Human Molecular Genetics, 2002, Vol. 11, No. 15 1731-1742
© 2002 Oxford University Press
Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant
1Department of Urology, The University of Tokyo, Tokyo 113-8655, Japan, 2Department of Cell Biology, Kobe University, Kobe 650-0017, Japan, 3Second Department of Internal Medicine, Gunma University, Maebashi 371-8511, Japan, 4Riken, Center for Developmental Biology, Kobe 650-0047, Japan, 5Mitsubishi Kasei Institute of Life Sciences, Machida 194-8511, Japan, 6Department of Cardiovascular Medicine, The University of Tokyo, Tokyo 113-8655, Japan, 7Department of Urology, Kyorin University, Mitaka 181-8611, Japan, 8Department of Nephrology, Mayo Clinic, Rochester, MN 55905, USA and 9National Institute for Basic Biology, Okazaki National Research Institutes, Okazaki 444–8585, Japan
Received March 28, 2002; Accepted May 24, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Mutations of either PKD1 or PKD2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The molecular function of the gene product of PKD1, polycystin-1, in vitro has been elucidated recently, but the molecular pathological consequences of the loss of polycystin-1 in vivo have remained unclear. We have generated a mouse with a targeted deletion of exons 2–6 of Pkd1 to study the molecular defects in Pkd1 mutants. Homozygote embryos (Pkd1-/-) developed hydrops, cardiac conotruncal defects and renal cystogenesis. Total protein levels of ß-catenin in heart and kidney and c-MYC in heart were decreased in Pkd1-/- embryos. In the kidneys of Pkd1-/-, the expression of E-cadherin and PECAM in basolateral membranes of renal tubules was attenuated, and tyrosine phosphorylation of epidermal growth factor receptor and Gab1 were constitutively enhanced when cystogenesis started on embryonic day (E) 15.5–16.5. Maternally administered pioglitazone, a thiazolidinedione compound, resolved these molecular defects of Pkd1-/-. Treatment with pioglitazone improved survival of Pkd1-/- embryos and ameliorated the cardiac defects and the degree of renal cystogenesis. Long-term treatment with pioglitazone improved the endothelial function of adult Pkd1+/-. These data indicated that molecular defects observed in Pkd1-/- embryos contributed to the pathogenesis of ADPKD and that thiazolidinediones had a compensatory effect on the pathway affected by the loss of polycystin-1. Pathways activated by thiazolidinediones may provide new therapeutic targets in ADPKD.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Autosomal dominant polycystic kidney disease (ADPKD) causes epithelial cysts, aneurysms, cardiac valvular insufficiency, hypertension and renal insufficiency (1). Mutations in PKD1 (2) and PKD2 (3) are present in ADPKD. The PKD1 and PKD2 genes encode polycystin-1 and polycystin-2, respectively. Recently, the molecular function of polycystin-1 in vitro has been reported. Interaction of polycystin-1 and -2 results in the formation of calcium-permeable non-selective cation channels (4). Overexpression of PKD1 inhibits apoptosis and promotes spontaneous tubulogenesis in MDCK cells (5). Targeted disruption of the murine ortholog of Pkd1 has helped elucidate the function of its protein product (6–10). Homozygotes of Pkd1 mutants, including Pkd1L (6), Pkd1del17–21geo (7), PKD1null (8) and Pkd1del34 (9), die in utero and develop renal and pancreatic cysts. Some homozygous Pkd1 mutants have cardiac conotruncal defects (7), vascular fragility associated with focal hemorrhage, or both (6–8). Heterozygous Pkd1 mutants develop adult-onset polycystic kidney as well as liver and pancreatic disease (7,8,10), which largely recapitulates the clinical pattern seen in ADPKD. Cystogenesis in ADPKD features perturbations of polarization and cellular hyperproliferation (11). Cultured ADPKD cells show impaired basolateral trafficking in membrane transport and abnormal localization of E-cadherin (12). The activation of tyrosine kinases mediated by growth factors such as epidermal growth factor (EGF) (13,14) and hepatocyte growth factor (HGF) (15,16) stimulates a tubular cell to change to a hyperproliferative state. The association of these in vitro findings with the phenotypes observed in Pkd1 mutants has not been fully examined.
