Gene therapy of Alport syndrome (hereditary nephritis) aims at the transfer of a corrected type IV collagen α chain gene into renal glomerular cells responsible for production of the glomerular basement membrane (GBM). A GBM network composed of type IV collagen molecules is abnormal in Alport syndrome which leads progressively to kidney failure. The most common X-linked form of the disease is caused by mutations in the gene for the α5(IV) chain, the α5 chain of type IV collagen. Full-length human α5(IV) cDNA was expressed in HT1080 cells with an adenovirus vector, and the recombinant α5(IV) chain was shown to assemble into heterotrimers consisting of α3(IV) and α4(IV) chains, utilizing a FLAG epitope in the recombinant α5(IV) chain. The results indicate that correction of the molecular defect in Alport syndrome is possible. Previously, we had developed an organ perfusion method for effective in vivo gene transfer into glomerular cells. In vivo perfusion of pig kidneys with the recombinant adenovirus resulted in expression of the α5(IV) chain in kidney glomeruli as shown by in situ hybridization and its deposition into the GBM was shown by immunohistochemistry. The results strongly suggest future possibilities for gene therapy of Alport syndrome.
Alport syndrome is an inherited kidney disease characterized by progressive hematuria, development of renal failure and frequently also hearing loss.1,2 The only available treatment is hemodialysis and/or kidney transplantation. The underlying cause of the disease is a defective structure of the type IV collagen framework of the glomerular basement membrane (GBM). The disorder results in deterioration of the GBM. The disease affects about 1:5000 males.1 It has been estimated that 85% of cases are caused by mutations in the X chromosomal gene encoding the α5(IV) collagen chain.3,4,5 The less frequent autosomal forms are caused by mutations in the α3(IV) or α4(IV) collagen chain genes located on chromosome 2.6,7
Type IV collagen is a basement membrane-specific collagen type which is the main structural component of these extracellular structures.8 Similarly to other collagens, type IV collagen is a triple-helical protein consisting of three α chains. The collagen α chains have (Gly-Xaa-Yaa)n repeats, glycine being the only amino acid small enough to fit into the center of the triple helix. The type IV collagen α chains have many interruptions in the Gly-Xaa-Yaa repeat which allows for flexible kinks in the triple helix. In addition to the collagenous domain, the type IV collagen molecules have a noncollagenous globular NC1 domain at the carboxyl end, and the aminoterminal has a noncollagenous 7S domain. Six genetically distinct type IV collagen α chains have been described. The α1(IV) and α2(IV) chains are ubiquitous and are present in triple-helical molecules in a 2:1 ratio.9 The other α chains have variable and more restricted tissue distribution. The current understanding of type IV collagen synthesis in the renal glomerulus is illustrated in Figure 1a and b. In the GBM, α1(IV) and α2(IV) dominate during embryonic development (Figure 1a), but after birth these are replaced by trimers containing α3(IV), α4(IV) and α5(IV) chains. Due to their high cysteine content the α3:α4:α5 trimers are thought to be necessary for forming a stronger, more cross-linked GBM collagen network (see Figure 1b).10,11,12 In X-linked Alport syndrome caused by a mutation in the α5(IV)αchain gene, the α3(IV) and α4(IV)αchains are usually absent from the GBM, even though their genes residing on chromosome 2 are intact.13 This is presumably due to intracellular degradation of the chains in the absence of α5(IV) that is essential for the formation of the α3:α4:α5 trimer. Instead, the Alport GBM contains the embryonic type of collagen IV molecules consisting of α1 and α2 chains (Figure 1c). However, since these apparently do not provide sufficient mechanical strength to the GBM and sufficient resistance to proteolysis,14 the consequence is deterioration of the structure, hematuria and development of Alport syndrome.
