Introduction

Anemia is a frequent complication of chronic renal failure (CRF). Insufficient production of glycoprotein hormone erythropoietin (Epo) is a main causative factor of uremic anemia. Currently, regular administration of recombinant erythropoietin (rEpo) and its newer derivatives, such as derivatives of rEpo-α and -β, is the main clinical treatment (Singh, 2008), and millions of patients have benefited from it. Yet recent studies revealed some adverse effects of this treatment, for example, mortality and cardiovascular complications due to the use of epoetin (Collins et al., 2000; Regidor et al., 2006) and pure red cell aplasia induced by newer derivatives (Casadevall et al., 2002; Bennett et al., 2004; Pollock et al., 2008). These thorny problems have not been settled so far.

Epo gene therapy is another treatment and still under investigation at present. Previous studies proved that direct transfer of Epo gene into the host via either viral or nonviral means could allow sustained Epo secretion to correct the renal anemia (Maione et al., 2000; Rizzuto et al., 2000; Maruyama et al., 2001). But this transfer may lead to constant and high-level production of Epo and even to potentially lethal polycythemia, because the transferred Epo gene was not under the control of physiologic hypoxia-inducible factor-1 (HIF-1) (Johnston et al., 2003; Fabre et al., 2008). Temporal control systems of transgene expression were proved to be able to avoid deleterious Epo secretion (Richard et al., 2005). Yet these systems are complicated in repeated administration and calculating the doses of therapeutic gene.

Hypoxia is a natural physiological condition to regulate Epo expression. When reduced oxygenation of blood reaches the kidney, the Epo gene expression and protein secretion are increased by the fibroblasts of the renal cortex and outer medulla to increase erythropoiesis. The induction of Epo gene transcription in hypoxia needs HIF-1 binding to a hypoxia response element (HRE) lying 3' to the Epo gene (Frede et al., 2011).

HIF-1 is an oxygen-sensitive transcriptional activator. Its primary function is to mediate the adaptation to hypoxia in cells and tissues, leading to the transcriptional induction of a series of genes that participate in angiogenesis, iron metabolism, glucose metabolism, and cell proliferation/survival (Ke and Costa, 2006). HIF-1 consists of a constitutively expressed subunit HIF-1β and an oxygen-regulated subunit HIF-1α. The stability and activity of the α subunit of HIF are regulated by its post-translational modifications such as hydroxylation, ubiquitination, acetylation, and phosphorylation. In normoxia, hydroxylation of two proline residues and acetylation of a lysine residue at the oxygen-dependent degradation domain of HIF-1α trigger its association with pVHL E3 ligase complex, leading to HIF-1α degradation via ubiquitin-proteasome pathway. In hypoxia, the HIF-1α subunit becomes stable and interacts with coactivators such as cAMP response element-binding protein binding protein/p300 and regulates the expression of target genes (Ke and Costa, 2006).

HRE is a key regulatory DNA sequence that controls gene expression specifically in response to low oxygen concentrations (Semenza et al., 1996). HRE can be identified in the 5'- or 3'-flanking regions of various genes, including tyrosine hydroxylase, Epo, vascular endothelial growth factor (VEGF), and several glycolytic enzymes including phosphoglycerate kinase (PGK) (Goldberg and Schneider, 1994; Semenza et al., 1994; Ataka et al., 2003). Utilizing the property that HRE is responsible for binding to the α subunit of HIF-1 to stimulate transcription in hypoxia, Binley et al. developed a hypoxia control system similar to the natural one that can switch Epo gene expression on and off. And this system avoided deleterious Epo secretion and maintained long-term normalization of hematocrit (Hct) in anemic Epo-deficient Epo-TAg transgenic mice (Binley et al., 2002). Yet it was unclear whether this system would work well under the influence of uremia. This study was to investigate this question.

This study adopted a strategy of PGK HRE in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter to construct the hypoxia-responsive promoter (HRE/CMV). Because PGK HRE has a relatively higher responsiveness to hypoxia (Boast et al., 1999) and CMV IE promoter is a good basal promoter that has been widely used as a useful component of eukaryotic expression vectors (Boshart et al., 1985). This study fused human Epo (hEpo) gene to the HRE/CMV promoter to develop the plasmid vectors and employed intramuscular injection as gene delivery method because plasmid injection can be repeated without apparent immune response to the DNA vector (Terada et al., 2002). To know whether this chimeric HRE/CMV promoter would be activated in hypoxic conditions and whether this promoter could direct hEpo gene expression to correct the renal anemia, this study first evaluated the hypoxia-inducible activity of this promoter in vitro and then in vivo in uremic rat model.