We have generated Pkd1-mutant mice with a targeted deletion of exon 2–6 to further delineate the molecular defects associated with loss of polycystin-1. In addition, we have explored the possibility of using thiazolidinediones to treat ADPKD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Generation of Pkd1-/- mice and analysis of the phenotypes
We designed a Pkd1 targeting vector to replace the BglII–BglII fragment including exons 2–5 and part of exon 6, which contains the neomycin-resistance gene (Fig. 1A). Embryonic stem (ES) cell clones containing the correct replacement (Fig. 1B) were injected into C57BL/6J blastocysts, and germline transmission was achieved. Heterozygous (Pkd1+/-) mice were crossed to produce homozygous (Pkd1-/-) offspring. The targeted Pkd1 alleles underwent a frameshift. By western blotting, we confirmed the absence of polycystin-1 expression in Pkd1-/- (Fig. 1C). Analysis of staged embryos showed that on embryonic day (E) 14.5, Pkd1-/- embryos started dying in utero (Table 1). Pkd1-/- embryos exhibited hydrops fetalis and focal hemorrhages (Fig. 2B). The hearts of Pkd1-/- embryos at E12.5 showed hemorrhagic pericardial effusion and had a double-outlet right ventricle (DORV) (Fig. 2E). In the Pkd1-/- kidney, progressive renal cystogenesis started around E15.5. At E18.5, the renal parenchyma of Pkd1-/- had numerous cysts (Fig. 3B).
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ß-Catenin and c-MYC decreased in Pkd1-/- heart
Previous studies showed that the C-terminal region of polycystin-1 protects soluble ß-catenin from degradation (17). We examined the amount of total ß-catenin in the hearts at E12.5. The western blot demonstrated that the ß-catenin protein level was 39% lower in Pkd1-/- than in wild-type (ß-catenin protein level normalized by ß-actin, arbitrary ratio; wild-type; 0.72±0.28, n=5; Pkd1-/-; 0.44±0.20, n=5, P=0.0047) (Fig. 2G). The protein level of c-MYC, a target molecule of ß-catenin (18), was 75% lower in Pkd1-/- heart than in wild-type heart (c-MYC protein level normalized by ß-actin, arbitrary ratio; wild-type, 0.68±0.18, n=3; Pkd1-/-, 0.13±0.13, n=3, P=0.021) (Fig. 2G).
Molecular defects in Pkd1-/- kidney
We examined the amount of total ß-catenin in the kidneys at E16.5. The ß-catenin protein level was 34% lower in Pkd1-/- than in wild-type (ß-catenin protein level normalized by ß-actin, arbitrary ratio; wild-type, 0.98±0.16, n=3; Pkd1-/-, 0.65±0.04, n=3, P=0.020) (Fig. 3E). However, the protein level of c-MYC was comparable between wild-type and Pkd1-/- kidney (c-MYC protein level normalized by ß-actin, arbitrary ratio; wild-type, 0.56±0.22, n=3; Pkd1-/-, 0.42±0.27, n=3, P=0.51) (Fig. 3E). To see if the loss of polycystin-1 affected the expression of adherens junction (AJ) proteins in vivo, we examined the expression of AJ proteins in renal tubules at E16.5. Anti-E-cadherin antibodies stained the basolateral membranes of the wild-type renal tubules (Fig. 4A). In Pkd1-/- tubules, the basolateral staining of E-cadherin was less prominent (Fig. 4B and G). The western blot showed that the total protein level of E-cadherin was lower in Pkd1-/- kidney than in wild-type kidney (Fig. 4H). In developing cysts, the height of an individual cyst epithelial cell was shorter compared to that of a normal tubule; and the basolateral expression of E-cadherin was lost to a remarkable extent (Fig. 4G). Basolateral expression of PECAM-1 (CD 31), another component of AJ, was also reduced in Pkd1-/- tubules (Fig. 4D and E) in association with the decrease in protein level (Fig. 4H). These data suggest that the loss of polycystin-1 affected the metabolism of AJ proteins. To investigate whether abnormal cell signaling is involved in the activation of EGF receptor (EGFR) and Gab1, an adapter cytoplasmic protein associated with activated EGFR and c-MET (19,20), we examined the phosphorylation status of EGFR and Gab1 in Pkd1-/- tubules. Immunohistochemistry with anti-tyrosine phosphorylated EGFR showed the positive staining of cells in renal tubules (Fig. 5B) and cyst epithelial cells (Fig. 5C) in Pkd1-/- kidneys at E16.5. On the other hand, tubular cells of wild-type kidney showed much weaker staining (Fig. 5A). The level of tyrosine-phosphorylated Gab1 was greater in Pkd1-/- kidneys at E16.5 than in wild-type kidneys, whereas the protein levels of Gab1 were similar (Fig. 5F).
