Study Of The Key Factors And Notch Signaling Pathway In MesenchymaMo-to-Epithelial Conversion During Kidney Development

IntroductionKidney disease has become the third major diseases that threaten human health. Acute injury, severe trauma patients prone to multiple organ failure, according to statistics, the mortality rate as high as45%. Studies have shown that the cytokine cascade effect can be caused by severe trauma, severe infection and strong stress. Kidney play an important role in stress and inflammatory response, and multiple organ dysfunction in patients with renal involvement the prognosis is obviously bad. Continuous renal replacement therapy (CRRT) as the main treatment, with expensive treatment costs and treatment of functional limitations, making the cure rate has been nearly50%. In the explicit study of kidney development and the inherent cell differentiation mechanism based on the development and application of cell therapy to repair kidney damage, will become a major focus and difficulty in treatment of acute kidney injury.On the other hand,10%of patients worldwide suffer by the distress of chronic kidney disease, of which10%of patients' progress to chronic renal insufficiency. High prevalence of the huge medical costs and poor prognosis, and the number of patients showed a trend of continued growth, causing serious social and economic burden. The cause of chronic kidney disease is complex and diverse, the pathogenesis is not yet completely clear, for the treatment of chronic kidney only limited symptomatic and supportive treatment without achieving a breakthrough. Recently, the ESRD patients increased7%-8%every year. The best treatment for ESRD is renal transplantation. There are nearly one million patients with chronic renal insufficiency, with an annual increase of12million people every year, and about500,000patients need kidney transplantation in China. However, the National kidneys available for transplant are only4000each year, and some patients have to stand to do hemodialysis with the enormous cost. Accompanied by the new immunosuppressive therapy on renal transplantation, the short term of allograft living increased, but10year living rate is only50%-61%. There was no breakthrough progress for kidney disease research in the pathogenesis and treatment. Thus proceed from the kidney embryonic development, to study the molecular mechanisms and signaling pathways of normal kidney development, and to clarify the development of mechanisms of kidney disease, the proposed new intervention repair strategy has become the nephropathy new focus.The aim of this study is to investigate intrinsic renal cell differentiation process, and clarify the signal transduction pathways and molecular mechanisms of the Notch signaling pathway. Our topic is expected to provide theoretical and experimental basis for kidney damage treatment and to provide some basis for future clinical research. The study may aid the development of human disease, and may aid the development of future clinical therapies and transplantation work. Part Ⅰ Screening for Candidate Genes in Notch Signaling Pathway During Mouse Embryonic Kidney DevelopmentObjective To investigate the profiles of embryonic kidney gene expressions at various stages of pregnancy in mouse, explore differential gene expressions during pregnancy, and to analyze the characteristics of key genes relevant to regeneration in Notch signaling pathway.Methods1. We set the mating cage at5Pm, when we found the Balb/c mice mice were pregnant next morning, it is gestation0.5day. Pregnant mice were sacrificed on gestation10.5/12.5/14.5/16.5/18.5days, and mouse embryonic kidney tissues were obtained under the microscope. With hematoxylin-eosin staining, we can compare embryo kidney developmental structure at various stages of pregnancy.2. Pregnant mice were sacrificed on gestation12.5/14.5/16.5/18.5days, and mouse embryonic kidney tissues were obtained under the microscope. Gene chips from SuperArray Bioscience Company were applied to explore gene expression profiles. Data, especially genes related to mouse embryonic kidney regeneration, were then analyzed by bioinformatical methodResultsWith hematoxylin-eosin staining, we compared embryo kidney developmental structure at various stages of pregnancy. Embryonic kidney tissues of mice were obtained during pregnancy at day12.5,14.5,16.5,18.5. By the Oligo GEArray (Notch signaling is pathway) screening candidate genes, three kinds of expression trends of genes differentially expressed were discovered. Expression of ADAM10, MFNG is gradual increase on gestation12.5-14.5, in the expression peaked at