Results

HRE/CMV promoter directs reporter gene expression in response to hypoxia in vitro

To assess whether HRE sequences of PGK gene could achieve hypoxic regulation in the context of a CMV IE basal promoter, we substituted the endogenous CMV IE enhancer with HRE sequences of PGK gene in the natural orientation (Figure 1A) and cloned the resulting chimeric HRE/CMV promoter into EGFP vector.

Figure 1
figure 1

(A) HRE/CMV and HREm/CMV promoter were constructed by substituting the endogenous CMV IE enhancer with HRE sequences of PGK gene or mutated HREs in the natural orientation. In the chimeric HRE sequences of PGK gene and HRE mutant oligonucleotides, the core consensus sequence of HRE responsible for HIF-α binding is underlined. A 3-bp substitution in each of the HRE consensus sites in the trimer is boxed. 5'-end Bgl II and 3'-end Sgf I are shown in italics. (B) Cytoplasmic fluorescence in normoxic and hypoxic condition of HeLa cells transfected with pHRE-, pCMV- and pHREm-EGFP (fluorescence microscope Olympus BX-60; magnification × 200; 24 h after transfection). (C) Luciferase activity in normoxic and hypoxic condition of HeLa cells 24 h after transfected with pHRE-, pCMV- and pHREm-Luc. Data represent the means of two independent experiments, each carried out in triplicate. **stands for P < 0.01.

Green fluorescent assay for hypoxia-inducible expression of EGFP showed that pHRE-EGFP-transfected HeLa cells that were incubated in hypoxic conditions had impressive cytoplasmic fluorescence. Whereas mutated HRE construct-transfected cells incubated in both hypoxic and normoxic conditions did not show significant EGFP fluorescence, and pCMV-EGFP transfected cells incubated in both hypoxic and normoxic conditions had similar cytoplasmic fluorescence (Figure 1B).

To determine to what extent the HRE/CMV promoter would induce gene expression in response to hypoxia, we cloned the chimeric HRE/CMV promoter into firefly luciferase receptor vector to examine the luciferase activity. Renilla luciferase activity from pRL-CMV served as an internal control for transfection efficiency. As shown in Figure 1C, a hypoxic induction ratio of chimeric HRE/CMV construct was 20-fold greater in hypoxic conditions than in normoxic conditions, whereas no statistical difference was observed in mutant- or pCMV-Luc-transfected cells. The maximum level of the luciferase activity induced by hypoxia reached the 40% level that the constitutive CMV IE promoter achieved in normoxia. These data indicate that the HRE/CMV promoter can direct hypoxia-induced expression of reporter genes.

HRE/CMV promoter drives the secreting expression of Epo gene in response to hypoxia in vitro

Human Epo gene with an NH2-terminal signal peptide sequence was introduced to the downstream of the chimeric HRE/CMV promoter to create pHRE-Epo, likewise for pHREm-Epo and pCMV-Epo (Figure 2A). After the HeLa cells were transiently transfected with pHRE, pHREm-Epo, pHRE-Epo and pCMV-Epo, and then incubated in hypoxia or normoxia, the mRNA expression level of Epo in HeLa cells were detected using RT-PCR (Figure 2B) and the protein expression level of Epo using western blot (Figure 2C), showing that the Epo mRNA and protein levels in the pHRE-Epo transfected cells were obviously higher in hypoxia than in normoxia, that the Epo levels in the pHREm-Epo transfected cells were not detected both in hypoxia and in normoxia, and that the Epo levels in the pCMV-Epo transfected cells were the highest but had no difference between in hypoxia and in normoxia. These findings could be confirmed by immunofluorescence assay in which the Epo-expressing cells displayed bright fluorescence in the cytoplasm (Figure 2D).

