Transgenic mice overexpressing glia maturation
factor-, an oxidative stress inducible gene, showpremature

aging

due

to

Zmpste24

down-regulation

Graduate School of Life Science, Kobe Women's
University
Rika Imai2015

Contents
page・Summaryi・Chapter 1. Introduction
1.1 Glia maturation factor-1

1.2 Glia maturation factor- and Chronic kidney disease2

1.3 Glia maturation factor- and Neurodegenerative disease3

1.4 Focus of this thesis

4・Chapter 2. Transgenic mice overexpressing GMF, an oxidative stressinducible

gene,

show

premature

aging

due

to

Zmpste24

down-regulation
2.1 Introduction

6

2.2 Materials and Methods

8

2.3 Results

14

2.4 Discussion

19

・Chapter 3. Concluding remarks

25

・Figures and Tables

26

・References

46

・List of publications

54

・Acknowledgments

55

Summary

This thesis begins with a brief introduction to glia maturation factor- (GMF), a
brain-specific protein, followed by a discussion of the physiological functions of GMF.
Next, the relation between GMF and chronic kidney disease (CKD) is introduced,
which will be referenced throughout this work. Briefly, GMF is ectopically induced inrenal tubules by CKD associated with proteinuria. GMF overexpression in non-brain
cells causes apoptosis in vitro via cellular vulnerability to oxidative stress. After that, thepathological roles of GMF in the neurodegenerative diseases are introduced. In thepresent study, we constructed transgenic mice overexpressing GMF (GMF-TG) andinvestigated the roles of GMF in non-brain tissues in vivo.

Results of the research conducted during this study are presented in Chapter 2. We
evaluated the GMF-TG mice for 155 weeks and compared with the wild-type mice. The
GMF-TG mice prematurely exhibited aging-associated symptoms including a lack ofhair glossiness, hair graying, alopecia, skin atrophy, and curvature of the spine as seen
in normal human aging. The GMF-TG mice also demonstrated short lifespans andreduced hair regrowth, suggesting an accelerated aging process.
The analysis of premature aging syndromes develops understanding of the molecularbasis of the physiological aging. The production of an abnormal lamin A, a nuclear
envelope protein, has been identified as the cause of accelerated aging diseases, known
as laminopathies, and might play a causal role in normal aging. Some phenotypes seenin the GMF-TG mice, such as alopecia and skin atrophy were similar to those ofilaminopathy-based premature aging. Here, we examined whether this accelerated aging
characteristics observed in the GMF-TG mice would be associated with laminopathies.
We identified the abnormal lamin A (prelamin A), accompanied by a down-regulation ofa lamin A processing enzyme (Zmpste24) in the kidney of the GMF-TG mice. The
GMF-TG mice showed accelerated aging in the kidney, compared with the wild-type
mice, showing the increased the transforming growth factor-1 (TGF-1) andconnective tissue growing factor (CTGF) gene expression and the decline of renal
function (e.g. increased serum creatinine). The mRNA expression of p21/waf1 wasincreased at an earlier stage of life, at 10 weeks, which was in turn down-regulated at alater stage, at 60 weeks. In this chapter, we demonstrated the GMF-TG mice showedvery mild accelerated aging phenotypes due to the abnormal lamin A, accompanied by
Zmpste24 down-regulation.

In chapter 3, we proposed a novel role of GMF overexpression in non-brain tissues
in vivo. This proposition suggests that the gene expression of Zmpste24 in the GMF-TG
mice might respond sensitively to oxidative stress, which may be associated with avulnerability to oxidative stress caused by GMF overexpression. The usefulness of the
GMF-TG mice will be presented.

iiChapter 1.

Introduction

1.1 Glia maturation factor-Glia maturation factor- (GMF) was first detected in 1972 by Lim et al. as aconsequence of search for growth or differentiation factors in the nervous system [1-3].
GMF is a 17-kDa highly conserved 141-amino acid protein, with 99% homology
between humans and rodents [4]. It is localized in the cytoplasm of glial cells, mainly
astrocytes, and some neurons [4-6]. GMF has no leader sequence and is not secreted by
the cells [5]. GMF mRNA is predominantly expressed in the brain and spinal cord,
although trace levels are found in other organs, including testis and ovary (Figure 1) [6].
It has been reported by Lim et al. that recombinant GMF can be phosphorylated invitro at the serine residue by protein kinase C, protein kinase A, and casein kinase II,
and at the threonine residue by p90 ribosomal S6 kinase, indicating that GMF possesses
several consensus phosphorylation sites [5,7]. Protein kinase A-phosphorylated GMF
inhibits the activity of mitogen-activated protein kinases (MAPK), ERK1 and ERK2
(extracellular signal-regulated kinase 1 and 2, respectively) [8], while it promotes theactivity of p38-MAPK [9]. MAPK is an important signaling pathway regulating avariety of physiological processes such as cell growth, proliferation, differentiation,
migration, and apoptosis [10]. The three major MAPK cascades; ERK cascade, c-Junamino-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) cascade and1p38-MAPK cascade are well-known [10]. Lim et al. [5] and Zaheer et al. [11] havedemonstrated that overexpression of GMF in brain cells stimulates p38-MAPK activity
and activates the transcription factor nuclear factor- (NF-B). Activation of NF-
plays a central role in the regulation of diverse cellular processes such as inflammation,
immune response, differentiation, proliferation and apoptosis [12]. GMF has been
currently described as an intracellular regulator of cell signal transduction.
GMF has also been identified as a member of the actin-depolymerizing factor
homology (ADF-H) family [13] which regulates actin filament dynamics at multiple
cellular locations [14]. Actin filament dynamics play important roles in fundamental
cellular processes, including cell motility, cell division, control of cell shape andendocytosis [13,15].

1.2 Glia maturation factor- and Chronic kidney disease
Previously Takenaka et al. [16] and Nakajima et al. [17] have shown that proteinuria
induces the ectopic expression of GMF, a brain specific protein, in renal proximal
tubular cells. Kaimori et al. [18] showed that GMF overexpression in non-brain cells led
to vulnerability to oxidative injury through p38-MAPK pathway and changes inantioxidant enzyme activities in vitro (Figure 2). This function was confirmed byevidence showing that GMF-null astrocytes increased resistance to oxidative stress [19].
Oxidative stress is broadly defined as an imbalance between reactive oxygen species(ROS) production and the cellular antioxidant defense system (Figure 3) [20,21]. ROS
are produced in all mammalian cells, as a consequence of normal cellular metabolism[20,22]. It is thought that oxidative stress causes damage to biomolecules such as

2

proteins, lipids and DNA, resulting in cellular damage and aging (Figure 3) [21,23,24].
Chronic kidney disease (CKD) is defined as abnormalities of kidney structure and/or
function, present for a period of three months or more (Table I) [25]. Chronic
proteinuria is not only a sign of CKD, it also plays an important role in the progressionof CKD [26]. It is known that oxidative stress is elevated in patients with CKD [27,28].
Taken together, it suggests that the induction of GMF in renal proximal tubular cells byproteinuria might play a key role in the pathogenesis of CKD by enhancing oxidative
injuries [18]. It is likely that GMF has the pathological functions as well as thephysiological functions.

