Hypoxic Kidney Research Articles

Evaluation of Renal Hypoxia in Diabetic Mice by BOLD MRI

Renal hypoxia has been proposed to be a pathophysiologic feature of diabetic kidney disease but it has been difficult to demonstrate in vivo, particularly in mouse models of diabetes. The objective of this work was to examine the sensitivity of blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) to assess renal oxygenation in vivo in a mouse model of diabetic kidney disease, the db/db mice. Kidney BOLD MRI studies were performed on a 3.0 T scanner using multiple gradient echo sequence with a custom-designed surface coil to acquire T2*-weighted images. Studies were performed in 10-week-old db/db mice (n = 7) and db/m controls (n = 6). R2* is a measure of the tissue deoxyhemoglobin concentration and higher values of R2* are associated with hypoxia. The db/db mice had higher medullary (43.1 ± 5.1 s⁻¹ vs. 32.3 ± 3.7⁻¹ s, P = 0.001) and cortical R2* (31.7 ± 3.1 s⁻¹ vs. 27.1 ± 4.1 s⁻¹, P = 0.04) values. Using pimonidazole staining as a marker of kidney hypoxia, in kidney sections from 10-week-old db/db mice neither cortex nor medulla had significant differences as compared with 10-week-old db/m mice (cortex: db/db 2.14 ± 0.05 vs. db/m 2.02 ± 0.28, medulla: db/db 2.81 ± 0.08 vs. db/m 2.6 ± 0.08). The db/db mice demonstrated further increased cortical and medullary hypoxia when scanned again at 15 weeks of age. The report shows that renal BOLD MRI is a sensitive method for the in vivo evaluation of renal hypoxia in a mouse model of diabetic kidney disease where progressive renal hypoxia can be documented over time. BOLD MRI may be useful to monitor therapeutic interventions that may improve tissue hypoxia in the diabetic kidney.

Protective Effect of Lifor Solution in Experimental Renal Ischemia-Reperfusion Injury

Improved kidney preservation methods are needed to reduce ischemia-reperfusion (IR) injury in kidney allografts. Lifor is an artificial preservation solution comprised of nutrients, growth factors, and a non-protein oxygen and nutrient carrier. The current study compared the effectiveness of Lifor to University of Wisconsin solution (UW) in protecting rat kidneys from warm IR and cold storage injury. In a warm IR model, rat kidneys were perfused in situ with either saline, UW, or Lifor for 45 min. Renal function and histology were assessed 24 h later. In a cold IR model, kidney slices were cold-stored in saline, UW, or Lifor at 4°C. Kidney injury was assessed by the release of lactate dehydrogenase (LDH) and immunoblot analysis for cleaved caspase-3. Lifor perfusion significantly mitigated renal dysfunction and tubular injury at 24 h compared with saline or UW. Lifor and UW prevented LDH release in hypoxic kidney slices in vitro, however activation of caspase-3 following hypoxia-reoxygenation was attenuated only with Lifor. Cold storage with Lifor or UW significantly decreased LDH release from kidney slices or normal rat kidney cells in comparison to storage in saline or culture media. After 24 h of cold storage there was a significant decrease in cleaved caspase-3 in Lifor stored slices compared that seen following cold storage in saline or UW solution. Lifor solution mitigates both warm and cold renal IR and appears to provide greater protection from apoptosis compared with UW solution.

Mice Deficient in Neuropeptide PACAP Demonstrate Increased Sensitivity to In Vitro Kidney Hypoxia