Figure 2
figure 2

(A) Hypoxia-activated Epo construct was created by inserting the Epo gene with NH2-terminal signal peptide sequence into the downstream of the chimeric HRE/CMV promoter. (B) mRNA expression levels of Epo in HeLa cells transfected with pHRE, pHRE-Epo, pCMV-Epo and pHREm-Epo in normoxic and hypoxic condition. (C) Protein levels of Epo in HeLa cells transfected with pHRE, pHRE-Epo, pCMV-Epo and pHREm-Epo in normoxic and hypoxic condition. (D) Immunofluorescence of the cells transfected with pHRE-Epo, pCMV-Epo and pHREm-Epo in normoxic and hypoxic condition (the Epo-expressing cells showing bright fluorescence in the cytoplasm; magnification: × 400). (E) Secreting expression level of Epo in the culture supernatants of HeLa cells transfected with pHRE-Epo, pCMV-Epo and pHREm-Epo in normoxic and hypoxic condition was analyzed with Epo enzyme-linked immunosorbent assay. **stands for P < 0.01. (F) Viable TF-1 cells incubated with the supernatants of HeLa cell transfectants were assessed by MTT assay. At the 4th day, the A490 value of the hypoxia-treated Hela-pHRE-Epo is significantly higher than that of the normoxia-treated Hela-pHRE-Epo (P < 0.05), and at the 7th day this difference is the most obvious (P < 0.01). Data represent the mean values of two independent experiments, each carried out in triplicate. **stands for P < 0.01.

The culture supernatant was harvested to examine the secreting expression of Epo in response to hypoxia. We found the Epo levels in the medium of HeLa-pHRE-Epo cells were much higher in hypoxia than in normoxia (P < 0.01), whereas the Epo secreted into the medium of HeLa-pCMV-Epo cells were at a similar level in both hypoxia and normoxia (Figure 2).

A growth factor-dependent cell line TF-1 cells were incubated together with the culture supernatants of HeLa-pHRE-Epo cells or HeLa-pCMV-Epo cells for evaluating the biological activity of Epo expression in vitro. As shown in Figure 2F, TF-1 cells exhibited promoted survival and sustained proliferation in the medium of hypoxia-treated HeLa-pHRE-Epo cells, but in the medium of normoxia-treated HeLa-pHRE-Epo cells they didn't exhibit. TF-1 cells continually proliferated in the medium of both hypoxia- and normoxia-treated HeLa-pCMV-Epo cells. These findings indicate this promoter can drive the expression of biologically active Epo when induced by hypoxia in vitro.

In vivo study shows pHRE-Epo may correct uremic anemia safely and stably

The serum creatinine (SCr), blood urea nitrogen (BUN) and Hct of the rats were recorded once every two weeks during the experimental period (16 weeks), showing that compared with the healthy rats, the levels of SCr (Figure 3A) and BUN (Figure 3B) of all the uremic rats kept going up since the adenine diet started (P < 0.05). Morphologic examination demonstrated that the kidneys of uremic rats were enlarged and pale. Renal pathology showed us the crystal deposition in renal tubules and interstitium, renal tubular atrophy and severe interstitial fibrosis in the renal cortex of uremic rats. Although the glomeruli of the uremic rats did not show crystalline deposition, but the glomeruli were sparse (Figure 3C). These indicate the construction of uremic model was successful.

Figure 3
figure 3

(A) Biweekly records of the SCr of all rats, uremic or healthy, show that the SCr of uremic rats goes up and keeps a high level since the 2nd week. (B) Biweekly records of the BUN of all rats show that the changes of BUN of uremic rats are similar to that of SCr of uremic rats. (C) The kidneys removed from the healthy and uremic rats display the obvious differences in the morphologic and histological observation in which the crystal depositions are marked with "white arrow". (D) One week after the end of intramuscular injection (in the 8th week), Epo expression in the muscular tissues around the injection location was detected. The muscular tissues (0.1 g) are collected from the untreated-, pHRE-Epo-treated-, pCMV-Epo-treated healthy and uremic rats (n = 2 per subgroup). (E) Biweekly records of HIF-1α in rat serum show no obvious alteration in healthy group. The HIF-1α levels of the uremic group keep going up in the first 4 weeks. But that of pHRE-Epo-treated or pCMV-Epo-treated uremic rats begins going down after the injection and gradually reaches the normal range. The HIF-1α levels of the untreated uremic rats elevate continuously. "↑" stands for the intramuscular injection of pHRE-Epo or pCMV-Epo. (F) The serum Epo of untreated healthy rat has no alteration but that of untreated uremic rats keep going down. In the pCMV-Epo-treated healthy or uremic rats, the serum Epo levels elevate continuously. In the pHRE-Epo-treated healthy rats, no change is observed. But in the pHRE-Epo-treated uremic rats, the serum Epo goes down before injection, goes up after injection and then fluctuates within a range. "↑" stands for the intramuscular injection of pHRE-Epo or pCMV-Epo. (G) Biweekly records of Hct demonstrate that only the Hct of pHRE-Epo-treated rats is better restored and maintained to normal physiological levels. "↑" stands for the intramuscular injection of pHRE-Epo or pCMV-Epo.

