published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

Cells sense and interact with their microenvironment to retrieve signals which include cell-matrix and cell-cell contacts. These signals account for the influence of culturing conditions and often control the local cellular phenotype and global functions of tissues. Here, I sought to understand if there is any information processed by cells in guiding cellular phenotype given the control of cell shapes and substrate rigidities. If there is, would these phenotypic changes achieve biomedical purposes? What is the strategy to engineer platforms that can handle the longstanding challenges in those fields? In this dissertation, the first chapter serves as an introduction which involves the origin of motivations, which mainly came from current challenges in biomedical researches of kidney podocytes. I have attempted to understand if it is possible to control podocyte differentiation through cell shape control which mimics their in vivo morphology. On the other hand, I have tried to reveal if it is possible that tissue stiffness can affect podocyte phenotype as a result of stiffness sensing. These two topics were rarely investigated for kidney podocytes, which is the critical component of human filtration barrier to perform renal functions. The effort that addresses the question how shape and substrate rigidity as in- formation repositories affect kidney podocytes phenotype has profound meaning in the understanding of renal physiological system and pathological mechanisms. The second chapter will focus on the methods to achieve successful long-term shape control on cells. Engineered cell-device interface using cross-linking biomaterial SU-8 plays a key role in this study. Compared with other previously used approaches summarized in this chapter, SU-8 provides various advantages both in the fabrication of micro- pattern architecture as well as its sustaining effectiveness in controlling cell shape. This approach has been proved very efficient and economic to facilitate single cell level manipulation. The chapter will describe in details the interface micro-fabrication and encountered technical challenges. The results that kidney podocytes were in good compliance with the micro-pattern proved the successful application of this technique. The third chapter will then transfer from micro-fabrication to biological experiments, which discusses in details how in intro kidney podocytes responded to their shapes by enforcing protein localization which characterizes a phenotype found in vivo. This phenotype among in vitro podocytes was further verified that it may contribute to podocytes differentiation and physiological functions. The information processed by shape was proved independent of tension-related processes and thus shape and tension could be regarded as separate contributors in cellular development. The interpretation of shape’s contribution could be referred to my previous publication in the journal of Cell: ”Decoding Information in Cell Shape”. In this study, the motifs of research were applied to other cell lines (Human vascular smooth muscle cell) as a step to generalize the ubiquity of shape’s contribution to cell differentiation. The study here was to differentiate shape and tension through investigating the difference between two major mechanosensors: β3 and β1 integrin receptors. The difference in cell phenotypes through integrin inhibition experiments demonstrated critical but unique role of integrin-based shape sensing in vitro. This chapter in dissertation covers most of the content in a previously submitted paper to Nature Cell Biology. In the fourth chapter, I further carried out a study that investigated if stiffness sensing can influence kidney podocyte phenotype. The fourth chapter will basically review the techniques in the fabrication of hydrogel-based cell culture platforms. In a similar manner to previous study using biomimetic shape for podocytes and find its phenotype, the target of this analysis was to use hydrogel-based biomimetic substrate with renal physiological stiffness and find if there is a differentiation phenotype. Since numerous materials have been reported in hydrogel studies, I will focus on the introduction to representative ones that have been most widely used. Their characteristics will be compared with the demands of kidney podocyte reasearch. Methodologies were the key to a successful research, and in this chapter I will describe in details what choices I made in choosing experimental methods that improved the efficiency and quality of cell culture platforms. A natural enzyme (microbial transglutaminase) cross-linked gelatin hydrogel was adopted here to provide ideal substrate rigidity control for podocytes. This method has demonstrated high efficiency and stability in making large cell culture surface. Moreover, it provides the hydrogel platform with an ideal range of elastic moduli suitable for renal tissue culture. The results will be discussed in detail in the fifth chapter. I successfully found a differentiation phenotype for podocytes cultured on the hydrogel platforms with a physiological stiffness. Similar phenotype, on the contrary, were not found in podocytes on platforms which were either too soft or too stiff. These resutls have formed one of my submitted paper to Scientific Report. The differentiation phenotype for kidney podocytes was characterized by up-regulation of differentiation markers. These findings were in a similar manner to a series of stem cells differentiation guided by regulated substrate stiffnesses. This phenotype of kidney podocytes was verified by microarray technique which confirmed the stiffness sensing using transcription factors. The enrichment analysis of kinases also showed significant response of Src, Fyn etc, of which the activities have been shown critical for podocytes to preserve their physiological functions. These results have successfully suggested the close relations between stiffness changes of glomeruli basement membrane (GBM) and progressive podocyte dysfunction. In summary, this dissertation covers interdisciplinary researches that decoded the information processed by cells from two separate aspects: shape and stiffness sensing. The details in each chapter cover a broader scope than the content selected for publications. Through this dissertation, readers will get in touch with the technique developed for plat- form and their applications to biomedical researches. I wish this will help people new in the field to get my hands-on experience.

