BACKGROUND: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is an ultra-rare disease caused by mutations in the ABCB11 gene. This study aimed to understand the course of PFIC2 during the native liver period. METHODS: From November 2014 to October 2015, a survey to identify PFIC2 patients was conducted in 207 hospitals registered with the Japanese Society of Pediatric Gastroenterology, Hepatology, and Nutrition. Investigators retrospectively collected clinical data at each facility in November 2018 using pre-specified forms. RESULTS: Based on the biallelic pathogenic variants in ABCB11 and/or no hepatic immunohistochemical detection of BSEP, 14 Japanese PFIC2 patients were enrolled at seven facilities. The median follow-up was 63.2 [47.7-123.3] months. The median age of disease onset was 2.5 [1-4] months. Twelve patients underwent living donor liver transplantation (LDLT), with a median age at LDLT of 9 [4-57] months. Two other patients received sodium 4-phenylbutyrate (NaPB) therapy and survived over 60 months with the native liver. No patients received biliary diversion. The cases that resulted in LDLT had gradually deteriorated growth retardation, biochemical tests, and liver histology since the initial visit. In the other two patients, jaundice, growth retardation, and most of the biochemical tests improved after NaPB therapy was started, but pruritus and liver fibrosis did not. CONCLUSIONS: Japanese PFIC2 patients had gradually worsening clinical findings since the initial visit, resulting in LDLT during infancy. NaPB therapy improved jaundice and growth retardation but was insufficient to treat pruritus and liver fibrosis.

2022年より欧米から「原因不明の小児急性肝炎」について多くの報告があがるようになり、アデノウイルスや新型コロナウイルス感染症(COVID-19)流行との関連が疑われている。これまで本邦では小児の急性肝炎のサーベイランスが行われていないことから、今回、日本小児科学会会員が所属する全国の病院小児科責任者を対象に、2017年1月～2022年6月までの期間における同疾患の実態について質問紙調査を行い、947名より回答を得た。その結果、COVID-19流行前の報告数は2017年260名、2018年257名、2019年243名で、COVID-19流行中の報告数は2020年164名、2021年192名、2022年1～6月で113名と、COVID-19流行中の方が少ない傾向がみられた。COVID-19流行前は0歳および1～4歳群の症例数が他の年齢群と比較して多い傾向がみられたが、流行中はこれらの年齢群で減少が著明であった。地域別では、COVID-19流行前・流行後ともに、京都府・愛知県・福島県からの報告が多かった。

Prostaglandin E-major urinary metabolite (PGE-MUM) is a urinary biomarker reflecting ulcerative colitis (UC) activity. This prospective observational study aimed to evaluate the usefulness of PGE-MUM via rapid chemiluminescent enzyme immunoassay in detecting endoscopic remission (ER) and histologic remission (HR) in pediatric UC (6-16 years) in comparison with fecal calprotectin (FCP). ER and HR were defined as Mayo endoscopic score (MES) of 0 and Matts' histological grades (Matts) of 1 or 2, respectively. A total of 104 UC and 39 functional gastrointestinal disorder (FGID) were analyzed. PGE-MUM levels were significantly higher in the UC group than in the FGID group (P < 0.001). FCP levels were significantly elevated in the group without ER and HR than in the group with ER and HR (P < 0.001 and P = 0.001), whereas PGE-MUM levels were significantly higher in the group without ER compared to the group with ER (P < 0.001). No significant differences were noted in the AUCs for PGE-MUM and FCP in detecting ER and HR. Although PGE-MUM was inferior to FCP for the detection of HR, it might have the potential for application as a biomarker of endoscopic activity in pediatric UC owing to its noninvasive and rapid method.

非典型溶血性尿毒症症候群(atypical hemolytic uremic syndrome:aHUS)は補体制御因子の異常による血栓性微小血管症で,生命予後に影響する腎外病変に対する関心が集まっている.症例は3歳女児.血尿,蛋白尿,浮腫を主訴に当院へ紹介され,精査によりaHUSと診断された.臨床経過中に消化管出血,難治性高血圧,肺水腫,そして可逆性の脳萎縮といった多彩な腎外病変を呈し,特に重篤な消化管出血と難治性高血圧の管理に難渋した.病初期におけるaHUSに対する血漿交換療法とエクリズマブの併用療法に加えて,難治性高血圧に対してレニン・アンジオテンシン系阻害薬,そして虚血性腸炎の体液管理に酢酸オクトレオチドがそれぞれ奏功した.多臓器にわたる腎外病変を有するaHUSは重症例が多く,適切な治療を行わなければ致死的な経過をたどる可能性があるため,集学的治療による全身管理が生命予後の改善に重要である.(著者抄録)

進行性家族性肝内胆汁うっ滞症(PFIC)は、乳児期より持続する進行性の胆汁うっ滞により肝不全に至るまれな疾患である。今回われわれは、本邦初報告となるPFIC 4型を含めた2例に対して生体肝移植を行った。【症例1】11歳男児、PFIC 1型。感染性腸炎を契機に急激な増悪を認め肝不全となり生体肝移植を施行した。完全外胆汁瘻を併施したが、移植肝から胆汁排泄が得られず術後6ヵ月で肝不全により死亡した。【症例2】4歳女児、PFIC 4型。肝生検における免疫染色で胆管上皮細胞のBSEP発現は確認できず。PFIC 2型の責任遺伝子は正常であったが臨床的にPFIC 2型としていた。経過中に肝硬変から肝不全に至り生体肝移植を施行。経過は良好で現在術後6年無病生存中である。移植後に改めて行われた遺伝子診断にて、TJP2遺伝子に変異を認めたため、PFIC 4型と診断された。PFICは遺伝子診断による病型判断が肝移植を始めとする治療を行ううえで重要である。(著者抄録)

進行性家族性肝内胆汁うっ滞症(PFIC)は、乳児期より持続する進行性の胆汁うっ滞により肝不全に至るまれな疾患である。今回われわれは、本邦初報告となるPFIC 4型を含めた2例に対して生体肝移植を行った。【症例1】11歳男児、PFIC 1型。感染性腸炎を契機に急激な増悪を認め肝不全となり生体肝移植を施行した。完全外胆汁瘻を併施したが、移植肝から胆汁排泄が得られず術後6ヵ月で肝不全により死亡した。【症例2】4歳女児、PFIC 4型。肝生検における免疫染色で胆管上皮細胞のBSEP発現は確認できず。PFIC 2型の責任遺伝子は正常であったが臨床的にPFIC 2型としていた。経過中に肝硬変から肝不全に至り生体肝移植を施行。経過は良好で現在術後6年無病生存中である。移植後に改めて行われた遺伝子診断にて、TJP2遺伝子に変異を認めたため、PFIC 4型と診断された。PFICは遺伝子診断による病型判断が肝移植を始めとする治療を行ううえで重要である。(著者抄録)

An 8-year-old boy was admitted to our hospital because of anorexia, jaundice, and liver dysfunction. Based on liver biopsy, the patient was initially diagnosed with noninfectious acute hepatitis. Treatment with predniso-lone (PSL) improved hepatobiliary enzyme levels. The patient was readmitted due to liver dysfunction, which recurred following PSL dose tapering. Although the re-administration of PSL was partly effective, cyclosporine and azathioprine had to be added because the liver function data did not improve sufficiently. Finally, liver biopsy revealed noncaseous epithelioid granulomas in the portal tracts. On the histopathological findings, serum angiotensin-converting enzyme and soluble interleukin-2 receptor levels were elevated thus, the patient was diagnosed with hepatic sarcoidosis. Combined steroid pulse therapy and azathioprine markedly improved liver function data and histological findings. Case reports of hepatic sarcoidosis in children are rare, but this disease should not be overlooked as a possible cause of acute liver dysfunction based on immunological abnormalities.

