Defect in Maintenance of Pulmonary Integrity during Postnatal Life |
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Abstract |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Homozygous mutant klotho (KL
/) mice exhibit multiple phenotypes resembling human aging. In the
present study, we focused on examining the pathology of the lungs of klotho mice and found that it closely
resembled pulmonary emphysema in humans both histologically and functionally. Histology of the lung of
KL/ mice was indistinguishable from those of wild-type littermates up to 2 wk of age. The first histologic changes appeared at 4 wk of age, showing enlargement of the air spaces accompanied by destruction
of the alveolar walls, and progressed gradually with age. In addition to these changes, we observed calcium deposits in type I collagen fibers in alveolar septa and degeneration of type II pneumocytes in 8- to
10-wk-old KL/ mice. Pulmonary function tests revealed prolonged expiration time in KL/ mice,
which is comparable with the pathophysiology of pulmonary emphysema. The expression level of messenger RNA for type IV collagen, surfactant protein-A and mitochondrial 
-adenosine triphosphatase was
significantly increased in KL/ mice, which may represent a compensatory response to alveolar destruction. Additionally, the heterozygous mutant klotho mice also developed pulmonary emphysema late in life,
around 120 wk of age. These findings indicate that klotho gene expression is essential to maintaining pulmonary integrity during postnatal life. The klotho mutant mouse is a useful laboratory animal model for
examining the relationship between aging and pulmonary emphysema.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pulmonary emphysema is defined anatomically as destruction of lung parenchyma distal to terminal bronchioles without fibrosis (1). Although cigarette smoking has been proved
as the most significant risk factor for pulmonary emphysema (2), only 10 to 20% of chronic smokers develop this
disease (3). This fact indicates that development of pulmonary emphysema is affected by factors other than smoking,
including genetic factors. In previous studies, several genes
have been shown as candidates that determine susceptibility to pulmonary emphysema: 
-1 antitrypsin (1-AT) (4),
gelatinase B (5), interstitial collagenase (5), macrophage
elastase (6), and microsomal epoxide hydrolase (7). However, precise mechanisms for determining the susceptibility to develop pulmonary emphysema have yet to be clarified.
We have recently developed a novel presenile mice
strain by insertional mutagenesis, named klotho (8). The
homozygous mutant klotho (KL/) mice, which have a
defect in klotho gene expression, show a short life span
and exhibit pulmonary emphysema, arteriosclerosis, osteoporosis, skin atrophy, and ectopic calcifications (8). The klotho gene encodes a novel single-pass membrane
protein that has sequence similarity with -glucosidase enzymes (8). Our current working hypothesis is that the
klotho gene product (KL protein) functions through a circulating humoral factor(s). This model is based on the following observations: (1) Despite the fact that KL/ mice
show systemic aging phenotypes, only limited organs express the klotho gene endogenously (8). (2) A splice variant encoding a putative secreted form of KL protein has
been detected. In addition, the secreted form is the major
klotho gene product in humans (9). (3) Arteries of heterozygous mutant klotho (KL+/) mice show decreased vasodilatation in response to acetylcholine due to impaired nitric
oxide (NO) production in endothelial cells. Parabiosis between wild-type (WT) KL+/+ mice and KL+/ mice improves
NO production in endothelial cells of KL+/ mice (10).
In this study, we characterized the lung pathology of
klotho mice and found that it closely resembles pulmonary
emphysema in humans. We also disclose that gene expression of type IV collagen and surfactant protein (SP)-A was
increased in the lung of KL/ mice compared with that of
WT mice. These results demonstrate that the klotho gene
is essential to maintaining pulmonary integrity during postnatal life.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals
All animals were generated from mating pairs of KL+/
mice. Newborns were weaned at 3 to 4 wk of age. Their
genotypes were determined by Southern blot analysis and
polymerase chain reaction (8).
Tissue Preparation and Histologic Analysis
Mice were anesthetized with urethane (1.5 g/kg, intraperitoneally) and killed by severing the abdominal aorta. Their lungs were fixed by intratracheal instillation of 4% paraformaldehyde at a constant pressure of 20 cm H2O for at least 24 h. Lung volume was measured by volume displacement.
