Precise Fabrication and Manipulation of Individual Polymer Nanofibers.

Polymer nanofibers hold promise in a wide range of applications owing to their diverse properties, flexibility, and cost effectiveness. In this study, we introduce a polymer nanofiber drawing process in a scanning electron microscope and focused ion beam (SEM/FIB) instrument with in situ observation. We employed a nanometer-sharp tungsten needle and prepolymer microcapsules to enable nanofiber drawing in a vacuum environment. This method produces individual polymer nanofibers with diameters as small as ∼500 nm and lengths extending to millimeters, yielding nanofibers with an aspect ratio of 2000:1. The attachment to the tungsten manipulator ensures accurate transfer of the polymer nanofiber to diverse substrate types as well as fabrication of assembled structures. Our findings provide valuable insights into ultrafine polymer fiber drawing, paving the way for high-precision manipulation and assembly of polymer nanofibers.

Epac induces ryanodine receptor-dependent intracellular and inter-organellar calcium mobilization in mpkCCD cells.

Arginine vasopressin (AVP) induces an increase in intracellular Ca2+ concentration ([Ca2+]i) with an oscillatory pattern in isolated perfused kidney inner medullary collecting duct (IMCD). The AVP-induced Ca2+ mobilization in inner medullary collecting ducts is essential for apical exocytosis and is mediated by the exchange protein directly activated by cyclic adenosine monophosphate (Epac). Murine principal kidney cortical collecting duct cells (mpkCCD) is the cell model used for transcriptomic and phosphoproteomic studies of AVP signaling in kidney collecting duct. The present study examined the characteristics of Ca2+ mobilization in mpkCCD cells, and utilized mpkCCD as a model to investigate the Epac-induced intracellular and intra-organellar Ca2+ mobilization. Ca2+ mobilization in cytosol, endoplasmic reticulum lumen, and mitochondrial matrix were monitored with a Ca2+ sensitive fluorescent probe and site-specific Ca2+ sensitive biosensors. Fluorescence images of mpkCCD cells and isolated perfused inner medullary duct were collected with confocal microscopy. Cell permeant ligands of ryanodine receptors (RyRs) and inositol 1,4,5 trisphosphate receptors (IP3Rs) both triggered increase of [Ca2+]i and Ca2+ oscillations in mpkCCD cells as reported previously in IMCD. The cell permeant Epac-specific cAMP analog Me-cAMP/AM also caused a robust Ca2+ mobilization and oscillations in mpkCCD cells. Using biosensors to monitor endoplasmic reticulum (ER) luminal Ca2+ and mitochondrial matrix Ca2+, Me-cAMP/AM not only triggered Ca2+ release from ER into cytoplasm, but also shuttled Ca2+ from ER into mitochondria. The Epac-agonist induced synchronized Ca2+ spikes in cytosol and mitochondrial matrix, with concomitant declines in ER luminal Ca2+. Me-cAMP/AM also effectively triggered store-operated Ca2+ entry (SOCE), suggesting that Epac-agonist is capable of depleting ER Ca2+ stores. These Epac-induced intracellular and inter-organelle Ca2+ signals were mimicked by the RyR agonist 4-CMC, but they were distinctly different from IP3R activation. The present study hence demonstrated that mpkCCD cells retain all reported features of Ca2+ mobilization observed in isolated perfused IMCD. It further revealed information on the dynamics of Epac-induced RyR-dependent Ca2+ signaling and ER-mitochondrial Ca2+ transfer. ER-mitochondrial Ca2+ coupling may play a key role in the regulation of ATP and reactive oxygen species (ROS) production in the mitochondria along the nephron. Our data suggest that mpkCCD cells can serve as a renal cell model to address novel questions of how mitochondrial Ca2+ regulates cytosolic Ca2+ signals, inter-organellar Ca2+ signaling, and renal tubular functions.

In Situ Spectroscopic Studies of NH3 Oxidation of Fe-Oxide/Al2O3.

