Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.

Mammals are well adapted to respond to changes in ambient oxygen concentration (O2) by activating homeostatic physiological and cellular responses which maintain cell function and survival. Although anemia has been associated with increased mortality in a number of clinical settings, surprisingly little is known about how anemia affects tissue PO2 and hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular response to acute hypoxia, we define the effects of NOS deficiency in acute anemia. Unlike wildtype (WT), endothelial NOS (eNOS) and inducible NOS (iNOS) deficient mice, only neuronal NOS (nNOS) deficient mice (nNOS-/-) demonstrated increased mortality during acute anemia. With respect to global tissue O2 delivery, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS -/- mice. At the cellular level, anemia increased expression of HIF-1α and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK) in the brain of WT, but not nNOS-/- mice. These date suggest that nNOS contributed to cardiovascular and cellular mechanisms which maintain oxygen homeostasis in anemia. To confirm the physiological relevance of these findings in a whole animal model of anemia, we utilized transgenic animals which express a reporter HIF-α(ODD)-luciferase chimeric protein. Using this model, we confirmed that nNOS is essential for anemia-induced increases in HIF-α protein stability in vivo in real-time whole animal images and brain tissue. With respect to the mechanism, nNOS-derived NO is known to affect S-nitrosylation of specific proteins, which may interfere with HIF-α and von Hippal Lindau protein (pVHL) interaction. Utilizing the biotin switch assay, we demonstrated that anemia caused a time-dependent increase in S-nitrosylation of pVHL in brain tissue from WT but not nNOS-/- mice. In addition, anemia also leads to a decrease in S-nitrosoglutathione (GSNO) reductase protein expression, an important enzyme responsible for de-nitrosylation of proteins. The combination of increased nNOS expression and decreased GSNO reductase expression would favor prolonged S-nitrosylation of proteins during anemia. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo. By contrast, after exposure to acute hypoxia, nNOS-/- mice survived longer, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. This comparative assessment provided essential mechanistic insight into the unexpected and striking difference between anemia and hypoxia. Understanding the adaptive responses to acute anemia will help to define novel therapeutic strategies for anemic patients.

This study investigated two hypotheses. First, that chronic cocaine treatment would mimic the changes in breathing that are associated with ventilatory acclimatisation to chronic hypoxia (VAH). Second, that pre-treatment with a hypoxic stressor would bring about cross-sensitisation to cocaine. To address the first hypothesis, rats were exposed to either chronically hypoxic or chronically normoxic conditions and treated with either cocaine or saline for a 14 day period. Following this period, acute breathing trials were performed to measure resting ventilation and ventilatory chemoreflexes. The results demonstrated that chronic cocaine treatment did not induce the changes in breathing associated with VAH. To address the second hypothesis rats were exposed to a hypoxic stressor for 10 days (either intermittent hypoxia or chronic hypoxia) after which cocaine sensitisation was measured via locomotor sensitisation trials. The results demonstrated that cross-sensitisation between a hypoxic stress and cocaine was observed for intermittent but not chronic hypoxia.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

This thesis sought to improve our understanding of how kidneys become injured as a consequence of organ donation, with the aim of improving the outcomes of transplantation. Every year, hundreds of patients on the waiting list die whilst awaiting a kidney transplant. With an ever-increasing demand for suitable organs, supply cannot keep up with the pressures on the transplant waiting list. As a consequence the transplant community are forced to use organs that previously would not have been considered suitable for transplant, including from older donors with additional comorbidities. This thesis aimed to develop an understanding as to how the kidney becomes injured during the donation process, identifying which key cellular homeostatic processes are disturbed as a consequence of donation. The thesis outlines the experimental development of rodent models of organ donation replicating the donation process for donation after brain death (DBD) and donation after circulatory death (DCD) donors and also the development of a kidney ischaemia reperfusion injury (IRI) model. Proteomics was subsequently used to identifying global protein alterations in the kidney as a consequence of brain death and ischemia reperfusion injury using bioinformatics tools to identify involvement of cellular pathways. The results indicated alterations in mitochondrial function and metabolic homeostasis occurring following brain death. Alterations in cellular metabolism and mitochondrial function were then confirmed using metabolomics and mitochondrial functional assays. I subsequently evaluated how alterations in cellular hypoxia and the hypoxia inducible factor system is altered in the brain dead organ donor kidney and aimed to target this system as a means of conditioning the brain dead organ donor to prevent mitochondrial and metabolic mediated injury to kidney cells following brain death. This involved exploring the role of prolyl hydroxylase inhibitors, including dimethyloxalylglycine, on mitochondrial function and whether this could be a therapeutic target in organ donation. This thesis provides important insights into the mechanism of injury of kidneys following brain death, providing evidence that even before procurement and preservation in the DBD donor alterations in mitochondrial function and metabolic homeostasis occur. I provide preliminary data on the use of prolyl hydroxylase inhibitors in altering mitochondrial function. I also outline my involvement in other ongoing projects in organ donation and machine perfusion that also aim to improve the outcomes of deceased donor kidney and liver transplantation.