1.3 Glia maturation factor- and Neurodegenerative disease
Recent studies had shown that GMF was up-regulated in the specific areas of
Alzheimer's disease brain [29-31]. It is considered that inflammatory response of glial
cells is closely intertwined with pathogenesis of neurodegenerative diseases, such as
Alzheimer's disease, Parkinson's disease and Multiple sclerosis [32-36]. It has beenreported that GMF induces granulocyte-macrophage colony-stimulating factor(GM-CSF) and interleukin-33 (IL-33) in astrocytes [4,34,35,37]. GMF also was
associated with the proinflammatory cytokine/chemokine production, including tumor
necrosis factor- (TNF-), interleukin-1 (IL-1), interleukin-6 (IL-6), and interferon
gamma-induced protein 10 (IP-10) in microglia [4,32-38]. It suggests that GMF
overexpression in brain tissue acts as a prominent mediator of inflammatory signaltransduction in the central nervous system, leading to the death of neurons in theneurodegenerative diseases (Figure 4) [4,32-38].

3

1.4 Focus of this thesis
Severalprevious

studies

have

shown

the

physiological[1-8,11]andpathophysiological [4,29-38] roles of the expression of GMF in the brain tissue.
However, as far as we know, the roles of GMF in the non-brain tissues have not been
fully clarified. Thus the aim of the present study was to investigate the roles of GMF
overexpression in non-brain tissues.

4Chapter 2.

Transgenic

mice

overexpressing

GMF,

an

oxidative stress inducible gene, show prematureaging due to Zmpste24 down-regulation

The thesis is prepared based on the quotations from our article published in Aging.
This has been already approved by the publisher.

5

2.1 Introduction
The nuclear lamina is a filamentous protein meshwork underlying the inner nuclear
membrane [39,40]. It plays important roles in maintaining the nuclear envelope andproviding anchorage sites for chromatin [40,41]. It has been recognized that the nuclear
lamina is also involved in various functions at the cellular level, including DNA
replication, transcription, and apoptosis [41-43]. The major components of the nuclear
lamina are intermediate filament proteins, the A- and B-type lamins [40,42]. The A-typelamins, such as lamin A and C, arise from the single gene (LMNA gene) by alternative
splicing of the transcript [40,41]. Lamin A is derived from its precursor prelamin A,
through a multi-step maturation process [39]. Zinc-metalloprotease, Zmpste24 is an
enzyme required for the correct processing and maturation of lamin A [39]. The B-typelamins, such as lamin B1 and B2, are encoded by separate genes (LMNB1 and LMNB2)[41].
Mutations in the gene coding the nuclear lamina components cause a wide variety of
diseases known as laminopathies [42]. For example, Hutchinson-Gilford progeria
syndrome (HGPS) [44] and restrictive dermopathy [45] are rare genetic accelerated
aging diseases caused by mutations of the LMNA or ZMPSTE24 gene. These mutations
produce mutant lamin A proteins, such as progerin or prelamin A, respectively [42],which can cause disassembly of the nuclear envelope proteins, subsequently
accompanied by accelerated aging due to laminopathies [42]. Laminopathies caused by
the alteration of the lamin A protein and their assembly can be further classified into theprimary and secondary laminopathies [42]. The primary laminopathies are due tomutations in the LMNA gene and the secondary laminopathies are caused by mutations

6

in the ZMPSTE24 gene [42]. Laminopathies exhibit clinical features mimicking
physiological aging, including sclerotic skin, joint contractures, bone abnormalities, andalopecia, but the clinical features of laminopathies are radical [42,44,45]. Interestingly,
the abnormalities associated with lamin A are normally observed in healthy humanaging, suggesting that the accumulation of abnormal lamin A protein is associated withorganismal physiological aging [46-49].
In the present study, the first step was to construct transgenic mice overexpressing
GMF (GMF-TG) in order to examine the roles of GMF in non-brain tissues in vivo.
During the breeding period, we found that the GMF-TG mice prematurely exhibited
phenotypes resembling human aging, such as alopecia and skin atrophy. Thesephenotypes appeared to be similar to those of laminopathy-based premature aging. We
investigated the development of accelerated aging phenotypes in the GMF-TG mice.

7

2.2 Materials and Methods
Ethics statement
All of the animal experiments employed in this study were conducted in accordancewith protocols approved by the Ethical Committee of Kobe Women’s University on
Animal Research (Permit Number: 154, 181, 209). All efforts possible were made tominimize animal suffering.

Production of transgenic mice overexpressing GMF
First, we prepared a construct to create transgenic mice overexpressing GMF(GMF-TG). The transgene construct was prepared by cloning the coding region of GMF(97-915 bp) to a pCAGGS vector [50]. The purified construct was used for
microinjection of fertilized oocytes from C57BL6/J mice. The transgenic mice were
established at Oriental BioService (Kyoto, Japan) and maintained on a C57BL6/J
genetic background. The GMF-TG mice were identified by PCR screening.

Animals and diets
C57BL6/J (wild-type) mice were purchased from Crea Japan Inc. (Tokyo, Japan).
The GMF-TG and wild-type male mice were used for the analyses. All of the mice
employed in the study were housed in standard cages with 3-4 mice per cage undercontrolled temperature (21 ± 2oC) and humidity (50) conditions, with a 12 h light/darkcycle. The mice were given free access to tap water and basal diets (CE-2, Crea-Japan
Inc., Tokyo, Japan) throughout the experiments.
The mice were kept until natural death and monitored twice a week for clinical signs,
morbidity or mortality during the experimental period of 155 weeks. The clinical signs

8used to assess health and welfare were characterized by obvious symptoms, like themoribund state, hypokinesia, fever, severe cachexia, loss of body weight, lack ofgrooming or nesting, not eating or drinking, alopecia, skin atrophy and spinal curvature[51,52]. Euthanasia was performed by an inhaled anesthetic overdose, followed by
sevoflurane (Sevofrane, Maruishi Pharma., Osaka, Japan) in order to minimize animal
suffering from distress or pain.
Histological analyses, PCR analyses and Western blot analyses: Tissue samples wereobtained after the euthanasia, which was performed by an overdose of the inhaledanesthetics sevoflurane.
PCR screening, hair-growth assay and creatinine levels: The experiments were
performed under anesthesia maintained by the inhalation of sevoflurane.