For histologic analysis, the paraffin-embedded tissues were sectioned (4 µm in thickness) and stained with hematoxylin and eosin (H&E) for light microscopy. Serial sections of the lung were also inspected by Kossa staining to detect calcification. Destructive index (D.I.) was calculated to estimate the degree of alveolar destruction (11). Mean linear intercept, the average distance between the opposing walls of a single alveolus, was also measured by the technique described by Dunnill (12).
For ultrastructural examination, the lung specimens were fixed at 4°C in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 2 h and postfixed at room temperature in 1% osmium tetroxide in 0.1 M cacodylate buffer. After fixation, the specimens were rinsed in buffer, stained with 1% uranylacetate in 50% ethanol overnight, dehydrated in graded series of ethanol, and finally embedded in agar 100 epoxy resin. The ultrathin sections (60 nm) were contrasted with lead citrate and examined.
Pulmonary Function Analysis
Physiologic parameters were measured in urethane (1.5 g/ kg)-anesthetized, spontaneously breathing mice according to the method for rats (13) with slight modification. In brief, a short polyethylene tube was inserted into the trachea and another was inserted into the lower third of the esophagus. Respiratory flow signal was measured through a Lilley-type pneumotachograph (TV-241T and TP-602T; Nihon Kohden, Tokyo, Japan) connected to the intratracheal tube. Lung volume was obtained by electric integration of the flow signal (14). Intraesophageal pressure was used as intrathoracic pressure. Body temperature was maintained at 37°C throughout the experiment. These data were fed into a computer through an A/D converter (MacLab 16/s; AD Instruments, Castle Hill, Australia). When the breathing became stable for more than 10 min, respiratory frequency, tidal volume, minute ventilation, expiration time, dynamic compliance, and total pulmonary resistance were measured (15). Arterial blood gas content was also analyzed.
Northern Blot Analysis
Total RNA was extracted from lungs of KL/ mice at 7 to
9 wk, from the WT mice at 7 to 9 and 120 wk, and from the
KL+/ mice at 120 wk of age. A total of 20 µg of RNA per
lane was separated on a formaldehyde-1% agarose gel,
transferred to a nylon membrane (Hybond-N; Amersham,
Arlington Heights, IL), and fixed to it by ultraviolet exposure. The 18S and 28S ribosomal RNA (rRNA) bands were stained with methylene blue in order to assess the
amount, quality, and size of the RNA. The membranes,
which were prehybridized in a solution of 50% formamide,
5× saline sodium phosphate ethylenediaminetetraacetic acid, 10× Denhardt's solution, 1% sodium dodecyl sulfate
(SDS), and 0.1 mg/ml herring sperm DNA, were hybridized with DNA probes labeled with [32P]deoxycytidine triphosphate (dCTP) (Amersham) at 42°C for 20 h using the
random primer labeling technique. The membranes were then washed twice in 2× saline sodium citrate (SSC) (0.15 M
NaCl and 0.015 M sodium citrate, pH 7.0) with 0.1% SDS
at room temperature for 10 min and twice in 0.1× SSC
with 0.1% SDS at 42°C for 10 min before autoradiography.
The membranes were exposed to Kodak XR films for 48 h
at 
80°C.
Differential Screening of Lung Messenger RNA
Poly (A) RNA was prepared from lung of both KL/
mice and WT mice using Oligotex-dT30 (Takara). A total
of 1 µg of poly (A) RNA from each sample was used as a
template for synthesizing radiolabeled probes in the
reaction mixture containing 5 mM MgCl2; 2.5 µM oligo (dT)12-18 primers; 0.25 U/µl avian myeloblastosis virus reverse transcriptase; 30 µCi [32P]dCTP; 1 mM each of deoxyadenosine triphosphate, deoxyguanosine triphosphate, and
deoxythymidine triphosphate; and 0.1 mM of dCTP. Reverse transcription was done at 42°C for 30 min. Rat lung
complementary DNA (cDNA) library constructed on 
gt10
was plated onto the plates at the density of 5 × 103 plaque-forming units per 150-mm plate and phage plaques were transferred to nylon membranes in duplicates as described
by Benton and Davis (16). The membranes were hybridized with the radioactive total cDNA probe at a specific
activity of 1 × 108 cpm/µg according to the standard procedures. The membranes were washed by final stringency
at 0.1× SSC at 42°C, then exposed to Kodak XR films at
80°C for 48 h. Plaques of different signal intensities between the two probes on autoradiograms were isolated and subjected to phage DNA preparation as described (17).
