Gene transfer to the rat kidney in vivo and ex vivo using an adenovirus vector: factors influencing transgene expression
Jun Fujishiro1,4,
Shin-ichi Takeda1,
Yuichi Takeno1,
Koichi Takeuchi3,
Yukiyo Ogata1,
Masafumi Takahashi1,
Yoji Hakamata1,
Takashi Kaneko1,
Takashi Murakami1,
Takashi Okada2,
Keiya Ozawa2,
Kohei Hashizume4 and
Eiji Kobayashi1
1 Division of Organ Replacement Research and 2 Division of Genetic Therapeutics, Center for Molecular Medicine, 3 Division of Anatomy, Department of Anatomy, Jichi Medical School, Tochigi and 4 Department of Pediatric Surgery, Faculty of Medicine, University of Tokyo, Tokyo, Japan
Correspondence and offprint requests to: Eiji Kobayashi, MD, PhD, Division of Organ Replacement Research, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan. Email: eijikoba{at}jichi.ac.jp
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Abstract
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Background. The characteristics of adenovirus-mediated gene transfer into the kidney are not well examined. We studied the effects of contact time and temperature on adenovirus-mediated transgene expression in rat kidneys, using catheter-based in vivo gene transfer and a rat renal transplant model ex vivo.
Methods. An adenovirus vector containing the luciferase (Ad-Luc) or ß-galactosidase (Ad-LacZ) gene was introduced in vivo into the kidney via a renal artery catheter. Various contact times and temperatures were evaluated. Ex vivo, the renal graft was injected with Ad-Luc through the renal artery, chilled for 60 min and then transplanted. Luciferase expression was evaluated periodically by a non-invasive bioimaging system or histology. Cells expressing the LacZ gene were identified by immunoelectron microscopy.
Results. In in vivo gene transfer, successful transgene expression was achieved; however, its efficiency was independent of contact time or temperature. In ex vivo gene transfer, transgene expression in the renal graft peaked early and gradually decreased. Strong gene expression was observed in the recipients' livers. LacZ expression was detected in fibroblasts, parietal epithelial cells of Bowman's capsule, mesangial cells, podocytes and tubular cells.
Conclusions. This study generated new information about in vivo and ex vivo gene transfer into the kidney, which would be useful for renal gene therapy.
Keywords: adenovirus; gene expression; gene transfer; renal transplantation
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Introduction
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Gene transfer into the kidney has great potential in the treatment of renal disease and in renal transplantation. However, gene therapy of the kidney is rather limited, because of the lack of an efficient gene delivery system [1]. Some vectors derived from viruses, such as adenovirus, adeno-associated virus (AAV) and retrovirus, were successfully transduced in renal cells [2,3]; however, little transduction occurred in the kidney in vivo when these recombinant virus vectors [1] or naked DNA [4] were used.
On the other hand, the adenovirus vector has been widely used clinically and experimentally [3,5,6]. When injected systemically, it has a high affinity to the liver; therefore, for gene transfer to other organs, direct local injection [7] or perfusion [3,5,6] has been attempted. There have been no reports on the efficacy of gene transfer to the whole kidney using an adenovirus vector delivered via blood vessels. In our previous study, we developed an in vivo method for gene transfer to the kidney via a catheter and we demonstrated that strong gene expression throughout the kidney was obtained with the AAV vector [8].
In this study, we tested adenovirus-mediated in vivo gene transfer into the rat kidney with varying contact time and temperature. We also applied ex vivo gene transfer to the renal graft. We evaluated LacZ gene expression by immunohistochemistry and immunoelectron microscopy and followed the intensity of luciferase transgene expression periodically using a luciferase-mediated non-invasive in vivo bioimaging technique.
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Subjects and methods
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Experimental animals
We used 52 male inbred Lewis rats (aged 810 weeks; Charles River Japan, Inc., Yokohama, Japan). All animals had access to standard chow and water ad libitum. All experiments were conducted in accordance with the Jichi Medical School Guide for Laboratory Animals.