Os lagostins são crustáceos decápodos límnicos que podem ser encontrados em água corrente, outros preferem água com pouca ou nenhuma corrente, como pequenos riachos, lagos, reservatórios e pântanos. Muitas espécies vivem em galerias subterrâneas com níveis mais baixos de oxigênio; assim, estas espécies podem mostrar adaptações metabólicas às condições hipóxicas. O objetivo desta pesquisa foi comparar o metabolismo de duas espécies de lagostins com diferentes hábitos, Parastacus defossus e Parastacus brasiliensis. P. defossus é uma espécie fossorial, vive em galerias com baixos níveis de oxigênio e P. brasiliensis é encontrado em ambientes lóticos com maiores níveis de oxigênio. Amostragens sazonais foram realizadas da primavera de 2006 ao inverno de 2007 para determinações metabólicas sazonais e posteriormente, amostragens foram realizadas durante o inverno de 2008 para análises metabólicas dos animais submetidos à hipóxia e recuperação pós-hipóxia. P. brasiliensis foi amostrado em Mariana Pimentel, Rio Grande do Sul (Brasil) e P. defossus foi amostrado no Lami, Porto Alegre, Rio Grande do Sul (Brasil). Nos experimentos de hipóxia, grupos de animais foram submetidos à hipóxia por 1, 2, 4 e 8 horas. Períodos de recuperação póshipóxia também foram analisados, após 4 hs de hipóxia, grupos de animais foram colocados em aquários com água aerada e foram removidos em intervalos de 1, 3, 6 e 9 hs. Após esse período foram extraídas amostras de hemolinfa e removidos o hepatopâncreas, o músculo, as brânquias e as gônadas para a determinação de glicose, lactato, glicose livre, glicogênio, proteínas totais, lipídios totais, colesterol total, arginina e arginina fosfato. Os resultados das análises sazonais mostraram diferentes respostas entre as estações do ano e entre as espécies, para todos os parâmetros metabólicos, com exceção das proteínas nas brânquias e do lactato na hemolinfa. As variações metabólicas em P. defossus foram principalmente relacionadas com o período reprodutivo e os períodos de baixa concentração de oxigênio nas galerias, enquanto os resultados em P. brasiliensis sugerem uma alocação significativa dos nutrientes da dieta para o tecido gonadal durante o período reprodutivo, com uma menor transferência das reservas de diferentes tecidos para as gônadas. Em relação ao metabolismo dos animais submetidos à hipóxia foi observado que em ambas as espécies, os níveis de glicose e de lactato aumentaram significativamente em hipóxia. Reduções de glicogênio, lipídios e colesterol foram registradas no hepatopâncreas e no tecido muscular, especialmente de P. defossus. Todos os tecidos de P. defossus e P. brasiliensis mostraram reduções nos níveis de glicose livre, mas essas reduções não foram significativas. Todas as reservas das brânquias anteriores e posteriores, com exceção das reservas de glicogênio, mostraram comportamento semelhante em ambas as espécies. As duas espécies de Parastacus armazenaram e utilizaram arginina fosfato, principalmente P. defossus. Entre os resultados do metabolismo dos animais submetidos à recuperação pós-hipóxia foram observadas que a restauração dos níveis de lactato foi mais rápido em P. defossus quando comparado com P. brasiliensis. Essa espécie restabeleceu suas reservas de glicogênio do hepatopâncreas e do tecido muscular. Já os níveis de glicose livre foram rapidamente restabelecidos em todos os tecidos das duas espécies. Em relação às reservas de arginina fosfato, P. defossus mostrou maiores concentrações que P. brasiliensis. As duas espécies mostraram capacidade de restaurar os níveis de arginina fosfato, mas também utilizaram essas reservas durante períodos de recuperação. Nas espécies, as reservas de lipídios totais e colesterol parecem ser uma importante fonte de energia durante a recuperação. / Some species of crayfish live in flowing water, and others prefer water with little or no current such as small streams, lakes, reservoirs, and swamps. Many species live in subterranean burrows with lower oxygen levels, and can show metabolic adaptations to hypoxic conditions. The aim of this study was to compare the metabolism of two crayfish species with different habitats, Parastacus defossus and Parastacus brasiliensis. P. defossus is fossorial, living in burrows with low oxygen levels, and P. brasiliensis lives in lotic environments with higher oxygen levels. Seasonal sampling was conducted from spring 2006 to winter 2007 for seasonal metabolic determinations, and samples were taken during the winter of 2008 for metabolic analyses of the animals subjected to hypoxia and during the post-hypoxia recovery. P. brasiliensis was collected in