Simple temperature-regulated chemical vapor deposition was used to disperse iron oxide nanoparticles on porous Al2O3 to create an Fe-oxide/Al2O3 structure for catalytic NH3 oxidation. The Fe-oxide/Al2O3 achieved nearly 100% removal of NH3, with N2 as a major reaction product at temperatures above 400 °C and negligible NOx emissions at all experimental temperatures. The results of a combination of in situ diffuse reflectance infrared Fourier-transform spectroscopy and near-ambient pressure-near-edge X-ray absorption fine structure spectroscopy suggest a N2H4-mediated oxidation mechanism of NH3 to N2 via the Mars-van Krevelen pathway on the Fe-oxide/Al2O3 surface. As a catalytic adsorbent-an energy-efficient approach to reducing NH3 levels in living environments via adsorption and thermal treatment of NH3-no harmful NOx emissions were produced during the thermal treatment of the NH3-adsorbed Fe-oxide/Al2O3 surface, while NH3 molecularly desorbed from the surface. A system with dual catalytic filters of Fe-oxide/Al2O3 was designed to fully oxidize this desorbed NH3 to N2 in a clean and energy-efficient manner.

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

Surface Structures of Fe-TiO2 Photocatalysts for NO Oxidation.

Commercial rutile TiO2 particles capped with Al2O3 and ZrO2 layers, which are widely used in white pigments, can serve as a starting material for the fabrication of visible light-responsive photocatalysts toward gas-phase NO oxidation. The as-received TiO2 with iron impurities exhibited reduced photocatalytic activity, and the activity was boosted by the deposition of additional iron comparable in quantity to the intrinsic iron impurity level. Analyses using X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy, and low-energy ion scattering spectroscopy revealed that the deposited iron and intrinsic impurity iron are dissimilar in terms of location, oxidation states, and interaction with TiO2. This suggests that tracking the structure and impurity levels of photocatalyst elements can be crucial for understanding structure-activity relationships of real catalysts.

All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission.

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

Site-specific opening of the blood-brain barrier by extracellular histones.

BACKGROUND: Increased extracellular histones in the bloodstream are known as a biomarker for vascular dysfunction associated with severe trauma or sepsis. There is limited information regarding the pathogenic role of circulating histones in neuroinflammation and cerebrovascular endothelial injury. Particularly, it remains unclear whether histones affect the blood-brain barrier (BBB) permeability function. METHODS: The direct effects of unfractionated histones on endothelial barrier properties were first assessed in brain microvascular endothelial cell monolayers by measuring transendothelial electrical resistance and solute flux. This was followed by in vivo mouse experiments, where BBB function was assessed by quantifying brain tissue accumulation of intravenously injected tracers of different molecular sizes, and comparison was made in mice receiving a sublethal dose of histones versus sterile saline. In parallel, the endothelial barrier ultrastructure was examined in histone- and saline-injected animals under transmission electron microscopy, corresponding to the expression of tight junction and adherens junction proteins. RESULTS: Histones increased paracellular permeability to sodium fluorescein and reduced barrier resistance at 100 µg/mL; these responses were accompanied by discontinuous staining of the tight junction proteins claudin-5 and zona ocludens-1. Interestingly, the effects of histones did not seem to result from cytotoxicity, as evidenced by negative propidium iodide staining. In vivo, histones increased the paracellular permeability of the BBB to small tracers of < 1-kDa, whereas tracers larger than 3-kDa remained impermeable across brain microvessels. Further analysis of different brain regions showed that histone-induced tracer leakage and loss of tight junction protein expression mainly occurred in the hippocampus, but not in the cerebral cortex. Consistently, opening of tight junctions was found in hippocampal capillaries from histone-injected animals. Protein expression levels of GFAP and iBA1 remained unchanged in histone-injected mice indicating that histones did not affect reactive gliosis. Moreover, cell membrane surface charge alterations are involved in histone-induced barrier dysfunction and tight junction disruption. CONCLUSIONS: Extracellular histones cause a reversible, region-specific increase in BBB permeability to small molecules by disrupting tight junctions in the hippocampus. We suggest that circulating histones may contribute to cerebrovascular injury or brain dysfunction by altering BBB structure and function.

