PRL is lacking. In order to fill this gap, an attempt was made to characterize the TRHR subtype existing in the PRL cells of the anterior pituitary gland of the bullfrog, Rana catesbeiana. We cloned cDNAs for three types of bullfrog TRHRs, namely TRHR1, TRHR2 and TRHR3, and confirmed that all of them are functional receptors for TRH by means of reporter gene assay. Analyses with semi-quantitative reverse transcription-PCR and in situ hybridization revealed that TRHR3 mRNA is expressed in the anterior lobe and that the signals reside mostly in the PRL cells. It was also noted that the expression levels of TRHR3 mRNA in the anterior pituitary as well as in the PRL cells of metamorphosing tadpoles elevate as metamorphosis progresses. Since the pattern of changes in TRHR3 mRNA levels in the larval pituitary is almost similar to that previously observed in the pituitary PRL mRNA and plasma PRL levels, we provide a view that TRHR3 mediates the action of TRH on the PRL cells to induce the release of PRL that is prerequisite for growth and metamorphosis in amphibians.

Contribution of nucleus accumbens core (AcbC) to behavior control during a learned resting period: introduction of a novel task and lesion experiments.

In recent years, the study of resting state neural activity has received much attention. To better understand the roles of different brain regions in the regulation of behavioral activity in an arousing or a resting period, we developed a novel behavioral paradigm (8-arm food-foraging task; 8-arm FFT) using the radial 8-arm maze and examined how AcbC lesions affect behavioral execution and learning. Repetitive training on the 8-arm FFT facilitated motivation of normal rats to run quickly to the arm tips and to the center platform before the last-reward collection. Importantly, just after this point and before confirmation of no reward at the next arm traverse, locomotor activity decreased. This indicates that well-trained rats can predict the absence of the reward at the end of food seeking and then start another behavior, namely planned resting. Lesions of the AcbC after training selectively impaired this reduction of locomotor activity after the last-reward collection without changing activity levels before the last-reward collection. Analysis of arm-selection patterns in the lesioned animals suggests little influence of the lesion in the ability to predict the reward absence. AcbC lesions did not change exploratory locomotor activity in an open-field test in which there were no rewards. This suggests that the AcbC controls the activity level of planned resting behavior shaped by the 8-arm FFT. Rats receiving training after AcbC lesioning showed a reduction in motivation for reward seeking. Thus, the AcbC also plays important roles not only in controlling the activity level after the last-reward collection but also in motivational learning for setting the activity level of reward-seeking behavior.

Phosphatidylglucoside: a novel marker for adult neural stem cells.

We investigated the expression of a novel glycophospholipid, phosphatidylglucoside (PtdGlc), in adult mouse brains. Immunohistochemical analysis with DIM21 antibody, a monoclonal anti-PtdGlc antibody, revealed robust PtdGlc staining in the two primary neurogenic regions of the adult rodent brain, the subventricular zone (SVZ) lining the lateral ventricle and the subgranular zone of the dentate gyrus. Intriguingly, the staining pattern of PtdGlc appeared to overlap that of glial fibrillary acidic protein, an adult neural stem cell marker in these regions. Further immunohistochemical analysis revealed that PtdGlc expression on the cell membranes of adult SVZ neural stem cells significantly overlapped with other proposed adult neural stem cell markers. Moreover, PtdGlc(+) cells isolated from adult mouse SVZs by fluorescence-activated cell sorting with anti-PtdGlc antibody efficiently generated neurospheres in cell culture. These cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, directly demonstrating that PtdGlc-expressing cells possessed multipotency. Our data suggest that PtdGlc could be a useful adult stem cell marker.

Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain.

In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L- and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.

Relationship between growth retardation and impaired glucose tolerance in hypothyroidal growth-retarded (grt) mice.

