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Endocrinology, doi:10.1210/en.2007-1806
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Endocrinology Vol. 149, No. 11 5803-5810
Copyright © 2008 by The Endocrine Society

Novel Treatment for Lithium-Induced Nephrogenic Diabetes Insipidus Rat Model Using the Sendai-Virus Vector Carrying Aquaporin 2 Gene

Hidetaka Suga, Hiroshi Nagasaki, Taka-aki Kondo, Yoshiki Okajima, Chizuko Suzuki, Nobuaki Ozaki, Hiroshi Arima, Tokunori Yamamoto, Noriyuki Ozaki, Masaro Akai, Aiko Sato, Nobuyuki Uozumi, Makoto Inoue, Mamoru Hasegawa and Yutaka Oiso

Department of Endocrinology and Diabetes (H.S., T.K., Y.Ok., C.S., Nobua.O., H.A., Y.On., Field of Internal Medicine, Department of Metabolic Medicine (H.N.), and Departments of Urology (T.Y.) and Functional Anatomy and Neuroscience (Nobuy.O.), Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan; Bioscience and Biotechnology Center (M.A., A.S., N.U.), Nagoya University, Nagoya 464-8601, Japan; Department of Biomolecular Engineering 1 (N.U.), Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan; and DNAVEC Research Inc. (M.I., M.H.), Ibaraki 305-0856, Japan

Address all correspondence and requests for reprints to: Dr. Hiroshi Nagasaki, Department of Metabolic Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan. E-mail: nagasaki{at}med.nagoya-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Congenital nephrogenic diabetes insipidus (NDI) is a chronic disorder involving polyuria and polydipsia that results from unresponsiveness of the renal collecting ducts to the antidiuretic hormone vasopressin. Either of the genetic defects in vasopressin V2 receptor or the water channel aquaporin 2 (AQP2) cause the disease, which interfere the water reabsorption at the epithelium of the collecting duct. An unconscious state including a perioperative situation can be life threatening because of the difficulty to regulate their water balance. The Sendai virus (SeV) vector system deleting fusion protein (F) gene (SeV/{Delta}F) is considered most suitable because of the short replication cycle and nontransmissible character. An animal model for NDI with reduced AQP2 by lithium chloride was used to develop the therapy. When the SeV/{Delta}F vector carrying a human AQP2 gene (AQP2-SeV/{Delta}F) was administered retrogradely via ureter to renal pelvis, AQP2 was expressed in the renal collecting duct to reduce urine output and water intake by up to 40%. In combination with the retorograde administration to pelvis, this system could be the cornerstone for the applicable therapies on not only NDI patients but also other diseases associate with the medullary collecting duct.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NEUROPEPTIDE ARGININE vasopressin (AVP; also known as antidiuretic hormone) is a key molecule in water homeostasis. In response to both body fluid depletion and hyperosmolality, AVP is released from the posterior pituitary into the circulation to bind vasopressin V2 receptors (V2Rs) located in the renal collecting ducts. The V2R stimulation increases intracellular cAMP, which activates protein kinase A. Protein kinase A phosphorylates both cAMP-responsive element binding protein and aquaporin 2 (AQP2) that is one of the water channel family (1). cAMP-responsive element binding protein induces AQP2 transcription, and phosphorylated AQP2 is translocated to the apical membrane, which transmits water from the ductal lumen to the epithelial cytosol (2, 3, 4).

Nephrogenic diabetes insipidus (NDI) is a renal tubular disorder resulting from malfunction of the collecting ducts, which do not respond sufficiently to AVP. This failure of proper water reabsorption causes characteristic diluted polyuria, which keeps NDI patients thirsty and in danger of dehydration throughout their life from birth. NDI is categorized into two forms, congenital and acquired (5). Congenital NDI is further classified into two forms caused by mutations in the V2R gene (6) and AQP2 gene (7), respectively. Acquired NDI is caused by certain medications, such as lithium (Li). Currently the medications for congenital NDI are limited, including a low-sodium diet, thiazide (8) alone, or thiazide/prostaglandin inhibitor (9) combination. These conventional treatments can substantially reduce the discomforts of polyuria and risks of dehydration, but there is a marked variability of therapeutic responses. Genetic therapies that will cure inherited NDI have long been desired (NDI Foundation; http://www.ndif.org/).