Mariana Pimentel, Rio Grande do Sul (Brazil) and P. defossus at Lami, Porto Alegre, Rio Grande do Sul. In the hypoxia experiments, groups of animals were subjected to hypoxia for 1, 2, 4, and 8 h. Periods of post-hypoxia recovery were also analyzed; after 4 h of hypoxia, groups of animals were placed in tanks with oxygenated water and were then removed at intervals of 1, 3, 6, and 9 h. The hemolymph was extracted, and the hepatopancreas, muscle, gills, and gonads were removed for determination of glucose, lactate, free glucose, glycogen, total proteins, total lipids, total cholesterol, arginine, and arginine phosphate. The results of the seasonal analysis showed, for all metabolic parameters, different seasonal responses between the species, with the exception of proteins in gills, and of lactate in hemolymph. The metabolic variations in P. defossus were mainly related to reproductive period and periods of low oxygen concentration in the burrows. The results for P. brasiliensis suggested a significant allocation of dietary nutrients to gonadal tissue during the reproductive period, with a smaller transfer of reserves from different tissues to gonads. In both species, glucose and lactate levels increased significantly in hypoxia. Reductions of glycogen, lipids, and cholesterol were recorded in hepatopancreas and muscle tissue, especially of P. defossus. In all tissues of P. defossus and P. brasiliensis were observed reductions in the free glucose levels, but these reductions weren’t significant. All reserves in the anterior and posterior gills, except glycogen, behaved similarly in both species. Both Parastacus species, mainly P. defossus, stored and used arginine phosphate. During post-hypoxia recovery, lactate was restored more rapidly in P. defossus than in P. brasiliensis. P. defossus restored its glycogen reserves in the hepatopancreas and muscle tissue. Free glucose was quickly restored in all tissues of both species. In relation to the reserves of arginine phosphate, P. defossus showed higher concentrations than P. brasiliensis. The two species showed ability to restore this metabolite, but they also used this metabolite during longer periods of recovery. In both species, the reserves of total lipids and cholesterol seemed be an important source of energy during the recovery period.

Os lagostins são crustáceos decápodos límnicos que podem ser encontrados em água corrente, outros preferem água com pouca ou nenhuma corrente, como pequenos riachos, lagos, reservatórios e pântanos. Muitas espécies vivem em galerias subterrâneas com níveis mais baixos de oxigênio; assim, estas espécies podem mostrar adaptações metabólicas às condições hipóxicas. O objetivo desta pesquisa foi comparar o metabolismo de duas espécies de lagostins com diferentes hábitos, Parastacus defossus e Parastacus brasiliensis. P. defossus é uma espécie fossorial, vive em galerias com baixos níveis de oxigênio e P. brasiliensis é encontrado em ambientes lóticos com maiores níveis de oxigênio. Amostragens sazonais foram realizadas da primavera de 2006 ao inverno de 2007 para determinações metabólicas sazonais e posteriormente, amostragens foram realizadas durante o inverno de 2008 para análises metabólicas dos animais submetidos à hipóxia e recuperação pós-hipóxia. P. brasiliensis foi amostrado em Mariana Pimentel, Rio Grande do Sul (Brasil) e P. defossus foi amostrado no Lami, Porto Alegre, Rio Grande do Sul (Brasil). Nos experimentos de hipóxia, grupos de animais foram submetidos à hipóxia por 1, 2, 4 e 8 horas. Períodos de recuperação póshipóxia também foram analisados, após 4 hs de hipóxia, grupos de animais foram colocados em aquários com água aerada e foram removidos em intervalos de 1, 3, 6 e 9 hs. Após esse período foram extraídas amostras de hemolinfa e removidos o hepatopâncreas, o músculo, as brânquias e as gônadas para a determinação de glicose, lactato, glicose livre, glicogênio, proteínas totais, lipídios totais, colesterol total, arginina e arginina fosfato. Os resultados das análises sazonais mostraram diferentes respostas entre as estações do ano e entre as espécies, para todos os parâmetros metabólicos, com exceção das proteínas nas brânquias e do lactato na hemolinfa. As variações metabólicas em P. defossus foram principalmente relacionadas com o período reprodutivo e os períodos de baixa concentração de oxigênio nas galerias, enquanto os resultados em P. brasiliensis sugerem uma alocação significativa dos nutrientes da dieta para o tecido gonadal durante o período reprodutivo, com uma menor transferência das reservas de diferentes tecidos para as gônadas. Em relação ao metabolismo