The adenovirus vector
We constructed a replication-deficient adenoviral vector containing the firefly luciferase gene under the control of cytomegalovirus promoter (AVC2Luc; Ad-Luc), according to a technique using a DNAprotein complex [9]. An adenovirus vector, AxCALacZ, that expresses ß-galactosidase (LacZ) under the control of the chicken beta-actin promoter with the CMV-IE enhancer was gratefully obtained from Dr Xiao-Kang Li (Department of Regeneration Surgery, National Research Institute for Child Health and Development, Tokyo, Japan) [10,11].
Detection and quantification of transgene expression using a non-invasive in vivo bioimaging system
Luciferase gene expression activity was measured by the Xenogen In Vivo Imaging System (IVIS). In this system, a charged-couple device camera, which is non-invasive, was used to detect bioluminescene emitted from D-luciferin (potassium salt; Xenogen Corporation, Alameda, CA, USA), which reacts with firefly luciferase in living animals. Both the IVIS and the analysis software are available commercially (Xenogen Corporation). While under isoflurane anaesthesia, the subjects received D-luciferin through a penile vein [30 mg/kg body weight, which was dissolved and diluted to 15 mg/ml in phosphate-buffered saline (PBS)] [12]. One minute after the infusion, the light emitted by luciferase was measured, with a 1 min integration time. The signal intensity was quantified as the sum of the photons detected in the region of interest.
Catheter-based in vivo gene transfer into the kidney
Gene transfer was performed with a slightly modified catheter-based method [8], which mimicked clinical angiography. Briefly, under ether anaesthesia, laparotomy was made by midline incision, a cut-down was performed on the iliac artery and a 2-Fr catheter (Solo-Cath; Solomon Scientific, Plymouth Meeting, PA, USA) was passed through to the abdominal aorta just distal to the origin of the left renal artery. The aorta was occluded gently just proximal to the left renal artery and just distal to the tip of the catheter. The right renal artery was also clamped if it originated between the occlusion points of the aorta. The left kidney then was injected with 2 ml lactated Ringer's solution and 1 ml PBS containing the adenovirus vector [5 x 108 plaque-forming units (p.f.u.) of Ad-Luc or 2 x 109 p.f.u. of Ad-LacZ] via the catheter. The left renal artery and vein were clamped at the renal hilum and the aortic occlusions were released. During its contact with the vector, to keep the kidney at the temperature specified by the protocol, the left kidney was pulled out from the abdominal cavity through a left lateral incision and was maintained in a water bath for the specified contact time and temperature. The clamp on the renal hilum then was released and the kidney was replaced into the abdomen.
Renal transplantation and ex vivo gene transfer
Orthotopic left renal transplantation was performed in a syngeneic combination of Lewis rats. Under ether anaesthesia, the left kidney and ureter was dissected free. After systemic heparinization (200 units), the left kidney, with the aortic cuff, was harvested and stored in lactated Ringer's solution at 4°C. Next, 2 ml cold lactated Ringer's solution and then 0.51 ml of PBS containing 5 x 108 p.f.u. of Ad-Luc or 4 x 109 p.f.u. of Ad-LacZ were injected into the renal graft via the renal artery. In the recipient, after removing the left kidney, the graft aorta and the renal vein were anastomosed to the recipient aorta and inferior vena cava in an end-to-side fashion by microsurgical suturing. The recipient's urinary tract was reconstructed by anastomosing the graft and recipient ureters.
Luc gene expression in the grafted kidney and the recipient's liver was measured periodically on days 2, 4, 7, 14 and 28 by IVIS. LacZ gene expression was histologically examined on day 2, as described below.
X-gal and immunohistochemical staining and immunoelectron microscopy
To detect gene-transduced cells in the kidney, the experimental rats were sacrificed 2 days after in vivo or ex vivo transduction. Thin fresh-frozen sections (610 µm) of the target kidneys, of their contralateral kidneys and of their livers were fixed in 0.2% glutaraldehyde for 10 min at room temperature and incubated in a solution (X-gal) containing 1 mg/ml 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-gal; Sigma), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 and 2 mM MgCl2 in PBS at 37°C for 1 h and counterstained with Nuclear Fast Red solution.
