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Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ

Departments of ¹Physiological Chemistry and Metabolism, ²Developmental Medical Technology (Sankyo), ³Urology, ⁴Respiratory Medicine, and ⁵Laboratory Medicine, Graduate School of Medicine and ⁶Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo; ¹⁰Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto; ¹¹Department of Biomedical Chemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan; and ⁷Division of Cardiology, Departments of Internal Medicine, ⁸Molecular Biology, and ⁹Internal Medicine and Pediatrics, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas

Submitted 28 April 2007 ; accepted in final form 31 December 2007

	ABSTRACT

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TAZ (transcriptional coactivator with PDZ-binding motif), also called WWTR1 (WW domain containing transcription regulator 1), is a 14-3-3-binding molecule homologous to Yes-associated protein. TAZ acts as a coactivator for several transcription factors as well as a modulator of membrane-associated PDZ domain-containing proteins, but its (patho)physiological roles remain unknown. Here we show that gene inactivation of TAZ in mice resulted in pathological changes in the kidney and lung that resemble the common human diseases polycystic kidney disease and pulmonary emphysema. Taz-null/lacZ knockin mutant homozygotes demonstrated renal cyst formation as early as embryonic day 15.5 with dilatation of Bowman's capsules and proximal tubules, followed by pelvic dilatation and hydronephrosis. After birth, only one-fifth of TAZ-deficient homozygotes grew to adulthood and demonstrated multicystic kidneys with severe urinary concentrating defects and polyuria. Furthermore, adult TAZ-deficient homozygotes exhibited diffuse emphysematous changes in the lung. Thus TAZ is essential for developmental mechanisms involved in kidney and lung organogenesis, whose disturbance may lead to the pathogenesis of common human diseases.

renal disease; knockout mice; transcription factor

In midgestation mouse embryos, TAZ is mainly expressed in the paraxial mesoderm, limb buds, and the neural tube (25). Later, TAZ is distributed more broadly in various tissues and organs (18 and UniGene's EST ProfileViewer). The broad distribution of TAZ and its interaction with different transcription factors essential for embryonic development led us to investigate the physiological role of TAZ by a gene targeting strategy.

Here, we demonstrate that inactivation of the mouse Taz gene in mice results in the formation of multiple cysts in the kidney and greatly enlarged air spaces in the lung. These phenotypes resemble human polycystic kidney diseases (PKD) and pulmonary emphysema, respectively. PKD is a human inherited renal disorder that is the most common genetic cause of renal failure in children and adults (3, 15). Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent form, with an incidence of 1–2 per 1,000 individuals and is characterized by bilateral formation of multiple cysts arising from any segment of the nephrons and collecting ducts. ADPKD is caused by mutations in either of two genes, PKD1 and PKD2, that encode membrane-associated proteins polycystin-1 and polycystin-2, respectively (23, 32, 33). Autosomal recessive polycystic kidney disease (ARPKD) is much less frequent (1/20,000 live births) and is caused by mutations of PKHD1 (29, 37), which encodes polyductin/fibrocystin, a large transmembrane protein. Although the causative genes have been identified, the molecular mechanism underlying cystic formation remains largely unknown.

TAZ-deficient mice also exhibited urinary concentration defects, polyuria, and hydronephrosis. Although urinary concentrating defects have been observed in human ADPKD and ARPKD, massive polyuria and hydronephrosis are uncommon. These findings suggest that the pathophysiological processes in TAZ-deficient kidneys and human PKD are different in some respects. The similarities and dissimilarities between TAZ-deficient mice and PKD may provide important clues to understanding the pathogenesis of a common human disease.

In addition, the dilatation of air spaces of the lung in TAZ-deficient mice is morphologically reminiscent of human pulmonary emphysema, which is a common disease that causes death and disability, especially in the aged (1, 12). As a manifestation of chronic obstructive pulmonary disease (COPD), pulmonary emphysema is characterized by enlargement of air spaces and destruction of the alveolar wall. Although -antitrypsin deficiency is known to cause a congenital form of pulmonary emphysema, little remains known concerning the molecular mechanisms underlying the pathogenesis of this disease. TAZ-deficient mice may also serve as a novel animal model for COPD as well as kidney diseases.

