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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 1, 2004 as doi:10.1096/fj.04-2758fje. |
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Laboratory of Animal Molecular Evolution, Institute of Evolution, University of Haifa, Mt. Carmel, Haifa, Israel
2 Correspondence: E-mail: aaron{at}esti.haifa.ac.il
SPECIFIC AIMS
We have previously cloned Epo and HIF-1
genes from blind subterranean mole rats (Spalax ehrenbergi superspecies) and have shown dramatic differences in mRNA expression levels compared with Rattus in adult kidneys under normoxic and hypoxic conditions. Under hypoxia, Spalax Epo mRNA reached significantly higher levels compared with Rattus and its response was time- and stress-dependent. HIF-1 mRNA was 2-fold higher in adult Spalax normoxic kidney and demonstrated a 2-fold increase at severe hypoxia of 3% O2.
However, in field measurements, 7.2% O2 and 6.1% CO2 were recorded in Spalax’s natural underground habitat in flooded heavy clay soils during the rainy season. The flooding season coincides with the breeding period (i.e., the gestation period and the delivery of pups occur in the most stressful hypoxic period). Therefore, we decided to study the adaptive mechanisms and strategies used by Spalax for tolerating hypoxia in different developmental stages compared with Rattus. Real-time PCR was used to compare the expression of Epo and HIF-1 mRMA in developing Spalax and Rattus.
PRINCIPAL FINDINGS
1. Epo expression
The expression patterns of Epo mRNA differ between the two rodents (Fig. 1
): 1) at prenatal stages Spalax liver and kidney produce higher Epo mRNA than Rattus; 2) at neonatal stages the expression level of Epo mRNA in Rattus exceeds the levels measured in Spalax under normoxic and hypoxic conditions and in both tissues; 3) adult Epo mRNA was undetectable in normoxic Rattus and Spalax livers and demonstrated very low expression in kidneys; 4) hypoxia induced a 2-fold higher response of Epo mRNA in adult Spalax kidney than in Rattus; 5) Epo mRNA response to hypoxia in adult liver is similar in both species (however, only 50% of the levels measured in the Spalax hypoxic kidney); and 6) in Rattus, the kidney becomes the major tissue of Epo mRNA production in response to hypoxia between 7 and 14 days after birth, but no similar switch was noticed in Spalax newborns at this stage.
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2. HIF-1 expression
Different from Epo mRNA expression, the overall pattern of expression of HIF-1 mRNA is similar in Spalax and Rattus with the exception of its expression pattern in Rattus liver (Fig. 2
). Nevertheless, quantitative differences between the two rodents have been noticed 1) generally, highest levels of HIF-1 mRNA were measured during neonatal stages in Spalax kidneys and livers and in Rattus kidneys; 2) levels of HIF-1 mRNA in Spalax kidneys were the highest among all comparisons of both rodents at all developmental stages studied; 3) a high expression level of HIF-1 mRNA was noticed in the normoxic kidney of 7- and 14-day-old Spalax newborns as well as the hypoxic kidney of 7-day-old Spalax newborns, which demonstrated 3- to 4-fold higher amounts than Rattus; 4) Spalax liver HIF-1 mRNA levels were notably higher than Rattus liver in fetal, neonatal, and normoxic adult stages; 5) under hypoxia, HIF-1 mRNA levels were down-regulated in neonatal stages of both rodents, with one exception in the Spalax liver of 7-day-old newborns; 6) in adults, Spalax kidney and Rattus liver responded to hypoxia by 
2-fold increase in HIF-1 mRNA levels; however, the level in the hypoxic adult Spalax kidney was higher than in the hypoxic adult Rattus liver.
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CONCLUSIONS AND SIGNIFICANCE
We investigated the expression of Epo and HIF-1 mRNA in fetal, neonatal, and adult kidney and liver of blind subterranean mole rats (Spalax) compared with white rats (Rattus). Spalax showed unique adaptive expression patterns of Epo and HIF-1 compared with Rattus at different developmental stages.
The results indicate higher Epo mRNA levels in Spalax fetal liver and kidney compared with Rattus. Seven days after birth, the levels of Rattus liver and kidney Epo were equal and demonstrated about an 3-fold increase in hypoxia. In 14-day-old pups, there was an increase in Epo mRNA in normoxic Rattus liver and a decrease in its kidney. However, the Rattus kidney response to hypoxia at this stage was higher than in the liver and achieved the maximum levels of Epo measured in this study. Unlike the sheep model, where the liver to kidney switch is initiated in utero in the last trimester of gestation, the switch in Rattus appears to start after birth. The Rattus liver ability to express Epo is retained, but the hypoxia-induced Epo transcription in the kidney exceeds that of the liver. Studies of developing Rattus showed that maximum Epo mRNA in the liver was on day 7 after birth in normoxic and hypoxic animals and declined during development, while kidney Epo mRNA increased during development. A similar decrease of Epo mRNA, as in 14-day-old pups of the normoxic Rattus kidney, was also reported in the porcine kidney. Our results demonstrate that under hypoxia, the adult liver and kidney Epo mRNA levels of Rattus are almost equal, though previous studies showed that nonrenal Epo protein levels in severe anemic nephrectomized Rattus are only 15% of the total Epo concentrations. It is noteworthy that Epo mRNA in human hepatoma cells Hep3B increased 50-fold under hypoxia, but only a 10-fold increase was noticed in nuclear run-off experiments, indicating the involvement of posttranscriptional mechanisms in Epo regulation.