dos animais submetidos à hipóxia foi observado que em ambas as espécies, os níveis de glicose e de lactato aumentaram significativamente em hipóxia. Reduções de glicogênio, lipídios e colesterol foram registradas no hepatopâncreas e no tecido muscular, especialmente de P. defossus. Todos os tecidos de P. defossus e P. brasiliensis mostraram reduções nos níveis de glicose livre, mas essas reduções não foram significativas. Todas as reservas das brânquias anteriores e posteriores, com exceção das reservas de glicogênio, mostraram comportamento semelhante em ambas as espécies. As duas espécies de Parastacus armazenaram e utilizaram arginina fosfato, principalmente P. defossus. Entre os resultados do metabolismo dos animais submetidos à recuperação pós-hipóxia foram observadas que a restauração dos níveis de lactato foi mais rápido em P. defossus quando comparado com P. brasiliensis. Essa espécie restabeleceu suas reservas de glicogênio do hepatopâncreas e do tecido muscular. Já os níveis de glicose livre foram rapidamente restabelecidos em todos os tecidos das duas espécies. Em relação às reservas de arginina fosfato, P. defossus mostrou maiores concentrações que P. brasiliensis. As duas espécies mostraram capacidade de restaurar os níveis de arginina fosfato, mas também utilizaram essas reservas durante períodos de recuperação. Nas espécies, as reservas de lipídios totais e colesterol parecem ser uma importante fonte de energia durante a recuperação. / Some species of crayfish live in flowing water, and others prefer water with little or no current such as small streams, lakes, reservoirs, and swamps. Many species live in subterranean burrows with lower oxygen levels, and can show metabolic adaptations to hypoxic conditions. The aim of this study was to compare the metabolism of two crayfish species with different habitats, Parastacus defossus and Parastacus brasiliensis. P. defossus is fossorial, living in burrows with low oxygen levels, and P. brasiliensis lives in lotic environments with higher oxygen levels. Seasonal sampling was conducted from spring 2006 to winter 2007 for seasonal metabolic determinations, and samples were taken during the winter of 2008 for metabolic analyses of the animals subjected to hypoxia and during the post-hypoxia recovery. P. brasiliensis was collected in Mariana Pimentel, Rio Grande do Sul (Brazil) and P. defossus at Lami, Porto Alegre, Rio Grande do Sul. In the hypoxia experiments, groups of animals were subjected to hypoxia for 1, 2, 4, and 8 h. Periods of post-hypoxia recovery were also analyzed; after 4 h of hypoxia, groups of animals were placed in tanks with oxygenated water and were then removed at intervals of 1, 3, 6, and 9 h. The hemolymph was extracted, and the hepatopancreas, muscle, gills, and gonads were removed for determination of glucose, lactate, free glucose, glycogen, total proteins, total lipids, total cholesterol, arginine, and arginine phosphate. The results of the seasonal analysis showed, for all metabolic parameters, different seasonal responses between the species, with the exception of proteins in gills, and of lactate in hemolymph. The metabolic variations in P. defossus were mainly related to reproductive period and periods of low oxygen concentration in the burrows. The results for P. brasiliensis suggested a significant allocation of dietary nutrients to gonadal tissue during the reproductive period, with a smaller transfer of reserves from different tissues to gonads. In both species, glucose and lactate levels increased significantly in hypoxia. Reductions of glycogen, lipids, and cholesterol were recorded in hepatopancreas and muscle tissue, especially of P. defossus. In all tissues of P. defossus and P. brasiliensis were observed reductions in the free glucose levels, but these reductions weren’t significant. All reserves in the anterior and posterior gills, except glycogen, behaved similarly in both species. Both Parastacus species, mainly P. defossus, stored and used arginine phosphate. During post-hypoxia recovery, lactate was restored more rapidly in P. defossus than in P. brasiliensis. P. defossus restored its glycogen reserves in the hepatopancreas and muscle tissue. Free glucose was quickly restored in all tissues of both species. In relation to the reserves of arginine phosphate, P. defossus showed higher concentrations than P. brasiliensis. The two species showed ability to restore this metabolite, but they also used this metabolite during longer periods of recovery. In both species, the reserves of total lipids and cholesterol seemed be an important source of energy during the recovery period.