	MATERIALS AND METHODS

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Generation and genotyping of mutant mice. A C57BL6/J-derived BAC clone containing the mouse Taz gene was obtained from BACPAC Resource Center (Oakland, CA). The nls-lacZ/PGKneo cassette was made by placing the lacZ gene with a nuclear localization signal (nls-lacZ) adjacent to the PGKneo gene and flanking it with lox71 at the 5'-end and lox2272 at the 3'-end to allow recombinase-mediated cassette exchange. A pKO Scrambler NTKV-1904 plasmid (Stratagene) was used as a backbone vector. For the targeting construct, a PCR-amplified 1.2-kb fragment extending from the promoter region to the 5'-untranslated region in exon 2 and an 8.2-kb NgoMIV-BamHI fragment from intron 2 were inserted on each side of the nls-lacZ/PGKneo cassette (Fig. 1A). The targeting vector was linearized and electroporated into the B6129F1-derived embryonic stem (ES) cell line ATOM1 (Amano T et al., unpublished observations). Clones that survived positive-negative selection with neomycin and 1-(2'-deoxy-2'-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil were screened for homologous recombination with diagnostic PCR primers. Targeted clones were injected in ICR blastocysts to generate germline chimeras. Mice homozygous for the Taz^lacZ allele were obtained by intercrossing F₁ heterozygotes. The genotypes of the offspring were determined by PCR or Southern blot analysis on tail-tip or amnion DNA. All animal experiments were reviewed and approved by the University of Tokyo Animal Care and Use Committee.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Targeting of the mouse Taz (transcriptional coactivator with PDZ-binding motif) gene. A: schematic representation of the targeting strategy employed to knockin an nls-lacZ cassette into the Taz locus. Two different probes for genotyping are indicated as 5'- and 3'-probes. B, BamHI; H, HindIII; S, SfuI; X, XbaI. B: representative genotyping of the offspring from an F1 intercross by Southern blot analysis. Genomic DNA samples were digested with BamHI and probed with the 5'-probe. Bands of 13.6 and 8.4 kb represent wild-type and mutant alleles, respectively. C and D: RT-PCR (C) and Western blot (D) confirming the absence of TAZ expression in TazlacZ/lacZ embryos. Total RNA and protein extracts were obtained from embryonic day 15.5 whole embryos. Lower band in D, top, represents nonspecific binding. E–G: lateral (E) and dorsal (F) views of an embryonic day 9.5 Taz+/lacZ embryo and a section at the level of the somites (G) stained with X-gal. FB, forebrain; DM, dermomyotome; M, myotome; NT, neural tube; r3 and r5, rhombomeres 3 and 5 of the hindbrain; Sm, somites.

Western blotting. Anti-TAZ rabbit polyclonal antibody was previously described (25). Western blotting was performed on lysates from embryonic day 13.5 whole embryos or 12-wk-old kidneys using a standard protocol.

Histology. For histological analysis, samples were fixed in formalin, embedded in paraffin, cut into 2-µm-thick sections, and stained with hematoxylin and eosin. Photomicrographs were obtained using a computer-assisted microscope (Nikon ECLIPSE 80i).

Urinary analysis. Urine volume was measured using the 48-h frequency/volume analysis system as previously described (4). Briefly, each mouse was placed in a metabolic cage connected to a digital scale and personal computer. Each mouse was provided with free access to food and water. After mice were acclimatized for 2 days in the cage, water intake, urine voiding frequency, and volume per void were recorded for 48 h.

Lectin and immunofluorescent staining. Lectin staining was performed on cryosectioned kidneys. Embryonic day 18.5 kidneys were dissected, embedded in optimum-cutting temperature (OCT) compound (Miles), and cut into 10-µm-thick sections. After being blocked with blocking buffer, the sections were incubated for 1 h at 37°C with 20 µg/ml biotin-conjugated Dolichos biflorus agglutinin (DBA) or Lotus tetragonolobus agglutinin (LTA) followed by extensive washing with blocking buffer. Lectin staining was visualized by reacting with fluorescein isothiocyanate-conjugated streptavidin, and nuclei were counterstained with propidium iodide. Photomicrographs were obtained using a computer-assisted confocal microscope (Nikon D-ECLIPSE C1).

For immunofluorescent staining, paraformaldehyde-fixed cryosections were stained with antibodies to aquaporin-2 (gift from M. Knepper, National Heart, Lung, and Blood Institute, Bethesda, MD) or aquaporin-3 (Chemicon), as described previously (31). Nuclei were stained with 4',6-diamidine-2-phenylindole (DAPI).