gestational day16.5, and follow on the expression of genes reduced at day18.5of pregnancy; the gene Ccnel expression with a gradual increase on gestation12.5-18.5; the gene Pparg expression with a gradual increase on gestation12.5-18.5.Conclusion On gestation14.5-16.5day, the comma and S-shaped bodies formatted, the glomerulus and nephron formatted and start to work. Expression trends of ADAM10, MFNG is important in the comma and S-shaped bodies formatted. Part Ⅱ The Role of Candidate Genes in the Notch Signaling Pathway of Mouse Embryonic Kidney in Vitro Mesenchymal-to-Epithelial ConversionObjective To verify the results of gene chips by RT-PCR and Western blot. By cultured mouse metanephric mesenchyme cell in the presence of ADAM10-selective inhibitor GI254023X, block Notch signaling, to observe the role of ADAM10in Mouse embryonic kidney Mesenchymal-to-Epithelial Conversion in vitro.Methods1. Pregnant mice were sacrificed on gestation12.5/14.5/16.5/18.5days, and mouse embryonic kidney tissues were obtained under the microscope.2. Expressions of genes were studied by RT-PCR to verify the results of gene chips; Expressions of protein were studied by Western blot to verify the results of gene chips.3. By cultured mouse metanephric mesenchyme cell in the presence of ADAM10-selective inhibitor GI254023X5days in vitro, block Notch signaling, to observe the role of ADAM10in Mouse embryonic kidney Mesenchymal-to-Epithelial. In the recovery experiments, both kidneys were initially cultured with DMSO (1μl/ml) plus Hydroxamate-based inhibitors GI254023X (GI1μM,5μM,10μM) for5days. Then mesenchyme stem cell markers PAX-2, WT1were detected in GI254023X-treated compared with control in immunohistochemistry. To investigate further the degree of epithelialization, and epithelial cell markers E-cadherin, Cytokeratin8was examined. In the end, expressions of WT1were studied by RT-PCR.Results1. By RT-PCR and Western blot method from the genetic and protein level, trend of expression were verified of ADAM10and MFNG gene, at E12.5days expression gradually increased to E14.5, E16.5reached peak, while E18.5was decreased.2. Cells immunohistochemistry:the Pax-2and WT1mesenchymal stem cells high expression, positive rate of the drug group compared with the control group significant low(P<0.05); positive rate of epithelial cell marker E-cadherin and Cytokeratin8the drug group than in the control group more low (P<0.05). The same as expressions of WT1were studied by RT-PCR.Conclusion Verification ADAM10and MFNG expression trends from the gene and protein level in mouse embryonic kidney development process.GI254023X, ADAM10-specific inhibitor, effectively blocking the Notch signaling pathway, inhibit the differentiation of embryonic kidney mesenchymal stem cells to renal intrinsic cells. Part Ⅲ Effects of Chlorpyrifos Exposure on Kidney Notch2-Jaggedl Pathway of Early Prenatal EmbryoObjective To evaluate the effects of this insecticide on the embryonic development of kidney, and to assess the important role of Notch2-Jagged1pathway in this duration.Methods We set the mating cage at5Pm, when we found the mice were pregnant next morning, it is gestation0.5day. Chlorpyrifos5mg/kg/d was administrated on gestation7.5-11.5day by subcutaneous injection. And the normal controls were injected0.9%sodium chloride at the same volume also on gestation7.5-11.5day.5fetuses were get from each CPF treaded pregnant mice (n=6) at random, and the same as the normal pregnant mice (n=6).Formalin-fixed paraffin-embedded tissue samples of the CPF treaded mice embryo kidneys of fetuses (n=30) and the normal embryo kidneys of fetuses (n=30) were obtained under the microscope after the pregnant Balb/c mice were sacrificed. With hematoxylin-eosin staining, we can compare the two groups embryo kidney developmental structure on gestation16.5day. ABC method (Avidin-biotin-peroxidase Complex method) was used to compare the two groups the expression of Notch2and Jagged1.Results On gestation16.5day, the normal embryo kidney developed through S shape duration to the original kidney which had the nephrons and could start to secret the urine. But for the chlorpyrifos treaded mice, the embryo kidney developed much more slowly, they did not show the S shape and the nephrons. The Notch2-Jagged1pathway should be expressed stronger in the normal embryo kidney on gestation16.5day, but for the chorpyrifos treaded mice we found the obvious weak pathway staining.Conclusion Chlorpyrifos broke the Notch2-Jagged1pathway during the embryo kidney development. And the Notch2-Jagged1pathway plays an important role in the S shape to original kidney formation duration.