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Maternal and Fetal Outcomes of Pregnancies
Complicated by Obesity
Xiaochu Cai
Bachelor of Science (Honours)
A thesis submitted for the degree of Doctor of Philosophy at
Monash University in March 2016
Department of Physiology
Monash University
Copyright notice Notice 1 Under the Copyright Act 1968, this thesis must be used only under the normal conditions of
scholarly fair dealing. In particular no results or conclusions should be extracted from it, nor
should it be copied or closely paraphrased in whole or in part without the written consent of
the author. Proper written acknowledgement should be made for any assistance obtained
from this thesis.
Notice 2 I certify that I have made all reasonable efforts to secure copyright permissions for third-party
content included in this thesis and have not knowingly added copyright content to my work
without the owner's permission.
Table of Contents
Abstract ....................................................................................................................... i
General Declaration ................................................................................................. iii
Acknowledgements .................................................................................................. iv
Publications and Conference Abstracts.................................................................. v
Abbreviations .......................................................................................................... vii
Chapter 1 GENERAL INTRODUCTION .................................................................. 1
1.1. THE MATERNAL ENVIRONMENT DETERMINES FETAL OUTCOMES ..... 4
1.2. HEMODYNAMIC ADAPTATIONS OF PREGNANCY ................................... 5
1.2.1. Cardiovascular Adaptations of Pregnancy .......................................................... 5
1.2.1.1. Systemic Vascular Resistance .................................................................... 5
1.2.1.2. Arterial Pressure ......................................................................................... 7
1.2.1.3. Cardiac Structure and Function .................................................................. 9
1.2.1.3.1. Stroke Volume, Heart Rate and Cardiac Output ................................... 9
1.2.1.3.2. Ventricular Remodelling ..................................................................... 10
1.2.2. Renal Adaptations of Pregnancy ...................................................................... 12
1.2.3. Hemodynamic Changes Post-partum ............................................................... 13
1.3. CARDIOVASCULAR AND RENAL ADAPTATIONS OF OBESITY ............ 15
1.3.1. Cardiovascular Adaptations of Obesity ............................................................. 15
1.3.1.1. Arterial Pressure ....................................................................................... 15
1.3.1.2. Cardiac Structure and Function ................................................................ 16
1.3.1.2.1. Stroke Volume, Heart Rate and Cardiac Output ................................. 16
1.3.1.2.2. Ventricular Remodelling ..................................................................... 16
1.3.2. Renal Adaptations of Obesity ........................................................................... 17
1.4. MATERNAL OUTCOMES OF PRE-PREGNANCY OBESITY .................... 18
1.4.1. Prevalence of Pre-pregnancy Obesity .............................................................. 18
1.4.2. Obstetric Complications of Obesity ................................................................... 18
1.4.3. Maternal Hemodynamic Adaptations In Obese Women ................................... 19
1.4.4. Long-Term Maternal Outcomes of Pre-pregnancy Obesity ............................... 20
1.5. MATERNAL OBESITY AND THE PROGRAMMING OF CARDIOVASCULAR AND RENAL DISEASE ...................................................... 22
1.5.1. Evidence from Human and Animal Studies ...................................................... 22
1.5.2. Maternal Obesity and The Intrauterine Environment......................................... 23
1.5.2.1. The Impact of Placenta ............................................................................. 23
1.5.2.2. Maternal Circulating Factors ..................................................................... 24
1.6. FETAL PROGRAMMING OF KIDNEY DEVELOPMENT AND ITS IMPACT ON CARDIOVASCULAR AND RENAL HEALTH ................................................. 25
1.6.1. Programming of Nephron Endowment ............................................................. 25
1.6.2. Impact of Low Nephron Endowment on Cardiovascular and Renal Health ....... 27
1.6.2.1. Nephron Endowment and Hypertension ................................................... 29
1.6.2.2. Nephron Endowment and Renal Dysfunction............................................ 29
1.6.3. Animal Model of Low Nephron Endowment ...................................................... 30
1.6.3.1. Programed and Congenital Models of Low Nephron Endowment ............. 30
1.6.3.2. GDNF Heterozygous Mice ........................................................................ 31
1.7. REGULATION OF RENAL FUNCTION IN AN ANIMAL MODEL OF LOW NEPHRON ENDOWMENT .................................................................................... 34
1.7.1. Role of NO in the Regulation of Renal Function ............................................... 34
1.7.1.1. Renal NO in Normal Kidneys .................................................................... 34
1.7.1.2. Role of NO in Nephron Deficient Kidneys ................................................. 35
1.8. HYPOTHESES AND AIMS .......................................................................... 36
Chapter 2 GENERAL METHODS .......................................................................... 39
2.1. ANIMALS ..................................................................................................... 39
2.1.1. Mouse Model of Pre-pregnancy Obesity: Chapter 3 & 4 ................................... 39
2.1.1.1. Mating....................................................................................................... 39
2.1.2. GDNF Heterozygous Mouse: Chapter 5 ........................................................... 40
2.1.2.1. Animal Origin ............................................................................................ 40
2.1.2.2. Housing and Diet ...................................................................................... 40
2.1.2.3. Genotyping ............................................................................................... 41
2.2. CONSCIOUS RENAL FUNCTION EXPERIMENTS .................................... 42
2.2.1. Assessment of Urinary Excretory Profile .......................................................... 42
2.2.2. Analysis of Urine and Plasma Samples ............................................................ 43
2.2.2.1. Urinary Osmolality and Electrolytes .......................................................... 43
2.2.2.2. Albumin Assay .......................................................................................... 43
2.2.2.2.1. Assay Principle .................................................................................. 43
2.2.2.2.2. Assay Procedure ............................................................................... 44
2.2.2.2.3. Data Analysis ..................................................................................... 44
2.2.2.3. Creatinine Clearance (Chapter 5) ............................................................. 44
2.2.3. Transcutaneous Measurement of GFR in Conscious Mice (Chapter 4) ............ 45
2.2.3.1. Principle of Transcutaneous Measurement of GFR................................... 45
2.2.3.2. Experimental Protocol ............................................................................... 46
2.2.3.2.1. Fur Depilation .................................................................................... 46
2.2.3.2.2. Experimental Procedure..................................................................... 46
2.2.3.3. Data Analysis ............................................................................................ 48
2.3. CONSCIOUS BLOOD PRESSURE MEASUREMENTS .............................. 49
2.3.1. Implantable Telemetry System for Mice............................................................ 49
2.3.2. Implantation Surgery ........................................................................................ 49
2.4. POST-MORTEM TISSUE COLLECTIONS .................................................. 51
2.4.1. Maternal and Fetal Tissues at GA19 (Chapter 3) ............................................. 51
2.4.2. Maternal tissues at 4WPW (Chapter 4) ............................................................ 51
2.4.3. GDNF HET Mice (Chapter 5) ........................................................................... 52
2.5. STEREOLOGY ............................................................................................ 53
2.5.1. Processing, Embedding and Sectioning of Fetal Kidneys ................................. 53
2.5.2. Sampling Sections ........................................................................................... 53
2.5.3. Histochemical Staining with A. hypogaea PNA ................................................. 54
2.5.4. Counting PNA-positive Glomeruli ..................................................................... 55
2.5.5. Estimating Kidney Volume................................................................................ 57
2.6. CARDIAC CINE-MAGNETIC RESONANCE IMAGING (Chapter 3) ........... 58
2.6.1. Animal Preparation ........................................................................................... 58
2.6.2. MRI scan .......................................................................................................... 59
2.6.3. Data Analysis ................................................................................................... 60
2.7. ASSESSMENT OF COLLAGEN CONTENT ............................................... 61
2.7.1. Hydroxyproline Colorimetric Assay (Chapter 4) ................................................ 61
2.7.1.1. Preparing Tissue Hydrolysates ................................................................. 61
2.7.1.2. Colorimetric Reaction and Absorbance Measurement .............................. 61
2.7.2. Histopathological and Microscopic Analysis of Collagen Content in Cardiac and Renal Tissue. ............................................................................................................... 62
2.7.2.1. Assessment of Collagen Content using PSR Staining .............................. 62
2.7.2.1.1. Picrosirius Red Staining ..................................................................... 62
2.7.2.1.2. Renal Collagen Content (Chapter 3) .................................................. 62
2.7.2.1.3. Cardiac Collagen Content (Chapter 3 & 4) ......................................... 62
2.7.2.2. Assessment of Glomerulosclerosis Using PAS Staining (Chapter 4) ........ 63
2.8. STATISTICAL ANALYSIS OF RESULTS ................................................... 65
Chapter 3 OBESITY LIMITS THE NORMAL CARDIOVASCULAR AND RENAL ADAPTATIONS OF PREGNANCY COMPROMISING FETAL KIDNEY DEVELOPMENT ....................................................................................................... 66
3.1. ABSTRACT ................................................................................................. 68
3.2. INTRODUCTION .......................................................................................... 69
3.3. METHODS ................................................................................................... 70
3.4. RESULTS .................................................................................................... 72
3.4.1. Diet-Induced Obesity in Female Mice ............................................................... 72
3.4.2. Arterial Pressure, Heart Rate and Activity ........................................................ 73
3.4.3. Cardiac MRI ..................................................................................................... 75
3.4.4. Renal Excretory Profile ..................................................................................... 76
3.4.5. Fetal Outcomes ................................................................................................ 78
3.4.6. Maternal Outcomes .......................................................................................... 81
3.5. DISCUSSION ............................................................................................... 83
3.6. CONCLUSION ............................................................................................. 87
Chapter 4 DOES PREGNANCY EXACERBATE THE CARDIOVASCULAR AND RENAL EFFECTS OF OBESITY? ............................................................................ 88
4.1. INTRODUCTION .......................................................................................... 89
4.2. METHODS ................................................................................................... 90
4.2.1. Animals ............................................................................................................ 90
4.2.2. Telemetry Recordings ...................................................................................... 90
4.2.3. Assessment of urinary excretory profile and renal function (GFR) .................... 90
4.2.4. Plasma and Tissue Collection .......................................................................... 90
4.2.5. Statistical Analysis ........................................................................................... 91
4.3. RESULTS .................................................................................................... 92
4.3.1. Post-partum Arterial Pressure and Heart Rate ................................................. 92
4.3.2. Post-partum Renal Function ............................................................................. 95
4.3.3. Maternal Outcomes .......................................................................................... 97
4.3.4. Renal and Cardiac Fibrosis ............................................................................ 100
4.4. DISCUSSION ............................................................................................. 103
4.5. CONCLUSION ........................................................................................... 106
Chapter 5 THE ROLE OF NITRIC OXIDE IN THE REGULATION OF RENAL FUNCTION AND ARTERIAL PRESSURE IN NEPHRON DEFICIENT MICE ........ 107
5.1. INTRODUCTION ........................................................................................ 108
5.2. METHODS ................................................................................................. 110
5.2.1. Animals .......................................................................................................... 110
5.2.2. Experimental Protocol .................................................................................... 110
5.2.3. Terminal Tissue Collection ............................................................................. 110
5.2.4. RT-qPCR ....................................................................................................... 111
5.2.5. Statistical Analysis ......................................................................................... 111
5.3. RESULTS .................................................................................................. 113
5.3.1. Basal Cardiovascular and Renal Excretory Profile ......................................... 113
5.3.2. Arterial Pressure and Renal Excretory Profile During NOS Inhibition ............. 115
5.3.3. Terminal Tissue Weights ................................................................................ 118
5.3.4. Gene Expression of Sodium and Water Channels .......................................... 120
5.4. DISCUSSION ............................................................................................. 122
5.5. CONCLUSION ........................................................................................... 126
Chapter 6 GENERAL DISCUSSION ................................................................... 127
6.1. BRIEF OVERVIEW AND KEY FINDINGS ................................................. 128
6.2. THE IMPACT OF PRE-PREGNANCY OBESITY ON THE MOTHER ....... 130
6.3. THE IMPACT OF PRE-PREGNANCY OBESITY ON FETAL HEALTH AND KIDNEY DEVELOPMENT ................................................................................... 132
6.4. THE RENAL FUNCTION IN NEPHRON DEFICIENT ANIMALS ............... 134
6.5. LIMITATIONS AND FUTURE DIRECTIONS ............................................. 135
6.6. CONCLUDING REMARKS ........................................................................ 137
i
Abstract
There is an increasing prevalence of maternal obesity in Western society. Maternal
obesity is associated with adverse maternal and fetal outcomes. Yet, our knowledge of the
impact of pre-pregnancy obesity on the hemodynamic adaptations of the mother during
pregnancy and postpartum is negligible. The fetal kidney is considered highly sensitive to an
adverse intrauterine environment. Surprisingly, the impact of maternal obesity on fetal kidney
development is poorly understood.
In this thesis, I established a mouse (C57BL6/J) model of diet-induced obesity and
used radiotelemetry, cardiac cine-MRI, and urine samples to characterize cardiovascular and
renal health. Obese mice demonstrated the major characteristics of human obesity pre-
pregnancy including 47% greater body weight, impaired glucose metabolism, hypertension,
cardiac hypertrophy, elevated cardiac output and albuminuria. Whilst mean arterial pressure
(MAP) and heart rate (HR) remained elevated over control mice throughout pregnancy, the
increases in MAP and HR of obese mice during late pregnancy were blunted. Obese dams
also failed to increase cardiac output, and left ventricular mass by late pregnancy and
albuminuria was exacerbated. These changes in obese dams were associated with greater
fetal loss, fetal growth restriction, altered renal morphology and, in male fetuses, a nephron
deficit (25%).
To determine the effect of pregnancy on the long-term cardiovascular and renal
health of obese mice, primiparous obese and control mice were examined 4-weeks post-
weaning and compared to time-matched nulliparous mice. Pregnancy led to greater visceral
obesity and exacerbated hypertension (light-phase) in obese mice postpartum. Total renal
and glomerular collagen content was greater in obese primiparous mice post-partum but this
was not related to renal dysfunction with GFR and albuminuria of obese mice unaffected by
pregnancy.
Maternal obesity in mice has been shown to lead to a nephron deficit in male fetuses
(Chapter 3). The ability to assess the contribution of a low nephron endowment to long-term
cardiovascular and renal health is often confounded by developmental programming of other
organs/systems in many models of nephron deficiency. Thus the choice of model is
important so that these confounding factors can be minimized. Interestingly, whilst a low
nephron endowment is associated with adult cardiovascular and renal disease, many models
of reduced nephron endowment demonstrate normal renal function and MAP. There is little
understanding of how renal function is maintained in states of nephron deficit, though nitric
oxide (NO) has been implicated. To investigate this, I used a genetic model of reduced
nephron endowment the GDNF heterozygous (HET) mouse. This model demonstrates two
ii
levels of nephron deficit. Renal function and MAP of GDNF HET mice were examined before
and after a 7-day systemic NOS inhibition (L-NAME). Nephron-deficient GDNF HET mice
with both moderate and marked nephron deficit were able to maintain normal GFR and
sodium balance in response to L-NAME. Further GDNF HET mice demonstrated a partial
escape from L-NAME-induced hypertension. These findings indicate that nephron deficient
GDNF mice do not rely heavily on NO to maintain renal function chronically.
In conclusion, findings of this thesis indicate that pre-pregnancy obesity not only
compromises the hemodynamic adaptations of pregnancy leading to poor fetal outcomes but
also has a long-term impact on the cardiovascular and renal health of the mother post-
partum. Early detection of the risk involved and the development of interventions that
enhance pregnancy-initiated hemodynamic adaptations may reduce the long-term impact of
pre-pregnancy obesity on the mother and offspring.
General Declaration
I hereby declare that this thesis contains no material which has been accepted for the award
of any other degree or diploma at any university or equivalent institution and that, to the best
of my knowledge and belief, this thesis contains no material previously published o
by another person, except where due reference is made in the text of the thesis.
Chapter 3 of this thesis was written in a format that was appropriate for submission to the
journal Hypertension. The inclusion of co
active collaboration between researchers and acknowledges input into team
Chapter Title
3 Obesity Limits The
Normal Cardiovascular
and Renal Adaptations
of Pregnancy
Compromising Fetal
Kidney Development
Candidate Name: Xiaochu Cai
Signature:
Date: 18/3/2016
Supervisor Name: Michelle Kett
Signature:
Date: 18/3/2016
iii
Declaration
his thesis contains no material which has been accepted for the award
of any other degree or diploma at any university or equivalent institution and that, to the best
of my knowledge and belief, this thesis contains no material previously published o
by another person, except where due reference is made in the text of the thesis.
Chapter 3 of this thesis was written in a format that was appropriate for submission to the
. The inclusion of co-authors reflects the fact that
active collaboration between researchers and acknowledges input into team
Publication
status
Nature and extent of
candidate’s contribution
Obesity Limits The
Normal Cardiovascular
and Renal Adaptations
of Pregnancy
Compromising Fetal
Kidney Development
Submitted
Performed a majority of
experiments, compiled and
analyzed majority of the data,
interpreted data and wrote the
manuscript 75%
Xiaochu Cai
Michelle Kett
his thesis contains no material which has been accepted for the award
of any other degree or diploma at any university or equivalent institution and that, to the best
of my knowledge and belief, this thesis contains no material previously published or written
by another person, except where due reference is made in the text of the thesis.
Chapter 3 of this thesis was written in a format that was appropriate for submission to the
authors reflects the fact that the work came from
active collaboration between researchers and acknowledges input into team-based research.
Nature and extent of
candidate’s contribution
Performed a majority of
experiments, compiled and
analyzed majority of the data,
interpreted data and wrote the
manuscript 75%
iv
Acknowledgements
First and foremost I would like to express my sincere gratitude to my supervisor, Dr
Michelle Kett. Michelle, I am so privileged to have this incredible opportunity to undertake my
research project with you. I really appreciate your patience, generosity, support, and
guidance throughout my candidature. Even in the darkest moment of this journey where the
freezer has broken down and I lost so many precious samples, your positivity and
encouragements have kept me going forward to complete this thesis after the horrific setback.
I would also like to extend my appreciation to Dr Luise Cullen-McEwen for her help
and expertise in the design and preparation of stereological analysis; to Dr James Pearson
for his time and input in cardiac MRI experiments and advice on histological analysis; and to
Prof Matthew Watt for his assistance in the measurement of plasma free fatty acids and
triacylglycerol. Thanks also go to Assoc. Prof David Nikolic-Paterson and Dr Frank Ma for
allowing me to perform collagen assays in your lab.
I also want to take this opportunity to acknowledge the scholarship funding agencies,
NHMRC and National Heart Foundation, for their financial support of my candidature.
To the people from the Renal Lab, Kate, Roger, Russell, Lucinda, Lisa, Katrina and
Rebecca, you are a wonderful group of people to be around with in the lab, in the office or in
the mouse room. Your support and guidance over the years are greatly appreciated. A
special thank-you to Katrina for helping me set up the gene expression analysis off-campus
and for your advice and feedback on this thesis. A big thank-you goes to Roger for your
brilliant advice on statistical analysis, and to Lisa for your helping hands and friendship over
the years. To Stacey and Heyley at the Ritchie centre, thank you for your assistance in my
gene expression experiments.
To Rachael Mason, thank you for being a wonderful friend to my family and me
during some of the toughest moments. Your time and effort in proofreading this thesis are
also greatly appreciated. To my personal mentor, Mark Lo, thank you for your
encouragements, time and prayers in recent years.
To my loving wife, Xiaoyu, thank you for your unconditional love, patience and
encouragements during this journey. I could not have completed this thesis without your
unreserved support. I would also like to thank my children, Muzhou and Muhan. You are both
inspiration to me on a daily basis so that I can be more than what I was the day before.
Finally, I would like to express my eternal gratitude to my parents. Thank you both for
the endless support, love and inspiration throughout my education. Mum, thank you for away
believing in me and being my biggest supporter in completing my PhD. Dad, your attitude
towards your career and life has always inspired me to be the best I can be.
v
Publications and Conference Abstracts
Publications
• Walker KA, Cai X, Caruana G, Thomas MC, Bertram JF, Kett MM, High nephron
endowment protests against salt-induced hypertension, AJP Renal Physiology 2012 July
15;303 (2): F:253-8
• Gurushighe S, Rrown RD, Cai X, Samuel CS, Ricardo SD, Thomas MC, Kett MM, Does a
nephron deficit exacerbate the renal and cardiovascular effects of obesity? PLos One
2013 Sep 3;8 (9):e73095
• Ellery, SJ, Cai, X, Walker DD, Dickinson,H, Kett MM, Transcutaneous measurement of
glomerular filtration rate in small rodents: Through the skin for the win? Nephrology
(Carlton) 2015 Mar 20(3) P:117-23
Conference Abstracts
International conferences
• Cai X, Brown RD, Thomas MC, Kett MM. The role of nitric oxide in the regulation of
arterial pressure and renal function in nephron deficient mice. (24th Scientific Meeting of
the International Society of Hypertension, Sydney, Oct 2012) (Poster)
• Cai X, Kett MM. Does obesity alter the cardiovascular and renal adaptation of pregnancy?
(AHA High Blood Pressure Research 2011 Scientific Sessions, New Orleans, USA, Sep
2013) (Poster)
• Cai X, Kett MM. Does obesity alter the cardiovascular and renal adaptation of pregnancy?
(3rd ISH Young Investigator Symposium, New Orleans, USA, Sep 2013) (Poster)
• Cai X, Kett MM. Does obesity alter the cardiovascular and renal adaptation of pregnancy?
(Annual Scientific Meeting of the Australian and New Zealand Obesity Society, Melbourne,
Oct 2013) (Oral)
vi
National conferences
• Cai X, Brown RD, Kett MM. The role of nitric oxide in the regulation of arterial pressure in
nephron deficient mice. (High Blood Pressure Research Council of Australia Annual
Scientific Meeting, Perth WA, Dec 2011) Selected student oral finalists
• Cai X, Kett MM. Does obesity alter the cardiovascular and renal adaptation of pregnancy?
(High Blood Pressure Research Council of Australia Annual Scientific Meeting, Melbourne
VIC, Dec 2013)
vii
Abbreviations
4WPW 4 week post-weaning
7NI 7-nitro-indazole
AQP2 aquaporin 2
AT1aR angiotensin type 1a receptor
AT1R angiotensin type 1 receptor
AT2R angiotensin type 2 receptor
BMI body mass index
BSA bovine serum albumin
BW body weight
Ccre creatinine clearance
cGMP cyclic guanosine monophosphate
CKD chronic kidney disease
CNS central nervous system
CO cardiac output
DNA deoxyribonucleic acid
DOHaD Developmental Origins of Health and Disease
ECG electrocardiogram
EDV end-diastolic volume
EF ejection fraction
ELISA enzyme-linked immunosorbent assay
eNOS endothelial nitric oxide synthase
ESV end-systolic volume
ET-1 endothelin-1
FFA free fatty acid
FITC-sinistrin fluorescein-isothiocyanate labeled sinistrin
FSA filtration surface area
GA gestational age
GDNF glial cell-derived neurotrophic factor
viii
GFR glomerular filtration rate
GTT glucose tolerance test
HCG Human chorionic gonadotropin
HET heterozygous
HFD high fat diet
HPLC high performance liquid chromatography
HR heart rate
HRP horseradish peroxidase
IUGR intrauterine growth restriction
L-NAME N-nitro L-arginnin methyl ester
LV left ventricle
LVM left ventricle mass
MAP mean arterial pressure
MRI magnetic resonance imaging
mRNA messenger ribonucleic acid
NHE3 sodium-hydrogen antiporter 3
NHP non-human primates
NKCC2 sodium-potassium-chloride cotransporter 2
nNOS neuronal nitric oxide synthase
NO nitric oxide
NOS nitric oxide synthase
PAS periodic acid-schiff
PBS phosphate-buffered saline
PCR polymerase chain reaction
PN postnatal day
PNA peanut agglutinin
PNA peanut agglutinin
PSR picrosirius red
RAS renin angiotensin system
RPF renal plasma flow
ix
RPM revolutions per minute
RSNA renal sympathetic nervous system
RT-qPCR real-time quantitative polymerase chain reaction
RVR renal vascular resistance
SGA small for gestational age
SNGFR single nephron glomerular filtration rate
SV stroke volume
SVR systemic vascular resistance
t1/2 plasma half-life
TAG triacylglycerol
TGF tubuloglomerular feedback
TGFβ Transforming growth factor beta
WT wild-type
Chapter 1 General Introduction
1
Chapter 1 GENERAL INTRODUCTION
The global prevalence of adult hypertension is expected to reach 30% by 2025.199
The latest Global Burden of Disease Study revealed that elevated arterial pressure (systolic
BP >115 mmHg) has become one of the biggest risk factors for global burden of disease and
morbidity, leading to 9.4 million deaths each year.228 The etiology of hypertension is complex
and multifactorial. Factors including the central nervous system, the vasculature, dietary salt
intake, genetics and the environment, have all been implicated in the development of
hypertension. Notably, the work of Guyton and colleagues established that the kidney plays a
dominant role in the pathogenesis of hypertension.147 Further, renal transplantation studies
have demonstrated that blood pressure follows the kidney,140 suggesting that structural and
functional changes in the kidney can initiate the development of hypertension. Whilst
historically the role of the environment on the risk for hypertension has focused on childhood
and adulthood, more recently the in utero and neonatal environments have been identified as
key factors in the etiology of hypertension.
The Developmental Origins of Health and Disease (DOHaD) hypothesis evolved from
early epidemiological studies conducted by Barker and colleagues demonstrating a strong
correlation between adult mortality due to coronary heart disease and low birth weight related
infant mortality.20,23 The DOHaD hypothesis states that “adverse environments during fetal
and early postnatal development alter the structure, function and metabolism of one or more
organ systems, predisposing an individual to a greater risk of developing diseases in adult
life”.363 This phenomenon has also been described as the “Fetal Programming” of adult
diseases. It has become increasingly clear that the fetal kidney development is very sensitive
to an adverse intrauterine environment leading to permanent structural and functional
abnormalities of the kidney (renal programming), and an increased risk in developing
cardiovascular and renal disease later in life.201 The consequences of maternal undernutrition
on renal programming have been extensively studied, particularly with respect to reduced
nephron endowment. Whilst maternal undernutrition remains a significant global issue, a
more pressing concern however is the increasing burden of maternal overnutrition. As a
result of the obesity epidemic in western society, there has been an alarming increase in the
rate of obesity among women of childbearing age. It has been shown that maternal obesity is
not only associated with significant complications during pregnancy, but it also impacts on
fetal outcomes leading to greater risk of cardiovascular and metabolic disorders in offspring.
Chapter 1 General Introduction
2
The impact of maternal obesity on fetal kidney development, specifically nephron endowment,
however has not been thoroughly investigated.
Pregnancy initiates a cascade of cardiovascular and renal adaptations that ultimately
lead to a marked increase in cardiac output.60 This significant elevation of cardiac output is
vital for maintaining adequate delivery of oxygen and nutrients to the fetoplacental unit.
Inadequate physiological adaptations during pregnancy lead to suboptimal fetal development
due to insufficient delivery of blood across the placenta and into the fetal circulation.360
Obesity, on the other hand, represents a state of hyperdynamic circulation, characterized by
increased cardiac output, tachycardia, hypertension, plasma volume expansion and
increased glomerular filtration rate.151 However, as this literature review will highlight, the
understanding of how this altered hemodynamic profile in obese women prior to conception
influences the hemodynamic adaptations of normal pregnancy is negligible. Such knowledge
is important when considering the implication of maternal obesity for fetal programming, and
will thus be examined in Chapter 3.
It is well recognized that normal pregnancy does not impact on the long-term
cardiovascular and renal health of the mother despite the marked adaptations of the
cardiovascular and renal systems during pregnancy.29 However pregnancy complications
such as hypertensive disorders of pregnancy, including preeclampsia, are associated with
increased risk of cardiovascular and renal disease later in life.240 Studies have also shown
that a compromised renal function prior to pregnancy not only limits the renal adaptation of
pregnancy but can also lead to the progression of chronic renal disease in the mother later in
life.32 Chronic obesity is associated with significant cardiovascular and renal abnormalities,136
yet whether pregnancy exacerbates these adverse outcomes of obesity post-birth is
unknown and will be investigated in Chapter 4.
A reduction in nephron endowment is a common outcome of adverse intrauterine
environment. Studies in Chapter 3 examined whether maternal obesity leads to a reduction
in nephron endowment in offspring. However, whilst a reduced nephron endowment is
associated with the development of hypertension and chronic renal disease, understanding
the impact of reduced nephron endowment on the cardiovascular and renal health in the
offspring is often confounded by the global effect of the adverse intrauterine environment to
other organ systems such as the heart and vasculature. There is little understanding of the
factors that control renal function in states of nephron deficit. Nitric oxide (NO) plays a
significant role in the regulation of arterial pressure and renal function in individuals with
normal nephron number. However, the role of NO in maintaining cardiovascular and renal
health in nephron deficient animal is not well understood and is the focus of studies in
Chapter 5. To eliminate the confounding global effect of intrauterine insult on cardiovascular
Chapter 1 General Introduction
3
system, a unique genetic mouse model of reduced nephron endowment with 2 levels of
nephron deficit will be used.
The following review will evaluate the current literature on the cardiovascular and
renal adaptations of normal pregnancy, the hyperdynamic circulation of obesity, and
gestational and long-term outcomes of pre-pregnancy obesity. It will also highlight the gaps
in our understanding of how pre-pregnancy obesity programmes cardiovascular and renal
disease in offspring. The impact of renal programming, in particular a low nephron
endowment on the development of hypertension and renal dysfunction will be discussed.
Lastly, the regulation of renal function in normal kidneys and nephron deficient kidneys will
also be discussed.
Chapter 1 General Introduction
4
A healthy maternal environment is the key for a successful pregnancy and optimal
fetal outcomes. The early epidemiological studies by Barker and colleagues facilitated the
establishment of the DOHaD Hypothesis.20-22 Initially low birth weight, as a clinical marker of
suboptimal intrauterine environment, was found to be associated with increased risk of
mortality from coronary heart disease.278 As the DOHaD hypothesis continued to evolve, it
has been recognized that low birth weight is not the only predictor of adult cardiovascular
and renal outcomes.181,182 In fact adverse intrauterine environments secondary to poor
maternal health, maternal malnutrition, maternal stress and placental dysfunction also have
significant impact on fetal outcomes.5,168,341 Maternal undernutrition has long been
recognized as an insult that leads to intrauterine growth restricted (IUGR) and small for
gestational age (SGA) babies,315,379 increasing the risks of developing cardiovascular and
metabolic disease later in life.39,315,377
Whilst it is clear that maternal nutrition contributes to adverse fetal outcomes, the
importance of adequate maternal hemodynamic adaptations for optimal fetal growth has
been largely overlooked in the literature. Maternal hemodynamic adaptations from early
pregnancy through to birth are critical for placental maturation and maximizing cardiac output
to facilitate rapid fatal growth during second half of the pregnancy.60,235,309 Further, studies
have demonstrated that poor fetal outcomes such as fetal loss, IUGR and SGA birth are
associated with inadequate maternal hemodynamic adaptations.19,103 Pre-pregnancy
cardiovascular and renal health directly influences the extent of maternal hemodynamic
adaptations, and thus impacts on maternal and fetal outcomes.7,42,128,163,360 It has been shown
that pre-existing cardiac dysfunction in the mother is associated with increased incidence of
pre-term birth, SGA babies and increased admittances to neonatal intensive care
unit.128,163,360 Women with advanced chronic kidney disease (very low glomerular filtration
rate) prior to conception also have significantly increased risk of pre-term birth, IUGR babies
and even fetal death.7 These findings highlight the importance of cardiovascular and renal
health prior to conception in the ability of cardiovascular and renal systems to adapt during
pregnancy and subsequent influence on fetal outcomes.
1.1. THE MATERNAL ENVIRONMENT DETERMINES FETAL
OUTCOMES
Chapter 1 General Introduction
5
1.2. HEMODYNAMIC ADAPTATIONS OF PREGNANCY
Pregnancy is associated with profound and reversible changes to the structure and
function of the cardiovascular and renal systems, ultimately leading to an elevated cardiac
output (CO) and thus adequate delivery of oxygen and nutrients to the fetus (Figure 1.1). As
highlighted above, studies indicate that inadequate increase in CO during pregnancy is
associated with poor maternal and fetal outcomes. In this part of the literature review, both
the cardiovascular and renal adaptations of normal pregnancy will be discussed.
Figure 1.1. Hemodynamic adaptations of pregnancy. Pregnancy initiates a cascade of cardiovascular and renal adaptations that leads to a marked increase in cardiac output and therefore delivery of oxygen and nutrients to fetoplacental unit. Figure modified from Conrad et al.75
1.2.1. Cardiovascular Adaptations of Pregnancy
Cardiovascular adaptations during pregnancy include a marked fall in systemic
vascular resistance, a mid-gestational dip in arterial pressure, an increase in plasma volume,
heart rate (HR), stroke volume (SV), and cardiac output (CO) in addition to marked cardiac
hypertrophy, and each of these will be discussed (Figure 1.1).
1.2.1.1. Systemic Vascular Resistance
The cascade of hemodynamic adaptations of pregnancy is initiated by a marked fall in
systemic vascular resistance (SVR), which is caused by a significant peripheral vasodilation
�Stroke Volume
Chapter 1 General Introduction
6
(Figure 1.1). Studies have shown that SVR starts to fall as early as 5 weeks gestation
reaching a nadir by 20 weeks gestation (-34%).309 SVR rises slightly during the second half
of pregnancy, however remains significantly lower than pre-pregnancy level at term (-
27%).60,236,309 This marked fall in SVR occurs prior to the completion of placentation, which
occurs between 6 to 12 weeks of gestation, suggesting that placental influence on
vasodilation during early pregnancy is negligible.50 As with humans, a persistent decrease in
SVR has been demonstrated in rats from early through to late pregnancy.53,129
The mechanisms underpinning the pregnancy-induced fall in SVR are not fully
elucidated, but studies have largely focused on the actions of relaxin and nitric oxide (NO).
