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. 2007 Jun 21;583(Pt 1):329–336. doi: 10.1113/jphysiol.2007.133454

Soluble erythropoietin receptor is present in the mouse brain and is required for the ventilatory acclimatization to hypoxia

Jorge Soliz 1, Max Gassmann 1,*, Vincent Joseph 2,*
PMCID: PMC2277219  PMID: 17584830

Abstract

While erythropoietin (Epo) and its receptor (EpoR) have been widely investigated in brain, the expression and function of the soluble Epo receptor (sEpoR) remain unknown. Here we demonstrate that sEpoR, a negative regulator of Epo's binding to the EpoR, is present in the mouse brain and is down-regulated by 62% after exposure to normobaric chronic hypoxia (10% O2 for 3 days). Furthermore, while normoxic minute ventilation increased by 58% in control mice following hypoxic acclimatization, sEpoR infusion in brain during the hypoxic challenge efficiently reduced brain Epo concentration and abolished the ventilatory acclimatization to hypoxia (VAH). These observations imply that hypoxic downregulation of sEpoR is required for adequate ventilatory acclimatization to hypoxia, thereby underlying the function of Epo as a key factor regulating oxygen delivery not only by its classical activity on red blood cell production, but also by regulating ventilation.

Erythropoietin (Epo) is a pleiotropic cytokine most commonly recognized as a factor that improves arterial oxygen carrying capacity (Gassmann et al. 2003; Sasaki, 2003). Upon sustained hypoxaemic conditions Epo synthesis in the kidney is accelerated, resulting in increased plasma Epo levels (Eckardt & Kurtz, 2005; Stockmann & Fandrey, 2006). Binding of Epo to its receptor (EpoR) on erythrocyte progenitor cells in the bone marrow maintains their viability, promotes cell division, and increases haemoglobin synthesis culminating in increased haematocrit levels (Fisher, 2003; Jelkmann, 2005). While this endocrine loop is essential, it is not sufficient to ensure fast and adequate oxygen supply under conditions of limited oxygen availability such as in high altitude or in patients suffering from impaired respiratory functions. Sustained hypoxic exposure also affects the respiratory control network, leading to a progressive, species-dependent increase in minute ventilation over minutes, hours or even weeks. This process is defined as the ventilatory acclimatization to hypoxia (VAH) and persists for a few days following return to normoxic conditions. VAH mainly relies on increased sensitivity of peripheral chemoreceptors to hypoxia, associated with central facilitation of the ventilatory output (Forster et al. 1981; Dempsey & Forster, 1982; Smith et al. 1986; Powell et al. 2000a; Prabhakar & Jacono, 2005). Here we show that the soluble form of EpoR (sEpoR) is involved in the VAH.

Endogenous expression of Epo has been reported in the nervous system (Digicaylioglu et al. 1995; Gassmann et al. 2003) and transgenic mice overexpressing human Epo in neural cells have shown that brain-derived Epo facilitates VAH (Soliz et al. 2005). Furthermore it was reported that the EpoR is present in respiratory areas of the brainstem, such as NK-1R positive neurons in the pre-Bötzinger complex (proposed as the respiratory rhythm generator), and the nucleus tractus solitarii, which relays input from peripheral chemoreceptors to the central respiratory areas to increase ventilation upon hypoxic exposure (Soliz et al. 2005). In analogy to several other members of the cytokine superfamily type I transmembrane proteins, EpoR is also synthesized in a soluble form (sEpoR) that corresponds to the extracellular domain of the complete receptor (Nagao et al. 1992; Harris & Winkelmann, 1996; Westphal et al. 2002). Synthesis of the sEpoR occurs by alternative splicing of EpoR mRNA. Following secretion into the extracellular fluid, sEpoR binds Epo, thereby limiting its ability to bind EpoR (Kuramochi et al. 1990; Baynes et al. 1993). While the presence of sEpoR has been reported in plasma and several tissues including liver, spleen, kidney, heart and bone marrow (Fujita et al. 1997), its cerebral expression and function has not been investigated yet. In the present work we show that sEpoR is expressed in the mouse brain. Moreover, down-regulation of sEpoR upon exposure to chronic hypoxia reflects the key role of sEpoR to regulate the VAH.

