Hours In Hypoxia Research Articles

Acute physiological response to a normobaric hypoxic exposure: sex differences.

Although preliminary studies suggested sex-related differences in physiological responses to altitude/hypoxia, controlled studies from standardised exposures to normobaric hypoxia are largely lacking. Hence, the goals of this study were to provide information on cardiorespiratory responses to a 7-h normobaric hypoxia exposure and to explore potential differences between men and women. In this crossover study, a total of 15 men and 14 women were subjected to a 7-h exposure in normoxia (FiO2: 21%) and normobaric hypoxia (FiO2: 15%). Values of peripheral oxygen saturation, heart rate, systolic and diastolic blood pressure and respiratory gases were recorded every hour (8 time points), and oxygen saturation every 30min (15 time points). Compared to normoxia, exposure to hypoxia significantly increased minute ventilation from baseline to hour 7 in males (+ 71%) and females (+ 40%), significantly greater in men (p < 0.05). A steeper decrease in peripheral oxygen saturation until 2.5h in hypoxia was seen in females compared to males (p < 0.05). In conclusion, the ventilatory response to hypoxia was more pronounced in men compared to women. Moreover, during the first hours in hypoxia, peripheral oxygen saturation dropped more markedly in women than in men, likely due an initially lower and/or less efficient ventilatory response to moderate hypoxia. Those findings should be considered when performing interventions for therapy or prevention in normobaric hypoxia. Nevertheless, further large-scaled and well-controlled studies are needed.

High Intraindividual Variability in the Response of Serum Erythropoietin to Multiple Simulated Altitude Exposures

Baranauskas, Marissa N., Timothy J. Fulton, Alyce D. Fly, Bruce J. Martin, Timothy D. Mickleborough, and Robert F. Chapman. High intraindividual variability in the response of serum erythropoietin to multiple simulated altitude exposures. High Alt Med Biol 00:000-000, 2022. Purpose: To evaluate within-subject variability in the serum erythropoietin (EPO) response to multiple simulated altitude exposures. Methods: Seven physically active men and women (age 27 ± 3 years, body mass index = 24.6 ± 4.0 kg/m2) were exposed to normobaric hypoxia (fraction of inspired oxygen [FiO2] = 0.14) for 12 hours on three separate occasions. Serum EPO concentrations were measured before exposure (0 hour), after 6 hours, and after 12 hours in hypoxia. The EPO response to hypoxia was calculated as percent change from 0 to 12 hours (ΔEPO0-12). Results: Exposure time had a significant effect on EPO (p < 0.001) with concentrations increasing 3.2 ± 1.3 mIU/ml from 0 to 6 hours (p = 0.034) and 4.7 ± 1.2 mIU/ml from 0 to 12 hours (p = 0.001). Group mean ΔEPO0-12 remained unchanged (p = 0.688) between the three exposures; however, there was considerable intraindividual variability in EPO responses. The intrasubject coefficient of variation for ΔEPO0-12 was 61% ± 28% (range: 17%-103%) with intrasubject associations ranging r = 0.052 to r = 0.651 between repeated exposures. Conclusions: Athletes who routinely supplement training with simulated altitude methods (e.g., hypoxic tents) should expect inconsistent EPO responses to intermittent exposures lasting ≤12 hours.

Abstract 12927: The Human Ductus Arteriosus Smooth Muscle Cell Transcriptome Indicates a Central Role for Mitochondria in Postnatal Oxygen Sensing