Relaxin is an ovarian hormone secreted by the corpus luteum. Relaxin circulates during the
late luteal phase of the menstrual cycle, and increases markedly after conception.334 Relaxin
has been identified as major hormone that mediates pregnancy-induced reduction in
SVR.75,94 Chronic administration of relaxin to conscious non-pregnant female and male rats
mimics the hemodynamic changes observed during pregnancy, including the marked fall in
SVR and the increase in arterial compliance.74,92,93 Relaxin has been shown to promote
vasodilation via increasing downstream NO production both acutely and chronically.76 NO, a
potent vasodilator is important in relaxin-mediated systemic and renal vasodilation during
pregnancy.75 Relaxin can not only activate endothelial nitric oxide synthase (eNOS) directly
via the activation of PI3K pathway, but also increase the expression and activity of eNOS.76
Systemic NOS inhibition in pregnant rats abolishes the fall in SVR and the rise in CO during
pregnancy (Figure 1.2).53 These studies suggest that the cardiovascular adaptations of
pregnancy that are vital for successful pregnancy are NO dependent.
Chapter 1 General Introduction
7
Figure 1.2. Cardiovascular and renal response to chronic NOS inhibition (L-NAME) during pregnancy. Effects of L-NAME on cardiac output (A), systemic vascular resistance (B), glomerular filtration rate (C) and renal plasma flow (D) in pregnant rats on gestational day 14. Figure from Schrier & Ohara.331 Data were originally published by Cadnapaphornchai et al 53
1.2.1.2. Arterial Pressure
Despite a marked increase in CO and plasma volume, mean arterial pressure (MAP)
falls from early gestation. This fall in MAP in early pregnancy is largely driven by the marked
fall in SVR such that the timing of the fall in these two parameters correlates with each other
during pregnancy in humans.60 Longitudinal studies in humans has found that MAP reaches
a nadir around mid-pregnancy (Figure 1.3A).152,236,309 Unlike the persistent reduction in SVR,
MAP rises post-nadir to pre-pregnancy levels by approximately 28 weeks of pregnancy.309
MAP continues to rise exceeding pre-pregnancy levels, such that at 38 weeks SBP and DBP
are 5.6% and 7.5% respectively above pre-pregnancy levels.309 Using radiotelemetry to
continuously measure MAP, studies have shown that mice also demonstrated the mid-
gestational dip in MAP (Figure 1.3B), indicating that the mouse is a suitable model to
investigate hypertensive disorders of pregnancy in the laboratory setting.48,52
A B
C D
Chapter 1 General Introduction
8
Figure 1.3 The fall in MAP during pregnancy in humans and mice. (A) MAP adaptation in human pregnancy, Graph from Hall et al 152; (B) MAP adaptation in mouse pregnancy measured using radiotelemetry. Graph from Butz et al 52.
Whilst the fall in MAP in early pregnancy may be largely driven by the relaxin/NO-
mediated fall in SVR,60 additional factors are also implicated in the control of MAP during
pregnancy, including the Renin Angiotensin System (RAS). Studies in humans have found
that although RAS is upregulated during pregnancy, the presser effect of Angiotensin II (Ang
II) via Angiotensin type 1 receptor (AT1R) is attenuated in pregnant women.126 Further the
depressor effect of Ang II via angiotensin type 2 receptor (AT2R) has been shown to play
important role in the regulation of arterial pressure during pregnancy.55,256 This was
demonstrated recently by Mirabito et al 256 who found that the fall in MAP during pregnancy
was completely abolished in AT2R knockout mice.
A
B
Chapter 1 General Introduction
9
1.2.1.3. Cardiac Structure and Function
1.2.1.3.1. Stroke Volume, Heart Rate and Cardiac Output
In humans, increases in both SV and HR contribute to the increase in CO during
pregnancy (Figure 1.4).279 However, SV is the primary determinant of CO during
pregnancy.179,279,309 SV increases sharply from early pregnancy resulting in a 32% rise by 20
weeks (Figure 1.4).279 Meanwhile, HR increases by 11-12% by mid-pregnancy (Figure
1.4).309 Although SV drops slightly during the third trimester, CO continues to rise up to 50%
of pre-pregnancy level by 32 weeks and remains stable towards term (Figure 1.4).179,309 The
maintenance of CO during third trimester is mainly attributed to a second increase in HR (17-
18%) in this period (Figure 1.4).179,309,322 The progressive increase in SV during early
pregnancy is mainly attributed to the increase in preload due to plasma volume expansion
(See Section 1.2.2).75,179
Figure 1.4. Change in cardiac function during human pregnancy. Percentage changes in cardiac output (CO), stroke volume (SV) and heart rate (HR) from pre-pregnancy values across pregnancy in humans. Figure modified from Ouzounian & Elkayam (2012) 279
This dramatic increase in SV and CO in humans has also been demonstrated in the
gravid rats. Slangen et al 342 reported a 28% and 20% increase in SV and CO, respectively
by gestational age (GA) 12, reaching 29% and 35% respectively by GA18, however, no
change in HR was detected in this model. In contrast, using radiotelemetry Butz and
Davisson52 showed that HR of pregnant C57BL6/J mice significant increased from early
gestation (14% GA6-8), through to mid- (18% GA11-13) and late-gestation (17% GA18-20),
an increase that was similar in timing and magnitude to that observed in humans. Using
Doppler ultrasonography, pregnant C57BL6/J mice also showed significant increase in CO
Inc
rea
se
(%
)
CO
SV
HR
Chapter 1 General Introduction
10
(48%) in late pregnancy, again an increase of similar magnitude and timing as observed in
humans.213 Measurement of SV and CO in mice using Doppler ultrasonography is limited by
the need to use a fixed ventricular geometry to calculate SV, resulting a less accurate
measurement in these parameters.344 This limitation can be overcome by using a cardiac
magnetic resonance imaging (MRI) protocol specific for mice, which allows more accurate,
reproducible and repeated measurements of cardiac geometry across multiple cardiac
cycles.211 Thus in Chapter 3 MRI will be used to determine SV and CO before and during
pregnancy.
1.2.1.3.2. Ventricular Remodelling
Following plasma volume expansion, the maternal heart undergoes significant
remodelling (ventricular hypertrophy). Ventricular hypertrophy is a compensatory
enlargement of the left ventricle in response to volume or pressure overload.37 Chronic
pressure overload, such as in states of hypertension and heart failure, often leads to
concentric hypertrophy. Concentric hypertrophy is generally classified as a pathological and
is characterized by greater chamber wall thickness and small reduction or no change in
chamber volume.37 Concentric hypertrophy is associated with a parallel addition of
sarcomeres leading to an increase in myocyte cell width (Figure 1.5).37 On the other hand,
persistent volume overload, as occurs in pregnancy or endurance exercise, results in
eccentric hypertrophy, which is characterized by a proportional enlargement of chamber
volume and wall thickness (Figure 1.5).37 This type of hypertrophy is a reversible
physiological remodelling that is associated with addition of sarcomeres in series leading to
increases in myocyte cell length.139
Chapter 1 General Introduction
11
Figure 1.5. Ventricular hypertrophy. Pressure overload such as hypertension and strength training causes thickening of left ventricular wall due to the addition of sarcomeres in parallel and leads to concentric hypertrophy. Volume overload such as pregnancy and endurance training leads to proportional enlargement of chamber size and wall thickness via addition of sarcomeres in series and results in a reversible eccentric hypertrophy. Figure modified from Bernardo et al 37
Ventricular hypertrophy during pregnancy is a transient and reversible process that is
not only associated with increases in left ventricular (LV) chamber size, but also involves a
significant increase in LV mass.339 Robson et al 309 showed that both LV mass and LV wall
thickness increased gradually throughout pregnancy in humans, reaching their maximum (53%
and 28% above pre-conception states, respectively) by 38 weeks of gestation. Pregnant
mice also demonstrate a marked increase in LV mass and chamber volume, however an
increase in wall thickness was not detected.105 This study in mice also confirmed that
eccentric hypertrophy at late pregnancy was associated with increase in myocyte cell
length,105 consistent with the characteristic of eccentric hypertrophy in humans.139
Chapter 1 General Introduction
12
1.2.2. Renal Adaptations of Pregnancy
Pregnancy-induced systemic vasodilation is accompanied by marked vasodilation
within the renal circulation, resulting in significant increase in renal plasma flow (RPF) and
glomerular filtration rate (GFR). The landmark human study by Chapman et al 60
demonstrated that both RPF (para-aminohippurate clearance) and GFR (inulin clearance)
increased significantly by 6 weeks of gestation, consistent with the timing of the marked fall
in renal vascular resistance (RVR). The magnitude of the rise in GFR was 45-55% by the
end of first trimester and up to 67% in late gestation.60,90,305 Whilst GFR continues to rise until
late gestation (36 weeks), RPF plateaus at 12 weeks of gestation, yet remains elevated until
late pregnancy.60,305 Anatomically, the kidneys also increase in length and volume to
accommodate the physiological adaptations of the renal system during pregnancy.17,63,65
Concomitant with systemic vasodilation, a progressive expansion of plasma volume
was detected as early as 6 weeks of gestation in humans.60,184 This maternal plasma volume
expansion in early pregnancy is mainly facilitated by marked renal sodium and water
retention, which is mediated by activation of the RAS.31,231 Schrier and Briner330 proposed
that the marked systemic vasodilation during early pregnancy leads to a relative arterial
underfilling which stimulates activation of the RAS. Within the kidney, the RAS promotes the
reabsorption of sodium and water from the renal tubules.332,373 In fact, the increase in plasma
renin activity and plasma aldosterone parallels the plasma volume expansion in humans.60,373
The increase in total plasma volume in the maternal circulation from early pregnancy not only
leads to increase in preload to the heart and thus SV,60 but also facilitates ventricular
hypertrophy via volume overload.198
The reduction in RVR and elevations in RPF and GFR in human pregnancy have also
been demonstrated in the conscious gravid rats, albeit to a lesser magnitude.73 An early
study by Baylis 28 found that the rise in GFR (25%) and RPF (31%) in the gravid rat are a
result of a parallel reduction of afferent and efferent arteriolar resistance that allows single
nephron GFR to increase without a rise in glomerular hydrostatic pressure. The finding that
glomerular pressure does not rise may explain why the sustained hyperfiltration in pregnancy
is not associated with renal injury, unlike other states of hyperfiltration such as diabetic
nephropathy289 and high salt intake239. Further, similar to human pregnancy, rats also
experience progressive plasma volume expansion17,73 and significant increase in kidney
weight28,369 and kidney volume66 by mid-gestation, suggesting a similar renal adaptation of
pregnancy occur in rodents.
Chapter 1 General Introduction
13
1.2.3. Hemodynamic Changes Post-partum
Functional and structural adaptations of the cardiovascular and renal systems
achieved during pregnancy return to pre-pregnancy levels during the post-partum period.
However, timing of the return of each parameter to its pre-pregnancy state is different and
often depends on the time when these parameters were assessed. In humans, MAP remains
relatively constant during the first 10 days post-partum period despite SVR increases by 30%
during this period.279 However, study by Clapp et al 70 showed that until 1 year post-partum
SVR was still 11% lower than pre-pregnancy level suggesting the adaptation of SVR during
pregnancy may take more than a year to return to pre-pregnancy state.70 The return of HR to
pre-pregnancy level has been consistently reported to occur between 12-17 weeks post-
partum.70,236 However, there is some controversy regarding the timing and magnitude of
change in SV and CO post-partum in humans. An early human study demonstrated that SV
and CO returns to pre-pregnancy level by 2 weeks post-partum.179 Mahendru et al 236 even
reported a small increase in SV 14-17 weeks post-partum compared with third trimester
measurement with CO back to pre-pregnancy levels at 17 weeks post-partum. However,
Clapp and Capeless70 found that SV falls moderately after birth and remains significantly
elevated over pre-pregnancy levels up to 1 year post-partum. Interestingly when the data for
SV was separated into first pregnancies (primigravida) and subsequent pregnancies
(multigravida), it was found that multigravida retained much higher SV at 1 year post-partum
than primigravida.70 CO followed a similar pattern in this study with CO of primigravida
dropping significantly by 12 weeks post-partum and almost completely back to pre-pregnancy
level by 24 weeks post-partum whilst CO of multigravida remained significantly higher than
pre-pregnancy levels at 24 weeks post-partum.70 Consistent with this, other studies of
predominantly primigravida women (>60%) have demonstrated that CO returns to normal
level by 17-24 weeks post-partum.236,308 These findings indicate that parity may impact the
timing and magnitude of changes in SV and CO post-partum, with multigravida more likely to
retain an elevated SV and CO for a longer period of time than primigravida post-partum. The
extensive ventricular hypertrophy that occurs during pregnancy including an increased LV
mass and LV thickness have also been shown to return to pre-pregnancy by 13 weeks post-
partum in humans.339 Further, the marked increase in GFR during human pregnancy has also
been shown to returns to pre-pregnancy level by 14-17 weeks post-partum.236
Despite the recognition that there is a vast difference in the timing of the return of
cardiovascular and renal parameters to pre-pregnancy levels, the vast majority of studies use
post-partum or even first trimester measurements as surrogate measure for pre-pregnancy
measurement.96,101,130,354 In doing so findings from these studies are confounded likely to
underestimate of the true extent of the pregnancy-induced changes in the cardiovascular and
renal systems. Thus, to properly investigate the cardiovascular and renal adaptation of
Chapter 1 General Introduction
14
pregnancy and post-partum returns of these variables, a pre-pregnancy cardiovascular and
renal profile should be established. Chapter 3 & 4 will examine the impact of pre-pregnancy
obesity on cardiovascular and renal systems before and during pregnancy and post-partum.
Chapter 1 General Introduction
15
1.3. CARDIOVASCULAR AND RENAL ADAPTATIONS OF OBESITY
Obesity and overweight have become a major global health burden affecting over 1.9
billion adults worldwide in 2014, with 600 million of those in the obese category (BMI over
30kg/m2).275 Findings from the Framingham Heart Study suggest more than 60% of essential
hypertension can be attributable to overweight and obesity.258 Apart from the direct link to
hypertension, obesity is known to associate with a range of cardiovascular, metabolic and
renal disorders. This section of the literature review will focus on the cardiovascular and renal
adaptations of obesity, as this is the hemodynamic background that pregnancy-induced
cardiovascular and renal adaptations must occur in pregnancies that are complicated by
obesity.
1.3.1. Cardiovascular Adaptations of Obesity
1.3.1.1. Arterial Pressure
Obesity has been recognized as one of the biggest contributors to the development of
hypertension. This phenomenon has been demonstrated in epidemiological studies208,258 and
many animal models including dogs,149 rabbits,49 rats,382 and mice.340 The mechanisms that
mediate obesity-induced hypertension are complex and multifactorial in nature. Alterations in
the central nervous system (CNS), renal sympathetic nervous activity (RSNA) and RAS have
been implicated to significantly contribute to obesity-induced hypertension. In a recent
landmark study, Simonds et al 340 demonstrated that leptin-mediated increase in sympathetic
overflow is one of the primary contributors to obesity-induced hypertension. However, the
exact mechanism linking the hypothalamic leptin signaling, sympathetic overflow and
development of hypertension remains to be elucidated. An increase in RSNA and activation
of the renal RAS also contribute to obesity-induced hypertension by promoting sodium
retention.149,150 Obese hypertensive humans demonstrated an elevation in renal
noradrenaline spillover compared to normotensive obese individuals suggesting that obesity-
induced hypertension is associated with increased RSNA.311 Diet-induced obese dogs have
increased tubular sodium reabsorption and require a higher arterial pressure to maintain
sodium balance.91,151 Renal denervation completely normalized arterial pressure in these
obese hypertensive dogs, suggesting that increased RSNA plays a significant role in obesity-
induced hypertension in this model.160,229 The contribution of RAS in obesity-induced
hypertension has also been implicated. This has been demonstrated by effective reduction of
arterial pressure by angiotensin type 1 receptor (AT1R) antagonists and angiotensin
converting enzyme inhibitors in obese animals43,307and humans,132,297 respectively.
Chapter 1 General Introduction
16
Animal models are used extensively in obesity research, however these studies
predominately use male animals. Recent studies have demonstrated that sex-difference
exists such that female mice appear to be partially protected from obesity-induced
hypertension.144,368 This sex-specific protection appears to be mediated by estrogen.144,368
Nevertheless, our understanding of the impact of pre-existing obesity-induced hypertension
on pregnancy-initiated cardiovascular and renal adaptations are negligible. Further, whether
pregnancy has any impact on the long-term arterial pressure regulation in obese females is
unknown. Chapter 3 & 4 of this thesis will address some of the gaps in our knowledge.
1.3.1.2. Cardiac Structure and Function
1.3.1.2.1. Stroke Volume, Heart Rate and Cardiac Output
Human obesity is associated with elevated CO, mediated largely by an increase in
SV,72,251,252 but tachycardia also contributes.208 The increase in SV in human obesity is
mainly driven by plasma volume expansion and thus an increase in preload.364 Diet-induced
obesity in mice also leads to significant increase in SV and CO, however this has only been
demonstrated in male mice using echocardiography.36 Obesity-induced tachycardia has also
been demonstrated in animal models146,185 including diet-induced obese male340 and
female144 mice. Thus diet-induced obese female mice are excellent model to investigate the
impact of pre-existing obesity-induced cardiovascular changes on the ability of the
cardiovascular system to adapt during pregnancy. Chapter 3 will address this issue using a
robust mouse model of obesity.
1.3.1.2.2. Ventricular Remodelling
Ventricular hypertrophy is one of the major cardiac adaptations associated with
obesity.85 However, whether obesity-induced cardiac hypertrophy is a physiological or
pathological hypertrophy is controversial. Studies in 1980s by Messerli et al have that shown
both eccentric and concentric hypertrophy occur in obesity, due to synergistically increased
preload (volume expansion) and afterload (hypertension).250,252 However, using 2D
echocardiography, more recent human studies suggested that concentric LV hypertrophy is
likely to be the predominant form of LV hypertrophy in obesity.15,219,376 This was then further
confirmed using cardiac MRI among obese individuals.202
Sex differences have been identified in obesity-induced LV remodelling. Rider et al 302
found that obese men predominantly have concentric LV hypertrophy without LV chamber
dilation. Conversely, obese women are more likely to exhibit both eccentric and concentric
hypertrophy.302 Irrespective of the type of cardiac hypertrophy obese women may have, it is
clear that the LV mass is significantly elevated. What is unknown is that whether pre-existing
Chapter 1 General Introduction
17
cardiac hypertrophy in obese women influences the structural and functional adaptations of
the heart during pregnancy. Chapter 3 of this thesis will address this gap in our knowledge.
1.3.2. Renal Adaptations of Obesity
During the early phase of human obesity the renal system is characterized by a state
of hyperfiltration and hyperperfusion that are associated with a reduction in RVR.58,296,301
Obesity also leads to structural changes to the kidney including renal and glomerular
hypertrophy, glomerulosclerosis and albuminuria191,196 and is an independent risk factor for
chronic kidney disease (CKD).136 In addition, animal models of obesity also demonstrated
increased renal lipid accumulation and renal collagen accumulation.95,189 It is unknown
whether pre-existing renal hyperfiltation and hyperperfusion in the kidneys of obese women
impacts the ability of the renal system to adapt during pregnancy. Further, whilst pregnancy
is known to have no long-term impact on renal function post-birth following normal pregnancy,
whether pregnancy-induced renal changes exacerbate obesity-related renal dysfunction (i.e.
albuminuria) is unknown. Studies in Chapter 3 & 4 will address these questions respectively.
Chapter 1 General Introduction
18
1.4. MATERNAL OUTCOMES OF PRE-PREGNANCY OBESITY
1.4.1. Prevalence of Pre-pregnancy Obesity
As the overall prevalence of obesity has risen, so too has the prevalence of obesity
among women of reproductive age. Over 50% of the women of reproductive age in Australia,
USA, UK and other European countries are currently overweight or obese.2,226,273 Despite the
reduced fertility rate and increased risk of miscarriage associated with obesity, the
prevalence of maternal obesity has been reported as approximately 20% in UK, and US and
13% in Australia.68,162,244 However, recent data suggests that the prevalence of maternal
obesity even reached 28-33% in rural and low socio-economic groups.84,164 Maternal obesity
is not only a significant epidemic in Western world, but has also started to impact urbanized
populations of developing countries, such as in India and China.294,386
1.4.2. Obstetric Complications of Obesity
Obesity is not only recognized as a major contributor to infertility but also increases
the risk of obstetric complications after successful conception.350 Maternal obesity is now
considered the most common preventable risk factor for complicated pregnancy in the
USA.69 The complications associated with maternal obesity include, but are not restricted to,
hypertensive disorders of pregnancy, preeclampsia, gestational diabetes, renal dysfunction,
venous thromboembolism, pre-term birth, macrosomia, congenital malformations,
miscarriage, stillbirth and maternal and neonatal death.69,188,232,245,283,316 Further, maternal
obesity also increases the risk of induction of labor, post-partum hemorrhage, caesarean
delivery, and caesarean wound complications leading to lengthy hospital stays for both the
mothers and their infants.161,232 The increase in obstetric and fetal complications has resulted
in an increased burden on resources and economics in managing pregnancies that are
complicated by obesity.161,310 In order to improve maternal and fetal outcomes of obese
women, greater understanding of the implication of pre-existing obesity on the hemodynamic
adaptations of pregnancy that are crucial to fetal health is needed.
In addition to the immediate risks associated with managing maternal and fetal health
during pregnancy, an understanding of the long-term effect of maternal obesity on the mother
and offspring is also imperative in disease prevention. Unfortunately, due to limited clinical
data, our knowledge of the long-term impact of maternal obesity on the mother and offspring
comes primarily from laboratory animal research. In general, animal studies indicate that
being obese prior to, or at the beginning of, pregnancy increases the risk of offspring
developing cardiovascular and metabolic disorders in adult life, consistent with the Barker
hypothesis. Yet, the mechanisms that lead to these programming outcomes by maternal
Chapter 1 General Introduction
19
obesity are unclear. Maternal environments, particularly maternal hemodynamic adaptations,
are critical in determining fetal nutritional supply and fetal growth, therefore are one of the
main focuses of this thesis.
1.4.3. Maternal Hemodynamic Adaptations In Obese Women
Adequate hemodynamic adaptations are vital for a successful pregnancy. Despite the
increasing prevalence of maternal obesity in the last two decades, only a handful of studies
have investigated the hemodynamic adaptations in obese women during pregnancy. The
mid-gestation dip in arterial pressure is a hallmark of cardiovascular adaptation of normal
pregnancy. However, whether obese women also experience this fall in arterial pressure
during pregnancy is contentious. This is largely due to the lack of pre-pregnancy data
recorded in these studies, a common limitation in studies that examine arterial pressure
during pregnancy.130,359 Further, these studies also differ in the timing and frequency of
arterial pressure measurement throughout pregnancy. Tomoda et al 354 monitored the
changes in MAP from 6 weeks of gestation through to term in normal weight and obese
women who were either primiparous or multiparous. It was found that both obese primigavida
and multigravida had significantly higher MAP over normal weight women across
pregnancy.354 Interestingly, only obese multigravida demonstrated a mid-gestational fall in
MAP whilst obese primigavida did not.354 This suggests that parity may contribute to the
adaptation of MAP in obese women. Further Abdullah et al 1 found that there was no change
in MAP between first and second trimester in morbidly obese women (BMI>45). However,
whether these women were primiparous or multiparaous was not reported.1 Importantly
studies have also demonstrated that the rise in MAP, particularly late in gestation, is
attenuated in women with greater BMI.1,123,352 This suggests that pre-existing obesity may
impact arterial pressure response in late pregnancy contributing to the complications at term.
However, no studies so far have specifically examined this matter.
The limited literature also suggests that the adaptation of CO in pregnancy
complicated by obesity might be compromised.96 Abdullah et al 1 demonstrated that morbidly
obese pregnant women had a blunted increase in CO across the trimesters compared to
non-obese controls. This appeared to be mainly due to blunted increase in SV with HR
increasing across pregnancy.1 Similarly, LV mass of obese women in this study also
remained unchanged indicating limited ventricular remodelling might have occurred during
pregnancy.1 Tomoda et al 354 also reported that the heart of obese pregnant women had a
limited capacity to increase SV in response to a mild exercise challenge. In contrast, a meta-
analysis have shown that obese women demonstrated a blunted rise in HR during pregnancy,
concomitant with the limited rise in CO.159 Further, pregnancy60 and obesity58,301 can
Chapter 1 General Introduction
20
independently lead to hyperfiltration or increase in GFR and renal plasma flow, however no
study to date has specifically examined changes in renal function in obese females during
pregnancy. In order to further elucidate the impact of obesity on the hemodynamic
adaptations that occur in normal pregnancy, a well-controlled animal study that examines the
cardiovascular parameters at pre-conception state and across pregnancy is needed. Chapter
3 of this thesis will address this gap in our knowledge.
1.4.4. Long-Term Maternal Outcomes of Pre-pregnancy Obesity
Women who were obese prior to conception are likely to remain obese post-birth
exposing them to greater risks of cardiovascular and renal morbidity and mortality later in
life.112 Surprisingly, we know very little about the long-term impact of pregnancy on the
cardiovascular and renal health in obese women. A recent epidemiological study from the UK
demonstrated that maternal obesity during pregnancy is strongly associated with premature
death and greater risk of a major cardiovascular event in women later in life.223 These
increased risks were independent of common maternal complications such as preeclampsia
and low birth weight.223 In supporting of this finding in the UK population, a study examining
women from a defined region of Israel found that those who were obese before conception
had greater risk of experiencing simple cardiovascular events, early occurrence of
cardiovascular morbidity, and greater number of cardiovascular related hospitalizations
during the 10 year follow-up period.384
Whilst our knowledge of the impact of pre-pregnancy obesity on the long-term
maternal cardiovascular and renal health is limited, the common maternal complications that
obese women experience during pregnancy such as chronic hypertension, preeclampsia and
gestational diabetes have been shown to cause significant cardiovascular and renal
morbidity post-birth. As mentioned earlier (Section 1.4.2) pre-pregnancy obesity leads to a
high incidence of hypertensive disorders of pregnancy, including chronic hypertension and
preeclampsia. It is known that women who experience hypertensive disorders of pregnancy
have greater risks of developing ischemic heart disease, myocardial infarction, heart failure,
ischemic cerebrovascular disease and chronic kidney disease (CKD) later in life.157,240
Further, preeclampsia is also associated with increased risk of chronic hypertension and
CKD.61,345 Interestingly, those women who experienced preeclampsia and remained
hypertensive 2 years post-partum had greater BMI than those who became normotensive
post-birth, indicating being overweight or obese during the post-partum period might
predispose them to chronic hypertension later in life.345 Gestational diabetes is not only
associated with early onset of type II diabetes, but also a significant risk factor for the
development of chronic hypertension and renal dysfunction post-birth.35,158 Further, studies
Chapter 1 General Introduction
21
have also shown that women with a compromised renal function prior to conception have
greater risks of developing CKD post-birth.32
Although obesity is associated with only mild proteinuria and glomerulosclerosis,8 it is
possible that pregnancy-related renal adaptions, particularly the glomerular hyperfiltration
could exacerbate the pre-existing renal abnormalities and lead to persistent renal injury post-
partum. Taken together, it is reasonable to predict that pre-pregnancy obesity increases the
risk of females developing chronic hypertension and renal dysfunction post-birth. To fully
elucidate the impact of pregnancy on the long-term risks of cardiovascular and renal health in
obese females, studies that incorporate both lean and obese females that are either
primiparous or virgin should be performed. Studies in Chapter 4 will address some aspects of
this issue in a mouse model of maternal obesity.
Chapter 1 General Introduction
22
1.5. MATERNAL OBESITY AND THE PROGRAMMING OF
CARDIOVASCULAR AND RENAL DISEASE
1.5.1. Evidence from Human and Animal Studies
Clinical studies examining the impact of pre-pregnancy obesity on cardiovascular and
renal health of offspring in the long term are lacking. A recent epidemiological study from the
UK found that obesity in pregnancy was associated with increased premature mortality and
hospital admissions for cardiovascular events in adult offspring.299 Limited human studies
suggested a strong association between maternal obesity and hypertension in young
offspring. Studies in the UK and the Netherlands found that pre-pregnancy BMI is
independently associated with elevation of arterial blood pressure in children at 5-6 years of
age.122,124,220 Strong associations between greater pre-pregnancy maternal BMI and elevated
arterial pressure in smaller cohorts of adolescents218 and adults166 have also been found.
Fetal cardiac dysfunction104,186 and increased incidence of congenital heart defect254 has also
been linked to maternal obesity in humans.
Extensive research using animal models has confirmed this strong association
between pre-pregnancy obesity and increased risk of offspring developing cardiovascular
and metabolic disorders later in life. Although the majority of studies using high fat diet to
induce obesity do not show major increase in body weight of the dams at conception, these
studies do demonstrate programmed hypertension in young and adult offspring.40,108,109,118,318
The mechanisms underlying this programming event are unclear, however studies in rats
and rabbits have demonstrated that an increased sympathetic activation in offspring of obese
mothers is likely to be a contributor for the development of hypertension.290,319 Further,
cardiac fibrosis, contractile dysfunction and pathological cardiac hypertrophy have also been
found in offspring of obese female rodents.40,118,174,367 These findings indicate that, similar to
models of maternal undernutrition, maternal overnutrition, in particular maternal obesity is
likely to program adult diseases through an adverse intrauterine environment.
Significant renal injury can also be programmed by maternal overnutrition. Jackson et
al 187 showed that male rat offspring born to high fat/fructose-fed dams and fed a control diet
postnatally had profound renal injury including albuminuria (209%) and glomerulosclerosis
(64%) compared to the offspring of control dams. Female offspring of this model seems have
less aggressive renal injuries compared to male offspring, with exacerbated albuminuria
(~175%), TGFβ protein overexpression (~100%) and mild increases in macrophage
infiltration (~60%), but no evidence of glomerulosclerosis compared to kidneys of control
offspring.119 An abnormal kidney development is likely to be a factor contributing to the
vulnerability of this model to renal injury. Currently, no study in the literature has examined
Chapter 1 General Introduction
23
the impact of pre-pregnancy obesity on fetal kidney development. Chapter 3 in this thesis will
address the gap in our knowledge using a model that has pre-existing obese phenotype.
1.5.2. Maternal Obesity and The Intrauterine Environment
Maternal obesity creates an adverse intrauterine environment with altered placental
function and abnormal maternal circulating factors such as glucose and cholesterol. This
section will discuss the evidence regarding the association of these two aspects of maternal
environment to the programming of cardiovascular and renal disease in offspring of obese
mothers.
1.5.2.1. The Impact of Placenta
The placenta plays a central role in fetal growth and development by acting as a
interface for maternal and fetal interactions. The placenta is also an important endocrine
organ crucial for a successful pregnancy. During pregnancy the placenta synthesizes
steroids such as HCG, progesterone and oestrogens, and growth hormones like placental
lactogen and placental growth hormone.78 Synthesis of these placental hormones not only
regulates reproductive function and maturation of the placenta from early pregnancy, but also
ensures an optimal intrauterine environment for adequate fetal growth during the later stage
of pregnancy.78 Impaired maturation of the placenta is associated with maternal
complications such as preeclampsia.51 In examination of term placentas of obese women
collected during caesarean section, there was increased expression of pro-inflammatory
mediators,59,304 increased oxidative stress,154,313 accumulation of macrophages,59
mitochondrial dysfunction,154 and an increased incidence of placental vascular lesions173,210
compared to the placenta of normal weight controls. Consistent with human studies, obese
female non-human primates had increased placental inflammatory cytokines, reduced blood
flow in both the uterine artery and fetal side of the placenta, and increased risk of placental
infarction contributing to the high incidence of stillbirth in this model.121,306 Despite the
structural differences between mouse and human placentas,238 similar placental pathology
has been demonstrated in mouse models of maternal obesity, confirming mouse placentas
can also be affected by maternal obesity.203,214 Placenta of obese mice demonstrated
decreased labyrinth thickness (cell proliferation), increased placental inflammation and
reduced placental efficiency.203,214 Interestingly, placentas that accompanying male fetuses
appeared to be most affected suggesting a sex-specific effect of maternal obesity in the
programming of adult disease in offspring via the placenta.203,298
Chapter 1 General Introduction
24
1.5.2.2. Maternal Circulating Factors
Abnormal maternal plasma profile such as increased pro-inflammatory cytokines,
maternal hyperglycemia, hypercholesterolemia and hyperleptinemia may also program
cardiovascular and renal disease in offspring. Excess level of these maternal circulating
molecules might either be transported across the placenta into the fetal circulation such as
glucose,349 or lead to placental dysfunction secondary to excess circulating pro-inflammatory
cytokines,203 thus increasing the risk of maternal complications and developmental
abnormalities of the fetus. Offspring exposed to intrauterine hyperglycemia, irrespective of
the etiology of maternal diabetes have increased risk of congenital malformations34,148 and
greater risk of developing cardiovascular and metabolic diseases in adulthood.197,222,333,383
Renal malformations are particularly prevalent among congenital malformations associated
with gestational diabetes in humans89,269 and mice167
Further, maternal hypercholesterolemia during pregnancy in humans has also been
associated with increased susceptibility to, and faster progression, of atherosclerosis in
young children.267 Similarly, treating obese dams with cholesterol-lowering agent statins have
been shown to reduce the risks of hypertension and dyslipidemia in offspring.108,110,
indicating that maternal hypercholesterolemia associated with obesity might contribute to the
programming of cardiovascular and metabolic disease in offspring. Further, maternal
hyperleptinemia has also been linked to the greater adiposity and development of
hypertension in offspring of dams fed on a high fat diet.205,317-319 These findings indicate that
abnormal plasma profile associated with obesity during pregnancy greatly impacts the
outcomes of offspring.