AIM: Bile salt export pump (BSEP) deficiency manifests a form of progressive intrahepatic cholestasis. This study aimed to establish a scoring system of liver histology for the uncommon genetic condition. METHODS: After a roundtable discussion and a histology review, a scoring system for BSEP deficiency was established. Eleven tissue samples were independently evaluated by three pathologists based on the proposed standard for an interobserver agreement analysis. In four cases with serial tissue samples available, correlation between changes in histology scores and clinical outcome was examined. RESULTS: Of 14 initially listed histopathological findings, 12 were selected for scoring and grouped into the following four categories: cholestasis, parenchymal changes, portal tract changes, and fibrosis. Each category consisted of two to four microscopic findings that were further divided into three to six scores; therefore, each category had a maximum score of 8 to 11. Interobserver agreement was highest for pericellular fibrosis (κ value: 0.849) and lowest for hepatocellular cholestasis (κ value: 0.241) with the mean and median κ values of the 12 parameters being 0.561 and 0.602, respectively. For two patients whose clinical features worsened, score changes between two time points were interpreted as deteriorated. In two patients, who showed a good clinical response to preprandial treatment with sodium 4-phenylbutyrate, histological changes were evaluated as improved or unchanged. CONCLUSIONS: The proposed histology-based scoring system for BSEP deficiency with moderate interobserver agreement may be useful not only for monitoring microscopic changes in the clinical practice but also for a surrogate endpoint in clinical trials.

Progressive familial intrahepatic cholestasis (PFIC), a rare inherited disorder, progresses to liver failure in childhood. We have shown that sodium 4-phenylbutyrate (NaPB), a drug approved for urea cycle disorders (UCDs), has beneficial effects in PFIC. However, there is little evidence to determine an optimal regimen for NaPB therapy. Herein, a multicenter, open-label, single-dose study was performed to investigate the influence of meal timing on the pharmacokinetics of NaPB. NaPB (150 mg/kg) was administered orally 30 min before, just before, and just after breakfast following overnight fasting. Seven pediatric PFIC patients were enrolled and six completed the study. Compared with postprandial administration, an approved regimen for UCDs, preprandial administration significantly increased the peak plasma concentration and area under the plasma concentration-time curve of 4-phenylbutyrate by 2.5-fold (95% confidential interval (CI), 2.0-3.0;P = 0.003) and 2.4-fold (95% CI, 1.7-3.2;P = 0.005). The observational study over 3 years in two PFIC patients showed that preprandial, but not prandial or postprandial, oral treatment with 500 mg/kg/day NaPB improved liver function tests and clinical symptoms and suppressed the fibrosis progression. No adverse events were observed. Preprandial oral administration of NaPB was needed to maximize its potency in PFIC patients.

AIM: Bile acid biosynthesis is strictly regulated under physiological conditions. The expression of fibroblast growth factor (FGF) 19 is induced when bile acids bind to the farnesoid X receptor in the intestinal epithelium. Fibroblast growth factor 19 is then transported by the portal flow, causing transcriptional inhibition of cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1), a key enzyme in bile acid biosynthesis, through the extracellular signal-regulated kinase (ERK) pathway. However, the regulatory mechanisms of these signaling pathways in hepatocytes under chronic cholestasis remain unclear. We investigated the regulation of these signaling pathways in patients with biliary atresia (BA). METHODS: We analyzed the regulation of molecules in these signaling pathways using liver and serum samples from eight BA children and four non-cholestatic disease controls. RESULTS: CYP7A1 mRNA expression was not inhibited in BA microdissected hepatocyte-enriched tissue (HET) despite high serum bile acid concentrations. The FGF19 protein was synthesized in BA HET, and its serum concentration was elevated. Fibroblast growth factor receptor 4 was phosphorylated in BA livers. However, ERK phosphorylation was significantly reduced. We examined SPRY2 expression to determine how the ERK pathway was inactivated downstream of the FGF receptor; the expression was significantly increased in BA HET. CONCLUSIONS: This is the first study to measure the CYP7A1 mRNA levels in human BA HET. Fibroblast growth factor 19 was increased in BA hepatocytes. By focusing on its regulation in hepatocytes, we showed that the FGF19 pathway did not suppress bile acid synthesis, probably due to an altered mechanism involving upregulated SPRY2 in BA patients.

Context: Citrin-deficient infants present neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), which resolves at 12 months. Thereafter, they have normal liver function associated with hypercholesterolemia, and a preference for lipid-rich carbohydrate-restricted diets. However, some develop adult-onset type II citrullinemia, which is associated with metabolic abnormalities. Objectives: To identify the causes of hypercholesterolemia in citrin-deficient children post-NICCD. Design and Setting: We determined the concentrations of sterol markers of cholesterol synthesis, absorption, and catabolism by liquid chromatography-electrospray ionization-tandem mass spectrometry and evaluated serum lipoprotein profiles. Subjects: Twenty citrin-deficient children aged 5 to 13 years and 37 age-matched healthy children. Intervention: None. Main Outcome Measures: Relationship between serum lipoproteins and sterol markers of cholesterol metabolism. Results: The citrin-deficient group had a significantly higher high-density lipoprotein cholesterol (HDL-C) concentration than did the control group (78 ± 11 mg/dL vs 62 ± 14 mg/dL, P < 0.001), whereas the two groups had similar low-density lipoprotein cholesterol and triglyceride concentrations. The concentrations of markers of cholesterol synthesis (lathosterol and 7-dehydrocholesterol) and bile acids synthesis (7α-hydroxycholesterol and 27-hydroxycholesterol) were 1.5- to 2.8-fold and 1.5- to 3.9-fold, respectively, higher in the citrin-deficient group than in the control group. The concentration of 24S-hydroxycholesterol, a marker of cholesterol catabolism in the brain, was 2.5-fold higher in the citrin-deficient group. In both groups, the HDL-C concentration was significantly positively correlated with that of 27-hydroxycholesterol, the first product of the alternative bile acid synthesis pathway. Conclusions: HDL-C and sterol marker concentrations are elevated in citrin-deficient children post-NICCD. Moreover, cholesterol synthesis and elimination are markedly enhanced in the liver and brain of citrin-deficient children.

