👤 Haruka Kasahara

The precise mechanism of sodium glucose co-transporter 2 (SGLT2) inhibitor on reno-protective effect has been still unclear. In this study, we hypothesised that SGLT2 inhibitor prevents diabetic kidney disease via reduction of hypoxia-induced factors. In this multicenter, prospective, randomised, double blinded clinical trial, people with type 2 diabetes and microalbuminuria were randomised equally to empagliflozin (10 mg/day) (n = 40) and placebo (n = 39) and followed 24 weeks. The primary endpoint was change in urinary albumin creatinine ratio (ACR) and urinary liver type fatty acid binding protein (L-FABP) excretion from baseline to 24 weeks. Major secondary outcome was change in serum vascular endothelial growth factor (VEGF), angiopoietin-like proteins 2 (ANGPTL2), angiopoietin-like proteins 4 (ANGPTL4), and adrenomedullin (AM) levels. Although the reduction of ACR was significantly greater in the empagliflozin group than the placebo group at 4 and 12 weeks, the difference of change at 24 weeks between the two groups was not statistically significant (Empagliflozin group-Placebo group: -0.3643, 95% CI: -0.7571 to 0.0285, p = 0.0686). There was no difference in urinary L-FABP excretion between the empagliflozin and placebo groups. Serum VEGF and ANGPTL2 decreased significantly more in the empagliflozin group, whereas there were no significant differences in AM and ANGPTL4. These results demonstrated that empagliflozin partially suppressed the hypoxia-induced angiogenic factors overproduction in addition to a declining trend in ACR in the early stage of diabetic kidney disease, which might contribute to the mechanisms of reno-protective effects of this agent (jRCTs051200147). Show less

Neuromuscular scoliosis is associated with cerebral palsy caused by metabolic diseases. Patients with scoliosis require meticulous consideration in abdominal surgery, as scoliosis can reduce the abdominal cavity volume, compress abdominal organs, and cause abdominal complications. Special attention should be paid to the graft position, especially in the setting of liver transplantation (LT). We herein describe a pediatric case of LT for carbamoyl phosphate synthetase I (CPS1) deficiency with severe scoliosis. A 13-year-old girl with CPS1 deficiency was transferred to our department as a candidate for liver transplantation. She underwent living donor liver transplantation with a left lobe from her mother. Following LT, portal vein (PV) complications occurred due to the kinking anastomosis, requiring several rounds of graft repositioning, PV reconstruction, thrombectomy, and finally stent placement due to severe scoliosis. Technical efforts were made to ensure PV blood flow with stent placement via the umbilical vein. Three months after LT, she was discharged from our hospital with sufficient PV flow. This report suggests the need for a careful surgical approach in patients with skeletal abnormalities, such as the management of complications arising from anatomical abnormalities and selection of the appropriate graft size. Preoperative assessment and surgical planning of both donors and recipients according to patient characteristics should be carefully conducted. Show less

Large-scale genome-wide association studies (GWASs) on bipolar disorder (BD) have implicated the involvement of the fatty acid desaturase (FADS) locus. These enzymes (FADS1 and FADS2) are involved in the metabolism of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are thought to potentially benefit patients with mood disorders. To model reductions in the activity of FADS1/2 affected by the susceptibility alleles, we generated mutant mice heterozygously lacking both Fads1/2 genes. We measured wheel-running activity over six months and observed bipolar swings in activity, including hyperactivity and hypoactivity. The hyperactivity episodes, in which activity was far above the norm, usually lasted half a day; mice manifested significantly shorter immobility times on the behavioral despair test performed during these episodes. The hypoactivity episodes, which lasted for several weeks, were accompanied by abnormal circadian rhythms and a marked decrease in wheel running, a spontaneous behavior associated with motivation and reward systems. We comprehensively examined lipid composition in the brain and found that levels of certain lipids were significantly altered between wild-type and the heterozygous mutant mice, but no changes were consistent with both sexes and either DHA or EPA was not altered. However, supplementation with DHA or a mixture of DHA and EPA prevented these episodic behavioral changes. Here we propose that heterozygous Fads1/2 knockout mice are a model of BD with robust constitutive, face, and predictive validity, as administration of the mood stabilizer lithium was also effective. This GWAS-based model helps to clarify how lipids and their metabolisms are involved in the pathogenesis and treatment of BD. Show less

Bipolar disorder is a common mental illness occurring in approximately 1% of individuals and requires lifelong treatment. Although genetic factors are known to contribute to this disorder, the genetic architecture has not yet been completely clarified. Our initial trio-based exome sequencing study of bipolar disorder showed enrichment of de novo, loss-of-function (LOF) or protein-altering mutations in a combined group with bipolar I and schizoaffective disorders, and the identified de novo mutations were enriched in calcium-related genes. These findings suggested a role for de novo mutations in bipolar disorder. The validity of these statistical associations will be strengthened if the functional impact of the mutations on cellular function and behavior are identified. In this study, we focused on two de novo LOF mutations in calcium-related genes, EHD1 and MACF1, found in patients with bipolar disorder. We first showed that the EHD1 mutation resulted in a truncated protein with diminished effect on neurite outgrowth and inhibited endocytosis. Next, we used CRISPR/Cas9 to establish two knock-in mouse lines to model the in vivo effects of these mutations. We performed behavioral screening using IntelliCage and long-term wheel running analysis. Ehd1 mutant mice showed higher activity in the light phase. Macf1 mutant mice showed diminished attention and persistence to rewards. These behavioral alterations were similar to the phenotypes in previously proposed animal models of bipolar disorder. These findings endorse the possible role of de novo mutations as a component of the genetic architecture of bipolar disorder, which was suggested by the statistical evidence. Show less