Introduction: The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increased oxygen (O 2 ) immediately post-partum, resulting in functional DA closure, with anatomic closure ensuing within the first days of life. Failure of DA closure results in patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA response to O 2 , though modulated by the endothelium, is intrinsic to DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondria-derived reactive oxygen species, which increase in proportion to PaO 2 . The molecular signature of the DASMC response to O 2 is not well understood. Methods &amp; Results: DASMC were isolated from DA obtained from 10 infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorted to eliminate fibroblasts (CD90+) or endothelial cells (CD31+). DASMC purity was confirmed by α-smooth muscle actin staining. Cells were grown for 96 hours in hypoxia (2.5% O 2 ) or normoxia (19% O 2 ) and confocal calcium imaging confirmed O 2 -responsiveness of all cell lines: O 2 exposure increased intracellular calcium 18.1%±4.4% and decreased cell length 27%±1.5% relative to hypoxic baseline. RNA sequencing (Seq) of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p&lt;0.05). Gene Ontology (GO) analysis provided an unbiased assessment of genes differentially regulated by O 2 , revealing enrichment of functional groups including mitochondria, cellular respiration and transcription. Multiple subunits of electron transport chain complex I, NDUF (NADH:ubiquinone oxidoreductase) family genes, are upregulated in hDASMC following normoxia. Upregulated NDUF genes were validated with qPCR (t-test p&lt;0.05): NDUFS5 (Seq = 0.62-fold, PCR = 3.8-fold), NDUFA9 (Seq = 0.8-fold, Seq = 4.3-fold), NDUFA13 (Seq = 0.7-fold, PCR = 6.1-fold), and NDUFS8 (Seq = 0.6-fold, PCR = 7.1-fold). Conclusion: This first examination of the effects of O 2 on human DA transcriptomics supports a putative role for mitochondria as O 2 sensors, with mitochondrial subunits as potential future therapeutic targets for PDA.

Hypoxia induces expression of angiotensin-converting enzyme II in alveolar epithelial cells: Implications for the pathogenesis of acute lung injury in COVID-19.

SARS‐CoV‐2 uptake by lung epithelial cells is a critical step in the pathogenesis of COVID‐19. Viral entry is dependent on the binding of the viral spike protein to the angiotensin converting enzyme II protein (ACE2) on the host cell surface, followed by proteolytic cleavage by a host serine protease such as TMPRSS2. Infection of alveolar epithelial cells (AEC) in the distal lung is a key feature in progression to the acute respiratory distress syndrome (ARDS). We hypothesized that AEC expression of ACE2 is induced by hypoxia. In a murine model of hypoxic stress (12% FiO2), the total lung Ace2 mRNA and protein expression was significantly increased after 24 hours in hypoxia compared to normoxia (21% FiO2). In experiments with primary murine type II AEC, we found that exposure to hypoxia either in vivo (prior to isolation) or in vitro resulted in greatly increased AEC expression of both Ace2 (mRNA and protein) and of Tmprss2. However, when isolated type II AEC were maintained in culture over 5 days, with loss of type II cell characteristics and induction of type I cell features, Ace2 expression was greatly reduced, suggesting that this expression was a feature of only this subset of AEC. Finally, in primary human small airway epithelial cells (SAEC), ACE2 mRNA and protein expression were also induced by hypoxia, as was binding to purified spike protein. Hypoxia‐induced increase in ACE2 expression in type II AEC may provide an explanation of the extended temporal course of human patients who develop ARDS in COVID‐19.

Natural Killer Cells Recognize Pulmonary Epithelial Stress Molecules during Primary Graft Dysfunction