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Maternally administered pioglitazone corrected the molecular defects of Pkd1-/- embryos
We hypothesized that decreased total protein levels of ß-catenin and c-MYC in Pkd1-/- embryonic hearts are associated with cardiac abnormalities which, in turn, result in embryonic death in mid-gestation. Recent studies have shown that thiazolidinediones, peroxisome proliferator-activated receptor
(PPAR) agonists, affect Wnt signaling by increasing ß-catenin levels in the colon of APC mutant mice (21). Thiazolidinediones also upregulate the expression of c-myc mRNA in thyroid cancer cells (22). We investigated whether maternally administered pioglitazone (PIO, Takeda Chemical Industries, Ltd, Osaka, Japan), a thiazolidinedione compound, increased the stability of ß-catenin and c-MYC levels in the heart and ameliorated the cardiac phenotype, thereby prolonging survival of the Pkd1-/- pups. We administered 80 mg/kg/day of PIO, which crosses the placenta, to pregnant Pkd1+/- dams during the period of embryonic days 7.5–9.5. The period of embryonic days7.5–9.5 was chosen because cardiac neural crest cells migrate to form the outflow tract septa within the lateral walls of the aortic sac and the truncus arteriosus at E9.5 (23). Pregnant Pkd1+/- dams were given freshly prepared paste food complemented with pioglitazone (PIO) daily, and its complete consumption was confirmed every day. At E16.5, the survival of Pkd1-/- embryos was not different between the control group and the PIO-treated group. Six of 51(11.8%) live embryos were Pkd1-/- without treatment, whereas 18 of 100 (18%) live embryos were Pkd1-/- after PIO treatment (P=0.32, 
2=0.98) (Table 1). However, significantly more Pkd1-/- embryos receiving PIO treatment lived to E18.5. At E18.5, only 2 of 68 (2.9%) live embryos were Pkd1-/- without treatment, whereas 6 of 41 (15%) live embryos were Pkd1-/- after treatment (P=0.023, c2=5.1). When PIO was administered to dams during the period 9.5–11.5 p.c., the number of Pkd1-/- embryos at E16.5 (2 of 33 total embryos, 6.1%) was lower than among those which had earlier treatment (18 of 100 total embryos, 18%), although the difference was not statistically significant (P=0.096, 2=2.8) (Table 1). PIO-treated Pkd1-/- embryos had less subcutaneous edema than the untreated embryos (Fig. 2B and C). Edema was quantified by measuring the thickness of the subcutaneous tissue of Pkd1-/- embryos at E16.5 in the control and PIO-treated groups (control, 820±282 µm; PIO-treated, 59.4±12.0 µm, P=0.0011) (Fig. 2F). Cardiac DORV, examined at E16.5, was not found in four live Pkd1-/- embryos from the PIO-treated dams, but did occur in four of five live control Pkd1-/- embryos (P=0.016, 2=5.76). In PIO-treated embryos, the levels of ß-catenin and c-MYC in the heart at E12.5 were similar in Pkd1-/- and wild-type embryos (Fig. 2G). These results indicated that maternal treatment with PIO corrected the molecular defects and reduced the cardiac defects of the Pkd1-/- embryos. PIO treatment significantly reduced the level of renal cystogenesis (Fig. 3B and C). PIO-treated Pkd1-/- kidneys showed only slightly dilated renal tubules even at E18.5, with much less cystic enlargement as compared to untreated Pkd1-/- kidneys. Another thiazolidinedione compound, troglitazone (TRO, Sankyo Co., Ltd, Tokyo, Japan) (2 g/kg/day administered during 7.5–9.5 p.c.) had a similar effect on the