Gene Delivery in Renal Tubular Epithelial Cells Using Recombinant Adeno-Associated Viral Vectors

Preparation of rAAV2-GFP Vector

The structure of the rAAV2-GFP vector (UF11) plasmid is shown in Figure 1A. The plasmid contains a “humanized” GFP as the transgene and is driven by a cytomegalovirus-chicken β-actin hybrid promoter (CBAp), as described previously (23). The rAAV2 production was performed according to the previously described method of double transfection of a permissive human cell line (HEK293) (24). Plasmid pDG containing both the AAV2rep and cap genes as well as a subset of adenovirus (Ad) 5 genes (E2a, E4, and VA-RNA) were co-transfected along with rAAV2-GFP vector plasmid into HEK293cells (Ela⁺, Elb⁺) grown in cell factories (Nalge Nunc, Rochester, NY). Cells were harvested and lysed by a freeze-thaw method to release virions. The virus containing supernatant was subsequently purified by iodixanol gradient ultracentrifugation followed by heparin sepharose affinity chromatography. The purity of the preparations was determined by silver-stained SDS acrylamide gel electrophoresis. Infectious center assays were used to determine rAAV virus titer, with dot blot analysis performed to quantify the titer of the rAAV physical particles and determine the particle to infectivity ratio. The particle-to-infectivity ratio averaged approximately 10 in all the rAAV preparations.

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Figure 1. Vector map of rAAV2-GFP (A). The elements of the cassette are as follows: ITR, the inverted terminal repeat of AAV serotype 2 (AAV2) virus; CBAp, the cytomegalovirus immediate early enhancer linked to chicken β-actin promoter and derived from pCacMam-1 (Novagen, Madison, WI); a chimeric intron from rabbit β-globin; GFP, a “humanized” green fluorescent protein cDNA (716 bp) from pTR-UF5 (23); poly(A), the bovine growth hormone polyadenylation site from pTR-UF2. Technique for intrarenal arterial administration of vector for transduction of rat kidneys (B through E). The infrarenal segment of the aorta was isolated and cannulated just below the origin of the renal arteries after clamping the suprarenal aorta, the right renal artery, and aortic branches near the left renal artery (B). An incision was made on the left renal vein. The left kidney was flushed with 1.0 ml of cold normal saline through the renal artery via the infrarenal aorta (C). The left renal vein was clamped thereafter and saline (0.125 ml/100 g body wt) or rAAV2-GFP (0.125 ml/100gm body weight, titer: 1 × 10¹⁰/ml infectious units) was injected through the left renal artery into the kidney. The artery and vein were clamped, and the vector was allowed to dwell in the kidney for 45 min (D). The cannula was removed, the incisions in the aorta and left renal vein were sutured (E), and the blood supply to the kidney was restored.

Intrarenal Artery Administration of Vector

Male Lewis rats weighing 80 to 100 g were anesthetized intraperitoneally with pentobarbital sodium (30 to 40 mg/kg body wt). A midline laparotomy incision was made, and administration of the vector was performed through the left renal artery as described in Figures 1B through 1E. Briefly, the left kidney was flushed with saline after clamping of the left renal vein, and saline (0.125 ml/100 g body wt; n = 7) or rAAV2-GFP (0.125 ml/100 g body wt; titer, 1 × 10¹⁰/ml infectious units, IU; n = 7) was injected through the left renal artery into the kidney. In initial pilot experiments, lower doses of 1 × 10⁸ to 1 × 10⁹ IU/ml showed no transduction. The vector was allowed to dwell in the kidney for 45 min before removal of the cannula and suturing of the incisions. The rats were sacrificed at 2 wk (n = 2 in saline and rAAV2-GFP group, respectively), 4 wk (n = 2 in each group), and 6 wk (n = 3 in each group) after transduction. Both the left and right kidneys of experimental animals were preserved by immersion in 10% neutral buffered formalin and processed for evaluation of transgene expression by immunohistochemistry for GFP and H⁺-ATPase, staining with hematoxylin and eosin for routine histologic evaluation, and in situ hybridization. We also performed experiments in which a higher titer of the vector was administered via intrarenal arterial administration. In these studies, male Lewis rats were injected with 0.125 ml/100 g body wt of 1 × 10¹¹ IU/ml of rAAV2-GFP or saline (n = 3 in each group). Animals were sacrificed at 6 wk after transduction, and the liver, spleen, heart, lung, and both kidneys were harvested for routine histologic examination and evaluation of transgene expression by PCR (qualitative and quantitative real-time PCR), immunofluorescence, and immunohistochemistry. For immunofluorescence, a slice of the kidney was fixed by immersion in 10% neutral buffered formalin overnight and then cryopreserved in 18% sucrose for 16 h. The tissue was embedded in OCT, and 5-μm-thick frozen sections were cut. Slides were mounted using Vector Shield (Vector Labs, Burlingame, CA) and examined under an Olympus Provis fluorescence microscope. The animal experiments were conducted in accord with the NIH Guide for the Care of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee, University of Florida, Gainesville, Florida.

