Experimental Study Of Heme Oxygenase-1 Gene Transfer Prevents Chronic Allograft Nephropathy

Background: Organ transplantation remains the main therapeutic option for patients with renal, pancreas, or cardiac chronic end-stage diseases. However, progressive impairment of graft function that begins months or years after organ transplantation significantly influences the long-term outcomes of organ transplantation. Despite the introduction of new immunosuppressive regimens, the incidence of the chronic allograft deterioration is not significantly altered. Although the precise nature of molecular and cellular events of chronic allograft deterioration is still unclear, it is generally believed that both immune and nonimmune factors are equally important in the pathogenesis of the disease. Heme oxygenase (HO) is the rate-limiting intracellular enzyme that degrades heme to biliverdin, free iron and carbon monoxide (CO). Three HO isoforms (HO-1, -2 and -3), products of three distinct genes, have been identified so far. The HO-2 and -3 isoforms are constitutively expressed. HO-1 isoform is inducible and can express in various types of cells. A wide range of stress-related stimuli can induce the expression of HO-1, which has potential cytoprotective effects that are likely to be mediated by three degradation products of heme: CO, biliverdin/bilirubin and free iron. Among them, CO, which has significant anti-inflammatory activities, is most extensively studied. In previous animal transplantation models, induction of HO-1 could protect grafts from ischemia reperfusion injury and acute graft rejection. In an animal model where anti-CD4 monoclonal antibody was used to induce long-term allograft survival, induction of HO-1 in the early days after transplantation could ameliorate antibody-associated graft arteriosclerosis. Chronic allograft nephropathy(CAN) is the predominant cause of allograft function loss during the first decade after transplantation. The pathogenesis remains uncertain, but both immune-mediated and nonimmune-mediated factors are thought to play a role. In this study, we transferred HO-1 gene into rat donor kidney using a recombinant adenovirous vector, an approach that might be potential for clinical transplantation. This allowed us to study whether HO-1 by itself could prevent chronic allograft nephropathy and lead to long-term survival of organ grafts. Objective: 1. To construct and identify HO-1 gene recombinant replication-deficient adenovirus (Ad-HO-1). 2. To observe the cytotoxicity of Ad-HO-1 and its capacity of mediating the expression of HO-1 in cultured cells. 3. To investigate the effects of HO-1 on renal allografts after HO-1 gene transfection in rat chronic allograft nephropathy model. Methods and Results: 1. The construction and identification of HO-1 gene recombinant replication deficient adenovirus. In this study, we used AdEasy system to construct HO-1 recombinant replication-deficient adenovirus. Firstly, the whole HO-1 gene was obtained from the PRHO-1 plasmid via XhoⅠ+HindⅢdigestion, and inserted into Puc18 plasmid. Then HO-1 gene was digested from Puc 18-HO-1 by KpnⅠ+HindⅢand subcloned into shuttle vector of pAdTrack-CMV. The resulting plasmid pAdTrack-CMV-HO-1 was linearized with PmeⅠand cotransformed into BJ5183 cells together with adenovirus genomic plasmid of pAdEasy-1. The pAdEasy-1 was E1 and E3 deleted and its E1 function could be complemented in 293 cells. Recombinants were selected with kanamycin and screened by restriction enzyme analysis. The recombinant adenoviral construct was then cleaved with PacI to expose its ITR (Inverted Terminal Repeats) and transfected 293 cells to produce viral particles. Recombinant adenoviruse was purified by Double Cesium Chloride Gradient and titered (infectious particles, IP) as TCID50 (tissue culture infectious dose 50). Adenovirus stocks were tested for the absence of replication-competent adenoviruses by PCR amplification of the E1 adenoviral region. The resulting recombinant adenovirus was identified by PCR of HO-1 gene and expression of HO-1 protein by western blot. 2. Cytotoxicity of Ad-HO-1 and its capacity mediating the expression of HO-1 in cultured cells. The cell lines HK-2 transfected by Ad-HO-1.Significant difference of the survivalrates of the cells were not observed between the transfected cells and the controls 2 and 4 days after transfection. The results suggest Ad-HO-1 has little cytotoxic affect on non-packaging cells. The expression of HO-1 and GFP in the Ad-HO-1 transfected cell lines was determined by Laser Scan Confocal Microscope.The results showed that the expression rates of HO-1 in HK-2 cell line transfected at 37℃were over 90%. Both GFP and HO-1 genes should be coexpressed in pAdTrack-pAdEasy system, so the experssion extent of GFP could represent the experssion extent of HO-1, were directly identified by fluorescence microscope. The results showed that there was strong expression of GFP in tubular epithelial cells, which suggested that Ad-HO-1 has the ability mediating HK-2 to express HO-1. 3. Ad-HO-1 mediated HO-1 gene transfection in rat donor kidney. The transfection efficiency of Adv HO-1 in kidney of F344 rats was determined with fluorescence microscope by observing the expression of GFP in kidney. Evident green fluorescence was observed in sections of kidneys when the vessel of liver, the aorta and vena cava below the renal vessel were ligated and Ad-HO-1 was injected systemically in vivo via the aorta for 3h. The transfection occurred in tubular cells and glomerular structure. The results suggested that the rat kidney could be transfected effeciently by high titer Ad-HO-1 in circulation at body temperature. The results showed that the expression had occurred on the third day after transplantation, and lasted for 3~4 weeks, which suggested that Ad-HO-1 gene could be expressed for a short time in HO-1 gene-transfected kidney after transplantation. 4. The effect of renal allografts transfected with HO-1 gene in chronic allograft nephropathy model. The effect of HO-1 gene transfection in kidney on the renal allografts was observed in the standardized Fisher/Lewis transplant model of CAN. The results showed that control recipients without HO-1 transfection developed histologic changes characteristic of CAN and that at the same time the inflammation in gene-transfected grafts was less sever than that in controls.The results presented here suggest that HO-1 gene transfection in kidney can reduce immune injury to renal allografts,It is a promosing candidate for the prevention of CAN,but the mechanism of HO-1 in preventiong the CAN warrants further studies.Conclusion: 1. HO-1 gene recombinant replication-deficient adenovirus was successfully constructed. 2. HO-1 gene recombinant replication-deficient adenovirus had the qualities of high efficiency, stable expression of HO-1 and safety in cultured tubular epithelial cells. 3. Ad-HO-1 could efficiently transfer HO-1 gene into rat donor kidney. 4. After transfected by HO-1 gene, the renal grafts showed lower expression of α-SMA, TGF-βand PDGFB, decreased level of glomerulosclerosis and tubulointerstitial fibrosis and better renal fanction. All above suggests that HO-1 can prevent CAN efficiently.