Epithelial-mesenchymal transition (EMT) plays a role in cancer metastasis as well as in drug resistance through various mechanisms, including increased drug efflux mediated by P-glycoprotein (P-gp). In this study, we investigated the activation mechanism of P-gp, including its regulatory factors, during EMT in hepatoblastoma-derived HepG2 cells. HepG2 cells were transfected with SNAI1 using human adenovirus serotype 5 vector. We quantified mRNA and protein expression levels using qRT-PCR and western blot analysis, respectively. P-gp activity was evaluated by uptake assay, and cell viability was assessed by an MTT assay. P-gp protein expression on plasma membrane was higher in SNAI1-transfected cells than in Mock cells, although there was no difference in P-gp protein level in whole cells. Among the scaffold proteins such as ezrin, radixin and moesin (ERM), only radixin was increased in SNAI1-transfected cells. Uptake of both Rho123 and paclitaxel was decreased in SNAI1-transfected cells, and this decrease was blocked by verapamil, a P-gp inhibitor. The reduced susceptibility of SNAI1-transfected cells to paclitaxel was reversed by elacridar, another P-gp inhibitor. Increased expression of radixin during SNAI1-induced EMT leads to increased P-gp membrane expression in HepG2 cells, enhancing P-gp function and thereby increasing drug resistance. Show less

The hepatocyte paraffin 1 (Hep Par 1) antibody is widely used as a hepatocyte marker, recognizing carbamoyl phosphate synthetase 1 (CPS1), an essential component of the urea cycle. Various missense, nonsense, and frameshift mutations occur in the CPS1 gene. In neonatal patients with homozygous CPS1 deficiency (CPS1D), urea cycle defects with resulting severe hyperammonemia can be fatal, though liver transplantation provides a complete cure for CPS1D. We performed Hep Par 1 immunostaining in the explanted livers of 10 liver transplant patients with CPS1D. Seven were negative for Hep Par 1 in the hepatocytes and the other three showed normal diffuse granular cytoplasmic staining. As expected, all three Hep Par 1-positive patients had at least one missense mutation, and all four patients who had only nonsense or frameshift mutations were Hep Par 1-negative. The other three patients were unexpectedly negative for Hep Par 1, even though each had one missense mutation. These results suggest that CPS1D can be related to the loss of Hep Par 1 reactivity due to the loss of protein production, a one amino acid substitution resulting in an abortive protein product, or both. Hep Par 1 immunohistochemistry can be used as a simple method to confirm CPS1D. Show less

Atrial natriuretic factor (ANF) is abundantly expressed in atrial cardiomyocytes throughout ontogeny and in ventricular cardiomyocytes in the developing heart. However, during cardiac failure and hypertrophy, ANF expression can reappear in adult ventricular cardiomyocytes. The transcription factor Nkx2-5 is one of the major transactivators of the ANF gene in the developing heart. We identified Nkx2-5 binding at three 5' regulatory elements (kb -34, -31, and -21) and at the proximal ANF promoter by ChIP assay using neonatal mouse cardiomyocytes. 3C analysis revealed close proximity between the distal elements and the promoter region. A 5.8-kb fragment consisting of these elements transactivated a reporter gene in vivo recapitulating endogenous ANF expression, which was markedly reduced in tamoxifen-inducible Nkx2-5 gene knockout mice. However, expression of a reporter gene was increased and expanded toward the outer compact layer in the absence of the transcription repressor Hey2, similar to endogenous ANF expression. Functional Nkx2-5 and Hey2 binding sites separated by 59 bp were identified in the -34 kb element in neonatal cardiomyocytes. In adult hearts, this fragment did not respond to pressure overload, and ANF was induced in the absence of Nkx2-5. These results demonstrate that Nkx2-5 and its responsive cis-regulatory DNA elements are essential for ANF expression selectively in the developing heart. Show less

CPS1 is a mitochondrial matrix enzyme that catalyzes the first committed step of the urea cycle, the primary system for removing nitrogen produced by protein metabolism using N-acetylglutamate. Patients with CPS1 deficiency have severe hyperammonemia that results in serious neurologic sequelae and sometimes death. LT has been indicated for neonatal-onset CPS1 deficiency. This study retrospectively reviewed five children with a diagnosis of CPS1 deficiency who underwent LDLT from heterozygous donors. Between November 2005 and May 2010, 124 children underwent LDLT with an overall patient and graft survival of 91.0%. Five patients were indicated for LDLT because of CPS1 deficiency. All recipients achieved resolution of their metabolic derangement, without donor complication, with a normal feeding regimen without medication for their original metabolic liver disease. LDLT, even from heterozygous donors, appears to be a feasible option, associated with a better quality of life for treating patients with CPS1 deficiency. Long-term observation may therefore be necessary to collect sufficient data to confirm the efficacy of this treatment modality. Show less