Alport syndrome is an attractive candidate disease for gene therapy due to its high kidney specificity and because the isolated blood circulation of the kidneys makes them a good target for organ-specific gene transfer. The principle of gene therapy of X-linked Alport syndrome is depicted in Figure 1d. This requires transfer of the appropriate type IV collagen α chain gene to the endothelial and epithelial cells of the glomerulus, expression of the protein and intracellular assembly of the exogenous recombinant chain into triple-helical molecules together with the endogenous or α3, α4 or α5 chains, and, finally, secretion of the protein and incorporation of the protein into the GBM type IV collagen network (see Figure 1d).15 For the development of gene therapy of Alport syndrome, we have previously developed an organ perfusion system for adenovirus-mediated gene transfer into renal glomeruli in vivo.16 Using this procedure, we obtained a transfer efficiency of up to 85% of pig glomeruli using an adenovirus containing the β-galactosidase reporter gene. Surprisingly, the kidney perfusion method resulted in efficient transfer only to glomerular cells, while cells in other regions of the kidney did not take up significant amounts of the virus.
The present study was undertaken to develop further a gene therapy procedure for Alport syndrome. The first objective was to obtain expression of full-length human α5 collagen cDNA with an adenovirus vector in cultured cells, and explore whether the recombinant α5(IV) chain is incorporated into triple-helical molecules. Two constructs were made. One construct contained full-length normal human α5(IV) chain cDNA, while the other one had an extra sequence, a FLAG-tag, that enables purification of the recombinant protein for functional studies, and allows for its localization in the tissue after in vivo gene transfer. The recombinant α5(IV) chain was shown to be incorporated into triple-helical type IV collagen molecules containing endogenous α3 and α4 chains, indicating that correction of the molecular defect in Alport syndrome might be possible. The second objective of this study was to explore if in vivo perfusion of pig kidneys with these viruses would result in expression of the recombinant α5(IV) chain and deposition of the polypeptide chain into the GBM. The experiments resulted in efficient gene transfer into glomeruli and expression of recombinant α5(IV) chain, as determined by in situ hybridization and immunolocalization. Importantly, the recombinant α5 chain was deposited into the pig GBM, indicating that it was assembled intracellularly into triple-helical molecules together with endogenous pig type IV collagen α chains and secreted from the cell. The results strongly suggest the feasibility of gene therapy for Alport syndrome by targeted gene transfer to renal glomeruli.
Adenovirus vectors for expression of the type IV collagen α5 chain
For expression of the α5 collagen chain two constructs were made (Figure 2). The first adenovirus Ad-A5wt vector contained the full-length human α5(IV) chain coding sequence,17 and the second one contained the same cDNA with an inserted FLAG-tag coding sequence. The FLAG tag was used to enable easy purification of the recombinant α5 chain, as well as to distinguish it from the endogenous one in tissues following in vivo gene transfer in pigs. The eight-residue FLAG tag sequence was placed in a 10-residue noncollagenous sequence in the fifth interruption in the collagenous domain of the α5 chain. This interruption coincides with interruptions in all other α(IV) chains, and it is not known to have any special role, other than to provide a kink in the molecule. The interruption does, for example, not contain cell binding sites or cross-linking amino acids and, furthermore, none of the over 300 different missense mutations now identified in the α5(IV) chain in Alport syndrome is located in this interruption.5,18 Consequently, it was considered likely that the FLAG sequence would not interfere with assembly of this chain into a triple-helical molecule. In addition to this modification, the translation initiation signal in both constructs was modified to contain the optimal context for initiation of translation according to Kozak.19 The two types of adenoviruses were made by homologous recombination, and the recombinant viral plaques were purified by plaque assays. The cDNA sequence encoding human α5(IV) cDNA was shown to be correct as determined by DNA sequencing (not shown).
Characterization of recombinant type IV collagen α5 chains expressed with adenovirus vectors in human cells
Expression of the α5(IV) chain was first detected by in vitro translation of a 180 kDa polypeptide demonstrating that the construct encodes a protein of the right size (data not shown). Adenoviral expression of the α5(IV) in infected 293A cells was detected by immunoblotting of the protein from cell lysates and media. The recombinant α5(IV) chain was detected as a band of about 200 kDa (Figure 3), using either an anti-FLAG antibody or monoclonal anti-α5(IV) antibody H53 made against a peptide sequence in the third interruption in the collagenous domain.20 The increase in size in vivo versus in vitro can be explained by glycosylation and other cellular post-translational modifications. The H53 antibody recognized polypeptide chains produced with both constructs, while the anti-FLAG antibody only recognized the α5(IV) chain with the FLAG marker. Both antibodies recognized two additional bands of about 100 kDa and 130 kDa in the medium. These bands probably represent degradation products.