Spalax, in contrast, showed higher Epo mRNA levels than Rattus in fetal stages but lower in neonates. Epo cannot cross the ovine and human placenta, and evidence for the physiological role of Epo in fetal erythropoiesis was shown earlier. This high expression of Epo mRNA in embryonic stages in Spalax may play a critical role since pregnancy and delivery occur under highly hypoxic/hypercapnic conditions in the most stressful rainy season, when flooding for long periods prevents ventilation of the burrows, restricts space, and increases hypoxic stress. Thus, developed fetal erythropoiesis may be critical for the adaptation of underground animals that encounter hypoxic/hypercapnic stress. Rattus gives birth in underground nests, where the ventilation is less effective than the normal atmosphere in its usual above-ground habitat. As the fetal erythropoietic system of Rattus is poorly responsive compared with that of Spalax, the newborns face a temporary hypoxic environment. This may explain why Epo levels in the liver and kidney of Rattus newborns under normoxic and hypoxic conditions are higher than in Spalax newborns. Spalax newborns are aerated in a nest above ground level, experiencing better ventilation than in adult underground tunnels.
Our results indicate higher levels of HIF-1 mRNA in Spalax kidney and liver than in Rattus from fetal to neonatal to adult stages. HIF-1 plays a key role in the establishment of complex physiological systems responsible for oxygen delivery during development, and elevated levels of HIF-1 mRNA may mediate adaptive responses to hypoxia via trans-activation of genes encoding angiogenic and erythropoietic factors. Due to the wide-ranging activities of HIF-1 that trans-activates a battery of different genes, some of our results of HIF-1 mRNA expression, especially the decreased levels under hypoxia in some pre-adult stages, are difficult to interpret. The decrease in HIF-1 mRNA expression under hypoxia was noted in developmental stages and tissues where HIF-1 mRNA is relatively highly expressed in ambient conditions. The decrease under hypoxia could result from feedback inhibition generated by one or more of the HIF-1-activated target proteins.
Our findings that elevated HIF-1 mRNA in the developing Spalax may be among the major events that contribute to its morphological and physiological properties allowing it to survive in the hypoxic environment to which it is born and spends its entire life. This expression may contribute to the significantly higher capillary density found in Spalax, as well as its higher erythrocyte count, and might explain a major adaptive strategy of Spalax to cope with hypoxia.
Rattus HIF-1 mRNA achieved its maximum in normoxic kidneys 7–14 days after birth and declined in adults. In contrast, Rattus liver levels increased gradually during development and, in adults, exceeded the levels of Rattus kidneys under normoxia and hypoxia. These data coincide with published investigations on mice, where the liver HIF-1 mRNA levels were lower than in the kidney during development. The opposite pattern was exhibited in adults.
Preconditioning to chronic hypoxia is known to improve cardiac ischemic tolerance, though the molecular mechanisms of this protection are poorly understood. Such adjustment may protect the heart under conditions that require enhanced work and, consequently, increased metabolism. Moreover, it may influence the cardiopulmonary system. Spalax embryos are exposed to chronic hypoxia in the pregnancy period during the rainy season, occasionally under flooding of its habitat. The pattern of Epo and HIF-1 mRNAs expression in Spalax starting with prenatal life described here may indeed suggest adaptation to the hypoxic stress beginning in the embryonic stages that prepare it for continuously enhanced digging efforts under abrupt fluctuations of O2 supply.
Some of the unique molecular mechanisms used by Spalax as adaptive strategies to cope with hypoxia were shown by: 1) a high Epo mRNA expression in Spalax kidney and liver in embryonic stages; 2) a strong response of Spalax kidney to hypoxia; 3) very high HIF-1 mRNA levels in the Spalax kidney at all developmental stages; and 4) the contribution of the Spalax liver to HIF-1 mRNA, much higher during all developmental stages, starting at embryonic stages (Fig. 3
).
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Functional genomic studies of comparative physiology of hypoxia are suggested as a potential approach to understand the genetic basis of the physiological processes and evolutionary adaptations of wild and domestic animals. Future studies in our laboratory will aim at probing the extensive adaptive battery of genetic responses of Spalax to variable hypoxic stress through comprehensive functional genomic studies and at unraveling the numerous genes cooperating in Spalax hypoxia tolerance. These could later be used in human gene therapy in the fight against ischemia and cancer and may contribute to life in environmental extremes such as outer space flights and deep-sea diving.
FOOTNOTES
1 These authors share senior authorship of this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2758fje;
This article has been cited by other articles:
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  M. Band, I. Shams, A. Joel, and A. Avivi Cloning and in vivo expression of vascular endothelial growth factor receptor 2 (Flk1) in the naturally hypoxia-tolerant subterranean mole rat FASEB J, January 1, 2008; 22(1): 105 - 112. [Abstract] [Full Text] [PDF]
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 O. Ravid, I. Shams, N. Ben Califa, E. Nevo, A. Avivi, and D. Neumann An extracellular region of the erythropoietin receptor of the subterranean blind mole rat Spalax enhances receptor maturation PNAS, September 4, 2007; 104(36): 14360 - 14365. [Abstract] [Full Text] [PDF]
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 A. Avivi, L. Brodsky, E. Nevo, and M. R. Band Differential expression profiling of the blind subterranean mole rat Spalax ehrenbergi superspecies: bioprospecting for hypoxia tolerance Physiol Genomics, January 12, 2007; 27(1): 54 - 64. [Abstract] [Full Text] [PDF]
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