One of the well-known effects of pituitary adenylate cyclase activating polypeptide (PACAP) is its neuroprotective and cytoprotective actions including renoprotective effects. Mice deficient in endogenous PACAP exhibit several behavioral, metabolic, and developmental alterations. Furthermore, PACAP-deficient mice have larger infarct volume in a model of cerebral ischemia, delayed axonal regeneration, and increased cell death in cerebellar oxidative stress. We have previously demonstrated that PACAP-deficient mice have increased susceptibility to in vitro oxidative stress, which can be counteracted by exogenous PACAP treatment. These results demonstrate that endogenous PACAP has a protective role against various stressors. The objective of the present study was to investigate whether endogenous PACAP has a protective effect in the kidney against in vitro hypoxia. Kidney cell cultures were isolated from wild-type and PACAP-deficient mice, and cell viability was assessed after in vitro hypoxia induced using CoCl 2. The sensitivity of cells from PACAP-deficient mice was increased to hypoxia: both after 24 and 48 hours of exposure, cell viability was significantly reduced compared with that in control wild-type mice. These results show that endogenous PACAP protects against noxious stimuli in the kidney and that PACAP may act as a stress sensor in renal cells.

Proteomic Analysis of Protein Expression Affected by Peroxiredoxin V Knock-Down in Hypoxic Kidney

Peroxiredoxin V, an atypical thioredoxin peroxidase, is widely expressed in mammalian tissues. In addition, Prdx V is localized in mitochondria, peroxisome, cytosol, and the nucleus. Prdx V has been reported to protect a wide range of cellular environments as an antioxidant enzyme, and its dysfunctions may be implicated in several diseases, such as cancer, inflammation, and neurodegenerative disease. Identification and relative quantification of proteins affected by Prdx V may help identify novel signaling mechanisms that are important for oxidative stress response. However, the role of Prdx V in the modulation of hypoxia-related cellular response is not studied yet. To examine the function of endogenous Prdx V in hypoxic condition in vivo, we generated a transgenic mouse model with Prdx V siRNA expression controlled by U6 promoter. Of many tissues, the knockdown of Prdx V expression was displayed in the kidney, lung, and liver but not the spleen and skin. We conducted on the basis of nano-UPLC-MS(E) proteomic study to identify the Prdx V-affected protein networks in hypoxic kidneys. In this study, we identified protein networks associated with oxidative stress, fatty acid metabolism, and mitochondrial dysfunction. Our results indicated that Prdx V affected to regulation of kidney homeostasis under hypoxia stress.

Uremia induces abnormal oxygen consumption in tubules and aggravates chronic hypoxia of the kidney via oxidative stress

In addition to causing uremic symptoms, uremic toxins accelerate the progression of renal failure. To elucidate the pathophysiology of uremic states, we investigated the effect of indoxyl sulfate (IS), a representative uremic toxin, on oxygen metabolism in tubular cells. We demonstrated an increase in oxygen consumption by IS in freshly isolated rat and human proximal tubules. Studies utilizing ouabain, the Na-K-ATPase inhibitor, and apocynin, the NADPH oxidase inhibitor, as well as the in vivo gene-silencing approach to knock down p22(phox) showed that the increase in tubular oxygen consumption by IS is dependent on Na-K-ATPase and oxidative stress. We investigated whether the enhanced oxygen consumption led to subsequent hypoxia of the kidney. An increase in serum IS concentrations in rats administered indole was associated with a decrease in renal oxygenation (8 h). The remnant kidney in rats developed hypoxia at 16 wk. Treatment of the rats with AST-120, an oral adsorbent that removes uremic toxins, reduced serum IS levels and improved oxygenation of the kidney. Amelioration of hypoxia in the remnant kidney was associated with better renal functions and less histological injury. Reduction of serum IS levels also led to a decrease in oxidative stress in the kidney. Our ex vivo and in vivo studies implicated that uremic states may deteriorate renal dysfunction via dysregulating oxygen metabolism in tubular cells. The abnormal oxygen metabolism in tubular cells by uremic toxins was, at least in part, mediated by oxidative stress.

Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney

Mitochondrial oxidative damage is a basic mechanism of aging, and multiple studies demonstrate that this process is attenuated by calorie restriction (CR). However, the molecular mechanism that underlies the beneficial effect of CR on mitochondrial dysfunction is unclear. Here, we investigated in mice the mechanisms underlying CR-mediated protection against hypoxia in aged kidney, with a special focus on the role of the NAD-dependent deacetylase sirtuin 1 (Sirt1), which is linked to CR-related longevity in model organisms, on mitochondrial autophagy. Adult-onset and long-term CR in mice promoted increased Sirt1 expression in aged kidney and attenuated hypoxia-associated mitochondrial and renal damage by enhancing BCL2/adenovirus E1B 19-kDa interacting protein 3-dependent (Bnip3-dependent) autophagy. Culture of primary renal proximal tubular cells (PTCs) in serum from CR mice promoted Sirt1-mediated forkhead box O3 (Foxo3) deacetylation. This activity was essential for expression of Bnip3 and p27Kip1 and for subsequent autophagy and cell survival of PTCs under hypoxia. Furthermore, the kidneys of aged Sirt1+/- mice were resistant to CR-mediated improvement in the accumulation of damaged mitochondria under hypoxia. These data highlight the role of the Sirt1-Foxo3 axis in cellular adaptation to hypoxia, delineate a molecular mechanism of the CR-mediated antiaging effect, and could potentially direct the design of new therapies for age- and hypoxia-related tissue damage.

Immunolocalization of phospho-Arg-directed protein kinase-substrate in hypoxic kidneys using in vivo cryotechnique

Protein kinases (PKs) phosphorylate proteins at active regions for signal transduction. In this study, normal and hypoxic mouse kidneys were prepared using an "in vivo cryotechnique" (IVCT) and examined immunohistochemically with specific antibodies against phospho-(Ser/Thr) PKA/C substrate (P-PK-S) and phospho-(Ser/Thr) Akt substrate (P-Akt-S) to capture their time-dependent regulation in vivo. Left kidneys were cryofixed with IVCT under normal blood circulation and after varying hypoxic intervals, followed by freeze-substitution with acetone containing paraformaldehyde. Deparaffinized sections were immunostained for P-PK-S, Na(+)/HCO(3) (-) cotransporter NBC1, and a membrane skeletal protein, 4.1B. The P-PK-S was diffusely immunolocalized in the cytoplasm of the proximal tubules in normal kidneys, whereas NBC1 and 4.1B were detected at the basal striations of S1 and S2 segments of the proximal tubule. After 10 or 30 s hypoxia, P-PK-S was still immunolocalized in the cytoplasm of kidneys, but it was detected at the basal striations after 1 or 2 min hypoxia. The immunolocalization of P-Akt-S was the same as P-PK-S in the normal and hypoxic kidneys. Immunoblotting analyses of the kidney tissues under normal or hypoxic condition clearly identified the same 40-kDa bands. The IVCT is useful for time-dependent analysis of the immunodistribution of P-PK-S and P-Akt-S.

Newborn resuscitation: should we oxygenate or not?

how should we oxygenate newborn and infants suffering from hypoxia and ischemia? Is it harmful to use 100% O2? It may seem odd to question a practice that has been carried out for so long, seemingly without any adverse effects. The case is, however, that after the practice of routinely using 100% O2

Adaptation to hypoxia in the diabetic rat kidney

Hypoxia of the kidney in diabetes could predispose it to develop acute and chronic renal failure. To examine the relationship between renal hypoxia and renal failure, we measured hypoxia (as a pimonidazole adducts), hypoxia-inducible factors (HIFs), and a hypoxia target gene heme oxygenase-1. The studies were performed in rats with streptozotocin (STZ)-induced diabetes, Cohen diabetes sensitive rats, and during short-term artificial hyperglycemia in rats induced by intravenous glucose and octreotide. STZ-treated rats received insulin, the superoxide dismutase mimetic tempol, or contrast medium. Radiocontrast media causes hypoxia and HIF induction. Hypoxia, HIFs, and heme oxygenase were undetectable in controls, but transiently activated in STZ-treated and the Cohen diabetes sensitive rats. Different patterns of HIFs and pimonidazole were observed between the three models. Insulin abolished pimonidazole and HIF induction, whereas tempol lead to increased HIFs and heme oxygenase induction at similar levels of pimonidazole. When compared with control rats, STZ-treated rats exhibited more intense and protracted renal pimonidazole, with augmented hypoxia inducible factor production and reduced GFR following contrast media. Our data suggest that both regional hypoxia and hypoxia adaptation transiently occur in early stages of experimental diabetes, largely dependent on hyperglycemia or after contrast media. Tempol may augment the HIF response in diabetes.

