Renal dysplasia is a disease characterized by developmental abnormalities of the kidney that affect 1 in 250 live births. Depending on the severity of the renal abnormalities, this disorder can lead to childhood kidney failure, adult onset chronic kidney disease, and hypertension. Currently, the best treatment options for patients with renal dysplasia are renal dialysis and kidney transplant. Our limited understanding of the pathogenesis of renal dysplasia has prevented the development of better treatment strategies for those patients. A hallmark of renal dysplasia is an expansion of loosely packed fibroblast cells, termed renal stroma. Markedly elevated levels of β-catenin have been reported in the expanded stromal population in patients with dysplastic kidneys. Yet, the contribution of stromal β-catenin to the pathogenesis of renal dysplasia is not known. Additionally, the role of stromal β-catenin in the developing kidney is not clear. The overall hypothesis of this PhD thesis is that β-catenin in stromal cells controls key signalling molecules that regulate proper kidney development. Furthermore, we hypothesize that elevated levels of β-catenin contribute to the pathogenesis of renal dysplasia. To mimic the human condition, we generated a mouse model that overexpresses β-catenin specifically in the stroma (termed β-catGOF-S). In addition, to gain a better understanding of its role in kidney development, we generated a second mouse model deficient for β-catenin exclusively in stromal cells (termed β-catS-/-). The goal of this study is to utilize these models to understand the role of stromal β-catenin in kidney formation and investigate its contribution to renal dysplasia. The first objective defines the contribution of stromal β-catenin to the genesis of renal dysplasia. We provide evidence for a mechanism whereby the overexpression of stromal β-catenin disrupts proper differentiation of stromal progenitors and leads to an expansion of stroma-like fibroblast cells and vascular morphogenesis defects. In the second objective, we establish a mechanism where stromal β-catenin modulates Wnt9b signaling in epithelial cells to control proliferation of the nephron progenitors. In the third objective, we define a role for stromal β-catenin in proper formation and survival of the medullary stroma. Finally, in a technical report, we outline a protocol to isolate stromal cells in the developing kidney and provide potential downstream applications to further our understanding of stromal β-catenin in the developing kidney. Taken together, our findings establish a crucial role for stromal β-catenin in the genesis of renal dysplasia and demonstrate, using two mouse models, that stromal β- catenin must be tightly regulated for proper formation of the stroma lineages and development of the kidney. / Thesis / Doctor of Philosophy (PhD)

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Mammalian kidney development proceeds through a series of interactions among metanephric mesenchyme, ureteric bud, extracellular matrix, growth factors and various other signalling molecules. These complex, but well integrated, networks control cell proliferation, differentiation, migration and survival and thus orchestrate kidney development. More and more molecules in these networks have been identified since the past few years. Therefore, it is crucial to explore their functions and the mechanisms by which they work to fill the research gaps. DACTs have recently been reported as pathway-specific regulators in WNT signalling, known to be important in kidney development, but their expressions and functions in mammalian kidneys are yet to be elucidated. The aims of this thesis are to describe the expression patterns of these two new genes, Dact1 and Dact2, in mouse embryonic kidneys and to further investigate their functional roles by applying RNAi to cell culture-based models. The first goal of this thesis is to establish the temporospatial expression patterns of Dact1 and Dact2 in kidneys by conventional end-point RT-PCR, quantitative real time PCR and RNA in situ hybridisation. Based on the expression patterns and preliminary observations in cell cultures, I hypothesize that Dact1 regulates cell proliferation while Dact2 governs cell migration. Experiments including siRNA transfection, BrdU proliferation assay, generation of stable cell lines expressing Dact2 shRNA and wound assay, are designed to test these hypotheses and the results may offer implications of functional roles of both molecules in kidney development. The results obtained are as follows. • Dact1 and Dact2 show different temporal expression patterns in mouse kidneys. In adult kidneys, Dact1 is greatly downregulated while Dact2 is still expressed at a comparable level to that at E14.5. • Dact1 is initially expressed in metanephric mesenchyme and, as development proceeds, shows a characteristic pattern of renal stroma whilst Dact2 is exclusively expressed in ureteric buds throughout embryonic stages. • Dact1 and Dact2 expressions are regulated by known regulators of kidney development including retinoic acid and chlorate. • Silencing of Dact1 facilitates proliferation of embryonic cells. • Silencing of Dact2 hinders migration of renal collecting duct cells. Taken together, I have characterised temporospatial expression patterns of Dact1 and Dact2 in kidneys and provided evidences of functional roles of both novel molecules in cell cultures. Based on this thesis, further studies on Dact1 and Dact2 using either in vitro or in vivo mammalian kidney models will offer more insights into their functions and regulations in renal organogenesis.