PCR screening
Genomic DNA was extracted from 1-2 mm sections of the tail tip. The DNA was
purified using the Wizard SV Genomic DNA Purification System (Promega, Madison,
WI), according to the manufacturer’s instructions. Briefly, mouse tail was incubatedovernight at 55oC with digestion solution master mix, and then added Wizard SV Lysis
Buffer. Lysate was transferred to the DNA binding column tube by centrifugation at
13,000 ×g for 3 min at 25oC. For elution of the DNA, the DNA binding filter column
was added a 250 μl Nuclease-Free Water. The DNA was eluted by centrifugation at
13,000 ×g for 1 min at 25oC. The PCR screening was carried out using the T3
and(5’-AATTAACCCTCACTAAAGGG-3’)(5’-GTAATACGACTCACTATAGGGC-3’) primer sequences

T7
and

TaKaRa Taq(TaKaRa, Shiga, Japan) under the following conditions: 94oC 90 s (1 cycle); 94oC 60 s,

955oC 60 s, 72oC 90 s (35 cycles) in the GeneAmp PCR System 9700 (Life technologies,
Carlsbad, CA). The PCR primers employed were obtained from Sigma-Aldrich (St.
Louis, MO).

Histological analyses
Mice were sacrificed at 30 weeks for the histological analyses. Tissue sections were
fixed in 10% neutral-buffered formalin solution, paraffin embedded, sectioned, andstained with hematoxylin-eosin (HE), periodic acid–schiff (PAS) and masson trichrome(MT), all of which were performed at Applied Medical Research (Osaka, Japan). The
sections were examined using an Olympus BX51 microscope & DP70 digital camera
system (Olympus, Tokyo, Japan).

Hair-growth assay
Employing age-matched mice, dorsal hair was removed by depilatory cream from asquare grid of skin measuring 1.5 cm×1.5 cm. Hair regrowth was scored 15 days laterfrom digital photographs and a semi-quantitative assessment was done using 6-9 hairsamples/animal. A square measuring 0.5 cm×0.5 cm was also used for the collection of
hair samples.

Creatinine levels
When the mice were 60-80 weeks old, the whole blood samples were collected fromthe fasting mice by venipuncture from the caudal vein into syringes without
anticoagulant, and the serum samples were acquired from the whole blood samples. The
serum samples were incubated for 30 min at RT and separated by centrifugation at
3,000 rpm for 15 min at 4oC after cooling with ice. Serum creatinine levels were
measured employing the CRE-EN kit (Kainos, Tokyo, Japan).

10RNA extraction and reverse transcription for PCR analyses
Total mouse tissue RNA was extracted with TRIzol reagent (Life technologies),
according to the manufacturer’s instructions, followed by DNase treatment to eliminate
contaminating genomic DNA. Briefly, mouse tissue was homogenized in 1 mL of
TRIzol reagent. The homogenized sample was added a 200 μl chloroform, and thencentrifuged at 12,000 ×g for 15 min at 4oC. The aqueous phase was transferred to a new
tube. The RNA pellet was eluted by centrifugation at 12,000 ×g for 10 min at 4oC after
adding a 500 μl 100% isopropanol to the aqueous phase. Total mouse tissue RNA wasprepared by resuspension of the RNA pellet in RNase-free water, and then eliminatedcontaminating genomic DNA using Recombinant DNase I (TaKaRa).
Single-stranded DNA was generated from the RNA with random hexamers primers
using the Transcriptor First Strand cDNA synthesis kit (Roche, Mannheim, Germany)
under the following conditions: 65oC 10 min; 25oC 10 min; 55oC 30 min; 85oC 5 min in
the Program Temp Control System PC-708 (ASTEC, Fukuoka, Japan).

Confirmation of gene expression of GMF
Quantitative PCR analyses of the GMF genes were performed with TaqMan
Universal PCR Master Mix (Life technologies) and TaqMan probe of GMF and mouse
GAPDH under the following conditions: 50oC 2 min (1 cycle); 95oC 10 min (1 cycle);
95oC 15 s, 60oC 1 min (40 cycles) in the 7500 Fast Real-Time PCR System (Lifetechnologies). The TaqMan probe for each GMF and mouse GAPDH employed was
obtained from Life technologies. The expression levels of the GMF mRNA werenormalized to those of the GAPDH mRNA.

11

Gene expression of Zmpste24, TGF-1, CTGF, and p21/waf1
Real-time PCR analyses were performed with SYBR Premix Reagent (TaKaRa)
under the following conditions: 95oC 10 s (1 cycle); 95oC 5 s, 60oC 34 s (40 cycles);
95oC 15 s, 60oC 1 min, 95oC 15 s (1 cycle) in the 7500 Fast Real-Time PCR System(Life technologies). The following primer pairs were used for real-time PCR analyses:
The GAPDH forward primer sequence 5’-AAATGGTGAAGGTCGGTGTG-3’, and its
reverse primer sequence 5’-TGAAGGGGTCGTTGATGG-3’, The Zmpste24 forward
primer sequence 5’-CCTTCAGCTTCTGGTCAGGACTCTA-3’, and its reverse primer
sequence 5’-CTGGTCCAAAGCCAGCAGAAC-3’, The TGF-1 forward primer
sequence 5’-GTGTGGAGCAACATGTGGAACTCTA-3’, and its reverse primer
sequence 5’-TTGGTTCAGCCACTGCCGTA-3’, The CTGF forward primer sequence5’-ACCCGAGTTACCAATGACAATACC-3’,

and

its

reverseprimersequence

5’-CCGCAGAACTTAGCCCTGTATG-3’, The p21/waf1 forward primer sequence
5’-CTGTCTTGCACTCTGGTGTCTCA-3’,

and

its

reverseprimersequence

5’-CCAATCTGCGCTTGGAGTGA-3’. The PCR primers employed were obtained
from TaKaRa. The expression level of each mRNA was normalized to that of thecorresponding GAPDH mRNA.