NDI is not a life-threatening disease as long as patients have access to drinking water. It is important to avoid severe dehydration that might cause fetal unconsciousness (10). And perioperative situations under general anesthesia could be dangerous because it requires expertise to maintain proper hydration and electrolyte balance due to the incurable polyuria (11). In the present study, we aimed to establish a fundamental technology for NDI treatment that can temporarily reverse the polyuria phenotype for application during the perioperative period.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of a FLAG-containing human aquaporin 2 cDNA
Normal human kidney tissue surrounding a renal carcinoma was obtained from a surgically dissected sample. The protocol was approved by the Ethics Committee of Nagoya University Graduate School of Medicine (protocol 05-76). Total RNA was prepared from the tissue by homogenization in TRIzol reagent (Life Technologies, Inc., Grand Island, NY). A first-strand cDNA library was synthesized from the human kidney RNA according to the manufacturer’s protocol. After amplification by PCR with human AQP2 gene-specific primers (sense with a FLAG epitope tag: 5'-CTACCATGGACTACAAAGACGATGACGACAAGGAATTCATGTGGGAGCTCCGCTCCATAGCCTTC-3'; antisense: 5'-CCTCTAGACTCGAGCGGCCGCC-3'), the resulting PCR product was subcloned into the pcDNA3.1/V5-His-TOPO vector (Invitrogen, Carlsbad, CA). The construct was confirmed by a DNA sequencing service provider (Macrogen, Rockville, MD).

Oocyte swelling assay
Capped FLAG-AQP2 cRNA was synthesized in pXβG plasmid (12, 13) by using T3 RNA polymerase. Defolliculated Xenopus laevis oocytes were injected with 10 ng of the synthesized cRNA or distilled water as a control, and incubated at 18 C in 200 milliosmolar modified Barth’s solution [containing 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% dimethylsulfoxide (1.51–1.83 Osm/kg) at 25 C] for 3 d. Oocytes were transferred into modified Barth’s solution diluted to 70 mOsm, and the time course of volume increase was monitored at room temperature by videomicroscopy. The relative volume (V/V0) was calculated from the area at the initial time (A0) and after a time interval (At): V/V0 = (At/A0)3/2. The coefficient of osmotic water permeability (Pf) was determined from the initial slope of the time course [d(V/V0)/dt], initial oocyte volume (V0 = 9 x 10–4 cm3), initial oocyte surface area (S = 0.045 cm2), and molar volume of water (Vw = 18 cm3/mol): Pf = [V0 x d(V/V0)/dt]/[S x Vw x (Osmin – Osmout)].

Plasmid construction
The FLAG-tagged human AQP2 cDNA was amplified with a pair of NotI site-tagged primers containing Sendai virus (SeV)-specific transcriptional regulatory signal sequences (end and start, underlined below), 5'-ATTGCGGCCGCCAAGGTTCAATGGACTACAAAGACGATGACG-3' and 5'-ATTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGGTCAGGCCTTGGTACCCCGTGGCAGGCTCTGCG-3'. The amplified fragment was introduced into the NotI site of the parental SeV vector cDNA, pSeV18+b(+)/{Delta}F (14), to generate pSeV18+AQP2-SeV/{Delta}F. pSeV18+AQP2-SeV/{Delta}F was transfected to LLC-MK2 cells that were preliminarily infected with psoralen- and long-wave UV-treated vaccinia virus vTF7–3, expressing T7 polymerase. The cells were then washed twice with MEM and cultured for 24 h in MEM containing cytosine β-D-arabinofuranoside (40 µg/ml) and trypsin (7.5 µg/ml). LLC-MK2/F7/A cells expressing the F protein were suspended in MEM containing cytosine β-D-arabinofuranoside and trypsin, layered onto the transfected cells, and cultured at 37 C for an additional 48 h. The recovered vector in the culture supernatants was propagated using the LLC-MK2/F7/A cells. The virus titer was determined by their infectivity and expressed using cell-infectious units (CIU). A green fluorescent protein (GFP) expression vector (GFP-SeV/{Delta}F) was prepared as previously described (15).