Growth-retarded (grt) mice exhibit congenital hypothyroidism and a characteristic growth pause followed by delayed onset of pubertal growth. This pattern of growth has never been reported in any other animal model exhibiting hypothyroidism; therefore, the growth retardation observed in grt mice is unlikely to be explained completely by the low plasma thyroid hormone levels. As growth is closely related to nutrient metabolism, we investigated the relationship between the appearance of growth retardation and glucose utilization, which is the main component of nutrient metabolism, in the peripubertal stage of grt mice. The relative weights of the organs involved in nutrient digestion and absorption were abnormal in grt mice. The intraperitoneal glucose tolerance test (IGTT) showed impaired glucose tolerance in grt mice. Moreover, this symptom appeared in parallel with the progression of growth retardation in grt mice. The impaired blood glucose levels on the IGTT in grt mice were considered to be attributable to decreased plasma insulin levels rather than to impaired insulin sensitivity. The pattern of anti-insulin antibody staining on sections of pancreatic islets from grt mice was almost the same as that in the corresponding sections from normal mice. Insulin treatment accelerated the growth of peripubertal grt mice. These findings suggest that the appearance of growth retardation in grt mice might be partially attributable to a reduction in glucose metabolism and impairment of insulin secretion during the early period of growth.

D2 Dopamine receptor subtype mediates the inhibitory effect of dopamine on TRH-induced prolactin release from the bullfrog pituitary.

Dopamine receptors in mammals are known to consist of two D1-like receptors (D1 and D5) and three D2-like receptors (D2, D3 and D4). The aim of this study was to determine the dopamine receptor subtype that mediates the inhibitory action of dopamine on the release of prolactin (PRL) from the amphibian pituitary. Distal lobes of the bullfrog (Rana catesbeiana) were perifused and the amount of PRL released in the effluent medium was measured by means of a homologous enzyme-immunoassay. TRH stimulated the release of PRL from perifused pituitaries. Dopamine suppressed TRH-induced elevation of PRL release. Quinpirole (a D2 receptor agonist) also suppressed the stimulatory effect of TRH on the release of PRL, whereas SKF-38393 (a D1 receptor agonist) exhibited no such an effect. The inhibitory action of dopamine on TRH-induced PRL release from the pituitary was nullified by the addition of L-741,626 (a selective D2 receptor antagonist) to the medium, but not by the addition of SCH-23390 (a selective D1 receptor antagonist). These data indicate that the inhibitory effect of dopamine on TRH-evoked PRL release from the bullfrog pituitary gland is mediated through D2 dopamine receptors.

Impaired insulin secretion from the pancreatic islets of hypothyroidal growth-retarded mice.

The growth-retarded (grt) mouse shows thyroid dysfunction-related hyporesponsiveness to TSH. Thyroid hormone is a critical regulator of metabolism in many cells; thus, derangement of thyroid function affects many organs and systems. Experiments were conducted focusing on the function of the pancreatic islets in grt mice. We showed occurrence of a fasting hyperglycemia and a decreased plasma insulin level response to a glucose load in grt mice, despite normal insulin molecules being stored in secretory granules of pancreatic islets. We also demonstrated a reduction of insulin secretion in response to glucose administration from islets of grt mice in vitro, while the insulin release in response to KCl stimulation was comparable to that in normal mice, indicating that the isolated islets from grt mice have normal ATP-sensitive K(+) channels and postchannel activity. The mRNA expression levels of glucose transporter 2 and glucokinase in the islets of grt mice were similar to those in normal mice. Triiodothyronine administration to grt mice improved insulin secretion very slightly. On the other hand, mRNA for tyrosylprotein sulfotransferase 2 (Tpst2) was found to be expressed in the pancreatic islets of grt mice. Considering that Tpst2 is the responsible gene of grt mice, mutation of which is associated with a poor function of TSH receptor, the findings raise a possibility of involvement of factors including Tpst2 in the insulin hyposecretion in grt mice.

Molecular cloning of bullfrog D2 dopamine receptor cDNA: Tissue distribution of three isoforms of D2 dopamine receptor mRNA.

The cDNA encoding D2 dopamine receptor was cloned from the distal lobe of the bullfrog pituitary. The deduced amino acid sequence of the bullfrog D2 dopamine receptor (bfD2A) spanned 444 amino acids and exhibited typical features of those of D2 dopamine receptors cloned in other animals to date. It showed a high similarity of 75-87% with rat, turkey, Xenopus and tilapia counterparts. Further analysis of nucleotide sequence of the cDNA revealed the presence of putative truncated D2 dopamine receptor isoforms, bfD2B and bfD2C, of which nucleotide sequences lacked 12 and 99 nucleotides of the coding region for bfD2A, respectively. The alignment analysis indicated that putative bfD2C isoform was close to D2(S) subtype cloned in mammals and birds, whereas bfD2A and putative bfD2B isoforms were close to mammalian and avian D2(L) subtype and homologous to two isoforms of Xenopus. This is the first report of the presence of mRNAs for two D2(L)-like isoforms and one D2(S)-like isoform in a single species. The amino acid sequence responsible for producing isoforms is present in the third intracellular loop, which has been shown to play an important role in the coupling with G protein. Accordingly, differences in the mode of coupling with G protein among three isoforms were suggested. The expression of three isoforms mRNA in organs and tissues was analyzed by RT-PCR. In the brain, pars distalis and pars neurointermedia, mRNAs for three isoforms were invariably expressed, whereas only putative bfD2C mRNA was expressed in peripheral organs and tissues.