Western Blot Analyses
The total protein obtained from each mouse kidney was extracted with RIPA buffer[53], and protease inhibitor (Roche). A total of 50 μg of each sample was separated by
5-20 polyacrylamide gel and transferred to a nylon membrane (Hybond-P: GE
Healthcare, Buckinghamshire, UK). Blots were blocked with 5% ECL Blocking Agent(GE Healthcare) in TBS (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 7.4) plus 0.112

Tween 20 (Sigma-Aldrich), and incubated overnight at 4oC with 1/400 anti-Lamin A/C
polyclonal antibody rabbit (Bio Vision, Milpitas, CA), 1/1,000 p21 monoclonal
antibody mouse (#60214-1-Ig, Proteintech, Chicago, IL), or 1/4000 alpha actin
polyclonal antibody rabbit (#23660-1-AP, Proteintech). Finally, the blots were incubatedwith 1/10,000 HRP-linked anti-rabbit IgG or anti-mouse IgG (GE Healthcare).
Antibody binding was detected with the ECL Prime chemiluminescence system (GEHealthcare), with subsequent exposure to LAS-3000 chemiluminescence (GEHealthcare). The protein expression of p21/waf1 was assayed employing the western
blot method and quantified using Image J densitometry software.

Statistical Analyses
The data analyses were performed using the Kaleida Graph software package(Synergy Software, Tokyo, Japan). Values were expressed as means ± S.E. Statisticalanalyses for the comparison of two groups were performed using Unpaired Student’s ttest. For the lifespan assessments, data were analyzed employing the Kaplan-Meiermethod and log-rank test using StatMate’3 software package (ATMS Inc., Tokyo,
Japan). P values < 0.05 were considered to indicate statistical significance.

132.3 Results
Transgenic mice overexpressing GMF
It is considered that GMF is normally expressed in the brain in a tissue-specificmanner [4-6]. However, GMF is also induced ectopically in renal proximal tubules byproteinuria [16,17]. In order to analyze the previously unknown roles of GMF
overexpression in non-brain tissues, we first created the GMF-TG mice, as described inthe ‘Materials and Methods’ section (Figure 5A). We confirmed that the specificsequence, which was incorporated in a genome, was expressed in the correct directionin established transgenic lines (data not shown). Next, we conducted quantitative PCR
analyses to confirm the expression of GMF in the GMF-TG mice. In this study, we usedkidney tissue because it has been shown that GMF overexpression was ectopicallyinduced in kidney tissue by proteinuria [16,17]. The results of the quantitative PCR
analyses, employing mRNA obtained from the kidneys of the wild-type and GMF-TG
mice, showed that GMF mRNA from the GMF-TG mice increased significantly(approximately 7-fold), compared with the wild-type mice (Figure 5B).

Phenotypes of GMF-TG mice
We bred the GMF-TG mice with the wild-type mice. During the breeding period,
only GMF-TG mice began to show signs of aging in appearance, including hair graying
and lack of hair glossiness, at about the age of 30 weeks (hereafter, 30 weeks, etc.). We
investigated whether the GMF-TG mice would develop features of accelerated aging.
Prematurely aged mice exhibit early aging-like appearance phenotypes, including
increased hair loss, lordokyphosis of the spine, a shortened lifespan and growth
retardation, compared to wild-type mice [54-58]. We monitored the aging-related14phenotypes of the GMF-TG and wild-type mice during an experimental period of 155
weeks. The GMF-TG mice developed alopecia early, by about 75 weeks, while thewild-type mice started to show alopecia after about 100 weeks (Figure 6A-B and Table
II). Some GMF-TG mice also exhibited skin atrophy and spinal curvature. Thesephenotypes were not detected in the wild-type mice (Figure 6 and Table II). We also
found that some of the GMF-TG mice died within 60 weeks. The average lifespan of the
GMF-TG mice was about 119 weeks, and that of the wild-type mice was about 126
weeks. Kaplan-Meier representations of the survival curves demonstrated that the
GMF-TG mice died significantly earlier than the wild-type mice (Figure 7). There wasno statistically significant difference in the body weight and size in mature-adult mice(at about 20 weeks) (data not shown).
Next, we examined aging-related changes in the tissue structure. Degenerativechanges in skin tissue are readily visible, so they can be detected easily [57]. Decreased
hair regrowth has often been reported in prematurely aged mice [55-57]. Because hair
growth assays can be employed to monitor degenerative changes without adversely
affecting the mice, we employed them to investigate the influence of aging on skintissue. When dorsal segments of skin were shaved on age-matched mice, the GMF-TG
mice showed sparse hair regrowth after 15-days. In contrast, at the same age, thewild-type mice displayed robust hair regrowth (Figure 8A-C). The hair regrowth ratio
significantly declined in the GMF-TG mice at 10, 60 and 80 weeks, compared with thewild-type mice at the same age (Figure 8D). In the kidney, liver, and abdominal aorta at
30 weeks, there were no histologically detectable changes between the GMF-TG andwild-type mice, as indicated in Figure 9. These results suggested that the GMF-TG mice15

developed mild premature aging phenotypes.

HGPS is associated with premature alopecia, which is one of the well-known
premature-aging syndromes due to laminopathies, seen in humans [44]. Cells and tissue
from HGPS patients exhibited an accumulation of abnormal lamin A (progerin) [46,47].
The phenotypes characterized in the skin of the GMF-TG mice, such as alopecia andskin atrophy, seemed to be similar to that of laminopathy-based premature aging (Figure10). Therefore, we hypothesized that an accumulation of abnormal lamin A resulted in
the accelerated aging phenotypes shown in the GMF-TG mice. In order to analyze theabnormalities of the lamin A in the tissue of the GMF-TG mice, we examined the lamin
A protein in the kidney by western blotting. At 10 weeks of age, no lamin A
abnormalities were exhibited in the kidney of either the GMF-TG or wild-type mice
(Figure 11A). However, at 60 weeks of age, an accumulation of abnormal lamin A(prelamin A) was detected in the kidneys of the GMF-TG mice, but not in the wild-typemice (Figure 11B). Next, we evaluated the expression levels of the cleaving enzyme ofprelamin A (Zmpste24) gene in the kidneys by real-time PCR analyses to confirm themechanism of the accumulation of prelamin A. At 10 weeks of age, the expression ofZmpste24 mRNA tended to decrease in the GMF-TG mice (Figure 11C). At 60 weeks ofage, a significant decrease was demonstrated in the expression of Zmpste24 mRNA in
the GMF-TG mice, compared with the wild-type mice at the same age (Figure 11D).
These results demonstrated that the GMF-TG mice exhibited an accumulation ofprelamin A, accompanied by a reduction of Zmpste24 gene expression in the kidney
tissue. On the basis of these results, we investigated the degree of aging in the kidney