No description available.

M.Sc. Thesis Dissertation, August 2019, McMaster University / Renal dysplasia, defined as the abnormal development of kidney tissue, is the leading cause of kidney disease in children. While there are numerous causes of renal dysplasia (i.e. genetic, environmental and epigenetic factors), there is no cure to this abnormal defect. Kidney development occurs by two main processes: branching morphogenesis, which forms the collecting duct system, and nephrogenesis, which generates the nephrons, the functional units of the kidney. Our previous studies have demonstrated that β-catenin, a dual-function protein involved in cell adhesion and gene transcription, regulates branching morphogenesis and nephrogenesis. Furthermore, we discovered that nuclear β-catenin levels are increased in kidneys from patients with renal dysplasia, suggesting β-catenin can be a potential therapeutic target to modulate kidney development and renal dysplasia. Quercetin is a flavonoid that reduces β-catenin levels and inhibits its transcriptional activity, leading to improved outcomes in cancer and in kidney fibrosis. The role of quercetin in kidney development and in abnormal defects that arise during kidney development is yet to be examined. Using embryonic mouse kidney organ culture, I found that quercetin treatment resulted in a dose-dependent disruption in branching morphogenesis and nephrogenesis. In addition, quantitative reverse-transcriptase PCR revealed a decreased expression of β-catenin target genes essential for kidney development (i.e. Pax2, Six2 and GDNF). Immunohistochemistry for β-catenin demonstrated that quercetin reduced nuclear β-catenin expression and increased cytoplasmic and membrane-bound expression in a dose-dependent manner. These results were confirmed by Western blot analysis. These novel findings demonstrate that quercetin treatment resulted in decreased levels of nuclear β-catenin, resulting in a decrease in its transcriptional activity which manifested in alterations in kidney developmental processes, suggesting quercetin is effective at reducing nuclear β-catenin in wild-type embryonic kidneys. Next, to determine whether quercetin has any effects on renal dysplasia, I utilized transgenic mice models that overexpress β-catenin in select cells of the embryonic kidney. These models recapitulate the defects observed in human renal dysplasia, including disorganized branching morphogenesis and disrupted nephrogenesis. Quercetin treatment of embryonic dysplastic kidneys resulted in a partial rescue of renal dysplasia which was evident in marked improvements in branching morphogenesis and nephrogenesis, as well as an increase in the number of properly-developing nephrons in the kidney tissue. Analysis of β-catenin expression in quercetin-treated dysplastic kidneys revealed a decrease in nuclear levels and an increase in cytoplasmic and membrane-bound levels, resulting in a reduced expression of target genes (Pax2, Six2, and GDNF). Finally, this partial rescue of renal dysplasia was associated with an improved and organized E-cadherin expression in quercetin-treated dysplastic kidneys, suggesting a possible molecular mechanism of quercetin action in resolving abnormal kidney development. Overall, my findings demonstrate, for the first time, that quercetin reduces β-catenin transcriptional activity in normal and dysplastic kidneys and reduces the severity of defects in renal dysplasia. / Thesis / Master of Science in Medical Sciences (MSMS)