<h3>Purpose</h3> Primary graft dysfunction (PGD) occurs in 1/3 of all lung transplants. It is defined by epithelial dysfunction and innate immune cell infiltration. Natural killer (NK) cells are innate lymphocytes that are activated by NKG2D receptor binding of specific stress molecules. We hypothesized that NK cells mediate PGD by recognizing stress molecules induced on lung epithelial cells and causing injury via direct cytotoxicity. <h3>Methods</h3> In an established experimental PGD model, left hilar clamp (HC) was compared to sham (S) surgery in C57BL/6 mice. Stress molecules (RAE-1, MULT1) and NK cell receptors were measured by flow cytometry median fluorescent intensity (MFI) on dissociated lung cells. Mice were given blocking anti-NKG2D or isotype control antibodies preceding HC. Human stress molecules (MICA, MICB, ULBP1, 3, and 2/5/6) were measured by flow cytometry on bronchial epithelial cells incubated for 4 hours in hypoxia (1% O₂) versus normoxia conditions. The Mann-Whitney U test was applied for pairwise comparisons. <h3>Results</h3> NK cells were increased as a percent of lymphocytes (p = 0.008) and by count (p = 0.04) in HC (n = 5) versus S (n = 5) lungs and infiltrated airways (Figure 1A). The NKG2D receptor was increased on NK cells following HC (p = 0.005). The stress molecules MULT1 (p = 0.0002) and RAE-1 (Figure 1B, p = 0.002) were increased on epithelial cells in HC compared to S lungs. NKG2D blockade (n = 9) resulted in less pulmonary edema following HC compared to isotype control antibody (Figure 1C, n = 9, p = 0.04). Hypoxic human bronchial epithelial cells (n = 6) had increased MICB (p = 0.008), ULBP-1 (p = 0.05), and ULBP-2,5,6 (Figure 1 D, p = 0.004) compared to normoxic cells (n = 6). <h3>Conclusion</h3> PGD induces NK cell stress ligands leading to NK cell recruitment that contributes to PGD. NKG2D blockade, under investigation in clinical trials of other diseases, resulted in decreased lung injury. The induction of human NKG2D stress molecules during hypoxia suggests that this NK cell receptor-ligand interaction may mediate PGD.

Symptom progression in acute mountain sickness during a 12-hour exposure to normobaric hypoxia equivalent to 4500 m.

The diagnosis and quantification of severity of acute mountain sickness (AMS) continue to be problematic. What symptoms should be included in a score and how to weigh any given symptom in the total score remain matter of debate. Seventy seven healthy male (n=43) and female (n=34) volunteers, aged between 18 and 42 years, were exposed to normobaric hypoxia (Fio2=12.6%≙4500 m) for 12 hours. Symptoms of AMS according to the Lake Louise Scoring system (LLS) were recorded before and after 30 min, 3, 6, 9, and 12 hours in hypoxia. AMS scores continued to increase steeply during the entire hypoxia exposure in subjects suffering from AMS (LLS>2). Headache was the predominant symptom and the severity of nausea progressed faster in subjects who left the hypoxia room prematurely (severely affected by AMS) compared to those moderately affected (LLS>2 but completing the 12-h hypoxia exposure). Whereas headache scores up to 6 hours in hypoxia were not correlated with other AMS symptoms, nausea was correlated with dizziness and fatigue (r=0.45 and 0.56, p<0.01). Cluster analysis identified three different distributions of symptom severity compatible with being very likely free of AMS (cluster 1), compatible with very likely suffering from AMS (cluster 3), and compatible with ambiguous allocation (cluster 2). In conclusion, our findings confirm that headache plus one or more of the symptoms nausea, dizziness, and fatigue of at least mild to moderate severity are required for diagnosis of AMS. The inter-relationship between nausea, dizziness, and fatigue, however, raises the question whether each of these symptoms should be given equal diagnostic weighting. The time course of symptom progression within the first hours at altitude may provide clinically important information on the severity of subsequent AMS development and will support the decision to start therapeutic intervention.

Iron exposure reduces viability and plasticity of brain microvascular endothelial cells after hypoxic injury (670.2)