For immunohistochemistry and immunoelectron microscopy, the kidneys were fixed with periodatelysine paraformaldehyde fixative for 4 h on ice and then incubated successively with 15%, 20%, and 30% sucrose for 4 h, embedded in OCT compound (Sakura Finetechnical Co. Ltd, Tokyo, Japan) and frozen in liquid nitrogen. Thin frozen sections (48 µm) were cut with a cryostat and blocked with avidin/biotin (Vector Laboratories, Burlingame, CA, USA) and 1% bovine serum albumin. Then they were incubated with rabbit anti-ß-galactosidase (Escherichia coli) polyclonal antibody (dilution 1:5000; CHEMICON International, Temecula, CA, USA) at 4°C overnight, with biotin-conjugated anti-rabbit IgG antibody (dilution 1:200; Rockland Immunochemicals, Gilbertsville, PA, USA) for 2 h at room temperature and with horseradish peroxidase-labelled streptavidin (dilution 1:200; Vector Laboratories) for another 2 h at room temperature. Immunoreactive cells were detected with 3,3'-diaminobenzidine tetrahydrochloride. Then, for immunoelectron microscopy, the sections were fixed with 2.5% glutaraldehyde at 4°C overnight, postfixed with 1% OsO4 for 3 min, dehydrated in ethanol and embedded in Epon. Ultrathin sections (100 nm) were cut and examined by a JEM 2000 Ex electron microscope.
Statistical analysis
The data are presented as means ± SD. Statistical analysis was performed using the MannWhitney test or the two-factor fractional analysis of variance (ANOVA). P-values of <0.05 were considered significant.
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Results
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Comparison between systemic injection and in vivo gene transfer into the kidney
The systemic injection of Ad-Luc resulted in gene expression only in the rat liver (Figure 1A), confirming the high affinity of the adenovirus vector for the liver. Using the catheter-based method, there was strong gene expression in the kidney on day 2, which decreased on day 7; in addition, expression was observed in the liver on day 7. Transgene expression in the liver tended to occur later than in the kidney with both systemic and catheter-based kidney-targeted injections. Using Ad-LacZ, transgene expression was observed only in the liver after systemic injection; however, by the catheter-based method, transgene expression was observed in renal interstitial cells as well (Figure 1B).

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Fig. 1. Transgene expressions with systemic injection and catheter-based in vivo gene transfer. (A) 5 x 108 p.f.u. of Ad-Luc was administered by either systemic injection through the penile vein or catheter-based in vivo gene transfer into the kidney. Luciferase gene expression was examined 2 and 7 days after gene transfer by the IVIS system. (See online Supplementary Material for colour figure.) (B) 2 x 109 p.f.u. of Ad-LacZ was administered by either systemic injection or in vivo gene transfer. Two days after gene transfer, kidneys and livers were examined histologically with X-gal staining. Original magnification: x100.
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Identification of gene-transduced cells in ex vivo gene transfer into the renal graft
To identify the cells gene-transfected by the ex vivo methods using the adenovirus vector, the LacZ gene was transduced into renal grafts. By X-gal staining, we observed positively stained cells mainly in the peritubular interstitium (Figure 2A) and sporadically in the glomeruli (Figure 2B). There were no X-gal positive cells in the kidneys contralateral to the grafts (data not shown). Immunohistological and immunoelectron microscopic studies showed that positively stained cells in the interstitium had long cytoplasmic processes and satellite-shaped nuclei (Figures 2C and 2H), features consistent with those of fibroblasts [13]. In the renal corpuscles, the parietal epithelial cells of Bowman's capsule were positively stained (Figures 2D and 2G). In the glomeruli, positively stained podocytes and mesangial cells were identified by electron microscopy (Figures 2D and 2G). Parts of the tubular epithelium also were positively marked by immunohistochemistry (Figures 2E and 2H). Contralateral kidneys had no similarly positive cells (Figure 2F).