The Epo mRNA expression in the hindlimb muscles was detected at the 8th week. The Epo mRNA expression was strong positive in both healthy and uremic pCMV-Epo-treated rats and at similar level between the healthy and the uremic. But in the pHRE-EPO-treated rats, the Epo mRNA expression was obviously higher in the uremic than in the healthy (Figure 3D). It means that the gene delivery by means of direct plasmid injection is efficacious and that our constructed hypoxia-regulated system can respond to the hypoxia or normoxia condition in vivo.

The biweekly records of the HIF-1α level in rat serum (Figure 3E) demonstrated that the HIF-1α in untreated uremic rats kept going up after adenine diet, but that in pCMV-Epo-treated or pHRE-Epo-treated uremic rats was first obviously elevated since adenine diet and then descended to the normal level since plasmids injection. The biweekly records of Epo presented a trend roughly opposite to the HIF-1α in the untreated uremic rats and the pCMV-Epo-treated healthy and uremic rats, namely increased HIF-1α vs. simultaneously decreased Epo (Figure 3F). Correlation analysis proved a negative correlation between them (P < 0.05). But in the pHRE-Epo-treated uremic rats, the changes of Epo in rat serum had a similar tendency to that of HIF-1α, presenting a positive correlation (P < 0.05). This indicates that the hypoxia-regulated system can provide a physiologically switching on-off the Epo production in vivo.

The Hct level of pHRE-Epo-treated uremic rats went up continuously for 6 weeks after pHRE-Epo injection and then gradually reached a plateau within normal range and maintained the normal level for 6 weeks. The Hct level of the pCMV-Epo-treated rats, both uremic and healthy, were continuously and dramatically elevated and exceeded the normal range for 4 or 8 weeks after pCMV-Epo injection (Figure 3G). This indicates that the HRE/CMV-directed Epo plasmids can safely and stably correct the anemia via restoring and maintaining HCT to the normal physiological level for a long time.

Discussion

Creatinine is a break-down product of creatine phosphate in muscle and chiefly filtered out of the blood by the kidneys. Measuring SCr is a simple test used as an indicator of renal function (Delanghe et al., 1989). The BUN test is also a measurement of renal function. It is to measure the amount of nitrogen in the blood in the form of urea that is a by-product from metabolism of proteins by the liver and is removed from the blood by the kidneys. Hct is the volume percentage (%) of red blood cells in blood which is considered an integral part of a person's complete blood count results, along with hemoglobin concentration, white blood cell count, and platelet count (Purves, 2004). Our results of SCr, BUN and Hct in uremic group show the rat model of uremic anorexia induced by adenine-diet was successful. The experiments in vitro prove that the hypoxia-inducible system of Epo expression that we constructed can drive the Epo gene and protein expression in response to hypoxia, and those in vivo confirm that this system can correct the uremic anemia and, more importantly, can maintain the Hct within the normal range for long time in the uremic rats. These results are basically congruent with Binley's study though the vectors and animal models used by the two studies are different (Binley et al., 2002).