Demonstration of the proopiomelanocortin signaling system in the primary immune organ of the quail.

There is growing evidence to suggest that proopiomelanocortin (POMC), a precursor of adrenocorticotropin and alpha-melanocyte-stimulating hormone (alpha-MSH), also exists in extrapituitary organs, including immune organs. We investigated the presence of the POMC signaling system in the avian-specific primary immune organ the bursa of Fabricius (BF) of the adult quail. Immunohistochemical staining revealed the presence of cells showing immunopositive reaction with anti-alpha-MSH antibody in the quail specimens. RT-PCR analysis revealed the expression of POMC, prohormone convertases (PC1/3 and PC2), and three melanocortin receptor subtype (MC1R, MC4R, and MC5R) mRNAs in total RNA specimens of the BF. These findings demonstrate that in the quail BF, just as in the pituitary, immunopositive alpha-MSH substances may be produced via specific cleavages of POMC by the sequential actions of PC1/3 and PC2. The observation of MCR expression within the BF suggests that the alpha-MSH substances may exert paracrine actions within the BF.

Selective upregulation of 3-phosphoglycerate dehydrogenase (Phgdh) expression in adult subventricular zone neurogenic niche.

In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-phosphoglycerate dehydrogenase (Phgdh), an enzyme involved in de novo synthesis of l-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that l-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. In the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of l-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain.

Sympathetic neural regulation of olfactory bulb blood flow in adult and aged rats.

Sympathetic adrenergic nerves originating in the superior cervical ganglia innervate cerebral blood vessels. The present study aimed to characterize olfactory bulb blood flow changes in response to cervical sympathetic trunk (CST) stimulation. Further, we compared the sympathetic control of olfactory bulb blood flow in adult (4-6 mo) and aged (18-21 mo) Wistar rats. Under urethane anesthesia, trains of electrical stimuli were applied to the CST for periods of 1 min while olfactory bulb blood flow was measured with laser Doppler flowmetry. In adult rats, stimulation at 5-30 Hz produced frequency-dependent decreases in CBF of as much as 31+/-4% (at 30 Hz). In aged rats, blood flow decreases occurred in response to stimulus trains ranging from 10-30 Hz, but the largest average decreases were 15+/-2% (at 20 Hz). Blood flow was significantly decreased from pre-stimulus flow in both adult and aged rats, and the stimulus-induced changes in flow were larger in adult compared with aged rats. Blood flow responses were abolished by i.v. administration of the alpha-adrenergic blocker phenoxybenzamine, in both age groups. These results indicate that blood vessels in the rat olfactory bulb are constricted by sympathetic nerve fibers via activation of alpha-adrenergic receptors, and the effectiveness of this regulation declines in aged rats.

Lipid rafts enriched in phosphatidylglucoside direct astroglial differentiation by regulating tyrosine kinase activity of epidermal growth factor receptors.

Membrane lipid rafts provide a specialized microenvironment enriched with sphingolipids and phospholipids containing saturated fatty acids and serve as a platform for various intracellular signalling pathways. PtdGlc (phosphatidylglucoside) is a type of glycophospholipid localized in the outer leaflet of the plasma membrane. Owing to PtdGlc's unique fatty acid composition, exclusively composed of C(18:0) at sn-1 and C(20:0) at sn-2 of the glycerol backbone, it tends to form PGLRs (PtdGlc-enriched lipid rafts). Previously, we demonstrated that PGLRs reside on the cell surface of astroglial cells from fetal rat brain [Nagatsuka, Horibata, Yamazaki, Kinoshita, Shinoda, Hashikawa, Koshino, Nakamura and Hirabayashi (2006) Biochemistry 45, 8742-8750]. In the present study, we observed PGLRs in astroglial lineage cells at mid-embryonic to early-postnatal stages of developing mouse cortex. This suggests that PGLRs are developmentally correlated with astroglial differentiation during fetal cortical development. Our cell culture studies with multipotent neural progenitor cells prepared from fetal mouse telencephalon demonstrated that treatment with EGF (epidermal growth factor) or anti-PtdGlc antibody caused recruitment of EGFRs (EGF receptors) into lipid raft compartments, leading to activation of EGFRs. Moreover, the activation of EGFRs by antibody triggered downstream tyrosine kinase signalling and induced marked GFAP (glial fibrillary acidic protein) expression via the JAK (Janus kinase)/STAT (signal transducer and activator of transcription) signalling pathway. These findings strongly suggest that PGLRs are physiologically coupled to activated EGFRs on neural progenitor cells during fetal cortical development, and thereby play a distinct role in mediating astrogliogenesis.

