1 Unité de Cardiologie d'Alfort, 2 UMR 955 INRA de Génétique Moléculaire et Cellulaire, Equipe de Génétique Virale, Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, 3 URA INRA de Physiopathologie et de Toxicologie Expérimentales, Ecole Nationale Vétérinaire de Toulouse, Toulouse, 4 Département de Physiologie et INSERM U492 and 5 Département de Pathologie, Faculté de médecine, CHU Henri-Mondor, Créteil, France
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Abstract |
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Methods. Dog puppies were injected with an adenoviral vector expressing the ß-galactosidase reporter gene in both kidneys via three different routes, i.e. intra-renalureteral route (IU) and intra-renalarterial route with (IAC) or without (IA) clamping of the renal vein. Toxicity of viral administration was assayed on day 4 at both physiological and histological levels. Renal samples were monitored for the presence of nuclear ß-galactosidase-expressing cells.
Results. All renal physiological parameters (glomerular filtration rate, effective renal plasma flow, and electrolyte excretion fractions) remained stable whatever the route of viral administration. No histological lesion was detected in any of the haematoxylineosin-stained kidney sections, and there was no evidence of ischaemiareperfusion injury in the kidneys subjected to venous clamping. Efficient transgene expression was obtained in dog kidneys following IAC and IU injection of adenoviral vectors. Gene transfer via the IAC route induced gene expression predominantly in the cortical interstitial cells. Retrograde IU adenoviral injection resulted in reduced transduction efficiency compared with the IAC route, with transgene expression occurring mainly in the distal tubular and pyelic epithelial cells.
Conclusions. The two major findings of this study were (i) the absence of acute histological and functional renal alteration following intra-arterial and intra-ureteral injections of adenoviral vectors in both kidneys of healthy dogs, and (ii) the efficiency of transgene expression with specific cellular targeting according to the route of administration.
Keywords: adenovirus; dog; gene transfer; glomerular filtration rate; kidney; safety
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Introduction |
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The efficiency of several vector systems has been demonstrated in kidney-targeted gene transfer including recombinant adenoviruses [13], retroviruses [4], and non-viral vectors [5,6]. In contrast, the safety of the routes of local gene delivery has never been documented and compared, to determine the benefit-to-risk ratio of such therapy in human patients. Most gene-transfer studies have been performed in rodents, and these are inadequate for the repeated assessment of renal function (especially glomerular filtration rate and effective renal plasma flow) in the conscious animal. The dog is a more appropriate model for such purposes.
The aim of this study was to compare the safety of three different routes of adenoviral administration (intra-renalureteral, and intra-renalarterial with or without renal vein clamping) in kidneys of healthy dogs, and to determine both the distribution of the transgene expression and the nature of the cell type transduced. In conventional experiments of kidney-targeted gene transfer, gene delivery is performed in one kidney and the contralateral kidney is used as an internal negative control [1,3]. In the present study, bilateral injections were performed to amplify the potential adverse effects on renal function of such delivery. The ultimate assessment of efficiency and safety of intra-renal gene delivery will have to be based on long-term studies. However, such investigations will only be possible after preliminary testing of the comparative efficiencies of the different routes of adenoviral administration for transgene expression, and after evaluation of the potential inoculation-associated complications, especially on renal function. We chose therefore to investigate only the short-term effects of local gene delivery (i.e. within 4 days of inoculation) on renal function, which could result both from the surgical technique and the viral vector. Adenovirus-mediated gene transfer may indeed be associated with non-specific inflammatory lesions localized in the target organ, which occur within the first 4 days following inoculation [7], and result from activation of macrophages, NK cells, complement, and cytokine release [8]. Moreover, the classical reporter gene of ß-galactosidase, which is known to be a powerful immunogen [9], was used to assess the efficiency of gene transfer in our study. This transgene product may create local inflammation 715 days after viral injection [10] which is incompatible with the long-term study of safety of adenoviral inoculation: such lesions would not necessarily be transposable to other genes of interest that encode self-proteins.
This experiment is the first to demonstrate both the efficiency of intra-renal inoculation of adenovirus vector in dogs, and the absence of short-term adverse effects on renal function.
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Subjects and methods |
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Nine 2-month-old male Beagle dogs, unvaccinated against canine adenoviruses, with a mean (±SEM) body weight of 3.5±0.4 kg, were studied. The absence of neutralizing antibody against adenovirus was checked 8 days before injection (day -8) and at day 0 (day of injection). The study started after an acclimatization period of 6 days. The dogs were weighed and examined clinically every day throughout the study. They were housed in individual metabolic cages in a room at 21°C with a 12-h light/dark cycle, allowed free access to tap water, and normal-salt dog food (Royal Canin A32, 0.35% NaCl) given twice a day (at 10.00 a.m. and 6.00 p.m.). The animal use procedures in this experiment were approved by the Ecole Nationale Vétérinaire dAlfort.