BACKGROUND: Citrin (mitochondrial aspartate-glutamate transporter) deficiency causes the failures in both carbohydrate-energy metabolism and the urea cycle, and the alterations in the serum levels of several amino acids in the stages of newborn (NICCD) and adult (CTLN2). However, the clinical manifestations are resolved between the NICCD and CTLN2, but the reasons are still unclear. This study evaluated the serum amino acid profile in citrin-deficient children during the healthy stage. METHODS: Using HPLC-MS/MS analysis, serum amino acids were evaluated among 20 citrin-deficient children aged 5-13 years exhibiting normal liver function and 35 age-matched healthy controls. RESULTS: The alterations in serum amino acids characterized in the NICCD and CTLN2 stages were not observed in the citrin-deficient children. Amino acids involved in the urea cycle, including arginine, ornithine, citrulline, and aspartate, were comparable in the citrin-deficient children to the respective control levels, but serum urea was twofold higher, suggestive of a functional urea cycle. The blood sugar level was normal, but glucogenic amino acids and glutamine were significantly decreased in the citrin-deficient children compared to those in the controls. In addition, significant increases of ketogenic amino acids, branched-chain amino acids (BCAAs), a valine intermediate 3-hydroxyisobutyrate, and β-alanine were also found in the citrin-deficient children. CONCLUSION: The profile of serum amino acids in the citrin-deficient children during the healthy stage showed different characteristics from the NICCD and CTLN2 stages, suggesting that the failures in both urea cycle function and energy metabolism might be compensated by amino acid metabolism. SYNOPSIS: In the citrin-deficient children during the healthy stage, the characteristics of serum amino acids, including decrease of glucogenic amino acids, and increase of ketogenic amino acids, BCAAs, valine intermediate, and β-alanine, were found by comparison to the age-matched healthy control children, and it suggested that the characteristic alteration of serum amino acids may be resulted from compensation for energy metabolism and ammonia detoxification.

Progressive familial intrahepatic cholestasis type 1 (PFIC1), a rare inherited recessive disease resulting from a genetic deficiency in ATP8B1, progresses to liver failure. Because of the difficulty of discriminating PFIC1 from other subtypes of PFIC based on its clinical and histological features and genome sequencing, an alternative method for diagnosing PFIC1 is desirable. Herein, we analyzed human peripheral blood monocyte-derived macrophages (HMDM) and found predominant expression of ATP8B1 in interleukin-10 (IL-10)-induced M2c, a subset of alternatively activated macrophages. SiRNA-mediated depletion of ATP8B1 in IL-10-treated HMDM markedly suppressed the expression of M2c-related surface markers and increased the side scatter (SSC) of M2c, likely via impairment of the IL-10/STAT3 signal transduction pathway. These phenotypic features were confirmed in IL-10-treated HMDM from four PFIC1 patients with disease-causing mutations in both alleles, but not in those from four patients with other subtypes of PFIC. This method identified three PFIC1 patients in a group of PFIC patients undiagnosed by genome sequencing, an identical diagnostic outcome to that achieved by analysis of liver specimens and in vitro mutagenesis studies. In conclusion, ATP8B1 deficiency caused incomplete polarization of HMDM into M2c. Phenotypic analysis of M2c helps to identify PFIC1 patients with no apparent disease-causing mutations in ATP8B1.

BACKGROUND: Growth hormone (GH) replacement therapy improves hypercholesterolemia in patients with GH deficiency, suggesting that GH modulates cholesterol metabolism. OBJECTIVES: We examined GH effects on lipid profiles and cholesterol-related markers reflecting hepatic and cerebral cholesterol metabolism in small-for-gestational age (SGA) children without catchup growth. METHODS: This study examined SGA children without catch-up growth (n = 22) and healthy children (controls, n = 11). Based on parents' choice, 11 SGA children received GH at 0.23 to 0.25mg/kg/d for 6 months, and at 0.34 to 0.36 mg/kg/d for the subsequent 6 months (GH (+) group). The other SGA children received no GH (GH (-) group, n = 11). We ascertained baseline and posttreatment lipid profiles and cholesterol-related markers reflecting hepatic and cerebral cholesterol metabolism. RESULTS: Baseline lipid profiles of SGA children and controls were similar. Serum 24S-hydroxycholesterol (marker for cerebral cholesterol metabolism) concentration was 19% lower in SGA children than in controls (P < .05). Compared with baseline, the GH (+) group low-density lipoprotein -cholesterol concentration had decreased by 6.6% during 6 months and 8.8% during 12 months (P <.01), whereas the high-density lipoprotein-cholesterol concentration had increased by 1.7% (P =.07) and 3.3% (P < .01). Serum 7a-hydroxycholesterol (marker for hepatic cholesterol elimination) concentration had increased by 34% at 6 months and 35% at 12 months (P < .01). In addition, 24S-hydroxycholesterol increased by 25% and 26% (P < .001). No marker for cholesterol synthesis or absorption changed. The GH (-) group lipid profiles and oxysterols remained unchanged during the observation period. CONCLUSION: GH activates hepatic and cerebral cholesterol metabolism in SGA children without catch-up growth. (C) 2017 National Lipid Association. All rights reserved.

Citrin deficiency causes adult-onset type II citrullinemia (CTLN-2), which later manifests as severe liver steatosis and life-threatening encephalopathy. Long-standing energy deficit of the liver and brain may predispose ones to CTLN-2. Here, we compared the energy-driving tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation cycle between 22 citrin-deficient children (age, 3-13 years) with normal liver functions and 37 healthy controls (age, 5-13 years). TCA cycle analysis showed that basal plasma citrate and alpha-ketoglutarate levels were significantly higher in the affected than the control group (p < 0.01). Conversely, basal plasma fumarate and malate levels were significantly lower than those for the control (p < 0.001). The plasma level of 3-OH-butyrate derived from fatty acid (3-oxidation was significantly higher in the affected group (p < 0.01). Ten patients underwent sodium pyruvate therapy. However, this therapy did not correct or attenuate such deviations in both cycles. Sodium pyruvate therapy significantly increased fasting insulin secretion (p < 0.01); the fasting sugar level remained unchanged. Our results suggest that citrin-deficient children show considerable deviations of TCA cycle metabolite profiles that are resistant to sodium pyruvate treatment. Thus, long-standing and considerable TCA cycle dysfunction might be a pivotal metabolic background of CTLN-2 development. (C) 2016 Elsevier Inc. All rights reserved.