Animals and surgical procedures
Male Sprague Dawley rats (10 wk of age; 250–300 g body weight; Chubu Science Materials, Nagoya, Japan) were individually housed in metabolic cages under controlled conditions (23.0 ± 0.5 C; lights on from 0900 to 2100 h). All animal experiments were approved by the Ethics Committee of Nagoya University Graduate School of Medicine (protocol 05-76). On d –14, the right renal artery, vein, and ureter were ligated, and the right kidney was removed under sufficient anesthesia with an ip injection of sodium pentobarbital (50 mg/kg body weight). After the operation, lithium chloride was added to the food to give a final concentration of 40 mmol/kg dry food for the first week and 60 mmol/kg thereafter, according to a previous report (16). On d 0, NDI rats were anesthetized and their abdominal cavities were opened. The left ureter was punctured with a 30G needle (BD Biosciences, Franklin Lakes, NJ). An aliquot (200 µl) of AQP2-SeV/{Delta}F or GFP-SeV/{Delta}F vector was injected from the left urinary duct into the left renal pelvis by retrograde infusion. The left urinary duct was clamped for 5 min just after the vector injection and then released. Finally, the abdominal wound was closed with stitches.

1-Deamino-8-D-AVP (dDAVP) test
Urine concentrating experiments were carried out by ip injection of dDAVP (0.4 µg/kg). Rats were injected twice in 30-min intervals, and urine was sampled twice for each animal: just before the first injection and 30 min after the second injection.

Measurement of plasma AVP
After decapitation, trunk blood was collected into chilled tubes containing EDTA (potassium salt). Plasma AVP was extracted through a Sep-Pak C18 Cartridge (Waters Associates, Milford, MA) and measured with a highly sensitive RIA kit (AVP-RIA kit, kindly provided by Mitsubishi Kagaku Iatron, Chiba, Japan). The sensitivity of the assay for AVP was 0.063 pg/tube (0.17 pg/ml), with less than 0.01% cross-reactivity with oxytocin.

Immunohistochemistry
For immunohistochemical analysis, isolated tissue pieces were fixed in 4% formaldehyde, embedded in paraffin, cut into 10-µm-thick sections, deparaffinized in xylene, and rehydrated. To suppress endogenous peroxidase activity, the sections were treated with 0.3% H2O2 in methanol for 30 min. The specimens were then blocked with 1% BSA or horse normal serum and incubated overnight with an anti-AQP2 antibody (Sigma, St. Louis, MO) or affinity-purified anti-FLAG antibody (Sigma) as the primary antibody. Next, the sections were incubated with appropriate secondary antibody (antirabbit or antimouse IgG) for 2 h. The reaction products were visualized using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) with diaminobenzidine as the substrate and fixed. Finally, the sections were rinsed, stained with Mayer’s hematoxylin (Sigma), dehydrated, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ).

Western blotting
Kidney samples were lysed in Laemmli buffer (2x Laemmli buffer contains 4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue, and 0.125 M Tris-HCl; solution has a pH ~6.8). For standardization, the total protein concentrations were measured using a commercially available protein assay system (BD Biosciences). Urinary samples were concentrated by dialysis and standardized by the urinary volume per day. After boiling for 5 min, the samples were separated by 15% SDS-PAGE and electrotransferred to Immobilon-P membranes (Millipore, Bedford, MA). After blocking with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 (Calbiochem, La Jolla, CA) for 1 h, the membranes were treated with an anti-AQP2 (Sigma) or anti-FLAG (Sigma) antibody, followed by incubation with horseradish peroxidase-conjugated antirabbit or antimouse IgG as appropriate for the primary antibody. The membranes were visualized by chemiluminescence using ECL-Plus (Amersham Pharmacia Biotech, Piscataway, NJ).