Data Analysis
Each parameter, either measured or calculated, is expressed as mean ± standard deviation. Differences between groups were assessed by analysis of variance (ANOVA).
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Lung Volume
The mean body lengths of KL/, KL+/, and WT mice
were 68.3 ± 4.7 mm, 91.8 ± 2.4 mm, and 91.5 ± 1.0 mm,
respectively, at 6 to 8 wk of age. This finding represents
the short stature of KL/ mice (P < 0.05). Inasmuch as
the lung volume of KL/ mice was not significantly different from that of KL+/ and WT mice (703 ± 306 µl versus
653 ± 180 µl, and 663 ± 122 µl, respectively), KL/
mice have a relatively large lung volume for their body
size. Therefore, the lungs of KL/ mice seem to be hyperinflated.
Light Microscopic Examination
Lungs of KL/ mice. At 2 wk of age, the lungs of KL/
mice (Figure 1A) were indistinguishable from those of WT
littermates (Figure 1B). Histologic examination of KL/
mice at 4 wk of age (Figure 1C) revealed obvious enlargement of alveolar ducts. Although alveolar structures were
preserved in some areas, the number of alveoli that were
contiguous with alveolar ducts was reduced. Neither inflammatory cell infiltration nor interstitial fibrosis was detected. These findings were comparable with those observed in patients with pulmonary emphysema. The lung of
WT littermates is presented in Figure 1D.
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At 10 wk of age, the emphysematous changes became
more prominent in the lung of KL/ mice (Figure 2A).
Normal alveolar structures were rarely found. At higher
magnification small homogeneous nodules were seen, attached to the alveolar septa (Figure 2B). These nodules
proved to be calcification because these structures were
stained black with von Kossa staining (Figure 2C).
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Lungs of KL+/ mice.
The lungs of the KL+/ mice
were comparable with those of WT mice at 2 to 10 wk of
age (Figures 3A and 3B). At 120 wk of age, when KL+/
mice exhibited kyphosis and hair loss (Figure 4), their
lungs showed emphysematous changes and alveolar calcification (Figures 3C and 3D). These findings were almost
identical to those in KL/ mice.
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Morphometry.
To assess emphysematous changes more
quantitatively, we did histomorphometric analysis. The D.I.,
which indicates the severity of destructive changes of alveolar walls, was not different among KL/, KL+/, and WT
mice at 2 wk of age (Figure 5). The D.I. of KL/ mice, however, increased rapidly thereafter. The D.I. of KL+/ mice
was not different from that of WT mice at 2, 4, or 10 wk of
age, but was raised at 120 wk of age. The mean linear intercept, which represents the size of alveolar air space, was approximately 1.8 times longer in KL/ mice than in WT
mice at 6 to 8 wk of age (104.8 ± 28.1 µm versus 58.3 ± 24.2 µm, P < 0.05). These results indicate that the lung of KL/
mice older than 4 wk of age and old KL+/ mice had both
destruction of alveolar walls and enlargement of air spaces.
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KL
KL+/Electron Microscopic Examination
Electron microscopic examination of the lung of KL/
mice revealed small deposits of high-density material on
type I collagen fibers in alveolar septa at 4 wk of age,
which increased both in number and in size at 8 wk of age
(Figures 6A and 6B). These deposits were most likely to
be composed of calcium because of their lamellar structure.
In contrast, the type IV collagen, a constituent of basement membrane, appeared to be normal. Degeneration of
some of the type II pneumocytes was also observed in the
lungs of KL/ mice (Figure 6C). These degenerated pneumocytes were negative for terminal deoxyribonucleotidyl
transferase-mediated deoxyuridine triphosphate-biotin nick-
end labeling staining, suggesting that they did not undergo
apoptosis (data not shown). The lungs of KL+/ mice
showed no remarkable ultrastructural changes at 8 wk of age (data not shown).
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Pulmonary Function Tests
Respiratory parameters of the mice at 6 to 8 wk of age are
summarized in Table 1. The KL/ mice had longer expiration time (Te) (P < 0.01) and higher dynamic compliance at tidal breathing (Ctb) (P = 0.09) than did WT mice. These data were comparable with those observed in patients with pulmonary emphysema. Although tidal volume
of the KL/ mice was smaller than that of WT mice (P < 0.01), minute ventilation per body weight (Ve) was comparable between the two. The arterial blood of KL/ mice
breathing room air showed normal oxygen and carbon dioxide partial pressure (data not shown).