BACKGROUND: Progressive familial intrahepatic cholestasis 2 (PFIC2) is the result of mutations in the ABCB11, which encodes for bile salt export pump (BSEP). An absence of BSEP in the canalicular membrane causes cholestasis and leads to the development of end-stage liver disease in the first decade of life. Liver transplantation (LT) has been considered curative for BSEP disease. However, patients with PFIC2 having undergone LT have recently been reported to develop recurrence of cholestasis together with the clinical and histological features of primary BSEP disease. CASE REPORT: We herein present a rare case of a patient with PFIC2 who developed post-transplantation recurrence of progressive intrahepatic cholestasis due to antibodies against BSEP after living-donor LT, which mimicked primary BSEP disease. The patient had mutations in the ABCB11 gene, resulting in the complete absence of BSEP in the native liver, explaining the lack of tolerance. Immunofluorescence staining of normal human liver sections with the patient's serum and using an anti-human immunoglobulin G antibody to detect serum antibodies showed reactivity to the BSEP epitope in the canalicular membrane. We suggest that the patients having undergone LT had been associated with a risk of autoantibody formation against the BSEP protein. The absence of primary tolerance for the BSEP epitopes may explain the formation of the anti-BSEP antibodies after LDLT.

Context: GH activates the expression of low-density lipoprotein (LDL) receptors, leading to decreased LDL-cholesterol (LDL-C). Apolipoprotein (apo) E4 carriers suppress LDL receptor expression, rendering high LDL-C concentrations. Objectives: We examined whether GH-deficient children carrying apoE4 exhibited a greater reduction in LDL-C after GH replacement therapy. Design and Setting: We determined lipoprotein profiles after 0, 4, and 12 months of GH treatment in children with an idiopathic GH deficiency. We compared the effects of GH treatment on LDL-C by apoE phenotype. Subjects: In total, 66 children with idiopathic GH deficiency and 89 healthy children were classified into subgroups according to apoE phenotype. Intervention: The intervention included GH replacement therapy for 12 months. Main Outcome Measures: The relationship between apoE phenotype and reduced LDL-C induced by OH treatment was measured. Results: Concentrations of LDL-C and apoB were highest in the apoE4/3 group (n = 13), second highest in the apoE3/3 group (n = 46), and lowest in the apoE3/2 group (n = 7), whereas apoE concentrations were highest in the apoE3/2 group and lowest in the apoE4/3 group. The apoE4/3 group had significantly reduced LDL-C and apoB concentrations at months 4 and 12, whereas the apoE3/3 and apoE3/2 groups showed no changes. LDL-C concentrations did not differ among the three groups after 12 months. The trend in apoE concentration did not change among the groups. Conclusions: Children with a OH deficiency carrying apoE4 had higher baseline LDL-C concentrations and experienced a greater reduction in LDL-C after GH replacement therapy than those without apoE4.

Aim: To characterize cholesterol regulation in the liver of patients with Alagille syndrome (AGS). Methods: Serum total cholesterol (TC) and total bile acid (TBA) levels were measured in 23 AGS patients. The expressions of genes involved in cholesterol regulation, including low-density lipoprotein receptor (LDLR), scavenger receptor class B type I (SR-B1), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), cholesterol 7 alpha-hydroxylase (CYP7A1), ATP-binding cassette transporter (ABC) A1. and ABCG1/5/8, were measured in liver tissues from five of these patients. Expression of regulators for these genes, including farnesoid X receptor/small heterodimer partner (SHP), liver X receptor a (LXR alpha) and mature Sterol regulatory element-binding protein 2 (SREBP2) was measured. The expression of mature SREBP2 protein was also examined. Results: Serum TC and TBA levels were correlated in the AGS patients. Liver cholesterol was also increased compared with controls, and correlated with bile acid contents. LDLR, SR-B1, HMGCR, and ABCGs mRNA expression were upregulated, while CYP7A1 mRNA expression was downregulated in AGS livers. SHP and LXRa mRNA expression was also increased, but maturation of SREBP2 was not suppressed in the patients. Conclusions: The major upregulators of liver cholesterol might be increased in AGS patients, indicating an impaired negative feedback mechanism and accelerated liver cholesterol accumulation. (C) 2014 Elsevier B.V. All rights reserved.

Background: Progressive familial intrahepatic cholestasis type 1 (PFIC1), an inherited liver disease caused by mutations in ATP8B1, progresses to severe cholestasis with a sustained intractable itch. Currently, no effective therapy has been established for PFIC1. Decreased function of the bile salt export pump (BSEP) in hepatocytes is suggested to be responsible for the severe cholestasis observed in PFIC1. We found a previously unidentified pharmacological effect of 4-phenylbutyrate (4PB) that increases the expression and function of BSEP. Here, we tested 4PB therapy in three patients with PFIC1. Methods: The therapeutic potency of 4PB in these patients was tested by oral administration of this drug with gradually increasing dosage (200, 350, and 500 mg/kg/day) for 6 months. Biochemical, histological, and clinical data were collected. Results: 4PB therapy had no beneficial effect on the patients' liver functions, as assessed by biochemical and histological analyses, despite an increase in hepatic BSEP expression. However, therapy with 4PB at a dosage of 350 or 500 mg/kg/day significantly relieved the intractable itch. Serum levels of potential pruritogens in cholestasis were much higher than the reference ranges during the 4PB therapy. Conclusions: 4PB therapy may be a new medication for patients with intractable cholestatic pruritus and may improve quality of life for patients and their families.

腸管不全患児において在宅静脈栄養(HPN)は社会・家庭復帰のために重要な役割を果たす。当科におけるHPNの現状と問題点について検討した。対象は1983年から2012年まで当科にてHPNを施行した40例。施行期間は8ヵ月〜27年、疾患は腸管運動障害19例、難治性下痢10例、短腸症候群6例、その他5例であった。カテーテル(CVC)はカフ付きもしくは埋め込み型リザーバーを用いた。HPN施行においては院外薬局による無菌調剤とし、輸液ラインはクローズドシステムを用いた。微量元素、ビタミンは年齢に応じて一日必要量を毎日投与し、必要に応じてSeを投与した。転帰は離脱による終了が11例、原疾患による死亡が8例、転医が3例で、1例で小腸移植を行った。17例が現在も継続中である。1,000日あたりのCVC感染頻度は0.26であった。カテーテル関連合併症はカフ付きCVCで少なかった。適切なHPNの施行により合併症も少なく、多くの患児で成長発達を維持し、QOLの向上が可能となった。(著者抄録)