HRE is a key regulatory DNA sequence to control gene expression in response to hypoxia. Many previous studies reported that they treated multiple diseases by delivering therapeutic genes under the control of chimeric hypoxia-responsive promoters constructed by diverse HREs in combination with heterologous promoters, such as tumor killing by delivering the apoptotic genes or "suicide genes" encoding prodrug-activating enzymes (Wang et al., 2005; Greco et al., 2006), the treatment of cardiovascular disease by VEGF gene delivery (Lee et al., 2003), the protection of tissues against I/R injury by heme-oxygenase-1 gene (Tang et al., 2005; Pachori et al., 2006), and correction of anemia by Epo gene (Binley et al., 2002). Therefore it is reliable to utilize HRE to regulate target gene expression. We selected PGK HRE strategy to construct the hypoxia responsive promoter, because murine PGK HRE has a relatively better hypoxia response mechanism (Boast et al., 1999) and incorporating PGK HREs in gene delivery vectors can provide an on-off physiological switch that renders the transcription of therapeutic gene completely responsive to hypoxia/ischemia. Such a pattern of endogenous regulation of transgene expression is similar to natural mechanism.

Hypoxia control system utilizes a highly-conserved signaling machinery involving endogenous transcription factor HIF (Pachori et al., 2004). Although Epo gene expression is cell-type specific, most cell types can activate the HIF-1 pathway in response to hypoxia. And many genes active in a broad range of cell types and tissues contain HREs and can respond to stabilization of the HIF-1 in hypoxia. Besides kidney and liver, muscle tissues are also believed to be able to sense the hypoxia as oxygen delivery is reduced, and this may be sufficient to activate gene expression from a hypoxia-responsive promoter (Binley et al., 2002). Considering that the vascularity of skeletal muscle allowed for the distribution of secreted proteins and that skeletal muscle was easily targeted by injection in a clinical setting, we selected intramuscular injection as the method of gene delivery.

At present, the delivery of Epo gene is mainly dependent either on introducing the Epo gene into autologous cells ex vivo and transferring the modified cells back into the individual (Bohl et al., 1997), or on directly transferring the Epo gene in vivo using an adenovirus vector, adeno-associated vector, or naked plasmid (Ye et al., 1999; Bohl et al., 2000). Ex vivo gene transfer is expensive and complicated to operate. Adenovirus vector induces the immunoresponse to adenovirus, which prevents long and stable gene expression in immunocompetent mice (Rivera et al., 1999). Plasmid injection, whereas, can be repeated without apparent immune response to the DNA vector (Terada et al., 2002), and has been successfully applied to deliver Epo gene into the muscles of mice to maintain long-term expression of Epo in the systemic circulation (Tripathy et al., 1996). Thereupon, naked plasmid injection becomes our preference although its transfection efficacy is lower than adenovirus vector's (Wolff et al., 1992). It was reported that the transfection rate of plasmid injection could be dramatically raised by means of in vivo electroporation (Maruyama et al., 2000; Terada et al., 2002). Yet we didn't use this technique, mainly because our primary purpose was to test whether our hypoxia-regulated system of Epo worked well in vivo or in vitro, rather than to contrive to raise the transfection rate of naked plasmid injection. Even if we didn't use this technique, our results of in vivo experiment confirmed that five consecutive injections of plasmids ensured the success of transfection.

Regulation of transgene expression is the utmost key factor for the safety and efficacy of gene transfer therapy. Scientists have developed the gene-switch system under pharmacological control of doxycycline (Bohl et al., 1998), rapamycin (Ye et al., 1999) and mifepristone (Terada et al., 2002). Comparing with them, the biggest advantage of the hypoxia control system is that it can switch transgene expression on and off without relying on the regular drug administration. Thus we think the hypoxia control system of Epo expression is more promising in the future clinical application, deserving more attention.

It has been proved that Epo may act as an angiogenic factor in malignant tumors (Ribatti et al., 2003b; Arcasoy et al., 2005; Jeong et al., 2008), and may stimulate proliferation and inhibit apoptosis of Epo-receptor-bearing tumor cells (Acs et al., 2001). Hypoxia may mediate the selection of neoplastic cells (Acs et al., 2003) and this may contribute to metastasis and treatment resistance of malignancies (Ribatti et al., 2003a). Whether the use of this hypoxia control system would induce, or on the contrary, inhibit tumor formation in uremic rats is still unclear. A recent report by Nairz et al. indicated that Epo-treatment had anti-inflammatory effect (Nairz et al., 2011). Whether this system would reduce the inflammatory response or protect against cardiac hypertrophy is still unclear. All these unsettled questions need more researches in future.