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Northern Blot Analysis
To obtain a clue to understanding molecular pathogenesis
of the emphysematous changes, we examined the expression of several genes known to be associated with the
pathophysiology of pulmonary emphysema. In the lung of
most KL/ mice, expression of type IV collagen and SP-A
messenger RNA (mRNA) was markedly increased at 7 to
9 wk of age when compared with that of WT mice (Figure
7A). The mRNA level of transcription factor specificity
protein 1 (Sp1) was significantly decreased. Expression of
the genes for manganese superoxide dismutase, glutathione peroxidase, catalase, transforming growth factor
(TGF)-1, Egr-1, and -actin were comparable between
KL/ and WT mice (data not shown). In KL+/ mice type
IV collagen mRNA levels were increased at 120 wk of age,
while SP-A mRNA levels were not changed (Figure 7B).
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Differential Screening
We performed differential screening using the lung mRNAs
to identify differentially expressed genes between KL/
and WT mice. Most mRNAs (> 98% of total population
expressed in the lung) were expressed at comparable
levels between KL/ and WT mice; however, levels of
some particular genes were selectively increased or decreased in KL/ mice. Among the mRNAs whose expression levels were upregulated in KL/ mice was the mitochondrial -adenosine triphosphatase (ATPase) mRNA,
which is encoded by nuclear DNA (Figure 7A). Northern
blot analysis of the lungs of 13 KL/ mice revealed that
most individuals (11 of 13, 85%) expressed higher levels of
mitochondrial -ATPase mRNA (Figure 7A). The other
two (15%), on the other hand, expressed lower levels of mitochondrial -ATPase mRNA than did the WT mice. In
these mice expression of a wide range of mRNA was downregulated in general, including mRNAs for TGF-1, type
IV collagen, and SP-A (Figure 7C).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The lungs of KL/ mice develop normally up to 2 wk of
age; by that time the alveolar structure should have almost
fully developed and become mature in mice (18). The emphysematous changes first appear at 4 wk of age in KL/
mice and progress with age thereafter until they die, at
around 8 to 10 wk of age. Therefore, the emphysematous
changes observed in KL/ mice are not caused by a developmental defect or hypoplasia of the lung. Rather, they
are a result of progressive destruction of normal alveolar
architecture after normal lung development. In addition,
KL+/ mice surviving for longer than 120 wk also showed
pulmonary emphysema, suggesting a gene-dose effect of
the klotho gene on the lung lesions. These observations indicate that the klotho gene expression is essential to maintaining normal alveolar architecture in adulthood.
Respiratory function of KL/ mice is also compatible
with that of patients with pulmonary emphysema. KL/
mice have longer expiration times than do WT mice. They
also show higher Ctb, which correlates with severity of
pulmonary emphysema, in animal models (19, 20). Despite
the impaired respiratory function, partial pressure levels
of oxygen and carbon dioxide in arterial blood of KL/
mice are normal, indicating that KL/ mice are not suffering from respiratory failure at rest.
The ultrastructural analysis of the lung of KL/ mice
detected calcium deposits in the type I collagen fibers in
alveolar septa and degeneration of the type II pneumocytes. Calcium deposits were not observed in the type
IV collagen fibers, which constitute basement membrane
and regulate pneumocyte differentiation (21). It is likely
that degeneration of type II pneumocytes may attenuate regeneration of alveolar cells in the KL/ mice, because
type II pneumocytes are known to play a critical role in restoring the damaged alveoli (21).