Chain composition of secreted type IV collagen molecules containing the recombinant α5(IV) chain
In order to study with which α chains the recombinant α5(IV) chain can assemble in a triple-helical collagen molecule, the chain was expressed in human HT1080 cells that normally express the α1(IV)–α5(IV) collagen chains, but not α6(IV). The α1(IV), α2(IV) and α5(IV) chains normally synthesized and secreted by these cells were easily detectable in Western blots from the medium, while the α3(IV) and α4(IV) chains were synthesized in such small amounts that they were only detectable in the cell lysate. Following infection of the cells with the adenovirus construct containing α5(IV) cDNA with the FLAG-tag, the α5(IV) chain was detectable in cell lysates and culture media by Western blotting using anti-FLAG antibodies. The recombinant α5-FLAG chain present in the medium was mainly observed as a single chain without assembling with the other endogenous α(IV) chains, but some of it was assembled with other α(IV) chains. To study the chain composition of trimers containing the α5-FLAG chain, medium protein was immunoprecipitated with the anti-FLAG antibody. The immunoprecipitate was found to contain α3(IV) and α4(IV) chains, in addition to the α5-FLAG chain, as shown by Western blotting with chain specific monoclonal antibodies21 (Figure 4). Furthermore, a minor band was visible by staining with an antibody against the α2(IV) chain. The α1(IV) chain was not contained at all in immunoprecipitates obtained with the anti-FLAG antibody. The α6(IV) chain was never observed, which can simply be explained by the fact that the α6(IV) chain is normally not expressed by this cell line. The immunoprecipitated medium from cells expressing the wild-type construct, used as a control in the immunoprecipitations, was negative for all α chains in immunostaining (Figure 4). These results demonstrated that the recombinant α5-FLAG chain is capable of incorporating into α3:α4:α5 chain trimers, the authentic type IV collagen isoform of adult GBM, while it does not assemble with α1(IV) and α2(IV) chains.
Analysis of expression of a recombinant α5(IV) chain mRNA and protein in the porcine kidneys following in vivo organ perfusion
To study if the recombinant α5(IV) can be expressed in kidney glomeruli in vivo, we used a perfusion method developed for adenovirus-mediated gene transfer in pigs.16 Briefly, the porcine kidney was isolated from the systemic blood circulation by clamping the renal artery and vein, after which the organ was perfused with a solution containing the adenovirus and red blood cells (hematocrit 17%) using an external pump and oxygenation system. Samples containing between 1.5 × 1010 and 1.6 × 1013 p.f.u. of Ad-A5FLAG adenovirus and same amounts of Ad5CMVlacZ adenovirus were injected into the renal artery before perfusion. The Ad5CMVlacZ vector was used as an internal control to evaluate general gene transfer efficiency in each experiment. Gene transfer efficiency, as measured by X-gal staining of tissue sections, varied between 10 and 50% of positive glomeruli, depending on the virus concentration (Table 1). The highest transfer efficiency was obtained with concentrations above 1.6 × 1013. The results were similar to those previously reported for the Ad5CMVlacZ virus, where transfer efficiency to pig glomeruli in vivo was as high as 80%.16 As previously reported,16 only glomerular cells showed expression of the transgene, while other kidney structures were practically completely negative for expression of β-galactosidase (not shown). In the experimental series of this study, virus concentration of 5 × 1011 p.f.u. gave the best results, with close to 50% gene transfer efficiency and no pathological changes observed in kidney morphology or function during the 4-day post-operation observation period (Table 1). In contrast, two gene transfer experiments with virus concentrations of 6 × 1012 and 1.6 × 1013 resulted in interstitial nephritis (Table 1).
To analyze expression of the recombinant α5(IV) chain in the kidney, mRNA for the α5 chain was visualized by in situ hybridization (Figure 5) using a 172 base pair probe from cDNA encoding the NC1 domain of the human α5(IV) collagen chain. The expression pattern in the perfused kidneys was similar to that obtained by X-gal staining, and no expression was seen in the control kidneys (Figure 5B). This means that the amount of endogenous α5(IV) chain mRNA is very low, or that porcine mRNA, which has not been sequenced, is not completely homologous with the human one.