Methods

Measurement of ventilation

A total of 20 adult male C57/Bl6 mice (Charles River Canada) at the age of 3 months were housed for 1 week at constant temperature (21 ± 1°C) and light (from 07.00 to 19.00 h). Brain tissue and blood were collected from 10 mice to measure cerebral and plasma Epo before (n = 5) and after (n = 5) VAH, as described below. The other 10 animals were used to measure ventilation in normoxia and acute hypoxia, before and after being surgically connected to a cannula (see below) and exposed to 10% O2 for 3 days. A whole-body flow-though plethysmography (EMKA Technologies, France) was used to monitor ventilation as described (Soliz et al. 2005). Briefly, mice were placed in a 600 ml chamber continuously supplied with airflow at 0.7–0.8 l min−1 using flow restrictors. Ventilation (V˙E) was calculated as the product of tidal volume (VT) and respiratory frequency (fR) and normalized to 100 g of body weight (e.g. ml min−1 (100 g)−1). Ventilatory measurements were performed in normoxia (21% O2). Acute hypoxia was achieved by flushing air balanced in N2. The fraction of inspired O2 (FIO2) in the chamber was gradually decreased from 21% to 10% O2 during 15 min. Respiratory recordings at 10% O2 were performed for 20 min. At the end of each experiment, body weight and body temperature (rectal thermocouple, Physitemp, USA) were measured. In parallel to ventilation measurements, O2 consumption (V˙o2, ml min−1 (100 g)−1) and CO2 production (V˙CO2, ml min−1 (100 g)−1) were determined by O2 and CO2 analysers (AEI technologies). Ventilatory measurements were performed under similar conditions before and after chronic hypoxic exposure. All animal procedures have been approved by the ethics committee of animal care at Laval University and followed the guidelines of the Canadian Council on Animal Care.

Intracerebroventricular infusions

For intracerebroventricular infusion, the animals were deeply anaesthetized with a gas mixture of 4% halothane, 70% N2 and O2, and maintained by reducing the inspired halothane concentration to 1–1.5%. Body temperature of the mice was maintained at 37°C using a temperature-controlled heating pad. They were then stereotaxically implanted with a permanent stainless-steel cannula (Alzet, Durect Corp., Toronto, Canada) into the left lateral ventricle of the brain at coordinates (Bregma: 0; L: 1 mm, and DV: −3 mm) according to Paxinos and Watson (Paxinos & Watson 2000). Subsequently an osmotic minipump (Alzet pump model 1003D; 100 μl; flow rate 1.4 μl h−1; Durect Corp.) was connected to the cannula with medical grade vinyl tubing and placed in a s.c. pocket in the dorsal region. Pumps were filled with soluble EpoR solution (50 μg; R & D systems, Inc., USA) or vehicle (phosphate buffer). After surgery, mice were allowed to recover in isolation and observed for at least 1 h until complete recovery from anaesthesia.

Quantification of Epo, EpoR and sEpoR

Mice were anaesthetized by an intraperitoneal injection of a mixture of ketamine (80 mg kg−1) and xylazine (10 mg kg−1). Blood samples were drawn by cardiac puncture into heparinized Ependorff tubes and plasma was collected after samples centrifugation at (18 000 g) Brain samples were obtained after transcardial perfusion of mice with phosphate buffer (0.1 m, pH 7.4) and immediately frozen in liquid nitrogen. For the extraction of total protein, brain tissues were complemented with lysis buffer, homogenized and centrifuged, and supernatants were harvested. Protein concentrations of both brain and plasma were determined by the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Epo levels in brain and plasma were quantified using an 125I-Epo-based radioimmunoassay (RIA) (Amersham, Zurich, Switzerland), according to previously published protocols (Kilic et al. 2005). The lower detection limit of our RIA was 4 U l−1, and the intrassay/interassay variances were < 2% and < 6%, respectively. For quantification of EpoR and sEpoR in brain samples, equal amounts of extracts were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Transfer and equal loading of proteins were confirmed by Ponceau S staining. For immunoblotting, the membranes were incubated with an EpoR antibody (H-194, Santa Cruz Biotechnology Inc., Lab Force ΔG CH-4208, Nummingen, Switzerland; 1: 200) and with the appropriate horseradish peroxidase-conjugated secondary antibody. EpoR and sEpoR were detected at the expected molecular weight (64 and 30 kDa, respectively; Nagao et al. 1992) by enhanced chemiluminescence and quantified using a Gel Doc 2000 scanner with Quantity One software (Bio-Rad). The membrane was then stripped (10 ml stripping buffer complemented with 70 μl β-mercaptoethanol) for 20 min and incubated with monoclonal anti-β-actin primary antibody (AC-15, Sigma, USA; 1: 5000), followed by suitable secondary antibody. The intensity of the Epo and sEpoR bands was then normalized to actin.