AimThe molecular mechanisms by which hepatocyte nuclear factor (HNF)4 regulates fetal liver development have not been fully elucidated. We screened the downstream molecules of HNF4 during liver development and identified sodium-coupled neutral amino acid transporter (SNAT)4. The aim of this study is to investigate the regulation of SNAT4 by HNF4 and to clarify its roles in differentiating hepatocytes. MethodsHNF4 was overexpressed in cultured liver buds using adenovirus, and suppression subtractive hybridization screening was performed. Temporal and spatial expression of SNAT4 during liver development was investigated. Regulation of SNAT4 by HNF4 was examined by promoter analyses and electrophoretic mobility shift assays (EMSA). Metabolic labeling and western blotting were carried out using primary hepatoblasts with SNAT4 overexpression. ResultsThe expression of Slc38a4 encoding SNAT4 showed a marked perinatal increase, and was predominant among system A amino acid transporters. It was first detected in embryonic day 18.5 liver, and found in most hepatocytes after birth. Three alternative first exons were found in the SNAT4 gene. Promoter analyses using approximately 3-kb fragments corresponding to each first exon (AP1, AP2, AP3) revealed that AP1 and AP2 exhibited strong promoter activity in mouse hepatoblasts with endogenous HNF4. Transactivation of AP2 was upregulated by HNF4 in HeLa cells without endogenous HNF4. EMSA has demonstrated that HNF4 directly binds to cis-elements in AP2. Overexpression of SNAT4 facilitated amino acid uptake and de novo protein synthesis in primary hepatoblasts. ConclusionSNAT4 functions downstream of HNF4 and plays significant roles in liver development through mechanisms of amino acid uptake and protein synthesis.

Prolonged-release tacrolimus allows for once-daily dosing. Although many adult recipients have been switched from standard tacrolimus, prolonged-release tacrolimus has not been popular for pediatric patients despite the potential benefits for medication compliance. We report on prolonged-release tacrolimus for 11 pediatric living related donor liver transplant (LRDLT) recipients. Patients under 18 years of age who were receiving standard tacrolimus-based immunosuppression and steroid taper underwent conversion from standard to prolonged-release tacrolimus. We monitored tacrolimus trough levels and liver function tests (LFTs). We also assessed adverse effects and satisfaction levels for prolonged-release tacrolimus. Mean age at transplantation was 4.3 years. The mean duration of follow-up was 12 months. The ratios of trough levels with prolonged-release vs standard tacrolimus were 0.97, 0.95, and 0.92 at 1, 2, and 4 weeks post conversion, respectively. Two patients discontinued prolonged-release tacrolimus owing to abnormal LFTs and neurological abnormalities, respectively; but symptoms resolved after reconversion. One patient returned to standard tacrolimus and the other was converted to cyclosporine. Once-daily administration satisfied 89% of patients. In the overall assessment, conversion to prolonged-release tacrolimus satisfied all patients. Prolonged-release tacrolimus was useful for pediatric patients after LRDLT. Trough levels after conversion were compatible with those before conversion. Most patients were satisfied with prolonged-release tacrolimus. However, some patients failed conversion because of unexpected responses. Close observation after conversion is required even if patients have previously had an uneventful course on standard tacrolimus.

Background: Maturity-onset diabetes of the young (MODY) is a subgroup of monogenic diabetes mellitus, of which MODY1, caused by HNF4A mutations, accounts for only 5% or less and has been rarely reported in East Asian countries. Here we report two novel HNF4A mutations in two Japanese families with MODY1. Methods: Proband 1 is an 8-year-old girl and proband 2 is a 14-year-old girl. Both were nonobese, demonstrated elevated HbA1c and negative serum anti-glutamic acid decarboxylase antibodies, and had a family history of diabetes. We directly sequenced HNF4A and performed functional analysis of the detected missense mutation. Results: Proband 1 had a heterozygous missense mutation, c.824A>G (p.Asn275Ser). Luciferase assay demonstrated a significant reduction in transcriptional activity. A heterozygous frame shift mutation, c.692-695delAGGA (p.Lys231ThrfsX5), was detected in proband 2. Affected family members shared the same mutations, showing high penetrance. Both mutations reside in the HNF4a dimerization domain and the corresponding amino acids are well conserved between species. Conclusions: These two mutations are most likely the cause of MODY1 in these families. Considering the effectiveness of sulfonylureas, it is important to correctly diagnose MODY1. Copyright (C) 2013 S. Karger AG, Basel

Although LT can be successful for treating end-stage liver disease in children, some patients develop fibrosis around the central vein area (PCF). This raises the possibility that PCF could lead to later cirrhosis and graft failure. Here, we report a retrospective immunohistochemical study of 28 patients who received a live donor liver transplant. We assessed the incidence and etiology of PCF using CD3, CD20, HLA-DR, and C4d-specific antibodies. Histological evidence of PCF was found in 13 cases (46.4%), of which 11 (84.6%) had experienced ACR and/or CP events post-transplant. Immunohistochemical evaluation revealed significantly stronger staining with these antibodies in the central vein area in PCF, especially for CD20 and C4d. This implies humoral immunopathology and suggests involvement of humoral immunity in the development of PCF. These results further imply that suppression of cellular immunity alone is insufficient to prevent PCF. We therefore suggest that suppression of both humoral and cellular immunity in combination would be required for prevention of PCF.

Outgrowth of the foregut endoderm to form the liver bud is considered the initial event of liver development. Hepatic stem/progenitor cells (HSPCs) in the liver bud are postulated to migrate into septum transversum mesenchyme at around embryonic day (E) 9 in mice. The studies of liver development focused on the mid-fetal stage (E11.5-14.5) have identified HSPCs at this stage. However, the in vitro characteristics of HSPCs before E11.5 have not been elucidated. This is probably partly because purification and characterization of HSPCs in early fetal livers have not been fully established. To permit detailed phenotypic analyses of early fetal HSPC candidates, we developed a new coculture system, using mouse embryonic fibroblast cells. In this coculture system, CD13(+)Dlk(+) cells purified from mouse early fetal livers (E9.5 and E10.5) formed colonies composed of both albumin-positive hepatocytic cells and cytokeratin (CK) 19-positive cholangiocytic cells, indicating that early fetal CD13(+)Dlk(+) cells have properties of bipotent progenitor cells. Inhibition of signaling by Rho-associated coiled-coil containing protein kinase (Rock) or by nonmuscle myosin II (downstream from Rock) was necessary for effective expansion of early fetal CD13(+)Dlk(+) cells in vitro. In sorted CD13(+)Dlk(+) cells, expression of the hepatocyte marker genes albumin and alpha-fetoprotein increased with fetal liver age, whereas expression of CK19 and Sox17, endodermal progenitor cell markers, was highest at E9.5 but decreased dramatically thereafter. These first prospective studies of early fetal HSPC candidates demonstrate that bipotent stem/progenitor cells exist before E11.5 and implicate Rock-myosin II signaling in their development.