Immunohistochemical Analyses

Additional pieces of formalin-fixed kidney and other organs (liver, heart, spleen, lung) were embedded in paraffin. Four-micron-thick sections were deparaffinized, blocked for endogenous peroxides, and incubated for 25 min at 95°C in Trilogy solution (Cell Marque, Hot Springs, AK) for antigen retrieval. After preliminary antibody titration studies, slides were stained for GFP using a rabbit anti-GFP antibody at 1:15 (Chemicon, Temecula, CA) for 1 h at room temperature. Normal rabbit IgG was used instead of the primary antibody as a negative control. Detection was achieved using the Vector Elite rabbit avidin-biotin conjugate (ABC) kit, with 3,3′-diaminobenzidine (DAB) nickel as the chromagen (Vector Labs). Sections were counterstained with Light Green (Sigma Chemical Co., St Louis, MO).

Identification of transduced intercalated cells was determined by two strategies. First, serial sections were stained for H⁺-ATPase using a rabbit polyclonal antibody against the 70-kD subunit of bovine brain clathrin-coated vesicle H⁺-ATPase (1:400 dilution; kindly provided by Dr. Dennis Stone, University of Texas Health Sciences Center, Dallas, Texas) or GFP using a rabbit anti-GFP antibody (1:1000 dilution; Novus, Littleton, CO). The H⁺-ATPase antibody is known to label intercalated cells in the rat, mouse, and rabbit kidney (25,26 ⇓). Detection was achieved using a Vector Elite Rabbit ABC kit, with DAB as the chromagen and hematoxylin QS as the counterstain.

Co-localization studies were also performed on 4-μm paraffin sections. Slides were deparaffinized and then blocked for both endogenous peroxides and biotin. Sections were then stained for GFP, followed by staining for H⁺-ATPase. Rabbit anti-GFP antibody (Novus, Littleton, CO) was diluted at 1:1000 and applied to the slides for 1 h at room temperature. Detection was achieved using a Vector Elite Rabbit ABC kit with 3-amino-9-ethylcarbazole (AEC) as the chromagen. Slides were re-equilibrated in buffer and incubated in 1:400 dilution of the H⁺-ATPase antibody for 1 h at room temperature. A Vector Elite Rabbit ABC Alkaline Phosphatase kit was used for detection. Vector Blue substrate was applied to the slides in the presence of levamisole to produce color contrast. No counterstaining agent was used. Slides were also stained with GFP alone using AEC as the chromagen as a color control. Images were taken with an Olympus Provis microscope using a digital color camera and Magnafire software.

In Situ Hybridization

A 657-bp fragment of the GFP transgene was amplified by PCR using DIG-labeled UTP (Roche Biochemicals, Germany) with the following primers: sense 5′-GGCGTGGTCCCAATTCTCGTGGAAC-3′ and antisense 5′-GCGGTCACAAACTCCAGCAGGACCA-3′. Four-micron-thick paraffin sections were immersed in xylene, 100% ethanol, and 95% ethanol and washed briefly with diethylpyrocarbonate-treated water. Tissue sections were then digested with proteinase K (10 μg/ml) at 37°C for 10 min. The DIG-labeled GFP probe was denatured and hybridization performed in CEP hybe solution (Vysis, Downer’s Grove, IL) at 37°C overnight. Slides were washed three times for 2 min each in 50% formamide/2× SSC, once for 2 min in 2× SSC and again for 2 min in 2× SSC + 0.1% Igepal (Sigma), and rinsed in TBS. Colorimetric detection was performed using an anti-DIG antibody conjugated to alkaline phosphatase (Roche) followed by incubation with Vector Blue Substrate and Nuclear Fast Red counterstaining. The slides were assessed by light microscopy, and images captured using a digital color camera and Magnafire software.