Statistical analysis

Analysis was performed using the StatView software (Abacus Concepts, Berkeley, CA, USA). The reported values are means ±s.d. For simple measurements, data were analysed by one-way ANOVA. For hypoxic ventilation responses, data were analysed by two-way ANOVA for repeated measurements. Differences were considered significant at P < 0.05.

Results

sEpoR is endogenously expressed in brain and is down-regulated upon chronic exposure to hypoxia

We performed Western blot and RIA analysis on mouse brain extracts. Both EpoR and sEpoR were detected under normoxia and after 3 days of hypoxic exposure (10% O2; Fig. 1). While the levels of EpoR (Fig. 1A) and Epo (Fig. 1C) synthesized in the hypoxic brain were not significantly altered compared to the normoxic control, hypoxia reduced the expression of sEpoR in the brain by 62% (Fig. 1B). Exposure to chronic hypoxia increased plasma Epo in mice (54%; Fig. 1D), confirming that the animals were indeed hypoxaemic. These data demonstrate that sEpoR is endogenously expressed in the mouse brain and that its expression is down-regulated during hypoxia.

Figure 1.

Figure 1

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sEpoR is expressed in the mouse brain and is down-regulated upon hypoxic exposure Brain tissue and plasma were collected from mice kept at normoxia (Nx; n = 5) and after 3 days of continuous exposure to hypoxia at 10% O2 (Hx; n = 5). EpoR and sEpoR were detected in the normoxic (Nx) and hypoxic (Hx) brain (A and B). Note that Hx but not Nx reduced the expression of sEpoR (B). The level of brain-derived Epo did not increase after Hx (C), while plasma Epo was increased after hypoxic stimulus as expected (D). Western blot analysis is exemplified at the top of A and B, including the negative control (no primary antibody). **P < 0.01, ***P < 0.001.

Intracerebroventricular infusion of sEpoR blocks the ventilatory acclimatization to hypoxia

Considering that mice achieve VAH within 3 days (Olson & Saunders, 1987; Malik et al. 2005), we hypothesized that the observed decrease in sEpoR expression in chronic hypoxia enhances the ability of Epo to facilitate VAH. We tested this hypothesis by performing respiratory measurements in normoxia and also acute hypoxia (10% O2) before and after acclimatization to chronic hypoxia (10% O2 for 3 days; Fig. 2A). Minute ventilation (V˙E), respiratory frequency (fR) and tidal volume (VT), as well as metabolic variables and rectal temperature were evaluated in 10 adult male mice before acclimatization. During the following 3 days mice received intracranial infusion of sEpoR (0.7 μg of sEpoR per hour over 3 days, n = 5) and were compared to vehicle-treated controls (n = 5). During the treatment animals were exposed to normobaric chronic hypoxia (10% O2) and then returned to room air for a second evaluation of ventilation under normoxic and acute hypoxic conditions. No differences were found between vehicle and sEpoR treated groups before acclimatization (Fig. 2B–G, normoxia before treatment). However, while control mice showed a large increase in normoxic ventilation after chronic exposure to hypoxia, this ventilatory acclimatization was completely blocked by sEpoR infusion (Fig. 2B–F). As metabolic rate was also increased following hypoxic exposure in control mice (but not in sEpoR-treated animals), we corrected ventilation to metabolic rate. We observed that chronic hypoxia-mediated hyperventilation (higher V˙E/V˙o2–Fig. 2F) was blunted by the sEpoR infusion. This is consistent with previous findings (Soliz et al. 2005) showing that Epo's effect on the ventilatory response to hypoxia is neither due to altered body metabolism (Fig. 2E) nor to alterations in body temperature (Fig. 2G).

Figure 2.

Figure 2

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Intracerebroventricular infusion of sEpoR abolishes VAH A, schematic diagram showing the sequential steps of the experiment protocol. Normoxic (Nx) and acute hypoxic (acute Hx) minute ventilation was evaluated before and after VAH (shaded area) in mice receiving continuous intracerebroventricular infusion of sEpoR or vehicle. Before and after acclimatization, mice were kept at normoxic conditions and the minute ventilation was determined. After acclimatization, control mice showed a large increase of normoxic ventilation (V˙E), respiratory frequency (fR), tidal volume (VT) and oxygen consumption (V˙o2). In contrast, this elevation in ventilation was abolished in the sEpoR group, showing that no acclimatization occurred (B, C, D and E). Accordingly, the oxygen convection ratio (V˙E/V˙o2) increased progressively in the control mice group but remained unaltered in sEpoR animals (F). These differences were not caused by altered body temperature (G). ***P < 0.001.