Defining Endothelial Cell-Derived Factors That Promote Pericyte Recruitment and Capillary Network Assembly.

OBJECTIVE: We sought to identify and investigate the functional role of the major endothelial cell (EC)-derived factors that control pericyte recruitment to EC tubes and pericyte-induced tube maturation during capillary network formation. Approach and Results: We identify PDGF (platelet-derived growth factor)-BB, PDGF-DD, ET (endothelin)-1, TGF (transforming growth factor)-ß, and HB-EGF (heparin-binding epidermal growth factor), as the key individual and combined regulators of pericyte assembly around EC tubes. Using novel pericyte only assays, we demonstrate that PDGF-BB, PDGF-DD, and ET-1 are the primary direct drivers of pericyte invasion. Their addition to pericytes induces invasion as if ECs were present. In contrast, TGF-ß and HB-EGF have minimal ability to directly stimulate pericyte invasion. In contrast, TGF-ß1 can act as an upstream pericyte primer to stimulate invasion in response to PDGFs and ET-1. HB-EGF stimulates pericyte proliferation along with PDGFs and ET-1. Using EC-pericyte cocultures, individual, or combined blockade of these EC-derived factors, or their pericyte receptors, using neutralizing antibodies or chemical inhibitors, respectively, interferes with pericyte recruitment and proliferation. As individual factors, PDGF-BB and ET-1 have the strongest impact on these events. However, when the blocking reagents are combined to interfere with each of the above factors or their receptors, more dramatic and profound blockade of pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition occurs. Under these conditions, ECs form tubes that become much wider and less elongated as if pericytes were absent. CONCLUSIONS: Overall, these new studies define and characterize a functional role for key EC-derived factors controlling pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition during capillary network assembly.

Endothelial-Stromal Communication in Murine and Human Corneas.

The purpose of this study is to identify and characterize interactions of corneal endothelial cells with the posterior stroma. Corneal endothelial-stromal interactions were examined in developing postnatal day 3 (P3) and mature postnatal day 30 (P30) C57BL/6 mice and adult human corneas. Flat mounts and cross-sections were studied using immunofluorescence microscopy. F-actin was labeled with phalloidin to evaluate cell processes traversing Descemet's membrane (DM). Dynamic cell-cell communication was evaluated with fluorescence recovery after photobleaching (FRAP) using calcein acetoxymethyl dye. Endothelial-stromal interactions were observed across the whole cornea transversing DM during early postnatal development (P3), while these interactions became restricted to the periphery in the mature murine cornea (P30). In adult human corneas, endothelial extensions through the DM were observed in the peripheral cornea. The pattern of FRAP in both mature mice and human central corneas demonstrated endothelial-endothelial cell communication. In contrast, in the human cornea 2, distinct patterns were observed consistent with endothelial-endothelial and stromal-endothelial communication. Endothelial-stromal interactions were observed in the entire cornea during early postnatal mouse corneas. This evidence of endothelial-posterior stromal contact contradicts the hypothesis that corneal endothelial cells are isolated from the stroma by the DM and provides direct data to support endothelial-stromal comunication that may directly influence posterior corneal structure and function. Anat Rec, 2020. © 2020 American Association for Anatomy.

Mapping of Sensory Nerve Subsets within the Vagal Ganglia and the Brainstem Using Reporter Mice for Pirt, TRPV1, 5-HT3, and Tac1 Expression.

