线粒体在cAMP/PKA信号调节肾小球壁层上皮细胞向足细胞分化中作用及机制

Podocytes are highly differentiated cells. The cells play a key role in the glomerular filtration barrier. It is well accepted that loss of podocyte triggered and promoted glomerular focal segmental sclerosis. Glomerular parietal epithelial cells (PECs) proliferation and differentiated into podocytes was the mainly source of supplement of loss of podocyte. In our primary studies, we found that activation of cAMP/PKA signaling pathway induced podocyte differentiation from PECs, as well as inhibited activation of PECs after podocyte injury. It’s unclear that the molecular mechanism of regulation of PECs differentiation or activation. PECs have a few of mitochondria and there are plenty of mitochondria in primary and secondary foot processes of podocyte. We also found that activation of cAMP/PKA with pCPT-cAMP promoted differentiation of podocytes companied with upregulation of mitochondria related proteins, suggesting that mitochondria may play a role in the cAMP/PKA-induced podocytes differentiation from PECs. To explore the mechanism of podocytes differentiation from PECs, we will check the relationship between number or function of mitochondria with cAMP/PKA-induced podocytes differentiation from PECs. We will also exam the role of ATP, mitochondrial ROS and ATP synthase in podocytes differentiation from PECs. At last, we are going to estimate the crosstalk between cAMP/PKA and signaling pathway which active PECs. We believe that these works will help us to understand the mechanism of podocytes differentiation from PECs and is helpful to find the new treatment for protection of podocytes, as well as prevention glomerular sclerosis.

肾小球足细胞受损伤后数量减少导致肾小球局灶节段硬化。肾小球壁层上皮细胞（PECs）既可向足细胞再分化以补充丢失的足细胞，也可能被激活分泌基质，导致肾小球硬化。然而还不清楚调控PECs向足细胞分化的具体机制。申请人前期研究发现激动cAMP/PKA信号可以促进PECs向足细胞分化并抑制PECs激活，调节未分化足细胞线粒体生成促进足细胞分化。PECs仅含有少量线粒体，而再分化的足细胞有大量的线粒体。这些均提示线粒体可能与PECs向足细胞分化相关。为了深入研究调节PECs向足细胞分化的分子机制，本项目拟通过体内和体外实验观察线粒体功能和数量，与PKA信号诱导的PECs向足细胞分化，抑制PECs活化的关系，并探索ATP、线粒体源ROS或ATP合成酶在PECs向足细胞分化中的作用。研究结果有助于深入理解肾小球硬化的发病与足细胞损伤后的修复机制，为探索防治肾小球疾病的新方法奠定基础。

肾小球壁层上皮细胞（PECs）是肾小球内潜在的干细胞，具有向足细胞分化的能力。本项目中我们使用ADR小鼠模型观察发现随着蛋白尿增加，肾小球足细胞标记蛋白表达减少，而肾小球内部分podocalyxin阳性的足细胞同时表达了PECs标记蛋白claudin-1。我们使用筛网+磁力吸附的方法分离肾小球，进行了PECs的原代培养。发现体外培养至第6代的PECs仍表达claudin-1、CD24和CD133，而不表达足细胞和系膜细胞的标记蛋白，和PECs体内特性一致。使用诱导分化培养基（VRADM）培养PECs后观察到PECs表达足细胞标记物，而claudin-1表达降低。进一步研究PECs向足细胞分化，细胞内总自由氧簇（ROS）和线粒体ROS均表达增多，而使用NAC和Mito Tempo分别阻断细胞内总ROS和线粒体ROS均可抑制PECs向足细胞分化。进一步研究ROS相关的分子Nrf2发现，Nrf2在PECs向足细胞分化过程中表达增高，使用NAC后抑制了VRADM诱导PECs细胞核内的Nrf2集聚。使用siRNA抑制Nrf2表达后阻断了VRADM诱导的PECs向足细胞分化。抑制Nrf2表达也可减弱Brg1表达，同时，PECs向足细胞分化过程中Brg1表达增高。抑制Brg1表达后，同样可以阻断PECs向足细胞分化。而过表达Brg1可促进PECs的分化。我们也在临床观察性研究中验证了PECs中Brg1的作用，免疫染色显示eGFR下降速率较快的FSGS患者Brg1阳性数较高，Brg1阳性数和蛋白尿呈正相关。. 这些结果提示了线粒体在PECs向足细胞分化中的重要作用。ROS，特别是线粒体ROS可能是调节PECs向足细胞分化的关键信号分子。PECs收到细胞外信号的刺激后，可能通过ROS-Nrf2-Brg1信号调节足细胞WT1-1转录因子高表达，从而向足细胞分化。