Diabetes increases the risk, causes bleeding into the brain, and worsens functional outcomes after ischemic stroke. We have previously shown that diabetic Goto‐Kakizaki (GK) rats have increased yet dysfunctional angiogenesis, profuse bleeding into the brain and poor vascular repair if subjected to stroke. The effect of bleeding on endothelial cell survival and repair is unknown. Here, we test the hypothesis that iron exposure reduces brain microvascular endothelial cell (BMVEC) viability and plasticity after ischemia/reperfusion in diabetes. To test this hypothesis, BMVECs isolated from Wistar (non‐diabetic) and GK rats were treated with 0.1 mM iron (III) sulfate, incubated 6 hours in hypoxia, and reoxygenated overnight. The cells were subjected to cell proliferation, migration and tube formation assays. GK BMVECs showed increased cell proliferation, migration and tube length under normoxia. Hypoxia reduced all three measures of angiogenesis for GK BMVECs. In addition, iron treatment further reduced migration and tube length of GK BMVECs exposed to hypoxia while it did not have an effect on control cells. These results suggest that iron has a deleterious effect on survival and angiogenic properties of GK BMVECs. More work is needed to investigate the exact mechanism of iron‐induced impairments but our work identifies iron chelation as a possible novel therapy to improve recovery after ischemic stroke.

Adipose-Derived Mesenchymal Stem Cells from Ventral Hernia Repair Patients Demonstrate Decreased Vasculogenesis

Introduction. In adipose tissue healing, angiogenesis is stimulated by adipose-derived stromal stem cells (ASCs). Ventral hernia repair (VHR) patients are at high risk for wound infections. We hypothesize that ASCs from VHR patients are less vasculogenic than ASCs from healthy controls. Methods. ASCs were harvested from the subcutaneous fat of patients undergoing VHR by the component separation technique and from matched abdominoplasty patients. RNA and protein were harvested on culture days 0 and 3. Both groups of ASCs were subjected to hypoxic conditions for 12 and 24 hours. RNA was analyzed using qRT-PCR, and protein was used for western blotting. ASCs were also grown in Matrigel under hypoxic conditions and assayed for tubule formation after 24 hours. Results. Hernia patient ASCs demonstrated decreased levels of VEGF-A protein and vasculogenic RNA at 3 days of growth in differentiation media. There were also decreases in VEGF-A protein and vasculogenic RNA after growth in hypoxic conditions compared to control ASCs. After 24 hours in hypoxia, VHR ASCs formed fewer tubules in Matrigel than in control patient ASCs. Conclusion. ASCs derived from VHR patients appear to express fewer vasculogenic markers and form fewer tubules in Matrigel than ASCs from abdominoplasty patients, suggesting decreased vasculogenic activity.

Investigation of Whole-Brain White Matter Identifies Altered Water Mobility in the Pathogenesis of High-Altitude Headache

Elevated brain water is a common finding in individuals with severe forms of altitude illness. However, the location, nature, and a causative link between brain edema and symptoms of acute mountain sickness such as headache remains unknown. We examined indices of brain white matter water mobility in 13 participants after 2 and 10 hours in normoxia (21% O2) and hypoxia (12% O2) using magnetic resonance imaging. Using a whole-brain analysis (tract-based spatial statistics (TBSS)), mean diffusivity was reduced in the left posterior hemisphere after 2 hours and globally reduced throughout cerebral white matter by 10 hours in hypoxia. However, no changes in T2 relaxation time (T2) or fractional anisotropy were observed. The TBSS identified an association between changes in mean diffusivity, fractional anisotropy, and T2 both supra and subtentorially after 2 and 10 hours, with headache score after 10 hours in hypoxia. Region of interest-based analyses generally confirmed these results. These data indicate that acute periods of hypoxemia cause a shift of water into the intracellular space within the cerebral white matter, whereas no evidence of brain edema (a volumetric enlargement) is identifiable. Furthermore, these changes in brain water mobility are related to the intensity of high-altitude headache.