Oil spills are well-known adverse anthropogenic events, as they can induce severe impacts on the environment and negative economic consequences. Still, much remains to be learned regarding the effects of crude oil exposure to aquatic organisms. The objectives of this dissertation were to fill some of those knowledge gaps by examining the effects of Deepwater Horizon (DWH) crude oil exposure on teleost kidney development and function. To this end, I analyzed how these effects translate into potential osmoregulatory impairments and investigated the interactive effects of ubiquitous natural factors, such as dissolved organic carbon (DOC) and ultraviolet (UV) light, on acute crude oil toxicity. Results demonstrated that acute early life stage (ELS) crude oil exposure induces developmental defects to the primordial kidney in teleost fish (i.e., the pronephros) as evident by alterations in: (1) transcriptional responses of key genes involved in pronephros development and function and (2) alterations in pronephros morphology. Crude oil-exposed zebrafish (Danio rerio) larvae presented defective pronephric function characterized by reduced renal clearance capacity and altered filtration selectivity, factors that likely contributed to the formation of edema. Latent osmoregulatory implications of crude oil exposure during ELS were observed in red drum (Sciaenops ocellatus) larvae, which manifested reduced survival in hypoosmotic waters, likely due to defective pronephros development and function. Finally, DOC-UV co-exposure slightly reduced acute crude oil photo-enhanced toxicity in red drum larvae. This dissertation provided novel information regarding crude oil toxicity that can be incorporated into environmental risk assessment and management for future oil spills.

Chronic kidney disease (CKD), defined as an irreversible reduction in glomerular filtration rate, is a large public health concern. Dissecting the genetic components of CKD is required to improve our understanding of disease pathogenesis. Researchers have identified that SHROOM3, has very high associations with kidney disease and function. Shroom3 encodes an actin-binding protein important in regulating cell and tissue morphogenesis. However, there is a lack of evidence supporting a role for Shroom3 in kidney function or disease. Here, I investigated the developmental and functional role of Shroom3 in the mammalian kidney. For the first time, I described the expression pattern of Shroom3 in the embryonic and adult mouse kidneys. By performing in situ hybridization and immunohistochemistry, I demonstrated that Shroom3 is expressed in the condensing mesenchyme, podocytes, and collecting ducts. I further showed that Shroom3 protein is localized in the foot processes of podocytes, utilizing immunogold labeling and transmission electron microscopy. In order to uncover a potential role of Shroom3 in the kidney, we utilized Shroom3 knockout mice. Shroom3 mutants demonstrated marked glomerular abnormalities including cystic and degenerating glomeruli, and reduced glomerular number. Scanning and transmission electron microscopic analyses of Shroom3 mutant glomeruli revealed disruptions in podocyte morphology characterized by disorganized foot processes with less interdigitation and segmental foot processes effacement. Furthermore, immunofluorescence analysis of mutant kidneys revealed aberrant distribution of podocyte actin-associated proteins. Elucidating the underlying molecular mechanism of this abnormal podocyte architecture; v we demonstrated that in the absence of Shroom3, Rho kinase is mislocalized in the apical membrane of podocytes. As a result, mislocalized Rho kinase failed to phosphorylate non-muscle myosin and induce actomyosin contraction resulting in a patchy granular distribution of actin in the podocytes of Shroom3 mutants. Taken together, our findings established that Shroom3 is essential for proper actin organization in the podocytes through interaction with Rock. Furthermore, we took advantage of a haploinsufficiency phenotype of Shroom3 heterozygote adult mice and demonstrated these mice develop glomerulosclerosis and proteinuria. In conclusion, our studies provided evidence to support a role for Shroom3 in kidney development and disease and support the GWAS studies that suggested a correlation between SHROOM3 variants and kidney function in humans. / Thesis / Master of Science (MSc)