Chapter 1 General Introduction
25
1.6. FETAL PROGRAMMING OF KIDNEY DEVELOPMENT AND ITS
IMPACT ON CARDIOVASCULAR AND RENAL HEALTH
While the field of fetal programming was initially focused on the development of
cardiovascular diseases such as coronary heart disease, the involvement of other organ
systems and physiological processes have also been explored, including the kidney. Fetal
kidney development is extremely sensitive to adverse intrauterine environments, such as
maternal low protein diet, glucocorticoid exposure, placental insufficiency, exposure to
maternal disease (hypertension, diabetes) and toxins (pharmaceuticals,
alcohol).5,25,168,201,379209 These insults have been found to program low nephron
endowment,22,24 alteration in renal RAS88,137 and renal sympathetic nerve activity (RSNA),6,86
all of which are known to contribute to the development of hypertension and renal disease in
offspring.25,277 Nevertheless, the most sensitive aspect of renal programming is nephron
endowment, which is the focus of this thesis.
1.6.1. Programming of Nephron Endowment
Nephron endowment refers to the number of nephrons an individual is born with or
the nephron number at the termination of nephrogenesis in species such as rats and mice.
Using gold standard, unbiased stereological technique, studies have found that nephron
number in humans varies widely. The Monash Series examined 420 kidneys obtained at
autopsy from Australian aborigines, Australian Caucasian, African-American, American
Caucasian and Senegalese Africans and revealed that there is at least 13-fold range in
nephron number in these populations.100,169,170,175,247,248,291 Of importance, Hughson et al 175
demonstrated a direct correlation between nephron number and birth weight in humans,
suggesting birth weight is a strong prediction of nephron endowment. Despite this vast range
in nephron number in humans, these autopsy studies could not determine the mechanisms
or maternal insults that led to reduced nephron endowment. The type of insults that result in
a reduction in nephron endowment have been identified through research in animal models.
Adverse intrauterine environments such as placental insufficiency/intrauterine growth
restriction,24,26 maternal low protein intake,315,379 maternal glucocorticoid
exposure,263,276,277,341,374 gestational diabetes9,167 and maternal alcohol exposure133,134 have all
been shown to program low nephron endowment.
Much attention on the programming of low nephron endowment has been focused on
maternal undernutrition, yet the impact of maternal overnutrition, particularly pre-pregnancy
obesity on nephron endowment is poorly understood. In fact, no study to date has
established any correlation between pre-pregnancy obesity and nephron endowment. In a
study using rats, Armitage et al 12 found no difference in kidney weight, glomerular number
Chapter 1 General Introduction
26
and glomerular volume in adult offspring of dams fed a high fat diet (HFD). However, female
rats in this study started the HFD only 10 days prior to conception and the appropriate
stereological technique (gold standard) was not used to estimate nephron number in
offspring. Although the direct evidence linking pre-pregnancy obesity and nephron deficiency
in offspring is lacking, common complications associated with pre-pregnancy obesity such as
maternal hyperglycemia and placental insufficiency, have been demonstrated to lead to a
programmed low nephron endowment.
Given pre-pregnancy obesity significantly increase the risk of maternal
hyperglycaemia and gestational diabetes,153 nephrogenesis in offspring of obese mother
could be greatly affected, resulting a reduced nephron endowment. Hokke et al 167 have
demonstrated that diabetic pregnancy in mice leads to a marked deficit in ureteric branching
morphogenesis and a reduced nephron endowment in offspring. This study also showed that
insulin therapy initiated following the onset of hyperglycemia could not reverse the deficit in
renal development, suggesting that early detection and correction of hyperglycemia might be
needed to prevent these developmental abnormalities in the kidney.167 This is particularly
relevant to those women have been screened and diagnosed with gestational diabetes late
in pregnancy, typically at 26-28 weeks, as nephrogenesis starts around 5 weeks of gestation
in humans (see Moritz & Wintour260 and Guron & Friberg145), long before the recommended
time for screening gestational diabetes Importantly, women who were obese prior to or at
time of conception are also likely to have uncontrolled hyperglycemia throughout pregnancy
and increased risk of developing gestational diabetes during pregnancy,18 implying offspring
of obese pregnant women might be at greater risk of being born with a nephron deficit and
consequently experiencing renal insufficiency later in life. Further, even without chronic
exposure to hyperglycemia from early pregnancy, Amri et al 9 have demonstrated that
glucose infusion from days 12-16 of gestation in rat pregnancy can led to a 20% reduction in
nephron number of offspring, but only among those dams whose hyperglycaemia was
transiently higher on day 13 of gestation. This suggests that even a small window of
exposure to hyperglycaemia during pregnancy could lead to nephron deficiency in offspring.
Human173 and mouse203,214 studies have shown that pre-pregnancy obesity increases
the risk of placental pathological lesions and leads to reduced placental efficiency. Placental
insufficiency in rats has been found to lead to significant growth restriction and associated
nephron deficit.24,262 It is not known whether obesity-induced placental insufficiency causes a
reduction in nephron endowment, however given the close association of pre-pregnancy
obesity and increased risk of maternal hyperglycemia and placental insufficiency, it is
reasonable to predict that pre-pregnancy obesity programmes a low nephron endowment
through an adverse intrauterine environment. Chapter 3 will examine this hypothesis in a
robust mouse model of pre-pregnancy obesity.
Chapter 1 General Introduction
27
1.6.2. Impact of Low Nephron Endowment on Cardiovascular and Renal Health
Although pre-pregnancy obesity could act through multiple mechanisms to program a
low nephron endowment in offspring, these mechanisms often affect the development of
other organs/systems. In doing so, it is difficult to determine the impact of nephron deficit per
se for adult offspring in these models. A low nephron endowment is associated with
increased risk of adult cardiovascular and renal disease, however the mechanisms are not
well understood. Brenner and colleagues were the first to proposed that a reduced nephron
number leads to the development and progression of hypertension and chronic kidney
disease (Figure 1.6).46 The hypothesis proposed that a reduced nephron number would lead
to reduced glomerular filtration surface area (FSA) and thus water and sodium retention.
Consequently, there would be a rightward shift of the pressure-natriuresis relationship to
restore the adequate sodium and water excretion, but at a cost of an elevation in arterial
pressure. Arterial hypertension in turn leads to an increase in glomerular capillary pressure in
remaining glomeruli. The remaining nephrons would adapt to the increase in glomerular
capillary pressure leading to glomerular hyperfiltration and glomerular and tubular
hypertrophy.216,312 Prolonged glomerular hypertension, glomerular hyperfiltration and
glomerular hypertrophy would lead to glomerular injury such as glomerulosclerosis and thus
further loss of nephron and FSA, continuing this vicious cycle (Figure 1.6).
Chapter 1 General Introduction
28
Figure 1.6. Brenner Hypothesis. A schematic illustration of a vicious cycle of progressive nephron loss, renal injury and hypertension.
The Brenner Hypothesis
Low Nephron Number
êFiltration Surface Area
Sodium and Water Retention
éArterial Pressure
éGlomerular Capillary Pressure
Renal Adaptations Glomerular Hypertrophy
Vicious Cycle
Glomerular Injury
Glomerular Hyperfiltation
Chapter 1 General Introduction
29
1.6.2.1. Nephron Endowment and Hypertension
Consistent with the Brenner hypothesis, being born with a low nephron endowment is
associated with increased risk of developing hypertension later in life. In a small German
cohort, it was found that nephron number was significantly lower in those with essential
hypertension compared to normotensive controls, linking the etiology of essential
hypertension to a reduced nephron number.200 Race is also an important determinant of
nephron number. For example, a reduced nephron number has been associated with
elevated blood pressure in indigenous Australians.170,171,192 From examining the kidneys
collected at autopsy from white and African Americans, only female white Americans who
suffered from hypertension were found to have significantly lower nephron number.178
Further, in a study that investigated children who were born with a solitary kidney or with
unilateral multicystic kidneys at birth, half of them were hypertensive by 9 years of age,
indicating the significance of nephron endowment in the determination of arterial pressure in
humans.329 Animal models of programmed low nephron endowment, such as maternal low
protein diet215,375 and prenatal dexamethasone treatment263,341,374 are also associated with the
development of hypertension in adult offspring.
Despite the evidence pointing to nephron deficiency as a contributor to the
development of hypertension, it has become evident that low nephron endowment does not
always associate with hypertension, and secondary insults might be necessary for the
development of hypertension.268 As mentioned earlier, the association of low nephron
number and hypertension was only found in female white Americans, but not in male white
Americans or African Americans.178 Some renal programming models of reduced nephron
endowment even demonstrated hypotension rather than hypertension.45,271 A study from our
laboratory found that mice born with a congenital nephron deficit, the GDNF heterozygous
mice were normotensive, even up to 1-year-old.312 In support of the secondary insult
hypothesis,268 both high salt intake312 and diet-induced obesity146 caused an increase in
arterial pressure in nephron deficient GDNF heterozygous mice in a manner proportion to the
level of nephron deficit.
1.6.2.2. Nephron Endowment and Renal Dysfunction
The kidney compensates for a nephron deficit functionally by hyperfiltration to
maintain adequate GFR, and structurally through glomerular hypertrophy to normalise the
deficit in FSA.338 However, whilst these compensatory adaptations may preserve adequate
renal function in short-term, they may also predispose kidneys to greater risk of renal injury
later in life, likely due to prolonged renal hyperfiltration and elevated glomerular capillary
pressure.282,295 This has been demonstrated both in patients born with a solitary functioning
kidney and in patients that underwent unilateral nephrectomy during childhood.282,295
Chapter 1 General Introduction
30
Although these patients demonstrated significantly elevated GFR in the remaining kidney,
they exhibited a gradual reduction in renal function reserve, the ability to increase GFR when
there is an increase in metabolic demand.282,295 Consistent with this abservation, Aboriginal
Australians, a population is known to have a significant nephron deficit (30%), are more likely
to suffer from chronic kidney disease and renal failure compared to non-aboriginal
Australians.170,172
Consistent with findings in human studies, some animal models of low nephron
endowment also demonstrated a gradual deterioration of renal function over time. Woods et
al 378 demonstrated that unilateral nephrectomy in neonatal rats (before completion of
nephrogenesis) led to a reduced GFR at 20 weeks of age compared to sham animals.
Similarly, in the ovine model of fetal unilateral nephrectomy, GFR of nephron deficient sheep
was lower than control sheep as early as 6 months of age.261 In contrast, it appears that
when unilateral nephrectomy is performed in adulthood, total GFR can be restored through
hyperfiltration (increased SNGFR).335,357 Further, a study from our laboratory, using a
congenital model of low nephron endowment, found that mice born with a 65% nephron
deficit exhibited normal GFR compared with controls mice when studied at 1 year of age.312
In fact, in order to maintain a normal GFR in mice with marked nephron deficit, the calculated
SNGFR (GFR divided by nephron number) in these mice was almost four fold greater than
control mice.312 Programming models of nephron deficiency also demonstrate
inconsistencies in GFR outcomes in offspring. For example, offspring of dams fed a low
protein diet were found to have reduced GFR comparing to the controls.24 However, when
nephron deficiency was induced by maternal dexamethasone treatment, offspring have a
normal GFR through adult life.203
1.6.3. Animal Model of Low Nephron Endowment
1.6.3.1. Programed and Congenital Models of Low Nephron Endowment
There is a large literature on animal models of low nephron endowment including
maternal protein restriction, maternal undernutrition, intrauterine growth restriction and
maternal glucocorticoid exposure. These models highlight how adverse maternal
environments increase the risk of offspring developing hypertension and renal disease.
However, these maternal insults are known to impact several organs and systems within the
fetus including the heart and vasculature,44,77,117,227,280 and the central nervous systems287 in
addition to the reduction in nephron number. As these extra renal systems can also influence
arterial pressure and renal function, the extent to which a reduction in nephron endowment
per se contributes to the development of hypertension and renal disease is not well
understood.201
Chapter 1 General Introduction
31
In order to investigate the impact of reduced nephron endowment per se on renal
function and arterial pressure, it is important to choose a model in which other organ systems
are less likely to be affected. The glial cell line-derived neurotrophic factor (GDNF)
heterozygous mouse is an ideal genetic model of reduced nephron endowment that offers 2
distinct levels of nephron deficiency within the same genotype to compare to wild-type
littermates.312 In addition, these mice have normal arterial pressure and renal function at
least up to 1 year old, avoiding the impact of elevated arterial pressure on renal function.312
1.6.3.2. GDNF Heterozygous Mice
Glial cell line-derived neurotrophic factor (GDNF) is essential to both the initiation of
nephrogenesis and the branching of the ureteric epithelium that ultimately determines final
nephron number.259,286,320 Due to these two distinct roles of GDNF in kidney development,
GDNF null mice fail to form kidneys and die shortly after birth.286 However, GDNF
heterozygous (HET) mice are viable and born with either 2 small kidneys (HET-2K) and a
moderate (~30%) nephron deficit, or a solitary kidney (HET-1K) and a marked (~65%)
nephron deficit compared to wild-type (WT) mice (Figure 1.7A).312 This unique model thus
provides opportunity for a within genotype comparison of how renal function and arterial
pressure are regulated in mice with both moderate and marked nephron deficiency.
Glomeruli of HET-2K and HET-1K kidneys undergo compensatory glomerular
hypertrophy as they age such that average individual glomerular volumes of HET-2K and
HET-1K mice are 33% greater than WT mice (Figure 1.7B).312 The greater individual
glomerular volume of HET-2K mice offsets the 30% nephron deficit, resulting a similar total
glomerular volume compared to WT mice (Figure 1.7C). However, glomerular hypertrophy in
the solitary kidney of HET-1K mice does not fully offset the 65% deficit in nephron number
and thus the total glomerular volume (likely total FSA) of HET-1K mice is only 50% that in
WT mice (Figure 1.7C).312 This compensatory glomerular hypertrophy in GDNF HET mice
completes by 30 weeks of age,336 and thus studies in Chapter 5 will commence at this time
point.
Whilst the urinary excretory profile is similar between HET-2K and WT mice, HET-1K
mice had significantly higher water intake, urine excretion, and sodium and osmolar excretion
than HET-2K and WT mice (Figure 1.7E-H).312 It has also been shown that both GDNF HET-
2K and HET-1K mice remain normotensive (Figure 1.7D) and have normal GFR up to 1 year
of age.312 Given the significant nephron deficit and normal total GFR in HET-2K and HET-1K
mice, it was calculated that HET-2K mice had double the single nephron GFR (SNGFR) that
of WT mice and SNGFR of HET-1K mice was nearly 4-fold higher than WT mice.312 However
this significant hyperfiltration is not associated with any renal injury such as albuminuria
(Figure 1.7I), interstitial fibrosis, or glomerulosclerosis in HET-2K and HET-1K mice.312,336
Chapter 1 General Introduction
32
The background strain of the GDNF colony, C57BL6/J mice, which are known to be
sclerosis-resistant, may contribute to this protection.115,224,234
Chapter 1 General Introduction
33
Figure 1.7. Characteristics of GDNF WT, HET-2K and HET-1K mice on a normal-salt diet at 1-year old. A: total number of nephrons B: mean glomerular volume (Vglom). C: total glomerular volume (Total Vglom). D: 24h mean arterial pressure (MAP). E: 24h water intake. F: 24h urine excretion. G: 24h Na+ excretion. H: 24h osmolar excretion. I: 24h albumin excretion. *P<0.05 vs WT, **P<0.001 vs WT; †P<0.05 vs HET2K, ††P<0.001 vs HET2K. Figure from Ruta et al 312
Chapter 1 General Introduction
34
1.7. REGULATION OF RENAL FUNCTION IN AN ANIMAL MODEL OF
LOW NEPHRON ENDOWMENT
As highlighted earlier, some models of nephron deficit demonstrate a reduction in
GFR including unilateral nephrectomized rats378 and sheep99, and in the rat model of
maternal protein restriction.379 There are also models where GFR is well maintained during
adulthood in animals with marked nephron deficiency including the GDNF HET mice.312 In
cases where GFR is normal, a significant elevation of SNGFR must occur to compensate for
the low nephron number. In fact, calculated SNGFR of nephron deficient GDNF HET-1K
mice was nearly fourfold higher than WT mice.312 To achieve this significantly elevated
SNGFR, regulation of GFR in the kidney of GDNF HET-1K mice must be altered to favor this
adaptation. Renal hemodynamic and excretory functions are tightly regulated by the
collective effort of several regulatory systems. Nitric oxide (NO), as an important signaling
molecule, plays a prominent role in the regulation of glomerular, vascular and tubular
function.207 In this section of the literature review, the functional contribution of NO in
regulation of renal function will be discussed, so will be the renal adaptations when NO
bioavailability is reduced, particularly in the setting of low nephron endowment.
1.7.1. Role of NO in the Regulation of Renal Function
1.7.1.1. Renal NO in Normal Kidneys
Nitric oxide (NO) was originally discovered as the endothelium-derived relaxing factor
that leads to vasodilation.237 NO is formed from oxygen and the amino acid L-arginine by
nitric oxide synthases (NOS) and cofactors.343 All 3 NOS isoforms, namely neuronal NOS
(nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) contribute to the synthesis of
NO in the kidney. However nNOS and eNOS are the two isoforms that are constitutively
expressed in the kidney.343 NO has been shown to regulate renal function through regulation
of renal hemodynamics and vascular resistance, mediation of pressure natriuresis, blunting
of tubuloglomerular feedback, inhibition of tubular sodium reabsorption and modulation of
renal sympathetic activity.264 In general, renal NO production promotes natriuresis and
diuresis.264 In addition, NO deficiency both in the kidney and systemically has been shown to
contribute to the pathogenesis and progression of hypertension and chronic kidney
disease.33,264 The local expression and activity of NOS in the kidney is positively linked with
the physiological action of NO.33 In rats, the highest NOS activity was found in the inner
medullary collecting duct,380 suggesting NO is strongly associated with regulation of water
and sodium reabsorption in distal tubules.
Chapter 1 General Introduction
35
1.7.1.2. Role of NO in Nephron Deficient Kidneys
Renal NO plays an important role in the regulation of renal hemodynamic and
excretory function in nephron deficient animals. A reduced NO bioavailability has been
implicated in the progression of renal disease and hypertension in the remnant kidney
models,4,346,347 as well as model of programmed renal insufficiency.348 Further, aged female
sheep with congenital renal mass reduction in utero demonstrated reduced renal blood flow
and GFR, impaired renal execratory function and renal vascular dysfunction that were
associated with reduced contribution of NO.216
An upregulation of NO-facilitated vasodilation has been demonstrated to play a role in
maintaining renal function following renal mass reduction. A study by Valdivielso et al 357
demonstrated that an increase in renal NO production mediates the increase in renal blood
flow via a reduction in renal vascular resistance in the remnant kidney 2 days after unilateral
nephrectomy in rats, facilitating an increase in SNGFR in nephrectomized rats.335 Consistent
with this finding, Sigmon et al 338 found that urinary excretion of cyclic guanosine
monophosphate (cGMP), a marker of renal NO production, had a 2.5 fold increase up to 4
weeks post-uninephrectomy. The involvement of NO in renal vasculature of the remnant
kidney of rats following uninephrectomy was also demonstrated by a sharper increase in
renal vascular resistance and a greater fall in renal plasma flow compared with shame rats
when L-NAME, a non-selective NOS inhibitor, was administered acutely.357 An elevation in
renal NO production observed in rats which remained normotensive following renal ablation
(75%) has been demonstrated to be responsible for the greater sodium excretion and the
normotensive phenotype.13 These findings indicate that enhanced NO production could be
one of the adaptive mechanisms that maintain normal renal function and arterial pressure in
individuals with a reduced nephron number. The important role of NO has also been
demonstrated in renal injury-resistant rodents following 5/6 nephrectomy. For example, both
Wistar Furth rats113,114 and C57BL6/J mice234,265 have demonstrated strong dependence in
adequate NO bioavailability to maintain normal renal function and resistance to renal injuries.
A previous study from our laboratory has shown that nephron deficient GDNF HET
mice remain normotensive and have normal total GFR even through old age.312 It is not
known whether enhanced NO bioavailability contributes to the normal renal function and
arterial pressure, particularly in the HET-1K mice with marked 65% nephron deficit. Chapter
5 of this thesis will address this gap in our understanding of the regulation of renal function in
nephron deficient animals.
Chapter 1 General Introduction
36
1.8. HYPOTHESES AND AIMS
Chapter 3: Obesity Limits the Normal Cardiovascular and Renal Adaptations of
Pregnancy Compromising Fetal Kidney Development
Despite the significant complications associated with pre-pregnancy obesity, the
impact of obesity on the cardiovascular and renal adaptations that occur during pregnancy is
not well understood. The altered hemodynamics in obesity has the potential to impose
significant constraint on the ability of cardiovascular and renal systems to adapt during
pregnancy. Like other adverse intrauterine environments, maternal obesity has also been
shown to increase the risk of offspring developing hypertension and renal dysfunction later in
life. Given the sensitivity of the kidney to maternal insults and the fact that common maternal
complications of maternal obesity such as hyperglycemia and placental dysfunction are
associated with reduced nephron endowment, it is reasonable to predict that nephron
endowment in offspring of obese mothers might be reduced, contributing the onset of
hypertension and renal disease in adulthood. Yet the impact of pre-pregnancy obesity on
fetal kidney development has not been investigated in a robust animal model of obesity or
using appropriate techniques and thus is poorly understood.
Hypotheses:
1) Pre-pregnancy obesity limits the normal cardiovascular and renal adaptations
of pregnancy, contributing to poor maternal and fetal outcomes
2) Pre-pregnancy obesity impacts fetal kidney development reducing nephron
endowment
Aim 1: To examine the impact of obesity on the cardiovascular and renal adaptations
during pregnancy and fetal outcomes in a robust mouse model of diet-induced obesity
Aim 2: To examine the impact of pre-pregnancy obesity on fetal kidney development
To address Aim 1, I established a mouse model of diet-induced obesity in C57BL6/J
females and used radiotelemetry (MAP and HR), cine-MRI (cardiac structure and function),
and 24 hr urine sample collected to characterize cardiovascular and renal physiology before
and during pregnancy. To address Aim 2, fetal kidneys of control and obese pregnant
C57BL6/J mice were collected at gestational age 19 and subjected to gold-standard
unbiased stereological analysis in order to estimate glomerular number, glomerular volume
and to examine general renal morphology.
Chapter 1 General Introduction
37
Chapter 4: Does Pregnancy Exacerbate the Cardiovascular and Renal Outcome
of Obesity?
Despite significant structure and functional adaptions to the cardiovascular and renal
systems during normal pregnancy, pregnancy has no long-term impact on the cardiovascular
and renal health in the mothers. In contrast, pregnancies that are complicated with
hypertension, preeclampsia and gestational diabetes are often associated with adverse
cardiovascular and renal outcomes in the mothers post-birth. Importantly, these detrimental
maternal complications are commonly observed in women who were obese prior to
conception. Recent epidemiological studies indicate that maternal obesity is an independent
risk factor for major cardiovascular events and premature mortality in women post-birth.
However, the mechanisms that how maternal obesity leads to these consequences are
unclear. Given obesity is associated with compromised cardiovascular and renal health,
whether pregnancy would exacerbate cardiovascular and renal phenotypes of obesity post-
birth is currently unknown.
Hypothesis: pregnancy exacerbates the cardiovascular and renal outcomes of obesity
post-partum.
Aim 3: To examine the impact of pregnancy on the long-term cardiovascular and renal
outcomes in obese female mice
To address Aim 3, I examined obese and control female mice at 4 weeks post-
weaning (or 7 weeks post-birth) and compared them with non-pregnant, age-matched obese
and control mice. Arterial pressure (telemetry; up to 5 weeks post-birth), conscious GFR
(FITC-sinistrin Clearance), renal excretory profile, and cardiac and renal fibrosis were
examined.
Chapter 5: The Role of Nitric Oxide in the Regulation of Renal Function and
Arterial Pressure in Nephron Deficient Mice
A reduced nephron endowment is arguably the most extensively studied renal
programming outcome and has been associated with development of hypertension and renal
disease later in life. Yet, some animal models of low nephron number including the GDNF
HET mice demonstrate remarkable renal adaptations such as a significantly elevated
SNGFR and greater natriuresis, contributing to the maintenance of normal total GFR and
arterial pressure even at an old age. However, the mechanisms that mediate these renal
Chapter 1 General Introduction
38
changes in nephron deficient kidneys are unclear. NO has been implicated as one of the
important regulators of renal hemodynamic and excretory function in both normal kidneys
and nephron deficient kidneys. In this Chapter, we used GDNF HET mouse, a genetic model
of reduced nephron endowment with two levels of nephron deficit, to investigate the
contribution of NO in the regulation of renal function and arterial pressure in nephron
deficient animals.
Hypothesis: Chronic NO deficiency leads to greater hypertension and renal
dysfunction in nephron deficient GDNF Heterozygous mice in a manner dependent on
the level of neohron deficit.
Aim 4: To determine the arterial pressure and renal function of GDNF WT, HET-2K and
HET-1K mice prior to and in response to systematic NOS inhibition with L-NAME.
To address Aim 4, arterial pressure (radiotelemetry) and renal excretory profile of
GDNF WT, HET-2K and HET-1K mice will be examined before and after systemic NOS
inhibition with L-NAME. Renal expression of RAS components, key water and sodium
channels will also be examined in L-NAME treated and untreated animals.
Chapter 2 General Methods
39
Chapter 2 GENERAL METHODS
2.1. ANIMALS
All experiments were conducted according to the Australian Code of Practice for the
Care and Use of Animals for Scientific Purposes and approved in advance by the Monash
University School of Biomedical Sciences Animal Ethics Committee.
2.1.1. Mouse Model of Pre-pregnancy Obesity: Chapter 3 & 4
For studies outlined in Chapter 3 & 4, a robust mouse model of diet-induced obesity
was established. Cardiovascular and renal phenotypes of these mice were characterized
before conception and the hemodynamic adaptations during pregnancy were assessed.
Cardiovascular and renal profiles of control and obese dams and time-controlled virgin
female control and obese mice were also assessed between 5-7 weeks post-birth.
Three week old female C57BL6/J mice were obtained from Monash Animal Research
Platform and housed in the experimental room maintained at 24-26 °C with 12:12 hour light-
dark cycle (light on at 6am & light off at 6pm). At 4 weeks of age, mice were separated into
individual cages and randomly allocated to receive either a control diet (CONT, Fat 7% w/w;
3.85kCal/g; AIN93G, Specialty Feeds, Australia) or high fat diet (HFD; Fat 23.5% w/w;
4.54kCal/g; SF04-001, Specialty Feeds, Australia). Mice received the diet treatment for a 10-
week period prior to baseline experiments. A subgroup of control and obese mice underwent
glucose tolerance tests (see Chapter 3 for details). Food intake and body weight of each
mouse were recorded weekly for the 10-week period. Separate cohorts of control and obese
mice were then allocated to each individual study accordingly. Mice continued to receive the
same diet until the end of the experimental protocol.
2.1.1.1. Mating
After baseline measurements female control and obese mice were allocated to be
mated or to remain virgin. For mating, female mice were paired with control-diet fed male
C57BL/6J mice from 12pm to 9 am the following day. Mice were examined for vaginal plugs
at 9 am. Gestational age (GA) 1 was defined as the 24hr period from the start of the light
cycle on the day of vaginal plug detection. Body weights were measured on the morning of
Chapter 2 General Methods
40
GA7, GA13 and GA19 (Chapter 3 only). Gestational length was recorded in mice allowed to
litter down.
2.1.2. GDNF Heterozygous Mouse: Chapter 5
For study outlined in Chapter 5, mice heterozygous for glial cell line-derived
neurotrophic factor (GDNF) were used as a congenital model of low nephron endowment.
GDNF wild-type (WT) mice were served as controls.
2.1.2.1. Animal Origin
Male GDNF heterozygous (HET) mice were initially received from Dr Heiner
Westphal (National Institutes of Health, Bethesda, MD, USA) to establish the colony at
Monash University through mating with C57BL/6J female mice.80,286 GDNF HET mice were
generated by replacing part of the third exon that encodes GDNF protein with a neo-cassette
expressing the selectable marker neomycin phosphotransferase.285,286 Tail tissue collected
from mice at 2 weeks of age were genotyped by polymerase chain reaction (PCR) analysis
(section 2.1.2.3). Genotypes of all mice were confirmed using tissue collected at post-
mortem. Whilst this study contained mice with only 2 genotypes, the GDNF WT and HET
genotypes, GDNF HET mice can be further divided into 2 groups based on their phenotype,
those born with 2 kidneys (HET-2K) and those born with a solitary kidney (HET-1K). Thus
study outlined in Chapter 5 contained 3 experimental groups, GDNF WT, GDNF HET-2K and
GDNF HET-1K groups. The separation of HET-2K mice and HET-1K mice could only be
achieved at post-mortem.
2.1.2.2. Housing and Diet
GDNF WT and HET mice were obtained from Monash Animal Research Platform at
26 weeks of age and group housed for 2 weeks in the experimental room maintained at 24-
26 °C with 12:12 hour light-dark cycle. Mice were allowed ad libitum access to tap water and
normal salt rodent chow (AIN93M, Specialty Feeds, Australia). A week later, littermates were
separated into individual cages and baseline experiments started at 30 weeks of age.
Chapter 2 General Methods
41
2.1.2.3. Genotyping
To distinguish and confirm GDNF WT and HET genotypes, tissues collected were
analysed by PCR.285,286 DNA extractions and PCR were performed using REDExtract-N-
AmpTM tissue PCR kit (Sigma-Aldrich, USA). Each PCR reaction consisted of following
components: 2 µl of H2O, 4 µl mixture of 4 primers (1 µg/µl, Geneworks, Australia), 4µl DNA
extract and 10 µl of REDExtract-N-Amp PCR Reaction Mix (supplied in REDExtract-N-AmpTM
tissue PCR kit, Sigma-Aldrich, USA). The sequences of primer 1-4 were285:
Primer 1 5’-CCAGAGAATTCCAGAGGGAAAGGTC-3’
Primer 2 5’-CAGATACATCCACACCGTTTAGCGG-3’
Primer 3 5’-GATCCCCTCAGAAGAACTCGT-3’
Primer 4 5’-CTGTGCTCGACGTTGTCACTG-3’
PCR amplifications were carried out using Mastercycler gradient (Eppendorf,
Germany) and amplification cycles were: 5 minutes at 95 °C; 40 cycles of 30 seconds at
94 °C, 55 seconds at 64.5 °C and 45 seconds at 72 °C; followed by 10 minutes at 72 °C and
held at 4°C until DNA gel electrophoresis.
PCR products were resolved into 3% agarose gel (Invitrogen, USA) stained with
SYBR SafeTM DNA gel stain (Invitrogen, USA) at 100 volts for 45 minutes and DNA gel was
viewed by GelDock viewer (Syngene, UK). Primer 1 & 2 generated a 338-bp fragment
representing WT GDNF allele and primer 3 & 4 generated a 567-bp fragment specifying the
mutant allele in HET GDNF genome (Figure 2.1).
Figure 2.1 DNA Bands of GDNF HET and WT genotypes.
338 bp
567 bp
HET HET WT WT HET HET
Chapter 2 General Methods
42
2.2. CONSCIOUS RENAL FUNCTION EXPERIMENTS
Conscious renal excretory profiles were assessed in Chapter 3, 4 & 5 from 24hr urine
samples collected in metabolic cages. In Chapter 4, glomerular filtration rate (GFR) was
assessed by transcutaneous FITC-sinistrin clearance method. In Chapter 5, GFR was
assessed by measuring renal creatinine clearance (Ccre) using urine obtained during 24hr
urine collection and plasma samples collected during radiotelemetry surgery (for baseline)
and tissue collection (for post-L-NAME).
2.2.1. Assessment of Urinary Excretory Profile
24hr urine samples were obtained by placing mice in glass metabolic cages purpose
build for mice (Figure 2.2). The metabolic cage consisted of a main chamber (5 cm in radius),
a food chamber, a water chamber, a splitter beneath the centre of the cage for separating
urine and faeces, and small collection vials for food shredding, unconsumed water, and urine
and faeces produced during the 24hr period (Figure 2.2). To acclimatize mice to the novel
environment of the metabolic cages, mice were placed in the cages for ~6 hours during the
daytime, 2 days prior to the 24 hours renal function experiments.
Figure 2.2 Image of Metabolic Cage
Main Chamber
Food Chamber Water Chamber
Urine Splitter
Urine Collection Vial Faeces Collection Vial
Food Shredding Vial Water Chamber
Chapter 2 General Methods
43
Prior to placing mice into the cage, food and water provided, and collection vials for
urine and faeces were weighed. Mice were weighed and placed in metabolic cages in the
morning (9-10 am) for 24 hours. At the end of the 24hr period, body weight of mice was
measured. The remaining food and water were measured to calculate 24hr food intake and
water consumption. Urine and faeces collection vials were weighed to calculate the volume
of urine and amount of faeces produced. Urine was then transferred to small centrifuge tubes
and clarified by centrifugation at 3000 RPM for 5 minutes at room temperature. Urine
samples were then analysed for urinary osmolality and electrolyte concentrations before
being stored at -20 °C for subsequent analysis of urinary albumin excretion and creatinine
clearance (See section 2.2.2).