Increased erythropoietin concentration after repeated apneas in humans

Hypoxia-induced increases in red blood cell production have been found in both altitude-adapted populations and acclimatized lowlanders. This process is mediated by erythropoietin (EPO) released mainly by the hypoxic kidney. We have previously observed high hemoglobin concentrations in elite breath-hold divers and our aim was to investigate whether apnea-induced hypoxia could increase EPO concentration. Ten healthy volunteers performed 15 maximal duration apneas, divided into three series of five apneas, each series separated by 10 min of rest. Apneas within series were separated by 2 min and preceded by 1 min of hyperventilation to increase apnea duration and arterial oxygen desaturation. When EPO concentration after serial apneas was compared to baseline values, an average maximum increase of 24% was found (P < 0.01). No changes in EPO concentration were observed during a control day without apnea, eliminating possible effects of a diurnal rhythm or blood loss. We therefore conclude that serial apneas increase circulating EPO concentration in humans.

Chronic hypoxia aggravates renal injury via suppression of Cu/Zn-SOD: a proteomic analysis

Accumulating evidence suggests a pathogenic role of chronic hypoxia in various kidney diseases. Chronic hypoxia in the kidney was induced by unilateral renal artery stenosis, followed 7 days later by observation of tubulointerstitial injury. Proteomic analysis of the hypoxic kidney found various altered proteins. Increased proteins included lipocortin-5, calgizzarin, ezrin, and transferrin, whereas the decreased proteins were alpha(2u)-globulin PGCL1, eukaryotic translation elongation factor 1alpha(2), and Cu/Zn superoxide dismutase (SOD1). Among these proteins, we focused on Cu/Zn-SOD, a crucial antioxidant. Western blot analysis and real-time quantitative PCR analysis confirmed the downregulation of Cu/Zn-SOD in the chronic hypoxic kidney. Furthermore, our laser capture microdissection system showed that the expression of Cu/Zn-SOD was predominant in the tubulointerstitium and was decreased by chronic hypoxia. The tubulointerstitial injury estimated by histology and immunohistochemical markers was ameliorated by tempol, a SOD mimetic. This amelioration was associated with a decrease in levels of the oxidative stress markers 4-hydroxyl-2-nonenal and nitrotyrosine. Our in vitro studies utilizing cultured tubular cells revealed a role of TNF-alpha in downregulation of Cu/Zn-SOD. Since the administration of anti-TNF-alpha antibody ameliorated Cu/Zn-SOD suppression, TNF-alpha seems to be one of the suppressants of Cu/Zn-SOD. In conclusion, our proteomic analysis revealed a decrease in Cu/Zn-SOD, at least partly by TNF-alpha, in the chronic hypoxic kidney. This study, for the first time, uncovered maladaptive suppression of Cu/Zn-SOD as a mediator of a vicious cycle of oxidative stress and subsequent renal injury induced by chronic hypoxia.

Involvement of Hypoxia-Inducible Transcription Factors in Polycystic Kidney Disease

