从单核-巨噬细胞角度探讨Kca3.1/TRPC3复合物在肾脏纤维化防治中的研究

Renal fibrosis is the primary pathologic change of chronic renal failure. Macrophages (MФ)-mediated inflammation plays an important role in the development of renal fibrosis.Our prioir studies find that transient receptor potential cation TRPC3 and intermediate conductance calcium-activated potassium ion channels Kca3.1 are all expressed in MФ and mesangial cell（MC）. Inhibition of Kca3.1 and/or TRPC3 can significantly increase the local amount of M2-type MФ in the renal fibrosis of mice and can reduce M1-type MФ infiltration and inherent cell proliferation. But the mechanism is not clear. It is the first time to propose the concept of "Kca3.1/TRPC3 complex", namely the Kca3.1/TRPC3 may affect the process of proliferation and renal fibrosis of inherent cells by regulating the transformation of five active isoforms of MФ and inflammation and the cytokine secretion. To test this hypothesis, we will use the co-immunoprecipitation and other techniques to determine the distribution of Kca3.1/TRPC3 in MФ, and use in vivo fibrosis model and in vitro cell culture experiments in combination with molecular biology and electrophysiology techniques to explore the effects of Kca3.1/TRPC3 mediation on the renal fibrosis, MФ activity and function and MФ electrophysiological parameters, to analyze the correlation between them, and to search for possible molecular signals. Thereby we hope to elaberate the effects of Kca3.1/TRPC3 on renal injury-repair and its mechanisms from a view of MФ, to seek new therapeutic targets for renal fibrosis prevention and to provide theoretical and experimental basis.

单核-巨噬细胞（MФ）及其介导的炎症在肾脏纤维化发生发展中起着重要作用。申请者前期研究发现钙激活钾通道Kca3.1和瞬时受体电位阳离子通道TRPC3同时定位于MФ和系膜细胞（MC）上，抑制Kca3.1和/或TRPC3表达，可减少MC增殖，并调控肾脏纤维化小鼠肾局部MФ活性，但具体机制不清楚。本课题首次提出"MФ上存在Kca3.1/TRPC3复合物"概念，并设想：Kca3.1/TRPC3可能通过调控MФ介导的炎症和细胞因子分泌，延缓肾固有细胞增殖及纤维化进程。为验证之，我们将利用免疫共沉淀与膜片钳等技术，观察MФ上Kca3.1/TRPC3定位、表达及电生理学变化，明确调控Kca3.1/TRPC3对MФ表型和功能以及肾纤维化指标的影响，并分析其相关性和可能信号转导通路。以期从MФ 角度阐释Kca3.1/TRPC3在肾脏损伤修复中的作用和机制，为肾脏纤维化研究提供理论和实验依据。

单核-巨噬细胞及其介导的炎症在肾脏纤维化发生发展中起着重要作用。本项目主要从体外和体内两部分实验进行，体外实验首先采用激光共聚焦观察巨噬细胞上Kca3.1与TRPC3的定位及表达。接着，光镜下观察了各型巨噬细胞形态学表现，并采用RT-PCR方法对其表面标志物进行了鉴定。利用Fluo-4AM技术检测了分别激活和抑制TRPC后，巨噬细胞胞内钙离子内流情况。最后，使用共培养技术，观察了巨噬细胞对HK-2及腹膜间皮细胞EMT的影响。体内实验，观察了TRPC各亚型在IgA肾病大鼠中的定位表达、对肾纤维化指标的影响及探索了可能的分子机制。结果发现: Kca3.1与TRPC3在巨噬细胞中均有表达；而单核细胞THP-1经 TPA 25 nM培养4h贴壁后分别加入LPS+IFN-γ、IL-4、 IL-10各20 ng/ml可诱导为M1、M2a、M2c，仅TPA诱导组为M0。在加入各刺激物24小时后：M1组较M2a、M2c组低表达炎症抑制因子IL-10，高表达IL-6、IL-12；M2a高表达CCL17、IL-10，低表达IL-6、IL-12；M2c高表达CXCL13、IL-10，IL-6、IL-12表达水平相对较低。对于M0组，IL-6、IL-12表达水平相对较低，CXCL13、CCL17、IL-10表达水平处于M2a及M2c表达水平之间，而高表达TGF-β1。Fluo-4AM结果：巨噬细胞在瞬时较高水平Ca2+刺激下，细胞出现Ca2+内流明显升高；在瞬时Ca2+刺激下，分别给予TRPC激动剂及拮抗剂，巨噬细胞仍出现Ca2+明显升高，但较未加拮抗剂组，Ca2+升高幅度明显降低；共培养结果发现：与空白对照组比，共培养组及2-APB处理组的HK-2细胞Vimentin及α-SMA的mRNA水平均升高，E-Cadherin的mRNA水平降低。同时，与正常共培养组比，2-APB预处理组HK-2细胞中Vimentin及α-SMA的mRNA升高程度减小，但E-Cadherin在两组中未见明显差别。腹膜间皮细胞：与对照组比，巨噬细胞共培养组的腹膜间皮细胞E-cadherin荧光强度均减低。与巨噬细胞共培养的腹膜间皮细胞α-SMA荧光强度均高于对照组。体内：IgA肾病模型中，TRPC亚型的基因表达均升高，而他克莫司可以下调TRPC、α-SMA的基因表达，其可能的信号通路与p-ERK1/2有关。