In situ hybridization. Isotopic in situ hybridization was performed on paraffin-embedded sections of embryonic day 14.5 and 16.5 embryos using ³⁵S-radiolabeled RNA probes for Taz/Wwtr1 as previously described (27).

β-Galactosidase staining. LacZ expression was detected by staining with X-gal (5-bromo-4-chloro-3-indoyl β-D-galactoside). Staining was performed as described by Nagy et al. (26) with minor modifications. Whole embryos and kidneys were washed in ice-cold PBS containing 2 mM MgCl₂ and fixed in 0.1 M phosphate buffer (pH 7.3) containing 0.2% glutaraldehyde, 5 mM EGTA, and 2 mM MgCl₂. Following rinsing three times with 0.1 M phosphate buffer containing 2 mM MgCl₂, 0.02% Nonidet P-40, 0.01% sodium deoxycholate, and 5 mM EGTA (washing buffer), samples were embedded in OCT compound and cryosectioned. Embryos or sections were incubated overnight at 37°C in X-gal-staining buffer (10 mM potassium ferrocyanide, 10 mM potassium ferricyanide and 2 mg/ml X-gal in washing buffer). For costaining with platelet/endothelial cell adhesion molecule, X-gal-stained sections were rinsed with PBS, incubated in the blocking buffer (see Lectin and immunofluorescent staining), and reacted with anti-CD31 antibody (BD Bioscience) (1:100) at 4°C overnight. Immunoreactivity was visualized with the VECTASTAIN ABC kit (Vector laboratories). Sections were counterstained with 1% Orange G (Sigma).

Morphometric analysis. The mean linear intercept, as a measure of interalveolar wall distance, was determined as described by Thurlbeck (34). Briefly, lines were drawn across light microscopic images of the lung section stained with hematoxylin and eosin. The mean linear intercept was calculated by dividing the total length of a line by the total number of intercepts encountered in 72 lines/lung.

Statistical analysis. Mann-Whitney nonparametric test was used to compare values between two groups for morphometric analysis. Student's t-test was used for the comparison of values in other experiments. Data were represented as means ± SD. Values of P < 0.05 were considered significant.

	RESULTS

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Generation of Taz-lacZ knockin mice. We disrupted the mouse Taz locus by replacing the coding region in exon 2 with an nls-lacZ/PGKneo cassette (Fig. 1A). Of 683 ES cell clones screened, three clones were positive for the mutant Taz^lacZ allele. All three clones were injected in ICR blastocysts and gave rise to male germline chimeras, which were subsequently bred with ICR females to produce Taz^+/lacZ heterozygous mice. The heterozygous mice appeared normal and were fertile. Offspring from Taz^+/lacZ intercrosses were genotyped by Southern analysis of tail genomic DNA (Fig. 1B). The absence of Taz transcripts and protein in Taz^lacZ/lacZ homozygous mice was confirmed by RT-PCR and Western blotting, respectively (Fig. 1, C and D).

To verify that Taz-directed lacZ expression reflected the pattern of authentic Taz expression, we stained for lacZ in embryonic day 9.5 Taz^+/lacZ embryos. At embryonic day 9.5, lacZ staining was observed in the somitic mesoderm and as in the neuroectoderm within the forebrain and hindbrain (Fig. 1, E and F). Within the somites, lacZ expression was detected in the myotome (Fig. 1G). These expression patterns coincide with that of authentic Taz expression revealed by in situ hybridization (25).

Early mortality in Taz^lacZ/lacZ mice. When 309 littermates derived from Taz^+/lacZ intercrosses were followed, 24 pups were found dead before 3 wk of age; 9 of 10 dead pups that could be genotyped were homozygous null. Genotyping of the remaining 285 littermates at 3 wk of age identified 90 wild-type (32%), 177 heterozygous (62%), and 18 homozygous (6%) mice, indicating that only one-fifth of the expected Mendelian ratio of Taz^lacZ/lacZ mice were alive at weaning (Table 1).