To identify the genes involved in chondrocytic differentiation, we applied gene trap mutagenesis to a murine mesenchymal chondrogenic cell line ATDC5 and isolated a clone in which the gene encoding vinculin was trapped. The trapped allele was assumed to express a fusion protein containing a truncated vinculin lacking the tail domain and the geo product derived from the trap vector. The truncated vinculin was suggested to exert a dominant negative effect. Impaired functioning of vinculin caused by gene trapping in ATDC5 cells or knockdown in primary chondrocytes resulted in the reduced expression of chondrocyte-specific genes, including Col2a1, aggrecan, and Col10a1. The expression of Runx2 also was suppressed by the dysfunctional vinculin. On the other hand, the expression of Sox9, encoding a key transcription factor for chondrogenesis, was retained. Knockdown of vinculin in metatarsal organ cultures impaired the growth of the explants and reduced the expression of Col2a1 and aggrecan. Gene trapping or knockdown of vinculin decreased the phosphorylation of ERK1/2 but increased that of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) and Akt during chondrocytic differentiation, suggesting a disturbance of signaling by insulin-like growth factor I (IGF-I). Knockdown of vinculin in the metatarsal organ culture abrogated the IGF-I-induced growth and inhibited the up-regulation of Col2a1 and aggrecan expression by IGF-I. Loss of vinculin function in differentiating chondrocytes impaired the activation of the p38 MAPK pathway also, suggesting its involvement in the regulation of chondrogenesis by vinculin. Our results indicate a tissue-specific function of vinculin in cartilage whereby it controls chondrocytic differentiation.

Aim: Hepatic steatosis accompanied by impaired protein synthesis is often observed in hepatic dysfunction. To assess whether protein synthesis inhibition directly induces hepatic steatosis, we investigated the molecular mechanisms of cycloheximide (CHX)-induced fatty liver mice. Methods: C57/BL6CR mice were i.p. administrated CHX (20 mg/kg) three times every 4 h to induce hepatic steatosis. Hepatic lipid secretion, fatty acid oxidation, hepatic lipogenesis and hepatic lipid uptake were evaluated. Results: Twenty-four hours after the first CHX injection, hepatic lipid levels increased in CHX-treated mice to 1.8-fold of that in controls but returned to normal within 48 h. The hepatic triglyceride (TG) secretion rate decreased significantly to 22% of controls, and the apolipoprotein B (apoB) protein level, but not microsomal TG transfer protein, decreased in CHX-treated mice. The apob gene expression was not significantly different between controls and CHX-treated mice. On the other hand, plasma free fatty acid and lipogenic protein levels did not increase and plasma beta-hydroxybutyrate level remained stable, suggesting that the coordinated balance between fatty acid oxidation, hepatic lipid uptake and lipogenesis was not disrupted in this model. Cellular lipid accumulation and decreased cellular and secreted apoB were also observed in CHX-treated HepG2 cells. Knockdown of apoB in HepG2 cells also resulted in the cellular TG accumulation. Conclusion: We demonstrated that decreased hepatic lipid secretion due to acute apoB reduction is involved in the pathogenesis of CHX-induced liver steatosis.

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced by bone and exerts its function in the target organs by binding the FGF receptor (FGFR) and Klotho. Since recent studies suggested that extracellular inorganic phosphate (Pi) itself triggers signal transduction and regulates gene expression in some cell types, we tested the notion that extracellular Pi induces signal transduction in the target cells of FGF23 also and influences its signaling, utilizing a human embryonic kidney cell line HEK293. HEK293 cells expressed low levels of klotho, and treatment with a recombinant FGF23[R179Q], a proteolysis-resistant mutant of FGF23, resulted in phosphorylation of ERK1/2 and induction of early growth response-1 (EGR1) expression. Interestingly, increased extracellular Pi resulted in activation of the Raf/MEK/ERK pathway and expression of EGR1, which involved type III sodium/phosphate (Na(+)/Pi) cotransporter PiT-1. Since the effects of an inhibitor of Na(+)/Pi cotransporter on FGF23 signaling suggested that the signaling triggered by increased extracellular Pi shares the same downstream cascade as FGF23 signaling, we further investigated their convergence point. Increasing the extracellular Pi concentration resulted in the phosphorylation of FGF receptor substrate 2 alpha (FRS2 alpha), as did treatment with FGF23. Knockdown of FGFR1 expression diminished the phosphorylation of both FRS2a and ERK1/2 induced by the Pi. Moreover, overexpression of FGFR1 rescued the decrease in Pi-induced phosphorylation of ERK1/2 in the cells where the expression of PIT-I was knocked down. These results suggest that increased extracellular Pi triggers signal transduction via PiT-1 and FGFR and influences FGF23 signaling in HEK293 cells. J. Cell. Biochem. 111: 1210-1221, 2010. (C) 2010 Wiley-Liss, Inc.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disease associated with demyelination of the central nervous system, adrenocortical insufficiency and accumulation of very long chain fatty acids. It is a clinically heterogeneous disorder ranging from a severe childhood cerebral form to an asymptomatic form. The incidence in Japan is estimated to be between 1:30,000 and 1:50,000 boys as determined by a nationwide retrospective survey between 1990 and 1999, which found no cases with Addison's form. We reviewed the medical records of eleven Japanese boys with X-ALD from 1990 to 2010 in our institute. Eight patients were detected by neuropsychological abnormalities, whereas a higher prevalence of unrecognized adrenocortical insufficiency (5/11: 45%) was observed than previously recognized. While no neurological abnormalities were demonstrated in two brothers, the elder brother had moderate Addison's disease at diagnosis and the presymptomatic younger brother progressed to Addison's disease six months after the diagnosis of X-ALD. Early detection of impaired adrenal function as well as early identification of neurologically presymptomatic patients by genetic analysis is essential for better prognosis. Addison's form might be overlooked in Japan; therefore, X-ALD should be suspected in patients with adrenocortical insufficiency.

Although the intermittent administration of PTH is known to stimulate the bone formation, the underlying mechanisms are not fully understood. Here we investigated the crosstalk between PTH/cAMP signaling and canonical Wnt signaling using the human osteoblastic cell line Saos-2. Treatment with PTH or forskolin, an activator of adenylate cyclase, facilitated T-cell factor (TCF)-dependent transactivation in a dose-dependent manner, which was abolished by pre-treatment with a PKA inhibitor, H89. Wnt3a and forskolin synergistically increased the TCF-dependent transactivation. Interestingly intermittent treatment with PTH enhanced the TCF-dependent transactivation more profoundly than continuous treatment. In addition to the effects on TCF-dependent reporter activity, treatment with PTH or forskolin resulted in the increased expression ofendogenous targets of Wnts, Wnt-induced secreted protein 2 (WISP2) and naked cuticle 2 (NKD2). We then investigated the convergence point of PTH/cAMP signaling and the canonical Wnt pathway. Western blotting demonstrated that GSK-3 beta was rapidly phosphorylated at Ser(9) on treatment with PTH or forskolin, leading to its inactivation. Moreover, overexpression of a constitutively active mutant of GSK-3 beta abolished the TCF-dependent transactivation induced by forskolin. On the other hand, overexpression of the Wnt antagonist Dickkopf-1 (DKK1) failed to cancel the effects of forskolin on the canocical Wnt pathway. Interestingly, treatment with Wnt3a markedly reduced the forskolin-induced expression of receptor activaror of NF-kappa B ligand (RANKL), a target gene of PTH/cAMP/PKA. these results suggest that cAMP/PKA signaling activates the canonical Wnt pathway through the inactivation of GSK-3 beta, whereas Wnt signaling might inhibit bone resorption through a negative impact on RANKL expression in osteoblasts. J. Cell. biochem. 104: 304-317, 2008. (C) 2007 Wiley-Liss. Inc.