In polycystic kidney disease (PKD), erythropoietin (EPO) production and interstitial vascularization are increased compared with other kidney diseases. EPO and several angiogenic factors are controlled by hypoxia-inducible transcription factors (HIFs), which are composed of a constitutive beta-subunit and two alternative alpha-subunits (HIF-1alpha, HIF-2alpha). We hypothesized that cyst expansion may result in pericystic hypoxia and consecutive up-regulation of HIF and thus examined the expression of HIF-alpha and HIF target genes in human PKD and in a rodent PKD model. HIF-1alpha and HIF-2alpha were found to be up-regulated in cyst epithelium and cells of cyst walls, respectively. The distinct expression pattern of the HIF-alpha isoforms closely resembles the respective pattern in normal kidneys under systemic hypoxia. Pimonidazole staining, a marker for tissue hypoxia, confirmed the existence of regional hypoxia in polycystic kidneys. Immunohistochemistry for selected target genes implicated a role for HIF-1alpha in vascular endothelial growth factor and Glut-1 activation and HIF-2alpha in endoglin and EPO stimulation. Polycystin-deficient cells showed physiological, oxygen-dependent HIF-alpha modulation, excluding a direct influence of polycystin deficiency on HIF-alpha regulation. In conclusion, HIF accumulation in human and rat PKD seems to be responsible for increased EPO production and pericystic hypervascularity and may have an impact on progression of PKD.

Hypoxia-inducible factors: where, when and why?

Hypoxia-inducible factor (HIF) is a family of transcription factors that regulate the homeostatic response to oxygen deprivation during development, physiological adaptation, and pathological processes such as ischemia and neoplasia. Our understanding of the function of different HIF isoforms is being advanced by understanding the processes that regulate their activity, learning where and when they are expressed and what genes they regulate.

Hypoxia in Renal Disease with Proteinuria and/or Glomerular Hypertension

Despite the increasing need to identify and quantify tissue oxygenation at the cellular level, relatively few methods have been available. In this study, we developed a new hypoxia-responsive reporter vector using a hypoxia-responsive element of the 5' vascular endothelial growth factor untranslated region and generated a novel hypoxia-sensing transgenic rat. We then applied this animal model to the detection of tubulointerstitial hypoxia in the diseased kidney. With this model, we were able to identify diffuse cortical hypoxia in the puromycin aminonucleoside-induced nephrotic syndrome and focal and segmental hypoxia in the remnant kidney model. Expression of the hypoxia-responsive transgene increased throughout the observation period, reaching 2.2-fold at 2 weeks in the puromycin aminonucleoside model and 2.6-fold at 4 weeks in the remnant kidney model, whereas that of vascular endothelial growth factor showed a mild decrease, reflecting distinct behaviors of the two genes. The degree of hypoxia showed a positive correlation with microscopic tubulointerstitial injury in both models. Finally, we identified the localization of proliferating cell nuclear antigen-positive, ED-1-positive, and terminal dUTP nick-end labeled-positive cells in the hypoxic cortical area in the remnant kidney model. We propose here a possible pathological tie between chronic tubulointerstitial hypoxia and progressive glomerular diseases.

Ontogenetic expression of erythropoietin and hypoxia‐inducible factor‐1 alpha genes in subterranean blind mole rats

Blind subterranean mole rats of the Spalax ehrenbergi superspecies in Israel have evolved multiple adaptive strategies to face underground hypoxia. Hypoxia-inducible factor-1alpha (HIF-1alpha) and erythropoietin (Epo) are key factors in the development of normal erythropoiesis and angiogenesis. Here, we demonstrate via real-time polymerase chain reaction (PCR) quantification that Spalax fetal liver and kidney express higher levels of Epo mRNA than Rattus, generating reinforcement of fetal erythropoiesis underground and adapting it to life underground in an atmosphere of abrupt and sharp fluctuations of O2 supply. In neonates, Rattus liver and kidney express higher Epo levels than Spalax under both normoxia and hypoxia, probably due to Rattus ineffective erythropoiesis during embryonic life and its birth in a poorly ventilated breeding nest under ground. Adult Rattus kidney and liver, and adult Spalax liver express similar levels of Epo mRNA under normoxia and hypoxia. However, adult Spalax hypoxic kidney, the major site of erythropoietin production in adult mammals, shows levels that were twice as high as that of Rattus. Spalax expresses remarkably higher levels of HIF-1alpha mRNA than Rattus at all developmental stages studied, which peaked in neonates, as an adaptation against hypoxia.

Gene Expression and Organ Damage in Sickle Transgenic Mouse Kidneys Exposed to Progressive Hypoxia.

