AngII/miR-30s/calcineurin信号通路在足细胞损伤中的作用

Primary glomerular disease is the leading cause of the end-stage renal disease (ESRD) in China. Podocytes are terminally differentiated epithelial cells lining the outer aspect of the glomerular capillaries. Podocytes function as a major barrier to prevent protein passing from the intravascular glomerular capillaries to the extravascular urinary space. Several studies reported that the abnormal activation of circulating and local renin angiotensin system (RAS) promoted the process of podocyte injury, proteinuria and glomerular sclerosis. Angiotensin II (AngII), as a principal effector molecules of the RAS, has already been confirmed to contribute to the development of glomerular diseases. Lots of evidences demonstrated that the abnormal activation of calcineurin signaling may play an vital role in AngII-induced podocyte injury, but the specific mechanism is not clear. MicroRNAs are endogenous small non-coding RNAs, which caused translational repression and/or mRNA degradation with sequence complementarities in the 3´UTR of the target mRNA. In our previous study, we found that the expression of miR-30 family members (through miR-30a to miR-30e) were significantly decreased during podocyte injury. Furthermore, we found that TRPC6, PPP3CA, PPP3CB, PPP3R1, and NFATC3, as the key components of the calcium/calcineurin signaling pathway, were the targets of the miR-30 family. Through experiments, we verificated that miR-30s could repress calcium/calcineurin signaling during podocyte injury. Considering that AngII could induce the activation of calcineurin signaling, we hypothesized that AngII may downregulate miR-30s and then induce the calcium/calcineurin signaling activation which lead to podocyte injury and kidney diseases. To verify this, we first examined the effect of AngII treatment on cultured human podocytes. Our results showed that AngII could repress miR-30 family levels. MiR-30 overexpression obviously ameliorated AngII-activated calcium/calcineurin signaling. In addition, we also found that the angiotensin type 1 (AT1) antagonists losartan could reverse miR-30s levels which were downregulated by AngII. However, it is not clear whether the AngII/miR-30s/ calcineurin play an important role in vivo. In this proposed project, we will establish AngII-induced kidney injury model, podocyte specific miR-30s knockdown and miR-30acd overexpression transgenic mouse model. Through the use of dynabead perfusion and flow cytometry cell sorting, we obtain EGFP-labeled podocyte from transgenic mouse. We also use FITC-inulin clearance measurements to evaluate the glomerular filtration rate. By using in vivo and in vitro experiments, we could deeply explore the role of AngII/miR-30s/calcineurin signaling during podocytes injury. This project will highlight the mechanism of AngII caused podocyte injury and provide theoretical basis for the treatment of glomerular diseases.

足细胞损伤是肾小球疾病发生和发展的关键环节，肾素-血管紧张素系统（RAS）异常活化在足细胞损伤中发挥重要作用。钙调磷酸酶（calcineurin）信号异常活化与血管紧张素（AngII）所致足细胞损伤密切相关，但其具体机制尚不明确。我们前期研究证实miR-30家族可通过靶向调控calcineurin信号通路关键组分维持足细胞稳态，并在细胞层面证实AngII可下调miR-30s从而激活calcineurin信号通路，而AngII受体拮抗剂（ARB）能够阻断AngII所致miR-30s下调。据此我们提出研究假说：AngII/miR-30s/calcineurin在足细胞损伤中发挥重要调控作用。本研究将利用一系列体内外模型深入阐明AngII异常表达所致足细胞损伤的分子机制，为发现新的蛋白尿治疗干预靶点提供理论依据。

原发性肾小球疾病是我国导致终末期肾脏疾病（ESRD）的首要因素，足细胞损伤是肾小球疾病发生和发展过程中的关键环节。肾素-血管紧张素系统（RAS）异常活化在足细胞损伤，蛋白尿产生及肾小球硬化过程中发挥重要作用。研究表明足细胞钙调磷酸酶（calcineurin）信号异常活化与血管紧张素（AngII）所致肾脏损伤密切相关，但其具体分子机制尚不明确。本课题组前期研究证实miR-30s在肾小球足细胞中含量丰富，并具有足细胞保护作用。miR-30s可作用于其靶基因钙通道蛋白TRPC6、calcineurin（PPP3CA、PPP3CB、PPP3R1）及下游转录因子NFATC3，调控calcineurin信号通路参与足细胞损伤过程。基于以上背景提出本研究假说: AngII可能通过下调miR-30s从而激活calcineurin信号诱导足细胞损伤发生。为验证该假说，本研究给予小鼠慢性输注AngII,发现造模小鼠出现血压升高、蛋白尿增加及足细胞损伤表型，提示AngII所致慢性肾损伤小鼠模型构建成功，同时检测到足细胞miR-30s水平下降和calcineurin信号通路核心分子（TRPC6、PPP3CA、PPP3CB、PPP3R1和NFATC3）表达上调。给予miR-30a慢病毒尾静脉注射或AngII受体拮抗剂氯沙坦治疗均能缓解AngII诱导的小鼠蛋白尿、肾小球系膜增宽、足突融合等损伤表型，并抑制AngII所致的calcineurin信号通路核心组分表达上调。进一步通过体外实验证实氯沙坦处理能够部分阻断AngII诱导的miR-30s水平下调及calcineurin信号异常活化，提示氯沙坦的足细胞保护作用是由miR-30s/calcineurin通路介导。综上，本研究聚焦疾病状态下足细胞损伤的分子机制，通过研究AngII对miR-30s/calcineurin 信号通路的调控作用揭示了AngII异常高表达所致肾损伤的机制，并通过体内外实验阐明AngII受体拮抗剂（ARB）对足细胞的直接保护作用，为寻找足细胞损伤和蛋白尿干预靶点提供了理论依据。