Translation of the recombinant α5 chain mRNA in the perfused kidneys was demonstrated by immunofluoresence staining using an FITC-conjugated anti-FLAG antibody (Figure 6a), while no staining was detected in the control kidneys (not shown). Similarly to the in situ hybridization results, the recombinant α5(IV) polypeptide chain was only seen in glomeruli. Importantly, the anti-FLAG staining was consistent with a linear GBM-like staining, indicating that the recombinant α5(IV) chain was truly incorporated into the GBM proper (Figure 6a). Co-staining of the same sections for the the type IV collagen α4 chain using an AlexaFluor-labeled secondary antibody (Figure 6b) indicated that the linear structures positive for the α5-FLAG chain were the GBM proper. Merge of the two colors (Figure 6c) gave a yellowish color of the GBM structures, but at two locations the α5(IV) chain staining did not overlap with that of the α4(IV) chain. This possibly represented mesangial regions.
The present work was carried out to explore the possibility of developing treatment for Alport syndrome by gene therapy. The basic disorder of this disease is abnormal structure of the GBM network made of triple-helical type IV collagen molecules that have an α3:α4:α5 chain composition. Mutations in any of the respective genes can lead to the disease.
Gene therapy of Alport syndrome aims at the transfer of a corrected type IV collagen α chain gene into renal glomerular cells that are responsible for production of the GBM. The prerequisites for gene therapy of Alport syndrome include: (1) availability of an appropriate gene delivery system into cells of renal glomeruli; (2) expression of the delivered type IV collagen gene in those cells; (3) proper post-translational modifications and folding of the respective α chain which facilitates intracellular association into an α3:α4:α5 trimer; and (4) incorporation of those trimers extracellulary into the GBM proper, which could restore the deteriorated GBM structure.
We have previously demonstrated the possibility of targeting expression of foreign genes into cells of the renal glomeruli in vivo, using adenovirus containing a reporter gene.16 The results of the present study represent two significant steps forward towards gene therapy of Alport syndrome. First, it was demonstrated that one can use adenovirus to produce the recombinant α5(IV) chain in cultured human cells so that this chain is incorporated into type IV collagen trimers with the α3:α4:α5 chain composition that is essential for normal structure and function of the adult GBM. Second, the adenovirus-mediated expression of the recombinant human α5(IV) chain in pig kidneys in vivo resulted in the production of an α5(IV) chain that also was deposited extracellularly into the GBM proper.
Type IV collagen trimers composed of α3, α4 and α5 chains in a 1:1:1 ratio are the predominant isoform of the GBM as shown in biochemical assays or indirectly by localization of these three chains in the GBM using chain-specific monoclonal antibodies.12,13 In this study, the recombinant human α5(IV) chain expressed in a human fibrosarcoma cell line was associated with α3(IV) and α4(IV) chains, but not with the α1(IV) or α2(IV) chains of type IV collagen. This was demonstrated by using a FLAG antibody that selectively immunoprecipitated the α3(IV), α4(IV) and α5(IV)-FLAG together. Although it was not possible to demonstrate if the three chains were present in the trimers in a 1:1:1 ratio, it can be considered likely, as the α5(IV) chain is known to assemble with α3(IV) and α4(IV) in basement membranes such as the GBM where these three chains are all present.12,13 The faint staining for the α2(IV) chain is not considered significant, because the α2(IV) chain is expressed in these cells in considerably higher amounts compared with that of the α3(IV) and α4(IV) chains. However, it is possible that minor amounts of the α2(IV) chain can be associated with the network composed of α3(IV), α4(IV), and α5(IV) chains by noncovalent interactions as shown by Gunwar et al.12 The present findings on chain composition were obtained under cell culture conditions, but the chain assembly of the α(IV)-FLAG chain in the perfused pig kidneys of this study is likely to occur in the same way.