Effect of corticosterone on developing hippocampus: short-term and long-term outcomes.

Many documents implicate that corticosterone plays a negative role in brain function, especially in learning and memory. However, less evidence confirms its direct actions on hippocampal development. In the work reported here, pro treatment, minimum corticosterone administration in infant mice, and con treatment, corticosterone deprivation by adrenalectomy, were used to examine the effects imposed by corticosterone on the structure and function of developing hippocampus. Our study shows that adrenalectomy induces decrease of plasma corticosterone levels and results in the impairment of learning performance and the degenerative changes not in CA regions of hippocampus but in dentate gyrus. Noteworthily, this damage effect is severer in 5-week-old mice than that in 10-week-old mice. In addition, the short-term effect of minimum corticosterone administration may accelerate the development of dentate gyrus of 10-day-old mice. Moreover, minimum corticosterone administration during infancy contributed to the learning performance and the structural integrity of hippocampal CA regions in different developing stages, while this phenomenon was not observed in dentate gyrus. In conclusion, corticosterone is necessary for the development of dentate gyrus, especially in relatively young individuals, and administration with minimum corticosterone in infancy has a long-term positive influence on the hippocampal structure and function in different developing stages.

Impaired Development of Somatotropes, Lactotropes and Thyrotropes in Growth-Retarded (grt) Mice.

Congenitally primary hypothyroid growth-retarded (grt) mice exhibit a characteristic growth pause followed by delayed onset of pubertal growth. We characterized the developmental pattern of somatotropes, lactotropes and thyrotropes in the anterior pituitary, as well as plasma levels of their secretory hormones, in grt mice. Compared with normal mice, the weight of grt pituitary gland was similar at 8 weeks of age but significantly heavier after 12 weeks of age. Compared with normal mice, there were significantly fewer somatotropes in the grt pituitary until 8 weeks of age, but the number gradually increased up to 48 weeks. The number of lactotropes in grt mice was consistently lower than that in normal mice from 2 through 48 weeks, whereas the number of thyrotropes in the grt pituitary was consistently higher than in the normal pituitary. Thyrotropes in the grt pituitary exhibited hypertrophy and hyperplasia with less intensive thyroid-stimulating hormone (TSH) immunoreactivity than normal thyrotropes. In normal mice, the sum of the relative proportions of these cells plateaued at 8 weeks, where it remained up to 48 weeks of age. In grt mice, these proportions almost reached normal levels at 12 weeks of age but gradually declined after 24 weeks. Plasma growth hormone concentrations did not differ between grt and normal mice until 24 weeks of age. Compared with normal mice, grt mice exhibited significantly lower plasma prolactin and thyroxine levels but higher TSH levels. These findings indicate that development of somatotropes, lactotropes and thyrotropes in grt mice is impaired, being followed by altered hormone secretion.

Effects of glucocorticoids on age-related impairments of hippocampal structure and function in mice.

Effects of glucocorticoids (GCs) on maze-learning performances and hippocampal morphology were observed in male C57BL/6Cr mice. Correlations between aging, GCs and maze-learning performances were also studied. (2) Eight-arm radial maze was used in maze-learning tests. Learning performance was assessed by the parameters of time of getting all the bait, number of reentry errors into the already-entered arm with bait, and number of missed entries into an unbaited arm. Brain sections, 8 mum thick, were Nissl-stained with cresyl violet or stained immunocytochemically with antibodies against neurofilaments. (3) With aging, normal pyramidal cells decreased gradually in amount, and degenerating cells increased since the age of 18 months, accompanied with the maze-learning deficit. Here we have suggested that these changes were associated with the age-related deficits in adaptation tolerance of neurons to stress. In addition, the age-related deficits in plasticity of hippocampal neurons to GCs in young mice (3 months of age) resulted in an increase in plasma corticosterone (CORT) concentrations, degeneration of hippocampal pyramidal cells, as well as maze-learning deficits. (4) In conclusion, our data indicated that CORT caused the degeneration of hippocampal pyramidal cells and the impairment of memory.

