[1]	袁晓菲，徐全福，李毅刚. 心房纤维化与组织结构重构：转化生长因子β1等多种神经体液介质的作用[J]. 中国组织工程研究, 2015, 19(7): 1036-1041.

In the growing mammalian embryonic kidney, ureteric bud (UB) that extends out from a pre-existing epithelial tube, gives rise to the branched collecting duct system while the surrounding metanephric mesenchyme undergoes mesenchymal-epithelial transition to form the proximal parts of the nephron. Tubulogenesis, a process by which disorganised mesenchymal cells become a highly organised epithelial tubule, can be summarily divided into four developmental stages [1]. ①Condensation, in which groups of undifferentiated mesenchymal cells condense tightly together to form a distinct mass; ② Epithelisation, in which condensed cells lose their mesenchymal characteristics and gain epithelial ones. At the end of this period they have formed a small epithelial cyst complete with a basement membrane, cell-cell tight junctions and a defined apico-basal polarity; ③Early morphogenesis, in which the cyst invaginates twice to form a comma and then an S-shaped body, one of these invagination sites later becoming the glomerular cleft. ④Tubule maturation, in which the specialized transporting segments of nephron differentiate. In this stage, tubules grow and extend in a straightforward, a convoluting or a branching manner to form the three-dimensional structures.
Observing the events of tubulogenesis within the growing kidney, one has to admit that despite intense investigations in the past decades, little information on the underlying mechanism is available. A major obstacle is the absence of a suitable in vitro culture model, upon which the absolutely controllable conditions are created to ensure not only the formation of tubules but also the maintenance of highly differentiated structures over a relatively long cultural period so that one can investigate the progression of tubule development. The present review focuses on two aspects: ①experimental systems now already available to analyze the tubulogenesis and their existing technical limitations. An innovative culture technique is recently established by us [2-4] and its advantages are briefly introduced. ②Applying this unique in vitro culture model, it was for the first time indicated that the steroid hormone aldosterone exerts a significantly inductive action on the tubular development of renal stem/progenitor cells [5-8]. Basing upon the so far achieved data, the author described the process of the aldosterone-mediated action and discussed the implication of these studies toward unveiling tubologenesis.

Then, how to explain that none of the growth factors, that have been already reported to promote tubulogenesis by applying other culture systems, is able to induce tubular formation in our Minu3D model? Recently, aldosterone synthase knockout mice were produced by Gomez et al. Despite abnormal electrolyte homeostasis and decreased blood pressure these mice have grossly normal kidney development and are still viable [51]. In fact, in many of the single growth factor knockout mice a defective kidney phenotype fails to demonstrate [52-53]. It is yet unknown whether all these regulatory factors are simultaneously indispensable to trigger tubulogenesis in vivo. It is most likely that different growth factors are needed in different developmental periods. We noticed differential expression profile of such factors. For example, transforming growth factor-α (TGF-α) is expressed in the embryonic kidney whereas EGF is expressed in the kidney mainly after birth. In addition, most of growth factors express in redundancy in developing tissues. Thus, compensatory pathways may exist in vivo in the case that a single factor lacks.
In our experiments, aldosterone-induced tubules can be seen only after the 5th culture day [6]. The applied embryonic explants are a mixture of different cell populations. During such a long culture period, it does not yet exclude the possibility whether other endogenous factors, which may be already over-expressed in neonatal kidneys, play a potential role to facilitate tubulogenesis as well in concert with aldosterone. Currently, by administration of aldosterone combined with different growth factors, we are searching for the possible candidates that are likely to improve or accelerate the action of aldosterone.   
Finally, the innovative Minu3D model allows us under absolutely controllable culture conditions to investigate the progression of tubulogenesis in different developmental phases. To understand the specific subcellular process, numerous cell biological and molecular biological approaches such as immunohistochemistry, real-time PCR, 2D western blotting and proteomics are being employed for analysis. Among them, antibody microarray may provide a promising tool to find out key signaling molecules/regulatory proteins for insights into the underlying mechanisms of tubulogenesis.  

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