Surgery and administration of viral vectors were performed on day 0. Dogs were anaesthetized by intravenous injection of ketamine (5 mg/kg) and diazepam (0.2 mg/kg). A mid-abdomen incision was made. The left, then right kidneys were exposed and injected similarly for each dog, either with AdCMV-ßgal, AdRSV-gD or a phosphate-buffered saline (PBS) solution. The incision was then closed. The renal artery was isolated for the intra-renalarterial injections. A 30-gauge needle (0.5/30 mm) was inserted into the renal artery. One millilitre of the viral suspension or PBS was infused over 1 min with (IAC route) or without (IA route) clamping of the renal vein. In the case of renal vein clamping, the renal blood flow was re-established 15 min later and the kidney examined for total homogeneous reperfusion. When retrograde intra-renalureteral injections were performed, the ureter was isolated. Using a 30-gauge needle, 1 ml of the viral suspension or PBS was directly infused over 1 min into the ureter (IU route). The distal portion of the ureter was clamped for 15 min, after which the urine-flow was re-established.
Five dogs received a single 1 ml injection of AdCMV-ßgal in each kidney at a dose of 109 p.f.u./kidney, by the intra-arterial route with (n=2) or without (n=1) clamping of the renal vein, and by the retrograde IU route (n=2). Three control dogs received 1 ml of PBS in each kidney by the same routes (by the intra-arterial route with (n=1) or without (n=1) clamping of the renal vein, and by retrograde IU route (n=1)). Another control dog received 1 ml of AdRSV-gD in each kidney at a dose of 109 p.f.u./kidney, by the IU route for the right kidney and by the IAC route for the left kidney.
The experiment was divided into three phases: phase 1: control period for 3 days (from day -2 to day 0); phase 2: immediate post-surgery period (days 1 and 2) to identify immediate surgical effects on renal function; phase 3: late post-surgery period (days 3 and 4). Plasma urea, creatinine, sodium and potassium concentrations were determined during the control (day -2 to day 0) and post-operative periods (day 0 to day 4). Twenty-four-hour-urine was collected from the metabolic cages every day at 8.00 a.m. Samples were stored at -80°C until assay (within 1 week). Urine osmolarity, urinary creatinine, sodium, potassium, and protein concentrations were determined. Plasma and urinary concentrations of electrolytes were determined using flame emission photometry (Ionocal 120, Hycel, Le Rheu, France). Plasma and urinary concentrations of urea, creatinine and protein were assessed using colorimetric methods (Cobasmira, Roche, Neuilly sur seine, France). Sodium (NaEF) and potassium (KEF) excretion fractions were calculated from plasma and urine concentrations of the given electrolytes and creatinine.
Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were determined before (day -2) and after surgery (day 4). Exo-iohexol and p-aminohippuric acid (PAH) were used respectively as GFR and ERPF markers, as previously described [12]. A commercially available iohexol formulation (Omnipaque 300, Nycomed Imaging AS, Oslo, Norway) and a PAH solution (100 mg/ml) extemporaneously prepared by dissolving PAH (Sigma, Saint-Quentin Fallavier, France) in sterile NaCl 0.9% were injected intravenously as a bolus. The nominal doses of iohexol and PAH were 64.7 mg/kg (which corresponds to 58.2 mg/kg of exo-iohexol) and 10 mg/kg, respectively. Blood (1 ml) was sampled before, and 2, 10, 30 min, and 1, 2, and 3 h after the administration, placed in a heparinized tube and centrifuged (1000 r.p.m., 10 min) at 4°C. Two aliquots (0.2 ml) of plasma were stored at -20°C until assayed. The plasma concentrations of PAH and iohexol were simultaneously determined by high-performance liquid chromatography [12]. The limits of quantification for the exo-iohexol and PAH assay methods were 9 and 1 µg/ml respectively. The within- and between-day reproducibilities were less than 6% for PAH and below 13% for iohexol (endo/exo). Exo-iohexol and PAH plasma clearances were determined using a non-compartmental approach with extrapolation to infinity [13].
Urinary excretion of viral particles was checked by incubation in 293 cells using X-gal staining and also by indirect immunofluorescence using a fibre specific monoclonal antibody. The detection limit of this virus excretion assay was 103 infectious particles/per millilitre.
The animals were euthanatized on day 4 by intravenous administration of an overdose of pentobarbitone. The kidneys were excised. Some pieces were immediately fixed in 10% buffered formalin for histological examination. A standard haematoxylineosin stain was used. Samples of kidneys, liver (right lateral lobe) and lungs (right caudal lobe) were fixed in 4% paraformaldehyde for 45 min, incubated overnight in a 30% sucrose solution at 4°C and then, in a reaction mix (400 µg/ml X-gal (Roche Molecular Biochemicals, Germany), 4 mmol/l potassium ferrocyanide, 4 mmol/l potassium ferricyanide, 2 mmol/l MgCl2) overnight at room temperature to detect ß-gal expression. Tissue samples were embedded in paraffin, cut into 5-µm sections, and then counterstained with haematoxylineosin.