In sum, we constructed the chimeric HRE/CMV promoter by fusing the tandem repeats of PGK HRE to the CMV IE basal promoter, and constructed the hypoxia control system of Epo expression by fusing Epo gene to the HRE/CMV promoter. Our data confirm that the HRE/CMV promoter can sense the hypoxia in skeletal muscles and switch Epo expression on and off accordingly, and that intramuscular injection of plasmids containing this system provides a long-term physiologically regulated Epo secretion in vivo to correct uremic anemia efficiently and safely. Though it is promising to use this technique to treat uremic anemia, there are still some unsettled questions, deserving more attention in future.

Methods

Plasmids

We synthesized complementary oligonucleotides that contained HRE sequences of the flanking region of murine PGK gene and HRE mutants with 3-bp substitution in each of the HRE consensus sites in the trimer (Figure 1A). The annealed HREs oligos with HREs flanked by 5'-end Bgl II and 3'-end Sgf I overhangs were linked to the CMV IE promoter in the pCI-neo vector (Promega) to create pHRE/CMV. The chimeric pHRE/CMV promoter cassette was then cloned into pGL3-Basic (Promega) and pEGFP-C2 (Clontech) respectively. hEpo gene obtained by reverse-transcription PCR from fetal liver tissue was cloned into multiple clone sites in pHRE/CMV to create pHRE-Epo. The hEpo PCR product was cloned into the pUC18 plasmid (Panvera) and was subsequently removed as an XbaI-EcoRI fragment and cloned in to the pCI-Neo (Promega) NheI-EcoRI sites to create pCMV-Epo. All constructs were confirmed by DNA sequencing.

Cell culture, transfection and hypoxic treatment

Human cervical carcinoma cell line HeLa (obtained from the Lab of Professor Angang Yang, Fourth Military Medical University (Jia et al., 2003)) was maintained in RPMI1640 medium in which 10% fetal bovine serum had been supplemented. The cells were seeded in 24-well plates (density 3 × 104 cells/well) 24 h before transfection. Transfection was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The transfected cells were transferred into hypoxic conditions (0.1% O2, 5% CO2, and 94.9% N2) for transient expression. 24 h after transfection the cells were harvested.

Green fluorescent protein and luciferase assay

HeLa cells were transfected with recombinant EGFP plasmids and then they were incubated in hypoxic conditions and visualized by EGFP expression for green fluorescent assay. The cells co-transfected with Firefly and Renilla luciferase vectors were incubated in hypoxia. The luciferase analyses were performed with Dual-Luciferase Reporter (DLR) Assay System (Promega) according to the manufacturer's protocol in a Turner Designs Luminometer TD-20/20 (Promega).

Immunofluorescence assay

Cells on coverslip were transfected with recombinant Epo plasmids and incubated in hypoxia. And then the cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and stained using anti-human erythropoietin antibodies (R&D Systems) and cy3-conjugated IgG secondary antibodies (sigma).

RNA isolation and determination of Epo gene expression

Epo-mRNA was determined by RT-PCR. RNA was extracted from cells or tissues using the RNeasy RNA isolation kit (Invitrogen) and was reverse transcribed into cDNA using Superscript III reverse kit (Invitrogen). Primers for Epo were 5'-GGAAGAGGATGGAGGTCG-3' (forward primer) and 5'-GCAGTGATTGTTCGGAGTG-3' (reverse primer), and the size of the amplified fragment was 253 bp. Primers for β-actin of HeLa cells were 5'-GTCACCAACTG GGACGACA-3' (forward primer) and 5'-CACAGCCTGGAT AGCAACG-3' (reverse primer), resulting in a 192 bp product. Primers for β-actin of rats were 5'-CATTGTCACCAACTGG GACG-3' (forward primer) and 5'-GGTACATGCATCGGTA GGTC-3' (reverse primer), resulting in 190 bp product.

PCR was performed with PE2400 instrument (Perkin Elmer). Cycling conditions were: a) for RNA extraction form cell culture: 95℃ for 5 min (1 cycle); 95℃ for 30 s, 53℃ for 30 s, 72℃ for 45 s (25 cycles); 72℃ for 7 min (1 cycle), and b) for RNA extraction form tissue: initial denaturation (95℃, 3 min), 40 cycles of denaturation (95℃, 10 s), annealing (60.5℃, 10 s), extension (72℃, 10 s), and a final extension (72℃, 10 min). PCR amplification products were analyzed in 1.5% agarose gels.

