Myocardin-related transcription factor (MRTF) mediates epithelial fibrogenesis in polycystic kidney disease

Background: Autosomal dominant polycystic kidney disease (PKD) is characterized by extensive cyst formation and progressive fibrosis. However, the molecular mechanisms whereby the loss/loss-of-function of polycystin 1 or 2 (PC1/2) provokes fibrosis are largely unknown. The small GTPase RhoA has been implicated in cystogenesis, while we have shown that the RhoA/cytoskeleton/myocardin-related transcription factor (MRTF) pathway is a key inducer of epithelium-induced fibrogenesis. Therefore, we hypothesized that MRTF 1) is activated by PC1/2 loss, and 2) plays a critical role in fibrogenic repogramming of the epithelium and the subsequent induction of fibrosis. Methods: PC1/2 loss was achieved by gene silencing in tubular cells (in vitro) and in PKD1 (RC/RC) and PKD2 (ws25/-) mice (in vivo). RhoA activation was measured by pull-down assays and immunofluorescence. MRTF localization (nuclear/cytosolic ratio, intensity) was quantified in large cell populations and in renal tissue using automated image analysis. PC1/PC2 loss-promoted, MRTF-dependent gene expression was followed by qPCR, RNAseq and RNAscope. MRTF-dependent paracrine effects were assessed by a bioassay. Results: Loss of PC1 or PC2 activated RhoA in vitro and in vivo, resulting in cytoskeletal remodelling and robust nuclear MRTF translocation, accompanied by increased MRTF expression. Nuclear translocation occurred predominantly in dilated tubules, while overexpression in the cyst-lining epithelium. A large cohort of PC1/PC2 downregulation-induced genes was MRTF-dependent, including cytoskeletal, integrin-related, and matricellular/fibrogenic proteins. Epithelial MRTF was necessary for paracrine priming of fibroblast-myofibroblast transition. Conclusion: The Rho/MRTF pathway is a critical novel mediator of PC1/2 loss-induced acquisition of the profibrotic epithelial phenotype and the ensuing fibrosis. Overall design: The porcine proximal tubule cell line LLC-PK1 was subjected to treatment with siRNAs (siPC1, siPC2, and in combination with siMRTFA) along with a non-related control siRNA (siNR). This was carried out to assess the impact of MRTFA in the context of polysystic kidney disease, particularly when the Polycystin (PC1 or PC2) gene is downregulated.

背景：常染色体显性遗传性多囊肾病（Autosomal dominant polycystic kidney disease, PKD）以广泛囊肿形成和进行性纤维化为特征。然而，多囊蛋白1或2（polycystin 1 or 2, PC1/2）功能缺失或功能丧失诱发纤维化的分子机制尚未完全阐明。此前研究表明小GTP酶RhoA（small GTPase RhoA）参与囊肿发生，而本团队已证实RhoA/细胞骨架/心肌素相关转录因子（myocardin-related transcription factor, MRTF）通路是上皮细胞诱导纤维化发生的关键诱导因子。因此，本研究提出如下假说：1）MRTF可因PC1/2功能缺失而被激活；2）MRTF在上皮细胞致纤维化表型重编程及后续纤维化诱导过程中发挥关键作用。
方法：通过基因沉默技术在肾小管细胞（体外实验）以及PKD1基因敲除（RC/RC）和PKD2基因敲除（ws25/-）小鼠（体内实验）中实现PC1/2功能缺失。采用蛋白下拉实验（pull-down assays）与免疫荧光（immunofluorescence）检测RhoA激活状态。通过自动化图像分析，在大量细胞群体及肾组织中定量分析MRTF的定位情况（核/胞质比值及荧光强度）。通过实时定量PCR（qPCR）、RNA测序（RNAseq）及RNAscope技术检测PC1/2功能缺失诱导的、依赖于MRTF的基因表达变化。采用生物测定法评估依赖于MRTF的旁分泌效应。
结果：在体外及体内实验中，PC1或PC2功能缺失均可激活RhoA，引发细胞骨架重构及显著的MRTF核转位，同时伴随MRTF表达水平升高。核转位主要发生在扩张的肾小管中，而MRTF在囊肿衬里上皮细胞中表达上调。PC1/2表达下调诱导的大量基因依赖于MRTF，其中包括细胞骨架蛋白、整合素相关蛋白及细胞外基质/致纤维化蛋白。上皮细胞MRTF是成纤维细胞-肌成纤维细胞转化的旁分泌启动过程所必需的。
结论：Rho/MRTF通路是介导PC1/2功能缺失诱导上皮细胞获得致纤维化表型并最终引发纤维化的关键新型介质。
整体实验设计：将猪近端肾小管细胞系LLC-PK1分别用小干扰RNA（siPC1、siPC2以及二者联合siMRTFA）以及无关对照小干扰RNA（siNR）进行处理，以评估在多囊肾病背景下，尤其是多囊蛋白（PC1或PC2）基因表达下调时，MRTFA所发挥的作用。