16tissue of the GMF-TG mice. In the kidney, aging-associated changes are characterized
by structural changes, including glomerulosclerosis and interstitial fibrosis [59,60], as
well as the decline of renal function [60]. It has been suggested that the transforming
growth factor-1 (TGF-1) gene is one of the factors that promote the process of renal
interstitial fibrosis associated with aging [59]. The connective tissue growing factor(CTGF) gene is known as a downstream mediator of TGF-1. In order to investigate
age-associated changes in the kidneys, we evaluated the expression levels of the
TGF-1 and CTGF genes by real-time PCR analyses. In the kidney tissue of the
GMF-TG mice at 10 weeks of age, the expression of TGF-1 mRNA increased
significantly, compared with the wild-type mice (Figure 12A). However, there was no
statistically significant difference between the expression of CTGF mRNA of the kidneyof the GMF-TG and wild-type mice at 10 weeks of age (Figure 12B). Importantly, in the
GMF-TG mice at 60 weeks of age, the expression of both TGF-1 and CTGF mRNA in
the kidney increased significantly, compared with that of the wild-type mice (Figure
12C and D). It has been reported that serum creatinine was increased in old micecompared with young mice, suggesting the decline of renal function with advancing age[60]. Figure 13 showed that serum creatinine was increased in the old GMF-TG mice
(Average age: 72.8 weeks of age) compared with the old wild-type mice (Average age:
84.8 weeks of age). These results demonstrated that the GMF-TG mice showedpremature-aging phenotypes in the kidney tissue, probably through an accumulation ofprelamin A. These findings suggested that the GMF-TG mice might show a tendency
for laminopathy-based premature aging.

17The mechanisms of laminopathy-based premature aging in the GMF-TG
mice
We attempted to demonstrate the mechanisms of laminopathy-based premature aging
in the GMF-TG mice. It has been suggested that premature aging in laminopathy model
mice is linked to p53 pathway activation [61]. The activated p53 pathway inducescell/tissue senescence and eventually leads to accelerated aging [23,24]. We examined
whether the expression of p21/waf1 gene, a p53 downstream target gene, would increasein the kidneys of the GMF-TG mice at 10 and 60 weeks of age, and found that theexpression of p21/waf1 mRNA and protein at 10 weeks increased significantly,
compared with the wild-type mice (Figure 14A and C). However, at 60 weeks, asignificant decrease in the expression of p21/waf1 mRNA was demonstrated in the
GMF-TG mice, compared with the wild-type mice at the same age (Figure 14B). There
was no statistically significant difference between protein expression of p21/waf1 in thekidney of the GMF-TG and wild-type mice at 60 weeks (Figure 14C). These results
suggested that the p53 pathway was activated only at an earlier age in the GMF-TG
mice, or that some compensative responses might be activated during a later stage inthese mice.

18

2.4 Discussion
In order to examine the roles of GMF overexpression in non-brain tissues in vivo, anovel line of mice, GMF-TG mice, was established in this study (Figure 5). We firstfound that the GMF-TG mice exhibited a premature onset of aging-associatedsymptoms seen in physiological human aging, including a lack of hair glossiness, hairgraying, alopecia, skin atrophy, and curvature of the spine (Figure 6 and Table II). We
hypothesized that GMF overexpression in non-brain tissues in vivo might play role inaging. Our findings demonstrated that the GMF-TG mice had short lifespans (Figure 7)
and showed premature degenerative changes in skin tissue (e.g., reduced hair regrowth)(Figure 8). However there were no visible age-related histological changes in the kidney,
liver, and abdominal aorta (Figure 9).
Mutations or altered post-translational lamin A processing lead to an accumulation oflamin A abnormalities, such as progerin or prelamin A [42]. Because these nuclear
intermediate filaments, such as lamin A/C, function as a mesh to protect the nucleus
from mechanical stress, the alteration of lamin A protein causes a loss of nuclear
stability and integrity, manifesting premature-aging syndromes (e.g., HGPS) [40-43].
The GMF-TG mice were characterized by changes in appearance, such as alopecia andskin atrophy from an earlier stage of life (Figure 6 and Table II). In appearance, these
phenotypes were similar to those seen in laminopathy-based premature aging (Figure10). In this study, we investigated whether or not the accelerated aging phenotypes thatwere observed in the GMF-TG mice were associated with laminopathies. First, wefound that the GMF-TG mice accumulated an abnormal lamin A (prelamin A) with age
in the kidney tissue (Figure 11A and B). Second, we demonstrated that the GMF-TG

19

mice showed a reduced expression of the cleaving enzyme of prelamin A (Zmpste24) inthe kidney tissue, from an earlier stage of life (Figure 11C and D), suggesting that the
GMF-TG mice would probably be affected by secondary laminopathy [42] due to an
accumulation of prelamin A, accompanied by a reduction in the expression of Zmpste24.
Finally, we confirmed the increased TGF-1 and CTGF gene expression and the declineof renal function (e.g. increased serum creatinine) (Figure 12-13). The GMF-TG mice
exhibited premature-aging phenotypes in the kidney tissue. The results of the present
study suggest that the GMF-TG mice might develop accelerated aging phenotypes due
to secondary laminopathy.

Asanimalmodelsemployedinstudiesonsecondarylaminopathies,

Zmpste24-knockout mice (Zmpste24-/- mice) are well-known [62]. Zmpste24-/- miceexclusively produce prelamin A as a consequence of Zmpste24 deficiency [62]. These
mice exhibit severe phenotypes consistent with human laminopathies (e.g. HGPS andrestrictive dermopathy), including growth retardation, jaw and bone abnormalities,
alopecia and shorter lifespan [62]. Intriguingly, the GMF-TG mice exhibited phenotypes
without the severe ones, such as growth retardation or extremely short lifespan during
the study (Figure 6-7 and Table II). Comparing to secondary laminopathies caused bygenetic mutation, the severity of the phenotypes observed in the GMF-TG mice wasvery mild. In regard to the contribution of prelamin A accumulation to the pathologicalaging phenotypes, several studies were conducted using Zmpste24-/- mice andZmpste24-/- Lmna+/- mice [61,63]. The accumulation levels of prelamin A of theZmpste24-/- Lmna+/- mice were significantly reduced, compared to that of Zmpste24-/-20