Multiple renal cysts and dilated calyces in Taz^lacZ/lacZ mice. To characterize the phenotype that may be related to early mortality, we performed macroscopic and histological examinations on surviving Taz^lacZ/lacZ mice. The most prominent abnormalities were first detected in the kidneys. Adult Taz^lacZ/lacZ mice showed bilaterally enlarged, pale kidneys (Fig. 2, C and D). The calyces were extremely dilated, leaving thinned parenchyma containing multiple cysts (Fig. 2E). Histological analysis of 10-wk-old Taz^lacZ/lacZ mice demonstrated numerous cysts of various sizes replacing most of the renal parenchyma (Fig. 2G), whereas no such changes were found in wild-type and Taz^+/lacZ kidneys (Fig. 2, F and H). Cysts were lined by a flattened epithelial monolayer and sometimes contained a glomerular tuft, indicating an origin from Bowman's capsule (Fig. 2, G and I). The surrounding tissues showed increased interstitial fibrosis in Masson's trichrome staining (data not shown). Notably, these renal changes in Taz^lacZ/lacZ mice shared a similar histology to human PKD.

View larger version (114K): [in this window] [in a new window]   Fig. 2. Enlarged kidneys containing multiple cysts in TazlacZ/lacZ mice. A-C: gross appearance of kidneys of Taz+/+ (A), Taz+/lacZ (B), and TazlacZ/lacZ (C) mice aged 10 wk. TazlacZ/lacZ kidneys are enlarged, pale, and filled with numerous cysts. D: comparison of TAZ+/lacZ and TazlacZ/lacZ kidneys. E: section of a TazlacZ/lacZ kidney showing dilated calyx and thinned parenchyma containing multiple cysts. F–I: histology of Taz+/+ (F and H) and TazlacZ/lacZ (G and I) kidneys at low (F and G) and high (H and I) magnification. Boxed areas in F and G are magnified in H and I, respectively. TazlacZ/lacZ kidneys exhibit numerous cysts of various sizes that are lined by a flattened epithelial monolayer. Cysts containing a glomerular tuft (arrowheads) are often observed. Scale bars indicate 500 µm (F and G) or 100 µm (H and I).

Extrarenal phenotypes in Taz^lacZ/lacZ mice. In human ADPKD, extrarenal manifestations are often observed in the liver, pancreas, blood vessels, heart, and other organs. However, examination of adult Taz^lacZ/lacZ mice did not reveal obvious abnormalities in these tissues (data not shown). Instead, the lung was unexpectedly affected in Taz^lacZ/lacZ mice. Histological examination revealed enlarged air spaces in Taz^lacZ/lacZ lungs at the age of 8–9 mo (Fig. 3, A and B). The mean linear intercept, as a measure of interalveolar wall distance, was significantly greater in Taz^lacZ/lacZ mice (149.6 ± 11.5 µm, n = 4) than in wild-type mice (51.6 ± 2.1 µm, n = 4) (Fig. 3C). There were no findings indicative of increased inflammation or fibrosis in Taz^lacZ/lacZ lungs. The phenotype of Taz^lacZ/lacZ lungs is highly reminiscent of human pulmonary emphysema.

View larger version (46K): [in this window] [in a new window]   Fig. 3. Pulmonary emphysema-like changes in TazlacZ/lacZ mice. A and B: hematoxylin and eosin staining of lung sections of Taz+/+ (A) and TazlacZ/lacZ (B) mice aged 9 mo. Enlarged air spaces and alveolar wall disruption are observed in the lung of TazlacZ/lacZ mice. Scale bars indicate 100 µm. C: morphometric analysis. Mean linear intercept values are significantly greater in TazlacZ/lacZ mice (n = 4) than wild-type mice (n = 4) aged 8–9 mo. A total of 288 lines drawn across the lung section were analyzed for each group. Error bars indicate SDs of the mean.

Urinary concentration defects in Taz^lacZ/lacZ mice. In addition to multiple cysts and dilated calyces, Taz^lacZ/lacZ mice showed signs of polyuria. Indeed, the urinary bladder in Taz^lacZ/lacZ mice was typically distended with a large volume of urine (data not shown). Measurement of the 48-h urine frequency and volume revealed that urine volume per void and total volume per day were much higher in Taz^lacZ/lacZ mice than in Taz^+/lacZ mice (Fig. 4). Measurements of urinary parameters showed polyuria and concentrating defects in Taz^lacZ/lacZ mice, as indicated by lower urinary osmolality (Table 2). Overall electrolyte excretion was not enhanced, although excretion of chloride was slightly increased (Table 2), indicating that electrolyte reabsorption remained grossly preserved. Urinary albumin excretion in Taz^lacZ/lacZ mice appeared to be increased, although the difference was not statistically significant (Table 2). The polyuria and concentrating defects in Taz^lacZ/lacZ mice were not improved by vasopressin administration (data not shown), suggesting that the abnormalities were likely to be nephrogenic rather than due to vasopressin deficiency. Overall, Taz^lacZ/lacZ kidneys were characterized by the concomitant occurrence of features that are typical of human PKD, including multicystic formation and urinary concentrating defects, and atypical features such as calyceal dilatation and hydronephrosis.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Representative 24-h urinary frequency/volume records. Mice aged 5–6 mo were subjected to the analysis. Urine volume per void, voiding frequency, and total volume per day are greatly increased in TazlacZ/lacZ mice (B) compared with Taz+/lacZ mice (A).