We have further examined the hypoxia-induced gene expression in the sickle transgenic mouse model with high expression of human βS and α globin that is homozygous for the mouse β-major deletion, NY1DD (Fabry et al, PNAS 89:12159, 1992) exposed to hypoxia (8% O2/92% N2) for 2 or 4 days vs normoxic conditions. Gene expression in the kidney was studied using cDNA expression microarrays, analyzed by GenePix software and the Significance Analysis of Microarrays (SAM) program, and validated by real-time PCR and immunocytochemistry. Data analyzed by hierarchical clustering (that segregates genes expressed in the hypoxic kidneys vs the normoxic kidney) and SOTA (self-organizing tree algorithm) revealed the effect of hypoxia on different clusters of genes. In one cluster, the effect of hypoxia on heme oxygenase-1 (HO-1), was particularly striking. HO-1 is slightly downregulated in normoxic kidneys and becomes up-regulated 2 and 4 fold on days 2 and 4 of hypoxia. HO-1 was also observed upon immunocytochemical staining with HO-1 antibodies (Calbiochem) in kidney sections from 4 kidneys harvested from 2 mice exposed to either 2 or 4 days hypoxia (4 mice total) and 2 kidneys each from a C57BL and a NY1DD mouse harvested under room air. Mice sacrificed under room air had negligible staining for HO-1. However, NY1DD mice maintained under hypoxia for 2 days had moderate HO-1 staining in some of the proximal tubules near the surface of the cortex and sickled cells in the vasculature. NY1DD mice exposed to 4 days of hypoxia had more intense staining for HO-1 in the proximal tubules that extend deeper towards the medulla. Histological examination of kidney sections confirmed red cell congestion and sickling predominantly in medullary vessels where hypoxia is maximal. HO-1, the rate-limiting enzyme in the heme degradation pathway, is inducible in the kidney (Pimstone et al, J Clin Invest 50:2042, 1971) and increased in sickle mouse kidneys (Nath et al, Am J Path 158:893, 2001). HO-1 also protects cells from oxidant damage. Other very up-regulated genes include myosin Vb (involved in membrane remodeling) and lipocalin 2 (present in distal tubules/collecting ducts and modulates the cellular response to injury). Genes that were down-regulated include calbindin 28K (a cytosolic vitamin D dependent calcium binding protein that regulates calcium homeostasis potentially disrupted by hypoxia), metallothionein 2 (a zinc binding protein and oxidant scavenger that blocks the most reactive oxidant species including peroxynitrite and superoxide radical), fructose bisphosphatase (a gluconeogenic enzyme located in the proximal nephron and a marker for glomerular damage) and the sodium/calcium exchanger (downregulated in renal ischemia/reperfusion injury as a mechanism to protect the cell from unregulated calcium influx). Identification of genetic modifiers of renal pathophysiology in sickle cell anemia is the first step in understanding the clinical heterogeneity among SS patients. These studies may also reveal novel metabolic pathways in the SS kidney and may, therefore, hold potential for therapeutic intervention.

Differential gene expression in the kidney of sickle cell transgenic mice: upregulated genes

The S+S-Antilles transgenic mouse used in this study has renal defects similar to those seen in sickle cell anemia patients: congested glomeruli, medullary fibrosis, renal enlargement, vasoocclusion, and a urine concentrating defect. We used gene expression microarrays to identify genes highly up-regulated in the kidneys of these mice and validated their expression by real-time PCR. Kidney hypoxia, as demonstrated by the presence of deoxyhemoglobin, was detected by blood oxygen dependent magnetic resonance imaging (BOLD-MRI). Some of the up-regulated genes included cytochrome P450 4a14, glutathione- S-transferase α-1, mitochondrial hydroxymethylglutaryl CoA synthase, cytokine inducible SH-2 containing protein, retinol dehydrogenase type III, arginase II, glycolate oxidase, Na/K ATPase, renin-1, and alkaline phosphatase 2. An increase in enzyme activity was also demonstrated for one of the up-regulated genes (arginase II). These genes can be integrated into several different pathophysiological processes: a hypoxia cascade, a replacement cascade, or an ameliorating cascade, one or all of which may explain the phenotype of this disease. We conclude that microarray technology is a powerful tool to identify genes involved in renal disease in sickle cell anemia and that the identification of various metabolic pathways may open new avenues for therapeutic interventions.