mice [61,63]. In response to this prelamin A reduction, the phenotypes observed in theZmpste24-/- mice were largely rescued in the Zmpste24-/- Lmna+/- mice [61,63].
Moreover, there were no differences shown in the body size, weight or lifespan between
the Zmpste24-/- Lmna+/- mice and the wild-type mice during the studies [61]. These
results suggested that the levels of accumulation of prelamin A might at least partiallycontribute to the phenotypes seen in the Zmpste24-/- mice, but that is not only thecritical determinant for the aging phenotype and the precise underlying pathogenic
mechanisms still need to be explored [61,63]. We detected an accumulation of prelamin
A only in the old GMF-TG mice (Figure 11A and B). The young GMF-TG mice tended
to show a reduced expression of the Zmpste24 gene (Figure 11C). It is conceivable thatthe amount of prelamin A in the GMF-TG mice might be less than that found in themouse models employed for secondary laminopathies. Here, we propose that thecharacteristic of the mild phenotypes shown in the GMF-TG mice might be because of
the slowly increased pattern of expression time course and/or the expression amount of
prelamin A.

Several studies showed that the presence of lamin A abnormalities, such as progerin
or prelamin A, was detected in normal human aging [46-49]. Furthermore, anaccumulation of prelamin A in healthy aging was associated with the down-regulationof Zmpste24 [48,49]. However, at present, the molecular mechanisms that regulate theexpression of Zmpste24 have not been fully clarified. Recent studies showed that thegene expression of Zmpste24 was reduced in response to oxidative stress [48,49]. Here,
we can speculate that the induction of GMF in non-brain tissues might cause an21

oxidative stress-related reduction of Zmpste24, presumably due to its vulnerability tooxidative stress [18]. We believe that GMF overexpression in local tissue might
contribute to the promotion of the local aging process by causing secondary
laminopathy. GMF is ectopically induced by proteinuria in renal tubules [16,17]. CKD
associated with proteinuria might accelerate the regular aging process in kidney tissue
and also enhance the progression of CKD, which consequently might have an impact on
systemic pathological changes, including organismal aging, cardiovascular damage andinflammatory changes.

Several in vivo studies have demonstrated that senescent cells accumulate with age[23]. Senescence is considered to be related to organismal aging, through the disruptionof tissue functions [23,24,64,65]. Senescence is regulated by the p53 or p16-Rb pathway,
both of which are activated in the presence of oxidative stress [23,24]. Some studies
have demonstrated that an accumulation of prelamin A induces a significant increase insenescence-associated biomarkers, such as SA--gal staining [48,61]. This suggests thatthe reduction of Zmpste24 levels and the accumulation of prelamin A might be linked tothe activation of the p53 pathway [61], leading to senescence and premature aging[23,24]. On the other hand, Kudlow et al. [66] reported that the expression of p53 target
genes was not highly up-regulated in laminopathy cases. Varela et al. [61] examined
whether or not the absence of p53 could result in a recovery of the premature-aging
phenotypes that were observed in the laminopathy model mice. Zmpste24-/- p53-/- mice
exhibit a gain in weight and an increased lifespan, compared with Zmpste24-/- mice[61]. The expression of p53 target genes, such as the p21/waf1 gene, decreased in22Zmpste24-/- p53-/- mice, compared with age-matched Zmpste24-/- mice [61]. In
Zmpste24-/- p53-/- mice, the phenotypes seen in the Zmpste24-/- mice were partially
improved by the absence of the p53 gene [61]. These findings indicate that theactivation of the p53 pathway might play a role in the progression of aging inlaminopathy cases.
In the present study, the expression of p21/waf1 mRNA and protein increased in the
GMF-TG mice at 10 weeks of age (Figure 14A and C), suggesting that premature aging
in the young GMF-TG mice might be associated with the p53 pathway. To our surprise,
the GMF-TG mice showed a reduced expression of p21/waf1 mRNA at 60 weeks of age(Figure 14B). There was no change in the expression of the p21/waf1 protein (Figure
14C). Because the p21/waf1 is a key mediator of the p53-dependent cell cycle arrest andsenescence process, the expression of p21/waf1 is exquisitely regulated bytranscriptional, post-transcriptional and post-translational mechanisms [67]. We canspeculate that the regulatory factors of the p21/waf1 might be influential in the old
GMF-TG mice in order to modulate p53-dependent senescence. For example, the p400
E1A-associated protein, which inhibits p53-dependent p21/waf1 transcription [68],modulates cell fate decisions (cell cycle progression, apoptosis, or senescence) by the
ROS homeostasis [69]. It is conceivable that the up-regulation of p400 might be
induced in the later age of GMF-TG mice in order to modulate increased oxidative
stress caused by the overexpressed GMF in non-brain cells [18]. Further studies are
required to elucidate these issues.

In summary, we demonstrated that the GMF-TG mice showed very mild accelerated

23

aging phenotypes, due to secondary laminopathy. In regard to the mechanisms involvedin this process, we propose that the ectopic GMF overexpression induces an oxidative
stress-related reduction of Zmpste24. This might be associated with the activation of the
p53 signaling-pathway, leading to senescence in cells and tissues.

24Chapter 3.

Concluding remarks
Lamin A, a component of the nuclear lamina, is spliced products of the LMNA genethat is synthesized as a precursor prelamin A [39,42]. Zmpste24, a zincmetalloproteinase, is involved in the maturation of lamin A [39,42]. Mutations in the
LMNA or Zmpste24 gene produce mutant lamin A proteins, such as progerin or prelaminA, respectively [42]. The abnormal lamin A has been identified as the cause oflaminopathies which are characterized by premature aging [42]. Interestingly, theZmpste24 gene expression has been shown to be decreased in response to oxidative
stress [48,49]. It suggests that the prelamin A accumulation might be not only caused bygenetic mutation, but also by stress signals, such as oxidative stress.
In this study, we identified an accumulation of prelamin A in the GMF-TG mice. It
suggests that accelerated aging phenotypes in the GMF-TG mice might be associatedwith secondary laminopathy, caused by Zmpste24 down-regulation. We propose that
GMF overexpression in non-brain tissues could cause down-regulation of Zmpste24
mRNA by through enhancing oxidative injuries due to its vulnerability to oxidative
stress (Figure 15) [18]. It is generally recognized that oxidative stress is one of the
major factors that promote the aging process in organisms [23,24]. The novel prematureaging model mice, the GMF-TG mice, may be useful to understanding molecularmechanisms of aging, and then applicable to anti-aging study.