Statistical analysis
The results were expressed as means ± SEM. The statistical significance of differences between two groups was determined using Student’s t test. Values of P < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction of a FLAG-containing human AQP2 cDNA and the effect of FLAG-AQP2 expression in Xenopus oocytes
Because the two types of congenital NDI arise through V2R or AQP2 gene mutations, both of the genes are considered candidates for therapeutic purpose. In the present study, we chose AQP2 because it is a key molecule in the final process of water reabsorption that might cure both types of genetic defects.

A human AQP2 cDNA was obtained from normal human kidney tissue. A FLAG epitope tag was fused to the N terminus of the AQP2 gene for later evaluation (sequences are shown in Fig. 1AGo).


Figure 1
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  		FIG. 1. Deduced amino acid sequence of FLAG-tagged human AQP2 (A) and the effect of FLAG-AQP2 cRNA transfection in oocyte (B and C). A, The FLAG epitope is shown in italic letters and the human AQP2 cDNA sequence is underlined. B, Osmotic Pf. Pf = [V0 x d(V/V0)/dt]/[S x Vw x (Osmin – Osmout)]. Xenopus oocytes were injected with the FLAG-AQP2 cRNA or distilled water as a control. Oocytes were transferred into hypotonic medium, and the time course of volume increase was monitored by videomicroscopy. The V/V0 was calculated from the area at the initial time (A0) and after a time interval (At): V/V0 = (At/A0)3/2. The coefficient of osmotic Pf was determined from the initial slope of the time course [d(V/V0)/dt], initial oocyte volume (V0 = 9 x 10–4 cm3), initial oocyte surface area (S = 0.045 cm2), and the molar volume of water (Vw = 18 cm3/mol). *, P < 0.0001 (two-tailed unpaired t test).

 
To test whether the FLAG-AQP2 works as a water channel, the osmotic Pf was measured in oocytes injected with transcribed cRNA. Pf was significantly higher in oocytes expressing the FLAG-AQP2 than control (Fig. 1BGo), which indicates that the fused FLAG-tag does not interfere the functional expression of the AQP2 as a water channel.

Therapeutic vector construction
As a delivery vehicle, we used recombinant SeV vector. For treatment of an NDI model, we generated a recombinant SeV/{Delta}F vector carrying a human AQP2 gene (AQP2-SeV/{Delta}F; Fig. 2Go). As previously described, a FLAG epitope tag was fused to the N terminus of the AQP2 gene to distinguish exogenous AQP2 from endogenous. The titer of AQP2-SeV/{Delta}F was 1.4 x 109 CIU/ml.


Figure 2
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  		FIG. 2. Schemata of the virus vectors. Schematic structure of the AQP2-SeV/{Delta}F and GFP-SeV/{Delta}F vectors. The target gene, human AQP2 fused with a FLAG epitope tag at its N terminus or GFP cDNA, is introduced into the 3'-proximal region of the SeV/{Delta}F vector, which is rendered nontransmissible by deleting the F gene from the wild-type SeV genome. NP, Nucleocapsid protein gene; P, phosphoprotein gene; M, matrix protein gene; HN, hemagglutinin-neuraminidase gene; L, large protein gene.

 
Li-induced NDI animal model
Li-induced NDI animal model is suitable for assessment of our therapy because Li is well known to cause severe NDI, mainly due to AQP2 depletion in the collecting ducts (17). Li was added to the food to give a final concentration of 40 mmol/kg dry food for the first week and 60 mmol/kg thereafter, according to a previous report (16).