Megalin is a multifunctional endocytic receptor that is expressed in renal proximal tubules and plays critical roles in the renal uptake of various proteins. It was hypothesized that megalin-dependent endocytosis might play a role in renal phosphate reabsorption. For addressing the short-term effects of altered megalin function, a recombinant protein for the soluble form of 39-kD receptor-associated protein (RAP) was administered intraperitorteally to 7-wk-old mice. Histidine (His)-tagged soluble RAP (amino acids 39 to 356) lacking the amino-terminal signal peptide and the carboxy-terminal endoplasmic reticulum retention signal was prepared by bacterial expression (designated His-sRAP). After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitorteal administration of His-sRAP (3.5 mg/dose). Immuno-staining and Western blot analyses demonstrated the uptake of His-sRAP and the accelerated internalization of megalin in proximal tubular cells 1 h after administration. In addition, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. The effects of three sequential administrations of His-sRAP (3.5 mg/dose, three doses at 4-h intervals) then were examined, and increased urinary excretion of low molecular weight proteins, including vitamin D-binding protein, was found, which is consistent with findings reported for megalin-deficient mice. It is interesting that urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border membrane was decreased. Serum concentration of 25-hydroxyvitamin D was decreased, whereas the plasma level of intact parathyroid hormone was not altered by the administration of His-sRAP. The results suggest that the His-sRAP-induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.

We present a 13-year-old boy who developed hyperthyroidism during the clinical course of idiopathic nephrotic syndrome treated with glucocorticoid. He had a second relapse of minimal change nephrotic syndrome, and complete remission of nephrotic syndrome was achieved immediately with oral glucocorticoid. However, when the steroid dosage was reduced, signs of hyperthyroidism such as systolic hypertension and tachycardia were observed. Laboratory findings revealed thyroid-stimulating hormone (TSH) below 0.05 muU/ml, free triiodothyronine of 16.1 pg/ml, free thyroxine of 5.6 ng/dl, and anti-TSH receptor antibody of 90%. Thus, a diagnosis of hyperthyroidism was made and treatment with thiamazol was started. Massive proteinuria may decrease the activity of hyperthyroidism due to urinary loss of thyroid hormones. A decrease in glucocorticoid dosage may also be involved in the development of hyperthyroidism due to a reduced immunosuppressive effect.

We found that in the liver from infants with biliary atresia, the expression level of beta-defensin, an antimicrobial peptide which activates innate immunity against gram-negative bacteria was upregulated compared to livers from diseased and normal controls. The expression level of its receptor, TLR4 was identified on hepatic dendritic cells. In the rotavirus-induced murine model of biliary atresia, when we kept the colony under bactrim diet, the incidence of obstruction of extrahepatic bile duct was reduced significantly after injection of rotavirus, and a part of gram negative bacteria was decreased in the stool of mouse from the bactrim-diet colony.

Regulatory mechanisms of bile acid synthesis under chronic cholestasis remain unclear. We analyzed molecules involving bile acid metabolism using liver and serum samples from 8 biliary atresia (BA) children and 4 non-cholestatic disease controls. CYP7A1 mRNA expression was not inhibited in BA microdissected hepatocyte-entriched tissue despite high serum bile acid concentrations. The FGF19 protein was synthesized in BA hepatocytes, and its serum concentration was elevated. FGFR4 was phosphorylated in BA livers. However, ERK phosphorylation was significantly decreased. his is the first study to demonstrated that the FGF19 pathway did not suppress bile acid synthesis due to downregulation of ERK pathway in BA patients.

Helicobacter hepaticus (H. hepaticus) is a gram-negative, spiral shaped microaerophilic bacterium. Several studies have suggested the possibility that infection of this bacterium plays a role in the pathogenesis of liver diseases in humans. We evaluated the association between H. hepaticus and children with liver disorders. Furthermore, we detect the antigenic H. hepaticus protein recognized by child sera using 2-DE immunoblot analysis. Positive rate of H. hepaticus in children without and with liver disorders was 30.3% (10/33) and 44.4% (4/9), respectively. Total 25 proteins were identified as candidate antigens by LS-MS/MS after the 2-DE immunoblot analysis. Among the proteins, flagelin A was the most reactive protein, particularly against highly reactive sera, although low reactive sera also detected the spot slightly.

We studied FGF19 signaling pathway in chronic cholestasis using blood sample and liver tissue from Biliary atresia (BA) patients. Serum concentration of bile acid was significantly higher in BA patients, and their serum and tissue concentrations of FGF19 were significantly higher in BA patients. IHC and ISH revealed that FGF19 were aberrantly synthesized in hepatocytes in BA liver. While, those target gene, CYP7A1mRNA was not suppressed in BA patients despite of high concentration of bile acid and FGF19. Next, FXR mRNA was significantly up-regulated in hepatocytes in BA patients, and FGFR4 and KLB mRNA were also up-regulated. Further, phosphorylation of FGFR4 in BA patients was increased, then phosphorylation of ERK was decreased. Whereas, SHP mRNA, which suppress CYP7A1 was up-regulated in BA patients. These results suggest that FGF19 was aberrantly expressed in chronic cholestatic hepatocytes. however, this signal was not able to suppress CYP7A1 via ERK pathway.

We screened the downstream molecules of HNF4a during liver development, sodium-coupled neutral amino acid transporter (SNAT) 4 was identified. The aim of this study is to investigate the regulation of SNAT4 by HNF4a and to clarify its roles in differentiating hepatocytes. SNAT4 mRNA showed a marked perinatal increase in mRNA levels, and was predominant among system A amino acid transporters. It was first detected in E18.5 liver in immunohistochemistry, and found in most hepatocytes after birth. Three alternative first exons were founding SNAT4 gene. Finally, over-expression experiments demonstrated that SNAT4 facilitated amino acid uptake and de novo protein synthesis in primary hepatoblasts.