25

Figures and Tables

Figure 1. The expression of GMF mRNA in rat organs at 3 months of age [6].
The expression of glia maturation factor- (GMF) is predominantly expressed in the
nervous system. Low levels of GMF mRNA 10-40 fold less than that in the brain.

26

Figure 2. Schematic representation of the effect of GMF overexpression on
non-brain cells [18].
GMF overexpression in non-brain cells causes an increase in the activity of the
H2O2-producing enzyme; copper/zinc-superoxide dismutase (CuZnSOD), a decrease in
the activities of the H2O2-reducing enzymes; catalase (CAT) and glutathione peroxidase(GPx), and a depletion of the content of the cellular glutathione peroxidase substrateglutathione (GSH) through the p38-MAPK. It promotes a sustained increase in
intracellular H2O2, leading to F-actin reorganization and apoptosis.

27

Figure 3. The illustration of relationship between ROS production and the cellularantioxidant defense system and the effects of oxidative stress [21].
Reactive oxygen species (ROS) including singlet oxygen (1O2), hydrogen peroxide(H2O2), superoxide anion radical (O2·-) and hydroxyl radical (·OH) are a byproduct of
the normal metabolism of oxygen [20,22]. Oxidative stress results from an imbalance
between ROS production and the cellular antioxidant defense system, such as
superoxide dismutase (SOD), CAT, GPx and GSH [20,21]. Oxidative stress induces
oxidative damage to proteins, lipids and DNA, resulting in apoptosis, cellularsenescence and aging [21,23,24].

28

Figure 4. Hypothetical sequence of events following GMF overexpression in brain
tissue by Zaheer et al. [4].
GMF overexpression in brain tissue leads to induction of granulocyte-macrophagecolony-stimulating factor (GM-CSF) through p38-MAPK and NF--mediatedpathway. GMF-dependent induction of GM-CSF activates microglia to produce TNF-,IL-1B, IL-6, and IP-10 and that finally results in the death of neuronal cells.

29

Figure 5. Preparation of transgenic mice overexpressing GMF (GMF-TG).
(A) This figure shows the construct used to prepare the transgenic mice. The construct
was prepared by cloning the coding region of GMF (97-915 bp) in a pCAGGS vector.
(B) This figure shows that the gene expression of GMF in the kidney of the GMF-TG
mice was significantly higher (about 7-fold) than that of C57BL6/J (WT) mice. The datais shown as means ± S.E. (WT; n=3, GMF-TG; n=3). *; P < 0.05 vs. WT mice.

30

Figure 6. Photograph of WT and GMF-TG mice.
(A-B) These photographs show the representative appearance of the WT (A) and
GMF-TG (B) mice at 80 weeks, respectively. The GMF-TG mice showed alopecia andskin atrophy. These phenotypes were not detected in the aged-matched WT mice. (C-D)
Indicators of aging phenotypes, such as spinal curvature, were detected in the GMF-TG(D) mice, but not in the WT (C) mice at 110 weeks.

31

Figure 7. Kaplan-Meier graph of WT and GMF-TG mice.
This figure shows a Kaplan-Meier representation of the survival curves, which revealed
that the GMF-TG mice died significantly earlier than the WT mice. The GMF-TG
mouse and the two WT mice were alive when the data analysis was performed. None of
the mice exhibited any signs of distress or pain due to the clinical symptoms used toassess health and welfare. (WT; n=41, GMF-TG; n=97). *P < 0.05 GMF-TG mice vs.
WT mice.

32

Figure 8. Hair regrowth phenotypes in WT and GMF-TG mice.
(A-C) These photographs show the representative appearance of the WT (A-C; Left)and GMF-TG (A-C; Right) mice at 10 (A), 60 (B) and 80 (C) weeks, 15 days after
shaving. Almost no hair regrowth was observed in the GMF-TG mice, whereas the WT
mice displayed robust hair regrowth. (D) The figure shows the results for the hairregrowth ratio in the WT and GMF-TG mice at 10, 60 and 80 weeks, 15 days after
shaving. The hair regrowth ratio for the GMF-TG mice declined significantly, comparedwith the WT mice at the same age. The mean of the hair regrowth results for the 10w
WT mice is shown as 1. The data is shown as means ± S.E. (10, 60, 80w WT; n=3, 10w
GMF-TG; n=6, 60, 80w GMF-TG; n=5). *; P < .0005 GMF-TG mice vs. WT mice. **;P < .0001 GMF-TG mice vs. WT mice

33

Figure 9. Histological appearance of the kidney, liver and abdominal aorta in WT
and GMF-TG mice.

34

(A-F) These photographs show an overview of the hematoxylin-eosin (HE) (A and D),
periodic acid–schiff (PAS) (B and E), and Masson trichrome (MTC) (C and F) stained
kidney sections in the WT (A-C) and GMF-TG (D-F) mice at 30 weeks. (G-L) These
photographs show an overview of the HE (G and J), PAS (H and K), and MTC (I and L)
stained liver sections in the WT (G-I) and GMF-TG (J-L) mice at 30 weeks. (M-R)
These photographs show an overview of the HE (M and P), PAS (N and Q), and MTC(O and R) stained abdominal aorta sections in the WT (M-O) and GMF-TG (P-R) miceat 30 weeks. These findings revealed no histological differences between the WT and
GMF-TG mice. Magnifications: ×100, Scale Bar = 200 μm.

35Figure 10. Photograph of mouse models of laminopathies [54,70] and GMF-TG
mice.
(A) This photograph shows the representative appearance of mouse models oflaminopathies, Lmna L530P/L530P mice (Right) and wild-type mice (Left) [54]. (B) This
photograph shows the representative appearance of mouse models of laminopathies,
Zmpste24-/- mice [70]. (C) This photograph shows the representative appearance of the
GMF-TG mice at 80 weeks. The appearance of the GMF-TG mice, such as alopecia andskin atrophy, seemed to be similar to that of mouse models of laminopathies.