inhibition of renal cystogenesis. The average area of individual renal cysts at E18.5 was significantly smaller in PIO- and TRO-treated Pkd1-/- embryos than in untreated Pkd1-/- controls (control, 124 cysts/5 kidneys; average area of individual renal cysts 179±197 µm2; PIO-treated, 73 cysts/5 kidneys; 71±76 µm2, P<0.0001 versus control; TRO-treated, 42 cysts/3 kidneys; 81±57 µm2, P=0.0003 versus control) (Fig. 3D). In PIO-treated embryos, the levels of ß-catenin and c-MYC in the kidneys at E16.5 were similar in Pkd1-/- and wild-type (ß-catenin protein level normalized by ß-actin, arbitrary ratio; wild-type, 0.68±0.18, n=3; Pkd1-/-, 0.79±0.02, n=3, P=0.24; c-MYC protein level normalized by ß-actin, arbitrary ratio; wild-type, 0.47±0.08, n=3; Pkd1-/-, 0.49±0.08, n=3, P=0.92) (Fig. 3E). Expression of AJ proteins in renal tubules was maintained in PIO-treated Pkd1-/- embryos (Fig. 4C,F and G). Enhanced tyrosine phosphorylation of EGFR and Gab1 observed in Pkd1-/- embryonic kidneys was not seen in PIO-treated Pkd1-/- (Fig. 5D–F).
Pioglitazone improved endothelial function in Pkd1+/- adults
Impaired endothelium-dependent relaxation has been demonstrated previously in small subcutaneous resistance vessels obtained from patients with ADPKD (24). To investigate whether endothelium-dependent relaxation was reduced also in large vessels, acetylcholine (ACh)-induced endothelium-dependent relaxation was measured in the aorta obtained from 30-week-old adult Pkd1+/- mice. The maximum relaxation rate (Emax) induced by ACh at the concentration of 10-5 M was significantly lower in Pkd1+/- than in wild-type (wild-type, 98.6±2.40%, n=5; Pkd1+/-, 84.8±5.80%, n=5, P=0.0011) (Fig. 6A). There was no difference in the sodium nitroprusside-induced (Emax) between Pkd1+/- and wild-type (data not shown). We next repeated this study in 10-month-old Pkd1+/- mice fed with PIO (40 mg/kg/day) and without PIO for the last 6 months to determine whether long-term treatment with PIO improves endothelial function. The systolic blood pressure of PIO-treated Pkd1+/- mice was not different from that of untreated Pkd1+/- mice untreated control, 119±10 mmHg; PIO-fed, 108±7 mmHg, P=0.33). However, comparing the animal growth rate, body weight was significantly decreased in PIO-fed Pkd1+/- mice compared to untreated Pkd1+/- mice untreated control, 37.0±0.7 g; PIO-fed, 33.3±1.3 g, P=0.017). PIO-treated Pkd1+/- mice showed a significantly increased Emax compared to untreated Pkd1+/- mice (control, 72.9±7.99%, n=7; PIO-treated, 96.8±2.50%, n=7, P=0.027) (Fig. 6B). Nitric oxide (NO) produced in vascular endothelium predominantly mediates ACh-mediated relaxation of the aorta (25). Indeed, the NO synthase inhibitor N
-nitro-L-arginine methylester completely abolished ACh-induced aortic relaxation in both groups (data not shown). Urinary concentration and total daily excretion of NOx were significantly lower in 30-week-old Pkd1+/- mice than in wild-type mice (concentration (mol/l): wild-type, 15.8±7.44, n=11; Pkd1+/-, 9.31±5.63, n=11, P=0.032) (total excretion (µM/day): wild-type, 2.44±0.83, n=11; Pkd1+/-, 1.58±0.78, n=11, P=0.021) (Fig. 6C). PIO treatment increased the urinary excretion of NOx in 10-month-old Pkd1+/- mice by 98% (µM/day: control, 1.00± 0.31, n=7; PIO-treated, 1.98±0.88, n=6, P=0.0019) (Fig. 6D). These data indicated that PIO improved the endothelial function of adult Pkd1+/- mice, presumably by increasing the production of NO.
