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The fundamental body functions that determine maximal O 2 uptake (V̇o 2max ) have not been studied in Aqp5 -/- mice (aquaporin 5, AQP5). We measured V̇o 2max to globally assess these functions and then investigated why it was found altered in Aqp5 -/- mice. V̇o 2max was measured by the Helox technique, which elicits maximal metabolic rate by intense cold exposure of the animals. We found V̇o 2max reduced in Aqp5 -/- mice by 20%-30% compared with wild-type (WT) mice. As AQP5 has been implicated to act as a membrane channel for respiratory gases, we studied whether this is caused by the known lack of AQP5 in the alveolar epithelial membranes of Aqp5 -/- mice. Lung function parameters as well as arterial O 2 saturation were normal and identical between Aqp5 -/- and WT mice, indicating that AQP5 does not contribute to pulmonary O 2 exchange. The cause for the decreased V̇o 2max thus might be found in decreased O 2 consumption of an intensely O 2 -consuming peripheral organ such as activated brown adipose tissue (BAT). We found indeed that absence of AQP5 greatly reduces the amount of interscapular BAT formed in response to 4 wk of cold exposure, from 63% in WT to 25% in Aqp5 -/- animals. We conclude that lack of AQP5 does not affect pulmonary O 2 exchange, but greatly inhibits transformation of white to brown adipose tissue. As under cold exposure, BAT is a major source of the animals' heat production, reduction of BAT likely causes the decrease in V̇o 2max under this condition.

Despite intense investigation, acute respiratory distress syndrome (ARDS) remains an enormous clinical problem for which no specific therapies currently exist. In this study, we used intratracheal lipopolysaccharide or Pseudomonas bacteria administration to model experimental acute lung injury (ALI) and to further understand mediators of the resolution phase of ARDS. Recent work demonstrates macrophages transition from a predominant proinflammatory M1 phenotype during acute inflammation to an anti-inflammatory M2 phenotype with ALI resolution. We tested the hypothesis that IL-4, a potent inducer of M2-specific protein expression, would accelerate ALI resolution and lung repair through reprogramming of endogenous inflammatory macrophages. In fact, IL-4 treatment was found to offer dramatic benefits following delayed administration to mice subjected to experimental ALI, including increased survival, accelerated resolution of lung injury, and improved lung function. Expression of the M2 proteins Arg1, FIZZ1, and Ym1 was increased in lung tissues following IL-4 treatment, and among macrophages, FIZZ1 was most prominently upregulated in the interstitial subpopulation. A similar trend was observed for the expression of macrophage mannose receptor (MMR) and Dectin-1 on the surface of alveolar macrophages following IL-4 administration. Macrophage depletion or STAT6 deficiency abrogated the therapeutic effect of IL-4. Collectively, these data demonstrate that IL-4-mediated therapeutic macrophage reprogramming can accelerate resolution and lung repair despite delayed use following experimental ALI. IL-4 or other therapies that target late-phase, proresolution pathways may hold promise for the treatment of human ARDS., (Copyright © 2016 the American Physiological Society.)

Acute respiratory distress syndrome (ARDS) is a devastating disease with distinct pathological stages. Fundamental to ARDS is the acute onset of lung inflammation as a part of the body's immune response to a variety of local and systemic stimuli. In patients surviving the inflammatory and subsequent fibroproliferative stages, transition from injury to resolution and recovery is an active process dependent on a series of highly coordinated events regulated by the immune system. Experimental animal models of acute lung injury (ALI) reproduce key components of the injury and resolution phases of human ARDS and provide a methodology to explore mechanisms and potential new therapies. Macrophages are essential to innate immunity and host defense, playing a featured role in the lung and alveolar space. Key aspects of their biological response, including differentiation, phenotype, function, and cellular interactions, are determined in large part by the presence, severity, and chronicity of local inflammation. Studies support the importance of macrophages to initiate and maintain the inflammatory response, as well as a determinant of resolution of lung inflammation and repair. We will discuss distinct roles for lung macrophages during early inflammatory and late resolution phases of ARDS using experimental animal models. In addition, each section will highlight human studies that relate to the diverse role of macrophages in initiation and resolution of ALI and ARDS.