The acute hypoxic ventilatory response upon VAH is blunted in mice treated with sEpoR

As ventilatory acclimatization to hypoxia is also manifested by an augmented response to subsequent acute hypoxia (Powell et al. 2000b; Malik et al. 2005; Soliz et al. 2005), acute hypoxic ventilation before and after the acclimatization period was compared (Fig. 3). In control mice acute hypoxic ventilation was doubled whereas in sEpoR treated mice it was completely abolished (Fig. 3A), due to increased tidal volume (VT; Fig. 3C) rather than respiratory frequency (fR; Fig. 3B) after acclimatization. These data demonstrate that sEpoR efficiently antagonizes Epo's ventilatory effect in brain. In agreement with the model of VAH proposed by Powell (Fig. 3D), control mice gradually increased minute ventilation from normoxia to acute hypoxia before acclimatization, and from hypoxia before acclimatization to acute hypoxia after acclimatization. In contrast, this effect was inhibited in sEpoR-infused animals (Fig. 3E). A similar response was observed in the oxygen convection ratio (Fig. 3F). Once again, these alterations were not caused by differences in body temperature after acclimatization (vehicle versus sEpoR in Nx: 37.2 ± 0.4 to 37.7 ± 0.7°C; in Hx: 35.9 ± 0.5 to 36.3 ± 0.4°C).

Figure 3.

Figure 3

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Exogenous application of sEpoR abolishes subsequent augmentation of acute hypoxic ventilation induced by VAH Acute hypoxic (acute Hx) ventilation was evaluated before and after VAH in mice infused with sEpoR or vehicle (A, B and C). In line with Powell (D), the VAH is observed in vehicle-infused mice as a supplemental increase of respiratory parameters measured in acute hypoxia after acclimatization (Hx after) compared to acute hypoxia before (Hx before) acclimatization (E). Soluble EpoR administration abolished this process (A, B, C and E). The oxygen convection ratio (V˙E/V˙o2) follows a similar dynamic (F). The 15 min gradual reduction of FIO2 from normoxia to acute hypoxia is represented by the black triangles. *P < 0.05, **P < 0.01, ***P < 0.001.

Intracerebroventricular infusion of sEpoR down-regulates the expression of cerebral Epo and EpoR

Following the ventilatory measurements described above, brain and plasma were collected to quantify Epo (by RIA) as well as EpoR and sEpoR (by Western blot). Treatment with sEpoR dramatically reduced the amount of detectable EpoR and Epo (Fig. 4A and C). The level of sEpoR did not increase (Fig. 4B) probably due to the rapid metabolism of exogenous sEpoR or because the resulting Epo–sEpoR complex was undetectable by either the Epo or EpoR antibody. Mice treated with sEpoR during chronic hypoxic exposure had higher levels of plasma Epo (Fig. 4D), a consistent sign of impaired VAH. These results imply that reduced brain-derived Epo availability, as induced by sEpoR infusion during chronic hypoxia, abolishes the progressive augmentation of ventilation, thus compromising the physiological acclimatization process to hypoxia.

Figure 4.

Figure 4

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Intracerebroventricular infusion of sEpoR decreases the levels of Epo and EpoR in the brain upon VAH Brain tissue and plasma were collected from mice infused with sEpoR or vehicle after VAH (3 days; 10% O2). Epo in brain and blood was analysed by RIA and EpoR variants were determined by Western blot. Western blot analysis is exemplified at the top of A and B, including the negative control (no primary antibody). Compared to the untreated control, the level of EpoR and Epo were decreased (A and C), while sEpoR (B) did not alter in sEpoR-infused mice. Plasma Epo was higher in the sEpoR group compared to control (D). *P < 0.01, **P < 0.001.