Impact of Hypoxia As Novel Microenvironmental Factor On Drug Susceptibility of Primary CLL Cells

AbstractAbstract 3918 Background:CLL cells circulating in the peripheral blood are sensitive to therapy while malignant cells residing in the microenvironment survive and are the source of relapse. One of the strongest microenvironmental stimuli is CD40 ligand (CD40L)-CD40 interaction, which induces proliferative/anti-apoptotic genes in CLL cells, protecting them from apoptosis and many cytotoxic drugs. Despite the evident importance of CD40 activation further stimuli have to be considered, especially hypoxia. Lymph nodes, particularly those being infiltrated by malignant cells, show a low oxygen tension (<1%). Prior CLL investigations never took this important factor into account, hence the impact of hypoxia on cell survival and drug-resistance is still unrevealed. Methods:We have established an in vitro model, which mimics hypoxic conditions and CD40L-CD40 interaction, in order to understand the molecular basis of drug resistance of CLL cells resident in the microenvironment. CLL cells were cultured on CD40L feeder cells and kept up to 96 hours in hypoxia (1% O2) or normoxia (21% O2). We determined how proliferation rates in CLL are affected by these conditions and subsequently applied several drugs to investigate differences in drug efficacy between normoxia and hypoxia. Apoptosis was determined by AnnexinV/7AAD-staining and subsequent flow cytometry. Expression of potential target molecules was determined by qRT-PCR and Western Blotting. Results:Hypoxia is known to protect malignant cells in solid cancers from chemotherapy. We made similar observations, since classical DNA-targeting drugs were inefficient to kill CLL cells under hypoxic conditions. However, we identified ABT-737, which affects mitochondrial integrity, to be even more efficient under hypoxic conditions compared to normoxia. In order to explain this discrepancy we investigated the expression of several mitochondrial localized anti-/proapoptotic genes on RNA and protein level. We show that the de-regulation of BclXL and Mcl-1 under hypoxic conditions is essential for ABT-737 sensitivity. BclXL deregulation depends on a general reduction in protein translation in hypoxic cells. Mcl-1 protein expression differs from its mRNA expression, hence we expected regulation subsequent to protein synthesis. Indeed we could identify an increased activity of the proteasome in hypoxia, as Mcl-1 is a short-lived protein with a rapid proteasomal turnover this is a feasible explanation for the observed downregulation. Interestingly, hypoxia has a great impact on proliferation of primary CLL cells under different stimuli in vitro. Conclusion:These are the first experiments investigating the impact of oxygen tension on survival and response to chemotherapy of CLL cells. We show that hypoxia renders CLL cells resistant to classical DNA-targeting agent Fludarabine and Bendamustine. Furthermore we point out that small molecules like ABT-737, which specifically target mitochondria, might be efficient in targeting CLL cells protected by hypoxia and CD40L-CD40 interaction within the microenvironment. Development of novel in vitro models like ours will help us understand the specific molecular changes induced by microenvironmental stimuli and their impact on drug efficacy. These findings will allow us to identify novel therapeutic targets.M.Hu. and L.P.F. contributed equally to this work Disclosures:No relevant conflicts of interest to declare.

Primed for Death: Hypoxia Sensitizes Primary CLL Cells towards Compounds Targeting Mitochondrial Integrity