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.

In animal models of acute lung injury (ALI), gene expression studies have focused on the acute phase of illness, with little emphasis on resolution. In this study, the acute phase of intratracheal lipopolysaccharide (IT LPS)-induced lung injury was similar in wild-type (WT) and recombinase-activating gene-1-deficient (Rag-1(-/-)) lymphocyte-deficient mice, but resolution was impaired and resolution-phase lung gene expression remained different from baseline only in Rag-1(-/-) mice. By focusing on groups of genes involved in similar biological processes (gene ontologies) pertinent to inflammation and the immune response, we identified 102 genes at days 4 and 10 after IT LPS with significantly different expression between WT and Rag-1(-/-) mice. After adoptive transfer of isolated CD4+CD25+Foxp3+ regulatory T cells (Tregs) to Rag-1(-/-) mice at the time of IT LPS, resolution was similar to that in WT mice. Of the 102 genes distinctly changed in either WT or Rag-1(-/-) mice from our 7 gene ontologies, 19 genes reverted from the Rag-1(-/-) to the WT pattern of expression after adoptive transfer of Tregs, implicating those 19 genes in Treg-mediated resolution of ALI.

Despite the associated morbidity and mortality, underlying mechanisms leading to the development of acute lung injury (ALI) remain incompletely understood. Frequently, ALI develops in the hospital, coinciding with institution of various therapies, including the use of supplemental oxygen. Although pathological evidence of hyperoxia-induced ALI in humans has yet to be proven, animal studies involving high oxygen concentration reproducibly induce ALI. The potentially injurious role of lower and presumably safer oxygen concentrations has not been well characterized in any species. We hypothesized that in the setting of a preexisting insult to the lung, the addition of moderate-range oxygen can augment lung injury. Our model of low-dose intratracheal LPS (IT LPS) followed by 60% oxygen caused a significant increase in ALI compared with LPS or oxygen alone with increased alveolar neutrophils, histological injury, and epithelial barrier permeability. In the LPS plus oxygen group, regulatory T cell number was reduced, and macrophage activation markers were increased, compared with LPS alone. Antibody-mediated depletion of neutrophils significantly abrogated the observed lung injury for all measured factors. The enhanced presence of alveolar neutrophils in the setting of LPS and oxygen is due, at least in part, to elevated chemokine gradients signaling neutrophils to the alveolar space. We believe these results strongly support an effect of lower concentrations of oxygen to augment the severity of a mild preexisting lung injury and warrants further investigation in both animals and humans.

We hypothesized that the influence of acute kidney injury (AKI) on the sensitivity of the lung to an injurious process varies with the severity of the injurious process. Thus, we thought that AKI would exacerbate lung injury from low degrees of lung trauma but attenuate lung injury from higher degrees of lung trauma. C57BL/6 mice underwent AKI (30-min kidney ischemia) or sham surgery, followed at 24 h by 4 h of spontaneous breathing (SB), mechanical ventilation with low tidal volume (7 ml/kg, LTV), or mechanical ventilation with high tidal volume (30 ml/kg, HTV). Compared with LTV, median bronchoalveolar lavage (BAL) protein leak was significantly lower with SB and greater with HTV in both sham and AKI mice. Compared with LTV, median Evans blue dye-labeled albumin extravasation in lungs (L-EBD) was also significantly lower with SB and greater with HTV. L-EBD showed a significant interaction between ventilatory mode and kidney health, such that AKI attenuated the L-EBD rise seen in HTV vs. LTV sham mice. An interaction between ventilatory mode and kidney health could also be seen in BAL neutrophil number (PMN). Thus, AKI attenuated the BAL PMN rise seen in HTV vs. LTV sham mice. These data support the presence of a complex interaction between mechanical ventilation and AKI in which the sensitivity of the lung to trauma varies with the magnitude of the trauma and may involve a modification of pulmonary neutrophil activity by AKI.