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Fig. 2. Histological analysis of ex vivo gene transduction into the renal graft. Two days after gene transfer using Ad-LacZ, sections of gene-transduced kidneys were stained with X-gal (A, B). Gene-transduced kidneys (CE) or contralateral kidneys (F) were immunohistochemically stained with anti-LacZ antibody. Immunoelectron micrographs (G, H) of gene-transduced kidneys stained with anti-LacZ antibody. F, interstitial fibroblast; T, tubular epithelium; M, mesangial cell; P, podocyte; black arrows, processes of a podocyte; white arrows, parietal epithelium of Bowman's capsule. Bars: 200 µm (A, F), 50 µm (B, E), 100 µm (C, D) and 5 µm (G, H).
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Effect of contact time and temperature on the efficiency of transgene expression in the catheter-based in vivo gene transfer into the kidney
To assess the effect of cooling and contact time on transgene expression in the kidney, in vivo gene transfer was performed under cold (4°C) or warm (37°C) conditions for several contact intervals (23, 30 and 60 min). We tested the short preservation time of 23 min to evaluate the effect of the surgical procedure (the clamping at the renal hilum and pulling out of the kidney). We conducted the experiment with the assumption that a prolonged contact time might improve the efficiency of gene transfer and that contact temperature might have some effect. However, we did not observe any consistent pattern in the effect of contact time and temperature on the efficiency of gene expression (Figure 3; P = NS, two-factor fractional ANOVA).

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Fig. 3. The effect of contact time and temperature on transgene expression with in vivo gene transfer to the kidney. Catheter-based in vivo gene transfer into the kidney was performed with 5 x 108 p.f.u. of Ad-Luc, with various contact times and temperatures. Luciferase gene expression was quantified 2 days after gene transfer using the IVIS system (n = 5 in each group; P = NS, two-factor fractional ANOVA).
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Transgene expression characteristics of ex vivo gene transfer into the graft kidney
Transgene expression in the transplanted kidney showed the same pattern as in vivo transduction (Figures 4 and 5). Gene expression in the graft kidney peaked at day 4 and decreased thereafter. Expression in the liver also occurred later, peaking between days 7 and 14, and it was much higher than in the graft kidney (Figure 5). Transgene expression in the grafted kidney on day 2 was about seven times lower (214 200±53 260 photons; n = 5) compared with in vivo gene transfer (1 450 000±668 431 photons; n = 5) under the same conditions (contact time: 60 min; temperature: 4°C).

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Fig. 4. Typical pattern of transgene expression in local gene transfer into the renal graft. After injection with 5 x 108 p.f.u. of Ad-Luc through the renal artery, the graft kidney was transplanted orthotopically. Luciferase gene expression was visualized by IVIS on day 4. (See online Supplementary Material for colour figure.)
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Fig. 5. Transgene expression in the graft kidney and host liver in ex vivo gene transfer. The graft kidney was injected with 5 x 108 p.f.u. of Ad-Luc and stored at 4°C for 60 min. Luciferase gene expression in the renal graft (A) and host liver (B) was measured quantitatively. *P<0.05 vs background, MannWhitney test.
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Discussion
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The major findings in the present study were that (i) transgene expression efficiency was independent of contact time or temperature in catheter-based in vivo gene transfer into kidney; (ii) transgene expression was observed in fibroblasts, mesangial cells, podocytes, the parietal epithelial cells of Bowman's capsule and tubular epithelium; (iii) gene expression in the ex vivo gene-transferred graft kidneys peaked early after gene transfer before decreasing; and (iv) strong gene expression was observed in the recipients' livers even with ex vivo gene transfer.
Some studies have attempted local gene transfer into the kidney by arterial injection with an adenovirus vector and the types of transduced cells have been different between these reports. Moullier et al. [3] reported a weak gene expression in the renal cortex, in tubular cells, and no expression in glomerular, vascular and interstitial structures by the simple injection of an adenovirus vector into the renal artery. Zhu et al. [5] demonstrated the transduction of a reporter gene to the interstitial vasculature, but not to the tubular cells by cooling and incubating the kidney with the adenovirus vectors for an extended period. In a porcine model, using continuous closed-circuit circulation of the kidney by a roller pump and membrane lung, Tryggvason and colleagues [14] reported transgene expression in 75% of the glomeruli, but not in parietal epithelial or tubular cells. The reason for the difference between previous reports and our result in the distribution of transduced cells is not clear, but differences in methodology may account for this discrepancy. And thus, by selecting the methods appropriately, gene transfer with an adenovirus vector to a specific type of cell in the kidney could be possible.