In the present study the adenoviral gene transfer into pig kidneys in vivo resulted in expression of human α5(IV) chain in the glomeruli. This was clearly demonstrated both by mRNA and protein analyses using in situ hybridization and indirect immunofluoresence, respectively. As previously described the perfusion method only directed the gene transfer to glomerular cells, which are the actual target cells for gene therapy of Alport syndrome. The recombinant α5(IV)-FLAG chains were clearly incorporated into the GBM proper, as demonstrated by the double staining for the recombinant α5(IV)-FLAG and endogenous α4(IV) chains. It can be considered likely that the α5(IV)-FLAG chains assembled intracellularly into trimers with endogenous porcine α3(IV) and α4(IV) chains, because this is the combination normally found in cells that simultaneously synthesize these chains. Furthermore, it is known that single α5(IV) chains do not form trimers, and that they are degraded inside the glomerular cells if they do not find their α3(IV) and α4(IV) chain partners, as happens in autosomal Alport syndrome where either the α3(IV) or α4(IV) chains are absent. The sequence encoding porcine α5(IV) chain is not known and, therefore, it is not known how homologous it its with the human sequence. However, in this study the deposition of the recombinant human α5(IV) chain into the GBM in swine kidneys implies that the swine α5(IV) can be replaced by the corresponding α chain of man. This is not unusual for heterotrimeric basement membrane proteins, as for example, recombinant mouse and human laminin chains expressed simultaneously in vitro and in vivo can form hybrid trimers.22,23 A large body of data has shown that only trimeric collagen molecules are incorporated into fibers or networks of the extracellular matrix. Therefore, it is likely that the deposition of the recombinant α5(IV)-FLAG chain in the GBM represents depostion of trimeric molecules. The most likely composition of these trimers is α3:α4:α5 because this isoform normally forms the type IV collagen network of adult GBM. However, direct evidence for such trimers would require isolation of such molecules and their biochemical analysis which could not be carried out within this study.
The current results indicate that a genetic collagen disease such as Alport syndrome might be treatable by targeted gene transfer to the renal glomeruli, and most of the criteria for carrying out actual gene therapy experiments have now been met. Several dog24,25 and mouse26,27,28 models for both X chromosome linked and autosomal recessive Alport syndromes are now available for gene therapy studies. The X-linked forms described in Samoyed dogs24 and in a family of mongrel dogs from Navasota, Texas25 render themselves useful for the type of gene transfer experiments carried out in this study, as the canine disease is essentially identical to that in humans. Affected dogs of both strains develop hematuria and proteinuria within a few months after birth, and progression to renal insufficiency by 8–15 months. Adenovirus-mediated gene transfer with α5(IV) cDNA initiated at the onset of hematuria symptoms might give a positive therapeutic response that could extend the life span of affected dogs. Such an experiment would be important as a ‘proof of principle approach’. Since adenovirus only provides expression of the transgene for about 6–8 weeks, it will not be a question of life-lasting treatment with such a vector. However, since the half-life of basement membrane proteins, such as type IV collagen, is up to 2 years, the life span of the affected dogs might be expanded significantly if the treatment turns out to be effective.
In conclusion, the present results provided important advances concerning two key prerequisites for gene therapy of Alport syndrome. First, a recombinant α5(IV) chain expressed by an adenovirus is capable of assembling with endogenous α3(IV) and α4(IV) chains of cultured cells into triple helical collagen molecules. Second, the recombinant α5(IV) chain expressed by adenovirus in swine renal glomerular cells in vivo is deposited into the GBM. These results together with the previous development of a glomerulus-specific gene transfer method strongly support the possibilities for gene therapy of Alport syndrome.
Materials and methods
DNA construction and analysis
A 5270 base pair plasmid containing full-length human α5(IV) collagen cDNA with an SV40 polyA signal was constructed from overlapping cDNA clones and PCR products into a pBluescriptSK− vector (Stratagene Cloning Systems, La Jolla, CA, USA). The SV40 poly A sequence was included in the early cloning steps, but not in the final expression constructs. First, a pBluescript plasmid containing the SV40 polyA signal was created. The SV40 polyA signal was amplified by PCR from a pSG5 vector (Stratagene) using a linker sequence in a forward primer to create EcoRI, MunI and BglII sites to the 5′ end, and an XhoI site to the 3′ end of the PCR fragment. The fragment was subsequently inserted into the pBluescript vector using the EcoRI and XhoI sites.