View larger version (119K): [in this window] [in a new window]   Fig. 5. Histological examination of developing kidneys in control (A, C, E, and G) and TazlacZ/lacZ (B, D, F, and H) embryos. A and B: at embryonic day 13.5, branching of ureteric buds and formation of renal vesicles and comma- and S-shaped bodies are observed in TazlacZ/lacZ (B) as well as wild-type and Taz+/lacZ (A) kidneys. C and D: at embryonic day 15.5, dilatation of Bowman's capsules (arrows) and adjacent tubules (arrowheads) are detected in TazlacZ/lacZ kidneys (D) but not in wild-type kidneys (C). E–H: at embryonic day 18.5, the atrophic medulla contains multiple cysts of various sizes in TazlacZ/lacZ kidneys (F and H). The pelvis is dilated and the papilla is not well formed (F). These changes are not observed in wild-type kidneys (E and G). The nephrogenic zone is present in the outer cortex just beneath the capsule in TazlacZ/lacZ (H) and wild-type (G) kidneys. Scale bars indicate 100 µm (A–D, G, and H) and 500 µm (E and F).

We examined the origins of the cysts using segment-specific markers. LTA, a lectin specific for the proximal tubule, stained the majority of epithelial cells lining dilated tubules and cysts in embryonic day 18.5 Taz^lacZ/lacZ embryos (Fig. 6, A–D). In contrast, no cysts were stained with DBA, a lectin specific for the collecting duct, at the same stage (Fig. 6, E–H). These results suggested that the cystic changes in Taz^lacZ/lacZ kidneys primarily originated from glomeruli and proximal tubules during the maturation of induced nephrons.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Proximal tubular dilatation leading to cyst formation in TazlacZ/lacZ embryos. Sections were stained with Lotus tetragonolobus agglutinin (LTA; A–D) or Dolichos biflorus agglutinin (DBA; E–H) in wild-type (A, B, E, and F) and TazlacZ/lacZ (C, D, G, and H) kidneys at embryonic day 18.5. B, D, F, and H show costaining with propidium iodide and lectins. Epithelial cells lining dilated tubules and cysts stain positive with LTA but not with DBA in TazlacZ/lacZ kidneys. Scale bars indicate 250 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 7. Taz expression during metanephric development. A–D: in situ hybridization of mouse embryonic kidneys with probes for Taz. Brightfield (A and C) and darkfield (B and D) images of embryonic day 14.5 (A and B) and embryonic day 16.5 (C–F) kidneys are shown. A and B: at embryonic day 14.5, Taz transcripts are diffusely detected in metanephric mesenchymal and epithelial cells and the ureteric bud. C and D: at embryonic day 16.5, Taz is expressed in mesenchymal and epithelial cells in the nephrogenic zone. Taz is also expressed in the collecting ducts. E–O: β-galactosidase activity in Taz+/lacZ (E, F, and J–L) and TazlacZ/lacZ (G–I and M–O) kidneys at embryonic day 16.5 (E–I) and day 0 postpartum (P0; J–O). E, F, and F', in embryonic day 16.5 Taz+/lacZ kidneys, X-gal staining (blue) is observed in glomeruli (arrows) and CD31-stained (brown) capillary endothelial cells (arrowheads). X-gal staining with (F) and without (F') CD31 staining is shown. G–I: in embryonic day 16.5 TazlacZ/lacZ kidneys, X-gal staining is observed in stromal(-like) cells (open arrowheads in I) as well as in glomeruli and capillary endothelial cells (filled arrowheads in H). J–L: in P0 Taz+/lacZ kidneys, scattered X-gal staining is observed in glomeruli (K) and capillary endothelial cells (L). M–O: in P0 TazlacZ/lacZ kidneys, X-gal staining is observed in outer cortical stromal cells, glomeruli, and capillaries (N). Some cysts are lined by X-gal-positive cells (O). Sections of P0 kidney were counterstained with Orange G. c, cyst; cd, collecting duct; g, glomerulus; me, mesenchyme; nz, nephrogenic zone; ub, ureteric bud. Scale bars indicate 50 µm (E and I) and 500 µm (J and M).