Abstract 1767 Background:CLL cells prefer to remain in the microenvironment since they feel safe. CD40 ligand (CD40L)-CD40 interaction induces proliferative/anti-apoptotic genes in CLL cells, which protects them from apoptosis and most cytotoxic drugs by the microenvironment.Research interested in identifying novel drugs that effectively target CLL cells within microenvironmental niches has to consider further microenvironmental stimuli, especially hypoxia. Lymph nodes, especially those being infiltrated by malignant cells show low oxygen tension (1%). Prior CLL investigations never took this important factor into account. The impact of hypoxia on survival and drug-resistance is still unknown. Methods:Therefore we have established an in vitro model, which mimics hypoxic conditions and CD40L-CD40 interaction, in order to understand the molecular basis of drug resistance of CLL resident in the microenvironment. CLL cells were cultured on CD40L feeder cells and kept up to 96 hours in hypoxia (1% O2 tension) or normoxia (21% O2 tension).We applied several drugs under these conditions to investigate the differences between normoxia and hypoxia. The miRNA expression was determined by using Illumina Bead Chip Arrays compromising 752 miRNAs. Gene expression was analyzed via mRNA-based Illumina microchip array. Target miRNAs and mRNAs were validated by qRT-PCR. Apoptosis was determined by AnnexinV-7AAD and JC-1 staining (mitochondrial outer membrane permeabilization) and subsequent flow cytometry. Results:In solid cancers hypoxia is expected to protect malignant cells from chemotherapy. We made similar observations, since classical DNA-targeting drugs were inefficient to kill CLL cells cultured on CD40L feeder cells under hypoxia and normoxia. However, we identified ABT-737, which affect mitochondrial integrity, to be even more efficient under hypoxic conditions and CD40L interaction compared to CD40L stimulation and normoxia (74,1% vs. 52,1% apoptotic cells, n=15; p<0.001). Interestingly, overall survival of primary CLL cells during CD40L-CD40 interaction without any cytotoxic treatment was higher under hypoxia compared to normoxia. To understand this discrepancy, we investigated the expression of several mitochondrial localized anti-/pro-apoptotic genes on RNA and protein level. We identified, that the de-regulation of BCLXL and MCL1 is crucial for ABT-737 sensitivity during hypoxic conditions. This de-regulation was also detectable during CD40L interaction. BCLXL deregulation could be attributed to differential NF-κB expression, as determined by EMSA. Since MCL1 protein expression differs from its mRNA expression, we expected regulation prior to protein synthesis. Indeed, we could identify miRNAs, which were upregulated during hypoxia and CD40L stimulation and regulate MCL1 expression. These miRNAs were validated by luciferase expression assays. Conclusion:Here we investigated for the first time the impact of oxygen tension on therapeutic response of CLL cells. We assume that small molecules like ABT-737, which specifically target mitochondria, might be efficient in targeting CLL cells protected by CD40L-CD40 interaction within the microenvironment.Development of novel in vitro models will allow us to understand the specific molecular changes induced by microenvironmental stimuli and to develop novel therapeutic targets.L.P.F. and M.Hue. contributed equally to this work. Disclosures:No relevant conflicts of interest to declare.

Heat shock attenuates VEGF expression in three-dimensional myoblast sheets deteriorating therapeutic efficacy in heart failure

SummaryBackgroundMyoblast sheet transplantation is a promising novel treatment for ischemic heart failure. The aim of this study was to test the hypothesis that heat shock (HS) pre-treatment affects the angiogenic properties of myoblast sheets in vivo and in vitro.Material/MethodsWe studied HS preconditioning of L6 myoblast sheets in relation to their apoptosis, proliferation, and vascular endothelial growth factor (VEGF)-associated responses under normoxia and under hypoxia in vitro. In vivo evaluation of their therapeutic effect was performed with 60 male Wistar rats divided into 3 groups (20 each): sole left anterior descending (LAD) ligation (control); LAD ligation and non-conditioned sheet transplantation (L6 No-Shock); and LAD ligation and L6-heat shock conditioned sheet transplantation (L6 Heat-Shock). Left ventricular function was evaluated by echocardiography after 3, 10, and 28 days.ResultsExpression of HSP70/72 was strongly induced 24 hours after HS, and thereafter it decreased notably during 72 hours in hypoxia. Under normal growth conditions, HSP70/72 expression remained stable. HS delayed apoptosis-associated caspase-3 expression during 24-hour hypoxia compared to non-treated controls. However, VEGF expression reduced significantly in the heat shock pretreated sheets. Ejection fraction of the L6-myoblast HS pre-treatment group (L6 Heat-Shock) decreased gradually during follow-up, in the same pattern as the controls. However, these functional parameters improved in the L6-myoblast normal sheet group (L6 No-Shock) at the tenth day and remained significantly better.ConclusionsHS protects myoblast sheets from hypoxia-associated apoptosis in vitro, but reduces VEGF expression of the sheet, leading to lower therapeutic effect in heart failure.