The rats presented obvious polyuria (about 180 ml/d, increased 5- to 10-fold larger than the urine volume of normal chow rats) and polydipsia after 10 d of the Li-containing diet. Urine osmolality after 14 d of Li-containing diet decreased significantly, but plasma osmolality and plasma AVP remained in a range that would be expected to concentrate the urine (Table 1Go). Even after 28 d of Li diet, both plasma osmolality and AVP levels stayed in the same range, which indicates the rats developed NDI after the Li diet during the period of observation (Table 1Go).


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  		TABLE 1. Effect of Li-containing diet

 
When administered to normal chow rats, a V2R-specific agonist, dDAVP led to a dramatic increase in urine concentration from 1202 to 3786 mOsm, whereas Li-fed (28 d) rats fail to concentrate their urine, from 179.8 to 172.0 mOsm (Fig. 3AGo). The unresponsiveness to dDAVP indicates Li ingestion developed severe NDI in unilaterally nephrectomized rats.


Figure 3
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  		FIG. 3. AQP2 suppression by Li feeding in the rat kidneys. A, Urine osmolality and concentrating ability in normal chow and Li-feeding rats, before (open bars) and after (closed bars) dDAVP treatment. Both groups of rats were nephrectomized of their right kidney before the feeding experiment. Before the dDAVP test, the Li-fed group received lithium containing food for 28 d (40 mmol/kg dry food for the first 7 d and thereafter 60 mmol/kg dry food for 21 d; n = 5, each group). *, P < 0.05, significant difference between the two groups (two-tailed unpaired t test). B, Immunohistochemistry using AQP2 antibody in the kidney medulla. Scale bars, 200 µm. C, upper panel, Western blotting with AQP2 antibody of the whole kidney lysates; lower panel, Western blotting with AQP2 antibody of the urine samples. AQP2 immunoreactivity are identified in the epithelium of the medullary collecting duct in normal chow rats and is almost completely suppressed in Li-fed rats.

 
The AQP2 immunoreactivity was almost absent in medullary collecting duct of the kidney and urine after the Li diet (Fig. 3Go, B and C). These data indicated that Li depleted AQP2 in the kidneys, which resulted in drug-induced NDI.

Retrograde infusion via the ureter
To establish a method for vector administration, a recombinant SeV/{Delta}F vector carrying a GFP gene (GFP-SeV/{Delta}F: 2.8 x 108 CIU in 200 µl per kidney) was injected in a retrograde manner into the renal pelvis of Li-fed rats (Fig. 4AGo). In the kidney removed for evaluation after 5 d, more than 50% of the collecting ducts expressed GFP in the cross-sectional slices of the inner medulla (Fig. 4BGo). The GFP expression was specific for the epithelium of collecting ducts and was not detected in the epithelium of thin tubules composing Henle’s loop. GFP was not expressed in any other slices of the outer medulla or kidney cortex, suggesting the vector infected in limited area of the nephron adjacent to the pelvis by retrograde perfusion. These results demonstrate that the combination of retrograde-perfusion and SeV/{Delta}F enabled a highly efficient and targeted gene transfer to the area of interest. GFP expression was not detected in other tissues, including the liver, spleen, heart, lungs, and intestine.


Figure 4
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  		FIG. 4. Schema of the vector administration route (A) and successful transfection to collecting duct by SeV/{Delta}F (B). A, The SeV vectors are injected into the urinary duct (i) in a retrograde fashion, reach the renal pelvis (ii), and infiltrate the collecting ducts in the renal medulla (iii), which is surrounded by the renal cortex. B, Immunohistochemistry using anti-GFP antibody of the kidney medulla of rats. After GFP-SeV/{Delta}F administration, GFP is specifically identified in most of the collecting ducts (arrowheads). Scale bars, 200 µm.

