他克莫司对FSGS足细胞保护作用的机制研究

Focal segmental glomerulosclerosis (FSGS) is the most common cause of end stage renal disease. which is podocyte dysfunction involving a massive loss of protein in the urine. Tacrolimus(FK506 )can reduce proteinuria in FSGS patients with steroid resistance.It has been assumed that T cells act on podocytes to cause proteinuria and that the antiproteinuric effect of FK506 results from the.inhibition of NFAT signaling in T cells. As it stands, experimental support of this hypothesis is missing. Recent studies highlighted the bene?cial effect of calcineurin inhibitor may be the result from the stabilization of the actin cytoskeleton in kidney podocytes. In our previous work, increased Cn expression was observed in the podocytes of FSGS patients . Further study of calcineurin activity in podocyte is needed. Therefore, we hypothesis that FK506 provided podocyte-direct protection via blocks the calcineurin activity to reduced proteinuria in FSGS. .To address the question of whether FK506 has a direct action on podocytes, an animal model of doxorubicin and immortalized podocyte injury was used to evaluate the effect of FK506 in vivo and in vitro study. Immunostaining, Cryo-immunogold electron microscopy, microdissected glomeruli and qRT-PCR，Western blot were performed to con?rm the direct protective effects of FK506 on podocyte injuries and to explore the underlying molecule mechanism of FK506 action. Based on these findings, we will find that abnormal calcinuerin in podocyte would be involved in the progression of FSGS. Modulation of podocyte-specific calcineruin signaling pathway may be a potential target in FSGS patients. These research will represent a new view of calcineurin signaling and shed further light on the treatment of FSGS.

局灶节段性肾小球硬化(FSGS)是导致终末期肾脏病的重要原因，钙神经蛋白抑制剂他克莫司(FK506)是治疗激素抵抗FSGS的主要药物，以往认为其主要通过抑制T细胞活化来治疗FSGS。近期研究发现足细胞钙神经蛋白(Cn)过度活化可导致足细胞损伤和蛋白尿。我们预实验发现FSGS患者足细胞 Cn表达增加，但其具体机制仍待进一步探索。为此，我们提出假说，FSGS足细胞存在Cn的过度活化，FK506可通过抑制足细胞Cn过度活化来治疗FSGS。我们将通过临床病理研究、阿霉素肾病和Cn基因敲除小鼠模型、体外人足细胞培养等方法，采用微分离肾小球，免疫荧光、qRT-PCR，Western blot等手段，从分子，细胞，组织及动物整体水平明确Cn活化在足细胞损伤中的作用，明确FK506治疗FSGS分子机制。本研究将从足细胞保护这一新视点，揭示FK506治疗FSGS的新机制，为FSGS的治疗研究提供新的思路。

局灶节段性肾小球硬化是导致终末期肾脏病的重要原因，钙神经蛋白抑制剂他克莫司(FK506)是治疗激素抵抗FSGS 的主要药物。足细胞钙神经蛋白(Cn)过度活化可能导致足细胞损伤和蛋白尿。我们研究发现仅半数FSGS 患者足细胞CnAα 表达明显增加，但未发现NFATc在足细胞核上的表达增加；FSGS足细胞 CnAα与synaptopodin 表达的关系也并非一致，且CnAα表达强度与他克莫司的疗效无关。因此，为进一步探讨FK506 足细胞保护作用机制，我们利用miR-30s在足细胞损伤和蛋白尿发生中的作用进行研究，探讨 FK506 足细胞保护作用机制是否与miR-30s家族调控足细胞calcineurin信号通路有关。在FSGS患者和嘌呤霉素（PAN）诱导的肾损伤模型中，肾小球miR-30s表达显著下降。体外试验证实刺激因子诱导足细胞损伤后，miR-30s表达明显下降。经miR30a质粒转染的足细胞可有效抵御刺激因子所引起的足细胞损伤。相反，敲除足细胞miR30s后，可观察到明显足细胞损伤。体内实验研究发现miR-30a治疗可明显减少PAN大鼠的蛋白尿。在培养的足细胞中，我们发现PAN大鼠模型或miR-30 sponge足细胞中TRPC6、PPP3ca、PPP3cb、PPP3r1及NFATc3蛋白表达上升，细胞内钙聚集， calcineurin信号通路活性增加。这一现象是由于miR-30减少引起的calcineurin 信号通路活化有关，能够被钙调蛋白抑制剂FK506所阻断。体内实验发现，足细胞特异miR-30 sponge转基因小鼠calcineurin信号通路活化，可以出现显著的蛋白尿，足突融合。而FK506可抑制miR-30 sponge诱导的足细胞骨架损伤， 抑制miR-30 sponge诱导的足细胞synaptopodin降解，并且抑制miR-30 sponge诱导的足细胞凋亡，从而达到足细胞保护作用。　因此， FSGS 患者足细胞受损的分子机制可能与肾小球中miR-30s表达显著下降有关。而FK506可抑制由于miR-30s 减少导致的calcineurin信号通路活化，足细胞骨架损伤和足细胞凋亡，从而达到足细胞保护作用，减少蛋白尿。