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. 2012 Nov 30;44(11):674-83.
doi: 10.3858/emm.2012.44.11.076.

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Jifeng Sun  1 , Yarong Wang, Jie Yang, Dewei Du, Zhanting Li, Junxia Wei, Angang Yang
Affiliations

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Jifeng Sun et al. Exp Mol Med. .

Abstract

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate- early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between- group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.

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Figures

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.
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.
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.
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