Genetic background-dependent abnormalities of the enteric nervous system and intestinal function in Kif26a-deficient mice.

The Kif26a protein-coding gene has been identified as a negative regulator of the GDNF-Ret signaling pathway in enteric neurons. The aim of this study was to investigate the influence of genetic background on the phenotype of Kif26a-deficient (KO, -/-) mice. KO mice with both C57BL/6 and BALB/c genetic backgrounds were established. Survival rates and megacolon development were compared between these two strains of KO mice. Functional bowel assessments and enteric neuron histopathology were performed in the deficient mice. KO mice with the BALB/c genetic background survived more than 400 days without evidence of megacolon, while all C57BL/6 KO mice developed megacolon and died within 30 days. Local enteric neuron hyperplasia in the colon and functional bowel abnormalities were observed in BALB/c KO mice. These results indicated that megacolon and enteric neuron hyperplasia in KO mice are influenced by the genetic background. BALB/c KO mice may represent a viable model for functional gastrointestinal diseases such as chronic constipation, facilitating studies on the underlying mechanisms and providing a foundation for the development of treatments.

A Thrombomodulin Promoter Gene Polymorphism, rs2239562, Influences Both Susceptibility to and Outcome of Sepsis.

Objective: Disseminated intravascular coagulation plays a key role in the pathophysiology of sepsis. Thrombomodulin is essential in the protein C system of coagulation cascade, and functional polymorphisms influence the human thrombomodulin gene (THBD). Therefore, we conducted a multicenter study to evaluate the influence of such polymorphisms on the pathophysiology of sepsis. Methods: A collaborative case-control study in the intensive care unit (ICU) of each of five tertiary emergency centers. The study included 259 patients (of whom 125 displayed severe sepsis), who were admitted to the ICU of Chiba University Hospital, Chiba, Japan between October 2001 and September 2008 (discovery cohort) and 793 patients (of whom 271 patients displayed severe sepsis), who were admitted to the five ICUs between October 2008 and September 2012 (multicenter validation cohort). To assess the susceptibility to severe sepsis, we further selected 222 critically ill patients from the validation cohort matched for age, gender, morbidity, and severity with the patients with severe sepsis, but without any evidence of sepsis. Results: We examined whether the eight THBD single nucleotide polymorphisms (SNPs) were associated with susceptibility to and/or mortality of sepsis. Higher mortality on severe sepsis in the discovery and combined cohorts was significantly associated with the CC genotype in a THBD promoter SNP (-1920*C/G; rs2239562) [odds ratio [OR] 2.709 (1.067-6.877), P = 0.033 and OR 1.768 (1.060-2.949), P = 0.028]. Furthermore, rs2239562 SNP was associated with susceptibility to severe sepsis [OR 1.593 (1.086-2.338), P = 0.017]. Conclusions: The data demonstrate that rs2239562, the THBD promoter SNP influences both the outcome and susceptibility to severe sepsis.

Influence of autophagy on acute kidney injury in a murine cecal ligation and puncture sepsis model.

The role of autophagy in the maintenance of renal homeostasis during sepsis is not well understood. We therefore aimed to determine the influence of autophagy on kidney during sepsis using a murine sepsis model, i.e. cecal ligation and puncture (CLP). In CLP treated animals, the number of autolysosomes observed by electron microscopy increased over time. The number of autophagosomes in CLP animals decreased relative sham operated controls at 24 hrs after CLP, indicating that autophagy flux is already diminishing by that time. Moreover, CLP induced an increase in LC3-II/LC3-I ratio at 6-8 hrs, demonstrated in western blots, as well as an increase in GFP-LC3 dots at 6-8 hrs and 24 hrs, using immunofluorescence and anti-LC3 and LAMP1 antibodies on tissue sections from GFP-LC3 transgenic mice. LC3-II/LC3-I ratio and the number of co-localized GFP-LC3 dots and LAMP1 signals (GFP LC3 + LAMP1 dots) in CLP mice at 24 hrs were significantly reduced compared with data obtained at 6-8 hrs. Notably, acceleration of autophagy by rapamycin resulted in improvement of renal function that was associated with improvement in the histologic severity of tubular epithelial injury in CLP treated animals. Autophagy in the kidney was significantly slowed in the kidney during the acute phase of sepsis; nonetheless, autophagy in kidney appears to play a protective role against sepsis.

Blocking Liver Autophagy Accelerates Apoptosis and Mitochondrial Injury in Hepatocytes and Reduces Time to Mortality in a Murine Sepsis Model.

Autophagy plays an important role in cell survival, sequestering, and degrading a wide variety of substrates. Although an increase of autophagosomes in liver has been reported in sepsis patients as well as in septic mice, the influence of autophagy on liver injury, the interaction between autophagy, and other types of cell death in sepsis remain unclear. The aim of this study was to elucidate the contribution of liver autophagy to the pathophysiology of sepsis. We performed a cecal ligation and puncture on liver-specific autophagy-deficient (Alb-Cre/Atg5) mice (6-8-week-old male). When compared with controls (C57BL/6), we found a significant accumulation of p62 in the liver and demonstrated a greater number of cleaved caspase-3 immunoreactive hepatocytes in these knockout (KO) mice. Additionally, we confirmed a significant increase in autophagic vacuoles in the control mice relative to KO mice; in contrast, cell shrinkage and nuclear fragmentation (morphological characteristics of apoptosis) were preferentially seen in the KO mice by transmission electron microscopy. Severe mitochondrial damage was also prominent in KO mice, relative to controls, associated with an increase of reactive oxygen species in hepatocytes. Serum aspartate transaminase levels (P = 0.005) and serum interleukin-6 levels (P = 0.020) were significantly increased in the KO mice compared with controls. Deficiency of autophagy in liver significantly decreased survival in the murine sepsis model (P = 0.025). In conclusion, blocking liver autophagy accelerates time to mortality in the murine sepsis model, suggesting that liver autophagy plays a protective role for organ failure through degradation of damaged mitochondria, as well as prevention of apoptosis.

Transcriptional repression of p27 is essential for murine embryonic development.

The Nczf gene has been identified as one of Ncx target genes and encodes a novel KRAB zinc-finger protein, which functions as a sequence specific transcriptional repressor. In order to elucidate Nczf functions, we generated Nczf knockout (Nczf-/-) mice. Nczf-/- mice died around embryonic day 8.5 (E8.5) with small body size and impairment of axial rotation. Histopathological analysis revealed that the cell number decreased and pyknotic cells were occasionally observed. We examined the expression of cell cycle related genes in Nczf-/- mice. p27 expression was increased in E8.0 Nczf-/- mice compared to that of wild type mice. Nczf knockdown by siRNA resulted in increased expression of p27 in mouse embryonic fibroblasts (MEFs). Furthermore, p27 promoter luciferase reporter gene analysis confirmed the regulation of p27 mRNA expression by Nczf. Nczf-/-; p27-/- double knockout mice survived until E11.5 and the defect of axial rotation was restored. These data suggest that p27 repression by Nczf is essential in the developing embryo.