 
Therapeutic effects of gene transfer
To assess the therapeutic effects of the transgene, the right kidney was surgically removed from the rats 14 d before the gene therapy (Fig. 5Go). After the surgery, the rats were fed with a Li-containing diet and housed in the metabolic cages to measure physiological parameters including water and food intake, urine output, and body weight. The rats presented obvious symptoms of NDI after 10 d of the Li-containing diet. In comparison with intact rat, the surgical resection of the kidney did not change the duration before the onset of drug-induced NDI (data not shown). As previously shown in Table 1Go, our Li ingestion rats kept in the NDI state from the vector transfection day (d 0) to the end of observation (d 11–14). The GFP-SeV/{Delta}F vector was placed as a control for the AQP2-SeV/{Delta}F vector. On the vector transfection day (d 0), anesthetized NDI rats were injected with 200 µl of AQP2-SeV/{Delta}F vector (1.4 x 109 CIU/ml) or the same amount of GFP-SeV/{Delta}F vector from the left urinary duct to the left renal pelvis by retrograde infusion as previously described.


Figure 5
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  		FIG. 5. Schema of time course. The right kidney was removed 14 d before the gene therapy (d –14). Li-containing diet was started at d –14, changed to higher concentration at d –7, and continued to the last. At d 0, anesthetized NDI rats were injected with AQP2-SeV/{Delta}F vector or GFP-SeV/{Delta}F vector.

 
After the vector infusion, both groups reduced urine output in the first few days, resulting from the damages after abdominal surgery. It took 3 d to recover to the previous levels in GFP-SeV/{Delta}F group. In AQP2-SeV/{Delta}F-treated rats, urine output remained reduced, significantly less than GFP-control group by up to 40% since d 4 to d 8 (Fig. 6AGoGo). Water intake was also decreased in AQP2-SeV/{Delta}F-treated rats from d 5 to d 8 by up to 40% (Fig. 6BGoGo). Food intake and body weight did not differ significantly (Fig. 6GoGo, C and D). Urinary osmolality at d 5 was significantly higher in AQP2-SeV/{Delta}F-treated rats than in control rats (Fig. 6EGoGo). Immunohistological examination of the renal tissues at d 5 revealed large numbers of AQP2- or FLAG-positive cells in the collecting ducts of the AQP2-SeV/{Delta}F-treated rats (Fig. 6GoGo, F and G). These findings were consistent with the results from Western blotting studies of whole-kidney lysates and urine samples at d 5 (Fig. 6GoGo, H and I). Li administration severely reduced endogenous AQP2 expression, and AQP2-SeV/{Delta}F treatment replenished the expression. The slight upward shift of the AQP2-positive band in AQP2-SeV/{Delta}F-treated rats (Fig. 6GoGo, H and I) compared with naïve AQP2 suggests that exogenous FLAG-tagged AQP2 predominantly expressed in the kidney.


Figure 6
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  		FIG. 6. Effects of AQP2-SeV/{Delta}F administration in a retrograde fashion via the ureter. A–D, Time-course measurements (mean ± SEM, n = 5). The GFP-SeV/{Delta}F (open circles) or AQP2-SeV/{Delta}F (filled circles) vector was administered at d 0. *, P < 0.05, significant difference between the two groups (two-tailed unpaired t test). Urine output (A) and water intake (B) are significantly lower in AQP2-SeV/{Delta}F-treated rats for several days. Food intake (C) and body weight (D) do not differ significantly. E, Urinary osmolality at d 5 (mean ± SEM, n = 5) is significantly higher in AQP2-SeV/{Delta}F-treated rats (filled bar) than in GFP-SeV/{Delta}F-treated rats (open bar). *, P = 0.0109 (two tailed unpaired t test). F and G, Immunohistochemistry of the kidney at d 5. AQP2 (F) and FLAG (G) immunoreactivites are detected in AQP2-SeV/{Delta}F-treated rats. Scale bars, 200 µm. H and I, Western blotting analysis of kidney lysates (H) and urine samples (I). Lane 1, Control Sprague Dawley rats; lane 2, Li-fed NDI rats; lane 3, Li-fed and GFP-SeV/{Delta}F-treated rats at d 5; lane 4, Li-fed and AQP2-SeV/{Delta}F-treated rats at d 5. In Li-fed NDI rats, AQP2 is suppressed to almost undetectable levels, whereas AQP2-SeV/{Delta}F administration compensated the AQP2 expression. FLAG immunoreactivity is specifically identified in AQP2-SeV/{Delta}F-treated rats.