36Figure 11. Western blot of lamin A/C and the expression of Zmpste24 in WT and
GMF-TG mice.
(A-B) These figures show the results from western blot analyses of the lamin A/Cprotein in the kidney of the WT and GMF-TG mice at 10 and 60 weeks. Prelamin A wasabsent in the WT mice at 10 and 60 weeks (A-B; Left) and the GMF-TG mice at 10
weeks (A; Right), but it was detectable in the GMF-TG mice at 60 weeks (B; Right).
There were no significant differences between the lamin C protein levels in the WT and
GMF-TG mice, confirming equal loading. (C) The expression of Zmpste24 mRNA
tended to decrease in the kidneys of the GMF-TG mice at 10 weeks, compared with theWT mice. The data is shown as means ± S.E. (10w WT; n=3, 10w GMF-TG; n=3). P <37

0.09 vs. 10w WT mice. (D) The expression of Zmpste24 mRNA decreased in thekidneys of the GMF-TG mice at 60 weeks, compared with the WT mice. The data isshown as means ± S.E. (60w WT; n=4, 60w GMF-TG; n=4). *; P < 0.01 vs. 60w WT
mice.

38

Figure 12. The expression of TGF-1 and CTGF in WT and GMF-TG mice.
(A-B) The expression of transforming growth factor-1 (TGF-1) (A) mRNA increasedin the kidneys of the GMF-TG mice at 10 weeks, compared with the WT mice.
However, no significant differences were shown in the expression of connective tissue
growing factor (CTGF) (B) mRNA in the kidney of the GMF-TG mice at 10 weeks,compared with the WT mice. TGF-1; The data is shown as means ± S.E. (10w WT;
n=4, 10w GMF-TG; n=5). CTGF; The data is shown as means ± S.E. (10w WT; n=3,
10w GMF-TG; n=4). *; P < 0.01 vs. 10w WT mice. N.S.; not significant versus 10w
WT mice. (C-D) These figures demonstrated a significant increase in TGF-1 (C) andCTGF (D) mRNA in the kidneys of the GMF-TG mice at 60 weeks, compared with theWT mice at the same age. The data is shown as means ± S.E. (60w WT; n=4, 60w
GMF-TG; n=4. *; P < 0.05 vs. 60w WT mice. **; P < 0.001 vs. 60w WT mice.

39

Figure 13. The serum creatinine levels in WT and GMF-TG mice.
This figure shows the result of the serum creatinine levels in the WT and GMF-TG mice,
which revealed that serum creatinine was increased in the GMF-TG mice, comparedwith the WT mice. The data is shown as means ± S.E. (WT; n=4, average age; 84.8 ±0.25 weeks, GMF-TG; n=4 average age; 72.8 ± 6.24 weeks). *; P < 0.05 vs. WT mice.

40

Figure 14. The expression of p21/waf1 mRNA and protein in WT and GMF-TG
mice.
(A) The expression of p21/waf1 mRNA increased in the kidneys of the GMF-TG miceat 10 weeks, compared with the WT mice. The data is shown as means ± S.E. (10w WT;
n=3, 10w GMF-TG; n=4). *; P < 0.05 vs. 10w WT mice. (B) In the kidneys at 60 weeks,
the expression of p21/waf1 mRNA in the GMF-TG mice was reduced significantly,
compared to that of the WT mice at the same age. The data is shown as means ± S.E.

41

(60w WT; n=4, 60w GMF-TG; n=4). **; P < 0.01 vs. 60 w WT mice. (C) This figure
shows the results from western blot analyses of the p21/waf1 protein in the kidney ofthe WT and GMF-TG mice at 10 and 60 weeks. The data shown is representative dataon the estimated ratios of p21/waf1 to -actin, in the case of equivalent protein loading
within a gel. Increased p21/waf1 protein levels were detected in the GMF-TG mice at
10 weeks, compared with the WT mice. However, there was no difference in the levelsof p21/waf1 protein between the GMF-TG and WT mice at 60 weeks. The data isshown as means ± S.E. (10, 60w WT; n=3, 10, 60w GMF-TG; n=3). *; P < 0.05 vs. 10w
WT mice. N.S.; not significant versus 60w WT mice.

42

Figure 15. Hypothetical schematic representation of the novel role of GMF
overexpression in non-brain tissues in vivo.
This study showed that the GMF-TG mice developed very mild accelerated aging
phenotypes due to lamin injury caused by Zmpste24 down-regulation. Zmpste24 isdown-regulated in response to oxidative stress [48,49], which appears to be associatedwith the activation of p53 and p16-Rb pathways [48]. Oxidative stress activates the p53
and p16-Rb pathways, inducing senescence and eventually leading to accelerated aging[23,24]. It is conceivable that the ectopic GMF overexpression might be a factor
connecting aging and lamin injury through oxidative stress.

43

Table I. Definition of chronic kidney disease [25].
Chronic kidney disease is diagnosed according to the criteria listed as follows.

44

Table II. Phenotypes seen in the appearance of WT and GMF-TG mice during theexperimental period of 155 weeks.

There were no detectable differences in body size, jaws or limbs between the WT and
GMF-TG mice during the study.

45

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List of publications
Rika Imai, Kanae Asai, Jun-ichi Hanai and Masaru Takenaka: Transgenic miceoverexpressing glia maturation factor-, an oxidative stress inducible gene, showpremature aging due to Zmpste24 down-regulation. Aging (Albany NY) 7: 486-499,(2015).

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Acknowledgements
First and foremost, I am deeply grateful to Dr. Masaru Takenaka, Professor of Kobe
Women’s University, for his continuous guidance, invaluable discussion, limitless
support, and critical reading of this thesis. At the same time, I would like to thank him
for giving me this opportunity to have this great experience of studying in Kobe
Women’s University.
I would also most grateful to Dr. Yasuhiko Okimura, Dr. Yuriko Kano and Dr.
Haruko Nagasawa, Professors of Kobe Women’s University and Dr. Yasuhiro Inoue,
Professor of Nara Women's University, for their valuable comments and advice.
I would also like to express my gratitude to Dr. Jun-ichi Hanai at Harvard Medical
School & Beth Israel Deaconess Medical Center Renal Division & IMBIO, for his
constructive opinion and suggestion throughout this work.
I am also indebted to Ms. Kanae Asai at Department of Metabolism and ClinicalNutrition, Kyoto University Hospital, for providing valuable experimental data.
I would also like to thank Ms. Yumiko Iwasaka, Assistant of Kobe Women’s
University, for her helpful advice.
This work was financially supported by the Grants-in-aid for Scientific Research
program sponsored by the Ministry of Education, Culture, Sports, Science andTechnology of Japan, and the Technology of Japan and Nestlé Nutrition Council, Japan(NNCJ). The experiments in this study were performed at the Laboratory of ClinicalNutrition and Internal Medicine, Department of Nutrition and Food Science, Graduate
School of Life Science, Kobe Women's University. I must also thank all those who gave55

me the possibility to complete this thesis.
Finally, I would like to thank my parents and family for their unconditional supportand encouragement.

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