Renal ischemia-reperfusion injury (IRI) is the leading cause of acute kidney injury [AKI; acute renal failure (ARF)] in native kidneys and delayed graft function in deceased donor kidney transplants. Serum creatinine rises late after renal IRI, which results in delayed diagnosis. There is an important need to identify novel biomarkers for early diagnosis and prognosis in renal IRI. Given the inflammatory pathophysiology of renal IRI, we used a protein array to measure 18 cytokines and chemokines in a mouse model of renal IRI at 3, 24, and 72 h postischemia. A rise in renal keratinocyte-derived chemokine (KC) was the earliest and most consistent compared with other molecules, with 3-h postischemia values being 9- and 13-fold greater than sham and normal animals, respectively. Histological changes were evident within 1 h of IRI but serum creatinine only increased 24 h after IRI. With the use of an ELISA, KC levels in serum and urine were highest 3 h postischemia, well before a significant rise in serum creatinine. The human analog of KC, Gro-alpha, was markedly elevated in urine from humans who received deceased donor kidney transplants that required dialysis, compared with deceased donor kidney recipients with good graft function and live donor recipients with minimal ischemia. Measurement of KC and its human analog, Gro-alpha, could serve as a useful new biomarker for ischemic ARF.

In vitro studies of cultured salivary gland cells and gland slices have indicated that there may be regulated translocation of aquaporin (AQP)-5 between the apical plasma membrane and intracellular compartments of the secretory cells. However, it remains unknown whether AQP-5 in salivary glands is subject to regulated trafficking in vivo. To examine this possibility, we have investigated the subcellular localization of AQP-5 in rat parotid and submandibular glands fixed in vivo under conditions of stimulated or inhibited salivary secretion. Immunofluorescence and immunoelectron microscopy was used to determine the subcellular distribution of AQP-5 in control conditions following the stimulation of secretion with pilocarpine (a muscarinic agonist) or epinephrine (an alpha-adrenoceptor agonist) or during inhibition of basal secretion with atropine (a muscarinic antagonist) or phentolamine (an alpha-adrenoceptor antagonist). Under control conditions, >90% of AQP-5 was associated with the apical plasma membrane of acinar and intercalated duct cells, with only rare gold particles associated with intracellular membrane domains. Pilocarpine treatment dramatically increased saliva production but had no discernible effect on AQP-5 distribution. However, the increased salivary secretion was associated with luminal dilation and the appearance of a markedly punctate AQP-5 labeling pattern due to clustering of AQP-5 at the microvilli (especially evident in the parotid gland) after 10 min of drug injection. No changes in the subcellular localization of AQP-5 were seen in response to epinephrine, atropine, or phentolamine treatment compared with control tissues. Thus AQP-5 is localized predominantly in the apical plasma membrane under control conditions, and neither the onset nor the cessation of secretion is associated in vivo with any significant short-term translocation of AQP-5 between intracellular structures and the apical plasma membrane.

The aquaporin-1 (AQP1) water channel protein is expressed in multiple mammalian tissues by several different developmental programs; however, the genetic regulation is undefined. The proximal promoter of mouse Aqp1 contains multiple putative cis-acting regulatory elements, and mouse erythroleukemia (MEL) cells are a well-characterized model for erythroid differentiation. Corticosteroid or dimethyl sulfoxide (DMSO) exposure induces AQP1 protein expression in MEL cells, and transcriptional regulation was investigated by transient transfections with Aqp1 promoter-reporter constructs. Dexamethasone induction is abrogated by deletion of two glucocorticoid response elements -0.5 kilobases (kb) from the transcription initiation site. Mutation of the GATA element at -0.62 kb has no effect, whereas mutation of the CACCC site at -37 bp significantly reduces DMSO-induced promoter activity. Hydroxyurea induces expression of AQP1 protein without acting through the proximal promoter. The MEL cell line is a reproducible erythroid model system for studying transcriptional regulation of the Aqp1 gene while determining the consequences on AQP1 protein biosynthesis.