DNA Extraction and PCR for Detection of Vector Genomes

Genomic DNA was extracted from the different tissues using a commercially available Qiagen kit (Valencia, CA) and quantified by spectrophotometry. GFP-specific primers, sense 5′-GGCGTGGTCCCAATTCTCGTGGAAC-3′ and antisense 5′-CTCCTGCACATAGCCCTCGGGCATG-3′, were used to amplify a single copy 258-bp fragment of the GFP transgene using 100 ng of genomic DNA as template and the following PCR parameters: 5 min at 94°C; 25 cycles of 30 s at 94°C; 30 s at 62°C (annealing); 1 min at 72°C; final extension period of 10 min at 72°C. Ten microliters of the PCR reaction was subjected to electrophoresis on a 2% agarose gel to verify the presence and the size of the amplified product. rAAV2-GFP plasmid DNA was used as a positive control in the PCR reactions.

For real-time quantitative PCR, the following primer probe set was used to determine the number of copies of rAAV2-GFP vector DNA: sense 5′-TTTCAAAGATGACGGGAACTACAA-3′ and antisense 5′-TCAATGCCCTTCAGCTCGAT-3′. Genomic DNA (100 ng) was used in each assay. Real-time PCR was performed using the conditions recommended by Perkin-Elmer/Applied Biosystems (Foster City, CA) as described previously (27). The standard curve for real-time PCR was generated by using known quantities of rAAV2-GFP plasmid DNA. An internal control consisted of a standard GAPDH primer-probe set provided by Perkin-Elmer/Applied Biosystems. The below threshold for any detectable signal for this assay was between 40 to 400 copies per sample. Results are expressed as mean ± SEM of vector genomes per microgram DNA.

Statistical Analyses

Data are expressed as the means ± SEM. Statistical analyses were performed using the t test or ANOVA and the Student-Newman-Keuls test. All results are considered significant at P < 0.05.

Histologic Evaluation by Hematoxylin and Eosin Staining

The protocol for intrarenal arterial administration of gene delivery involved an ischemia time of approximately 45 min, during which time vector was allowed to dwell in the kidney, followed by reperfusion. To evaluate the renal morphologic changes secondary to the procedure, we examined hematoxylin and eosin-stained kidney sections by light microscopy. Focal areas of chronic interstitial infiltration with mononuclear cells, tubular atrophy, and scar formation (Figure 2, arrowheads) were observed in both saline- and rAAV2-GFP (1 × 10¹⁰ IU/ml)-injected kidneys at 6 wk after injection. Tubular cell regeneration and occasional casts were observed at earlier time points (2 to 4 wk; data not shown). No difference was observed between the saline- and rAAV2-GFP-injected kidneys, suggesting that these changes were most likely the result of ischemic injury inherent in the procedure and not a consequence of vector administration or an adverse immune response to rAAV or the transgene.

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Figure 2. Histologic evaluation of transduced kidneys. Hematoxylin and eosin-stained sections of the left kidney transduced with either saline (control; A and B) or rAAV2-GFP 1 × 10¹⁰ IU/ml (C and D). Representative low magnification (×25) images of cortex and medulla are shown in A and C. Higher magnifications (×100) of the outer medullary region are shown in B and D. Arrowheads indicate areas of mononuclear cell infiltration, tubular atrophy, and scar formation.

Immunohistochemical Detection of Transgene Expression

We examined the kidneys for expression of the GFP transgene by immunohistochemistry. Only minimal expression was identifiable in tubular cells at 2 wk after transduction. The number of positive cells increased to approximately 8 to 10 cells/40× field at 4 wk (data not shown). However, at 6 wk after transduction, we observed significant expression of GFP in renal tubules in all kidneys injected with rAAV2-GFP (1 × 10¹⁰ IU/ml; n = 3), while no expression was observed in the non-injected right kidney or the saline-injected left kidneys (Figure 3). The expression of GFP was restricted to the outer medullary region of the kidney and was localized to proximal tubule epithelial cells identified by the presence of a brush border (Figure 3, C through F). No transduction was observed in glomeruli, blood vessels, or interstitial cells.