Vagal afferent sensory nerves, originating in jugular and nodose ganglia, are composed of functionally distinct subsets whose activation evokes distinct thoracic and abdominal reflex responses. We used Cre-expressing mouse strains to identify specific vagal afferent populations and map their central projections within the brainstem. We show that Pirt is expressed in virtually all vagal afferents; whereas, 5-HT3 is expressed only in nodose neurons, with little expression in jugular neurons. Transient receptor potential vanilloid 1 (TRPV1), the capsaicin receptor, is expressed in a subset of small nodose and jugular neurons. Tac1, the gene for tachykinins, is expressed predominantly in jugular neurons, some of which also express TRPV1. Vagal fibers project centrally to the nucleus tractus solitarius (nTS), paratrigeminal complex, area postrema, and to a limited extent the dorsal motor nucleus of the vagus. nTS subnuclei preferentially receive projections by specific afferent subsets, with TRPV1+ fibers terminating in medial and dorsal regions predominantly caudal of obex, whereas TRPV1- fibers terminate in ventral and lateral regions throughout the rostral-caudal aspect of the medulla. Many vagal Tac1+ afferents (mostly derived from the jugular ganglion) terminate in the nTS. The paratrigeminal complex was the target of multiple vagal afferent subsets. Importantly, lung-specific TRPV1+ and Tac1+ afferent terminations were restricted to the caudal medial nTS, with no innervation of other medulla regions. In summary, this study identifies the specific medulla regions innervated by vagal afferent subsets. The distinct terminations provide a neuroanatomic substrate for the diverse range of reflexes initiated by vagal afferent activation.

AKT2 maintains brain endothelial claudin-5 expression and selective activation of IR/AKT2/FOXO1-signaling reverses barrier dysfunction.

Inflammation-induced blood-brain barrier (BBB) dysfunction and microvascular leakage are associated with a host of neurological disorders. The tight junction protein claudin-5 (CLDN5) is a crucial protein necessary for BBB integrity and maintenance. CLDN5 is negatively regulated by the transcriptional repressor FOXO1, whose activity increases during impaired insulin/AKT signaling. Owing to an incomplete understanding of the mechanisms that regulate CLDN5 expression in BBB maintenance and dysfunction, therapeutic interventions remain underdeveloped. Here, we show a novel isoform-specific function for AKT2 in maintenance of BBB integrity. We identified that AKT2 during homeostasis specifically regulates CLDN5-dependent barrier integrity in brain microvascular endothelial cells (BMVECs) and that intervention with a selective insulin-receptor (IR) agonist, demethylasterriquinone B1 (DMAQ-B1), rescued IL-1ß-induced AKT2 inactivation, FOXO1 nuclear accumulation, and loss of CLDN5-dependent barrier integrity. Moreover, DMAQ-B1 attenuated preclinical CLDN5-dependent BBB dysfunction in mice subjected to experimental autoimmune encephalomyelitis. Taken together, the data suggest a regulatory role for IR/AKT2/FOXO1-signaling in CLDN5 expression and BBB integrity during neuroinflammation.

Endothelial microvesicles carrying Src-rich cargo impair adherens junction integrity and cytoskeleton homeostasis.

AIMS: Microvesicles (MVs) conduct intercellular communication and impact diverse biological processes by transferring bioactive cargos to other cells. We investigated whether and how endothelial production of MVs contribute to vascular dysfunction during inflammation. METHODS AND RESULTS: We measured the levels and molecular properties of endothelial-derived MVs (EC-MVs) from mouse plasma following a septic injury elicited by cecal ligation and puncture, as well as those from supernatants of cultured endothelial cells stimulated by inflammatory agents including cytokines, thrombin, and complement 5a. The mouse studies showed that sepsis caused a significant increase in total plasma vesicles and VE-cadherin+ EC-MVs compared to sham control. In cultured ECs, different inflammatory agents caused diverse patterns of EC-MV production and cargo contents. When topically applied to endothelial cells, EC-MVs induced a cytoskeleton-junction response characterized by myosin light chain phosphorylation, contractile fibre reorganization, VE-cadherin phosphorylation, and adherens junction dissociation, functionally measured as increased albumin transendothelial flux and decreased barrier resistance. The endothelial response was coupled with protein tyrosine phosphorylation promoted by MV cargo containing c-Src kinase, whereas MVs produced from c-Src deficient cells did not exert barrier-disrupting effects. Additionally, EC-MVs contribute to endothelial inflammatory injury by promoting neutrophil-endothelium adhesion and release of neutrophil extracellular traps containing citrullinated histones and myeloperoxidase, a response unaltered by c-Src knockdown. CONCLUSION: Endothelial-derived microparticles cause endothelial barrier dysfunction by impairing adherens junctions and activating neutrophils. The signalling mechanisms underlying the endothelial cytoskeleton-junction response to EC-MVs involve protein phosphorylation promoted by MV cargo carrying c-Src. However, EC-MV-induced neutrophil activation was not dependent on c-Src.