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) (n=5), 30-week-old mice. *, P<0.005, t-test, relative to wild-type, mean±SD. (B) Effect of long-term PIO treatment in Pkd1+/- adult, 10-month-old mice. Comparison of untreated control (
) (n=7) and PIO-treated (
) (n=7). *, P<0.05, t-test, relative to untreated control, mean±SD. (C and D) Amount of NOx excreted in urine. (C) Comparison of wild-type (Pkd1+/+) (n=11) and Pkd1+/- (n=11), 30-week-old mice. *, P<0.05, t-test, relative to wild-type, mean±SD. (D) Effect of long-term PIO treatment in Pkd1+/- adult, 10-month-old mice. PIO(-), untreated control (n=7); PIO (+), PIO-treated (n=6). *, P<0.05, t-test, relative to untreated control, mean±SD. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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We have generated Pkd1-mutant mice by targeted disruption of exon 2–6, which resulted in a frameshift. The phenotype of a Pkd1-/- embryo in this study was almost identical to those of Pkd1L (6), Pkd1del17–21geo (7) and Pkd1null (8). Among mutants with a C57BL/6-129 background (Table 2), Pkd1L/L has the most severe disease, judging by its embryonic lethality, since it does not survive to E16.5. Mutant polycystin-1 of Pkd1L/L, L3946*, is much longer than the truncated proteins created by a frameshift in Pkd1null/null and Pkd1-/-. Thus the relationship between disease phenotype and severity and the length of the respective mutant polycystin-1 is not linear.
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We then went on to demonstrate biochemical defects in these animals and the effects of a potential treatment. First, we found that there were decreased levels of ß-catenin and c-MYC protein in Pkd1-/- embryonic hearts which had conotruncal defects. Cardiac neural crest cells contribute to the development of the proper outflow tract septation (26). In a mouse model of conotruncal heart defects, Splotch, Wnt signaling is reduced in cardiac neural crest cells (27). Previous studies showed that Wnt signaling regulates the stability of ß-catenin (reviewed in 28), and the C-terminal portion of polycystin-1 protects soluble ß-catenin from degradation (17). Present findings suggest that polycystin-1 might play an essential role in the cardiac conotruncal development by modulating the expression of ß-catenin and c-MYC. Treatment with maternally administered PIO often corrected the conotruncal defects and increased the levels of ß-catenin and c-MYC compared to those of wild-type littermates. The targeted disruption of retinoic X receptors (RXRs)
causes conotruncal defects (29), almost identical to the cardiac phenotype of Pkd1-/-. PPAR forms heterodimers with RXRs, which then bind to PPAR-responsive elements (PPRE) in the promoters of PPAR target genes (30). Since no DORV was observed in PIO-treated Pkd1-/- embryos, transcriptional regulation by PPRE may have a compensatory effect on the development of cardiac conotruncal defects in response to loss of polycystin-1. The kidneys of Pkd1-/- embryos showed the following molecular defects: decreased levels of ß-catenin, perturbation of basolateral expression of AJ proteins in tubules, and enhanced tyrosine phosphorylation of EGFR and Gab1. Recent studies suggest that an aberrant ß-catenin signaling pathway may be one of the cellular defects in cystogenesis. Transgenic mice that overproduce an oncogenic form of ß-catenin in the epithelial cells of the kidneys develop severe polycystic lesions in the glomeruli, proximal and distal tubules and collecting ducts (31). Mice deficient in the Bcl-2 gene (Bcl-2-/-) develop polycystic kidney disease (32), and the epithelial cells that form the cysts in the Bcl-2-/- mice accumulate ß-catenin in their nuclei (33). These data clearly demonstrate that increased transcriptional activity induced by ß-catenin in nuclei mediates renal cystogenesis which features cellular hyperproliferation and dedifferentiation (11). Several lines of evidence suggest that the ß-catenin signaling pathway is a downstream effector of polycystin-1. Polycystin-1 has been found in a