线粒体功能变化在mGluR1/cAMP信号防止足细胞损伤中的作用

Podocytes are highly differentiated cells with numerous foot processes. The foot processes of neighboring podocytes interdigitate sharing filtration slits bridged by the slit diaphragm. Foot processes and their slit diaphragms constitute an architectural element of the permeability barrier and become effacement during podocyte injury due to genetic or acquired factor. Intercellular and intracellular signaling plays an important role in stability maintenance for foot processes. .In our previous studies, we found that podocyte express metabotropic glutamate receptor 1 and 5. Activation of mGluR1/5 with DHPG results in generation of cAMP, activation of PKA and CREB, which protect against puromycin aminonucleod and adriamycin-induced albuminuine, podocyte apoptosis, foot processes effacement and nephrin downregulation. Not only our group, Rastaldi MP developed mGluR1 gene mutation mouse (Grm1crv4/crv4 mice) and demonstrated that Grm1crv4/crv4 mice showed albuminuria, podocyte foot process effacement and reduced levels of nephrin. These data suggest that mGluR1 may regulate foot process morphology and intercellular signaling, also contribute the integrity of glomerular filtration barrier. .We, however, don't know the mechanism of protection of mGluR1/5 signal and intercellular signaling pathway. In our preliminary studies, an agonist for cAMP/PKA, but no cAMP/Epac prevented mitochondrial membrane potential collapse when podocytes were injuried, suggesting that cAMP/PKA protect podocyte via regulate mitochondrial function. The role of mitochondrial dysfunction in podocyte injury/repair is poorly investigated. Several line evidences implicated that cAMP/PKA signaling regulate mitochondrial fusion/fission dynamic balance, mitochondrial oxidative phosphorylation and mitochondrial biogenesis by transcript factor dependent and independent manner. Therefore, we hypothesis that mGluR1/cAMP/PKA signaling protect against podocyte injury via regulation of mitochondrial function. .In the present project, we will focus on the role of mitochondrial dysfuncation, including mitochondrial fusion/fission, oxidative phosphorylation, biogenesis and mitochondrial DNA transcription and the transcript factor dependent and independent regulation of mGluR1/cAMP/PKA in podocyte apoptosis in vitro. We will also evaluate mitochondrial function and cAMP/PKA signaling in ADR nephrosis mice podocyte and db/db mice podocyte. Additionally, we will explore the role of mitochondria on the podocyte differentiation and dedifferentiation/proliferation. .We believe that these work will help us to understand the role of mitochondrial dysfunction in podocyte injury/repair and the regulation of mGluR1/cAMP/PKA signaling.

我们前期工作原创性地发现足细胞表达代谢型谷氨酸受体（mGluR）1和5，激活mGluR1/5通过cAMP信号途径防止嘌呤霉素和阿霉素诱导的足细胞损伤。但是还不清楚mGluR1/cAMP信号激活后保护足细胞的具体机制。预初实验发现cAMP/PKA激动剂改善足细胞损伤时的线粒体膜电位降低，提示cAMP/PKA信号通过影响线粒体功能发挥其保护作用。为深入研究其具体的分子机制，本项目拟通过分子生物学方法，从体内和体外实验中观察cAMP/PKA转录依赖和非转录依赖信号对线粒体分裂/融合、氧化磷酸化、线粒体生成及线粒体基因组转录的影响，研究其在防止足细胞损伤凋亡中的作用。也将探索线粒体功能变化在cAMP/PKA信号调节足细胞分化与增殖中的作用。本项目的实施不仅让我们进一步了解谷氨酸信号在足细胞中的作用机制，更深入理解线粒体功能改变在足细胞损伤/修复中的作用，为寻找特异性保护足细胞方法提供新思路。

足细胞损伤是蛋白尿，特别是大量蛋白尿发生的细胞学基础。本项目中我们观察发现激活cAMP/PKA信号防止ADR或PAN诱导的足细胞数量减少凋亡，抑制ADR诱导的足细胞内线粒体缩短。体外实验显示ADR诱导足细胞线粒体变化与Drp1磷酸化增加和Mfn1表达降低相关，而相关变化可以被cAMP信号抑制。进一步进行体外实验显示pCPT-cAMP（PKA特异性激动剂）孵育足细胞导致细胞核内的磷酸化CREB表达上升，并防止 ADR诱导的被切割的半胱氨酸蛋白酶-3表达升高和足细胞的数量减少。RNA干扰（RNAi）可使得足细胞CREB蛋白表达下降，激动PKA信号失去了保护的作用。应用Agilent表达谱芯片检测小鼠足细胞全基因组表达发现，ADR刺激可使足细胞内线粒体基因编码的呼吸链复合体I各亚基基因表达明显下调，预孵育pCPT-cAMP可以防止ADR诱导的足细胞内呼吸链复合体I的ND1-5亚基的基因下调。激活cAMP/ PKA可防止ADR引起的线粒体呼吸链复合体I亚基ND1/3/4蛋白的表达下降。pCPT-cAMP预处理可以防止ND3，而非ND1/4蛋白的降低，这种作用在抑制足细胞内CREB蛋白表达之后消失。同时，CREB siRNA也可减弱pCPT-cAMP引起的足细胞ATP产生增加以及过氧化物酶体增殖活化受体 γ共激活因子-1α(PGC-1α)的表达上升。.我们也使用PKA信号激动剂治疗ADR肾病小鼠，发现PKA信号激动剂减轻8周时肾小球硬化。免疫荧光染色发现，pCPT-cAMP可显著防止ADR诱导足细胞特异性蛋白表达下降，降低足细胞损伤标志desmin 的表达。相比于ADR组小鼠，pCPT-cAMP治疗的小鼠壁层上皮细胞增殖显著减轻，抑制了ADR小鼠PECs的细胞活化，并促进了PECs向足细胞的分化。.这些结果提示足细胞mGluR1/cAMP主要通过下游的PKA/CREB信号途径，防止线粒体缩短，促进线粒体生物合成和ATP生成。其可能的机制是通过改善线粒体氧化磷酸化蛋白ND3的生成。而PKA信号也能够抑制ADR小鼠PECs的增殖和活化，促进PECs向足细胞分化。这些研究为进一步寻找特异性保护足细胞，改善足细胞损伤后的修复的措施提供了基础。