A subclone A5–3′XX containing the 3′ end of the α5(IV) cDNA linked to SV40 poly A sequence was created by inserting an EcoRI(2934)–ApaI(3969)fragment from the PL-31 cDNA clone and an ApaI(3969)–EcoRI(5270) fragment from the MD-6 cDNA clone14 into a pBluescript vector cut with EcoRI. The resulting EcoRI(2934–5270) fragment was ligated into the SV40 polyA signal containing plasmid that was digested with MunI and EcoRI. The subclones in which the EcoRI site (5270) was mutated, due to ligation to the MunI site, were selected and subsequently cut with XbaI and EcoRI. The XbaI(2404)–EcoRI(2934) fragment from the HT14 cDNA clone17 was then inserted to create the plasmid A5–3′XX, that contained the 3′ half of the cDNA. The numbering of the nucleotides starts from base 1 at the 5′ of the cDNA.17
A plasmid A5–5′NX containing the 5′ half of the cDNA was created by ligating the following three DNA fragments into the pBluescript vector that was linearized with NotI and XbaI: (1) NotI–AvaI(420) from the JZ-4 cDNA clone17 subcloned to the pBluescript vector; (2) AvaI(420)–AccI(768) PCR fragment amplified from the HT14 cDNA template using a forward primer extending to the AvaI(420) site (HT14 clone starts at site 444); (3) AccI(768)–XbaI(2404) from the HT14 cDNA clone.
To generate plasmid construct pA5-UFL that contains the full-length coding sequence for the α5(IV) chain, the XbaI(2404)–XhoI fragment was recovered from the A5–3′XX plasmid and ligated into the A5–5′NX plasmid that was cut with XbaI and XhoI.
To facilitate recombinant protein purification and distinction from the endogenous α5(IV) collagen the full-length α5(IV) cDNA was modified to contain a nucleotide sequence encoding the FLAG epitope, an octapeptide with the amino acid sequence DYKDDDDK. The FLAG sequence was added to the sequence encoding the fifth interruption in the collagenous domain by oligonucleotide-directed mutagenesis. A forward primer homologous to fragment 1033–1052 in the cDNA sequence and a mutagenic primer 5′-TTCTCCTATAGTTATCTTGTCA- TCGTCGTCCTTGTAGTCTCTAGGAATTACAAGTCCA-3′ containing the FLAG encoding sequence were used to amplify a 254 bp megaprimer which after purification was used as a primer in a second PCR reaction with a reverse primer homologous to nucleotides 1702–1721. The HT14 cDNA clone was used as a template. The 700 bp PCR product was digested with MscI(1120) and HincII(1683) and inserted using the same enzymes into A5–5′NX which was earlier modified by deleting the HincII site from the polylinker. The sequence of the PCR fragment and the cloning sites of the resulting plasmid were verified by sequencing, and the NotI–XbaI(2404) insert was ligated into the A5–3′XX plasmid to generate the pA5-UFLAG plasmid. The inserts of the pA5-Uwt and pA5-UFLAG plasmids were sequenced to check the presence of possible mutations, and then used in an in vitro translation assay (TNT coupled reticulocyte lysate system; Promega, Madison, WI, USA) to ensure the translation of a full-length polypeptide chains (185 kDa).
To increase the expression level of the recombinant protein, the translation initiation signal of α5(IV) cDNA was replaced by a modified Kozak translation initiation sequence.19 A forward primer 5′-AAGGAAAAAAGCG- GCCGCAAGCTTGCCGCCACCATGGAACTGCGTGG- AGTCAGCCT-3′ homologous to α5(IV) cDNA at position 203–225, plus containing a modified translation initiation signal and restriction sites for NotI and HindIII, and a reverse primer homologous to 417–440, was used to amplify a 270 bp PCR fragment using the HT14 cDNA clone as a template. The PCR product was digested with NotI–AvaI(420) and subcloned, together with the AvaI(420)–XbaI(2404) insert from the pA5-Uwt and pA5-UFLAG plasmids into the A5–3′XX plasmid that was linearized with NotI–XbaI to generate plasmids pA5-Uwt-205 and pA5-UFLAG-205, respectively.