2.2.2. Analysis of Urine and Plasma Samples
2.2.2.1. Urinary Osmolality and Electrolytes
Urinary osmolality was determined by freezing point depression method using the
osmometer (Advanced instrument 2020, Needham Heights, MA, USA). Urinary sodium (Na+),
potassium (K+) and chloride (Cl-) concentrations were measured by ion-selective electrodes
using EasyElectrolytes analyser (MEDICA, MA, USA). Values of urinary osmolality
(mOsmol/Kg H2O) and electrolyte concentrations (mmol/L) of each sample were then
multiplied by 24 hr urine volume to obtain 24hr excretion data, which was used to present
urinary excretory data.
2.2.2.2. Albumin Assay
Urinary albumin concentration was determined by Albuwell M kit (Exocell, PA, USA).
Albuwell M is a widely used enzyme-linked immunosorbent assay (ELISA) for quantitative
determination of albumin in mouse urine.224,312,366
2.2.2.2.1. Assay Principle
To complete the assay, albumin standard solution, urine sample and rabbit anti-
murine albumin antibody (primary antibody) are added to albumin-coated wells in a 96-well
plate. This primary antibody competitively binds to either the albumin immobilised to the
stationary phase or with that in the fluid phase (urine sample or standards). Following
washing, an anti-rabbit horseradish peroxidase (HRP) conjugate antibody (secondary
antibody) is added to the well to label the primary antibody in the stationary phase. This
Chapter 2 General Methods
44
antibody conjugate is then detected using a chromogenic reaction. The colour intensity of the
reaction is inversely proportional to the logarithm of albumin concentration in the samples.
2.2.2.2.2. Assay Procedure
Prior to the assay, urine samples were thawed to room temperature, and well mixed
before being clarified by a quick centrifugation (30sec at 3000x RPM).314 Diluted Murine
Serum Albumin (MSA) standards and urine samples were added to the 96-well plate in
triplicates using a manual micropipette. Rabbit anti-murine albumin antibody was then added
to all wells across the plate except the blank and the plate was incubated for 30 min at room
temperature. The plate was then wash with wash buffer (pH 6.8, 0.15M NaCl, 0.01M
triethanolamine, 0.05% Tween 20) for 10 cycles using a plate washer (TriContinent, CA, USA)
before each well was incubated with anti-rabbit HRP conjugate for another 30 min at room
temperature. A 10-cycle plate washing was performed again. Colour developer solution was
then added to each well and incubated for 10 min in dark at room temperature before the
colour stopper solution was added to each well. Absorbance was measured using a plate
reader at 450nm (BioRad 3350, Tokyo, Japan). Urine samples were diluted in a range of 1:3
to 1:5 to ensure values of absorbance fell within the range of the standard curve.
2.2.2.2.3. Data Analysis
The best-fit line was plotted with the log10 [MSA] on the x-axis and mean absorbance
(A450) on the y-axis in Microsoft Excel and equation of the standard curve was obtained as
shown below.
Log10 [MSA] = m A450 + b
Where m is the gradient of the standard curve generated and b is where the standard
curve intersects the y-axis. The absorbance of each sample measured was subjected to the
above standard curve to obtain log10 [MSA], which was then converted to albumin
concentration of each sample added to the well via the semi-logarithmic relationship. This
concentration of albumin was adjusted for dilution factor and then multiplied by 24hr urine
volume to obtain 24hr albumin excretion.
2.2.2.3. Creatinine Clearance (Chapter 5)
Creatinine concentrations of urine and plasma samples obtained at baseline and
post-L-NAME treatment (Chapter 5) were measured using high performance liquid
Chapter 2 General Methods
45
chromatography (HPLC) by A/Prof Merlin C Thomas’s group at the Baker IDI (Melbourne,
Australia).54,102 The creatinine clearance (Ccr) was determined using following equation:
Ccr =����Urine creatinine concentration ����µmol/L���� x Urine volume(ml/min)����
Plasma creatinine concentration (µmol/L)
2.2.3. Transcutaneous Measurement of GFR in Conscious Mice (Chapter 4)
The transcutaneous measurement of GFR using the FITC-sinistrin clearance is a new
technique that allows researchers to repeatedly measure GFR in conscious rodents including
mice, without serial sampling of urine or plasma, or laboratory assays of renal markers.326-328
Our optimised experimental protocol and methodological considerations of this newly
developed technique were published recently.111 This technique was used in study outlined in
Chapter 4. This technique, however, was not adopted in the study outlined in Chapter 5 as it
was not available in our laboratory at the time the study commenced.
2.2.3.1. Principle of Transcutaneous Measurement of GFR
Fluorescein-isothiocyanate labeled sinistrin (FITC-sinistrin) is a commercially
available exogenous marker of renal function. Sinistrin, like inulin, is freely filtered by the
glomerulus, but is not secreted or reabsorbed by the renal tubules, and not metabolized by
the body, making it an ideal renal marker to measure GFR. The NIC-Kidney Device
(Mannheim Pharma and Diagnostics, Mannheim, Germany) is a miniaturized optical device
that records the fluorescent emission of FITC-sinistrin through the depilated skin of the
mouse. Data recorded on the device over an experimental period was used to generate an
elimination kinetics curve of FITC-sinistrin (Figure 2.5). The half-life of FITC-sinistrin (t1/2) was
determined by the relative fluorescent emission signal detected by the device in the
corresponding to the single exponential excretion phase of the elimination kinetics curve
(Figure 2.5). As FITC-sinistrin is exclusively filtered by the glomerulus, the linear relationship
between the t1/2 of FITC-sinistrin and GFR can be calculated using a pre-established
conversion factor (14616. 8).328
GFR ����(μl/min)/100gBW���� =14616.8
t1/2 (min)
Chapter 2 General Methods
46
2.2.3.2. Experimental Protocol
2.2.3.2.1. Fur Depilation
One day prior to GFR measurement, mice were briefly anaesthetized by isoflurane
inhalation (~8min; Phodia, Australia), which was delivered by an anaesthesia unit (Univentor
Ltd, Zehtun, Malta) at a concentration of 4.9 % for induction and 2.1-2.4 % for maintenance
during the procedure. A small area of fur (~ 2 x 2 cm) on the flank of the mouse was shaved
using an electric shaver. Hair removal cream (Veet, Reckitt Benckiser, Australia) was then
applied and removed less than 2 minutes later with the use of a spatula. Warm water was
used to wash off the residual hair removal cream. If black pigmentation (commonly seen in
C57BL6/J mice) was present in the area where the hair was depilated, the opposite flank of
the mouse was used as this pigmentation may affect the detection of florescent signal
through the skin.
2.2.3.2.2. Experimental Procedure
The NIC-Kidney Device was adhered to one side of a double-sided adhesive patch
positioning the LEDs within the transparent window of this patch (Figure 2.3). FITC-sinistrin
was dissolved in sterile saline to make stock solution of 10-15 mg/ml. The dose of FITC-
sinistrin administered was 6-10 mg/100 g bodyweight.
Figure 2.3 Adhesive patch for device, NIC-Kidney Device and Battery. Figure from Ellery et
al 111
Chapter 2 General Methods
47
Mice were lightly anaesthetized (isoflurane). A strip of adhesive tape (10-15 cm long
& 2cm wide; Leukosilk® tape; BSM Medical, Luxembourg) was placed under the abdomen of
the mouse. The battery was then connected to the NIC-Kidney Device (Figure 2.3) and was
then mounted on top of the device using a small piece of the double-sided tape. The
powered NIC-Kidney Device was then adhered to the depilated skin area (Figure 2.4A), and
secured in place by the tape strip underneath the body to minimize movement artifact (Figure
2.4B).
Figure 2.4 Example of securing NIC-Kidney Device and battery on the back of the mouse. A: NIC-Kidney Device and battery adhered to depilated skin on the back. Battery is connected and mounted on top of the device. B: The NIC-Kidney Device was secured in place by adhesive tape while data were recording in free moving mouse in its home cage.
While the background signal was recorded (3-4 min), a latex glove filled with warm
water (50-60°C) was placed on top of the tail to warm the tail and assist dilation of the tail
vein for injection. FITC-sinistrin was then injected intravenously (tail vein) and mice were
placed back in their home cage (Figure 2.4B). Elimination kinetics of FITC-sinistrin was then
recorded in the conscious mouse for 1 hour (Figure 2.4B). At the end of the 1hr-recording
period, the tape and device were removed under brief anesthesia (isoflurane; <3 mins). Mice
were then returned to home cage for recovery.
Chapter 2 General Methods
48
2.2.3.3. Data Analysis
Device was connected to a computer and the Data analyzed by NIC-Kidney Device
software (Mannheim Pharma and Diagnostics, Mannheim, Germany) to generate the
elimination kinetics curve (Figure 2.5). A horizontal dotted line was placed along the
background signal to set the baseline signal level and a vertical dotted line was placed ~15
min post-injection to define the point where the analysis of elimination started (Figure 2.5).111
Signal was cropped at 1hr post-injection. The t1/2 of FITC-sinistrin was then determined from
the area between the vertical line (~15 min post-injection) and 1hr post-injection time by the
NIC-Kidney Device software. In cases of FITC-sinistrin solution was injected subcutaneously,
experiments were aborted and repeated the next day.
Figure 2.5 Example of FITC-sinistrin elimination kinetics curve
Chapter 2 General Methods
49
2.3. CONSCIOUS BLOOD PRESSURE MEASUREMENTS
2.3.1. Implantable Telemetry System for Mice
The implantable telemetry system and PhysioTel®PA-C10 transmitter developed by
Data Sciences International (DSI, St Paul, MN, USA) provide accurate and reliable
measurements of systolic, diastolic, and mean arterial pressures (MAP) as well as heart rate
(HR) and locomotor activity in free moving mice in their home cages.52 The receivers placed
underneath the home cages detects digitized data via radio frequency signal sent from the
transmitters and converts data into a form readily accessible by Dataquest A.R.T.TM System
(DSI, St Paul, MN, USA).
The PhysioTel®PA-C10 transmitter consists of a 5 cm-long fluid-filled catheter with a
thick-walled tip, a thermoplastic body contains electronics, sensor and battery, weighing 1.4
gram in total.255 The lumen of the catheter is filled with a low viscosity fluid. The distal 2mm of
the thin-walled tip is filled with a blood-compatible silicone gel that prevents blood from
entering the catheter lumen and clotting, allowing efficient transmission of high frequency
components of the pressure signal into the lumen of the catheter.255
2.3.2. Implantation Surgery
Mice were anaesthetised (isoflurane) and placed on a heated surgical pad to maintain
body temperature during surgery. A ventral midline incision (1-1.5cm) was made over the
neck region. Through this incision, a subcutaneous pouch was created along the right flank
using blunt dissection, for placement of the transmitter body. The left common carotid artery
was isolated and placed over curved forceps (tips 0.5 cm apart) to occlude blood flow. Three
sterile silk ligatures (Size 6-0, Dynek, Australia) were passed underneath the artery and tied
loosely around the artery. The silk tie most proximal to the head (back tie) was used to
permanently ligate the artery. A tiny incision on the artery was made using microscissors. A
small blood sample (~75 µl) was collected in a haematocrit tube through the incision and
plasma sample was used for measurement of creatinine clearance (section 2.2.2.3, for
Chapter 5 only). The catheter of the transmitter was then inserted through the incision on the
artery and advanced towards to the heart until the notch on the catheter matches the position
of the back tie. This placement ensures that the pressure-sensing tip of the catheter sits
inside of the aorta arch. The sound of pulse that generated by an AM radio was used to
Chapter 2 General Methods
50
confirm the catheter is in the artery. The catheter was secured with two silk ligatures proximal
to the heart and with the back tie at the notch to secure the catheter in place (Figure 2.5).
Figure 2.5 Implantation of Radio-telemetry Transmitters in Mice and Position of the End of Transmitter Catheter Within the Aorta Arch. Figure from Butz & Davisson52
Sterile saline (~ 1 ml) was infused into the subcutaneous pouch in the right flank and
the transmitter body inserted into the pouch (Figure 2.5). The incision in the neck region was
closed with 6-8 interrupted sutures (5-0 with needle; Dynek; Australia). Before the last suture,
10 µl of antibacterial solution (80 mg/ml of Trimethoprim, 400 mg/ml Sulfadiazine,
Trabactral®, JUROX, Australia) were administered under the skin. Mice also received
Carprofen (5 mg/kg body weight; Rimadyl® Injection, Pfizer Animal Health Group, Australia)
subcutaneously to reduce pain and inflammation. Mice were allowed to recover from surgery
in their home cage on a heated pad before they were returned to the experimental room.
Sutures were checked daily and body weights of mice were closely monitored post-surgery.
Mice were allowed to recover for 10 days before commencing baseline recording.
Chapter 2 General Methods
51
2.4. POST-MORTEM TISSUE COLLECTIONS
2.4.1. Maternal and Fetal Tissues at GA19 (Chapter 3)
At GA19, control and obese dams were weighed and anaesthetized (isoflurane). A
midline incision was made to expose the abdominal cavity. The number of viable and non-
viable fetuses and implantation sites were recorded and their location in the uterus identified.
One embryonic sac at a time, the fetus and placenta were rapidly removed and weighed.
Presence of meconium of fetal membranes and visible placental thrombosis were recorded.
Fetuses were sexed and the left fetal kidneys fixed in 4% paraformaldehyde (Sigma-Aldrich
Corp. St. Louis, MO, USA) for 2 hours then transferred to 70% ethanol until processing for
stereological analysis (see section 2.5). Fetal head width was measured between the ears
using a digital caliper (EDC-153, PI Manufacturing, Walnut, CA, USA).
Following collection of all fetal tissues, an arterial blood sample was taken from the
carotid artery of the dam for measurement of plasma free fatty acid and triacylglycerol levels
(see Chapter 3 supplement methods for details). Kidneys were rapidly excised, decapsulated,
weighed and cut into 4 equal portions transversely and immersion fixed in 10% buffered
formalin (Sigma-Aldrich, USA). One of middle portions of left kidney was processed for
histological analysis of collagen deposition (Picrosirius red staining; Section 2.7.2.1.2). The
heart was excised, and atria and ventricles were separated and weighed and all values
reported relative to tibia length.246 Ventricles (left and right) were cut in half transversely. Top
half of the ventricles were fixed in 10% buffered formalin for histological analysis of collagen
deposition (Picrosirius red staining, Section 2.7.2.1.3). Pericardial fat and liver were also
weighed. Age matched virgin control and obese mice were used as time controls.
2.4.2. Maternal tissues at 4WPW (Chapter 4)
At 4 weeks post-weaning (4WPW) or 7 weeks post-birth, control and obese dams and
age matched virgin mice (time controls) were weighed and anaesthetized (isoflurane). An
arterial blood sample was taken from the carotid artery to determine maternal plasma free
fatty acid and triacylglycerol levels (see Chapter 4 for details). Kidneys were rapidly excised,
decapsulated, weighed and cut into 4 equal portions transversely and immersion fixed in 10%
buffered formalin (Sigma-Aldrich, USA). One of middle portions of left kidney was processed
for histological assessment of glomerulosclerosis (PAS staining; Section 2.7.2.2). All 4
portions of the right kidney were snap-frozen in liquid nitrogen. One of the two middle
portions of the right kidney was used for measurement of collagen content by hydroxyproline
Chapter 2 General Methods
52
assay (section 2.7.1). The heart was excised and both atria were removed. Left ventricles
were separated and weighed and cut through the transverse plane into 3 equal portions.
Basal and apical portions of the left ventricle were then snap-frozen in liquid nitrogen. Apical
portions of the left ventricle were used for measurement of collagen content by
hydroxyproline assay (Section 2.7.1). The middle portion of the left ventricle was fixed in 10%
buffered formalin for histological analysis of collagen accumulation (Picrosirius red staining;
Section 2.7.2.1.3). Pericardial fat pad, gonadal fat pad (visceral fat pad), inguinal fat pad
(subcutaneous fat) and liver were also weighed.
2.4.3. GDNF HET Mice (Chapter 5)
Following post-L-NAME renal function experiments, mice were weighed and
anaesthetised (isoflurane). A terminal arterial blood sample was taken from the right common
carotid artery and plasma was used to analyse creatinine clearnace (Section 2.2.2.3). For
animals with two kidneys (GDNF WT and HET-2K mice), left kidney was excised, weighed,
and cut into 4 equal portions through transverse plane and immersion fixed in 4%
paraformaldehyde (Sigma-Aldrich, USA) for examination of general renal histology. The right
kidney was excised, weighed, and cut into equal 4 portions transversely. All portions of the
right kidneys were frozen in liquid nitrogen and one of the middle portions was used for RT-
PCR analysis (Section 5.2.4). In the case of the solitary kidney (GDNF HET-1K mice), the
kidney was excised, weighed, cut through transverse plane into 3 portions. The middle
portion of the kidney was frozen in liquid nitrogen for PCR analysis and the remaining
portions were immersion fixed in 4% paraformaldehyde for examination of general renal
histology. The heart was excised and atria removed, and left and right ventricles were
separated and weighed. Toes were collected to confirm the genotypes by PCR (Section
2.1.2.3).
Chapter 2 General Methods
53
2.5. STEREOLOGY
In chapter 3, fetal kidneys collected at GA19 were processed for stereological
analysis. The physical disector/fractionator stereological technique is considered the gold-
standard method for estimating nephron number in the adult kidney.83 In this project, a
recently developed variation of this technique was used for estimating number of developing
nephrons.82 This method utilizes the lectin peanut (Arachis hypogaea) agglutinin (PNA) to
histochemically identify podocytes within the glomeruli, from early S-shaped bodies through
to mature glomeruli. Once these PNA-positive structures are unambiguously identified in
systemically selected sections, the physical disector/fractionator method is then used to
accurately estimate total glomerular number (Nglom), and thus total nephron number.
2.5.1. Processing, Embedding and Sectioning of Fetal Kidneys
Kidneys were placed into microcassettes and dehydrated through a series of graded
ethanol baths and then infiltrated with paraffin wax. Before kidneys were embedded in the
mold with paraffin, a thin layer of paraffin was placed on the base of the mold to ensure that
the full block face will be obtained prior to collecting the first tissue sections. The mold was
then filled with paraffin and left to solidify.
The entire block was exhaustively sectioned at 4 µm and every section was collected.
The ribbons of sections were then transferred onto warm water bath. All sections were
collected in a similar orientation on Poly-L-Lysine-coated glass slides and allowed to dry. The
serial sectioning was performed by Dr Luise Cullen-McEwen.
2.5.2. Sampling Sections
Pairs of sections evenly spaced across the kidney were required to estimate
glomerular number. A sampling fraction was determined from the total number of sections
cut to achieve approximately 10-12 pairs of sections per kidney. Each pair of sections
consists of the n (reference section) and the n+2 (lookup section) sections. For example, if
250 sections were cut, every 25th and 27th (n=25) sections were selected, for 300 sections
every 30th and 32nd (n=30) were selected, and so on. The first section was chosen at
random (with use of a random number table) within the interval selected (i.e. 1 to n). Slides
with required sections were then selected for PNA staining.
Chapter 2 General Methods
54
2.5.3. Histochemical Staining with A. hypogaea PNA
1) Sections were deparaffinised through a series of three xylene washes and brought to
water through a series of two 100% alcohol washes and one 70% alcohol wash.
Slides were then rinsed in phosphate-buffered saline (PBS, 0.01M pH7.4, made from
PBS tablets, Medicago AB, Uppsala, Sweden).
2) Slides were incubated for 10 minutes in 2% H2O2 (Sigma-Aldrich, St Louis, MO, USA)
methanol solution and then followed with two 5-min washes in PBS.
3) Slides were then incubated for 30 min at 37°C with 0.1u/ml Neuraminidase (Sigma-
Aldrich, St Louis, MO, USA) with 1% CaCl2 in PBS, 100µl per slide. Slides were then
rinsed three times with PBS.
4) Non-specific binding was blocked by incubating sections with 2% BSA (Albumin from
Bovin serum; Sigma-Aldrich, St Louis, MO, USA) and 0.3% Triton X-100 (Sigma-
Aldrich, St Louis, MO, USA) in PBS for 30 min at room temperature (RT).
5) Without washing off BSA, slides were incubated for 1-1.5h with 20µg/ml biotinylated
PNA (Sigma-Aldrich, St Louis, MO, USA) diluted in 0.3% Triton X-100 in PBS, with
1mM CaCl2/MnCl2/MgCl2, at 37°C (also can incubate for 2h at RT or overnight at 4°C),
100µl per slide. Slides were then rinsed three times with PBS.
6) Slides were incubated with Avidin/biotin complex (ABC, from Vectastain ABC kit,
Vector Laboratories, Burlingame, CA, USA) for 1h at RT, 100µl per slide.
7) PNA staining was then developed with DAB and 0.01% H2O2 in PBS, 100µl per slide.
The color developing was confirmed under the microscope and was stopped by
placing slides in PBS washing chamber.
8) Slides were then rinsed two times with PBS before being conterstained with
haematoxylin for 20 sec.
9) Slides were washed under running tap water until the water runs clear and then
placed in Scott’s tap water for 1 min to turn sections blue.
10) Sections were then dehydrated through three washes of 100% alcohol and followed
by three washes of xylene before being coverslipped with DPX mounting media.
11) Slides were allowed to dry at room temperature before counting PNA-positive glomeruli.
Chapter 2 General Methods
55
2.5.4. Counting PNA-positive Glomeruli
Section pairs (n & n+2) were projected one at a time, at magnification of 180x onto a
piece of paper on a table in a darkened room using a microscope modified for projection
(Figure 2.6A). Developing glomeruli (PNA-positive structures in brown) from S-shaped
bodies to fully developed glomeruli were identified and marked with open circles on the paper
(Figure 2.6B). For those sections that were too large to be fully projected within the field of
view, this step was taken in stages by moving section from one side to the other using
already circled glomeruli as reference points until the entire section was analyzed. Once all
PNA-positive structures were marked on reference section, the projection was replaced with
the lookup section.
The glomeruli present in the reference section (circles on paper) were used as
reference to align the lookup section. Glomeruli that were present in the reference section
that were no longer present in the lookup section were identified as disappearing glomeruli.
The disappearing glomeruli were identified by filling the original open circles (black close
circles; Figure 2.6D). Glomeruli that were present in the reference section that were still
present in the lookup section were not counted and remained as open circles. Glomeruli that
were present in the lookup section that were not present in the reference section were
identified as appearing glomeruli. The appearing glomeruli were marked as close circles in
an alternate color (red close circles; Figure 2.6D). This process was repeated for each of the
10-12 pairs of sections selected for each kidney.
Chapter 2 General Methods
56
Figure 2.6 Estimating PNA-positive nephrons. A: micrograph of PNA-positively stained reference kidney section (n). B: All nephrons with PNA-positive podocytes are marked with open circles. C: micrograph of PNA-positively stained lookup kidney section (n+2). D: micrograph of lookup section with overlay of glomeruli identified on the reference section. Glomeruli present in the reference section not present in the lookup section are marked (disappearing glomeruli; 3 filled black circles). Glomeruli not present in the reference section but present in the lookup section are also marked (appearing glomeruli; filled red circles). Bar
= 150µm. Figure from Cullen-McEwen et al. 81
Sum of the total number of disappearing and appearing glomeruli (��� in all the
section pairs were calculated and used to calculate the total nephron number (Nglom) using
the following equation:
Nglom=1
SFF×
1
2×
1
2×Q
-,
Where Nglom is the total number of PNA-positive developing nephrons in the kidney and
1/SSF is the reciprocal of the section-sampling fraction (number of sections advanced
between section pairs). The first ½ accounts for the fact that the dissector pair of the sections
consisted of the n and the n+2 sections. The second ½ accounts for the fact the PNA-
positive structures were counted in both directions between the section pair. �� is the sum of
the total number of disappearing and appearing glomeruli in all section pairs.
Chapter 2 General Methods
57
2.5.5. Estimating Kidney Volume
Kidney volume was estimated from PNA stained kidney sections using the Cavalier
Principle.80,141 Each reference section used for estimating glomerular number was projected
at 72x magnification with full view of the each kidney on a table in the darkened room. A
stereological grid (2 x 2 cm) printed on a transparent sheet was overlaid on the projected
image. The total number of grid points landing on renal tissue was counted. Kidney volume
was then estimated using following formula:
Vkid = ∑P× a����p���� × T × 1
f
Where Vkid is the total kidney volume, ΣP is the total number of grid points counted, a(p) is
the area associated with each grid point and T is the section thickness, and 1/f is the inverse
of the section sampling fraction used.
2.6. CARDIAC CINE
In Chapter 3, female control and obese mice underwent cardiac cine
ventricle chamber dimensions and cardiac function at baseline and on GA14. This MRI
technique designed specific for
measurements of cardiac geometry across multiple cardiac cycles with minimal observer
dependency.206,211 These experiments were performed by Dr Michelle Kett and Dr James
Pearson at the Monash Biomedical Imaging Facility.
2.6.1. Animal Preparation
All experiments were conducted using a 9.4 Tesla small Animal MRI system (Agi
Technologies, CA, USA) at Monash Biomedical Imaging facility. Mice underwent cardiac
cine-MRI experiment before conception (baseline) and on GA14. Mice were anaesthetised
(isoflurane) and placed in a prone position on the cradle (Figure 2.7). Two fine
electrodes were placed subcutaneously in the right arm and left leg to detect
electrocardiogram (ECG) signals through an ECG/Temperature Module sensor (SA
Instruments Inc, NY, USA). A transducer sensor was placed on the lower back of the mouse
to monitor respiration via a Respiration/2
Once clear ECG and respiration signals were established, the cradle was then inserted into
the MR scanner for imaging. Mice were kept at an ambient temperature of 30
MRI Scanner.
Figure 2.7 Animal setup on the 9.4 Tesla small Animal MRI system. Mice were anaesthetised and placed in the cradle in a prone position. ECG electrodes were implanted subcutaneously into the right fore limb and left hind limb, and alower back of the mouse for respiratory recording.
Chapter 2 General Methods
58
CARDIAC CINE-MAGNETIC RESONANCE IMAGING (Chapter 3)
In Chapter 3, female control and obese mice underwent cardiac cine
ventricle chamber dimensions and cardiac function at baseline and on GA14. This MRI
technique designed specific for mice provides accurate, reproducible and repeated
measurements of cardiac geometry across multiple cardiac cycles with minimal observer
These experiments were performed by Dr Michelle Kett and Dr James
Pearson at the Monash Biomedical Imaging Facility.
Animal Preparation
All experiments were conducted using a 9.4 Tesla small Animal MRI system (Agi
Technologies, CA, USA) at Monash Biomedical Imaging facility. Mice underwent cardiac
MRI experiment before conception (baseline) and on GA14. Mice were anaesthetised
(isoflurane) and placed in a prone position on the cradle (Figure 2.7). Two fine
electrodes were placed subcutaneously in the right arm and left leg to detect
electrocardiogram (ECG) signals through an ECG/Temperature Module sensor (SA
Instruments Inc, NY, USA). A transducer sensor was placed on the lower back of the mouse
onitor respiration via a Respiration/2-CH IBP module (SA Instruments Inc, NY, USA).
Once clear ECG and respiration signals were established, the cradle was then inserted into
the MR scanner for imaging. Mice were kept at an ambient temperature of 30
Animal setup on the 9.4 Tesla small Animal MRI system. Mice were anaesthetised and placed in the cradle in a prone position. ECG electrodes were implanted subcutaneously into the right fore limb and left hind limb, and a sensor was placed on the lower back of the mouse for respiratory recording.
Chapter 2 General Methods
MAGNETIC RESONANCE IMAGING (Chapter 3)
In Chapter 3, female control and obese mice underwent cardiac cine-MRI to assess left
ventricle chamber dimensions and cardiac function at baseline and on GA14. This MRI
mice provides accurate, reproducible and repeated
measurements of cardiac geometry across multiple cardiac cycles with minimal observer
These experiments were performed by Dr Michelle Kett and Dr James
All experiments were conducted using a 9.4 Tesla small Animal MRI system (Agilent
Technologies, CA, USA) at Monash Biomedical Imaging facility. Mice underwent cardiac
MRI experiment before conception (baseline) and on GA14. Mice were anaesthetised
(isoflurane) and placed in a prone position on the cradle (Figure 2.7). Two fine needle
electrodes were placed subcutaneously in the right arm and left leg to detect
electrocardiogram (ECG) signals through an ECG/Temperature Module sensor (SA
Instruments Inc, NY, USA). A transducer sensor was placed on the lower back of the mouse
CH IBP module (SA Instruments Inc, NY, USA).
Once clear ECG and respiration signals were established, the cradle was then inserted into
the MR scanner for imaging. Mice were kept at an ambient temperature of 30-32°C within the
Animal setup on the 9.4 Tesla small Animal MRI system. Mice were anaesthetised and placed in the cradle in a prone position. ECG electrodes were implanted
sensor was placed on the
Chapter 2 General Methods
59
2.6.2. MRI scan
Once the mouse was placed in the centre of the magnetic field, a series of steps was
carried out to determine the long axis of the heart before establishing a short axis for imaging
the left ventricle.
1) An initial scout scan was performed to adjust the position of the mouse within the
scanner to maximize sensitivity. A 3D Shimming was then conducted to fine-tune the
magnetic field of the gradient and transmitter coils at the center of the view, in order
to optimizes the radiofrequency intensity and contract of the signal from the heart.
2) A short axis view from the oblique angle was then obtained for the localization of the
long axis of the left ventricle in the sagittal plan.
3) The long axis of the left ventricle was located through the sagittal plane, running from
the apex of the heart thought to the aortic valve.
4) The short axis plane perpendicular to the long axis was then determined.
5) Finally, a 4-chamber view was established through the sagittal and coronal planes
and final adjustment was made to finalize the orientation of the short axis.
The images of the heart were captured in across 7 to 8 slices of 1 mm thickness
covering the entire ventricle from the aorta to the apex of the heart (Figure 2.8 A & B). An
evenly spaced 25 frames within each slice were captured across one cardiac cycle (Figure
2.8 C). These images was then used to analyze left ventricular mass, regional wall thickness
(anterior, posterior and septum), end diastolic volume, and end systolic volume.
Figure 2.8 The 4-chamber view of mouseslices of 1mm thickness covering the aorta to the apex of the heart (represents an individual slice the image taken. From each slice (across a cardiac cycle (C)
2.6.3. Data Analysis
The MRI images were
Sweden; http://segment.heiberg.se
outlined, and end-diastolic and end
calculated as the differenc
(ESV). Ejection fraction was determined by the fraction of EDV that were ejected from each
heart beat (percentage of SV/EDV). Cardiac output was calculated by multiplying SV with HR.
Left ventricular mass (LVM) was derived from chamber wall volume in assuming a density of
the cardiac muscle of 1.06g/ml. The middle short
determine wall thickness of the septum, anterior and posterior wall.
Chapter 2 General Methods
60
chamber view of mouse heart in determination of short axis plane for 7slices of 1mm thickness covering the aorta to the apex of the heart (represents an individual slice the image taken. From each slice (B), 25 frames were captured
The MRI images were analyzed using Segment software (V1.9 R2626, Medviso,
http://segment.heiberg.se).156 The epicardium and endocardium of all slices were
diastolic and end-systolic volumes determined. Stroke volume (SV) was
calculated as the difference between end-diastolic volume (EDV) and end
(ESV). Ejection fraction was determined by the fraction of EDV that were ejected from each
heart beat (percentage of SV/EDV). Cardiac output was calculated by multiplying SV with HR.
ular mass (LVM) was derived from chamber wall volume in assuming a density of
the cardiac muscle of 1.06g/ml. The middle short-axis slice of each heart was used to
determine wall thickness of the septum, anterior and posterior wall.
Chapter 2 General Methods
heart in determination of short axis plane for 7-8 slices of 1mm thickness covering the aorta to the apex of the heart (A). Each blue line
), 25 frames were captured
using Segment software (V1.9 R2626, Medviso,
The epicardium and endocardium of all slices were
systolic volumes determined. Stroke volume (SV) was
diastolic volume (EDV) and end-systolic volume
(ESV). Ejection fraction was determined by the fraction of EDV that were ejected from each
heart beat (percentage of SV/EDV). Cardiac output was calculated by multiplying SV with HR.
ular mass (LVM) was derived from chamber wall volume in assuming a density of
axis slice of each heart was used to
Chapter 2 General Methods
61
2.7. ASSESSMENT OF COLLAGEN CONTENT
2.7.1. Hydroxyproline Colorimetric Assay (Chapter 4)
Hydroxyproline is a common nonproteinogenic animo acid and it represents ~14.4%
of the amino acids in collagen.125 Therefore, hydroxyproline content in tissue hydrolysates is
a direct representation of the total collagen content of the tissue. Using the Hydroxyproline
Colorimetric Assay Kit (BioVision, CA, USA), hydroxyproline concentration was determined
by the reaction of oxidized hydroxyproline with 4-benzaldehyde (DMAB), which results in a
colorimetric (579nm) product, proportional to the hydroxyproline present in the tissue.