Background: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by increased pulmonary artery pressure, leading to right ventricle hypertrophy and ultimately heart failure and death. Sirtuin 1 (SIRT1) is an NAD+ dependent protein deacetylase that has been strongly implicated as a crucial link between longevity, stress response and maintenance of vascular health. In this thesis, we investigated the role of SIRT1 in the pulmonary vascular hypoxic response and the pathogenesis of pulmonary hypertension (PH) working under the hypothesis that SIRT1 plays a protective role in the pulmonary vasculature and that lack of SIRT1 would lead to worsening of PH in a model of chronic hypoxia (CH). Results: We determined that global SIRT1 knockout or SIRT1 catalytic inactivation resulted in a marked increase in right ventricle pressure and remodeling compared to wildtype mice in CH. Furthermore, hypoxia-induced erythrocytosis and pulmonary vascular remodeling were profoundly increased in both SIRT1 mouse lines. Subsequent molecular assessment revealed that SIRT1 knockout, but not inactivation, led to a significant increase in mRNA levels of hypoxia inducible factor (HIF)-1α and significantly higher activity in hypoxia, leading to elevated lactate dehydrogenase A (LDHA) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) in the lungs. Interestingly, both knockout and inactivation of SIRT1 enhanced the activity of HIF2α in the hypoxic lungs and kidneys, leading to increased erythropoietin (EPO) and plasminogen activator inhibitor-1 (PAI-1). Moreover, SIRT1 knockout or inactivation was associated with a trend towards hypoxic-independent increases in HIF3α mRNA in the lungs. Prevention of glycolytic shift using dichloroacetate (DCA) did not result in improvement in this model, yet resveratrol (RSV), a SIRT1 activator/mimic, partially prevented PH only in absence of SIRT1 activity. Finally, selective endothelial cell SIRT1 deletion was sufficient to cause worse PH in the CH model. Conclusions: SIRT1 plays a protective role in the hypoxic response through transcriptional and non-transcriptional control of the hypoxia inducible factors, thus protecting against worse hypoxia-induced PH. SIRT1 could be a novel target for future therapies in PAH.

No description available.

BACKGROUND:We tested the hypothesis that ventilatory drive in hypoxia and hypercapnia is inversely correlated with the number of hypopneas and obstructive apneas per hour of sleep (obstructive apnea hypopnea index, OAHI) in children.METHODS:Fifty children, 6 to 12 years of age were studied. Participants had an in-home unattended polysomnogram to compute the OAHI. We subsequently estimated ventilatory drive in normoxia, at two levels of isocapnic hypoxia, and at three levels of hyperoxic hypercapnia in each subject. Experiments were done during wakefulness, and the mouth occlusion pressure measured 0.1 seconds after inspiratory onset (P0.1) was measured in all conditions. The slope of the relation between P0.1 and the partial pressure of end-tidal O2 or CO2 (PETO2 and PETCO2) served as the index of hypoxic or hypercapnic ventilatory drive.RESULTS:Hypoxic ventilatory drive correlated inversely with OAHI (r = -0.31, P = 0.041), but the hypercapnic ventilatory drive did not (r = -0.19, P = 0.27). We also found that the resting PETCO2 was significantly and positively correlated with the OAHI, suggesting that high OAHI values were associated with resting CO2 retention.CONCLUSIONS:In awake children the OAHI correlates inversely with the hypoxic ventilatory drive and positively with the resting PETCO2. Whether or not diminished hypoxic drive or resting CO2 retention while awake can explain the severity of sleep-disordered breathing in this population is uncertain, but a reduced hypoxic ventilatory drive and resting CO2 retention are associated with sleep-disordered breathing in 6-12 year old children.