| Ischemia/reperfusion injury(IRI) and acute rejection(AR) remain two important factors affecting kidney allograft after transplantation. IRI is inherent in transplantation because every donor vascularized organ by definition suffers varied degree ischemia and reperfusion. The short-term consequences of IRI may range from clinically inapparent to severe, with delayed graft function(DGF) or primary nonfunction. Although the majority of the renal changes are reversible, hospital stay is increased, administration of immunosuppression is more difficult, dialysis is prolonged, and cost is amplified. IRI may lead to increased immunogenicity of the graft, resulting in acceleration of Ag-specific processes such as acute rejection episodes and decreased long-term graft survival due to the progression of chronic-type rejection. There is now a growing recognition that long-term graft failure is at least in part influenced by the consequence of transplantation-related IRI. With current immunosuppressive protocols resulting in significant reduction in the rate of acute rejection, increased attention has been directed toward Ag-independent factors influencing allograft survival. Hence, it is important to develop novel strategies to limit or prevent the graft injury, which in turn should ultimately result in the improvement of the long-term allograft outcome. Last years, with the application of gene therapy techniques to transplantation, local cytoprotection and/or immunosupression in graft may be obtained, and gene therapy has been a promising way to prevent renal from IRI. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme, also known as heat shock protein (hsp) 32. It is one of the three HO isoforms that catalyze the degradation of heme to biliverdin, Fe2+, and carbon monoxide (CO), but it is the only one that is inducible. Like other hsp, upregulation of HO-1 may be among the critical cytoprotective mechanisms that are activated during times of cellular stress such as inflammation, ischemia, hypoxia, hyperoxia, hyperthermia, or radiation. A growing body of evidence suggests that overexpression of heme oxygenase-1 (HO-1) may protect organs/tissues from immune-mediated injury either through prevention of oxidative damage or via a local immunomodulatory influence on infiltrating inflammatory cells. This protective property has been observed in models of organ transplant rejection. Although HO-1 can be induced by various stimuli, such as heme and Coprotoporphyrin(CoPP) and so on, exogenous administration of HO-1 by gene transfer seems a more specific and attractive approach. The adenovirus has several features that make it be studied best. Adenovirus is a mildly pathogenic human virus, rapidly infect a broad range of human cells including proliferation and nonproliferating cells and tend to yield high levels of adenoviral doses. In addition, adenoviral vectors are relatively easy to manipulate, purify and manufacture using recombinant DNA techniques, and inserted foreign genes are generally maintained without change through successive rounds of viral replication. These give us a gurantine to succeed in transfecting renal cells in vivo. Gene transfer provides distinct advantages over conventional systemic administration of therapeutics. Local delivery avoids systemic complications and is economical since lesser amounts of therapeutics are required. The general barriers of gene transfer in medicine include imprecise site-targeted expression and transient expression. However, these are not barriers for gene transfer in transplantation. Imprecise site (organ) targeting the kidney do not apply to transplantation, since gene transfer into harvested organs is readily achievable. Furthermore, the second barrier, transient expression, may not necessarily be a problem in transplantation, since the period of renal ischemia-reperfusion injury hasn't last a long time, and the long-time expression of interest gene in gene therapy is harmful and unnecessary. These are the reasons that we study on gene therapy with recombinant adenovirus carrying human HO-1 in renal I/R model. Methods and Results 1. Construction of a recombinant adenovirus carrying human HO-1 gene The sequence of human HO-1 gene was acquired by PCR from pOTB7-hHO-1 with the primers VT341 and VT342, and the 891bp fragment was obtained. This DNA fragment digested with endonuclease EcoRⅠand BglⅡand got the 879 bp DNAfragment which named hHO-1/ EcoRⅠ+ BglⅡ. It was ligated into adenovirus shuttle vector pSGCMV after digested with EcoRI and BglⅡby standard procedure. The recombinant adenoviral plasmid pSGCMV-HO-1 was identified correct by sequencing and the correct clones containing target gene were selected for further study. The pSGCMV-HO-1 and backbone vector pBHGloxP△E1E3(containing the right arm sequence of adenovirus type 5), each bearing a loxP site and the latter containing a cre site, were cotransfered into the adenoviral packaging 293 cells by lipofectamine mediated gene transfer method to recombine and pack the virus (site-specific recombination). The virus plaque was sublimated three times. After the recombinant adenoviruses were verified by PCR, we name it Ad-hHO-1. The viruses were propagated in 293 cells and purified by cesium chloride density purification, titrated by TCID50 method. Their titer was 2.0×1010pfu/ml. In order to observe the efficiency of the recombinant adenovirus carrying human HO-1 gene in transfecting target organ in ischemia/reperfusion model in rats, we transfected the Ad-hHO-1 to 293 cells(human embryon kidney cells, HEKC)in vitro after confirmed it's security by excluding the existence of wide-type adenovirus. The results showed that the recombinant adenovirus carrying hHO-1 gene could transfect the human embryon kidney cells (293 cells) and non-prolification cells as MRC-5 cells(human fibroblast cells) effectively by assay of RT-PCR and Western Blotting. 2,The protection of recombinant adenovirus carrying human HO-1 gene against renal ischemia-reperfusion in rats In order to study the protection against renal ischemia/reperfusion with the recombinant adenovirus , the model of kidney ischemia-reperfusion was established with Sprague-Dawley rats. In brief, the right kidney was cut off firstly, and the left kidney was flushed via abdominal aorta with 0～4℃HC-A organ storage solution. 1 hour late, the left kidney was reperfused, and blood samples were collected at 1d, 2d and 7d after the kidney reperfusion for the BUN,Cr and TNF-αassaying, meanwhile, the left kidney was cut off at 2d and 7d after the kidney reperfusion and prepared for further analyses. In the therapy team, every left kidney was perfused and preserved with Ad-hHO-1 at 2.5×109pfu after flushed with 0～4℃HC-A organ storage solution via renal artery. The rats in control groups were perfused with 0.9% normal saline solution or Ad-EGFP2.5×109pfu instead of Ad-hHO-1. BUN and Cr in serum were measured byslide chemical methods, the change of TNF-αin serum among the groups was checked with ELISA. The kidney samples of rats were harvested for assaying of histology, immunohistochemistry and quantification of HO enzymatic activity. The results showed that perfusion and preservation with Ad-hHO-1 via renal aorta could transfect renal cells of rats effectively. By immunohistochemical and enzymatic activity of HO assay, we found that the rats treated with Ad-hHO-1 expressed hHO-1 in kidneys at a high level. According to this, the level of BUN,Cr and TNF-αin serum, and the number of apoptosis cells(by TUNEL) were decreased, and the change in histology(including HE staining and transmission electron microscoping) was ameliorated. Conclusion: We draw a conclusion that recombinant adenovirus carrying hHO-1 can be introduced into kidney during cold preservation effectively in IRI model. The target organ can overexpress cytoprotective protein(HO-1), which can attenuate the kidney ischemia/reperfusion injury in rats. These findings provide some experimental evidences that gene delivery of sequences encoding graft cytoprotection can be full considered for clinical application in renal transplantation.
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