2.7.1.1. Preparing Tissue Hydrolysates
Kidneys (middle quadrant) and left ventricle tissue (apical portion) were dried
overnight at 62°C. Dried tissues were weighed and homogenized in a volume of dH2O
according to the dry weight of the each sample (200µl of dH2O for every 10mg of dry tissue).
Homogenates were then centrifuged for 5 min at 1250 RPM at room temperature. To a 100µl
of sample homogenate, 100µl of concentrated HCl was added. Samples were then
hydrolyzed at 120°C for 3 hours.
2.7.1.2. Colorimetric Reaction and Absorbance Measurement
Serial dilutions of hydroxyproline standard solution were preformed and each
concentration of standard was added to a 96- well plate in duplicate. Each hydrolysates was
then added to 96-well plate and subsequently dried to eliminate HCl. Chloramine T reagent
was added to each well and incubated for 5 min at room temperature. DMAB reagent was
then added to each well and incubated at 60°C for 20 minutes. A plate reader was used to
determine absorbance at 570 nm. A standard curve was generated using the absorbance
obtained and known concentration of diluted hydroxyproline standard. The slope of this
standard curve was used to convert absorbance to hydroxyproline concentration for each
sample. The dilution factors and tissue weights were then used to calculate hydroxyproline
content in the tissue.
Chapter 2 General Methods
62
2.7.2. Histopathological and Microscopic Analysis of Collagen Content in
Cardiac and Renal Tissue.
2.7.2.1. Assessment of Collagen Content using PSR Staining
2.7.2.1.1. Picrosirius Red Staining
To visualise the accumulation of collagen in the cardiac and renal tissue, picrosirius
red (PSR) was used to stain for collagen fibres. The fixed left kidneys and left ventricles were
processed and embedded into paraffin block. Tissue blocks were sectioned at 4 µm and full-
face sections were collected, flattened and dried on a glass slide. Tissue sections were
submerged in Bouin’s fixative for 1 hr at 60°C to stain the cytoplasm in yellow. Excess
Bouin’s fixative was washed off using running water and counterstained in 0.1% picrosirius
red solution for 1 hr at room temperature. The sections were washed in running water and
rehydrated through 3 changes of graded ethanol and xylene. Slides were coversliped with
DPX mounting media and left to dry overnight at room temperature.
2.7.2.1.2. Renal Collagen Content (Chapter 3)
Renal interstitial collagen content was assessed using PSR stained kidney sections
as described previously.180 PSR stained kidney sections was visualised using a polarizing
microscope (Abrio, Hinds Instrument, USA) and images acquired (CCD camera). Eight
consecutive non-overlapping fields were selected across each kidney section. In the LPS
processed image, light retarded by collagen bundles appeared white, with a maximum
retardance set at 34nm. All images were semi-quantified based on the proportional area of
collagen birefringence to tissue area. Total collagen was calculated on the polarized images
with a minimum threshold of 35 (4.67nm). Proportional area of PSR stained collagen in all
selected fields was quantified using ImageJ analysis software (Version 1.49c; National
Institutes of Health, USA). Regions of PSR staining larger than 100 pixels were defined as
renal tubular dilation and were excluded from selected fields. Results were expressed as
percentage of PSR stained area in total area chosen.
2.7.2.1.3. Cardiac Collagen Content (Chapter 3 & 4)
PSR stained left ventricle sections were scanned by Aperio AT Turbo digital Scanner
(Leica Biosystems, IL, USA) and visualized in ImageScope Viewing Software (Leica
Biosystems, IL, USA). Eight non-overlapping fields (~0.16mm2) from the subendocardium
region around the inner lumen of the left ventricle were selected (Figure 2.9). Blood vessels
Chapter 2 General Methods
63
contained in selected fields were excluded. The percentage of area with PSR-postive
staining within the selected fields in each section was quantified using ImageScope software
(Leica Biosystems, IL, USA).127
Figure 2.9 Selection of 8 non-overlapping fields in the subendocardium region of the left ventricle for analysis of cardiac collagen content. Bar = 2mm.
2.7.2.2. Assessment of Glomerulosclerosis Using PAS Staining (Chapter 4)
The transverse middle quadrant of the fixed left kidney was processed and
embedded into paraffin block. Tissue blocks were sectioned at 4 µm and full-face sections
were collected, flattened and dried on a glass slide. Slides were then stained for Periodic
acid-Schiff (PAS) and scanned using Aperio AT Turbo digital Scanner (Leica Biosystems, IL,
USA) and visualized in ImageScope Viewing Software (Leica Biosystems, IL, USA). All
glomeruli on one full-face section were selected and the tuft circled (Figure 2.10). Number of
glomeruli within each section ranged from 90-160 and did not differ between groups. The
percentage of area with PAS-positive staining within the total glomerular tuft area selected in
each section was quantified using ImageScope software.385
Chapter 2 General Methods
64
Figure 2.10 Selection of glomerular tuft area for analysis of glomerulosclerosis using PAS staining. Bar = 100μm.
Chapter 2 General Methods
65
2.8. STATISTICAL ANALYSIS OF RESULTS
Data were plotted and statistically analysed using GraphPad Prism 6 (GraphPad
software, CA, USA). The specific statistical analysis conducted for each study was detailed in
the Methods section of each chapter. All values were expressed as Mean ± SEM. P value
<0.05 were considered statistically significant.
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
66
Chapter 3 OBESITY LIMITS THE NORMAL
CARDIOVASCULAR AND RENAL ADAPTATIONS
OF PREGNANCY COMPROMISING FETAL KIDNEY
DEVELOPMENT
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
67
OBESITY LIMITS THE NORMAL CARDIOVASCULAR AND RENAL ADAPTATIONS OF
PREGNANCY COMPROMISING FETAL KIDNEY DEVELOPMENT
Xiaochu Caia, James T Pearsona,b,c, Luise Cullen-McEwend, Jeremy Colea, Kevin Yaod,
Tracey A Gasparie, Matthew J. Wattf and Michelle M Ketta
Affiliations:
aCardiovascular Disease Program, fMetabolic Disease and Obesity Program, Biomedicine
Discovery Institute and Department of Physiology, bMonash Biomedical Imaging Facility,
dDepartment of Anatomy and Developmental Biology, eDepartment of Pharmacology,
Monash University, cAustralian Synchrotron, Clayton, Victoria, Australia.
Short title: Obesity and hemodynamic adaptations of pregnancy
Publication status: Submitted
Corresponding author:
Dr. Michelle Kett
Department of Physiology,
26 Innovation Walk
Monash University,
Clayton, Victoria, Australia, 3800.
Phone: +61 3 9905 4284
Email: Michelle.Kett@monash.edu
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
68
3.1. ABSTRACT
Maternal obesity is associated with poor maternal and fetal outcomes, yet the impact
of pre-pregnancy obesity on the normal cardiovascular and renal adaptations of pregnancy
and fetal kidney development is largely unknown and thus was the focus of this study. Four-
week old female C57BL/6J mice were fed control (7%w/w fat) or high fat (23.5%w/w fat)
chow for 10 weeks. Mean arterial pressure (MAP) and heart rate (HR; radiotelemetry),
cardiac structure and function (cine MRI) and 24hr urinary albumin excretion was assessed
before and during pregnancy. Maternal and fetal tissues were collected at GA19 and fetal
nephron number estimated (stereology). High fat feeding led to diet-induced obesity, glucose
intolerance, hypertension, tachycardia, ventricular hypertrophy and fibrosis, elevated cardiac
output and albuminuria, consistent with the obese phenotype in humans. Whilst MAP and HR
of obese mice remained elevated over control mice throughout pregnancy, the increases in
MAP and HR of obese mice during the second half of pregnancy, particularly prior to birth,
were blunted. Obese dams also failed to demonstrate an increase in cardiac output, stroke
volume or left ventricular mass by GA14, and albuminuria was exacerbated. Obesity led to
fewer viable fetuses and suboptimal fetal development at GA19 with lower body weight,
smaller kidneys, higher incidence of abnormal glomerular morphology in male and female
fetuses, and a 25% nephron deficit in male fetuses. These data indicate that obesity limits
the hemodynamic adaptations of pregnancy and leads to adverse fetal outcomes and sub-
optimal kidney development.
Key Words: obesity, hemodynamics, pregnancy, fetal outcomes, kidney development,
nephron number
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
69
3.2. INTRODUCTION
Over 50% of women of reproductive age in Australia, the USA, and the UK are
overweight or obese.2,226,273 Despite the reduced fertility rate and increased risk of
miscarriage associated with obesity, the prevalence of maternal obesity has been reported
as approximately 20% in the USA, and 13% in Australia.68,244 However recent data suggests
that the prevalence of maternal obesity may be higher (28-33%) in rural and low socio-
economic groups.84,164 Maternal obesity is associated with hypertensive disorders of
pregnancy, gestational diabetes, renal dysfunction and, importantly, higher rates of
congenital malformations, and maternal and fetal death.79 Apart from the immediate risks
during pregnancy and perinatally, maternal obesity is associated with the programming of
adult cardiovascular disease in humans299 and animals.40,116,318 Whilst the sensitivity of the
fetal kidney to adverse intrauterine environments is well recognized, the impact of maternal
obesity on fetal kidney development is poorly understood.
Pregnancy involves significant cardiovascular and renal adaptations to facilitate blood
flow to the fetoplacental unit. These changes include increases in blood volume, heart rate
(HR), stroke volume (SV), cardiac output (CO), and cardiac hypertrophy secondary to
volume load,60,75 whilst blood pressure falls from early pregnancy reaching a nadir mid-
pregnancy.60 Glomerular filtration rate (GFR) increases due to a fall in renal vascular
resistance, however this hyperfiltration is not associated with renal injury.60 Obesity also
induces significant cardiovascular and renal adaptations including increased blood volume,
HR, CO and GFR.151 However, in contrast to pregnancy, obesity is associated with
hypertension, pathological cardiac hypertrophy, and renal dysfunction including
albuminuria.151
Despite the significant complications associated with maternal obesity, no study to
date has examined the impact of obesity on the cardiovascular and renal adaptations that
occur during pregnancy. Further, few studies have examined the effects of maternal obesity
on kidney development in offspring. We hypothesized that obesity would limit the
cardiovascular and renal adaptations of pregnancy contributing to poor fetal outcomes. To
address this hypothesis we established a robust mouse model of diet-induced obesity in
female C57BL/6 mice and characterized the cardiovascular and renal phenotypes of these
mice and the hemodynamic adaptations during pregnancy. We also assessed fetal outcomes
with a focus on kidney development.
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
70
3.3. METHODS
All experiments were approved by Monash University Animal Ethics Committee and
conducted in accordance with the Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes. Four-week-old female C57BL/6J mice received either a
control or a high fat diet (HFD) for 10 weeks prior to experimentation and throughout the
study (See Section 2.1.1.1 for details).
Glucose metabolism was examined via glucose tolerance tests in a sub-cohort of
mice. After 9 weeks of diet treatment, mice were fasted for 4hrs before undergoing glucose
tolerance tests (GTTs) after intraperitoneal administration of glucose. Fasting blood glucose
(tail tip blood sample) was measured (Accu-Chek Performa blood glucose meter; Roche
Diagnostics Gmbh, Germany) before all mice received 46mg of glucose ip. This dose is
equivalent to 2mg/g BW for CONT mice at this age (23g).243 Blood glucose was monitored
every 15 minute for 90 minutes.
Separate cohorts of obese (n=53) and control (n=32) mice were allocated to each
individual study. Arterial pressure, Heart rate and activity prior to conception and throughout
pregnancy were assessed using radiotelemetry (See Section 2.3) Cardiac MRI was used for
assessing left ventricle mass and cardiac function at baseline and GA14 (See Section 2.6).
Renal Excretory Profile was examined at baseline and GA13 (See Section 2.2.1). Following
baseline measurements female mice were mated overnight and gestational age (GA) 1 was
defined as the 24hr period commencing the start of the light cycle (6am) on the day of
vaginal plug detection.
Fetal and maternal outcomes were examined at GA19 (See Section 2.4.1 for greater
detail). Briefly, dams were anaesthetized (isoflurane) and the number of viable, non-viable
fetuses and implantation sites were recorded and their location in the uterus identified.
Fetuses and placentas were rapidly removed and weighed. Fetuses were sexed and the left
fetal kidney fixed in 4% paraformaldehyde for 2 hours then transferred to 70% ethanol until
processing for stereological analysis. Fetal head width was measured between ears.
Total glomerular number was estimated using unbiased stereology (See Section 2.5
for greater details).81,82 Briefly, fetal kidneys were embedded in paraffin and exhaustively
sectioned at 4 µm. 10-15 evenly spaced section pairs were systematically sampled and
histochemically stained with the lectin peanut agglutinin (PNA) to identfy glomerular
podocytes in early S-shaped bodies through to mature glomeruli. Sections were then
counterstained with hematoxylin. Section pairs were then used to estimate PNA-positive
glomeruli using the physical disector/fractionator combination as previously described.81,82 All
glomeruli in section pairs were assessed for evidence of abnormal characteristics,
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
71
specifically glomerular capillary dilation and enlarged Bowman’s space, and the percentage
of abnormal glomeruli calculated. Kidney volume was estimated from kidney sections using
the Cavalier Principle.80,141
Following collection of fetal tissues, a carotid arterial blood sample was taken from
the dam for determining plasma free fatty acids (FFAs) and triacylglycerol (TAG) using
enzymatic colorimetric assays (FFAs, Wako Pure Chemical Industries, Japan; TAG, GP-PAP
reagent, Roche Diagnostic, Germany respectively). Maternal kidneys were rapidly excised,
decapsulated and weighed. The left kidney was immersion fixed in 10% buffered formalin for
histological analysis of collagen deposition. The heart of the dam was excised and weighed.
The atria were removed and both atria and ventricles of the heart were weighed and all
values reported relative to tibia length.246 Top third of the ventricles were fixed in 10%
buffered formalin for histological analysis of collagen deposition. Pericardial fat and liver were
also weighed. Age-matched virgin control and obese mice were used as time controls.
Data were analyzed by unpaired t-test or two-way ANOVA, repeated measure where
appropriate. For studies that examined bodyweight and food intake, glucose metabolism,
MAP and HR, renal excretory profile and cardiac functions, two-way repeated measure
ANOVA were conducted, the factors of diet (Pd) and time (Pt), and interaction between those
factors were examined (Pd*t). For GA19 maternal tissue data, two-way ANOVA was
conducted; the factors of obesity (Pd) and pregnancy (Pp), and interaction between those
factors (Pd*p) were examined. For GA19 fetal outcome data, two-way ANOVA was
performed; the factors of diet (Pd) and sex of the fetus (Ps), and interaction between these
factors (Pd*s) were examined. Sidak post-hoc analyses were conducted where appropriate.
Values are mean ± SEM.
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
72
3.4. RESULTS
3.4.1. Diet-Induced Obesity in Female Mice
HFD fed mice had greater weight gain (Pd*t <0.001) and were 47% heavier than
controls prior to mating (33.3±0.6, 22.7±0.2g respectively, P<0.001; Figure 3.1A) due to
greater food and caloric intake (Pdiet<0.01-0.001; Pd*t <0.001; Figure 3.1B&C). HFD fed
obese mice had significantly higher fasting blood glucose compared to controls (8.98±0.25,
6.88±0.37mmol/L respectively; P<0.001) and impaired glucose tolerance (Pd*t <0.001,
Figure 3.1D).
Figure 3.1. Weekly body weight (A) of control (open symbols, n=32), and obese (closed symbols, n=53) mice, food intake (B) and caloric intake (C) of control (open symbols, blue line, n=19) and obese (closed symbols, red line, n=15) mice during 10-week diet treatment; Glucose tolerance tests (GTT; D) in control (n=18) and obese (n=25) mice. Data analyzed by two-way repeated measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t). Mean ± SEM.
0 1 2 3 4 5 6 7 8 9 100
5
10
15
20
25
30
35
Weeks
g
Body Weight
ControlObese
Pdiet <0.001Ptime <0.001Pd*t <0.001
0 1 2 3 4 5 6 7 8 9 100
20
40
60
80
100
120
Weeks
kC
al /
we
ek
Caloric Intake
ControlObese
Pdiet <0.001Ptime <0.001Pd*t <0.001
0 1 2 3 4 5 6 7 8 9 100
5
10
15
20
25
30
g / w
ee
k
Food Intake
ControlObese
Pdiet <0.001Ptime <0.001Pd*t <0.001
0 15 30 45 60 75 900
5
10
15
20
25
Minutes
Blo
od
Glu
co
se
(m
mo
l/L)
Glucose Tolerance
ControlObese Pdiet <0.001
Ptime <0.001Pd*t <0.001
A
D
B
C
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
73
3.4.2. Arterial Pressure, Heart Rate and Activity
Diet-induced obesity led to significantly higher MAP and HR during light and dark
periods (Figure 3.2A,B), but locomotor activity was not significantly different from control
mice (Figure 3.2C). Mice showed the expected changes in MAP across pregnancy
(Ptime<0.001) including the mid-gestation dip reaching a nadir on GA9 (Figure 3.2A). MAP of
obese mice remained elevated over controls for most of pregnancy (Pdiet<0.001), however
the post-dip rise of MAP in control mice was more profound, such that the groups no longer
differed from GA18. Analysis of the delta MAP across pregnancy highlighted that the timing
and the magnitude of the dip (~11mmHg) was similar between obese and control mice.
However, the rise in MAP post-dip was blunted in obese mice and, in contrast to control mice,
MAP did not rise above pregnancy levels towards term (Pd*t<0.01, Figure 3.2A).
HR increased across pregnancy as expected (Ptime<0.001, Figure 3.2B). Whilst
obese mice had elevated HR compared to control mice (Pdiet<0.01, Figure 3.2B), this effect
was lost with time and HR was no longer different between the groups from GA11 during
dark phase (GA12 light phase). The HR of obese mice changed little early in pregnancy with
the surge in HR both delayed (GA13 vs GA11) and diminished in magnitude, being
approximately half that of control mice (Figure 3.2B). Locomotor activity was lower in obese
animals (dark period only; Pdiet<0.05), decreased with pregnancy (Ptime <0.001), but the
decrease was similar in both groups (Figure 3.2C).
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
74
Figure 3.2. Mean arterial pressure (MAP; A), heart rate (HR; B) and locomotor activity (C)
for light period (left panel), dark period (center panel) and the change from basal levels in
dark period (∆; right panel) for control (open symbols, blue line; n=9) and obese (closed
symbols, red line; n=6) mice during pregnancy. Data analyzed by two-way repeated
measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t). Mean ±
SEM.
LIGHT
80
90
100
110
120
MAP
mmHg
Pdiet <0.001Ptime <0.001
Pd*t NS
450
500
550
600
650
HRbpm
Pdiet <0.01Ptime <0.001Pd*t NS
Basal 1 7 13 190
4
8
12
16
Gestational day
ActivityCounts/min
Pdiet NS
Ptime <0.001Pd*t NS
DARK
Pdiet <0.001Ptime <0.001
Pd*t <0.01
Pdiet <0.001Ptime <0.001Pd*t =0.05
Basal 1 7 13 19Gestational day
Pdiet <0.05Ptime <0.001
Pd*t NS
DARK
-15
-10
-5
0
5
10
15
∆
Pdiet NS
Ptime <0.001Pd*t <0.001
-50
0
50
100
150
∆
Pdiet <0.01
Ptime <0.001Pd*t NS
1 7 13 19
-8
-4
0
4
Gestational day
∆
Pdiet NS
Ptime <0.001
Pd*t NS
A
B
C
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
75
3.4.3. Cardiac MRI
Obese mice had significantly greater left ventricle mass (LVM) and CO compared to
controls. LVM and CO increased significantly with pregnancy but the effects were blunted in
obese mice (Ptime<0.001; Pd*t<0.01; Figure 3.3A,B). Post-hoc analysis demonstrated that
while the pregnancy-induced increases in LVM and CO for control mice were profound and
highly significant (26% and 25%; P<0.001), obese mice did not show significant increases
with pregnancy (6% and 7%). Indeed at GA14, LVM and CO were no longer different
between obese and control mice (Figure 3.3A,B). The changes in CO were reflected in
changes in SV with only control mice showing a significant increase with pregnancy (21%;
P<0.01; Figure 3.3C). The increase in stroke volume with pregnancy in control mice was
due to increases in end-diastolic volume (EDV) with end-systolic volume (ESV) not changing
significantly with pregnancy (Figure 3.3D,E). ESV however tended to be higher in obese
mice. Neither obesity nor pregnancy impacted ejection fraction (Figure 3.3F). Obesity did not
impact wall thickness (data not shown).
Figure 3.3. Left ventricle mass (LVM; A), cardiac output (CO; B), stroke volume (SV; C), end-diastolic volume (EDV; D), end systolic volume (ESV; E), and ejection fraction (EF; F) in control (open symbols, blue line; n=6) and obese (closed symbols, red line; n=5) mice at baseline and GA14. Data analyzed by two-way repeated measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t). *P<0.05, ** P<0.01, ***P<0.001. Mean ± SEM.
50
60
70
80
mg
LVM
Pdiet NSPtime <0.001Pd*t <0.01
***
5
10
15
20
25
ml
/ m
in
CO
Pdiet NSPtime <0.001Pd*t <0.01
***
20
30
40
50
ul
SV
Pdiet NSPtime <0.01Pd*t =0.08
**
Baseline GA1430
40
50
60
ul
EDV
Pdiet NSPtime <0.05Pd*t NS
*
Baseline GA140
5
10
15
ul
ESV
Pdiet =0.06Ptime NSPd*t NS
Baseline GA1460
70
80
90
%
EF
Pdiet NSPtime NSPd*t NS
A CB
D FE
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
76
3.4.4. Renal Excretory Profile
At baseline there were no differences in 24hr water or food intake, urine or sodium
excretion, or urinary osmolality between control and obese mice, however obese mice had
significantly higher albumin excretion (P<0.001; Figure 3.4F). There was a tendency
(P=0.055; Figure 3.4A) for pregnancy to increase water intake, yet neither obesity nor
pregnancy impacted urine excretion and food intake. Sodium excretion decreased
significantly with pregnancy (Ptime<0.01, Figure 3.4E), however post-hoc analysis
demonstrated that only control mice showed a significant decrease (P<0.05). Urinary
osmolality decreased with pregnancy in both groups (Ptime<0.05, Figure 3.4C). Pregnancy
resulted in a profound increase in albuminuria in obese but not control mice (Pdiet<0.001;
Pd*t<0.05; Figure 3.4F).
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
77
Figure 3.4. 24hour water intake (A), food intake (B); urine osmolality (C); urinary excretion
(D); sodium excretion (E); albumin excretion (F) in control (open symbols, blue line; n=8) and
obese (closed symbols, red line; n=7) at baseline and GA13. Data analyzed by 2-way
repeated measures ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t).
*P<0.05. Mean ± SEM.
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
78
3.4.5. Fetal Outcomes
Gestational length (control 19.6±0.1; obese 19.8±0.2 days) was similar between
groups. The number of implantation sites between groups was similar (control 7.8±0.7;
obese 7.0±1.0). However obese dams had significantly fewer viable fetuses per litter (control
7.5±0.9; obese 4.0±0.8; P<0.05).
The fetal sex ratio was similar between control and obese dams. Male and female
fetuses of obese dams had significantly lower average bodyweights per litter than fetuses of
control dams (Pdiet<0.01; Table 3.1). Whilst diet-induced obesity did not affect placental
weight, male fetuses had heavier placentas (Psex<0.01; Table 3.1). The fetal:placental
weight ratio was significantly reduced with obesity (Pdiet<0.01; Table 3.1). Post-hoc analysis
demonstrated that obesity-induced reduction in fetal:placental weight ratio was only
significant for male fetuses (P<0.05). Fetuses from obese dams had a lower average fetal
head width per litter (Pdiet<0.01), and profoundly higher incidence of placental thrombosis
(Pdiet<0.05) and meconium of fetal membranes (Pdiet<0.001; Table 3.1).
Fetal glomerular number was reduced with pre-pregnancy obesity (Pdiet<0.001;
Table 3.1). Post-hoc analysis demonstrated that this obesity-induced reduction in fetal
glomerular number was only significant for male fetuses and profound at 43% (P<0.001;
Table 3.1). The low glomerular number in male fetuses remained after correction for fetal
body weight (25% lower; P<0.01; Table 3.1). Maternal obesity also led to smaller fetal
kidney volume (Pdiet<0.05; Table 3.1), however kidney volume to bodyweight ratio was not
different between groups (Table 3.1).
Three of the 5 kidneys from male fetuses of obese dams showed either limited or
absent renal papilla formation (Figure 3.5). Further, glomeruli of male and female fetuses of
obese dams showed significant abnormalities, including glomerular capillary dilation and/or
grossly enlarged Bowman’s space. The percentage of glomeruli affected was 5 times greater
in kidneys from obese compared to control dams (Pdiet<0.001; Table 1; Figure 3.5).
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
79
Table 3.1. Litter Characteristics and Stereological Analysis of Fetal Kidneys at GA19. Vkid indicates kidney volume; BW, body weight and Nglom, glomerular number. Data analyzed by two-way ANOVA with factors of diet (Pdiet), sex (Psex) and interaction (Pint). *P<0.05, †P<0.01 and ‡P<0.001 vs control within the same gender. Values are mean ± SEM.
Male Female P
Control Obese Control Obese diet sex int
Litter Characteristics
Fetal Wt g 1.01±0.03 0.83±0.06 0.98±0.06 0.84±0.06 <0.001 NS NS
Placental Wt g 0.086±0.002 0.095±0.005 0.077±0.003 0.082±0.004 NS <0.01 NS
Fetal/Placental Ratio 11.8±0.6 9.0±1.0* 12.9±0.6 10.4±0.7 <0.01 NS NS
Placental Thrombosis % 0 45.8±18.7‡ 8.3±8.3 31.0±15.6‡ <0.05 NS NS
Meconium of fetal membranes % 5.6±5.6 91.7±8.3‡ 2.4±2.4 67.9±16.1‡ <0.001 NS NS
Head Width mm 6.62±0.09 6.11±0.11* 6.47±0.08 6.01±0.18* <0.01 NS NS
No. of Litters 6 6 6 7
Stereology
Vkid mm3 1.77±0.09 1.22±0.24 1.72±0.13 1.53±0.13 <0.05 NS NS
Vkid/gBW 1.62±0.04 1.44±0.20 1.70±0.11 1.79±0.07 NS NS NS
Nglom (PNA+ve) 1604±58 914±94‡ 1397±124 1100±101 <0.001 NS NS
Nglom/gBW 1474±39 1107±95† 1373±73 1280±47 <0.01 NS =0.052
Nglom/Vkid 912±30 815±102 822±62 724±45 NS NS NS
Abnormal Glomeruli % 1.6±0.4 9.2±1.6‡ 2.1±0.7 11.6±1.0‡ <0.001 NS NS
No. of Kidneys 5 5 6 7
Figure 3.5. Light micrographs of fetal kidney sections of male and female fetuses of control and obese dams at lower magnification (LP; top panel; scale bar 500μm) and higher magnification (HP
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
80
Light micrographs of fetal kidney sections of male and female fetuses of control and obese dams at lower magnification (LP; top panel; scale bar 500μm) and higher magnification (HP- bottom panel; scale bar100 μm).
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
Light micrographs of fetal kidney sections of male and female fetuses of control and obese dams at lower bottom panel; scale bar100 μm).
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
81
3.4.6. Maternal Outcomes
Weight gain was significantly lower in obese dams (Pd*t <0.01; Figure 3.6) due to
reduced weight gain between GA13-19 (11.9±0.4 vs 13.7±0.7g; P<0.05; Figure 3.6).
Pregnancy and obesity led to significantly greater kidney, heart and liver weights
(Ppreg<0.05, Pdiet <0.001; Table 3.2), however obesity did not exacerbate the pregnancy
induced increases in organ weights. Cardiac collagen content of obese primiparous
(4.08±0.36%area) and nulliparous (5.07±0.80%area) mice was significantly greater than
control primiparous and nulliparous hearts (1.96±0.44, 2.04±0.42 %area respectively;
Pdiet<0.001), however there was no effect of pregnancy. Pregnancy did not impact on
pericardial fat but obese animals had an approximately 5 fold greater pericardial fat mass
than control mice (Table 3.2). Neither obesity nor pregnancy impacted renal collagen content
(data not shown). Obese nulliparous mice had significantly greater plasma FFA compared to
control mice. Pregnancy did not affect the FFA levels in obese mice however control mice
demonstrated an increase with pregnancy (Pd*t<0.05; Table 3.2).
Figure 3.6 Body weight gain in control (open symbols, blue line) and obese (closed symbols, red line) dams at GA7 (n=12,14), GA13 (n=12,10) and GA19 (n=6,6). Data were analyzed by two-way ANOVA with factors of diet (Pdiet), time (Ptime) and interaction (Pd*t), followed by Sidak post hoc tests. *P<0.05 compared control and obese groups. Values are mean ± SEM.
7 13 190
5
10
15
Gestational day
g
Body Weight Gain
*
P diet NSP time <0.001P d*t <0.01
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
82
Table 3.2. Maternal body weight, total kidney weight, kidney weight/tibia ratio, heart weight, heart weight/tibia ratio, left ventricle (LV) weight, LV weight/tibia ratio, combined atria weight, atria weight/tibia ratio, pericardial fat weight, liver weight, tibia length, plasma free fatty acids and plasma triacylglycerol of control and obese dams (primiparous) at GA19 and age matched non-pregnant control and obese mice (nulliparous). Data were analyzed by two-way ANOVA with factors of Pregnancy (Ppreg), Obesity (Pdiet) and interaction (Pp*d), followed by Sidak post hoc tests comparing control and obese mice within the nulliparous groups and within the primiparous groups *P<0.05, †P<0.01 and ‡P<0.001. Values are mean ± SEM.
Nulliparous Primiparous P
Cont (10) Obese (10) Cont (6) Obese (6) Preg Diet p*d
Body Wt (g) 25.8±0.9 42.2±1.6‡ 37.2±0.7 51.8±1.6‡ <0.001 <0.001 NS
Total Kidney Wt (mg) 249±7 264±7 279±11 315±14* <0.05 <0.001 NS
Kidney Wt/Tibia ratio 14.3±0.4 15.2±0.4 16.2±0.6 18.0±0.7 <0.001 <0.05 NS
Heart Wt (mg) 105±2.5 118±3.0* 123±2.4 145±7.5† <0.001 <0.001 NS
Heart Wt/Tibia ratio 6.03±0.15 6.80±0.18* 7.18±0.15 8.28±0.38† <0.001 <0.001 NS
LV Wt (mg) 98.2±2.3 109.0±2.5* 112.3±2.5 132.4±6.9† <0.001 <0.001 NS
LV Wt/Tibia ratio 5.63±0.14 6.27±0.14* 6.54±0.16 7.57±0.35† <0.001 <0.001 NS
Atria Wt (mg) 5.44±0.33 6.69±0.35* 7.22±0.18 8.93±0.67* <0.001 <0.01 NS
Atria Wt/Tibia ratio 0.31±0.02 0.39±0.02* 0.42±0.01 0.51±0.03* <0.001 <0.01 NS
Pericardial Fat Wt (mg) 42.2±5.8 167.2±19.1‡ 28.3±6.2 171.9±23.8‡ NS <0.001 NS
Liver Wt (g) 1.11±0.06 1.53±0.13† 1.77±0.08 2.15±0.11 <0.001 <0.001 NS
Tibia Length (mm) 17.5±0.1 17.4±0.2 17.2±0.1 17.5±0.3 NS NS NS
Free Fatty Acid (mM) 0.27±0.02 0.40±0.03 0.36±0.03 0.36±0.03 NS <0.05 <0.05
Triacylglycerol (mM) 0.52±0.04 0.66±0.06* 0.57±0.06 0.61±0.08 NS NS NS
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
83
3.5. DISCUSSION
This study determined the impact of diet-induced obesity on the hemodynamic
adaptations of pregnancy, and fetal and maternal outcomes in late pregnancy. Our mouse
model had profound diet-induced obesity, glucose intolerance, hypertension, tachycardia,
cardiac hypertrophy and fibrosis, elevated CO and albuminuria prior to mating, consistent
with human obesity. Whilst MAP and HR of obese mice remained elevated over control mice
throughout pregnancy, the increases in MAP and HR of obese mice towards term, were
blunted. Obese dams also failed to increase CO, SV and LVM at GA14, and albuminuria was
exacerbated. Obesity led to greater fetal death and suboptimal fetal development at GA19
with lower body weight, smaller kidneys, abnormal glomerular morphology and, in males,
nephron deficiency. These results are consistent with the hypothesis that obesity limits the
normal cardiovascular adaptations of pregnancy and leads to adverse fetal outcomes.
Few studies have performed prospective longitudinal cardiovascular measurements
in women from preconception, through pregnancy and birth, and none to date in obese
women. Mice,48 like humans,236 show an early fall in arterial pressure, reaching a nadir mid-
pregnancy before rising to, or exceeding, pre-pregnancy levels near term. MAP of obese
mice remained elevated over control mice throughout pregnancy consistent with the literature
in obese women.1,354 Interestingly, both the timing and magnitude of the mid-gestation fall in
MAP were similar in obese and control mice indicating that the mechanisms driving the fall in
MAP were not affected by pre-existing obesity. The presence of the dip in arterial pressure in
obese women is contentious, most likely due to lack of pre-pregnancy values, differences in
timing and frequency of measurements during pregnancy, but parity might also contribute.