Effects of nicotine on regional blood flow in the olfactory bulb in rats.

Effects of nicotine on blood flow in the olfactory bulb were examined in anesthetized rats. Nicotine administered intravenously at 100 microg/kg increased regional blood flow in the olfactory bulb, irrespective of changes in systemic arterial pressure. Nicotine administered locally into the internal carotid artery at 10 microg increased blood flow, without changing arterial pressure; this response was abolished by hexamethonium. These results indicate that nicotine produces vasodilatation in the olfactory bulb via activation of nicotinic receptors located close to the olfactory bulb. Nicotine may be of therapeutic value in improving blood flow in the olfactory bulb.

Topographic analysis of cell proliferation in the hippocampus of the adult mouse.

The dentate gyrus of the hippocampus generates new neurons throughout life. The distribution of neural progenitor cells in the hippocampus along the anteroposterior axis is, however, not known. To determine whether mitotic activity differs along the anteroposterior axis, we examined mitotic activity in serial hippocampal sections of two experimental groups: normal bred (control) and exercised groups. In both groups, the number of 5-bromo-2'-deoxyuridine-positive cells was markedly decreased in the caudal dentate gyrus; exercised groups also had mitotic activity of about 1.5 times that of control groups. Mitotic activity tended to increase in the suprapyramidal blade of the middle dentate gyrus of exercised groups. These results indicate that the adult hippocampus has regional differences in mitotic activity.

Thyroid-stimulating hormone receptor levels and binding affinity in the thyroid gland of growth-retarded mice.

Growth-retarded (grt/grt) mice are congenitally primary hypothyroid. Our previous study indicated that thyroid-stimulating hormone (TSH) responsiveness was defective in the grt/grt thyroid gland. We now report additional studies of impaired grt/grt thyroid function. Semiquantitative RT-PCR confirmed that TSH receptor (TSHR) mRNA expression in the grt/grt thyroid was significantly decreased compared with +/+ thyroids. Scatchard analysis revealed that grt/grt and +/+ mice have only one type of TSH binding site. grt/grt thyroids had fewer TSH binding sites, although this did not apparently affect the affinity of TSH for its receptor. The present data suggest that reduced TSHR levels or defects in TSHR signaling could be one of the possible defective sites in the grt/grt thyroid gland.

Citrullination preferentially proceeds in glomerular Bowman's capsule and increases in obstructive nephropathy.

BACKGROUND: Peptidylarginine deiminases (PADs) are a group of posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues, yielding citrulline residues. Citrullination of arginine residues abolishes their positive charge, markedly altering their structure. We undertook this study to investigate the actions of PADs in the kidney. METHODS: In male rats, we ligated the unilateral ureter, then analyzed the obstructed and contralateral kidneys 1 week later. Controls were rats simultaneously given sham operations. In another experiment, we ligated unilateral ureters of eight rats, four of which received a ureter-bladder anastomosis 1 week later. These rats were subjected to histologic examinations 5 weeks after unilateral ureteral obstruction (UUO). RESULTS: Reverse transcription-polymerase chain reaction (RT-PCR) revealed that, of PADs (type I, II, III, and IV), only PAD type II was expressed in kidneys. Western blot study showed that PAD type II expression and citrullinated protein content increased greatly in kidneys that underwent unilateral ureteral ligation compared to that in contralateral or sham-operated kidneys. Immunohistochemical analyses revealed that PAD type II was preferentially expressed by parietal epithelial cells and that only in Bowman's capsule were proteins citrullinated. Additionally, these PAD type II and citrullinated proteins in obstructed nephropathy were significantly attenuated by the release of the obstruction. Proteome analysis revealed that one of citrullinated proteins in the kidney should be actin. CONCLUSION: This result indicates that PAD type II and citrullinated proteins are suitable markers of Bowman's capsule. Not only are these markers preferentially expressed in Bowman's capsules but their expression is also increased in damaged kidneys by UUO, features that promise the further clarification of kidney diseases.