Adsorption and Oxidative Desorption of Acetaldehyde over Mesoporous Fe x O y H z /Al2O3.

Fe x O y H z nanostructures were incorporated into commercially available and highly porous alumina using the temperature-regulated chemical vapor deposition method with ferrocene as an Fe precursor and subsequent annealing. All processes were conducted under ambient pressure conditions without using any high-vacuum equipment. The entire internal micro- and mesopores of the Al2O3 substrate with a bead diameter of ∼2 mm were evenly decorated with Fe x O y H z nanoparticles. The Fe x O y H z /Al2O3 structures showed substantially high activity for acetaldehyde oxidation. Most importantly, Fe x O y H z /Al2O3 with a high surface area (∼200 m2/g) and abundant mesopores was found to uptake a large amount of acetaldehyde at room temperature, and subsequent thermal regeneration of Fe x O y H z /Al2O3 in air resulted in the emission of CO2 with only a negligibly small amount of acetaldehyde because Fe x O y H z nanoparticles can catalyze total oxidation of adsorbed acetaldehyde during the thermal treatment. Increase in the humidity of the atmosphere decreased the amount of acetaldehyde adsorbed on the surface due to the competitive adsorption of acetaldehyde and water molecules, although the adsorptive removal of acetaldehyde and total oxidative regeneration were verified under a broad range of humidity conditions (0-70%). Combinatory use of room-temperature adsorption and catalytic oxidation of adsorbed volatile organic compounds using Fe x O y H z /Al2O3 can be of potential application in indoor and outdoor pollution treatments.

Mesoporous SiO2 Particles Combined with Fe Oxide Nanoparticles as a Regenerative Methylene Blue Adsorbent.

Mesoporous SiO2 adsorbents were combined with Fe oxide nanoparticles (∼10 nm) that can catalyze thermal oxidation of organic compounds at low temperatures. Fe oxide nanoparticle (∼10 nm)-incorporated SiO2 adsorbents were prepared via a temperature-regulated chemical vapor deposition method followed by a thermal annealing process. The removal efficiency and reusability of Fe oxide/SiO2 particles were examined and compared to those of bare SiO2. Upon deposition of Fe oxide nanoparticles, not only the equilibrium adsorption capacity of mesoporous SiO2 for methylene blue (MB) was improved but also the reusability of SiO2 adsorbent was increased significantly. The adsorption ability of fresh Fe oxide/SiO2 particles can be almost fully recovered by simple thermal annealing at atmospheric conditions (400 °C), whereas that of bare SiO2 reduced significantly under same conditions. In addition, full recovery of initial MB adsorption ability of Fe oxide/SiO2 can be achieved by a 100 °C annealing process. Fourier transform infrared, thermogravimetric analysis, and X-ray photoelectron spectroscopy analyses indicated that Fe oxide nanoparticles catalyzed thermal degradation of adsorbed MB molecules, resulting in the improved reusability of the Fe oxide/SiO2 adsorbent. In addition to reusability, the equilibrium adsorption capacity of mesoporous SiO2 particles for various cationic dye molecules, such as MB, malachite green, and rhodamine B, can be improved by combining Fe oxide nanoparticles.

TOF-SIMS Analysis Using Bi3 + as Primary Ions on Au Nanoparticles Supported by SiO2/Si: Providing Insight into Metal-Support Interactions.