To produce the transfer plasmids for generation of the adenoviruses to be used for gene transfer of the α5(IV) cDNAs, the NotI–BglII inserts of pA5-UFL-205 and pA5-UFLAG-205 were ligated into a dephosphorylated pAdBM5pAG vector (Quantum Biotechnologies, Quebec, Canada) digested with BamHI. The unligated ends were treated by Klenow, phenol extracted and subsequently blunt-end ligated to form circular transfer plasmids called pAdBM5pAG+Uwt and pAdBM5pAG+UFLAG.
Construction and purification of recombinant adenoviruses
Recombinant adenoviruses (Ad-A5FLAG and Ad-A5wt) containing the 5.2 kb cDNA encoding the human α5(IV) type IV collagen chain with and without a FLAG tag were produced using an Adeno-Quest Adenovirus expression system (Quantum Biotechnologies). Briefly, E1/E3-deleted replication-defective serotype 5 human adenoviral (AdCMVlacZΔE1/ΔE3) DNA and the recombinant transfer vectors pAdBM5pAG+Uwt and pAdBM5pAG+UFLAG linearized with ClaI were co-transfected into 293A cells. The recombinant virus plaques purified by consecutive plaque assays and viral clones expressing the α5(IV) chain were identified first by PCR and then by Western blotting using anti-FLAG-M2 and anti-α5(IV) chain H53 antibodies. The resulting adenoviruses contained the α5(IV) chain cDNA under the control of the adenovirus major late promoter and enhancer and the rabbit globin poly A sequences. The adenoviral stocks were purified twice by ultracentrifugation through a CsCl2 gradient, followed by desalting with Econopac columns (Bio-Rad Laboratories, Hercules, CA, USA). The titers of the virus stocks were assessed by plaque assays. Human adenovirus AdCMVlacZ, used as a control, was amplified and purified as above. The viral preparations were tested for replication competence by extended cultivation on HeLa cells.
Infection of human cells in vitro
HT1080 or 293A cells were cultured in DMEM (Life Technologies, Gaithersburg, MD, USA) containing 10% FCS and penicillin/streptomycin until 80% confluency. The cells were infected with adenoviruses Ad-A5FLAG or Ad-A5WT at MOI 1000 in serum-free medium for 3 h. The virus containing medium was rinsed off and the cells were fed by serum-free medium containing 100 μg/ml ascorbate (Sigma, St Louis, MO, USA). Ascorbate (50 μg/ml) was added daily to ensure appropriate post-translational modifications of collagenous proteins.
Protein analysis, purification, antibodies and immunoblotting
Three days after infection, the cells were collected and suspended in an SDS sample buffer. The medium was collected and centrifuged at 8000 r.p.m. for 10 min, and used in immunoprecipitation or concentrated by precipitation with 70% ethanol at −20°C for 1 h. After centrifugation at 8000 r.p.m. for 1 h at 4°C, the pellets containing the precipitated proteins were suspended in a SDS sample buffer and analyzed by SDS-PAGE.
To purify intact, recombinant α5(IV) chains containing a FLAG tag, medium from adenovirus-infected cells was immunoprecipitated using an anti-FLAG M2 antibody covalently conjugated to Sephadex matrix (Eastman Kodak Scientific Imaging Systems, New Haven, CT, USA), as described in the manufacturer's instructions. The protein was eluted into reductive SDS sample buffer and analyzed by SDS-PAGE using 6% gels. Western blot analysis was performed using standard methods. The primary antibodies used were rat monoclonal antibodies H11, H22, H31, H44, H53 and H63 against the type IV collagen α1 to α6 chains, respectively,20,21 or antibodies raised against the FLAG epitope (Eastman Kodak Scientific Imaging Systems). The blots were incubated with the primary antibodies, followed by incubation with the secondary antibody and chemiluminescent detection (Du Pont NEN, Boston, MA, USA). The secondary anti- bodies were peroxidase-conjugated (DAKO, Glostrup, Denmark).