Some investigators have tried to improve transduction efficiency; however, their results, especially regarding transduction temperature, have been controversial. Parpala-Sparman et al. [14] reported that marked gene expression was observed when the continuous circulation was carried out at 37°C, but no expression was observed by circulation at room temperature. Those studies implied that prolonged contact time at a normal (not cold) temperature could result in highly efficient local gene transfer into the kidney by the arterial perfusion of the adenovirus vector. On the other hand, as described above, Zhu et al. [5] mentioned the superiority of cooling for adenovirus-mediated renal gene transfer. As for in vitro processing, it is well known that the adhesion of the adenovirus to cell surface receptors occurs at 4°C, whereas its endocytosis requires 37°C. Therefore, we performed the infection of the adenovirus vector at 4°C to estimate the effect of the contact time on the transduction efficiency in vivo. Furthermore, the cooling has advantages, including the fact that transduction at low temperature allows the organs to be better preserved. We then conducted the present study to clarify the effect of contact time and temperature on transgene expression in the kidney using our catheter-based in vivo gene transfer model. Nevertheless, our results do not demonstrate time- or temperature-dependency
Compared with in vivo gene transfer, studies that focused on ex vivo gene transfer into graft kidneys have achieved more positive results. In experimental renal transplantation, gene therapy was attempted by ex vivo gene transfer using an adenovirus vector and considerable success was obtained in preventing allograft rejection [15,16] or ischaemiareperfusion injury [17]. In those studies, transgene expression was detected by reverse transcriptasepolymerase chain reaction (RTPCR) during the first 57 days, which was consistent with our results that transgene expression in the graft kidney is observable by IVIS on days 2 and 7, but is very weak on day 14. However, the transgene expression efficiency of the ex vivo gene transfer was lower than that of in vivo transfer. Gene expression in the host liver was demonstrated in our ex vivo transfer experiment. Previous studies of ex vivo gene transfer into the renal graft did not report transgene expression in the host liver. One of the major limitations of adenovirus-mediated gene therapy is the immune reactions especially in the liver, that can cause death [18], our findings that transgene expression can occur in a host liver even when gene transfer is performed ex vivo is clinically important.
In our study we used a non-invasive bioimaging system to quantify transgene expression. This enabled us to evaluate periodically the same animals without sacrificing them. Moreover, the quantification of luciferase gene expression by this technique correlates well with luciferase activity measured by the conventional biochemical assay in vitro [19]. Previous studies that attempted gene transfer into kidneys in vivo or ex vivo relied on histological examination using the LacZ gene [36,14] or RTPCR [15,16] to investigate the efficiency of gene transfer. These methods need tissue samples and periodic evaluation in the same animal is impossible, for multiple biopsies from a single animal in separate timings are very difficult in small animal experiments. Considering the large interindividual variations in gene transfer, this non-invasive bioimaging system is suitable for quantitative evaluation of transgene expression in each animal.
In summary, we evaluated the effect of contact time and temperature on transgene expression in catheter-based in vivo gene transfer to the rat kidney and found that the efficiency of transgene expression was independent of both these factors. We also exposed the characteristics of gene expression in the renal graft and host liver in ex vivo gene transfer into the graft kidney. Transgene expression was observed in mesangial cells, podocytes, the parietal cells of Bowman's capsule and tubular cells. These findings may help improve gene therapy, in particular when targeting the kidney in vivo and ex vivo.
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Acknowledgments
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The authors thank Ms Harumi Kawana and Megumi Hata for their excellent technical assistance. This work was partially supported by 21st Century Center of Excellence (COE) programme of the Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan).
Conflict of interest statement. None declared.