Northern blot analysis disclosed that the expression of
the type IV collagen and the SP-A was markedly upregulated in KL/ mice at 7 to 9 wk of age, when pulmonary
emphysema was fully developed. These proteins are
thought to exert favorable effects on preventing emphysematous changes because type IV collagen fiber is one of
the important components of extracellular matrix and because SP-A has been reported to have a protective function against development of elastase-induced pulmonary
emphysema (22). Hence, the increase in type IV collagen
and SP-A expression may be a compensatory response to
the destructive changes of the lung. Recently, we have
found that pulmonary emphysema induced by cigarette smoking resulted in upregulation of the type IV collagen
gene in KL+/ mice (unpublished data). This finding may
support our hypothesis that an increase in expression of
the type IV collagen gene is a compensatory genetic response to lung injury. Another interesting finding to be
noted here is that the Sp1 mRNA level was rather decreased in KL/ mice, despite the fact that the promoter
region of both type IV collagen and SP-A gene contains
multiple Sp1 sites that positively regulate their expression
(23, 24). These observations suggest that the upregulation
of type IV collagen and SP-A gene expression observed in
KL/ mice is independent of the transcriptional regulation with Sp1.
Mitochondrial -ATPase gene was identified as one of
the selectively upregulated genes in the lung of KL/ mice
through the differential screening. Mitochondrial -ATPase, a subunit of adenosine triphosphate (ATP) synthase, is an
essential enzyme for ATP synthesis. Northern blot analysis of 13 KL/ mice disclosed that expression of mitochondrial -ATPase was increased in most individuals.
However, some KL/ mice exhibited marked downregulation of this gene. Because mitochondrial -ATPase plays
an essential role in ATP synthesis, it is likely that a decreased expression of this gene eventually leads to cell death. In support of this notion, these mice showed downregulation of all mRNA levels tested, including TGF-1,
type IV collagen, and SP-A. Thus, a decrease in mitochondrial -ATPase mRNA level may represent extensive cell
damage and failure of compensatory response to destruction of lung parenchyma. Despite indistinguishable histopathologic findings, a decreased gene expression of mitochondrial -ATPase may represent a more advanced stage
of pulmonary emphysema at the molecular level.
Several animal strains have been reported to develop
pulmonary emphysema so far. However, klotho mice are
different from the previously reported mice models in various ways. The blotchy mice show progressive panlobular
emphysema due to affected lysyl oxidase activity with defect of normal collagen crosslinking (25). The tight-skin mice, which have systemic connective tissue disorder, demonstrate increased numbers of neutrophils and macrophages in the lower respiratory tract before development of pulmonary emphysema (28, 29). The pallid mice
have markedly low levels of serum 1-AT associated with
a severe deficiency in serum antielastase capacity (30), and
show disruption of alveolar septa with air space enlargement beginning at 12 mo of age. In platelet-derived growth
factor A chain null mice, alveolar septation is disturbed due to the lack of alveolar myofibroblasts (31). Although
not only klotho mutant mice but also these animal models
imitate pulmonary emphysema in humans, the klotho mutant mouse is unique because it accompanies various aging
phenotypes simultaneously.
Smoking and aging are the two major risk factors for
pulmonary emphysema. Studies on the synergistic effects
of smoking and the klotho gene mutation on the emphysematous changes in KL+/ mice are in progress in our laboratory. Analysis of pathophysiology of pulmonary emphysema in klotho mice will provide a unique insight into the
relationship between pulmonary emphysema, smoking,
and aging.
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Footnotes |
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Address correspondence to: Ryozo Nagai, M.D., Ph.D., Second Dept. of Internal Medicine, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan. E-mail: nagai{at}news.sb.gunma-u.ac.jp
(Received in original form September 14, 1998 and in revised form July 6, 1999).
Abbreviations: adenosine triphosphate, ATP; adenosine triphosphatase, ATPase; complementary DNA, cDNA; dynamic compliance at tidal breathing, Ctb; destructive index, D.I.; deoxycytidine triphosphate, dCTP; hematoxylin and eosin, H&E; heterozygous mutant klotho, KL+/; homozygous
mutant klotho, KL/; messenger RNA, mRNA; sodium dodecyl sulfate,
SDS; surfactant protein, SP; specificity protein 1, Sp1; saline sodium citrate, SSC; transforming growth factor, TGF; wild-type, WT.
Acknowledgments: The authors thank Dr. S. Asano (Teijin Institute for Biomedical Research, Tokyo, Japan) for electron microscopy; Drs. T. Akino and Y. Kuroki (Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan) for providing SP-A cDNA; and Ms. Y. Nonaka, M. Yamazaki, and K. Ishihara for excellent technical assistance. This study was supported in part by research grants from the Japanese Ministry of Education, Science and Culture; by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan; and by the Smoking Research Foundation.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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