complex containing E-cadherin and catenins (34). The C-terminal tail of polycystin-1 protects cytoplasmic ß-catenin from degradation by the proteasome, and activates Wnt/ß-catenin-dependent transcription (17). In differentiated epithelia, the amount of ß-catenin is tightly regulated and its steady-state level outside the cadherin–catenin complex is low. The loss of polycystin-1 might disrupt a polycystin-1–E-cadherin–ß-catenin complex at the adherens junctions and might alter the cellular metabolism of ß-catenin. Therefore, the decreased amounts of ß-catenin in Pkd1-/- kidneys seen in this study would not necessarily exclude the possibility that transcriptional activity mediated by ß-catenin in the nuclei was increased.
Cyst epithelium features a hyperproliferative state and loss of cellular polarity (11). In a developing cyst in Pkd1-/- kidneys, the cyst epithelial cells were shorter in height compared to normal tubular cells and there was a remarkable decrease in the expression of E-cadherin (Fig. 4G). The precise relationship between the disruption of cell polarity and the induction of cell proliferation remains unclear. However, it has recently been reported that ErbB2 affects cell polarity and induces cell proliferation in growth-arrested mammary acini (35). This report emphasizes that uncontrolled activation of tyrosine kinase-mediated signaling leads to the modification of cytoarchitecture. Hence, the decrease in AJ proteins and constitutive activation of EGFR and Gab1 in the Pkd1-/- kidney might be mutually associated with the loss of polycystin-1. As mentioned before, molecular defects were corrected in the kidney of PIO-treated Pkd1-/- embryos. The mechanisms remain unclear. However, since the EGFR tyrosine kinase inhibitor EKI-785 inhibits renal cystogenesis in bpk mice (36), the effect of PIO on the quenching of tyrosine phosphorylation of EGFR and Gab1 might play a significant role in the prevention of renal cystogenesis in Pkd1-/-. Recent studies show that polycystin-1 is targeted to the basolateral membrane, where it complexes with polycystin-2, which is either in the plasma membrane or in the endoplasmic reticulum in close apposition to the plasma membrane. Hanaoka et al. (4) have found that co-expression and co-assembly of the two polycystins induce a novel cell-surface cation channel activity. Gonzalez-Perrett et al. (37) have reported the first direct evidence that polycystin-2 is indeed a cation channel. Recently, Koulen et al. (38) have shown that polycystin-2 behaves as a calcium-activated, high-conductance endoplasmic reticulum channel that is permeable to divalent cations in vivo. They demonstrated that C-terminal truncation or the introduction of a disease-causing missence mutation led to the loss of intracellular calcium-release signals. Thus they propose that ADPKD results from the loss of a regulated intracellular calcium-release signaling mechanism (38). These studies show that activation of the complex of polycystins results in increased local [Ca2+]. Whether PIO compensates for the signaling pathway of polycystin-1 as being mediated by Ca2+ is an intriguing question, and warrants further studies.
Expression of polycystin-1 in vascular smooth muscle cells and in endothelium has been previously reported (39,40). Hypertension and vascular disease develop in patients with ADPKD well before renal function starts to decline. A previous study (24) showed that endothelial dysfunction was present in small-resistance vessels obtained from normotensive ADPKD patients and that eNOS impairment in ADPKD endothelium may be responsible. In this study, adult heterozygous mice revealed the subtle endothelial dysfunction and the defective production of NOx. Moreover, PIO improved endothelial function in older adult heterozygotes and increased NOx production. Our data suggest that adult heterozygous mice might recapitulate the endothelial dysfunction seen in human subjects. The effect of PIO in adult heterozygous Pkd1 mutants may indicate that thiazolidinediones improve the endothelial dysfunction in ADPKD patients.
