Stereological, functional and molecular studies of development and disease : a collection of published works 1981 to 2013

The unifying theme throughout this Doctorate of Science thesis is the development, refinement and utilisation of stereological techniques to study tissue structures in development, and in adult health and disease. Stereology is the discipline based on geometric probability theory that enables us to quantify structures in three-dimensional space. Stereological techniques are often applied in material science as well as biomedical science. When applied to histology and pathology, stereology can be used to estimate such parameters as the number of glomeruli in the kidney, surface areas of capillary networks, the number of neurons in the brain, and the proportion of liver volume occupied by cancerous tissue. With stereology, we not only obtain accurate and precise quantitative estimates of structures of interest, but we can identify morphological differences between specimens that cannot be seen or appreciated by simple visual observation. Moreover, by quantifying microanatomical structures, we can explore correlations between morphological data and quantitative physiological and molecular data, and thereby discover mechanisms responsible for normal development, for maintenance of homeostasis in healthy adult tissues and organs, and for development of disease. Accordingly, most of the studies in this thesis, in addition to the use stereological methods, involve the use of one or more imaging, physiological, molecular or genetic techniques. This thesis incorporates 209 research studies conducted from the early 1980's. Early studies investigated aspects of lung biology. However, the majority of studies explore kidney development and disease, with specific focus on human nephron number and size, kidney development, kidney pathology, and the consequences of suboptimal kidney development for adult renal and cardiovascular health. Other publications in this thesis emanated from collaborative projects where my expertise in stereo1ogy and imaging contributed to new insights into aspects of spinal cord, brain, heart, vascular, skin, platelet, liver, prostate gland and mammary gland development and disease. The work is divided into 10 chapters. Chapter 1 (Publications 1-11) presents studies on lung cancer (PhD studies at the Unit of Human Morphology, Flinders University), asthma (postdoctoral studies at the Department of Pathology, University of Western Australia) and pulmonary cell biology (Department of Biological Structure, University of Washington, Seattle). These studies involved stereological assessments at the light and/or electron microscopic levels in animal and human tissues. Chapter 2 {Publications 12-26) presents studies which developed and evaluated new stereological techniques. The majority of these techniques are based on the new generation of design-based or unbiased stereological estimators such as the disector, fractionator and Cavalieri estimator. These studies were conducted at the University of Washington, University of Melbourne, and Monash University. The disector-based methods presented in this thesis for estimating glomerular number and size in developing and adult kidneys are considered the gold-standard in the field. Chapter 3 {Publications 27-35) presents stereological studies of neurons and synapses in the spinal cord, cortex and pituitary gland. These collaborative studies were conducted at the University of Washington, University of Melbourne and Monash University. Chapter 4 (Publications 36-43) presents studies on glomerular podocytes conducted at the University of Melbourne and at Monash University. Podocytes are a key cell of the glomerular filtration barrier. In this chapter, both in vitro and in vivo studies are described. The studies in Chapter 5 (Publications 44-66) mostly represent collaborative studies with researchers at the University of Melbourne, the Baker Institute and Monash University. In these studies, my stereological expertise was required for analyses of the heart, aorta, resistance vessels and kidney in investigations of the causes and consequences of high blood pressure (hypertension). Chapter 6 (Publications 67-147) details animal studies on the genetic and environmental regulation of nephron endowment, as well as the consequences of suboptimal kidney development for adult kidney and cardiovascular health. Almost all of these studies were conducted at Monash University. These studies arguably represent the most comprehensive compilation of studies yet conducted on mammalian kidney development and links with adult disease - the so-called developmental origins of health and disease (DOHAD)hypothesis. The genetic studies focused on the roles of glial cell line-derived neurotrophic factor {GDNF), transforming growth factor B1 and B2 (TGF-B1 and TGF-B2), bone morphogenetic protein 4 (BMP4) and the fibroblast growth factors (FGFs) and receptors (FGFRs) in kidney development. The reports on