 

Figure 6A
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  		FIG. 6A. Continued

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we invented the method for targeted expression in the collecting duct of the AQP2 gene that is deficient in recessive NDI. The recombinant SeV/{Delta}F vector, which is admitted as highly efficient and safe, successfully transmitted the target gene specifically in the collecting duct by retrograde ureter administration. This method would be the cornerstone on the development of gene therapies for NDI, especially in the perioperative periods.

Gene transfer to renal tissues has been difficult due to their low mitotic index and structural complexity, being composed of glomeruli, tubules, vasculature, and interstitium. Recent studies claimed several vectors or reagents, including adenoviruses (18, 19), lentiviruses (20), liposomes (21, 22, 23), electroporation (24, 25, 26), and ultrasound (27, 28), as potential mediators for kidney-targeting gene therapies. However, these methods are not applicable to NDI patients for the following reasons. First, most of the reports targeted the kidney glomeruli or tubules, not crucial for water reabsorption (29). Second, no successful transductions to alleviate a hereditary deficit proteins in the kidneys in vivo have been reported to date. Only small reverse-complementary nucleotides were reported to suppress pathogenic genes, including inflammatory cytokines, in glomerulonephritis model animals (30, 31, 32). Gene therapy for NDI is required to compensate for the function of the mutated molecules (V2R or AQP2) in water reuptake. Therefore, efficient vehicles and administration methods for targeting the renal medulla need to be developed.

The SeV vector infects and multiplies its genome in most mammalian cells and directs high-level transgene expression. There is no risk of SeV vector integration into the host genome because its replication is restricted to the cytoplasm with the RNA phase and independent of nuclear functions. Thus, the SeV vector is completely free from genotoxicity. Deletion of the F gene, which encodes a viral glycoprotein inserted into the viral envelope and essential for penetration of the vector genome into the cytoplasm through cell fusion, renders the resulting SeV/{Delta}F vector nontransmissible (33). Therefore, SeV/{Delta}F vector works only in the primarily infected host cell. Because SeV/{Delta}F vector degrades gradually, the effect is normally waning. This short duration of the vector suits the purpose of this study, and the nontransmissible character makes the method much safer.

The Li-induced NDI as well as those caused by mutations of the AQP2 or V2R genes is associated with a deficiency of functional AQP2. In theory, the reversing the AQP2 deficiency might provide a new treatment for all types of NDI. Therefore, we generated a recombinant SeV/{Delta}F vector carrying a human AQP2 gene for treatment of AQP2-defective NDI model. It has been known that the addition of various epitope-tag to AQP2 moieties (poly-His, c-myc, or VSV-G), either on the N or C terminus, do not interfere the bioactivity (34, 35). In this paper, we chose FLAG-tag because it is highly selective and efficient. When expressed in the Xenopus oocyte, the chimeric FLAG-hAQP2 cDNA enormously increased water permeability.

The genetic interventions in both the V2R and AQP2 genes had shown neonatal mortality in animal models (36, 37). More recently mice strains that survive to adulthood were established. But they develop severe hydronephrosis within few months after birth (38, 39). These changes will destroy the collecting duct structure, which would make the gene therapy difficult. For these reasons, we used a Li-induced NDI animal model.

For the vector administration route, we chose retrograde infusion via the ureter so that the vector would infiltrate the collecting ducts of the renal medulla via the renal pelvis. The retrograde administration enabled the efficient GFP expression in the collecting duct in limited area of renal medulla. Although the control GFP-SeV/{Delta}F vector does not have any special sequences for tissue-specific targeting, it is considered that vector infection occurred by direct contact to the epithelium in the adjacent area surrounding the renal pelvis. Alternative routes via the renal artery or renal vein were not tested due to the risk of systemic perfusion of the vector. Furthermore, the retrograde administration route would be beneficial in humans because vector administration will be carried out using a ureterocystoscope, which is a low-invasive and routine technique in urology.

