Transgenic up-regulation of Claudin-6 decreases fine diesel particulate matter (DPM)-induced pulmonary inflammation.

Claudin-6 (Cldn6) is a tetraspanin transmembrane protein that contributes to tight junctional complexes and has been implicated in the maintenance of lung epithelial barriers. In the present study, we tested the hypothesis that genetic up-regulation of Cldn-6 influences inflammation in mice exposed to short-term environmental diesel particulate matter (DPM). Mice were subjected to ten exposures of nebulized DPM (PM2.5) over a period of 20 days via a nose-only inhalation system (Scireq, Montreal, Canada). Using real-time RT-PCR, we discovered that the Cldn6 gene was up-regulated in control mice exposed to DPM and in lung-specific transgenic mice that up-regulate Cldn-6 (Cldn-6 TG). Interestingly, DPM did not further enhance Cldn-6 expression in Cldn-6 TG mice. DPM caused increased cell diapedesis into bronchoalveolar lavage fluid (BALF) from control mice; however, Cldn-6 TG mice had less total cells and PMNs in BALF following DPM exposure. Because Cldn-6 TG mice had diminished cell diapedesis, other inflammatory intermediates were screened to characterize the impact of increased Cldn-6 on inflammatory signaling. Cytokines that mediate inflammatory responses including TNF-α and IL-1ß were differentially regulated in Cldn6 TG mice and controls following DPM exposure. These results demonstrate that epithelial barriers organized by Cldn-6 mediate, at least in part, diesel-induced inflammation. Further work may show that Cldn-6 is a key target in understanding pulmonary epithelial gateways exacerbated by environmental pollution.

Up-Regulation of Claudin-6 in the Distal Lung Impacts Secondhand Smoke-Induced Inflammation.

It has long been understood that increased epithelial permeability contributes to inflammation observed in many respiratory diseases. Recently, evidence has revealed that environmental exposure to noxious material such as cigarette smoke reduces tight junction barrier integrity, thus enhancing inflammatory conditions. Claudin-6 (Cldn6) is a tetraspanin transmembrane protein found within the tight junctional complex and is implicated in maintaining lung epithelial barriers. To test the hypothesis that increased Cldn6 ameliorates inflammation at the respiratory barrier, we utilized the Tet-On inducible transgenic system to conditionally over-express Clnd6 in the distal lung. Cldn6 transgenic (TG) and control mice were continuously provided doxycycline from postnatal day (PN) 30 until euthanasia date at PN90. A subset of Cldn6 TG and control mice were also subjected to daily secondhand tobacco smoke (SHS) via a nose only inhalation system from PN30-90 and compared to room air (RA) controls. Animals were euthanized on PN90 and lungs were harvested for histological and molecular characterization. Bronchoalveolar lavage fluid (BALF) was procured for the assessment of inflammatory cells and molecules. Quantitative RT-PCR and immunoblotting revealed increased Cldn6 expression in TG vs. control animals and SHS decreased Cldn6 expression regardless of genetic up-regulation. Histological evaluations revealed no adverse pulmonary remodeling via Hematoxylin and Eosin (H&E) staining or any qualitative alterations in the abundance of type II pneumocytes or proximal non-ciliated epithelial cells via staining for cell specific propeptide of Surfactant Protein-C (proSP-C) or Club Cell Secretory Protein (CCSP), respectively. Immunoblotting and qRT-PCR confirmed the differential expression of Cldn6 and the pro-inflammatory cytokines TNF-α and IL-1ß. As a general theme, inflammation induced by SHS exposure was influenced by the availability of Cldn6. These data reveal captivating information suggesting a role for Cldn6 in lungs exposed to tobacco smoke. Further research is critically necessary in order to fully explain roles for tight junctional components such as Cldn6 and other related molecules in lungs coping with exposure.

The impact of decellularization agents on renal tissue extracellular matrix.

The combination of patient-specific cells with scaffolds obtained from natural sources may result in improved regeneration of human tissues. Decellularization of the native tissue is the first step in this technology. Effective decellularization uses agents that lyse cells and remove all cellular materials, leaving intact collagenous extracellular matrices (ECMs). Removing cellular remnants prevents an immune response while preserving the underlying structure. In this study, the impact of five decellularization agents (0.1 N NaOH, 1% peracetic acid, 3% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 0.05% trypsin/EDTA) on renal tissue was examined using slices of porcine kidneys. The NaOH solution induced the most efficient cell removal, and resulted in the highest amount of cell viability and proliferation after recellularization, although it also produced the most significant damage to collagenous fiber networks, glycosaminoglycans (GAGs) and fibroblast growth factor (FGF). The SDS solution led to less severe damage to the ECM structure but it resulted in lower metabolic activity and less proliferation. Peracetic acid and Triton X-100 resulted in minimum disruption of ECMs and the most preserved GAGs and FGF. However, these last two agents were not as efficient in removing cellular materials as NaOH and SDS, especially peracetic acid, which left more than 80% of cellular material within the ECM. As a proof of principle, after completing the comparison studies using slices of renal ECM, the NaOH process was used to decellularize a whole kidney, with good results. The overall results demonstrate the significant effect of cell lysing agents and the importance of developing an optimized protocol to avoid extensive damage to the ECM while retaining the ability to support cell growth.

Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix.

Conditional pulmonary over-expression of Claudin 6 during embryogenesis delays lung morphogenesis.

Claudin 6 (Cldn6) is a tetraspanin protein expressed by barrier epithelial cells. In order to assess the effects of persistent tight junctions involving Cldn6 during lung development, a doxycycline (dox)-inducible conditional transgenic mouse was generated that up-regulates Cldn6 in the distal lung. Pups had unlimited access to dox from conception until sacrifice date at embryonic day (E) 18.5. Quantitative PCR, immunoblotting, and immunohistochemistry revealed significantly elevated Cldn6 expression in transgenic mice compared to non-transgenic controls. There were no differences in terms of lung size, lung weight, or whole body weight at the time of necropsy. Histological evaluations led to the discovery that E18.5 Cldn6 transgenic pups appeared to be in the early canalicular stage of development coincident with fewer, thickened respiratory airspaces. In contrast, controls appeared to have entered the saccular stage characterized by thin airspace walls and spherical architecture. Immunostaining for transcriptional regulators including TTF-1 and FoxA2 was conducted to assess cell differentiation and specific cell types were identified via staining for pro-surfactant protein C (alveolar type II epithelial cells) or Clara Cell Secretory Protein (cub or Clara cells). Lastly, cell turnover was qualitatively measured via staining for cell proliferation or apoptosis. These data suggest that Cldn6 is an important junctional protein potentially involved in the programming of epithelial cells during lung development. Furthermore, genetic down-regulation of Cldn6 as development proceeds may influence differentiation observed in the transition from the canalicular to the saccular lung.