Discusion

Our results demonstrated for the first time that endogenously synthesized Epo and sEpoR in the central nervous system play a crucial role in the physiological process related to long-term oxygen homeostasis, thereby ultimately contributing to the same goal as plasma Epo, i.e. increasing the overall capacity of oxygen delivery. This finding is in agreement with previous data obtained in genetically modified mice overexpressing Epo in the brain, in which the hypoxic ventilatory response persists after carotid body denervation (Soliz et al. 2005). Our data provide convincing evidence that the regulation of Epo and sEpoR in the central nervous system and at a systemic level is finely tuned to establish a balance between ventilatory and haematopoietic control of oxygen carrying capacity as shown by the proposed model for hypoxic acclimatization presented in Fig. 5. Acute hypoxia triggers during the first minutes the response of the chemoreflex loop that leads to an immediate increase in ventilation termed hypoxic ventilatory response (short-term regulation). If hypoxia persists, the organism enters a process of acclimatization in which long-term regulatory responses are initiated. Thereby, central and systemic availability of Epo is increased. In mice, hypoxic induction of Epo mRNA expression in the kidneys is maximal after 2 h, while plasma Epo protein level peaks after 20 h, and despite continuous hypoxia both mRNA and protein are quickly lowered (Abbrecht & Littell, 1972; Chikuma et al. 2000). Most probably, the elevated Epo concentration in the blood immediately impacts the carotid bodies thereby contributing to the modulation of ventilation. Note that we recently showed a dense staining of EpoR in glomus cells (Soliz et al. 2005). At the same time, increased Epo plasma levels progressively augments the number of circulating red cells to augment the oxygen carrying capacity. In brain, the Epo mRNA reaches a peak expression after 4 h of hypoxia. Additionally, we observed that brain-derived Epo protein levels peaks after 24 h (unpublished data) and then decreased to basal level after 72 h, this kinetic being consistent with the HIF-1 expression in the hypoxic brain (Stroka et al. 2001). However, during the decay of brain Epo, sEpoR expression is decreased to maintain continuously high Epo availability. The mechanism by which cerebral Epo controls VAH remains to be elucidated. We previously showed that Epo regulates breathing by altering catecholaminergic metabolism in the brainstem (Soliz et al. 2005). However we do not exclude the involvement of other factors, such as nitric oxide (Lipton et al. 2001) and neuroglobin (Hundahl et al. 2005), both up-regulated in brain after hypoxic induction (Fig. 5).

Figure 5.

Figure 5

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Model of ventilatory acclimatization to hypoxia (VAH) showing the contribution of cerebral and plasma Epo During the first minutes of hypoxia, carotid bodies sense the drop of arterial oxygen pressure thus leading the short-term response to hypoxia by promoting a fast increase of ventilation. Persisting hypoxia activates long-term regulation mechanisms. In mice, Epo synthesis in brain and kidney is initiated a few hours after hypoxic exposure. Plasma Epo augments the oxygen carrying capacity (by gradual increase of the haematocrit) as well as contributes to the regulation of ventilation (binding of the EpoR present in the carotid body glomus cells). While we recently showed that catecholamines are involved in central regulation of ventilation, other molecules such as nitric oxide and neuroglobin might be involved, too.

On the other hand, although the mechanism involved are not completely understood, it has been well established that hypoxia causes changes in body metabolism (Gautier, 1996). As such, acute hypoxia induces a decrease in oxygen demand and an increase of ventilation mediated by an augmented chemosensitive drive (Kline et al. 1998; Kline et al. 2000; Kline et al. 2002). In parallel, VAH reestablishes tissue oxygenation by offsetting minute ventilation to a new basal level. Furthermore, we and others reported in previous studies that the metabolic rate is increased after VAH (Malik et al. 2005; Soliz et al. 2005), but the identity of this event is unknown so far. At present, we cannot explain why metabolic rate was not increased in sEpoR infused mice. We speculate that the metabolic cost of elevated minute ventilation, as seen in control mice, is not occurring in sEpoR-treated animals due to their unaltered ventilation. In line with this, we hypothesize that high Epo plasma levels reflect a hypoxaemia that in turn hinders higher metabolic rate.

Our data suggest that cerebral expression of Epo and/or sEpoR in brain is implicated in respiratory disorders occurring at high altitude such as acute and chronic mountain sickness (Leon-Velarde et al. 1998; Joseph et al. 2000; Joseph et al. 2002). Similarly, it is tempting to speculate that this system could also play a role in the respiratory plasticity induced by exposure to intermittent hypoxia, either in adults or in newborn mammals. Therefore, expression and regulation of cerebral Epo and sEpoR are of central importance in the physiological response to hypoxia providing new insights into disease pathogenesis and to generate novel therapeutic approaches.

Acknowledgments

The authors wish to thank Van Diep Doan and Beat Grenacher for their superb assistance and Csilla Becskei, Edith Schneider, Johannes Vogel and Michelle Scott for fruitful discussions. J.S and M.G. are supported by grants of RoFAR and M.G. by the Swiss National Science Foundation and the EU-project ‘Pulmotension’. Animal experiments in Canada were supported by a NSERC grant to V.J.

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