open access

https://doi.org/10.14943/doctoral.k9951

https://hdl.handle.net/2115/44980

Clarification of molecular network during nephrogenesis in mice - Hepatocyte nuclear factor 4 alpha plays a central role in nephrogenesis-

日本語

2011-03-24

Hokkaido University

92p.

Kidney development is started by invasion of the uretelic bud (UB) arising from the Wöllfian duct into the metanephric mesenchyme (MM). Then the MM concentrates around the UB tips and forms the condensed mesenchyme (CM). The CM transforms sequentially to the renal vesicles, comma-shaped body (CSB), S-shaped body (SSB) and finally the nephron. In these processes, MM cells turn to nephron resulting by the mesenchymal-epithelial transition (MET). Although the biological mechanism of nephrogenesis and MET have been clarified gradually, a lot of uncertain points still remain, owing to the complexity of kidney structure. Hepatocyte nuclear factor 4 alpha (Hnf4 protein is one of the Zinc finger type-nuclear transcription factors expressing in the liver, intestine, pancreatic beta cells and kidney. Hnf4 has a relationship among large amount of gene regulation in hepatocytes and pancreatic beta cells, playing a key role in fat metabolism, insulin secretion and maintenance of the homeostasis. However, since disruption of the Hnf4a gene results in embryonic lethality due to emblyonal endodermal cell death and failure of gastrulation before kidney development, its renal function is still unknown. In this research, to clarify the dynamics and the function of Hnf4 during nephrogenesis, following analyses were performed. In chapter 1, the localization of Hnf4 in several organs and the dynamics of this gene during nephrogenesis were investigated. In chapter 2, to assess the function during nephrogenesis, gene-silencing of Hnf4a was performed in organ-cultured kidney by RNAi method. Additionally, micro-environmental gene expression of Hnf family was investigated. In chapter 3, to elucidate the details of MET during nephrogenesis, the embryonic kidneys were analyzed by electron microscopy, immunohistochemistry, and molecular biology. In addition, cellular model inducing the expression of Hnf4a gene into fibroblast cell line was produced and analyzed, in order to assess the relationship between Hnf4 and MET,.In chapter 1, it was clarified that Hnf4aP1 or P2 was expressed dominantly in kidney and in stomach and pancreas respectively, while both types were expressed in liver, small intestine and large intestine. In immunohistochemical analysis, Hnf4 protein was located only in the segment of the proximal tubules in kidney, but not in the other segment. In the developing kidney, Hnfwas detected first at the epithelial cell nuclei in part of the CSB/SSB, distributed widely throughout the developing nephron and finally restricted to proximal tubules. Interestingly, it was noted that Hnf4aP1 and P2 were detected from stomach, pancreas and kidney tissues in embryonic periods. Additionally, Hnf4 was expressed earlier than Hnf1 in the same tubule, giving the impression that Hnf4 played the more fundamental role in Hnf network during nephrogenesis.In chapter 2, as a result of gene-silencing, the cellular organization in the CM fell into disorder and many apoptotic cells appeared in cultured kidney. LMD-RT-PCR revealed that both of Hnf4aP1 and P2 were expressed in the CM and only P1 was expressed in the CSB/SSB, while both were not detected in the MM. Additionally, only Hnf1b which are up-stream genes of Hnf4a was expressed in the CM and Hnf1a and Hnf1b were expressed in the CSB/SSB. These results suggested that Hnf4 played an important role during cell survival in the CM and this gene expression was induced by Hnf1.In chapter 3, the TEM observation and immunohistochemistry in developing kidney showed that the intercellular junction, but not the basal lamina, was present in the CM. Additionally, immediately after Hnf4a gene expression, the expression of epithelial genes (Krt8, Tjp1, and Cdh1) increased, and those of mesenchymal genes (Acta1 and Vim) decreased, in the CM compared to the MM. In the cellular model analysis, it was noted that Hnf4 induced increasing epithelial and decreasing mesenchymal gene expression. In this analysis, up-regulation of Pvrl1, -2, and Mllt4 genes which mediate the formation of apico-basal polarity, were found. These results suggested that the cells in the CM showed the intermediated forms between epithelial and mesenchymal and Hnf4a proteins induced the MET in typical fibroblast. In conclusion, following dynamics model was provided; Hnf4appears in the CM stage and in the CSB/SSB stage. In the CM stage, both of P1 and P2 induced by Hnf1 relate to the cellular survival and the initiation of MET via activating Nectin-Afadin. In the CSB/SSB stage, only P1 is expressed and up-regulates the expression of Hnf1a. Hnf1 and Hnf4 activate the gene expression each other and relate to the characterization of proximal tubules.

2011-03-24

甲第9951号

博士(獣医学)

博士論文

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