In vivo administration of adenovirus into the pig kidney by organ perfusion
In vivo perfusion was carried out essentially as previously reported.16 Briefly, young farm pigs were anesthesized, and the left kidney was exposed via paramedial incision and using extraperitoneal dissection. The renal artery, vein and ureter were clamped and cannulated. The cannules were connected to silicon tubings connected to the perfusion device. Perfusion was performed in a closed-circuit mode where the oxygenated and heated (37°C) perfusate was recirculated continuously through the kidney. The perfusate solution (about 250 ml) contained previously separated and blood group compatible porcine red blood cells (17% hematocrit value), 10 000 U heparin (Lövens, Malmö, Sweden), 100 mg cefuroxim (Zinacef; Glaxo Welcome, Mölndahl, Sweden) in Krebs-Ringer solution. Before connection of the renal arterial inlet to the perfusion device, 18 ml of 0.9% NaCl solution, containing 180 mg lidocain (Xylocain; Astra-Zeneca, Södertalje, Sweden) and 5000 U heparin, was infused into the renal artery followed by infusion of the adenovirus dissolved in 9–14 ml final volume of phosphate-buffered saline pH 7.4. The kidney was exposed to 15 min warm ischemia before perfusion with the oxygenated perfusate. The urine was collected continuously and added back to the perfusion fluid. Perfusions were carried out for 120 min with a perfusion flow of 53 ml/min and an arterial pressure varying between 150 and 200 mmHg. Thereafter, the cannules were removed and the incisions in the vessels and ureter were sutured. Heparin 1000 U was given i.v. immediately before reperfusion and 15 min after reperfusion. Fragmin (2500 U) (low molecular weight heparin, Pharmacia Upjohn, Stockholm, Sweden) was administered s.c. at the end of the operation and the following morning. Solu-Cortef (hydrocortisone, 100 mg; Pharmacia Upjohn, Stockholm, Sweden) was given i.v. at reperfusion. To avoid circulatory instability, calciumchloridehexahydrate 100 mg/ml (Apoteksbolaget, Umeå, Sweden) and a dose of 0.3 ml/kg i.v. was administrated 5–10 min before reperfusion. Zinacef was given at a dose of 100 mg i.v. at the start of the operation and after 8 and 16 h as a prophylactic antibiotic treatment. The animals were maintained on i.v. fluids overnight after which they gradually resumed oral feeding. Four days later, the animals were killed and the kidneys were harvested for later analysis by histochemistry and immunohistochemistry. The study was approved by the local Animal Ethics Committee and was performed in accordance with the Swedish law concerning animal welfare and treatment of research animals.
In situ hybridization
For in situ hybridization, tissues from pig kidneys were fixed in paraformaldehyde, dehydrated, embedded in paraffin and sectioned. The sections were post-fixed, incubated in PBS containing 0.1% active diethyl pyrocarbonate (Sigma), equilibrated in 5 × SSC and prehybridized for 2 h at 55°C. The sections were hybridized overnight at 55°C with a 172 bp fragment encoding the NC1 domain of the α5(IV) chain. Digoxygenin (DIG)-11-UTP-labeled antisense and sense riboprobes were generated by in vitro transcription with T7 and T3 RNA polymerase (Boehringer Mannheim Biochemicals, Mannheim, Germany). After washing in 50% formamide and standard sodium citrate, the sections were incubated with an alkaline phosphatase-coupled anti-DIG antibody and developed using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solutions (Boehringer Mannheim).Tissues from the right untreated kidney were used for control analysis.
Immunohistochemical and histochemical analysis of kidney tissues
Kidney tissues were studied for β-galactosidase expression by X-gal staining.16 In immunohistological studies, anti-FLAG-M2 FITC conjugate (Sigma) and anti-α4(IV) chain antibody H4320,21 were used. Cryosections were fixed using acetone/methanol (1:1), and blocked using 20% normal goat serum. The M2 antibody FITC conjugate was diluted to 10 μg/ml and the H43 antibody to 1:50, and the staining was carried out using AlexaFluor 546 labeled goat secondary antibody (Molecular Probes, Eugene, OR, USA). Tissues from the right untreated kidney were used as a control.
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We are grateful to Tiina Berg, Margareta Andersson and Dr Ehab Rafael for assistance, and thank Ingvild Halbig for care of the animals. This work was supported in part by grants from the Swedish Medical Research Council, the Novo Nordisk Foundation, Hedlund's Foundation, and by a EU grant No. BIO4-CT96–0537.
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