Wnt-4 and Ets-1 signaling pathways for regeneration after acute renal failure

Ischemic acute renal failure (ARF) is the most common form of ARF in the adult population. The molecular mechanisms of tubular regeneration after ischemic renal injury remain largely unknown. An understanding of the mechanisms that lead to renal cell proliferation and regeneration will be necessary for the exploration of novel therapeutic strategies for the treatment of ARF. It has been suggested that regeneration processes may recapitulate developmental processes in order to restore organ or tissue function. The adult tubular epithelial cells have a potent ability of regenerate after cellular damage. We examined functional role of two developmental genes, Wnt-4 and Ets-1, in renal tubular regeneration in ARF. The Wnt-β-catenin pathway plays key roles in embryogenesis. Wnt-4 is known to be expressed in the mesonephric duct in the embryonic development. To clarify the significance of the Wnt-4-β-catenin pathway in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. After clamping left rat renal artery for 1 hour, we examined whole kidney homogenate and total RNA extracted at 3, 6, 12, 24, 48, and 72 hours after reperfusion by Western blot analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR). Wnt-4 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after ischemia, respectively. In immunohistologic examination, Wnt-4 was expressed in the proximal tubules and coexpressed with aquaporin 1 and proliferating cell nuclear antigen (PCNA). Cyclin D1 and cyclin A were expressed at 12 to 48 hours after reperfusion. Furthermore, overexpression of Wnt-4 and β-catenin promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 and cyclin A in LLC-PK1 cells. These data suggest that the Wnt-4-β-catenin pathway plays a key role in the cell cycle progression of renal tubules in ARF. The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. To clarify the significance of Ets-1 in ARF, we used a rat ARF model in vivo and LLC-PK1 cells as an in vitro model. Ets-1 mRNA and protein expression were strongly increased at 3 to 12 hours and 6 to 24 hours after the ischemia, respectively. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with PCNA. Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 hours and 6 hours in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell cycle progression of renal tubules in ARF. Our data suggest that Wnt-4-β-catenin and Ets-1 pathways regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF. These developmental genes may play key roles in dedifferentiation and regeneration of the renal tubular cells after ARF.

322 Prenatal Glucocorticoids: Effects on Lung of Newborn Rats Exposed to Hypoxia and Recovered in Hyperoxia or Air.

Aims: To examine the morphometric changes produced on lung of neonatal rats treated with prenatal administration of betamethasone or dexamethasone, and test the hypothesis if glucocorticoids would produce any difference when pups are exposed to hypoxia and its recovery with oxygen or air ambient. Material and Methods: Dexamethasone, Bethametasone or equivalent saline solution was administered i.v. To gestant Wistar rats on 20 and 21 day of gestation. All rats delivered their pups within 48 hours after the last injection. The groups of rat pups were randomly assigned to either. At 4–8 hours of life newborn rats were exposed in sequential way during 2 hours in hypoxia, and recovered in > 95% oxygen or 21% oxygen. Lung and body weight were measured. For the morphometric study the lungs were fixed by intratracheal instilation of formol at a pressure of 20 cm.H2O. Quantitative morphometric assessment was done on coded slides with 400x magnification and a eye piece with a sample square grid pattern (model CPLW 1018, Zeiss Optical, Hannover Md). Morphmetric changes are shown in table 1. Statystical Analyses: ANOVA test was performed followed by Bonferroni test. P< 0,05 was considered. Result: Comparative wet lung weight/dry lung weight and morphometric study is show in table I DEX (dexamethasone) BETA (bethametasone) W.(weight), Sa (alveolar surface), SV (surface density of alveolar walls), Nv (numerical density of alveoli), rv (alveolar radius) * Significant differences with saline group. Conclusion: Prenatal administration of dexametasone and bethametasone causes a reduction in alveolar number in newborn rats. Lungs of treated pups showed a longer alveoli radius, lower values of surface density of alveolar walls and lower numerical density of alveoli than the control group. Probably, prenatal glucocorticoids could produce a partial suppression of the formation of secondary septa and decreased final alveoli number.