肾小管形态及功能发生发育的细胞生物学研究

Proper renal tubulogenesis is based on a strict temporal-spatial sequence of cellular proliferation, migration and differentiation. According to developmental theories, positional information plays an instructive role in tubulogenesis, and hundreds of determinants and proteins are involved in its modulation, but the mechanism remains a mystery. The proposed project will explore the temporal-spatial features of cellular proliferation, migration and differentiation during the tubulogenesis occurring in developing mouse and human kidneys using multi-disciplinary techniques, such as digitalized medicine for the 3D reconstruction of developing renal tubules and molecular biological techniques for the detection of proteins and RNA, based on theories from various disciplines, such as developmental genetics, embryology and cell biology. The applicant completed a Ph.D. degree and studied in Nephrology in Denmark for seven years. In previous projects funded by grants from the National Natural Science Foundation of China (NSFC), the applicant developed 3D reconstructions of hundreds of nephrons and collecting ductules from adult and developing mouse kidneys using multi-disciplinary techniques. Thereafter, the epithelial transporters were localized in part, and a database of microscopic images of human fetal renal tissues was established using improved tissue preparation methods.. These research findings have been reference many times in authoritative international journals on Nephrology, e.g. Kidney International and the Journal of the American Society of Nephrology. Using previous renal development research combined with multi-disciplinary techniques - such as laser capture microdissection and PCR, in situ hybridization, realtime PCR, immunohistochemistry and western blot - the proposed project aims to improve upon current theories of renal development.

肾小管上皮细胞严格瞬时、定位、多样性分化是肾小管形态和功能正常发生的基础，发生发育学研究指出，"位置信息"在此过程中起到指令性作用，数百因子和蛋白质起调控作用，其机制尚是个谜。本项目拟运用发育遗传学、胚胎学及细胞生物学理论，采用多学科交叉技术手段，如数字医学微细结构三维重建技术，分子生物学蛋白质及RNA检测技术，探索小鼠及人肾小管形态发生时细胞生命活动如增殖、迁移、功能分化等现象的时空规律。项目申请人有多年肾结构与功能相关性研究并取得博士学位的留学背景。在前期国家自然科学基金资助下，利用数字医学技术三维重建了数百个发生发育和成年小鼠肾单位及集合管，进而定位其上皮重要转运体，并通过改良技术建立了胎儿肾发生发育显微图像数据库。研究结果多次发表在肾学权威杂志KI和JASN上。此次申请的项目是前期发生发育肾结构和功能研究从小鼠到胎儿，从组织水平向细胞分子水平的深入，旨在完善肾发生发育的理论。

自从100多年前Barker首次提出“胎儿期发生的事件可能对出生后的整个生命过程的健康状况都将产生影响”，器官发生发育进入了病因学研究的范畴。就肾脏而言，尽管机制不清楚，但得到普遍共识的是，肾小球的数量与高血压的发病率、肾脏的衰老速度直接相关。什么决定了肾小球数量？显而易见的答案却留下重重疑点，即输尿管芽在后肾的反复二杈分支，其终末在被膜下生肾区诱导了后肾间充质细胞分化成肾单位，然而，肾单位的数目几倍甚至几十倍于二杈分支的次数；此外，在肾单位发生的同时，肾血管系统的发生也与之呈现一定时空关系，包括参与肾滤过及重吸收功能的肾微循环，而微循环损伤是急肾及慢肾的主要病因或结果。无疑，肾发生发育的研究有助于肾病因学的发展。本研究借助微细结构三维可视化技术平台，结合细胞蛋白检测技术及体视学方法，在详尽记录集合管、肾小管及血管形态发生及毗邻关系的同时，对各节段细胞增殖、凋亡及转运功能分化进行了研究，发表了5篇SCI文章，得出了若干重要结论：①集合管系的发生以输尿管芽的二杈分支为基础，在此基础上以“出芽”发式形成芽泡，后者诱导1~2个肾单位发生；②肾单位最初与芽泡相连是以后肾间充质细胞索为始发，而直到S小体期出现了血流的灌注才与芽泡相通，同时球内外微循环相连；③髓质逆流倍增产生浓缩尿液的结构基础是，肾微血管与肾小管在发生发育中形成的伴行关系；④发生发育中，细胞增殖显示出时空及肾小管节段的依赖性；⑤肾近端及远端小管线粒体结构不同，提示其对小管缺氧耐受及损伤修复的差异性；⑥远端小管及连接小管与微动脉存在致密斑之外的又一可能的“crosstalk”；⑦肾小管对蛋白、水、离子等的转运功能早于结构的成熟。综上，肾脏在输尿管芽与后肾间充质相互诱导中，瞬时、定位产生了肾单位及血管系统，并逐渐形成有机整体行使生理功能，这其中的影响因素既有高度保守的基因遗传又有肾脏发生时组织环境中的分子因素，如分子的种类及浓度，这为接续的研究指出了方向。