Although it has been revealed that several molecules are involved in the regulation of renal phosphate reabsorption, the functional relationship between these molecules remains to be elucidated. In the current study, we aimed to clarify the mechanisms by which these molecules coordinate to regulate renal phosphate reabsorption. We have previously found that megalin-dependent endocytosis is involved in renal phosphate reabsorption via the regulation of subcellular distribution of type IIa Na^+/Pi co-transporter. Therefore, we first examined whether megalin-dependent endocytosis affect the action of FGF23 as a phosphatunc hormone. The disturbance of megalin-dependent endocytosis using soluble form of receptor associated protein(RAP) resulted in the reduced serum level of FGF23 in mice. This observation recapitulates the reduced serum FGF23 in human patients with Fanconi syndrome, suggesting that FGF23 is filtered in glomerulus and excreted in urine. We also examined the effect of extracellular phosphate(Pi) itself on the FGF23 signaling using HEK293 human embryonic kidney cells. We found that increased extracellular Pi resulted in the signal transduction in the cells, and it shared the downstream signaling network with FGF23. These results suggest that extracellular Pi itself affect the responsiveness of renal tubular cells to FGF23.

マウス発生初期肝臓に発現する遺伝子のスクリーニングを行うため,まず肝臓が発生して1日目の胎生9.5日胚から肝臓を単離し,器官培養することに成功した。この器官培養系を用いて,申請者は,このHnf4αを器官培養胎仔肝に強制的に発現させて細胞内遣伝子を撹乱し,対照として同じくLacZを導入して24時間器官培養後,mRNAを抽出した。このmRNAを鋳型としてcDNA合成を行い,この2つのcDNAに対し,suppression subtractive hybridization法を用いてmRNAの発現プロファイルの違いを比較しmRNAの量が増加または減少したものを同定した。その結果,肝特異的アミノ酸トランスポーターsodiuim-coupled neutral amino acid transporter 4(SNAT4)や蛋白修飾因子Spcs 2,クロマチン構成因子H2afzが肝発生期に発現している有望な分子として同定できた。申請者は,SNAT4につき更なる解析を進め,定量PCRにて肝発生過程におけるmRNAレベルの経時的な発現の変化を調べると,SNATファミリーの中でSNAT4が最も優位に発現しており,少なくともE9.5日から発現し,胎生後期に急激に増加することを見いだした(2007年米国肝臓学会にて発表)。また申請者は,マウスSNAT4をコードする遺伝子Slc38a4の上流約3.7kbの領域をクローニングした。レポーターベクターを構築し,レポーターアッセイにて転写活性化能を測定すると,Slc38a4はHnf4αにより転写が活性化されることが判明した(2007年米国肝臓学会にて発表)。この成果に基づいて,現在,論文投稿準備中である。

The Grb10 expression in human pathological cirrhotic liver tissues and the animal models with liver damages were assessed. Dimethylnitrosamine (DMN) administration and CCI4 administration mice models were constructed according to the former reported protocol but they were not utilized for the following experiments because of their high lethality and hepatocyte damage rather than portal fibrosis. Then immunohistochemical staining for Grb10 was performed on the common bileduct ligation (CBDL) model, but no significant staining was obtained. Examination of mRNA expression by RT-PCR in the liver tissue of the CBDL mouse and the normal adult mouse demonstrated coordinative expressions of Grb10. The result was different from the former report that the Grb10 expression in the adult mouse liver was very low as compared with that in the embryo. On the other hand, in Western blot, protein expression in the adult mouse liver was very low compared to those in fetus and CBDL mouse liver. Then hepatic parenchymal cells and hepatic stellate cells (HSC) were extracted from the rat liver, the mypfibroblastic transformation of HSC was assessed and the expressions of Grb10 were examined by RT-PCR. In result, the mRNA expressions were detected in myofibroblast (HSC), while very low in hepatocyte. In addition, mRNA expression was demonstrated in the damaged liver tissue of DMN-treated SD rats which is identical to the localization found in human cirrhotic liver. Though that does not reflect the pathological state in human cirrhotic liver, in vitro examination utilizing the cultured rat HSC can be a efficient tool for the assessment of the role of Grb10 in the mechanism of hepatic fibrosis in future.

Megalin is a multifunctional endocytic receptor expressed in renal proximal tubules, and plays critical roles in the renal uptake of various proteins. We hypothesized that megalin-dependent endocytosis might play a role in renal reabsorption of inorganic phosphate. To address the short-term effects of altered megalin function, we administered a recombinant protein for the soluble form of 39-kD receptor associated protein (RAP) intraperitoneally to 7-wk-old mice. The recombinant protein was prepared as histidine-tagged soluble RAP (aa 39-356) lacking the amino-terminal signal peptide and the carboxyl terminal endoplasmic reticulum (ER) retention signal, which was designated as His-sRAP. After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitoneal injection of His-sRAP (3.5 mg/dose). Immunostaining and Western blot analyses demonstrated that the uptake of His-sHAP and the accelerated internalization of megalin in proximal tubular cells 1 hour after administration. Interestingly, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. Three sequential administrations of His-sRAP (3.5 mg/dose, three doses with 4-hr intervals) increased the urinary excretion of low molecular weight proteins including vitamin D-binding protein. In addition, the urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border was decreased. Serum 25-hydroxyvitamin D was decreased, while the plasma level of intact parathyroid hormone was not altered. These results suggest that the His-sRAP induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.

トロンボスポンジン1アンタゴニスト・LSKLペプチドの持つ肝線維化抑制作用を、肝臓における構成細胞ごとに明らかにする為、ラット培養小型肝細胞を用いた肝細胞の3次元培養系に着目した。ラット培養小型肝細胞は、1つの細胞からコロニーを形成し、肝細胞に成熟する細胞と胆管上皮系のマーカー(CK19)を発現する細胞とに分かれるが、これを培養液中にLSKLペプチドを添加群とペプチド非添加(対照)群の2群に分けてCK19陽性細胞率を比較検討すると、対照群では54.6%であったのに対し、LSKLペプチド添加群では31.3%と有意に抑制されていた。胆管上皮系への分化が抑制され肝細胞への分化が促進されたことは、LSKLペプチドを投与した肝線維化モデルラットの肝組織像において線維化抑制および肝再生が観察されたことと一致した。 肝再生と肝発生における分子メカニズムには、共通する部分が多くあると考えられた為、申請者は、マウス胎生9.5日胎仔肝の器官培養系を確立した。3日間培養を行った結果、培養胎仔肝は、培養3日目に約3倍の体積に成長し、組織像においても肝芽細胞が増殖し、器官発生が進んでいることが観察された。また、肝細胞のマーカーであるアルブミン遺伝子転写産物の発現も増強した。この系を用いて肝細胞の分化・成熟に重要なHepatocyte nuclear factor 4αを過剰発現させた胎仔肝と対照としてLacZを発現させた胎仔肝のmRNAのプロファイルをsubtractive suppression PCR法で比較したところ、いくつかの遺伝子を同定することが出来た。その中には、TGF-β1の標的遺伝子と思われる遺伝子も存在した為、今後さらなる検討を進める予定である。