No abstract

Claudin-7, a member of the claudin family, is highly expressed in distal nephrons of kidneys and has been reported to be involved in the regulation of paracellular Cl(-) permeability in cell cultures. To investigate the role of claudin-7 in vivo, we generated claudin-7 knockout mice (Cln7(-/-)) by the gene-targeting deletion method. Here we report that Cln7(-/-) mice were born viable, but died within 12 days after birth. Cln7(-/-) mice showed severe salt wasting, chronic dehydration, and growth retardation. We found that urine Na(+), Cl(-), and K(+) were significantly increased in Cln7(-/-) mice compared with that of Cln7(+/+) mice. Blood urea nitrogen and hematocrit were also significantly higher in Cln7(-/-) mice. The wrinkled skin was evident when Cln7(-/-) mice were approximately 1 wk old, indicating that they suffered from chronic fluid loss. Transepidermal water loss measurements showed no difference between Cln7(+/+) and Cln7(-/-) skin, suggesting that there was no transepidermal water barrier defect in Cln7(-/-) mice. Claudin-7 deletion resulted in the dramatic increase of aldosterone synthase mRNA level as early as 2 days after birth. The significant increases of epithelial Na(+) channel alpha, Na(+)-Cl(-) cotransporter, and aquaporin 2 mRNA levels revealed a compensatory response to the loss of electrolytes and fluid in Cln7(-/-) mice. Na(+)-K(+)-ATPase alpha(1) expression level was also greatly increased in distal convoluted tubules and collecting ducts where claudin-7 is normally expressed. Our study demonstrates that claudin-7 is essential for NaCl homeostasis in distal nephrons, and the paracellular ion transport pathway plays indispensable roles in keeping ionic balance in kidneys.

Animal models exhibiting high homology with humans at the genetic and pathophysiological levels will facilitate identification and validation of gene targets underlying asthma. In the present study, a nonhuman primate model of allergic asthma was developed by sensitizing cynomolgus monkeys to dust mite antigen. Sensitization elevated allergen-specific serum IgE and IgG levels, and peripheral blood mononuclear cells isolated from sensitized animals released IL-4, IL-5, and IL-10, but not IFN-gamma. Aerosolized allergen decreased dynamic compliance and induced airway inflammation and hyperresponsiveness to aerosolized histamine. Albuterol and dexamethasone inhibited the airway constriction and allergen-induced inflammation, respectively. Airway wall remodeling that included goblet cell hyperplasia, basement membrane thickening, and smooth muscle hypertrophy was particularly evident in neonatally sensitized animals. In contrast to animals sensitized as adults, neonatally sensitized animals exhibited increased sensitivity to adenosine and larger allergen-induced changes in airway resistance and dynamic compliance. These results demonstrate that sensitization of cynomolgus monkeys with dust mite induces asthmalike symptoms, some of which may be dependent on age at the time of sensitization.

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry-driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell-based phenotypic screening revealed that noncytotoxic concentrations of (Z)-(+/-)-2-(1-benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2. 2.2]octan-3-ol (analog I) and (Z)-(+/-)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (analog II) inhibited endothelial cell migration and the ability to form capillary-like structures in Matrigel by > or =70%. The ability to undergo neoangiogenesis, as measured in a window-chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC(50) = 10 microM). Retroviral-mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small-molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation-mediated Caspase3-activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X-irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.