Tomodo et al 354 enrolled Japanese women at 6 weeks of gestation, measuring arterial
pressure every 3 weeks and found that multiparous obese women demonstrated the mid-
gestation dip in arterial pressure whilst primiparous obese women did not. Following the mid-
gestation dip, MAP of control mice returned to pre-pregnancy level by GA15 and continued to
rise until GA18, consistent with human studies that found that at 38 weeks of pregnancy,
systolic and diastolic blood pressure were 5.6% and 7.5% respectively above pre-conception
levels.309 In contrast, the rise of MAP in obese mice late pregnancy was significantly blunted
such that MAP did not exceed pre-pregnancy level. Human studies have also suggested that
the rise in arterial pressure, particularly late in gestation, is attenuated with increasing
BMI.1,123,352
Control mice showed a 3% rise in dark-phase HR early in pregnancy with a profound
increase (~20%) commencing GA11. These values are consistent with human data that
demonstrate an early increase in HR (4%; 6 weeks gestation)236 reaching ~20% late in
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
84
pregnancy.236,279 In contrast, obese mice showed negligible change in HR in early pregnancy,
with the later rise delayed in onset (GA13), and blunted in magnitude, rising by only 10%.
Indeed although HR of obese mice started off 12% greater than controls, HR was no longer
significantly different between the groups from GA11. Consistent with our mouse studies,
findings for women have shown that HR is not different between obese and normal weight
women late in gestation.1,96 However given that obesity is associated with tachycardia,362 the
similarity in HR between obese and normal weight women suggests a blunted adaptation to
pregnancy. Although no study to date has measured the change in HR in obese women
relative to preconception levels, a meta-analysis found that the increase in HR of obese
women between the first and last trimester was significantly less than non-obese women (+3
vs +14bpm).159
As in human pregnancy, control mice showed significant increases in CO (25%), SV
(21%) and LVM (26%) from pre-pregnancy to GA14, the beginning of the final trimester.
Obese mice had elevated CO (17%), SV (12%) and LVM (15%) compared to control mice
prior to conception, however the increase in these parameters (5-7%) with pregnancy was
negligible such that at GA14, CO, SV and LVM were indistinguishable between obese and
control mice. Consistent with this finding, Dennis et al 96 found that at 36 weeks gestation,
obese women had similar CO, SV and HR compared to non-obese women, though in
contrast to our study, they found LVM was greater in obese women at this time. The
changes in cardiac function from pre-pregnancy have not been measured in obese women,
however Abdullah et al 1 demonstrated that morbidly obese pregnant women had a limited
increase in CO across the trimesters compared to non-obese controls.
The mechanisms underpinning the blunted cardiovascular responses in obese mice
are unclear. Normal pregnancy is characterized by increased sympathetic drive and reduced
baroreceptor sensitivity near term, changes that are thought to contribute to the rise in
arterial pressure and heart rate at this time.135 Our data thus supports the contention of
Helmreich et al 159 that obesity results in diminished autonomic responsiveness during
pregnancy. In humans the late rise in HR is thought to maintain the elevated CO as SV
decreases towards term,279 thus the blunted HR response in obese mice may contribute to
the limited increase in CO. It is also possible that obese dams did not have the expected
plasma volume expansion, and thus increase in preload, and ultimately stroke volume with
pregnancy. Although control mice demonstrated evidence of the sodium retention expected
in pregnancy (i.e. a marked fall in sodium excretion in the absence of a change in
food/sodium intake), this was not present in obese dams where the fall in sodium excretion
was not significant. The failure to increase plasma volume would also explain the blunted
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
85
cardiac remodeling in obese mice, as volume overload is the primary stimulus for
hypertrophy in pregnancy.105
Obese dams exhibited exacerbated albuminuria with pregnancy compared to control
dams. This may be associated with underlying pre-existing renal injury in obese mice prior to
pregnancy. Obesity in humans and animal models is associated with increased GFR and
renal blood flow, albuminuria and renal pathology.146,151,301 Given that pregnancy also leads
to a marked increase in GFR and renal blood flow,32 this aggravated renal hyperfiltration is
likely to contribute to exacerbated albuminuria with pregnancy. Future studies that assess
GFR and renal pathology are warranted to address the gaps in our understanding of the
impact of obesity on renal health during pregnancy and post-partum.
Obesity had no impact on the number of implantation sites, however the incidence of
fetal death and resorption was increased. This is consistent with studies in human and non-
human primates (NHP) that show markedly increased risk (5-fold in the Danish Cohort)270 of
late gestation miscarriage and stillbirth with maternal obesity.121,270,316 In contrast, most
mouse models of maternal obesity do not show a reduced litter size or adverse fetal
outcomes, though for the majority of these studies, female mice were only marginally (10-
20%), albeit significantly, heavier than control mice.40,203 However, where HFD fed mice were
more than 30% heavier than control mice prior to conception, the litter size was reduced as
in our model.62,204 Maternal obesity also led to significantly reduced fetal body weight and
head width. Although pre-pregnancy obesity in humans increases the risk of large for
gestation age babies, it is also recognized as a strong independent risk factor for small for
gestation age and IUGR babies,10,293 consistent with our finding in mice. Further, placentas of
obese dams had significantly higher incidence of thrombosis, and males had lower placental
efficiency. These findings are consistent with a recent human study that found pre-pregnancy
obesity is associated with placental insufficiency and higher risk of placental pathological
lesions, including placental thrombosis.173
The greater fetal loss and low fetal weight in obese dams is consistent with our
findings of a blunted cardiovascular response in obese dams through pregnancy. Studies
have found that fetal outcomes, including birth weight, are poorer in women who fail to
demonstrate an increase in stroke volume (index of preload or plasma volume expansion),
CO or GFR with pregnancy.7,19,103 Although our study did not demonstrate a direct link
between the blunted rise in CO in obese dams and reduced blood flow to the fetoplacental
unit, Frias et al 121 found that uterine blood flow was decreased in a NHP model of maternal
obesity, contributing to the high rate of stillbirth observed in their study. Strikingly, maternal
obesity in these NHP also led to a reduction in blood flow on the fetal side of the placenta,
increased expression of placental inflammatory cytokines and higher incidence of placental
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
86
infarcts.121 Our finding that the reduced placental efficiency was restricted to male fetuses of
obese dams, suggests that fetal sex is a determinant of fetal outcomes with maternal obesity.
The finding that male placenta of high fat diet-fed mice had greater inflammation and
macrophage activation than female placenta,203 suggests male offspring could be more
vulnerable to maternal obesity.
Given the cardiovascular adaptations have been shown to differ between primiparous
and multiparous obese females,354 the present study focused on maternal cardiovascular
adaptations in primiparous dams. The disadvantage of investigating primiparous dams is that
it is common for them to eat their first litter, known as maternal infanticide. This was evident
in early cohorts in this study with litters eaten within the first 24 hours post-birth. Thus, to
maintain a consistent post-partum experience in these dams for the post-partum studies in
Chapter 4, all pups were removed 24 hour post-birth. Due to the loss of offspring shortly after
birth, long-term studies in the offspring of control and obese dams could not be completed in
this thesis. Future studies should investigate the programming effects of pre-pregnancy
obesity on the long-term cardiovascular and renal health of adult offspring from second or
third litters.
This is the first study to assess the impact of maternal obesity on nephron
endowment using gold-standard stereological techniques in a model representative of human
obesity, that is, where body weight is markedly elevated prior to conception. One of most
extraordinary findings of the present study was that pre-pregnancy obesity in mice led to
lower nephron number at GA19, an effect primarily seen in kidneys of male fetuses. In
addition, kidneys of both male and female fetuses from obese dams demonstrated significant
structural abnormalities including dilated glomerular capillaries and Bowman’s space, and
limited formation of renal papilla. These structural abnormalities are likely to have a long-term
impact on GFR and sodium and water homeostasis of offspring from obese dams. Together
these findings indicate that fetal kidney development is significantly impacted by pre-
pregnancy obesity. The low nephron number in offspring of obese dams is not unexpected in
light of their lower body weight. Hughson et al 175 found a linear relationship between
nephron number and birth weight in humans. The presence of reduced nephron endowment
in male fetuses after adjusting for body weight however suggests an additional sex specific
mechanism. This is consistent with the finding that nephron endowment of male offspring
was more sensitive to exposure to a low protein diet, suggesting kidney development of male
offspring might be more vulnerable to maternal insults.379 Although the mechanisms that
mediate the nephron deficit in male offspring in the present study are unclear, it is possible
that the structural and functional differences in placentas of male offspring might be
Chapter 3 Obesity & Hemodynamic Adaptations of Pregnancy
87
important, including, the greater pro-inflammatory profiles previously demonstrated in male
placentas with maternal obesity.203
3.6. CONCLUSION
In summary, findings from the present study have demonstrated, for the first time,
that pre-pregnancy obesity limits the cardiovascular and renal adaptations of pregnancy
ultimately resulting in a blunted increase in CO, and poor fetal outcomes. The present study
also revealed the detrimental impact of pre-pregnancy obesity on fetal kidney development,
particularly nephron endowment. The lower fetal weight in male offspring could not explain
the reduction in nephron number, suggesting mechanisms other than global growth
restriction may be involved, and further examination of the placenta from these fetuses may
provide deeper understanding of this sex-specific effect. In conclusion, our findings indicate
that pre-pregnancy obesity prevents normal cardiovascular and renal adaptations of
pregnancy and leads to fetal loss, growth restriction and suboptimal fetal kidney development.
The consequence of these developmental abnormalities may contribute to the development
of cardiovascular and renal diseases later in life. Interventions that enhance the
cardiovascular adaptations in pregnancies complicated by obesity may reduce the incidence
of fetal loss and growth restriction, and the long-term impact of renal programming on
cardiovascular and renal health of the offspring.
Chapter 4 Obesity & Hemodynamic Changes Post-partum
88
Chapter 4 DOES PREGNANCY EXACERBATE THE
CARDIOVASCULAR AND RENAL EFFECTS OF
OBESITY?
Chapter 4 Obesity & Hemodynamic Changes Post-partum
89
4.1. INTRODUCTION
An uncomplicated pregnancy has no long-term adverse effects on the cardiovascular
and renal health (see Section 1.2.2). However recent studies have reported that obesity
during pregnancy is strongly associated with premature death, greater risk of major and
simple cardiovascular events, and greater number of hospitalizations of these women due to
cardiovascular events 10 years post-partum.223,384 Obesity, per se, is associated with
significant cardiovascular and renal consequences (see Section 1.3). Further, pre-pregnancy
obesity is closely associated with significant maternal complications, including gestational
hypertension, preeclampsia, and gestational diabetes.79 Importantly, these complications
have been recognized as independent risk factors for the development of significant
cardiovascular and renal disorders, such as chronic hypertension, type 2 diabetes, and CKD
in women later in life.35,61,158,240 Surprisingly, no study to date has formally investigated the
cardiovascular and renal health of females following a pregnancy complicated by obesity.
In Chapter 3 we reported that the obese dams demonstrated blunted increases in
MAP and HR towards term, and limited adaptations in SV, CO and LVM in late pregnancy.
Further, pregnancy also led to an exacerbation of albuminuria in obese dams during
pregnancy. In this chapter, we will use this mouse model of pre-pregnancy obesity to
examine the impact of pregnancy on obese female mice post-partum. We hypothesized that
pregnancy exacerbates the cardiovascular and renal outcomes of obesity and leads to
greater cardiovascular and renal morbidities. To address this hypothesis, we examined
arterial pressure, heart rate, GFR, renal excretory profiles, and cardiac and renal fibrosis in
control and obese mice 4 weeks post-weaning (4WPW) and compared them with non-
pregnant (nulliparous), age-matched control and obese mice.
Chapter 4 Obesity & Hemodynamic Changes Post-partum
90
4.2. METHODS
4.2.1. Animals
All experiments were approved by Monash University Animal Ethics Committee and
conducted in accordance with the Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes. Four-week-old female C57BL/6J mice received either a
control or a high fat diet (HFD) for 10 weeks prior to experimentation and throughout the
study (See Section 2.1.1.1 for details). Following baseline (pre-pregnancy) measurement, a
subgroup of control and obese mice were then mated with male mice and became pregnant
(see Section 2.1.1.1). The rest of control and obese remained virgin and served as time-
matched non-pregnant (nulliparous) groups to be compared with primiparous control and
obese mice post-partum.
4.2.2. Telemetry Recordings
Radiotelemetry transmitters were implanted in mice used in Chapter 3 at the
completion of the initial 10-week diet treatment and pre-pregnancy data were recorded
before mating (see section 2.3.2). Mean arterial pressure (MAP), heart rate (HR) and
locomotor activity were continuously recorded throughout pregnancy and post-partum until
PN36. Recording ceased at this time point as the battery life of the telemetry transmitters
were exhausted.
4.2.3. Assessment of urinary excretory profile and renal function (GFR)
Urinary excretory profile (see section 2.2.1) was assessed at before mating (pre-
pregnancy) and then again at 4WPW in primiparous and time-matched nulliparous control
and obese mice. Transcutaneous measurements of GFR were performed only at 4WPW in
conscious primiparous and time-match nulliparous control and obese mice (see Section 2.2.3
for details).111
4.2.4. Plasma and Tissue Collection
At 4WPW, plasma and tissues were collected from primiparous and time-matched
nulliparous control and obese mice (see Section 2.4.2). Briefly mice were anaesthetised
(Isoflurane) and an arterial blood sample was taken from the carotid artery for the
measurement of plasma FFA and TAG concentrations. Plasma free fatty acids (FFAs) and
triacylglycerol (TAG) were determined by enzymatic colorimetric assays (FFAs, Wako Pure
Chemical Industries, Japan; TAG, GP-PAP reagent, Roche Diagnostic, Germany
Chapter 4 Obesity & Hemodynamic Changes Post-partum
91
respectively). The kidneys and heart were rapidly excised and weighed. One of middle
portions of the right kidney and the apical portions of the left ventricle were used for
hydroxyproline assay to assess total collagen content in the tissue (see Section 2.4.2 &
2.7.1). One of middle portions of the left kidney was subject to histological analysis of
glomerulosclerosis (PAS staining; see Section 2.7.2.2). The middle portion of the left
ventricle was subjected to histological analysis of cardiac fibrosis (Picrosirius red staining;
see Section 2.7.2.1.3). Peri-cardial fat pad, gonadal fat pad (visceral fat) and inguinal fat pad
(subcutaneous fat) were also weighed.
4.2.5. Statistical Analysis
Data were analyzed by unpaired t-tests and two-way ANOVA, repeated measure
where appropriate. Sidak post-hoc analyses were conducted where appropriate. To assess
the change in MAP, HR and locomotor activity from pre-pregnancy levels in control and
obese primiparous mice, two-way repeated measure ANOVA was conducted, factors of
obesity (Pobe) and time (Ptime), and interaction (Pint) between those factors were examined.
For MAP and HR of obese mice at pre-pregnancy and PN36, two-way repeated measure
ANOVA was conducted, factors of pregnancy (Ppreg) and time (Ptime), and interaction (Pint)
between those factors were examined. The change in MAP and HR from pre-pregnancy to
PN36 of primiparous and time-matched nulliparous obese mice was analyzed by unpaired t-
tests. For GFR, urinary excretion profile, maternal plasma lipid profile, maternal tissue data,
total collagen contents and histological analysis of collagen accumulation, two-way ANOVA
was conducted, factors of obesity (Pobe) and pregnancy (Ppreg), and interaction (Pint)
between those factors were examined. Values are mean ± SEM. P<0.05 was considered
statistically significant.
Chapter 4 Obesity & Hemodynamic Changes Post-partum
92
4.3. RESULTS
4.3.1. Post-partum Arterial Pressure and Heart Rate
Obese dams had elevated 24h MAP and HR over control dams throughout the post-
partum period up to PN36 (Figure 4.1A&G). MAP fell rapidly by approximately 15 mmHg in
both groups post-partum reaching pre-pregnancy levels by approximately day 6 (Figure
4.1A&B), whilst HR did not change significantly with time post-partum (Figure 4.1G&H). The
change in 24h MAP and HR from pre-pregnancy levels were not significantly different
between obese and control mice across this period (Figure 4.1B&H). Similar results were
found in 24h diastolic blood pressure and systolic blood pressure (Data not shown).
Locomotor activity of control and obese dams were indistinguishable over the post-partum
period (Figure 4.1I&J).
When examining the MAP data closely, control and obese mice appear to drift apart
from PN29, with 24h MAP of obese mice rising gradually, whilst 24h MAP of control mice
remained relatively stable until the end of recording (Figure 4.1A&B). Interestingly, this effect
was only significant in light-phase MAP (Pint<0.05; Figure 4.1C&D) and not present in dark-
phase MAP (Figure 4.1E&F). In comparing the light-phase MAP at PN36, the rise in light-
phase MAP from pre-pregnancy in control mice was 2.1± 1.9 mmHg (Figure 4.1D). However
in obese mice this rise was more than 6 fold higher at 14.2 ± 3.0 mmHg (P<0.05; Figure
4.1D).
To examine whether the greater rise in MAP was simply an effect of obesity, we
compared the change in MAP from pre-pregnancy to PN36 of primiparous obese mice with
nulliparous obese mice over the same period. The change in light phase MAP of primiparous
obese mice at PN36 was 78% higher than nulliparous obese mice (8.0 ± 1.1 mmHg; P<0.05;
Figure 4.2D). However, this effect of pregnancy on MAP in obese mice was not found in 24h
MAP or dark-phase MAP (Figure 4.2A,B,E&F). Interestingly, we found that whilst the rise in
HR in primiparous obese mice from pre-pregnancy levels was minimal and similar to
primiparous control mice (Figure 4.1H), nulliparous obese mice demonstrated a greater
increase in HR over this time compared to primiparous obese mice (Pint<0.05; P<0.05;
Figure 4.2G&H).
Chapter 4 Obesity & Hemodynamic Changes Post-partum
93
Figure 4.1 MAP(24hr, light & dark phases), HR and locomotor activity at pre-pregnancy state and postnatal day 1 to 36, and the change from pre-pregnancy levels for control (blue line) and obese (red line) primiparous mice. A) 24h MAP; B) Delta 24h MAP;C) MAP in light phase; D) Delta MAP in light phase; E) MAP in dark phase; F) Delta MAP in dark phase; G) 24h HR; H) Delta 24h HR; I) 24h locomotor activity;J) Delta 24h locomotor activity. Data analyzed by two-way repeated measures ANOVA with factors of obesity (Pobe), time (Ptime) and interaction (Pint), followed by Sidak post-hoc analysis. Mean±SEM
1 4 8 12 16 20 24 28 32 3680
90
100
110
120
130
140
mm
Hg
24h MAP
CONT (n=5)
Obese (n=3)
Pre-Preg
1 4 8 12 16 20 24 28 32 3670
80
90
100
110
120
130
mm
Hg
Light MAP
Pre-Preg
1 4 8 12 16 20 24 28 32 3680
90
100
110
120
130
140
mm
Hg
Dark MAP
Pre-Preg
1 4 8 12 16 20 24 28 32 36400
450
500
550
600
650
bp
m
24h HR
Pre-Preg
1 4 8 12 16 20 24 28 32 360
5
10
Post-natal Day
Co
un
ts/m
in
24h Activity
Pre-Preg
1 4 8 12 16 20 24 28 32 36-10
0
10
20
30
∆m
mH
g
Delta 24h MAP
CONT (n=5)
Obese (n=3) P int NS P time <0.001P obe NS
1 4 8 12 16 20 24 28 32 36-10
0
10
20
30
∆m
mH
g
Delta Light MAP
P int <0.05 P time <0.001P obe NS
1 4 8 12 16 20 24 28 32 36-10
0
10
20
30
∆m
mH
gDelta Dark MAP
P int NSP time <0.001P obe NS
1 4 8 12 16 20 24 28 32 36-100
-50
0
50
100
∆b
pm
Delta 24h HRP int NS P time NSP obe NS
1 4 8 12 16 20 24 28 32 36-10
-5
0
5
10
Post-natal Day
∆c
ou
nts
/min
Delta 24h ActivityP int NSP time NSP obe NS
A B
C D
FE
I J
G H
Chapter 4 Obesity & Hemodynamic Changes Post-partum
94
Figure 4.2 MAP (24h, light & dark phases) and HR at pre-pregnancy state and postnatal day 36 for nulliparous (n=5) and primiparous (n=3) obese mice. A) 24h MAP; B) Delta 24h MAP; C) Dark-phase MAP; D) Delta dark-phase MAP; E) Light-phase MAP; F) Delta light-phase MAP; G) 24h HR; H) Delta 24h HR; N: nulliparous; P: primiparous; Absolute data were analysed by two-way ANOVA with factors of pregnancy (Ppreg), time (Ptime) and interaction (Pint), followed by Sidak post-hoc analysis. Delta data was analysed by unpaired t-tests. *P<0.05; **P<0.01. Data are presented as Mean±SEM.
24h MAP
Pre-preg PN360
9090
100
110
120
130
140
mm
Hg
Nulliparous
Primiparous
P int NS
P time <0.001P preg <0.05
Dark MAP
Pre-preg PN360
9090
100
110
120
130
140
mm
Hg
Nulliparous
Primiparous
P int NS P time <0.05P preg NS
Light MAP
Pre-preg PN360
9090
100
110
120
130
140
mm
Hg
Nulliparous
Primiparous
P int <0.05 P time <0.001P preg <0.05
**
24h HR
Pre-preg PN360
400500
550
600
650
700
bp
m
Nulliparous
Primiparous
P int <0.05 P time <0.01
P preg NS
N P0
5
10
15
20
∆m
mH
g
Delta 24h MAP
N P0
5
10
15
20
∆m
mH
g
Delta Dark MAP
N P0
5
10
15
20
∆m
mH
g
Delta Light MAP
*
N P0
10
20
30
40
50
∆b
pm
Delta 24h HR
*
A
HG
FE
DC
B
Chapter 4 Obesity & Hemodynamic Changes Post-partum
95
4.3.2. Post-partum Renal Function
Neither obesity nor pregnancy had an impact on 24h urine or sodium excretion at
4WPW, with values similar across the groups of primaparous and nulliparous control and
obese mice (Figure 4.3A&C). The changes in urine and sodium excretion from pre-
pregnancy level were also not impacted by obesity or pregnancy (Figure 4.3B&D). Obese
primiparous and nulliparous mice had significantly elevated albumin excretion compared to
control mice at 4WPW (Pobe <0.001: Figure 4.3E). Pregnancy had no effect on albumin
excretion whether assessed as absolute excretion at 4WPW (Figure 4.3E) or as the change
in albumin excretion from pre-pregnancy levels (Figure 4.3F).
GFR was measured only at 4WPW using the transcutaneous FITC-sinistrin clearance
technique. There was no evidence of renal dysfunction in any of the groups with T1/2 and
calculated GFR were similar across all 4 groups of mice (Figure 4.4A&B).
Chapter 4 Obesity & Hemodynamic Changes Post-partum
96
Figure 4.3 Urinary excretory profiles at 4WPW and change from pre-pregnancy level. A) Urine volume; B) Delta Urine volume; C) Urinary sodium excretion; D) Delta sodium excretion; E) Albumin excretion; F) Delta Albumin excretion. N: nulliparous; P: primiparous; N-Control (n=8); N-Obese (n=7); P-Control (n=7); P-Obese (n=8); Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. Data are presented as Mean±SEM.
Urine Excretion
N P N P0.0
0.5
1.0
1.5
2.0
2.5
ml
P Int NSP Preg NS P Obe NS
Control Obese
Na+ Excretion
N P N P0
50
100
150
µm
ol
P Int NSP Preg NS P Obe NS
Control Obese
Albumin Excretion
N P N P0
5
10
15
20
25
µg
/24h
P Int NSP Preg NS P Obe <0.001
Control Obese
Delta Urine Excretion
N P N P-0.4
-0.2
0.0
0.2
0.4
0.6
∆m
l
P Int NSP Preg NS P Obe NS
Control Obese
Delta Na+ Excretion
N P N P-40
-20
0
20
40
60
∆µ
mo
l P Int NSP Preg NS P Obe NS
Control Obese
Delta Albumin Excretion
N P N P0
5
10
15
∆µ
g/2
4h
P Int NSP Preg NS P Obe <0.05
Control Obese
A
E
DC
F
B
Chapter 4 Obesity & Hemodynamic Changes Post-partum
97
Figure 4.4 Transcutaneous measurement of GFR. A) T1/2; B) Calculated GFR. N: nulliparous; P: primiparous; N-Control (n=7); N-Obese (n=9); P-Control (n=5); P-Obese (n=7); Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. Data are presented as Mean±SEM.
4.3.3. Maternal Outcomes
Plasma FFA and TAG concentrations were measured at 4WPW. Plasma FFA level
was not affected by pregnancy or obesity (Figure 4.5A). In control groups, primiparous mice
had significantly lower plasma TAG level than nulliparous mice (P<0.05; Pint<0.05; Figure
4.5B). However, this effect was not observed in obese mice (Figure 4.5B).
Figure 4.5 Plasma lipid profile at 4WPW. A) Plasma FFA, B) Plasma TAG. N: nulliparous; P: primiparous; N-Control (n=9); N-Obese (n=9); P-Control (n=7); P-Obese (n=9). Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05. Data are presented as Mean±SEM.
N P N P0
5
10
15
20
25
min
T1/2
P Int NSP Preg NSP Obe NS
Control Obese
N P N P0
300
600
900
1200
1500
1800
ul/m
in/1
00
gB
W
Calculated GFR
P Int NSP Preg NSP Obe NS
Control Obese
A B
N P N P0.0
0.2
0.4
0.6
0.8
1.0
mM
Plasma FFA
Pint NSPpreg NSPobe NS
Control Obese
N P N P0.0
0.2
0.4
0.6
0.8
1.0
mM
Plasma TAG
Pint <0.05Ppreg NSPobe <0.01
*
Control Obese
A B
Chapter 4 Obesity & Hemodynamic Changes Post-partum
98
At 4WPW, obese mice were 75% heavier than control mice (Pobe<0.01; Figure 4.6A).
This large difference was due to a greater weight gain in obese mice, 16.8± 0.9 g from pre-
pregnancy values, compared to 6.5± 0.5 g in control mice (Pobe<0.01; Figure 4.6B).
Pregnancy also increased weight gain, but this effect was only present among obese mice
(P<0.05; Figure 4.6B). Differences in gonadal fat may have contributed to this greater weight
gain. Gonadal fat mass was significantly increased with pregnancy (Pint<0.01) such that
primiparous obese mice (5.4 ± 0.3 g) had 74% greater gonadal fat mass than nulliparous
obese mice (3.1± 0.3 g; P<0.001; Figure 4.6C). Obesity had significant effects on inguinal
and peri-cardial fat mass with values 3 fold greater than control mice (Pobe<0.001 Figure
4.6D&E). However, pregnancy had no impact on inguinal or peri-cardial fat mass (Figure
4.6D&E). Obese mice had significantly greater kidney and ventricle weights, however
pregnancy did not impact these organ weights at 4WPW (Pobe<0.001; Figure 4.6F-I).
Chapter 4 Obesity & Hemodynamic Changes Post-partum
99
Figure 4.6 Maternal body weight (BW) and tissue weight at 4WPW for primiparous (P) and time-matched nulliparous (N) control and obese mice. A) BW at 4WPW; B) Delta BW against pre-pregnancy BW; C) Gonadal fat pad; D) Inguinal fat pad; E) Peri-cardial fat pad; F) Total kidney weight; G) Total ventricle weight; H) Left ventricle weight; I) Right ventricle weight. N: nulliparous; P: primiparous; Data analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05; ***P<0.001. Data are presented as Mean±SEM.
N P N P0
10
20
30
40
50
60
70
80
g
BW Pint NSPpreg <0.05Pobe <0.001
n =10 n =11n =15n =13
Control Obese
N P N P0
1
2
3
4
5
6
7
g
Gonadal Fat Pint <0.01Ppreg <0.001Pobe <0.001
***
n = 8
n = 3n = 6n = 6
Control Obese
N P N P0
100
200
300
400m
gPeri-cardial fat
Pint NSPpreg NSPobe <0.001
n = 7n = 8n = 9n = 8
Control Obese
N P N P0
100
200
300
400
500
mg
Total KidneyPint NSPpreg NSPobe <0.001
n =10 n =11n =15n =13
Control Obese
N P N P0
20
40
60
80
100
120
140
mg
Left VentriclePint NSPpreg NSPobe <0.001
n =10 n =11n =14n =13
Control Obese
N P N P0
10
20
30
∆g
∆BW
Pint NSPpreg <0.05Pobe <0.001 *
n =10 n =11n =15n =13
Control Obese
N P N P0
1
2
3
g
Inguinal Fat
Pint NSPpreg NSPobe <0.001
n = 8 n = 3n = 6n = 6
Control Obese
N P N P0
20
40
60
80
100
120
140
160
mg
Total Ventricle Pint NSPpreg NSPobe <0.001
n =10 n =11n =14n =13
Control Obese
N P N P0
10
20
30
40
mg
Right VentriclePint NSPpreg NSPobe <0.001
n =10 n =11n =14n =13
Control Obese
A CB
D E F
IHG
Chapter 4 Obesity & Hemodynamic Changes Post-partum
100
4.3.4. Renal and Cardiac Fibrosis
Pregnancy resulted in a profound increase in renal hydroxyproline content that was
dependent on group (Pint <0.05; Ppreg<0.05; Pobe<0.05; Figure 4.7A). Post-hoc analysis
showed that this effect of pregnancy was only significant among obese mice with renal
collagen content 44% greater in primiparous obese compared with nulliparous obese mice,
(P<0.01; Figure 4.7A). Neither pregnancy nor obesity had any impact on hydroxyproline
content of the LV (Figure 4.7B). In examining the level of glomerulosclerosis using PAS
staining, there was no global effect of obesity or pregnancy (Figure 4.8A&B). However, post-
hoc analysis showed that percentage area of PAS-positive staining in glomeruli of
primiparous obese mice was greater than that of the nulliparous obese mice (P<0.05; Figure
4.8B). Percentage area of picrosirius red (PSR) staining within transverse sections of the
subendocardium region of the mid-ventricle wall was used as marker of cardiac fibrosis
(Figure 4.8A&B). Percentage area of PSR staining in the subendocardium of mid-ventricle
was significantly elevated with pregnancy at 4WPW (Ppreg<0.05; Figure 4.9B). However,
post-hoc analysis demonstrated that this effect of pregnancy was only present among obese
mice, but not in control mice (P<0.05; Figure 4.9B).
Figure 4.7 Total renal and left ventricular (LV) collagen content. A) Renal hydroxyproline
content; B) LV hydroxyproline content. N: nulliparous; P: primiparous; N-Control (n=7); N-
Obese (n=8); P-Control (n=5); P-Obese (n=5); Data were analyzed by two-way ANOVA with
factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-
hoc analysis. **P<0.01. Data are presented as Mean±SEM.
Chapter 4 Obesity & Hemodynamic Changes Post-partum
101
Figure 4.8 Assessment of glomerulosclerosis. A) Representative Brightfield images of renal PAS staining in control and obese mice that were either nulliparous or primiparous; Scale bar = 100 µm B) Quantification of percentage PAS positive staining within the glomeruli. N: nulliparous; P: primiparous; N-Control (n=6); N-Obese (n=8); P-Control (n=5); P-Obese (n=5); Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05. Data are presented as Mean±SEM.
Nulliparous Primiparous C
ontr
ol
Obe
se
A
B
N P N P0
10
20
30
%PAS positive staining
GlomerulosclerosisPint NSPpreg NSPobe NS
Control Obese
*
Chapter 4 Obesity & Hemodynamic Changes Post-partum
102
Figure 4.9 Assessment of cardiac fibrosis. A) Representative Brightfield images of PSR staining in the subendocardium region of the left ventricle in control and obese mice that were either nulliparous or primiparous. Scale bar = 300 µm B) Quantification of percentage PSR positive staining within the fields selected. N: nulliparous; P: primiparous; N-Control (n=5); N-Obese (n=8); P-Control (n=4); P-Obese (n=5). Data were analyzed by two-way ANOVA with factors of obesity (Pobe), pregnancy (Ppreg) and interaction (Pint), followed by Sidak post-hoc analysis. *P<0.05. Data are presented as Mean±SEM.
Nulliparous Primiparous
Con
trol
O
bese
A
N P N P0.0
0.5
1.0
1.5
2.0
% P
SR p
osit
ive
stai
nin
g
LV Fibrosis
*
Pint NSPpreg <0.05Pobe NS
Control Obese
B
Chapter 4 Obesity & Hemodynamic Changes Post-partum
103
4.4. DISCUSSION
This study aimed to determine the impact of pregnancy on cardiovascular and renal
outcomes post-partum in a model of obesity. There were three major findings in the present
study. Firstly, pregnancy exacerbated obesity-induced hypertension post-partum. Secondly,
pregnancy led to greater renal and glomerular fibrosis in obese mice, in the absence of an
exacerbated effect on albuminuria or renal function post-partum. Lastly, pregnancy led to
greater weight gain, greater accumulation of visceral fat, and a small but significant elevated
cardiac fibrosis in obese mice post-partum. These findings indicate that pregnancy has a
mild impact on cardiovascular and renal health in obese females post-partum. Evidence of
exacerbated hypertension and greater renal and cardiac fibrosis may however predispose
obese females to greater risks of cardiovascular and renal morbidity later in life.