Au nanoparticles with a mean diameter of 20 nm with a coverage of ∼20% of the surface were distributed on a Si wafer surface and studied both before and after being annealed (at 100 and 300 °C). The two types of samples were analyzed using secondary ion mass spectroscopy (SIMS) with Bi3 + clusters as the primary ions combined with surface etching using Ar1000 + clusters. We observed a substantial difference in the SIMS spectra combined with a relatively short sputtering time of Ar1000 +. In the nonannealed samples, bare Au cluster cations and Si+ were observed in the SIMS spectra; AuSi+ clusters were also observed in the annealed samples. These results indicate Au-silicide formation at a part of the periphery of the Au nanoparticles upon annealing. We suggest that SIMS experiments using cluster ions such as Bi3 + can not only be used for surface elemental analyses but also provide information on local chemical environments of elements on the surface. This is an important issue in heterogeneous catalysis (e.g., strong metal-support interactions). We also advise that one should be careful interpreting the SIMS data combined with a longer Ar1000 + sputtering time because this can deteriorate the surfaces from their original structures.

A murine neonatal model of necrotizing enterocolitis caused by anemia and red blood cell transfusions.

Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants. Clinical studies have linked NEC with antecedent red blood cell (RBC) transfusions, but the underlying mechanisms are unclear. Here we report a neonatal murine model to investigate this association. C57BL/6 mouse pups rendered anemic by timed phlebotomy and then given RBC transfusions develop NEC-like intestinal injury with prominent necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon within 12-24 h. The anemic intestine is infiltrated by inflammatory macrophages, which are activated in situ by RBC transfusions via a Toll-like receptor (TLR)-4-mediated mechanism and cause bowel injury. Chelation of RBC degradation products with haptoglobin, absence of TLR4, macrophage depletion, and inhibition of macrophage activation is protective. Intestinal injury worsens with increasing severity and the duration of anemia prior to transfusion, indicating a need for the re-evaluation of current transfusion guidelines for premature infants.

A mouse model of renal ischemia-reperfusion injury solely induced by cold ischemia.

Transplanted kidneys usually experience several episodes of ischemia, including cold ischemia during allograft storage in preservation solution. However, previous studies focusing on cold renal ischemia were only carried out in vitro or ex vivo. In the present study, we developed and characterized an in vivo mouse model of renal ischemia-reperfusion injury (IRI) induced exclusively by cold ischemia. C57BL/6 mice underwent right kidney nephrectomy, and the left kidney was kept cool with circulating cold saline in a kidney cup, while body temperature was maintained at 37°C. We clamped the renal pedicle and flushed out the blood inside the kidney with cold saline via an opening on the renal vein. The severity of renal IRI was examined with different ischemic durations. We found that the mice with <2 h of cold ischemia exhibited no significant changes in renal function or histopathology; animals with 3 or 4 h of cold ischemia developed into mild to moderate acute kidney injury with characteristic features, including the elevation in plasma creatinine concentration and reduction in glomerular filtration rate and tubular necrosis, followed by a subsequent recovery. However, mice with 5 h of cold ischemia died in a few days with severe acute kidney injury. In summary, we generated a mouse model of renal IRI induced exclusively by cold ischemia, which mimics graft cold storage in preservation solution, and renal function can be evaluated in vivo.

A new low-nephron CKD model with hypertension, progressive decline of renal function, and enhanced inflammation in C57BL/6 mice.

Chronic kidney disease (CKD) is a major health issue in the US. The typical five-sixths nephrectomy (typical 5/6 NX) is a widely used experimental CKD model. However, the typical 5/6 NX model is hypertensive in rats but strain dependent in mice. In particular, C57BL/6 mice with the typical 5/6 NX exhibits normal blood pressure and well-preserved renal function. The goal of the present study was to create a new hypertensive CKD model in C57BL/6 mice. We first characterized the vascular architecture originated from each renal artery branch by confocal laser-scanning microscopy with fluorescent lectin. Then, a novel 5/6 NX-BL model was generated by uninephrectomy combined with 2/3 renal infarction via a ligation of upper renal artery branch on the contralateral kidney. Compared with 5/6 NX-C, the 5/6 NX-BL model exhibited elevated mean arterial pressure (137.6 ± 13.9 vs. 104.7 ± 8.2 mmHg), decreased glomerular filtration rate (82.9 ± 19.2 vs. 125.0 ± 13.9 µl/min) with a reciprocal increase in plasma creatinine (0.31 ± 0.03 vs. 0.19 ± 0.04 mg/dl), and significant renal injury as assessed by proteinuria, histology with light, and transmission electron microscopy. In addition, inflammatory status, as indicated by the level of proinflammatory cytokine TNFα and the leukocyte counts, was significantly upregulated in 5/6 NX-BL compared with the 5/6 NX-C. In summary, we developed a new hypertensive CKD model in C57BL/6 mice with 5/6 renal mass reduction by uninephrectomy and upper renal artery branch ligation on the contralateral kidney. This 5/6 NX-BL model exhibits an infarction zone-dependent hypertension and progressive deterioration of the renal function accompanied by enhanced inflammatory response.