Expression of cystic disease transcripts and proteins in Taz^lacZ/lacZ kidneys. To find clues to the mechanism underlying the renal phenotype of Taz^lacZ/lacZ mice, we analyzed the expression of genes involved in human PKD. No alterations in the levels of Pkd1 and Pkd2 mRNA and their products, polycystin-1 and polycystin-2, were observed at embryonic day 15.5 and 18.5 by real-time RT-PCR (Fig. 8, A and B) and Western blotting (Fig. 8D). Also, the expression of Pkhd1, a gene linked to ARPKD, was not different between wild-type and Taz^lacZ/lacZ kidneys (Fig. 8C). These results indicated that renal cyst formation induced by Taz-null mutation was not due to changes in the expression of Pkd1, Pkd2, and Pkhd1 at the perinatal stage.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Real-time RT-PCR analysis and Western blotting of cystic disease genes and proteins. A: real-time RT-PCR. Total RNA samples were prepared from embryonic day 15.5 and 18.5 wild-type (+/+) and homozygous (lacZ/lacZ) kidneys. The abundance of transcripts for Pkd1, Pkd2, and Pkhd1 was measured relative to the internal control Gapdh. Error bars indicate SDs of the mean (n = 5). For all threes genes at both stages, the expression in homozygous mutant kidneys is not statistically different from wild-type kidney (P > 0.05). B: Western blotting for polycystin-1 and polycystin-2. Protein levels of polycystin-1 and -2 are not different among wild-type, Taz+/lacZ, and TazlacZ/lacZ kidneys at embryonic day 18.5. Blotting for β-galactosidase shows intensities corresponding to the copy number of the lacZ gene. Blotting for -tubulin serves as an internal control.

View larger version (60K): [in this window] [in a new window]   Fig. 9. Expression of genes involved in water transport. A: real-time RT-PCR. Total RNA samples were prepared from embryonic day 18.5 and 2-wk-old wild-type (+/+) and homozygous (lacZ/lacZ) kidneys. The abundance of transcripts for Avpr2 was measured relative to the internal control Gapdh. Error bars indicate SDs of the mean; n = 5 (+/+) and 5 (lacZ/lacZ) at embryonic day 18.5; n = 4 (+/+) and 3 (lacZ/lacZ) at 2 wk; n = 8 (+/+) and 6 (lacZ/lacZ) at 8–12 wk; n = 4 (+/+) and 4 (lacZ/lacZ) at 20–26 wk. No statistically significant difference is detected in the expression of Avpr2 between +/+ and lacZ/lacZ kidneys (P > 0.05). B: RT-PCR analysis of Aquaporin-1, -2, and -3 expression. Total RNA samples were prepared from embryonic day 18.5 wild-type (+/+), heterozygous (+/lacZ), and homozygous (lacZ/lacZ) kidneys. The levels of Aquaporin-1, -2, and -3 transcripts are not different among wild-type, heterozygous, and homozygous kidneys. RT-PCR for Gapdh served as an internal control. C–F: immunofluorescence staining of aquaporins. Sections were stained for aquaporin-3 (C and D) or aquaporin-2 (E and F) in Taz+/lacZ (C and E) and TazlacZ/lacZ (D and F) kidneys at embryonic day 18.5. All sections were costained with 4',6-diamidine-2-phenylindole. Aquaporin-3 is localized in the basolateral membrane of epithelial cells in some cysts (arrow) as well as in noncystic tubules (arrowheads) in TazlacZ/lacZ kidneys. Aquaporin-2 is also expressed similarly in collecting ducts in heterozygous and homozygous kidneys. Scale bars indicate 10 µm.