Based on these findings, we tested the physiological effects of AQP2-SeV/{Delta}F injection, using GFP-SeV/{Delta}F as a control. Urine output and water intake were decreased by up to 40% in AQP2-SeV/{Delta}F-treated rats temporally for several days. At that time, urinary osmolality was significantly higher in AQP2-SeV/{Delta}F-treated rats than control rats. These results strongly suggest that forced AQP2 expression by AQP2-SeV/{Delta}F compensates for the function of water reabsorption in the renal medulla of Li-induced NDI rats, thereby resulting in urine concentration, decreased urine output, and lower water intake. To the best of our knowledge, this is the first report of successful gene therapy targeting the renal collecting ducts to restore the function of a deficient gene.

However, it requires some notice to interpret these physiological data because they were obtained in drug-induced NDI model using Li. We confirmed Li almost depleted endogenous AQP2 in this model. Li is also known to interfere with the adenylate cyclase system (40, 41, 42) that is pivotal for AQP2 translocation. In this context, it is supposed that the considerable amount of exogenous AQP2 protein from the therapeutic vector might not be phosphorylated properly and might fail to be translocated to apical membrane. The reason AQP2-SeV/{Delta}F partially rescued polyuria might be explained by the fact that guanylate cyclase pathways phosphorylate AQP2 in addition to the attenuated adenylate cyclase system (43). Detailed studies both in vivo and in vitro will be required to elucidate the intracellular mechanisms.

Therefore, the clinical applications of the method have to be considered deliberately. In this study, the effect observed in the Li-induced animal model was not sufficient enough to normalize urine output and was no greater than that achievable with conventional therapy in patients with NDI. However, we have to keep the possibility in mind that Li might affect the physiological effect of the vector, and the method could be more effective in the NDI patients with AQP2 gene defect. There is also a fear the gene therapy is too effective in clinical use; excessive expression of AQP2 due to the therapy might develop nephrogenic syndrome of inappropriate antidiuresis. To solve these problems, it might be helpful to engineer the vector to be induced by circulating AVP. Another method to solve the problem is the use of ribavirin, a mutagen and inhibitor of viral RNA polymerase (44, 45). It shows antiviral activity against a variety of RNA viruses and is used to treat infections of hepatitis C virus. This drug would be a tool to regulate SeV-mediated gene expression. Accordingly, if NDI patients were treated in this method, they would still require the expert attention to their perioperative fluid balance.

In the current study, V2R dysfunction models were not tested. However, our oocyte-swelling assay (Fig. 1BGo) revealed that the overexpression of AQP2 itself increased water permeability in the circumstance where AVP is absent. This may indicate that AQP2-SeV/{Delta}F is also beneficial for other types of congenital NDI arising from V2R mutations.

In conclusion, this method is a new approach for correcting the fatal imbalance of water homeostasis in NDI patients, especially in the perioperative period. Further improvements are required in many aspects, including its efficacy, inducibility, the optimal dosage, and clinical safety. By exchanging the therapeutic gene or using other vectors aiming for persistent expression, our method has the potential to cure other congenital kidney diseases of the renal distal tubules or collecting ducts, including polycystic kidney disease and nephronophthisis.


   Acknowledgments
 
We thank Michiko Yamada and Keiko Shimamoto for excellent technical assistance.


   Footnotes
 
Current address for M.A. and A.S.: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan.

Disclosure Statement: The authors have nothing to declare.

First Published Online July 24, 2008

Abbreviations: AQP2, Aquaporin 2; AVP, arginine vasopressin; CIU, cell-infectious units; dDAVP, 1-deamino-8-D-AVP; GFP, green fluorescent protein; Li, lithium; NDI, nephrogenic diabetes insipidus; Pf, water permeability; SeV, Sendai virus; V2R, vasopressin V2 receptor; V/V0, relative volume.

Received December 31, 2007.

Accepted for publication July 15, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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