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References
|
---|
- Imai E. Gene therapy approach in renal disease in the 21st century. Nephrol Dial Transplant 2001; 16 [Suppl 5]: 2634[Medline]
- Bosch RJ, Woolf AS, Fine LG. Gene transfer into the mammalian kidney: direct retrovirus-transduction of regenerating tubular epithelial cells. Exp Nephrol 1993; 1: 4954[ISI][Medline]
- Moullier P, Friedlander G, Calise D, Ronco P, Perricaudet M, Ferry N. Adenoviral-mediated gene transfer to renal tubular cells in vivo. Kidney Int 1994; 45: 12201225[ISI][Medline]
- Maruyama H, Higuchi N, Nishikawa Y et al. Kidney-targeted naked DNA transfer by retrograde renal vein injection in rats. Human Gene Ther 2002; 13: 455468[CrossRef][ISI][Medline]
- Zhu G, Nicolson AG, Cowley BD, Rosen S, Sukhatme VP. In vivo adenovirus-mediated gene transfer into normal and cystic rat kidneys. Gene Ther 1996; 3: 298304[ISI][Medline]
- Chetboul V, Klonjkowski B, Lefebvre HP et al. Short-term efficiency and safety of gene delivery into canine kidneys. Nephrol Dial Transplant 2001; 16: 608614[Abstract/Free Full Text]
- Ikeda Y, Gu Y, Iwanaga Y et al. Restoration of deficient membrane proteins in the cardiomyopathic hamster by in vivo cardiac gene transfer. Circulation 2002; 105: 502508[Abstract/Free Full Text]
- Takeda S, Takahashi M, Mizukami H et al. Successful gene transfer using adeno-associated virus vectors into the kidney: comparison among adeno-associated virus serotype 15 vectors in vitro and in vivo. Nephron Exp Nephrol 2004; 96: e119e126[CrossRef][ISI][Medline]
- Okada T, Ramsey WJ, Munir J, Wildner O, Blaese RM. Efficient directional cloning of recombinant the adenovirus vectors using DNAprotein complex. Nucleic Acids Res 1998; 26: 19471950[Abstract/Free Full Text]
- Kanegae Y, Lee G, Sato Y et al. Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res 1995; 23: 38163821[Abstract]
- Kita Y, Li XK, Ohba M et al. Prolonged cardiac allograft survival in rats systemically injected adenoviral vectors containing CTLA4Ig-gene. Transplantation 1999; 68: 758766[ISI][Medline]
- Inoue S, Hakamata Y, Kaneko M, Kobayashi E. Gene therapy for organ grafts using rapid injection of naked DNA: application to the rat liver. Transplantation 2004; 77: 9971003[CrossRef][ISI][Medline]
- Kaissling B, Hegyi I, Loffing J, Le Hir M. Morphology of interstitial cells in the healthy kidney. Anat Embryol (Berl) 1996; 193: 303318[ISI][Medline]
- Parpala-Sparman T, Lukkarinen O, Heikkila P, Tryggvason K. A novel surgical organ perfusion method for effective ex vivo and in vivo gene transfer into renal glomerular cells. Urol Res 1999; 27: 97102[CrossRef][ISI][Medline]
- Swenson KM, Ke B, Wang T et al. Fas ligand gene transfer to renal allografts in rats: effects on allograft survival. Transplantation 1998; 65: 155160[ISI][Medline]
- Tomasoni S, Azzollini N, Casiraghi F, Capogrossi MC, Remuzzi G, Benigni A. CTLA4Ig gene transfer prolongs survival and induces donor-specific tolerance in a rat renal allograft. J Am Soc Nephrol 2000; 11: 747752[Abstract/Free Full Text]
- Blydt-Hansen TD, Katori M, Lassman C et al. Gene transfer-induced local heme oxygenase-1 overexpression protects rat kidney transplants from ischemia/reperfusion injury. J Am Soc Nephrol 2003; 14: 745754[Abstract/Free Full Text]
- Marshall E. Gene therapy death prompts review of the adenovirus vector. Science 1999; 286: 22442245[Free Full Text]
- Wu JC, Inubushi M, Sundaresan G, Schelbert HR, Gambhir SS. Optical imaging of cardiac reporter gene expression in living rats. Circulation 2002; 105: 16311634[Abstract/Free Full Text]
Received for publication: 12. 4.04
Accepted in revised form: 14. 1.05