Modulation of mesenteric collecting lymphatic contractions by σ1-receptor activation and nitric oxide production.

Recently, it has been reported that a σ-receptor antagonist could reduce inflammation-induced edema. Lymphatic vessels play an essential role in removing excess interstitial fluid. We tested the hypothesis that activation of σ-receptors would reduce or weaken collecting lymphatic contractions. We used isolated, cannulated rat mesenteric collecting lymphatic vessels to study contractions in response to the σ-receptor agonist afobazole in the absence and presence of different σ-receptor antagonists. We used RT-PCR and Western blot analysis to investigate whether these vessels express the σ1-receptor and immunofluorescence confocal microscopy to examine localization of the σ1-receptor in the collecting lymphatic wall. Using N-nitro-l-arginine methyl ester (l-NAME) pretreatment before afobazole in isolated lymphatics, we tested the role of nitric oxide (NO) signaling. Finally, we used 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence as an indicator to test whether afobazole increases NO release in cultured lymphatic endothelial cells. Our results show that afobazole (50-150 µM) elevated end-systolic diameter and generally reduced pump efficiency and that this response could be partially blocked by the σ1-receptor antagonists BD 1047 and BD 1063 but not by the σ2-receptor antagonist SM-21. σ1-Receptor mRNA and protein were detected in lysates from isolated rat mesenteric collecting lymphatics. Confocal images with anti-σ1-receptor antibody labeling suggested localization in the lymphatic endothelium. Blockade of NO synthases with l-NAME inhibited the effects of afobazole. Finally, afobazole elicited increases in NO production from cultured lymphatic endothelial cells. Our findings suggest that the σ1-receptor limits collecting lymphatic pumping through a NO-dependent mechanism.NEW & NOTEWORTHY Relatively little is known about the mechanisms that govern contractions of lymphatic vessels. σ1-Receptor activation has been shown to reduce the fractional pump flow of isolated rat mesenteric collecting lymphatics. The σ1-receptor was localized mainly in the endothelium, and blockade of nitric oxide synthase inhibited the effects of afobazole.

Palmitoyl acyltransferase DHHC21 mediates endothelial dysfunction in systemic inflammatory response syndrome.

Endothelial dysfunction is a hallmark of systemic inflammatory response underlying multiple organ failure. Here we report a novel function of DHHC-containing palmitoyl acyltransferases (PATs) in mediating endothelial inflammation. Pharmacological inhibition of PATs attenuates barrier leakage and leucocyte adhesion induced by endothelial junction hyperpermeability and ICAM-1 expression during inflammation. Among 11 DHHCs detected in vascular endothelium, DHHC21 is required for barrier response. Mice with DHHC21 function deficiency (Zdhhc21dep/dep) exhibit marked resistance to injury, characterized by reduced plasma leakage, decreased leucocyte adhesion and ameliorated lung pathology, culminating in improved survival. Endothelial cells from Zdhhc21dep/dep display blunted barrier dysfunction and leucocyte adhesion, whereas leucocytes from these mice did not show altered adhesiveness. Furthermore, inflammation enhances PLCß1 palmitoylation and signalling activity, effects significantly reduced in Zdhhc21dep/dep and rescued by DHHC21 overexpression. Likewise, overexpression of wild-type, not mutant, PLCß1 augments barrier dysfunction. Altogether, these data suggest the involvement of DHHC21-mediated PLCß1 palmitoylation in endothelial inflammation.