The acquisition of renogenic competence (the ability to give rise to kidney) during embryonic development is not yet fully understood. Clarifying the temporal and molecular aspects of this process is equally essential for understanding excretory system development and for devising methods for successful differentiation of embryonic stem cells (ESCs) to renal cells for disease modeling, toxicology screening and potential cell replacement therapies. In embryo development, the metanephric (permanent) kidney arises as a result of inductive interactions between two embryonic structures that arise in the intermediate mesoderm - the ureteric bud (UB, a diverticulum of the Wolffian duct) and the metanephric mesenchyme (MM). The UB develops into the collecting duct system and the MM undergoes an epithelial-to-mesenchymal transition to form the secretory units of the kidney - the nephrons. In this thesis, I used a tissue disaggregation-reaggregation method that allows the reconstruction of mouse organotypic kidney rudiments to place different embryonic cells in the environment of a developing kidney and assess their potential to integrate into kidney epithelia and differentiate to renal cells. First, the suitability of this method was evaluated and a quantitative assay for evaluating the numbers of test cells integrating in various renal compartments was developed. Second, the reaggregation method was used to characterise the renogenic potential of undifferentiated mouse ESCs, ESC-derived cells after Notch inhibition, and cells derived from the presumptive nephrogenic regions of embryos at various stages of development. ESCs are isolated from the inner cell mass of an embryo and have the potential to differentiate to any tissue of the body when injected into mouse blastocysts. Strategies have successfully been devised for ESC differentiation to many lineages, but very few studies reported any success with the differentiation of ESCs to a renal lineage. Undifferentiated ESCs showed a very good ability to form chimeric structures with developing kidney tubules (both nephrons and extending UBs). Nevertheless, the resulting structures were morphologically different from renal epithelia in most cases and integrated ESC-derived cells were not positive for several combinations of kidney markers. These results suggested that the influence of the niche was not sufficient for a successful ESC differentiation to renal cells. Treatment of ESC with an inhibitor of the Notch pathway to increase the proportion of mesodermal cells did not improve this outcome. On the basis of these results, it was speculated that the earliest lineage to which embryonic stem cells must be differentiated in order to become competent to make renal cells should first be identified. I addressed this by determining the developmental stage at which cells able to contribute to the formation of metanephric epithelia first appear in mouse embryo development. When mixed in embryonic kidney reaggregates labelled cells isolated from the nephrogenic regions of E9.5 embryos integrated into various renal compartments. These cells were seen in UBs, nephrons, glomeruli and the condensing mesenchyme. Marker expression studies showed that the exogenous E9.5 cells expressed an array of kidney markers - Pax2 in renal epithelia and the condensing mesenchyme, Wt1 in glomeruli and Six2 in the condensing mesenchyme. Furthermore, exogenous E9.5 cells also co-expressed Pax2/Wt1 in the condensing mesenchyme, Megalin/Ecadherin in the proximal tubule and Pax2/E-cadherin in renal epithelia. This provides evidence that challenges the existing model and suggests that some cells from the intermediate mesoderm at a stage where the metanephric blastema is yet formed are competent to contribute to kidney structures. Furthermore, experiments with E8.5 embryos showed that such a renocompetence could be acquired even before the specification of intermediate mesoderm. These findings contribute to our knowledge about kidney cell specification and provide valuable information to guide future attempts to develop an efficient method for deriving renal cells from ESCs. Furthermore, the reported ability of ESC-derived non-kidney cells to form chimeric structures with renal tubules provides a proof-of-principle that it might be possible to use exogenous types of cells for physiological support to injured kidney tubules, thus offering a possible novel approach for cell replacement therapies.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.

The renal capsule is a flattened layer of cells which surround the kidney. Expression of the transcription factor Foxd1 is required for normal development of the capsule. Furthermore, current evidence suggests that during development the capsule progenitors are in a state of active hedgehog signaling. We hypothesize that hedgehog plays a role in modulating capsule morphogenesis in the embryonic kidney. To test the hypothesis hedgehog signaling was inhibited in the capsule via Foxd1Cre mediated deletion of Smoothened (Smo), the activator of the pathway. Mutant kidneys were approximately 48% smaller in volume and had a 42% decrease in nephron number. Furthermore, mutants displayed abnormal patterning of the capsule where regions on the surface of the kidney had no capsule cells. The discontinuous capsule phenotype was observed only after E13.5. Additionally, capsule cells progressively lost expression of known markers Foxd1 and Raldh2 and their proliferative capacity was decreased by 54% at E13.5.