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Figure 3. Immunohistochemical staining of rat kidneys 6 wk after transduction with either saline (control; A) or rAAV2-GFP 1 × 10¹⁰ IU/ml (B through G). Staining for GFP (arrows, black color) was performed using a rabbit anti-GFP antibody (1:15 dilution; Chemicon, Temecula, CA) detected by a vector Elite ABC kit using DAB nickel as the chromagen (Vector Labs, Burlingame, CA). Sections were counterstained with Fast Green. As shown in C through G, significant positive staining for GFP was present in renal tubular epithelial cells in animals that received rAAV2-GFP. No staining was observed in blood vessels and glomeruli (C, D, and E). Saline-injected animals demonstrated background staining reaction (A). In addition, a section from the opposite right kidney (H) had background staining. Rabbit IgG was used instead of the primary antibody as an additional control (B). Representative low (A, B, C, and H [×20]) and higher magnification (D, E, and G [×80] and F [×250]) images are shown with dense cytoplasmic staining in the regions of the S₃ segment of the proximal tubule (C through F) and cells with the morphologic appearance of type A intercalated cells in the medulla (C and G).

Interestingly, a subpopulation of collecting duct cells in the medullary region of the kidney with the histologic appearance of intercalated cells was also positive for GFP (Figure 3G). These cells were identified as type A intercalated cells by positive apical immunoreactivity for H⁺-ATPase in serial sections that were also positive for GFP (Figure 4, A through D). Co-localization of GFP with H⁺-ATPase staining was also confirmed by double labeling, as shown in Figure 4F. H⁺-ATPase was also expressed in the proximal convoluted tubule cells, where it localized to the base of the brush border as reported previously (28). Examination of serial sections revealed that proximal tubules with apical H⁺-ATPase staining were not positive for GFP, while tubules that contained GFP positive cells were negative for H⁺-ATPase. These findings led to the conclusion that the cells of the S₃ segment of the proximal tubule were the most frequently transduced cell population. The presence of GFP was also confirmed by in situ hybridization. As shown in Figure 5A, no signal was observed in the kidney of saline-injected animals. However, positive signal was detected in proximal tubular and intercalated cells in the rAAV2-GFP-injected kidneys (Figure 5, B and C).

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Figure 4. Co-localization of GFP with H⁺-ATPase. Serial sections were stained with either GFP (A and C) or the intercalated cell marker H⁺-ATPase (B and D) as described in the Materials and Methods. For easy comparison, both GFP and H⁺-ATPase were stained brown (arrows) in the serial sections (A through D). There were no GFP-positive cells in the saline-injected kidney (A). Some but not all intercalated cells in rAAV2-GFP-transduced kidneys were selectively stained by the GFP antibody (C). For co-localization studies, dual-labeling with GFP and H⁺-ATPase (E and F) was performed as described in Materials and Methods. The inset in F shows an rAAV2-GFP-injected kidney section stained with GFP alone (red). The apical dark blue staining of cells in (E) represents H⁺-ATPase-positive cells, and the purple-stained cells (arrows) in F represent cells positive for both GFP and H⁺-ATPase, indicating transduction of intercalated cells. The saline-injected kidney has no red or purple staining (E). Significant number of doubly positive purple cells was observed in rAAV2-GFP-transduced kidneys (F). All kidneys were positive for H⁺-ATPase staining. Magnification, ×400 for all images.

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Figure 5. GFP expression detected by in situ hybridization. A 657-bp DIG-labeled GFP fragment was amplified by PCR, and hybridization was performed as described in the Materials and Methods. Colorimetric detection was done using an anti-DIG antibody conjugated to alkaline phosphatase (Roche) followed by incubation with Vector Blue Substrate and Nuclear Fast Red counterstaining. Saline-injected kidneys showed no signal (A; ×400), while vector-injected animals showed positive signal (blue color) in proximal tubublar (B, arrowhead; ×600) and intercalated cells (C, arrow; ×600).