	DISCUSSION

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Comparison of the renal phenotype of TAZ-deficient mice to human cystic kidney diseases. In human ADPKD, the epithelial-lined cysts originate from any segment of the nephron and collecting ducts, and in ARPKD the cysts mainly originate from the collecting ducts (3, 10). In contrast, the cysts in TAZ-deficient kidneys mainly originate from the glomeruli and proximal tubules. However, patients with early onset ADPKD may develop glomerular cysts, suggesting that cyst formation in the proximal nephrons may be an early manifestation of ADPKD (11. 15). Consistently, Pkd1-null mice start to exhibit cyst formation at embryonic day 15.5 in the proximal tubule (20). Other animal models of PKD, such as the cpk/cpk mice and the Han:SPRD cy/+ rats, have also shown cysts originating predominantly from the proximal tubule (39). Studies of human fetuses with ARPKD have shown cysts originating from proximal tubules. Thus the renal phonotype of TAZ-deficient mice may recapitulate the early phase of human PKD.

However, no apparent differences in the expression of PKD genes, Pkd1, Pkd2, and Pkhd1, are detected in the kidneys of wild-type and TAZ-deficient embryos before birth. This finding indicates that cyst formation in TAZ-deficient kidneys is not due to changes in the expression of these cystic disease genes. Polycystin-1 and -2, proteins encoded by ADPKD genes PKD1 and PKD2, respectively, are membrane glycoproteins that can associate with each other to form a complex in the primary cilium of renal epithelial cells (2, 10, 19). The polycystin complex is implicated in cell cycle regulation, intracellular calcium regulation, and maintenance of cellular polarity (2, 10, 19). Polyductin/fibrocystin, the protein encoded by PKHD1, is also a large transmembrane protein. Considering the possible interaction of TAZ with membrane-associated PDZ domain-containing proteins, it may still be possible that TAZ and the cystic disease proteins share a common pathway involved in normal epithelial function and structural integrity. However, taken together with the pathological differences from human PKD, distinct mechanisms may be involved in the renal phenotype of TAZ-deficient mice.

Recently, Hossain et al. (16) also reported the development of cystic kidney disease in TAZ/Wwtr1-deficient mice. Their study suggested that the loss of renal cilia integrity and downregulation of several genes, including Pkhd1, might be associated with the development of renal cysts in TAZ-deficient mice. Independently, Tian et al. (35) have reported that polycystin-2 is overexpressed in adult TAZ-deficient kidneys as a result of decreased ubiquitin-mediated degradation. In the present study, no changes in fibrocystin or polycystin-2 expression are observed in TAZ-deficient kidneys during the prenatal stage, when cyst formation starts. Abnormalities in fibrocystin and polycystin-2 may be involved in later stages of disease progression rather than initial cystogenesis.

Comparison of the renal phenotype of TAZ-deficient mice with human nephrogenic diabetes insipidus. A major difference between the renal phenotype of TAZ-deficient mice and human PKD is the presence of severely dilated calyces and massive polyuria. Although humans with ADPKD and ARPKD may have urinary concentration defects (6, 21), severe polyuria and hydronephrosis are not typical clinical features. TAZ-deficient mice have normal daily excretion of sodium and potassium, indicating that tubular electrolyte reabsorption is well preserved. The relatively low urine osmolality compared with wild-type together with impaired response to exogenous vasopressin are characteristic of nephrogenic diabetes insipidus. Hydronephrosis may be secondary to polyuria, as seen in congenital progressive hydronephrosis (cph) mutant mice (22). Thus the renal phenotype of TAZ-deficient mice is characterized by two distinct pathophysiological processes, cyst formation and urinary concentration defects.

Nephrogenic diabetes insipidus is caused by the inability of the renal collecting ducts to reabsorb water in response to vasopressin. About 90% of affected patients have mutations in the AVPR2 gene, whereas the remaining 10% of patients are caused by AQP2 gene mutations (28). However, TAZ-knockout kidneys do not show major abnormalities in the levels of expression of Avpr2, Aqp2, and Aqp3. In addition, the apical-basolateral polarity of aquaporin-2 and aquaporin-3 is preserved in both cystic and noncystic collecting ducts at embryonic and postnatal stages. These findings suggest that the polyuria and hydronephrosis in TAZ-deficient mice do not arise from major defects in the expression or localization of the V₂ vasopressin receptor, aquaporin-2, or aquaporin-3. Elucidation of the mechanism underlying the concentration defects in TAZ-deficient kidney may reveal novel pathways regulating renal water transport.

