CHL1在足细胞发育及损伤中的作用

Podocyte are highly specialized cells, with an important role in maintaining the glomerular filtration barrier. With their foot process they form slit diaphragms with neighboring podocytes. The podocyte has gained much attention, because of its pivotal role in glomerular function, and the substantial effects of loss of podocytes or podocyte function on the kidney. Injury to the podocytes is the initiating cause of many renal disease, leading to proteinuria, and is the determining factor for the progression toward glomerulosclerosis and end-stage renal disease. It is possible that progression to glomerulosclerosis can be diverted with prevention of the podocyte loss from injury. The main problem is that mature podocytes cannot proliferate or regenerate once apoptosis happens. Research in the last decades has demonstrated great progress in understanding the molecular mechanisms on slit diaphragm proteins leading to podocytopathies. For example, the podocyte and neuron share many cell biological characteristics in both structure and regulatory proteins. Many "neuron-specific" factors have been found expressing on the slit diaphrams of podocytes. The exploration of neurual molecules on podocytes may provide certain clues for understanding the pathophysiological mechanism, as well as for opening a new way to novel therapeutic approaches. CHL1 is demonstrated to be an inductive factor in neural development and a survival factor in neural injury. Whole genescan of mouse kidney during embryonic development discloses that Chll seems appearing in nephrogenic mesenchyme and thereafter glomeruli. However the function of Chl1 in the kidney is unknown. Our preliminary investigations firstly localised CHL1 to cultured podocytes and also found a close association between CHL1 and podocyte differentiation states. Furthuremore, we will investigate whether CHL1 is also an inductive factor to podocyte differentiation and a survival factor for injuried podocytes. We will upregulate CHL1 expression by introduction of exogenous Chl1 cDNA or downregulate CHL1 expression by selective silencing of Chl mRNA, to deeply explore the role of CHL1 in the different differentiation states of podocytes. We will use adriamycin injury model to observe the roles of CHL1 in the injuried podocytes in vitro. We will also take advantage of Chl1 knockout mouse models to examine in vivo the effect of Chl1 deletion on glomerular filtration formation in embryonic mice and proteinuria in mature mice, as well as to observe the clinical and biological manifestations in adriamycin-injuried Chl1 transgenic mouse models. This investigation will provide us a better understanding of the molecular mechanisms that control the development and survival of podocytes and lead us to novel therapeutic avenues for treating podocytopathies.

足细胞损伤是肾小球硬化起始和进展的关键因素，探索足细胞损伤的机制，对减慢重大疾病尿毒症群体的增加速度有重要意义。长期以来围绕着足细胞的难题是它损伤后得不到修复，难以再生。由于裂孔隔膜的异常是足细胞损伤的重要标记，所以裂孔隔膜上的分子是我们研究足细胞损伤的切入点。近年研究提示，足细胞和神经细胞具有许多相似特点，并且许多曾被认为只在神经细胞"特异"表达的粘附分子还在足细胞裂孔隔膜上表达。我们的前期研究在体外培养的足细胞上首次定位了神经粘附、存活、诱导分子CHLl，同时发现CHL1的表达与足细胞的分化状态密切相关。但CHL1对不同分化状态的足细胞起什么样的作用及其意义并不清楚，国内外未见报道。本研究拟通过体内、体外实验，利用靶向基因技术，制造损伤模型等手段，来深入探讨CHL1对不同分化、损伤状态的足细胞的影响，有望解答CHL1对足细胞发育及损伤修复的作用及其意义，为防治足细胞病提供新的分子靶点。

大量蛋白尿是慢性肾脏病进展和预后不良的独立危险因素。而导致大量蛋白尿的重要始动环节是构成肾小球滤过屏障最外层的足细胞的损伤。目前的治疗难点在于足细胞是终末分化的细胞，数量恒定不能再生，一旦损伤修复困难。为解决这个问题，我们从足细胞发育得到启发，后者代表了足细胞从未分化到分化的过程，而足细胞损伤首先是逆向发育的过程。由于发育和损伤的密切关联，我们假设，调控足细胞发育的分子可能在足细胞损伤后修复中发挥作用。为了寻找这种调控足细胞发育的分子，我们通过文献查询发现足细胞和神经细胞在结构和功能上有许多相似性，一些神经特异性分子被证实在足细胞的发育及损伤修复中重要作用（如GDNF）。我们在小鼠不同时期胚肾发育的基因扫描热图上发现，一种以往认为是神经特异性表达的分子CHL1在后肾间充质时期高度表达。随着间充质向肾小球分化，CHL1的表达显著减少；且CHL1仅在肾小球出现，未出现于肾小管；CHL1的出现先于足细胞成熟的标志基因Nphs1/2的出现。以上现象提示，CHL1是与肾小球发育密切相关的基因；而足细胞作为肾小球不同结构类型细胞的组织者和管理者，我们推测，CHL1与足细胞发育相关。本项目证实了部分我们的推测。首先，我们在大鼠上验证了CHL1随肾脏发育不同阶段的表达规律。通过real-time RT-PCR、westernblot、免疫荧光发现，CHL1在大鼠E11.5天的后肾开始出现，E12.5天达到峰值，随着胚肾发育CHL1的表达不断降低，于E18.5天达到最低，出生后CHL1在肾脏组织几乎检测不到。然而，通过免疫电镜胶体金技术我们观察到未成熟和出生后的大鼠CHL1肾小球足细胞足突上表达。我们推测这些少量表达的CHL1对于维持足突形态及足细胞存活起重要作用。我们进一步利用小鼠永生化足细胞系，观察到33℃未分化的足细胞CHL1呈高度表达，而随着足细胞在37℃分化成熟，CHL1表达水平显著减少。在33℃未分化细胞CHL1在足突远端表达，而在37℃分化足细胞，CHL1表达于核周。构建嘌呤霉素足细胞损伤模型，发现CHL1在足细胞的表达有从核周向足突远端移行的趋势。本项目的意义在于，首次揭示了神经特异性分子CHL1在大鼠肾脏发育的时间空间规律，首次发现了CHL1在足细胞上表达，并与足细胞分化状态密切相关，CHL1的位置移行有可能与保护足细胞损伤有关，进一步需要转染实验的验证。