In the present study, we found that MAP of obese mice remained elevated over the
MAP of control mice during the post-partum period. The Initial response in MAP post-partum
was similar, with MAP of control and obese mothers returning to pre-pregnancy levels by
PN6. Similarly, studies in humans have found that arterial pressure returned to pre-
pregnancy values when measured at 12-14 weeks post-partum70,236. Whilst MAP of control
mice remained stable post-partum, obese mice showed a rise in MAP beginning on day
PN29. Moreover, this increase in arterial pressure was only present during the light phase
when mice are at rest. This finding is consistent with what has been shown in human obesity,
where SBP, DBP and pulse pressure of obese individuals during night-time were higher than
lean subjects. 208 Further, study by Kang et al193 also demonstrated that obese women had
more profound elevation of night-time blood pressure than obese men when compared to
respective lean counterparts, indicating that obese hypertensive women might have a greater
risk of developing cardiovascular events during night-time. It has also been reported that
night-time high blood pressure in humans was related to an increased risk of chronic kidney
disease and all-cause death, suggesting that night-time blood pressure has significant
clinical relevance.194 This greater rise in light phase MAP of obese compared to control
primiparous mice at PN36 was profound (14.2 versus 2.1 mmHg respectively). Importantly
the present study also demonstrated that pregnancy exacerbated hypertension in obese
mice with light-phase MAP of primiparous obese mice was greater than nulliparous obese
mice over the same period (14.2 versus 8.0 mmHg respectively). This indicates that the
impact of pregnancy and obesity on MAP was greater than the impact by obesity alone.
The findings of this chapter are consistent with studies demonstrating that women
who experience all forms of hypertensive disorders of pregnancy not only have higher pre-
pregnancy BMI but also exhibit greater risk of developing subsequent hypertension, stroke,
CKD and diabetes compared with normotensive controls.240,372 Interestingly, Goel et al 131
Chapter 4 Obesity & Hemodynamic Changes Post-partum
104
demonstrated that women who developed hypertension post-partum, regardless of whether
they were hypertensive or normotensive during pregnancy, had significantly elevated BMI
compared with women who were normotensive post-partum. Taken together, these studies
suggest that being overweight or obese contributes significantly to the development of post-
partum hypertension. No study has prospectively examined cardiovascular parameters in
obese women prior to and during pregnancy, and chronically post-partum. Future studies
should address this gap in our knowledge.
Whilst tachycardia in obese mice was not exacerbated by pregnancy in present study,
persistent tachycardia is an independent risk factor for the development atherosclerosis and
coronary heart disease.281 In examining the impact of pregnancy and obesity in the
development of cardiac fibrosis using a hydroxyproline assay, we found neither pregnancy
nor obesity had an impact on total collagen content between the groups. However, using
PSR staining we demonstrated a greater level of cardiac fibrosis with pregnancy in obese
mice. It is important to note that the portion of the tissue used in hydroxyproline assay was
restricted to the one-third of the LV tissue at the apical end, which is generally considered to
be less prone to fibrosis. In contrast, the histological analysis of collagen deposition using
PSR staining was performed on transverse sections of the mid-ventricle of the heart. The
fields selected for this analysis was restricted to the subendocardium region of the ventricular
wall, the region that experiences the greatest force and stress during contraction and filling in
a cardiac cycle, and thus more likely to develop fibrosis. Given the myocardial shortening in
subendocardium region is the dominant force generator of myocardial contraction,57,71 the
development of fibrosis in this region would have a substantial impact on the contractile
function of the heart. Given that the increase in cardiac fibrosis was only observed in obese
mice during the post-partum period suggests that obese females might be at a high risk of
developing cardiac dysfunction post-partum due to a greater level of cardiac fibrosis.
However, the mechanisms that mediate the greater collagen deposition in the heart of obese
mice post-partum are yet to be elucidated.
To the best of my knowledge, this is the first study that has specifically and
comprehensively examined maternal renal structure and function pos-tpartum in an animal
model of pre-pregnancy obesity. In contrast to what we hypothesized, pregnancy had
minimal impact on renal function in obese mice post-partum. At 4WPW, we found that neither
obesity nor pregnancy had an impact on renal excretory function or GFR. Although albumin
excretion was greater in obese mice, pregnancy did not exacerbate albuminuria. Pregnancy
however increased renal collagen content in obese mice. Obese primiparous mice had 44%
greater total renal collagen deposition and a 28% increase in glomerular collagen content
compared with nulliparous obese mice, suggesting pregnancy leads to some degree of renal
Chapter 4 Obesity & Hemodynamic Changes Post-partum
105
and glomerular injury in obese mice post-partum. Whilst no human studies have specifically
addressed the impact of pregnancy on renal health in obesity, similar studies have been
performed post-partum in preeclamptic women who demonstrate hypertension and
albuminuria. Consistent with our study, preeclamptic women have demonstrated comparable
GFR to control subjects by 4 weeks post-partum, despite a lower GFR detected at post-
partum day 1.165 Although preeclamptic women had normal GFR, a high percentage of these
women experienced persistant hypertension (16%) and overt proteinuria (22%) 3 months
post-partum.209 Further, some women who had preeclampsia exhibited permanent renal
injury including glomerulosclerosis and focal interstitial scarring.155 These findings indicate
that whilst renal function in women who have had preeclampsia might be preserved short-
term post-partum, the presence of renal injury and persistent hypertension may increase the
rise of CKD later in life.
Another major health risk in women post-partum is weight retention and this is
exacerbated with obesity. In the present study, we demonstrated that pregnancy facilitates
excess weight gain in obese mice to a large extent by promoting accumulation of excess
visceral fat. Our result is consistent with findings in humans that high pre-pregnancy BMI is a
strong predictor of post-partum weight retention and high prevalence of obesity both short-
term (3 months) and up to 2 years post-partum.112,142,241,337 Further, although obese mice in
the present study had only relatively mild cardiovascular and renal phenotype at 4WPW, it is
recognized that visceral obesity is associated with a greater risk of comorbidities such as
type 2 diabetes, chronic hypertension, coronary heart disease, renal dysfunction and chronic
inflammation.351 Thus, having excess abdominal fat in addition to persistent obesity and
hypertension in women post-partum, is likely to predispose women to a greater risk of
cardiovascular and renal morbidity and mortality later in life, supporting the epidemiological
findings.223,384
Several limitations of the present study should be considered. Firstly, renal function
including GFR and albuminuria were examined at a single time point (4WPW) as a snapshot
of the renal health during the post-partum period. Multiple measurements of renal function
post-partum in a longitudinal setting would likely to provide further understanding into the
potential for developing CKD in this model. Secondly, we did not obtain any radiotelemetry
data in nulliparous control mice, thus we were unable to differentiate the impact of pregnancy
on arterial pressure post-partum between control and obese mice. However the literature
suggests that arterial pressure is not adversely affected by parity in uncomplicated
pregnancies.143 Thirdly, although we detected a clear difference in the level of fibrosis in the
kidney, the evidence of fibrosis in heart was inconsistent. This inconsistency is likely due to
the differences in the section of the ventricle used for each analysis. Future studies should
Chapter 4 Obesity & Hemodynamic Changes Post-partum
106
focus on the development of fibrosis in the mid-ventricular wall of the heart. In both the heart
and kidney, it would be also interesting to examine the subtypes of collagen present in these
tissues and abundance of collagen-producing myofibroblasts using biochemical and
immunohistochemical techniques.249,281 Lastly, our study only followed cardiovascular
phenotype for a short period post-partum. Unfortunately due to limitations in battery life of the
radiotelemetry transmiters we were unable to track arterial pressure beyond PN36. In the
current protocol mice were recorded continuously for 85-100 days. Future studies could use
intermittent recording to increase the longevity of the radiotelemetry transmitters to assess
the impact of pregnancy on arterial pressure of obese females in the long-term. Further, it
would be also important to investigate whether obese primiparous mice are more vulnerable
to secondary cardiovascular and renal insults such as diabetes and high salt diet during later
life.
4.5. CONCLUSION
The present study demonstrates that pregnancy exacerbates hypertension in obese
mice during light phase over a short period post-partum, but does not lead to overt renal
dysfunction during this period. However, excess weight gain and greater level of renal and
cardiac fibrosis in obese mice post-partum would likely contribute to the progression of
cardiovascular and renal morbidities if these mice were to followed for a longer period post-
partum. Our findings suggest that pregnancy is likely to be detrimental for obese women in
terms of long-term cardiovascular and renal health. Early detection and appropriate
interventions should be considered by health professionals in managing the risk factors for
the early onset of cardiovascular and renal morbidities in obese mothers post-partum.
Chapter 5 Renal Function & Nephron Deficiency
107
Chapter 5 THE ROLE OF NITRIC OXIDE IN THE
REGULATION OF RENAL FUNCTION AND
ARTERIAL PRESSURE IN NEPHRON DEFICIENT
MICE
Chapter 5 Renal Function & Nephron Deficiency
108
5.1. INTRODUCTION
Human studies have shown that nephron number varies up to 13 fold among
populations.38,248,291 Studies in animal models would suggest this is due to both genetic
influence and the intrauterine environment which is highly susceptible to maternal
malnutrition, disease, drug exposure and, as identified in Chapter 3, overnutrition,201 Brenner
and colleagues suggested that individuals born with a low nephron number are at a greater
risk of developing hypertension and progressive renal disease.46 This hypothesis has been
supported by findings obtained in animal models of nephron deficiency87,216,277,374 and human
studies where offspring show elevated arterial pressure, low glomerular filtration rate (GFR)
and renal fibrosis.170,176,200,321,329 However not all studies in this field support the Brenner
hypothesis with studies in humans177 and animal45,212,234,312 with low nephron number
demonstrating normal arterial pressure and renal function even at an old age. One such
study from our laboratory demonstrated that both phenotypes of GDNF heterozygous (HET)
mice (HET-2K -30% deficit; HET-1K -65% deficit) had a normal total GFR up to 14months of
age.312 With a normal GFR but low nephron number, this study indicated that single nephron
GFR (SNGFR) of HET-2K mice was doubled, and HET-1K mice almost fourfold higher than
WT mice.312 However, the mechanism by which the single nephron hyperfiltration is
maintained in this state of nephron deficit is unclear.
One of the most prominent regulators of renal function is nitric oxide (NO). NO is
formed during the conversion of L-arginine to L-citrulline by NO synthases (NOS) and
cofactors.343 In the kidney, NO plays a significant role in the regulation of renal
hemodynamics, blunting tubuloglomerular feedback (TGF), inhibition of tubular sodium
reabsorption and modulation of renal sympathetic activity.264 In general, renal NO production
promotes natriuresis and diuresis.183,264 Neuronal NOS (nNOS) and endothelial NOS (eNOS)
are the main NOS isoforms abundantly expressed in the kidney. Whilst nNOS is primarily
expressed in macula densa, thick ascending limb, vasa recta and throughout collecting duct
of the nephron,16,242,380 eNOS has been found mainly in renal vasculature, thick ascending
limb, as well as collecting duct of the nephron.16,264,356 Of importance, the high abundance of
nNOS in the macula densa has been implicated as the source of NO production that is
responsible for blunting TGF mechanism, which is a potent regulator of GFR and arterial
pressure.47,230,370,371 Indeed, reduced NO bioavailability has been shown to contribute to the
pathogenesis and progression of hypertension and chronic kidney disease in humans.11,27,233
A recent study using an ovine model of congenital nephron deficiency demonstrated that
age-related renal dysfunction was associated with a reduction in NO-mediated regulation of
renal hemodynamics and sodium excretion, and significant vascular dysfunction due to
reduced contribution of NO.216 Further, Muller et al 265 demonstrated that chronic systematic
Chapter 5 Renal Function & Nephron Deficiency
109
NOS inhibition with either the non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester
(L-NAME) or the nNOS selective inhibitor, 7-nitro-indazole (7NI) rendered the renal injury
resistant C57BL/6J mice susceptible to renal mass reduction-induced chronic kidney disease.
These findings led us to hypothesize that NO contributes significantly to the maintenance of
normal GFR and excretory function in animals with a nephron deficit, and in the absence of
NO, renal function deteriorates.
To test our hypothesis, we used GDNF HET mice for two reasons. Firstly, this genetic
mouse model of low nephron endowment allows us to examine the regulation of renal
function and arterial pressure without the confounding influence of globally programmed
cardiovascular dysfunction that often results from maternal undernutrition and overnutrition.
Secondly, this model provides two distinct levels of nephron deficit within one genotype and
both GDNF HET-2K and HET-1K mice have normal GFR and remain normotensive even
through old age. The aim of present study was to determine the arterial pressure and renal
function of GDNF WT, HET-2K, and HET-1K mice prior to and in response to systematic NO
inhibition with L-NAME. We predicted that chronic NO deficiency would lead to greater
hypertension and renal dysfunction in GDNF HET mice in a manner dependent on the level
of nephron deficit.
Chapter 5 Renal Function & Nephron Deficiency
110
5.2. METHODS
5.2.1. Animals
All experiments were approved by Monash University Animal Ethics Committee and
conducted in accordance with the Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes. Thirty-week-old male GDNF HET mice and WT littermates
were obtained from Monash Animal Research Platform, Monash University. Genotype of the
mice was determined by PCR as described in Section 2.1.2.3. Mice had ad libitum access to
a maintenance diet (AIN93M, Specialty Feed, Australia) and tap water, and were housed in
the experimental room maintained at 24-26 °C with 12:12 hour light-dark cycle.
5.2.2. Experimental Protocol
At 30 weeks of age, mice were placed in metabolic cages to obtain 24hour urine
samples for assessment of basal renal excretory profile (electrolytes, osmolality, albumin
excretion and creatinine concentration; see Section 2.2.1). Mice were then anaesthetized
(Isoflurane, 2.2-2.6%) and radiotelemetry probes (PA-C10, DSI, MN, USA) implanted for the
measurement of conscious arterial pressure (see Section 2.3).312,365 A small blood sample
(~60μl) was collected from the left carotid artery to determine basal plasma creatinine
concentration. Mice were given a 10-day recovery period following which basal arterial
pressures, heart rate and locomotor activity were recorded continuously for 7 days. At the
end of the 7 days of basal recording, mice were administered L-NAME (0.5mg/ml L-NAME
Hydrochloride; Sigma-Aldrich, USA) in the drinking water for a further 7 days.138 Mice were
then placed in metabolic cages again to obtain 24hr urine samples for assessment of renal
excretory profile.
5.2.3. Terminal Tissue Collection
Following the final 24hour urine sample collection, mice were anaesthetised
(Isoflurane) and an arterial blood sample taken from the right carotid artery for the
measurement of plasma creatinine concentration. The kidneys were rapidly excised,
decapsulated, weighed and cut into equal 4 portions transversely. All portions of the right
kidney were frozen in liquid nitrogen and a middle portion used for RT-PCR analysis. In the
case of the solitary kidney (GDNF HET-1K mice), the kidney was excised, weighed, cut
through transverse plane into 3 portions. The middle portion of the kidney was frozen in liquid
nitrogen for PCR analysis. The heart was excised and the left ventricle isolated and weighed.
Chapter 5 Renal Function & Nephron Deficiency
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Tissues from non-treated, age-matched cohort (time controls) of GDNF WT, HET-2K and
HET-1K mice were collected in the same manner.
5.2.4. RT-qPCR
To assess relative renal gene expression levels, total RNA was extracted from frozen
kidney tissues of L-NAME treated and untreated GDNF WT, HET-2K and HET-1K mice using
an RNeasy Mini Kit (Giagen, Hilden, Germany). RNA was then reverse transcribed into
cDNAs using iScript Reverse Transcription Supermix for RT-qPCR (BIORAD, Hercules, CA,
USA) according to manufacturer’s instructions. Gene expression analysis for the NOS
isoforms (NOS1, NOS2 & NOS3), aquaporin 2 (AQP2), sodium-hydrogen antiporter 3
(NHE3), sodium-potassium-chloride cotransporter 2 (NKCC2), angiotensin type 1a receptor
(AT1aR), angiotensin type 2 receptor (AT2R) were performed with TaqMan gene expression
assays using the Applied Biosystems 7900HT Fast Real-Time PCR system (Applied
Biosystems by Life Technologies, Foster City, CA, USA). TaqMan gene expression assays
(Dye: FAM-MGB; Applied Biosystems) used for genes of interest are Nos1
(Mm00435175_m1) for NOS1/nNOS, Nos2 (Mm00440502_m1) for NOS2/iNOS, Nos3
(Mm00435217_m1) for NOS3/eNOS, Slc12a1 (Mm01275821_m1) for NKCC2, Slc9a3
(Mm01352473_m1) for NHE3, Aqp2 (Mm00437575_m1) for AQP2, Agtr1a
(Mm01957722_s1) for AT1aR, and Agtr2 (Mm01341373_m1) for AT2R. RT-qPCR reactions
were run in triplicate and duplexed with 18S as endogenous housekeeping gene (Dye: VIC-
Primer limited; Eukaryotic 18S rRNA endogenous control for Agtr1a and Agtr2; Rn18s,
Mm03928990_m1 for other genes; Applied Biosystems by Life Technologies, CA, USA).
Reactions were assembled on a 384-well PCR plate using an automated liquid handler
(CAS-1200 liquid handler, Giagen, Germany). RT-qPCR Data was analyzed by Applied
Biosystems Sequence Detection (SDS) version 2.4 software. The relative expression of
mRNA levels was calculated using the comparative ΔCt (threshold cycle number) method.323
The relative fold changes in gene expression for each target gene was calculated against
untreated GDNF WT group.
5.2.5. Statistical Analysis
Basal telemetry data and renal excretory profile data were analyzed using one-way
ANOVA, followed by Tukey post-hoc analysis. Change in MAP, HR and locomotor activity
during L-NAME treatement was analyzed by two-way repeated measure ANOVA considering
factors of group (Pg) and time (Pt) and interaction between group and time (Pgxt), followed
by Bonferroni post-hoc analysis. Renal excretory profile before and after L-NAME treatement
Chapter 5 Renal Function & Nephron Deficiency
112
was analyzed by two-way repeated measure ANOVA considering factors of group (Pg) and
treatment (Pt) and interaction between group and treatment (Pgxt), followed by Bonferroni
post-hoc analysis. For terminal tissue weight and gene expression analysis, two-way ANOVA
were conducted considering factors of group (Pg) and treatment (Pt) and interaction between
group and treatment (Pgxt), followed by Bonferroni post-hoc analysis. Values are mean ±
SEM. P<0.05 was considered statistically significant.
Chapter 5 Renal Function & Nephron Deficiency
113
5.3. RESULTS
5.3.1. Basal Cardiovascular and Renal Excretory Profile
WT, HET-2K and HET-1K mice did not differ in 24hr mean arterial pressure (MAP;
104.1±10, 103.0±2.1, 101.1±2.1 mmHg respectively; Figure 5.1A) or heart rate (HR, 486±11,
500±9, 510±17 bpm Figure 5.1B) under basal conditions. There were also no differences in
systolic or diastolic pressures between the groups, or any differences between groups during
light or dark phase of the day (data not shown). Locomotor activity of HET-2K and HET-1K
mice (3.6±0.3, 5.1±0.4 Counts/min respectively) was not different to WT mice (4.3±0.5
Counts/min) though HET-1K mice were more active than HET-2K mice (P<0.05).
Water intake and urine excretion were not significantly different between WT and
HET-2K mice. Both water intake and urine excretion were significantly greater in HET-1K
mice compared with WT and HET-2K mice under basal conditions (P<0.01-0.001; Figure
5.1C&D). HET-1K mice excreted significantly more sodium than WT and HET-2K mice
during 24 hr urine collection period (P<0.01 and P<0.05 respectively, Figure 5.1F). The high
sodium excretion was reflected in higher food intake in HET-1K mice compared with WT
mice (P<0.05; Figure 5.1E), however the comparison with HET-2K mice did not reach
statistical significance. In addition, there were no differences between WT, HET-2K and HET-
1K mice in creatinine clearance, nor in 24hr albumin excretion under basal conditions (Figure
5.1G&H).
Chapter 5 Renal Function & Nephron Deficiency
114
Figure 5.1 Basal arterial pressure and heart rate (WT n=8, HET-2K n=8 and HET-1K n=7) and renal excretory profile (WT n=11, HET-2K n=14 and HET-1K n=8). A: Basal 24hr mean arterial pressure (MAP). B: Basal 24hr heart rate (HR). Water intake. C: Water intake. D: Urine excretion. E: Food intake. F: Sodium excretion. G: Creatinine clearance (Ccre). H: Albumin excretion. Date analyzed using one-way ANOVA with Tukey post-hoc analysis. *P<0.05; **P<0.01; ***P<0.001.
24h MAP
WT HET-2K HET-1K
0
20
40
60
80
100
120
mm
Hg
24h HR
WT HET-2K HET-1K
0
100
200
300
400
500
600
bp
m
A
E
C
B
F
DWater Intake
WT HET-2K HET-1K
0
30
60
90
120
150
180
µl/2
4h
/gB
W
*****
Urine Excretion
WT HET-2K HET-1K
0
20
40
60
80
100
µl/2
4h
/gB
W
******
Food Intake
WT HET-2K HET-1K
0.00
0.03
0.06
0.09
0.12
0.15
0.18
g/g
BW
*
Sodium Excretion
WT HET-2K HET-1K
0
2
4
6
8
µm
ol/2
4h
/gB
W
***
Ccre
WT HET-2K HET-1K
0
2
4
6
8
10
12
µl/m
in/g
BW
Albumin Excretion
WT HET-2K HET-1K
0.00
0.02
0.04
0.06
0.08
0.10
0.12
µg
/24
h/g
BW
HG
Chapter 5 Renal Function & Nephron Deficiency
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5.3.2. Arterial Pressure and Renal Excretory Profile During NOS Inhibition
In response to systematic NOS inhibition with L-NAME, there were similar rises in
MAP of WT, HET-2K and HET-1K mice during the first 24 hours (17.9±0.6, 16.1±0.9,
18.4±1.4 mmHg respectively; Figure 5.2A&B). However the groups differed in their response
over the following 6 days (Pgxt<0.001; Figure 5.2B). The initial rise in MAP of WT mice was
maintained over the next 6 days (Figure 5.2B). However, the MAP response to L-NAME in
HET-2K and HET-1K mice was gradually attenuated over the following 6 days. By the last
day of recording, the rise in MAP of WT mice from the basal level was 14.0±1.3 mmHg whilst
the rise in MAP of HET-2K and HET-1K mice had lessened to 12.0±1.7 and 7.6±2.0 mmHg
respectively (Figure 5.2B). However, post-hoc analysis demonstrated that only HET-1K mice
had significantly lower MAP than WT mice on day 7 of L-NAME treatment (P<0.01). HR of all
groups demonstrated an initial fall in response to L-NAME (Figure 5.2C) before gradually
returning to basal level (Pt<0.01; Figure 5.2D). There was no significant difference in HR
response to L-NAME between groups (Figure 5.2D). Locomotor activity was not altered in
any of the groups following L-NAME treatment. (Data not shown)
Figure 5.2 Mean arterial pressure (MAP) and heart rate (HR) during 7 day of L-NAME treatment in WT (n=8), HET-2K (n=8) and HET-1K (n=7) mice. A: Absolute MAP from basal condition to day 7 of NOS inhibition (L-NAME treatment). B: Change in 24hr MAP in response to NOS inhibition. C: Absolute HR. D: Change in 24hr HR in response to NOS inhibition. Data (B&D) were analyzed using two-way repeated measures ANOVA with factors of group (Pg), time (Pt) and interaction (Pgxt) and Bonferroni’s post-hoc analysis was conducted. NS, not significant.
24h MAP during NOS inhibition
Days of NOS Inhibition
mm
Hg
Basal 1 2 3 4 5 6 790
100
110
120
130
HET1K (n=7)
WT (n=8)
HET2K (n=8)
24h HR during NO inhibition
Basal 1 2 3 4 5 6 7400
450
500
550
Days of NOS Inhibition
bp
m
WT (n=8)
HET2K (n=8)
HET1K (n=7)
Change in 24h MAP
1 2 3 4 5 6 70
5
10
15
20
Days of NOS Inhibition
∆ m
mH
g
Pgxt <0.001Pg <0.05Pt <0.001
Change in 24h HR
1 2 3 4 5 6 7
-80
-60
-40
-20
0
20
Days of NOS Inhibition
∆ b
pm
Pgxt NSPg NSPt <0.001
A
C
B
D
Chapter 5 Renal Function & Nephron Deficiency
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At the completion of arterial pressure recordings, 24h urine samples were collected
again while mice continued L-NAME treatment. There was a significant effect of L-NAME on
water intake, food intake, urine and sodium excretion (Pt<0.05-0.001; Figure 5.3A-D),
however this effect was primarily found in HET-1K mice. The higher 24h water intake and
urine excretion of HET-1K mice were abolished by L-NAME treatment (water intake Pgxt
<0.01, urine excretion Pgxt <0.001; Post-hoc analysis P<0.001; Figure 5.3A&B). Post-hoc
analysis showed that urine excretion of HET-2K was also significantly reduced with L-NAME
treatment (P<0.01), however this was not reflected in a significant fall in water intake (Figure
5.3A&B). The greater 24h sodium excretion of HET-1K mice was abolished by L-NAME
treatment such that they no longer differed from WT and HET-2K mice (Pgxt <0.01, Post-hoc
analysis P<0.001; Figure 5.3D). Food intake in HET-1K mice reflected the changes in
sodium excretion such that L-NAME treatment normalized the differences in food intake
between the WT and HET-1K mice (Pt<0.05; Post-hoc analysis P<0.05; Figure 5.3C). L-
NAME treatment did not alter creatinine clearance among groups (Figure 5.3E). Albumin
excretion was reduced with L-NAME treatment (Pt<0.01), but no difference was detected
among groups (Figure 5.3F).
Chapter 5 Renal Function & Nephron Deficiency
117
Figure 5.3 Renal excretory profile (WT n=11, HET-2K n=14 & HET-1K n=8) and creatinine clearance (WT n=6, HET-2K n=9 & HET-1K n=7) under basal conditions and after L-NAME treatment. A: 24h water intake. B: 24h urine excretion. C: 24h food intake. D: 24h sodium excretion. E: Creatinine clearance (Ccre). F: 24h albumin excretion. Date analyzed using two way repeated measures ANOVA with factors of group (Pg), treatment (Pt) and interaction (Pgxt), followed by Bonferroni’s post-hoc analysis. NS = not significant. *P<0.05; **P<0.01; ***P<0.001.
Chapter 5 Renal Function & Nephron Deficiency
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5.3.3. Terminal Tissue Weights
Body weights of GDNF WT, HET-2K and HET-1K mice was not influence by L-NAME
treatment and no difference was detected among groups within each cohort (Table 5.1). As
expected, total kidney weight of untreated control HET-2K and HET-1K mice was
significantly lower than WT mice (Pg<0.001, Post-hoc analysis P<0.001; Table 5.1). Although
the solitary kidney of HET-1K was substantially heavier than a single kidney of HET-2K and
WT mice (data not shown), the solitary kidney of HET-1K mice weighed significantly less
than the total kidney weight of HET2K and WT mice (Post-hoc analysis P<0.01-0.001; Table
5.1). L-NAME treatment did not alter the differences in kidney weight observed in untreated
control cohort (Table 5.1). Similar effects of group and L-NAME treatment were found for
kidney weights when corrected for body weight (Table 5.1). Left ventricle weight was not
affected by group or L-NAME treatment (Table 5.1).
Chapter 5 Renal Function & Nephron Deficiency
119
Table 5.1 Terminal tissue weights. Kid: B Wt, total kidney to body weight ratio; LV, left ventricle; LV:B Wt, left ventricle to body weight ratio; WT, wild-type; HET-2K, GDNF HET mice born with 2 kidneys; HET-1K, GDNF HET mice born with 1 kidney; Data were analyzed by Two-way ANOVA considering factors of treatment (t), group (g), and interaction (gxt), followed by Bonferroni’s post-hoc analysis. For all the parameters of L-NAME treated group, WT (n=16), HET-2K (n=14) and HET-1K (n=10). For untreated control group, WT (n=18), HET-2K (n=10) and HET-1K (n=11) except for LV Wt and LV:Bwt Ratio of untreated control group, WT (n=15), HET-2K (n=5) and HET-1K (n=9). § P<0.05, # P<0.01, * P<0.001, compared to WT within group. † P<0.01 ^ P<0.001 compared to HET-2K within group. NS, not significant.
Untreated control L-NAME treated P
WT HET-2K HET-1K WT HET-2K HET-1K t g g x t
Body Wt, g 36.0±1.0 34.4±1.3 34.8±1.5 34.7±1.1 34.0±1.0 33.6±0.8 NS NS NS
Kidney Wt, mg 347±8 283±11* 223±9*† 339±13 296±12§ 228±12*^ NS <0.001 NS
Kid:B Wt, mg/g 9.79±0.36 8.26±0.26# 6.46±0.27*† 9.76±0.25 8.72±0.26§ 6.81±0.35*† NS <0.001 NS
LV Wt, mg 107±3 106±8 102±3 105±2 109±4 105±5 NS NS NS
LV:B Wt, mg/g 3.06±0.13 3.09±0.15 2.96±0.13 3.05±0.08 3.21±0.10 3.14±0.17 NS NS NS
Chapter 5 Renal Function & Nephron Deficiency
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5.3.4. Gene Expression of Sodium and Water Channels
There were no differences in relative gene expression of renal NOS isoforms (NOS1,
NOS2, NOS3), NHE2, NKCC2 and AT2R in L-NAME treated and untreated GDNF WT, HET-
2K and HET-1K mice (Table 5.2). There was a tendency for L-NAME to increase renal AQP2
expression in GDNF HET-2K and HET-1K mice, however these differences did not reach
statistical significance (Pt=0.08; Table 5.2). There was an increase in AT1aR gene
expression in response to L-NAME, but this effect was dependent on group (Pg<0.05,
Pgxt<0.01, Table 5.2). Post-hoc analysis showed that L-NAME resulted in a 1.5 fold increase
in AT1aR expression in kidneys of WT mice (P<0.05), but L-NAME did not change AT1aR
gene expression in the kidneys of HET-2K and HET-1K mice (Table 5.2).
Chapter 5 Renal Function & Nephron Deficiency
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Table 5.2 Relative gene expression. NOS, nitric oxide synthase; AQP2, aquaporin 2, NHE3, sodium-hydrogen antiporter 3; NKCC2, sodium-potassium-chloride cotransporter 2; AT1aR, angiotensin type 1a receptor; AT2R, angiotensin type 2 receptor; WT, wild-type; HET-2K, GDNF HET mice born with 2 kidneys; HET-1K, GDNF HET mice born with 1 kidney; Data analyzed by Two-way ANOVA considering factors of treatment (Pt), group (Pg) and interaction (Pgxt), followed by Bonferroni’s post-hoc analysis. For untreated cohort, WT (n=6), HET-2K (n=7) and HET-1K (n=6). For L-NAME treated cohort, WT (n=5), HET-2K (n=5) and HET-1K (n=5). †P<0.05 when compared to untreated WT mice. NS, not significant.
Untreated control L-NAME treated P
Genes WT HET-2K HET-1K WT HET-2K HET-1K t g g x t
NOS1 1.00±0.18 0.96±0.09 1.03±0.11 1.10±0.24 1.22±0.13 1.20±0.15 NS NS NS
NOS2 1.00±0.16 0.83±0.17 0.76±0.11 0.91±0.09 0.71±0.03 0.84±0.10 NS NS NS
NOS3 1.00±0.10 1.11±0.11 1.05±0.14 1.09±0.08 1.34±0.11 1.08±0.12 NS NS NS
AQP2 1.00±0.15 1.00±0.21 0.88±0.15 1.11±0.29 1.66±0.70 1.68±0.45 NS NS NS
NHE3 1.00±0.07 1.01±0.09 0.93±0.08 1.01±0.06 1.07±0.08 0.95±0.04 NS NS NS
NKCC2 1.00±0.18 0.88±0.04 0.73±0.07 0.86±0.07 1.19±0.21 0.98±0.14 NS NS NS
AT1aR 1.00±0.03 1.27±0.14 0.98±0.04 1.46±0.18† 0.91±0.07 0.93±0.10 NS P<0.05 P<0.01
AT2R 1.00±0.17 0.89±0.18 0.66±0.19 0.89±0.09 0.98±0.16 0.61±0.04 NS NS NS
Chapter 5 Renal Function & Nephron Deficiency
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5.4. DISCUSSION
The aim of the present study was to examine the arterial pressure and renal function
responses to systematic NOS inhibition in nephron deficient GDNF HET mice. There are
three major findings of the present study. Firstly, in stark contrast to our hypothesis, L-
NAME-induced hypertension was not exacerbated in GDNF HET mice, instead GDNF HET
mice, particularly HET-1K mice demonstrated a marked escape from the initial hypertensive
response to L-NAME treatment over the 7 days. Secondly, L-NAME treatment abolished
greater diuresis and natriuresis present in the GDNF HET-1K mice, and did not reduce GFR
or induce albuminuria. Lastly, the upregulation of renal AT1aR gene expression in WT mice
in response to NOS inhibition was absent in GDNF HET-2K and HET-1K mice. Taken
together, these findings indicate that maintenance of normal arterial pressure and renal
function in nephron deficient GDNF HET mice may not be strongly dependent on NO
bioavailability.