Possible requirement of TAZ for normal kidney development. The kidney develops through reciprocal interactions between the metanephric mesenchyme and the ureteric bud epithelium during embryogenesis (8, 36, 40). The ureteric bud grows into the metanephric mesenchyme and branches to form the collecting duct system while the mesenchyme adjacent to the tips of the ureteric bud is induced to condense and undergo a mesenchymal-to-epithelial transition. The resultant renal vesicle further differentiates into comma- and then S-shaped bodies. Morphogenesis and patterning of the epithelial structures lead to the formation and functional maturation of nephron segments including the glomerulus, the proximal tubule, the loop of Henle, and the distal tubule. Recent advances in gene targeting experiments have greatly contributed to the understanding of molecular mechanisms underlying the early "inductive" phase of kidney development. However, it remains largely unknown how each nephron segment is specified and functionally matures during the late phase of kidney development.

In situ hybridization and β-galactosidase staining demonstrated that Taz is diffusely expressed throughout the kidney. Taz expression is most intense in the nephrogenic zone during kidney development and is found in both mesenchymal and epithelial cells. Taz is not restricted to a specific segment of the nephron. Capillary endothelial cells also express Taz. LacZ expression is relatively scant and scattered compared with the pattern of in situ hybridization. This difference may be caused by deletion or disruption of critical enhancer element(s) during the generation of the targeted mutation.

In TAZ-deficient kidneys, most, but not all, of the cyst epithelium is lacZ-negative. In contrast, stromal cells, especially in the nephrogenic zone, show strong lacZ expression in TAZ-deficient kidneys. This finding indicates that TAZ may be crucial for gene expression in stromal cells supporting normal nephric development.

Pulmonary emphysematous changes in TAZ-deficient mice. In addition to renal cyst formation, TAZ-deficient mice demonstrate severely enlarged air spaces in the lung. This finding is morphologically reminiscent of human pulmonary emphysema, whose genetic pathogenesis is poorly understood. Only a congenital form of emphysema is known to be caused by a deficiency of -antitrypsin, but its expression was not affected in TAZ-deficient mice (our unpublished data).

Pulmonary emphysema, as a manifestation of COPD, is regarded as a multifactorial disorder triggered by environmental factors such as cigarette smoking and pollutants. Although genetic factors involved in protease/antiprotease balance have been considered as possible determinants of susceptibility to emphysematous changes, the molecular pathogenesis is still unknown. In mice, emphysema-like pulmonary changes can be caused by deficiency in surfactant proteins SP-C or SP-D, possibly through increased activity of matrix metalloproteases (9, 38). Interestingly, TAZ has been reported to be a coactivator for the transcription factor TTF-1/Nkx2.1 and upregulates the expression of SP-C in respiratory epithelial cells (5). However, SP-C expression was not apparently affected in TAZ-deficient lungs (unpublished observations), so different mechanisms may generate the emphysematous changes in TAZ-deficient lungs.

The coexistence of renal and pulmonary abnormalities observed in TAZ-deficient mice has not been described before. However, there are similarities in the embryonic development of the kidney and lung. Both involve common processes, e.g., branching morphogenesis and common signaling pathways such as sonic hedgehog, fibroblast growth factor, and bone morphogenetic protein (17, 41). These similarities raise the possibility that TAZ may be an effector in a common pathway that is involved in both lung and kidney development. Although the apical-basolateral polarity of epithelial cells in collecting ducts is not impaired in TAZ-deficient kidneys, further examination of the processes of epithelial tubule formation in TAZ-deficient kidneys and lungs may reveal a common mechanism for organogenesis and pathogenesis of human diseases.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, Grants-in-Aid for Scientific Research from the Ministry of Health, Labour, and Welfare of Japan, a Research Grant from Uehara Memorial Foundation, the University of Texas Southwestern O'Brien Kidney Research Core Center (National Institute of Diabetes and Digestive and Kidney Diseases Grant P30DK-079328), and a Basil O'Connor Research Grant from the March of Dimes Birth Defects Foundation.

	ACKNOWLEDGMENTS

 
We thank Dr. Mark Knepper (National Heart, Lung, and Blood Institute) for the aquaporin-2 antibody.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. Kurihara, Dept. of Physiological Chemistry and Metabolism, Graduate School of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (e-mail: kuri-tky{at}umin.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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