the role of the feto-maternal environment in kidney development included studies on the effects of low protein maternal diet, maternal exposure to natural and synthetic glucocorticoids, certain antibiotics and alcohol, as well as gestational diabetes. In vivo and in vitro studies were conducted in wildtype as well as genetically modified animals. Taken together, these studies increase our understandings of the roles of GDNF, TGF-Bs, BMP4 and FGFs in kidney development, and show that a range of perturbations to the feto-maternal environment can result in a permanent nephron deficit (often involving perturbed ureteric branching morphogenesis), leading in adulthood to increased blood pressure. Chapter 7 (Publications 148-168) includes qualitative and quantitative studies of renal histopathology in animal models and humans. These include important findings in animal models of diabetic nephropathy and studies of glomerular size and glomerulosclerosis in Australian Aborigines. Most studies were conducted at Monash University. Chapter 8 (Publications 169-194) presents landmark studies on glomerular (nephron) number and size in human kidneys. In this so-called Monash Series, my laboratory used design-based stereological methods to estimate the number and volume of glomeruli in 420 kidneys obtained at autopsy from white and Aboriginal Australians, white and African Americans, and Senegalese Africans. This is the seminal study of glomerular number and size in normal human kidneys. Amongst many key findings we have shown that: (1) glomerular (nephron) number varies up to 13-fold in normal human kidneys; (2) the kidneys of Australian Aborigines have fewer nephrons than whites from the same geographical region (we believe this is associated with low birth weight due to the poor fetal environment, which in turn is the result of substandard socioeconomic conditions); (3) nephron number is directly correlated with birth weight, with each kilogram increase in birth weight associated with approximately 230,000 additional nephrons per kidney; (4)nephron number is inversely correlated with blood pressure in American whites and Aborigines (lower nephron number is associated with higher blood pressure), but this is not the case in African Americans; (5) mean glomerular volume varies more than 6-fold in normal kidneys; and (6) larger mean glomerular volume and greater heterogeneity in glomerular volumes within normal human kidneys is associated with lower nephron number, lower birth weight, larger adult body size and hypertension. Chapter 9 (Publications 195-197) presents the first three papers ever published on the use of magnetic resonance imaging (MRI) to image, count and size all glomeruli in a kidney. These studies examined whole ex vivo rat kidneys. We believe this is the first step towards non-invasive quantitation of glomerular number and size in vivo in animal models of kidney disease, and in humans with, or at risk of, kidney disease. Chapter 10 (Publications 198-209) mostly consists of collaborative projects in which I provided stereological or imaging expertise. These projects include studies on skin, liver, prostate gland, platelets and cell organelles. Extent to Which the Work Contributes to the Advancement of Knowledge The experimental questions investigated in this work are diverse. Numerous tissues and organs have been studied in a range of experimental animals as well as humans. Developing organs have been studied, as well as healthy and diseased adult organs. Diseases studied include cancer, asthma, motor neuron disease, hypertension, diabetes and chronic kidney disease. A broad range of research techniques has been employed. Despite this diversity of topics and research approaches, the clear unifying theme throughout this work is the development, refinement and implementation of stereological methods for quantifying tissue structures. These stereological studies have frequently been a component of larger multidisciplinary studies involving physiological, molecular and/or genetic analyses. The majority of studies in this work (>150 of 209 publications) have concerned aspects of kidney development, structure and disease. These kidney studies have contributed to the advancement of knowledge of kidney development ·and disease in many ways. For example, the studies of human glomerular number and size in this work are unparalleled in size, scope and impact. Studies at Monash on kidneys obtained at autopsy from 420 men, women and children from five racial groups have provided novel and unexpected insights into variability both between and within subjects, and associations with birth weight and chronic kidney disease risk factors (age, race, adult body size, hypertensive status). With further development of MRI approaches for sizing and counting glomeruli in vivo, we predict these parameters will be incorporated into the clinical work-up of patients with, or at risk (premature babies and babies born with low birth weight) of developing chronic kidney disease.