Using radiotelemetry, the present study found that 30-week old GDNF HET-2K and
HET-1K mice had very similar basal MAP and HR as WT mice, consistent with previous
studies.312,336 Brenner et al 46 suggested that a reduced filtration surface area (FSA) is
responsible for the development of hypertension in individuals born with low nephron
endowment. We have previously shown that glomeruli of HET-2K mice undergo significant
hypertrophy such that by 30 weeks of age total glomerular volume and, therefore total FSA
are not different from WT mice.336 Thus, it is perhaps not surprising that HET-2K mice remain
normotensive. However, although glomeruli of HET-1K mice undergo the same magnitude of
hypertrophy, this does not offset the 65% nephron deficit leaving total glomerular volume
(therefore FSA) only 50% of WT and HET-2K mice.312 Yet, we have consistently
demonstrated that the markedly reduced nephron number and FSA are not associated with
hypertension in HET-1K mice under basal conditions. Again consistent with our previous
studies, we found that urine and sodium excretion were significantly higher in 30-week old
HET-1K mice compared to WT and HET-2K mice.312 GFR and albumin excretion were also
similar among the 3 groups of mice, in agreement with our previous findings.312
In response to L-NAME, urine and sodium excretion of WT mice were not altered
from basal levels. Carlstrom et al 56 also found that L-NAME given at the same dose as the
current study did not affect diuresis in rats after 7 days. However in contrast to our study,
they found L-NAME for 7 days reduced sodium excretion.56 Given that NO promotes
natriuresis, it is likely that other natriuretic mechanisms might be upregulated to maintain a
sodium balance in WT mice when NO synthesis is blocked. Sodium excretion of HET-2K
mice was also not changed in response to L-NAME treatment. However, we did observe an
anti-diuretic effect of L-NAME on HET-2K mice. The effect of L-NAME was however more
Chapter 5 Renal Function & Nephron Deficiency
123
profound in HET-1K reducing urine and sodium excretion of GDNF HET-1K mice to the
levels seen in HET-2K and WT mice. This anti-diuretic and anti-natriuretic effect of L-NAME
indicates NO plays a dominant role in promoting diuresis and natriuresis in HET-1 mice.
However, the anti-diuretic and anti-natriuretic effects of L-NAME found in HET-1 mice could
not be explained by changes in the level of mRNA expression of water (AQP2) and sodium
channels (NHE2 & NKCC2) in the renal tissues. Although not statistically significant, AQP2
mRNA expression in L-NAME treated HET-1K kidneys was 90% higher than untreated HET-
1K kidneys, indicating that AQP2 gene expression might be upregulated during L-NAME
treatment, potentially contributing to the anti-diuretic effects. Whilst the mRNA expression of
renal AQP2 did not change, we cannot exclude the possibility that changes may have
occurred at the level of AQP2 protein abundance, particularly at the apical membrane of the
collecting duct, which is likely to be responsible for the changes in renal excretory profile
seen in HET-1K mice. To further investigate the role of AQP2 in the diuresis, abundance of
membrane-bound AQP2 protein should be assessed.64,257,381 The present study also could
not detect any effect of nephron deficiency nor L-NAME on gene expression of two sodium
channels, NHE3 and NKCC2, despite demonstrating a significant anti-natriuretic effect of L-
NAME in HET-1K mice. Further examination of the abundance and activity of other major
renal sodium channels in the renal tubule might provide insight into the possible mechanisms
that may explain the anti-natriuretic effect of L-NAME in HET-1K mice.
Consistent with our previous finding in 1-year old mice,312 a normal GFR was found in
30-week old nephron deficient HET-2K and HET-1K mice in the present study, indicating
these mice must have a significantly elevated SNGFR to compensate for the nephron deficit.
Previous reports calculated SNGFR (GFR divided by nephron number) of HET-1K mice to be
4 fold greater, and HET-2K mice doubled, that of WT mice.312 However, the mechanism(s)
that maintains this elevated SNGFR in HET-2K and HET-1K mice is unclear. An altered TGF
response has been suggested to play a role in the regulation of SNGFR in nephron deficient
rodents. It was reported that the sensitivity of TGF was reduced following uninephrectomy in
rats, which facilitated an increase in SNGFR in remaining kidney preventing a fall in total
GFR.266,335 Therefore there is a possibility that the sensitivity of TGF in nephron deficient
GDNF HET mice is significantly decreased compared with WT mice, allowing a marked
elevation in SNGFR to occur in HET mice.
Macula densa derived NO is known to blunt the sensitivity of the TGF mechanism
facilitating a higher SNGFR.47,274,353,370 Acute NOS inhibition with L-NAME or nNOS-specific
inhibition with 7-NI in control rats results in a significant fall in SNGFR and total GFR due to
an enhanced TGF sensitivity.47,274 Following nephron loss, there is evidence of greater NO
bioavailability.357 Further, acute blockade of NOS with L-NAME led to a fall (~30%) in
SNGFR in the remaining kidney of rats following uninephrectomy.41 We predicted that there
Chapter 5 Renal Function & Nephron Deficiency
124
would be a greater dependence on NO to maintain normal GFR in nephron deficient GDNF
HET mice. Thus the current study investigated the impact of NOS inhibition with L-NAME on
GFR predicting that GFR would be reduced in a manner dependent on the level of nephron
deficit. However after 7 days of L-NAME, GFR of all three groups was not different from
baseline measurements indicating that NO may play little role in the chronic regulation of
GFR in these mice. In examining the literature it is clear that the chronic responses to L-
NAME is different to the acute setting. Although Ollerstam et al 274 demonstrated a reduction
in SNGFR and enhanced TGF sensitivity in rats in response to acute NOS inhibition, they
found that after chronically treating rats with 7NI for 4 weeks, GFR was not different to
untreated rats. Further, the enhanced TGF sensitivity that was present in the acute setting of
nNOS inhibition was no longer present after chronic NOS inhibition.274 These findings
suggests that other factors are involved in the maintenance of GFR within the normal range
in chronic NOS deficiency. These factors may include formation of prostaglandins and
adenosine receptor signaling in the control of renin production in macula densa cells,120,284,358
as well as the contribution of RAS in the regulation of TGF.325
Future studies should clarify the role of NO, particularly nNOS at the macula densa in
in determining TGF sensitivity and the control of SNGFR and thus total GFR. In addition,
treating rats with L-NAME for extended periods (4-8 weeks) has been shown to cause a
reduction in GFR in addition to the significant renal injury such as glomerulosclerosis and
proteinuria.30,300 Thus future studies should also examine the impact of long-term NOS
inhibition on GFR and renal excretory function in nephron deficient GDNF mice in order to
determine whether the progression to renal injury is enhanced in GDNF HET mice
The most unexpected finding in the present study was the blood pressure response
to L-NAME in GDNF HET mice. MAP of GDNF WT mice had risen by 18mmHg during the
first day of L-NAME treatment, consistent with what has been shown in the literature.225 HET-
2K and HET-1K mice showed a similar initial rise in MAP. The elevated MAP of WT mice
was well maintained during next 6 days. Interestingly, instead of further exacerbation of
hypertension as we hypothesized, the increase in MAP of GDNF HET-2K and HET-1K mice
was gradually attenuated over the next 6 days. The escape from hypertension was more
profound in HET-1K mice than HET-2K mice, such that by the 7th-day post-L-NAME
treatment, MAP of HET-1K mice fell to be less than half of the initial rise. This fall in MAP in
HET-1K animals could not be explained by any further enhancement of diuresis and
natriuresis in these animals, instead we actually observed a reduction of sodium and water
excretion to WT levels.
One potential explanation for the escape of L-NAME-induced hypertension in HET-2K
and HET-1K mice may be related to the difference in response of the renin-angiotensin
system (RAS) following L-NAME treatment. WT mice showed an increase in AT1aR
Chapter 5 Renal Function & Nephron Deficiency
125
expression in response to L-NAME treatment, consistent with what has been demonstrated
previously.292,303 Interestingly L-NAME-induced hypertension is prevented when rats were
treated with the AT1 receptor antagonist Losartan,190 suggesting activation of the AT1
receptor directly contributes to L-NAME-induced hypertension. Thus, the upregulation of
AT1aR gene expression in WT mice may facilitate the sustained L-NAME-induced
hypertension in these mice. In contrast, the upregulation of renal AT1aR gene expression
was absent in HET-2K and HET-1K mice. This may explain the partial escape of L-NAME-
induced hypertension. It is unclear why L-NAME does not lead to upregulation of the AT1a
receptor in GDNF HET mice. Future studies may require examination of plasma renin activity
and renal angiotensin II level to further confirm the status of RAS activation in L-NAME
treated WT and HET mice. Apart from the RAS, collecting duct-derived endothelin (ET-1) has
also been shown to play a role in pressure-induced changes in diuresis and natriuresis in L-
NAME-induced hypertension.3,324 Treatment with L-NAME leads to a rather moderate
increase in blood pressure in collecting duct-specific ET-1 knockout mice compared to a
marked hypertension in controls, suggesting that NO derived from ET-1 signaling in the
collecting duct contribute greatly to the regulation of blood pressure.324 Investigation into the
status of endothelin-mediated NO pathway before and after NOS inhibition in GDNF HET
mice should be considered in future studies.
Our results indicate that NO is critical in maintaining elevated diuresis and natriuresis
in HET-1K mice. However, to our surprise, NO seems less important in the long-term
maintenance of arterial pressure and GFR in GDNF HET mice. There are several limitations
to the present study that should be considered. Firstly, renal excretory profiles were only
examined at baseline and at the end of 7 days L-NAME treatment. It would be ideal to collect
urine samples daily from the beginning of the L-NAME treatment in addition to the conscious
arterial pressure measurement, in order to monitor potential changes in pressure-natriuresis
relationship throughout L-NAME treatment. Secondly, we only examined the blood pressure
response to 7 days of L-NAME treatment.. It is possible that the partial escape of
hypertension and well-maintained real function in response to NOS inhibition observed in
nephron deficient GDNF HET mice were only transient. Studies that have treated rodents
with L-NAME for a longer period (e.g. 6-12 weeks) result in significant hypertension,265
profound renal injury including albuminuria,98,195 glomerulosclerosis,265,300 and a fall in GFR300.
Future studies should consider examining the cardiovascular and renal outcomes of GDNF
HET mice in response to long-term NOS inhibition in order to verify the longevity of this
protection against hypertension and renal dysfunction in these mice. Thirdly, in the present
study the gene expression analysis was limited to renal NOS isoforms, RAS receptors, and
key water and sodium channels. As mentioned earlier, further investigation of the protein
abundance of water and sodium channels in the apical membrane of the distal tubules, and
Chapter 5 Renal Function & Nephron Deficiency
126
the state of renal RAS activation may help to elucidate the mechanisms behind our
physiological observations.
5.5. CONCLUSION
In the present study, we predicted that renal function would deteriorate in nephron
deficient GDNF HET mice in response to NO deficiency in a manner dependent on the level
of nephron deficit. In contrast, our findings indicate NO may have little contribution in the
long-term regulation of renal function and blood pressure in nephron deficient GDNF HET
mice. Further elucidation of the mechanisms underpinning may help us to understand why
some nephron deficient animals are able to maintain a normal GFR and be protected from
hyperfiltration-related renal injury. A better understanding of these protective mechanisms in
nephron deficient kidneys would be useful in developing therapeutic strategies for individuals
with a significant nephron deficit who are at a greater risk of developing progressive renal
disease and chronic hypertension.
Chapter 6 General Discussion
127
Chapter 6 GENERAL DISCUSSION
Chapter 6 General Discussion
128
6.1. BRIEF OVERVIEW AND KEY FINDINGS
There is an increasing prevalence of pregnancies complicated by obesity, with more
than one in five pregnancies in Western society affected.226,273 Pregnancies that occur in
obese women are often associated with adverse maternal and fetal outcomes that are likely
to have a persistent impact. In the last decade, numerous studies have demonstrated a
strong association between maternal obesity and the development of cardiovascular and
renal dysfunction in young and adult offspring. Nevertheless, the mechanisms that are
involved in the programming of cardiovascular and renal disease in offspring by the adverse
intrauterine environment generated by obesity are not well understood. Importantly, the lack
of knowledge of this process means the strategies in managing the wellbeing of the mother
and fetus in pregnancies complicated by obesity is very limited. Thus, this thesis was aimed
to address this gap in the field of research.
One overlooked aspect of obesity-induced fetal programming in the literature is
whether obesity alters the cardiovascular and renal adaptations of pregnancy, and thereby
contributes to adverse fetal outcomes. Therefore, in Chapter 3, I investigated the impact of
pre-pregnancy obesity on pregnancy-induced cardiovascular and renal adaptations,
specifically examining the changes in MAP, HR, SV, CO and renal excretory function.
Chapter 3 also examined the impact of maternal obesity on fetal development. Given
complications that are commonly associated with maternal obesity such as placental
insufficiency and gestational diabetes are also associated with a reduced nephron
endowment in offspring, we focused our attention on examining fetal kidney development, in
particular, nephron number in this mouse model of pre-pregnancy obesity. The key findings
include:
� The diet-induced obesity model used in this thesis was appropriate to test the
hypothesis. This model of pre-pregnancy exhibited a similar phenotype to human
obesity including profound diet-induced obesity, glucose intolerance, hypertension,
tachycardia, cardiac hypertrophy and fibrosis, elevated CO and albuminuria.
� Pre-pregnancy obesity limited the cardiovascular adaptations of pregnancy including
blunting the increases in MAP, HR, SV, CO and LVM during late pregnancy, and
exacerbated obesity-induced albuminuria.
� Pre-pregnancy obesity led to increased fetal death, fetal growth restriction, small
head size and kidneys, and reduced placental efficiency.
Chapter 6 General Discussion
129
� Pre-pregnancy obesity led to significantly developmental abnormalities of the fetal
kidney including an abnormal renal and glomerular morphology, and in males, a
reduced nephron number.
These findings indicate that adaptations of cardiovascular and renal excretory systems were
indeed limited by pre-existing obesity, and may contribute to fetal loss, growth restriction,
significant glomerular abnormalities, and, in male fetuses, a nephron deficit.
In examining the impact of obesity during pregnancy, studies in the literature have
focused on the programming of adult disease in the offspring. Few studies have considered
the wellbeing of the mother post-birth with only handful of epidemiological studies that have
specifically investigated the cardiovascular outcomes in obese women more than 10 years
post-partum. These epidemiological studies did however indicate that there is a close
association between cardiovascular-related mortality and pre-pregnancy obesity.223,384 Thus
studies in Chapter 4 followed control and obese gravid mice post-partum and compared the
cardiovascular and renal phenotypes of these mice with non-pregnant time controls. The key
findings include:
� Pregnancy led to greater visceral obesity and exacerbated hypertension (light-phase)
in obese mice post-partum.
� Total renal and glomerular collagen content was greater in obese primiparous mice
post-partum but this was not related to renal dysfunction with GFR and albuminuria
of obese mice unaffected by pregnancy.
These findings indicate that pregnancy exacerbates obesity-induced hypertension in the
inactive phase of the day, but did not lead to overt renal dysfunction post-birth.
A variety of adverse intrauterine environments can program low nephron endowment
in the offspring, including as demonstrated in Chapter 3, maternal obesity. A low nephron
endowment is associated with the development of hypertension and renal insufficiency later
in life. However, not all models of reduced nephron endowment demonstrate hypertension or
renal dysfunction. One such model is the GDNF heterozygous (GDNF HET) that has been
well characterized by our laboratory as normotensive when examined at 14months of age
with no evidence of renal dysfunction or disease. An important question to ask is how
nephron deficient kidneys function in order to maintain normal renal function. Chapter 5
investigated the role of NO in maintaining renal function and normal arterial pressure in
nephron deficient GDNF HET mice. The key findings include:
Chapter 6 General Discussion
130
� Nephron-deficient GDNF HET mice with both moderate and marked nephron deficit
were able to maintain a normal GFR and sodium balance in response to 7 days of
L-NAME treatment.
� Nephron-deficient GDNF HET mice demonstrated a partial escape from L-NAME-
induced hypertension when compared with WT mice. The absence of an
upregulated renal AT1R expression that was demonstrated in WT mice in response
to NO inhibition might underpin the partial escape of GDNF HET mice to L-NAME
induced hypertension and well-maintained renal function in nephron deficient GDNF
Het mice.
� NOS inhibition normalized the exaggerated diuresis and natriuresis in GDNF HET-
1K mice suggesting NO plays a dominant role in promoting diuresis and natriuresis
in HET-1 mice.
These findings indicate that GDNF HET mice do not rely heavily on NO to maintain normal
blood pressure and renal function.
6.2. THE IMPACT OF PRE-PREGNANCY OBESITY ON THE MOTHER
Research into the detrimental impact of pre-pregnancy obesity has to a large extent
focused on the programming of cardiovascular and metabolic morbidities in offspring.
However, the mechanisms that contribute to this programming process are poorly
understood. An important aspect missing in the literature is what happens to the obese
mother during pregnancy and post-birth. The vital role of adequate adaptations in the
cardiovascular and renal systems during pregnancy has been overlooked in the field of fetal
programming and certainly has not been specifically examined in the context of pre-
pregnancy obesity. Studies in Chapter 3 of this thesis have addressed this gap in the field,
and for the first time, demonstrated that obesity indeed limits the cardiovascular and renal
adaptations that occur in pregnancies complicated by obesity. We used gold-standard
radiotelemetry to follow MAP and HR before and throughout pregnancy and found that the
rises of both MAP and HR were blunted towards term. We also adopted sophisticated
cardiac MRI technique to evaluate the cardiac function and structure at the pre-pregnancy
state and during late pregnancy. We found that obese dams failed to increase SV, CO and
LVM to the same magnitude as achieved by control mice at GA14, the time when cardiac
adaptations should be completed. To the best of my knowledge, these findings have not
been demonstrated in any animal model in the literature to date.
Chapter 6 General Discussion
131
This study had several distinct advantages. Our mouse model of pre-pregnancy
obesity had profound diet-induced obesity (47% heavier) and significantly compromised
glucose metabolism prior to mating, reflecting its clinical relevance. In contrast, many studies
in the literature claimed they have used a model of maternal obesity. In fact, many of these
models were only fed a high fat diet for a few weeks, and many had very little rise in body
weight compared to controls, before conception.40,118,317,318 Further, the significant obesity
achieved prior to conception in our model allowed us to establish a profound cardiovascular
and renal phenotype in obese female mice including hypertension, tachycardia, cardiac
hypertrophy and fibrosis, elevated cardiac output and albuminuria, consistent with human
obesity.
Another key advantage of this study is that cardiovascular and renal profiles of control
and obese mice were well characterized before conception. This approach has been largely
absent in human studies where early first trimester or post-partum measurements are used
to calculate the change in cardiovascular and renal parameters during pregnancy.101,130,359 It
was only recently that a longitudinal study by Mahendru et al 236 prospectively and
comprehensively examined the maternal cardiovascular and renal function changes from
pre-pregnancy until 17 weeks post-partum. This study demonstrated that not all
cardiovascular parameters return to pre-pregnancy levels at the same time with stroke
volume reported as still significantly elevated at 17 weeks post-partum.236 Thus, this study
highlighted that the use of post-partum measurements as a surrogate for pre-pregnancy
values would likely confound the interpretation in the extent of the cardiovascular adaptations
during pregnancy. Surprisingly, no study to date has prospectively examined the adaptations
of the cardiovascular and renal system in obese women prior to and during pregnancy and
post-partum. The existing literature in women suggests the adaptations of heart rate, stroke
volume and cardiac output in pregnancies complicated by obesity are likely to be
compromised.1,96,159,354 Taken together with the data generated in this thesis, the findings
highlight the need for a prospective longitudinal study into the cardiovascular and renal
adaptations of obese women during pregnancy and post-partum. In addition, parity has been
shown to influence whether obese women experience the mid-pregnancy dip in arterial
pressure.355 Thus, parity should be considered in the design of future studies.
Our characterization of the intrauterine environment in obese dams was not without
limitations. One aspect beyond the scope of this thesis was the assessment of maternal
plasma profiles before and throughout pregnancy. Glucose metabolism was only assessed
pre-pregnancy but not during pregnancy. Although glucose metabolism was not examined
during pregnancy, this impaired glucose tolerance would likely persist into pregnancy given
mice were continually receiving the high fat diet throughout the pregnancy. Further,
assessment of other maternal circulating factors such as plasma insulin, leptin, adiponectin,
Chapter 6 General Discussion
132
cholesterols and inflammatory makers might also be helpful to further characterize this model.
Given our results suggest that adequate volume expansion might not be achieved in obese
dams during pregnancy, future studies should measure hematocrit and plasma volume in
obese dams during pregnancy. Examination of the RAS including plasma renin activity and
aldosterone levels and tissue levels of the RAS and receptors during early pregnancy might
be beneficial in determining the contribution of this system to deficits in volume expansion in
obese dams.332,373 Further characterization of the maternal circulating profile would not only
be helpful in extending our understanding of the maternal and fetoplacental phenotypes of
our model but may also provide opportunities to identify possible therapeutics targets and
develop strategies to improve maternal and fetal outcomes in the future. Another piece of the
puzzle missing from our examination of the adaptation of renal system in Chapter 3 is how
GFR changes throughout pregnancy in obese dams. Future studies should measure GFR
before and at various stages throughout the pregnancy.111
Significant complications associated with obesity not only lead to poor maternal
outcomes during pregnancy but are also likely to have a prolonged impact in the mother.
Alarmingly, very little is documented in the literature regarding the long-term cardiovascular
and renal risks in this population of women post-partum. What we currently know is that
women who were obese before conception have a greater risk of mortality due to
cardiovascular events and these events occur earlier than women who had normal weight
before conception.223,384 Chapter 4 demonstrated that obesity-induced hypertension can be
exacerbated within a short period post-partum. Although renal function was largely preserved
at the time of assessment (4WPW), there was evidence of greater renal and glomerular
collagen deposition in obese primiparous mice. Although these mild renal and glomerular
injuries have not yet manifested in a detectable difference in albuminuria or GFR, they may
predispose these obese mice to early onset renal dysfunction and aggravated hypertension if
they were followed for a longer period or subjected to renal or cardiovascular insults. Again,
a longitudinal human study that examines the mother from pre-pregnancy, during gestation
and long after birth is desperately needed in improving our understanding of the impact of
pre-pregnancy obesity to a greater detail, therefore facilitating the development of
intervention for those women at risk.
6.3. THE IMPACT OF PRE-PREGNANCY OBESITY ON FETAL
HEALTH AND KIDNEY DEVELOPMENT
Pre-pregnancy obesity in humans is strongly associated with macrosomic infants.106,361
However, the literature also highlights that obesity in pregnancy is associated with greater
incidence of miscarriage, intrauterine growth restriction and particularly late gestational
Chapter 6 General Discussion
133
stillbirth.14,67,270,293,316 Our model of pre-pregnancy is consistent with the later situation. We
demonstrated that pre-pregnancy obesity lead to higher fetal loss per litter and fetal growth
restriction in the remaining viable fetuses that manifest as lower body weight, shorter head
width and smaller kidneys. The greater fetal loss and growth restriction in the fetus of obese
dams might be a results of a blunted cardiovascular adaptation such as the limited rise in HR,
SV and CO we observed in later pregnancy. Although our study did not measure uterine
blood flow in pregnant mice, a study by Frias et al 121 showed that obesity in pregnancy led to
a reduced uterine blood flow in a non-human primate model of maternal obesity, contributing
to the high rate of stillbirth found in these dams. The fetal growth restriction found in our
model is also likely to be consequence of a compromised placental function in our model. We
found that placentas of obese dams had a greater incidence of placental thrombosis and a
reduced fetal/placental weight ratio, a surrogate measure of placental efficiency, specifically
in male fetuses. These findings are consistent with evidence in humans that high risk of
placental injury was associated with pre-pregnancy obesity.173
Kidney development, particularly a programmed low nephron endowment has been
associated with the development of hypertension and renal dysfunction in offspring.201 We
demonstrated that pre-pregnancy obesity leads to fetal renal and glomerular abnormalities,
and a nephron deficit in male fetuses at term. This novel finding was generated using the
gold-standard stereological analysis for estimating nephron number in developing kidneys.
Importantly, the fact that the nephron deficit found in male fetuses was independent of the
low fetal weight suggests that factors other than fetal weight must contribute to the significant
nephron deficit (25%). One possible explanation is that our model of pre-pregnancy obesity
may be complicated by an uncontrolled hyperglycemia. It has been shown that even a short
period of exposure to hyperglycemia during pregnancy could have significant impact on fetal
kidney development leading to a reduced nephron endowment.9 Assessment of glucose
metabolism during pregnancy would be helpful in determining whether our model of pre-
pregnancy obesity lead to hyperglycemia during pregnancy, therefore lead to nephron
deficiency in the offspring.167 Similarly, plasma level of corticosterone has also been shown
to be significantly elevated in obese rats during pregnancy.97 Numerous studies have shown
that elevated maternal plasma corticosterone programs low nephron number.263,272,277,341
Further evaluation of maternal plasma corticosterone level could provide possible
mechanism that how nephron deficiency might be programmed in our model.
Another factor that is likely to contribute greatly to this nephron deficit, but was not
fully elucidated in this thesis is placental dysfunction. Placental insufficiency has been shown
to cause nephron deficit in offspring. 24,262 Further both mouse 203 and human173 studies have
demonstrated that maternal obesity lead to placental injury and dysfunction. Consistent with
the literature,203 we found that there was a reduced placental efficiency restricted to male
Chapter 6 General Discussion
134
fetuses of obese dams, which is likely to be associated with nephron deficit found in male
fetuses in our model. Further, similar to significant placental pathology observed in human
placentas collected at birth,173 we also observed a high incidence of placental thrombosis,
indicating these placental injuries might contribute to reduced placental efficiency. Gene
expression of pro-inflammatory cytokines had been found to be elevated in placentas of both
mouse models of maternal obesity203 and in humans,173 which may contribute to the placental
dysfunction. Unfortunately, placental pro-inflammatory cytokines expression was not
examined in our study. Histological analysis of placental morphology and evaluation in the
inflammatory status of the placenta are needed in assessing the extent of the placental
damage in this model.
6.4. THE RENAL FUNCTION IN NEPHRON DEFICIENT ANIMALS
In order to investigate the impact of reduced nephron endowment per se on renal
function and arterial pressure, it is important to choose a model in which other organ systems
that might influence renal function are less likely to be affected. We used GDNF HET mice as
this genetic mouse model of low nephron endowment allows us to examine the regulation of
renal function and arterial pressure without the confounding influence of globally
programmed cardiovascular dysfunction that often results from maternal undernutrition or
overnutrition, such as demonstrated in Chapter 3. Further, this model provides two distinct
levels of nephron deficit (30% and 65%) within one genotype and both GDNF HET-2K and
HET-1K mice have normal GFR and remain normotensive even through old age. Chapter 5
of this thesis examined whether NO, an important regulator of renal function and system
vasodilation, contributes to the maintenance of normal renal function and arterial pressure in
GDNF HET mice. To our surprise, our findings suggest NO might not be as critical as we
expected in regulating GFR and arterial pressure in the chronic setting. We found that GFR
of GDNF WT, HET-2K and HET-1K mice were not altered following 7-day of NOS inhibition
with L-NAME. And even more surprisingly, GDNF HET mice, and HET-1K in particular, had a
marked escape from L-NAME-induced hypertension. At the level of mRNA expression, we
showed that upregulation of AT1R gene expression during NO inhibition in WT mice was
absent in GDNF HET mice. This might explain the partial escape of hypertension and well-
maintained GFR in these nephron-deficient mice. Although our findings could not suggest
which compensatory mechanisms might be involved in mediating these effects, it certainly
warrants further investigations in the search for these mechanisms. Using microarray-based
gene expression profiling to examine renal tissues collected before and at various time points
following NO inhibition could be more efficient in narrowing down which pathways are likely
to be associated with the phenotypes observed in vivo.288
Chapter 6 General Discussion
135
6.5. LIMITATIONS AND FUTURE DIRECTIONS
Model of pre-pregnancy obesity
Human studies have demonstrated that pre-pregnancy obesity is strongly associated
with high prevalence of large for gestational age babies or macrosomic infants.106,361
However, it should be noted that majority of the babies born to obese mothers are not
macrosomic infants.221 In fact, pre-pregnancy obesity is also considered an independent
factor for miscarriage, IUGR babies and stillbirth.14,67,217,253,270,293,316 In the present study,
rather than resulting in large for gestation age fetuses as a common fetal outcome of pre-
pregnancy obesity in humans, fetus of obese dams in the model presented in Chapter 3
resembled the characteristics of IUGR fetus in humans. Further, our model also lead to
significant fetal loss and late reabsorption of fetus, consistent with increased incidence of
miscarriage217,253 and stillbirth14,67,316 associated with maternal obesity in humans. Thus it is
important to acknowledge that the findings from our model may be only relevant to proportion
of women who are at risk of adverse fetal outcome such as IUGR, miscarriage and stillbirth.
As mentioned above, it was not possible to measure the many important maternal
circulating factors such as glucose, insulin, leptin, maternal hormones (renin, aldosterone)
and inflammatory markers during pregnancy. Future studies should address these gaps in
order to further characterize this model. Further characterization of placental dysfunction in
our model may hold the clue to explain many of the fetal phenotypes in our models such as
nephron deficiency, IUGR fetuses and greater incidence of fetal loss. One aspect missing in
the characterization of cardiovascular system in our model post-partum was measurement of
cardiac structure and function using cine-MRI. Future studies should examine whether the
return of SV, CO and LV hypertrophy to pre-pregnancy levels post-partum were impacted by
pre-existing obesity in a chronic setting.
The impact of pre-pregnancy obesity on kidney development was only examined at
GA19 in the present thesis. It is known that mice complete nephrogenesis between postnatal
day 5-7.260 There is a possibility that the low nephron number observed in the present study
reflected a delay in nephrogenesis rather than a reduction, however the finding that the
nephron deficit in male fetuses was independent of fetal weight suggests this nephron deficit
is likely to persist post-birth until the completion of nephrogenesis. Future study should
examine nephron number and renal morphology at the end of nephrogenesis and later in
adulthood to further characterize the impact of pre-pregnancy obesity on kidney development.
Further, there is large body of work regarding the programming effect of pre-pregnancy
obesity on cardiovascular and renal outcomes of the offspring that could not be completed
within the scope of this thesis. Studies that characterize the blood pressure and renal
function in offspring of obese dams from weaning through to adulthood are needed to
Chapter 6 General Discussion
136
investigate whether renal insufficiency programmed by pre-pregnancy obesity lead to
progressive renal dysfunction and hypertension in offspring.
Clearly, a longitudinal human study that prospectively and comprehensive examines
the maternal and fetal outcomes of pre-pregnancy obesity is desperately needed to gain
greater understanding of the pathophysiology associated with pre-pregnancy obesity. Future
studies in the field of research should focus on the understanding of the mechanisms
underpinning the adverse maternal and fetal outcomes of pre-pregnancy in order to identify
potential targets for intervention. The findings from this thesis suggest that interventions that
enhance the cardiovascular and renal adaptations in pregnancies complicated by obesity
may reduce the incidence of fetal loss, stillbirth and small for gestational age babies for
obese women, and minimize the long-term impact of fetal programming on the
cardiovascular and renal health of the offspring.
GDNF HET mice
Findings from Chapter 5 indicate that NO may play less important role in the
regulation of renal function and arterial pressure in nephron deficient GDNF HET mice.
However, as mentioned in Chapter 5 we only examined renal function at a single time point
following short-term (7 days) NO inhibition. Future studies should examine the impact of
acute and long-term (6-8 weeks) NOS inhibition on TGF sensitivity, GFR and renal excretory
function in nephron deficient GDNF HET mice. It is also important to acknowledge the fact
that we used creatinine clearance (HPLC) as a measure of GFR in Chapter 5. Although we
have demonstrated previously that measurement of creatinine using HPLC methods was
consistent with the results of inulin clearance, we could not fully ignore the possibility that
using creatinine clearance could overestimate GFR in mice due the contribution of tubular
secretion of creatinine.107 Future studies could adopt the newly developed transcutaneous
FITC-sinistrin clearance method (used in Chapter 4) to repeatedly examine GFR before and
over the course of NOS inhibition. Further, the study presented in Chapter 5 did not examine
NO bioavailability or NOS protein levels in the kidney of GDNF WT and HET mice at baseline
and during NOS inhibition. Future study should address this issue accordingly.
Taken together, future studies should continue to expand our understanding in the
protective mechanisms by which GDNF HET mice maintain a normal renal function through
to old age and the ability of these mice to escape the hypertension induced by NO deficiency.
If these protective mechanisms were identified, we may be able to develop intervention to
treat those individuals with a low nephron number who are at a greater risk of further
deterioration of renal function later in life.
Chapter 6 General Discussion
137
6.6. CONCLUDING REMARKS
The findings in this thesis demonstrated for the first time that pre-pregnancy obesity
compromises hemodynamic adaptation during pregnancy and leads to significant fetal loss,
growth restriction, and suboptimal fetal kidney development. Early awareness of the risks
involved and the development of interventions that enhance pregnancy-induced
cardiovascular and renal adaptations may reduce the detrimental impact of pre-pregnancy
obesity in the mother and offspring. This thesis also revealed that pregnancy could be
harmful to obese women in the long-term management of cardiovascular and renal health
post-birth. Moreover, the identification of the mechanisms involved in the surprising ability of
nephron deficient animal to avoid overt hypertension and preserve renal function in a nitric
oxide deficient state could be beneficial in managing cardiovascular and renal health in
individuals with a low nephron number. Taken together, our findings provide valuable insight
into the possible mechanisms that could be responsible for the programming of hypertension
and renal insufficiency in offspring by pre-pregnancy obesity. The understanding of the
impact of pre-pregnancy obesity during pregnancy and post-partum presented in this thesis
allows us to be one step closer towards developing effective interventions that may in the
long-term benefit the cardiovascular and renal health in obese women and their offspring.
References
138
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