The present study determined whether dietary supplementation with reduced glutathione (GSH), glutathione ester (GSHE) or N-acetylcysteine (NAC) increased the mitochondrial level of GSH, the major antioxidant inside cells, in rat kidney cortex and medulla. Nine month-old female Lewis rats were given daily intraperitoneal injections of isotonic saline (n=6), or saline containing GSH (250mg or 0.81mmol/Kg of body wt; n=7), GSHE (12mg or 0.03mmol/Kg; n=8), or NAC (200mg or 1.22mmol/Kg; n=8) for four weeks. At the end of the injection period, the rats were anesthetized and the kidneys removed. The kidneys were separated into cortical and medullary sections, weighed, and homogenized. The sections were separated into cytosolic and mitochondrial fractions by differential centrifugation. The GSH levels were determined by a colorimetric assay. Cortical and medullary mitochondrial GSH levels were significantly increased by all three supplements. Cytosolic GSH levels were also significantly increased in both cortical and medullary sections. Thus, dietary supplementation can significantly increase the mitochondrial pool of GSH in the rat kidney. / Department of Physiology and Health Science

This study investigated whether exogenous supplementation with reduced glutathione (GSH) increased kidney mitochondrial and cytosolic GSH levels in young and old female Lewis rats. The young rats were 3 months of age and old rats were 22 months old. The rats were divided into a young control group (n=8), an old control group (n=5), a young experimental group (n=7), and an old experimental group (n=7). Rats in the young and old control groups did not receive any treatment, while rats in both the young and old experimental groups were injected with GSH (250 mg/Kg of body weight) into the peritoneal cavity once a day for a week. At the end of the injection period, the rats were anesthetized and kidneys were harvested. The mitochondrial and cytosolic fractions were separated from rat cortex and medulla by differential centrifugation. GSH concentrations were measured using a spectrophotometric assay. Both mitochondrial and cytosolic GSH levels in kidneys from young and old female Lewis rats were significantly increased with GSH supplementation. The results indicate that kidneys from both young and old rats respond to exogenous dietary supplementation with GSH. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science

Reptiles are polyphyletic, and previous studies of renal anatomy and physiology in reptiles have covered a wide diversity of species of different phylogeny and habitat. To date, no study has examined the renal morphology and function of a group of closely related reptiles from different environments, yet this design has a number of advantages. Firstly, phylogenetic effects are reduced while adaptive specialisations in renal function or structure can be elucidated, and secondly, the variation in renal form and function between closely related species may be quantified in an effort to appreciate better the variation between more distantly related species. In this thesis, kidney morphology and renal function were studied in three Western Australian agamid lizards inhabiting environments differing in the availability of water. These key species were Pogona minor, Ctenophorus nuchalis and Ctenophorus salinarum. The renal anatomy of the three key lizards was characterised by determining glomerular diameter, volume density, surface area and number in each. Allometric relationships between kidney, colon and body mass were investigated in these and an additional 11 species of agamid lizard. Patterns of response to osmotic challenge were recorded by measuring renal variables such as urine flow rate, glomerular filtration rate and fractional reabsorption of filtrate among the three key species, and concurrent measurements of circulating arginine vasotocin in P. minor and C. nuchalis allowed the response of this hormone to homeostatic imbalance in these species to be gauged. The gross morphology and the glomerular characteristics of the kidneys was remarkably similar between species. Glomerular number and other characters varied as a function of body size rather than species, contrasting with reports in the literature suggesting that a given species has a particular number of glomeruli. ... Thus, kidney morphology is constrained among species and the response of each species to osmotic perturbation is similar. However, the mechanisms underlying antidiuresis and the hormonal control of this process differ subtly between species, and there is some evidence to suggest that P. minor is more adapted to a mesic environment than the other two lizards examined in this study. The hypothesis that renal form and function reflect the environment in which a lizard lives therefore receives partial support, although the reptilian bauplan is able to mitigate many of the forces that could potentially lead to renal specialisation.

Thesis (M.Sc. (Physiology)) -- University of Limpopo, 2021 / The purpose of this study was to investigate the effect of cART on renal function and assess the association between renal function and cardiovascular risk factors in a black rural HIV-positive population in Limpopo Province, Mankweng district. We have conducted a cross-sectional study which included both male and female cART-treated patients (n=84), cART-naïve patients (n=27) and HIV-negative controls (n=44). We have measured biomarkers of renal function (plasma cystatin C, clusterin, retinol binding protein 4 [RBP4]) and determined the estimated glomerular filtration rate (eGFR) using the chronic kidney disease-epidemiology collaboration formula (CKD-EPI). We have also measured blood pressure (BP), body mass index (BMI) and fasting blood glucose (FBG). The prevalence of renal dysfunction was similar among the study groups. A significant difference in RBP4 was found among the groups after controlling for covariates (age, gender, alcohol consumption, BMI, systolic blood pressure and FBG) (F (2, 146) = [4.479], p=0.010). The significant difference in RBP4 was specifically observed between the cART-treated and cART-naïve groups (p=0.008). Cystatin C, clusterin and eGFR were not significantly different among the study groups after controlling for the covariates. The cardiovascular risk factors age (β=0.207; p=0.039), CD4+ T-cell count (β=-0.236; p=0.040), and duration of cART (β=0.232; p=0.043) were independently associated with cystatin C. The use of cART independently associated with RBP4 (β=0.282; p=0.004). Age (β=-0.363; p=0.001), CD4+ T-cell count (β=0.222; p=0.034) and duration of cART (β=-0.230; p=0.034) independently associated eGFR. Renal dysfunction is common in this HIV-positive population, with similar rates as the HIV-negative population. Plasma cystatin C as a promising alternative renal biomarker need to be re-evaluated in this HIV-positive population. RBP4 may be a more promising renal function biomarker in the HIV-positive population. Cardiovascular risk factors are associated with renal dysfunction in this rural HIV-positive population and CD4+ T-cell count may be an independent predictor for renal function.