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Results |
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Plasma biochemical parameters remained stable and within the normal canine range throughout the study period in all dogs (data not shown). Visual inspection of the data (Figure 1) showed that there was no decrease in GFR and ERPF during the late post-operative period (phase 3) as compared with the pre-operative phase 1, in dogs injected with adenoviral vectors (AdCMV-ßgal and AdRSV-gD), by either IA or IU routes. NaEF and KEF appeared similarly unaffected (Figure 2
). Other urinary parameters including the urinary protein/creatinine ratio and osmolarity were less than 0.003 and between 200 and 400 mOm/l respectively, i.e. within the normal limits. All kidneys (n=18) appeared normal on macroscopic examination. No histological lesions, in particularly no inflammatory infiltration, were apparent on examination of any of the haematoxylineosin stained kidney sections.
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Discussion |
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Recombinant adenovirus is one of the most efficient vectors for in vivo gene delivery. However, adenovirus-mediated gene transfer is associated with some negative side-effects such as early toxicity localized at the site of injection, and CD4+/CD8+-mediated immune response. This specific immune response is directed against both viral proteins and the transgene product. However, early local toxicity is a general component of an adenoviral administration. Such in vivo toxicity may be a limiting factor for the successful use of adenoviral vectors and requires clinical investigation to clearly define both the limits and efficacy of adenoviral-mediated gene therapy [14]. In our study, gene transfer was not associated with short-term nephrotoxicity, as assessed by renal histology and function. The examination of haematoxylineosin stained sections of all kidneys did not show any histological lesions, and particularly no inflammatory reactions. No evidence of ischaemiareperfusion injury was observed in the kidneys subjected to venous clamping. Moreover, although adenoviral injections were performed in both kidneys, the urea and creatinine plasma levels, GFR, ERPF, sodium and potassium excretion fraction, protein/creatinine ratio, and osmolarity remained stable whatever the route of viral administration. These results demonstrate that in vivo adenovirus-mediated gene transfer into the two kidneys of normal dogs leads to significant gene expression without any short-term deleterious effects, this being a pre-requisite for kidney-targeted gene therapy of renal diseases.
Efficient transgene expression was obtained in canine kidneys following IAC and IU injections of adenoviral vectors. Gene transfer by IAC injection induced cortical gene expression located predominantly in the interstitial cells, and also in some endothelial cells. In contrast to the IAC route, retrograde IU adenoviral injection resulted in reduced transduction efficiency, with transgene expression mainly in the distal tubular and pyelic epithelial cells. The absence of transduction observed following intra-renalarterial injection of an adenoviral vector without clamping of the renal vein is consistent with previous results obtained for rat kidneys [3] and pig kidneys [15]. One way to achieve gene transfer by IA route is to increase the time that the kidney is exposed to the viral solution. In our protocol, we clamped the renal vein for 15 min, which resulted in gene transfer in the cortical area without renal damage. Such a procedure has already been reported in the rat [2].
Some of our results differ slightly as regards transduced cell distribution from those published in other animal models. It was previously reported that injecting a replication-defective adenoviral vector into the rat renal artery resulted in a selective proximal tubular cell expression, with no evidence of any gene transfer into interstitial and vascular cells [1]. In the same report, transduction of the medulla was observed, when the IU route was used. The inefficient gene transfer to the medulla by IU route observed in our study may reflect the inability of the adenoviral vector to enter the canine medullar tubular cells. This could be explained by the lack of high-affinity fibre receptors [16] on their apical face and/or the absence of Vß3/5 integrins reported to partially mediate the internalization of the virus into the cell [17,18]. The limited entry of adenovirus into well-differentiated epithelium has not in fact been studied in renal tissue, but has already been proved for intestinal and airway epithelial cells [19,20].
The duration of the post-operative follow-up and the number of animals were the two limits of our study. Certainly it would have been interesting to assess the safety of intra-renal adenoviral administration over a longer period. However, the goal of our study was to investigate the tropism of gene delivery using three different routes of Ad-vector administration and the potential short-term (or acute) adverse effects induced by both the surgical procedure and the intra-renal inoculation of the adenovirus vector. Moreover, lacZ is not a reporter gene of choice to study the duration of expression after gene delivery, because ß-galactosidase is a strong immunogenic protein which induces a host immune response deleterious to transgene expression and is thus incompatible with a long-term study. In further long-term experiments, lacZ will be replaced by clinically relevant genes to study not only the long-term tolerance of such a gene transfer, but also the duration of gene expression and its potential beneficial effect.
The number of dogs used in our study (n=9) could have been greater. However, this number was consistent with previous gene transfer experiments involving large animals, and represented an optimal compromise for testing our hypothesis and respecting ethical issues, as previously described for this kind of study in cats [21], dogs [11], and monkeys [22].
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Conclusion |
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Acknowledgments |
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Notes |
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Correspondence and offprint requests to: Marc Eloit, UMR 955 INRA de Génétique Moléculaire et Cellulaire, Equipe de Génétique Virale, Ecole Nationale Vétérinaire d'Alfort, F-94704 Maisons-Alfort Cedex, France.
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References |
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