Western blot analysis and ELISA assay

Transfected cells were lysed in 50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM MgCl2, 0.5% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluoride. The samples (10 µg protein) were subjected to electrophoresis using a 15% SDS-polyacrylamide gel and the separated proteins were transferred to a polyvinylidene difluoride membrane. The membrane was probed with anti-human erythropoietin antibodies or β-actin antibodies (Sigma) and detected with an enhanced chemiluminescence system (Pierce). Epo levels in culture supernatants of the transfected cells were determined using the human Epo ELISA Kit (ExCell Biology).

Cell viability assay

TF-1 cells (from ATCC) were plated in 96-well dishes (density 5 × 103 cells/well) and then followed by incubation with culture supernatants that had been harvested from HeLa cells 24 h after transfection with recombinant Epo plasmids. 20 µl aliquots of MTT solution (5 mg/ml in PBS) were added to each well, followed by 4 h incubation and addition of 150 µl of DMSO (Sigma). Sunrise microplate reader (Tecan) tested A490 values. TF-1 cells without culture supernatants or incubated with culture supernatants of pCMV-Epo or pHRE-Epo transfectant were used as controls.

Experimental animals and in vivo study

The Ethics Committee of Fourth Military Medical University approved animal use (approval ID: 2009-LS-0617). Animal care and treatment were conducted in conformity with institutional guidelines that are in compliance with international laws and politics. 60 SD rats (180 ± 10 g, 6-8 weeks) were purchased from Laboratory Animal Center in Fourth Military Medical University and randomly classified into two groups, uremic and healthy group (n = 30 per group). Each group was further assigned into three subgroups, pHRE-Epo-treated, pCMV-Epo-treated and untreated subgroup (n = 10 per subgroup).

The whole experiment lasted 16 weeks during which all rats in healthy group were housed under standardized conditions in plastic cages (light-dark cycle 12/12 hrs, temperature 22 ± 2℃, humidity 50 ± 10%), had free access to tap water and fed with standard diet (provided by the Laboratory Animal Center in Fourth Military Medical University). The rats in uremic group had the same conditions of housing and water-drinking, but they were fed with 4 weeks of 0.75% adenine diet and then 12 weeks of standard diet. In this study, adenine diet was used to induce chronic renal failure and severe anemia based on the protocol and rationale of adenine diet of previous studies (Yokozawa et al., 1986; Ataka et al., 2003).

Biweekly blood sample harvests from rat tail vein (0.5-1 ml) were performed (totally 9 times during the experiment) for detection of SCr, BUN, Hct, and the levels of HIF-1α and Epo in rat serum. SCr and BUN were determined using Cobas Inegra 400 Plus automatic biochemical analyzer (Roche), and Hct using CA620 blood analyzer (Medonica). The levels of HIF-1α and Epo in rat serum were quantitatively measured using rat HIF-1α ELISA kit (Shanghai Kanu Biotechnology Co., Ltd., China) and ELISA Kit for Epo in rat serum (E90028Ra, Uscn Life Science Inc., US) respectively. Specific procedures were performed according to the manufacturers' directions.

From the 4th week on, 100 µl lipofectamine-encapsulated 50 µg pHRE-Epo and 50 µg pCMV-Epo were intramuscularly injected into the quadriceps of the left or right hindlimb of the pHRE-Epo-treated and the pCMV-Epo-treated subgroups respectively at 3-d intervals for 3 weeks (totally 5 injections). At the 8th week, namely one week after the end of the injections, the Epo mRNA expression in the muscles of hindlimb around the injection location were detected. At the 16th week, all rats were killed for morphologic examination of kidney and renal pathology. The pCMV-Epo-treated- and untreated-rats served as controls.

Statistical analysis

Data were presented as means ± SD. Statistical analysis was done using one way analysis of variance (ANOVA) for multiple samples, Student's t-test for comparing paired sample sets, and correlation analysis for determining a relationship between two variables. P < 0.05 were considered statistically significant.