Proper development of the nephron, the functional unit of the kidney, is essential for kidney function. The nephron develops from a pool of cap mesenchymal cells, as defined by a cluster of cells adjacent to the ureteric bud tips of branching ureteric epithelium, giving rise to two subset populations: the self renewing cells and the nephron progenitors. These nephron progenitors undergo mesenchymal-epithelial transition (MET) to develop into polarized renal vesicles (RV), and eventually fuse with the epithelial tubule to develop into a mature nephron. Although these processes are essential for the formation of functional kidneys, little is known about the molecular mechanisms that regulate them. In this study, we characterize several steps during cap mesenchyme and renal vesicle formation using our Shroom3 knockout mouse kidney as our model. Previous researchers have associated Shroom3 with chronic kidney disease. Detecting and analyzing the genetic components of CKD is needed to improve our understanding of its pathogenesis. Shroom3 encodes an actin-binding protein that regulates cell shape changes through induction of apical constriction. However, there is a lack of evidence about Shroom3’s expression pattern and functional role upstream of developed nephrons. Here, I defined the spatial and temporal expression of Shroom3 within the cap mesenchyme region. I investigated the nephron progenitors between Shroom3 wildtypes and mutants. Lastly, I analyzed the renal vesicle polarity in mutants, by analyzing apical membrane markers on RVs to characterize any abnormalities in their orientation and establishment of polarity.

Thesis

Master of Science in Medical Sciences (MSMS)

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)

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…

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)