Evaluation of Transgene Expression after High-Dose Vector Administration

To evaluate whether rAAV-mediated transduction was associated with any toxicity, we examined the kidney and other organs at 6 wk after a higher dose (0.125 ml/100 g body wt, 1 × 10¹¹ IU/ml) of rAAV2-GFP or an equal volume of saline administered via intrarenal arterial injection as described above. The weight gain in the saline-injected animals was similar to the weight gain in the animals treated with the high dose vector during the 6 wk period (saline, 164.3 ± 9.9 g; vector, 177 ± 15.9 g; n = 3 in each group; P > 0.05). As shown in Figure 6, significantly higher transduction was observed in renal tubular cells by immunofluorescence for GFP, which was also confirmed by immunohistochemistry (Figure 7A, lower panel). Again, the predominant cells transduced were proximal tubular (Figures 6B, 6D, and 7A, lower panel) and intercalated cells (Figures 6C and 7A, inset). Glomeruli, vasculature, and interstitial cells were not transduced. Histologic examination of the transduced left kidney revealed focal mononuclear infiltration and tubular atrophy (Figure 7A, upper panel), which was also seen in the left kidneys of saline-injected animals. No histologic changes in the form of immune cell infiltration or cell injury were observed in other organs, including the right kidney, spleen, lung, liver and heart in animals injected with the vector (Figure 7, B through F, upper panel) compared with saline-injected animals. Evaluation for transgene expression by immunohistochemistry failed to detect any GFP in the other organs (Figure 7, B through F, lower panel). We also evaluated other organs for the presence or absence of vector DNA by both qualitative and quantitative real-time PCR. As expected, a positive PCR product (258 bp) was amplified from the transduced left kidney as well as from the liver, lung, and spleen of rAAV2-GFP-injected animals (data not shown), while no product was obtained in the saline-injected animals. High copies of vector genomes were detected from the transduced left kidney (Figure 8). Vector DNA was also detectable in the liver (approximately threefold lower) and to a lesser extent in the lung and spleen; none was detectable in the heart and the right kidney of vector-treated animals (Figure 8B). The number of vector genomes was below the threshold for detection in the saline-injected animals.

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Figure 6. Immunofluorescence for GFP in rat kidneys 6 wk after transduction with either saline (control; A) or rAAV2-GFP 1 × 10¹¹ IU/ml (B through D). Frozen sections of the kidney were examined by immunofluorescence microscopy. Positive green fluorescence is seen in renal tubular cells in animals transduced with rAAV2-GFP (B through D), while faint background autofluorescence is seen in the saline-injected kidney (A). Proximal tubular cells in the outer medullary region (B) and intercalated cells in the medullary collecting ducts (C) were positive (A through C, ×80). A higher magnification (×600) of a proximal tubule with positive green fluorescence is shown in D.

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Figure 7. Histologic evaluation of tissues from animals injected with high dose of the vector (1 × 10¹¹ IU/ml). Upper panel (A through F): Representative images (×25) of hematoxylin and eosin-stained sections of the left kidney, right kidney, spleen, lung, liver, and heart at 6 wk after transduction. Lower panel (A through F; ×25): Immunohistochemical staining for GFP was performed using a rabbit anti-GFP antibody as described in Materials and Methods. As shown in A, significant positive staining for GFP was present in renal tubular epithelial cells in the left kidney of animals that received the high dose of rAAV2-GFP. The inset in A (×80) is a representative image from the medullary region showing positive GFP staining of intercalated cells. No staining for GFP was observed in the opposite right kidney, spleen, lung, liver, or heart (B through F).

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Figure 8. Quantitative PCR for detection of vector DNA in animals injected with high-dose rAAV2-GFP (1 × 10¹¹ IU/ml). Real-time PCR was used to quantify the number of vector genomes per microgram of genomic DNA isolated from tissues of saline and rAAV2-GFP vector injected animals at 6 wk after transduction. 1, liver; 2, lung; 3, spleen; 4, heart; 5, right kidney; 6, left kidney. Values are mean ± SEM, n = 3 in each group. *P < 0.001 by ANOVA and Student-Newman-Keuls test.