In summary, Pkd1-/- embryos had specific molecular defects, including decreased levels of total ß-catenin in the developing hearts and kidneys, decreased levels of c-MYC in the developing hearts, abnormal metabolism of E-cadherin and PECAM-1 in maturing renal tubules, and enhanced tyrosine phosphorylation of EGFR and Gab1 in the developing kidneys. Maternally administered PIO at 7.5–9.5 p.c. corrected these molecular abnormalities and ameliorated the resulting phenotypes. Long-term treatment with pioglitazone improved the endothelial function in adult Pkd1+/- mice. We propose that these molecular defects contribute to the phenotype of ADPKD. The effects of thiazolidinediones on the molecular pathogenesis of ADPKD warrant further studies.
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METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Generation of Pkd1-/- (del2–6) mice
We screened a 129/sv mouse genomic library (Stratagene, La Jolla, CA, USA) with the use of mouse Pkd1 cDNA fragments (41, Gene bank accession # NM_013630). To generate the targeting construct, we subcloned a 10.2 kb HincII–KpnI fragment containing exons 2–6 into pBluescript (Stratagene). A 1.9 kb BglII–BglII fragment was replaced with a 1.6 kb BglII–BglII fragment containing a neo gene derived from pMC1neo–polyA (Stratagene), and a 1.0 kb fragment containing the DT-A gene for negative selection (a gift from Dr S Aizawa; 42) was inserted at the 5' end of a 6.5 kb HincII–BglII fragment (Fig. 1A). BglII cuts base position 1547 in exon 6 of Pkd1 mRNA, and the targeted Pkd1 alleles resulted in a frameshift. ES cells (E14, 129/sv background, provided by A. Smith) were transfected with the targeting vector as described previously (43). Homologous recombinants were identified by southern blot analysis using a 5' probe and a 3' probe as illustrated (Fig. 1A). Two independently targeted ES clones were injected into C57BL/6J blastocysts to generate chimeric mice, and the mutation was transmitted into the germline, resulting in a 129/sv/C57BL/6J background.
Histology and immunofluorescence
For morphological evaluation, mouse tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin. Sections (3–5 µm thick) were stained with hematoxylin and eosin (H&E) according to standard protocols. Morphological analysis was performed with a BX51 Olympus microscope (Olympus, Tokyo, Japan). For immunohistochemistry, after deparaffinization through graded xylene and ethanol series, sections were washed in PBS (pH 7.4), and treated with 0.3% hydrogen peroxide in PBS for 30 min. After incubation for 30 min with 10% normal goat serum to block non-specific binding of the antibodies, the sections were incubated with goat polyclonal anti-phospho-EGFR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After overnight incubation with the primary antibody at 4°C, the sections were reacted with biotinylated secondary antibody for 30 min. Subsequently, the sections were allowed to react for 30 min with avidin–biotin–peroxidase complex (ABC) by using a Vectastain ABS kit (Vector Laboratories, Burlingame, CA, USA) and subjected to the peroxidase reaction with 0.02% 3,3'-diaminobenzidine tetrahydrochloride as a chromogen in PBS constaining 0.007% hydrogen peroxide. For immunofluorescence, frozen sections were subjected to permeabilization with 0.2% Triton X-100 in PBS, blocked with 1% bovine serum albumin (BSA), and stained with anti-PECAM-1 (BD PharMingen, San Diego, CA, USA) or anti-E-cadherin (Takara Shuzo, Otsu, Japan) antibodies. Sections were washed with Tris-buffered saline (TBS)–Triton X-100 (0.01%) and subjected to secondary, CyTM3-conjugated goat anti-rat IgG antibodies (Jackson ImmunoResearch, West Grove, PA, USA), and mounted for confocal laser scanning microscopy (MRC-1024, Bio-Rad, Hercules, CA, USA).