Indiana University-Purdue University Indianapolis (IUPUI) / Polycystic kidney diseases (PKD) are genetic disorders characterized by fluid filled cysts in the kidney tubules and liver bile ducts. There are two forms of PKD, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). The focus of the studies in this thesis has been on ADPKD. The disease progresses slowly and the fluid-filled cysts grow in size due to increased rates of cell proliferation and fluid secretion into the cyst lumen. The expanding cysts compromise the normal kidney function and result in a decrease of renal function to the point of end-stage renal failure in midlife. Cyst enlargement is due, at least in part, to chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Currently therapy is limited to renal cyst aspiration, dialysis, and eventually renal transplantation after organ failure, thus it has critical to determine possible drug therapies for the treatment of PKD. Previous studies showed that cyst fluid caused a secretory response in cells lining the cysts. We hypothesized that once the cyst have expanded and become so large that they burst or leak, which could also occur due to renal injury or aging, the cyst fluid may stimulate additional cyst growth. Lysophosphatidic Acid (LPA) was determined to be the active component of human cyst fluid, and we investigated the LPA stimulated signaling pathway. Our data suggest that the LPA stimulates chloride and fluid secretion by a combination of CFTR and Calcium-Activated chloride channels (CaCC) and that the two channels may functionally be linked to each other. The secretion is not occurring through a cAMP stimulated pathway, and it is possible that TMEM16A, a CaCC, plays a larger role than previously expected. Previous studies demonstrated that PPARγ agonists, insulin sensitizing drugs used to treat diabetes, inhibit chloride secretion by the collecting duct principal cells by decreasing CFTR synthesis. It was logical therefore to considered PPARγ agonists as long-term treatment for PKD. The first preclinical studied showed that high (20 mg/kg BW) dose pioglitazone, a PPARγ agonist, inhibited cyst growth in the PCK rat model, a slow progressing model, of PKD. To continue to look at the effects of the PPARγ agonists another preclinical study was completed, which tested if there was a class action of PPARγ agonists and if a lower dose was effective in treating the cystic burden. Using the PCK rat model, and another PPARγ agonist, rosiglitazone, a 24 week study was completed using 3 doses (4, 0.4, and 0.04 mg/kg BW). 4 mg/kg BW rosiglitazone is analogous to 20 mg/kg BW pioglitazone. The data indicated that the rosiglitazone is effective in lowering the cystic burden, and importantly the low dose proved to be effective. An additional rat model, the W-WPK rapidly progressing model was used to determine efficacy across multiple models, and to determine if there was a way to track the progress of the disease in a manner analogous to that used in human patients. The animals were treated with pioglitazone using 2 doses (2 and 20 mg/kg BW), and were imaged using CT scans to track the progress of the disease. The data suggest that pioglitazone was not as effective in the W-WPK rat model as it was the PCK rat model. There was a trend however, that low dose PPARγ agonist was as effective ad high dose. Even more important, the CT scans proved to be an effective way to track the progress of the disease in animal models.

Abstract not available

Indiana University-Purdue University Indianapolis (IUPUI) / FGF23 is the central regulator for phosphate homeostasis. Both FGF23 and phosphate dysregulation are highly related with the progression of chronic kidney disease (CKD), which is a global health problem. In previous studies, FGF23 was found to be produced in bone and targeting the kidneys to regulate phosphate reabsorption and excretion. In the FGF23 signaling axis, it binds a receptor complex (αKlotho and FGFRs) in the distal convoluted tubules (DCT) and causes its biological effects in the proximal tubules (PT). The mechanism of how the signals passing on from DCT to PT is not clear. In my research, experiments were focused on the FGF23 signaling pathway within the kidney to study the communication steps between tubular cells. HBEGF treatment was given to FGF23 signaling impaired mouse models resulting in significant change of genes regulated by FGF23, indicating that HBEGF was important in the FGF23 signaling axis. Then high quality rabbit anti-mouse HBEGF antibodies were made to better study HBEGF activity in vivo and in vitro. A new cell model was characterized to test FGF23 effects on HBEGF signaling using Western blots and immunofluorescence. Lastly, the location of HBEGF activity was examined in the kidney in vivo. Immunostaining suggested that HBEGF activated the mitogen activated protein kinase (MAPK) pathway. This mapping may provide important information for the molecular relationships between FGF23 and HBEGF.