Developmental expression of aquaporin water transport proteins is not well understood in respiratory tract or secretory glands; here we define aquaporin protein ontogeny in rat. Expression of aquaporin-3 (AQP3), AQP4, and AQP5 proteins occurs within 2 wk after birth, whereas AQP1 first appears before birth. In most tissues, aquaporin protein expression increases progressively, although transient high-level expression is noted in distal lung (AQP4 at postnatal day +2) and trachea (AQP5 at postnatal day +21 and AQP3 at postnatal day +42). In mature animals, AQP5 is abundant in distal lung and salivary glands, AQP3 and AQP4 are present in trachea, and AQP1 is present in all of these tissues except salivary glands. Surprisingly, all four aquaporin proteins are highly abundant in nasopharynx. Unlike AQP1, corticosteroids did not induce expression of AQP3, AQP4, or AQP5 in lung. Our results seemingly implicate aquaporins in proximal airway humidification, glandular secretion, and perinatal clearance of fluid from distal airways. However, the studies underscore a need for detailed immunohistochemical characterizations and definitive functional studies.

The molecular pathways for fluid transport in pulmonary, oral, and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of four aquaporins in the respiratory tract and glandular epithelia, where they reside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is present in apical and basolateral membranes of bronchial, tracheal, and nasopharyngeal vascular endothelium and fibroblasts. AQP5 is localized to the apical plasma membrane of type I pneumocytes and the apical plasma membranes of secretory epithelium in upper airway and salivary glands. In contrast, AQP3 is present in basal cells of tracheal and nasopharyngeal epithelium and is abundant in basolateral membranes of surface epithelial cells of nasal conchus. AQP4 resides in basolateral membranes of columnar cells of bronchial, tracheal, and nasopharyngeal epithelium; in nasal conchus AQP4 is restricted to basolateral membranes of a subset of intra- and subepithelial glands. These sites of expression suggest that transalveolar water movement, modulation of airway surface liquid, air humidification, and generation of nasopharyngeal secretions involve a coordinated network of aquaporin water channels.

The causes of respiratory distress in O2 toxicity are not well understood. The purpose of this study was to better define the airway abnormalities caused by breathing 100% O2. Sheep were instrumented for measurements of dynamic compliance (Cdyn), functional residual capacity by body plethysmography (FRC), hemodynamics, and lung lymph flow. Each day Cdyn and FRC were measured before, during, and after the application of 45 min continuous positive airway pressure (CPAP) at 15 cmH2O. The amount of aerosol histamine necessary to reduce Cdyn 35% from baseline (ED35) was measured each day as was the response to aerosol metaproterenol. Cdyn decreased progressively from 0.083 +/- 0.005 (SE) 1/cmH2O at baseline to 0.032 +/- 0.004 l/cm H2O at 96 h of O2. Surprisingly, FRC did not decrease (1,397 +/- 153 ml at baseline vs. 1,523 +/- 139 ml at 96 h). The ED35 to histamine did not vary among days or from air controls. Metaproterenol produced a variable inconsistent increase in Cdyn. We also measured changes in Cdyn during changes in respiratory rate and static pressure-volume relationships in five other sheep. We found a small but significant frequency dependence of compliance and an increase in lung stiffness with O2 toxicity. We conclude that in adult sheep O2 toxicity reduces Cdyn but does not increase airway reactivity. The large reduction in Cdyn in O2 toxicity results from processes other than increased airway reactivity or reduced lung volume, and Cdyn decreases before the development of lung edema.