Immunoblotting
Embryonic hearts and kidneys were sonicated in RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 50 mM NaF, 1 mM sodium orthovanadate, 20 µg/ml, aprotinin, and 20 µg/ml leupeptin). The protein concentration was determined by bicinchinonic acid assay (Pierce, Rockfold, IL, USA). Proteins were separated by SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Gels were stained with Coomasie blue to check that comparable amounts of proteins were loaded on the gel and also to check the homogeneity of the transfer. Immunodetection was performed after the membranes were blocked in blocking solutions (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton-X, and 3% BSA) and blotted with the following antibodies: anti-c-myc (NeoMarkers, Fremont, CA, USA), anti-ß-catenin (BD Transduction Laboratories, Lexington, KY, USA), anti-E-cadherin, anti-PECAM-1, anti-Gab1 (Upstate Biotechnology, Lake Placid, NY, USA), anti-ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse anti-polycystin-1 (7e12) (44). The filters were washed with TBS/0.1% Triton-X, and immunoreactive bands were visualized with an enhanced chemiluminescence (ECL) blotting system (Amersham Pharmacia Biotech, Uppsala, Sweden). Blots were quantified by densitometric analysis with the NIH Image 1.61/ppc program.
Immunoprecipitation and detection of tyrosine phosphorylation
Pre-cleared total lysates (50–100 µg) from the indicated organs were incubated with 2 µg of anti-Gab1 antibody with protein A–Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) at 4°C overnight. Immunocomplexes bound to Sepharose beads were washed with RIPA buffer, resuspended in Laemli sample buffer, and boiled before loading. Proteins were resolved by 7.5% SDS–PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking, immunodetection was performed with an anti-phosphotyrosine antibody (anti-PY) linked to horseradish peroxidase (RC20; BD Transduction Laboratories, Lexington, KY, USA). Signals were detected by ECL blotting.
Analysis of aortic relaxation in response to acetylcholine
Aortic ring (2 mm long) segments were mounted between two stainless steel wires and placed in an organ bath containing Krebs' bicarbonate solution bubbled with a mixture of 95% O2 and 5% CO2. One wire was connected to a force-displacement transducer (UR-50G, Minebea Co., Ltd, Nagano, Japan) (45). The preparation was allowed to equilibrate for 90 min and was then preconstricted by phenylephrine (10-7 M). To obtain a dose–response curve for acetylcholine (10-8 to 10-5 M) and sodium nitroprusside (10-10 to 10-7 M), agents were added cumulatively to the organ bath. Data are expressed as percentage relaxation of phenylephrine-induced preconstriction.
NOx (NO2 and NO3) assay
Concentrations of NO2 and NO3 in urine were measured by an autoanalyzer (TCI-NOX 1000, FIA Instruments Co., Ltd, Tokyo, Japan). Deproteinized urine samples were premixed with carrier solution (0.007% EDTA and 0.03% NH4Cl). Samples were passed through a cadmium reducer and reacted with Griess reagent (1% sulfonamide and 0.1% N-1-naphthylethylene-diamine dihydrochloride in 5% HCl). Absorbance was detected at 540 nm using a flow-through visible spectrophotometer (S/3250, Soma-Kogaku, Tokyo, Japan) (45).
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ACKNOWLEDGEMENTS |
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We thank K. Katsuki and T. Etoh for their excellent technical assistance, Y. Chida and M. Emoto for cell culture work and H. Yoshikura, K. Kurokawa, Y. Asano, T. Fujita, T. Igarashi, S. Sasaki, K. Miyazono, T. Suda, T. Koike and R. Nishinakamura for critical comments on this study. This work was supported in part by grants from the Ministry of Health, Labor and Welfare, and the Ministry of Education, Culture, Sports Science and Technology of Japan.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Urology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo, Japan 181-8611. Fax: +81 422428431; Email: shorie-kkr{at}umin.ac.jp
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REFERENCES |
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