Differential gene expression in the kidney of sickle cell transgenic mice: upregulated genes

The S+S-Antilles transgenic mouse used in this study has renal defects similar to those seen in sickle cell anemia patients: congested glomeruli, medullary fibrosis, renal enlargement, vasoocclusion, and a urine concentrating defect. We used gene expression microarrays to identify genes highly up-regulated in the kidneys of these mice and validated their expression by real-time PCR. Kidney hypoxia, as demonstrated by the presence of deoxyhemoglobin, was detected by blood oxygen dependent magnetic resonance imaging (BOLD-MRI). Some of the up-regulated genes included cytochrome P450 4a14, glutathione- S -transferase α-1, mitochondrial hydroxymethylglutaryl CoA synthase, cytokine inducible SH-2 containing protein, retinol dehydrogenase type III, arginase II, glycolate oxidase, Na/K ATPase, renin-1, and alkaline phosphatase 2. An increase in enzyme activity was also demonstrated for one of the up-regulated genes (arginase II). These genes can be integrated into several different pathophysiological processes: a hypoxia cascade, a replacement cascade, or an ameliorating cascade, one or all of which may explain the phenotype of this disease. We conclude that microarray technology is a powerful tool to identify genes involved in renal disease in sickle cell anemia and that the identification of various metabolic pathways may open new avenues for therapeutic interventions.

Molecular cloning and characterization of a hypoxia-responsive CITED3 cDNA from grass carp

We have isolated a 1586-bp full-length CITED3 cDNA from grass carp which specifies for a cAMP-responsive element-binding protein/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain protein. The cDNA, designated as gcCITED3, has an open reading frame of 762 bp and encodes a protein of 253 amino acids with a predicted molecular mass of 28.3 kDa and p I of 6.4. Pairwise comparison showed that gcCITED3 shares high sequence identity with the CITED3 of zebrafish (94%), chicken (72%) and Xenopus (59%). Northern blot analysis indicated that gcCITED3 is most highly expressed and responsive to hypoxia in the carp kidney. Hypoxic induction was also observed in heart, albeit at a lower level. This is the first report on the isolation of a hypoxia-responsive CITED3 gene from fish.

Isolation, characterization and expression analysis of a hypoxia-responsive glucose transporter gene from the grass carp, Ctenopharyngodon idellus.

Glucose transporters (GLUTs) have been implicated in adaptive and survival responses to hypoxic stress in mammals. In fish, the expression and regulation of GLUT in relation to hypoxia remains unexplored. Here we describe the identification of a hypoxia-responsive glucose transporter gene (gcGLUT) and the corresponding full-length cDNA from the grass carp. The gene spans approximately 11 kb of genomic sequence and consists of 12 exons and 11 introns, and an open reading frame (ORF) of 1599 bp encoding a polypeptide of 533 amino acids, with a predicted molecular mass of approximately 57 kDa and a pI of 8.34. blastx analysis showed that the ORF shared high sequence identity with the GLUT1 (57-59%), GLUT3 (59-60%) and GLUT4 (55-59%) proteins from different vertebrates. Comparative analysis of GLUT genomic structures showed that the arrangement of exons and position of split codons are highly conserved amongst members of the class I GLUTs suggesting that these genes share a common ancestor. Phylogenetic analysis indicated that gcGLUT is most closely related to the GLUT3 proteins. Northern blot analysis showed that the 3.1-kb gcGLUT transcript was most abundantly expressed and responsive to hypoxia in kidney. Up-regulated expression by hypoxia was also evident in eye and gill, but differential patterns of expression were observed. Low expression levels detected in brain, heart, liver and muscle were not responsive to hypoxic stress.