植烷酸氧化代谢异常导致原纤毛形成缺陷和肾囊肿发生的分子机制研究

Primary cilia have been found to play important roles in renal cystogenesis. In our preliminary study, we were surprised to find the primary cilia defect in skin fibroblast of a patient with Refsum’s disease, which is characterized by phytanic acid а-oxidation impairment and accumulation of phytanic acid in the body, and also presented with bilateral renal medullary cysts and renal failure. The ciliogenesis defect was also validated in a patient with α- Methylacyl CoA racemase (AMACR) deficiency disease, which is another type of phytanic acid oxidation impairment disease with accumulation of pristanic acid. Our finding suggested the novel link between phytanic acid oxidation pathway and ciliogenesis, the mechanism of which may be due to long chain fatty acid (palmitic acid and stearic acid) deficiency secondary to phytanic acid and pristanic acid accumulation, which further affect palmitoylation of ciliary proteins. In this grant proposal, we plan to verify the above hypothesis by finding the cystic protein/proteins with palmitoylation defect in the skin fibroblast of Refsum diease patient by Acyl-biotinyl exchange (ABE) method followed by LC-MS analysis. Palmitoylation of the cystic protein found by ABE and also its ciliary localization in Refsum’s disease patient fibroblast will further be observed by transfection of WT and palmitoylated site point mutation construct of the above ciliary protein/proteins into both patient and control skin fibroblasts. For in vivo study, we plan to make zebrafish model of Refsum’s disease by knocking out PHYH gene using CRISPR-CAS9 system. The ciliopathy phenotype such as pronephros cysts will be observed and ciliogenesis as well. The aim of this study is to provide novel insights on the mechanism of ciliogenesis and renal cystogenesis, and to find potential novel therapeutic target of ciliopathy and renal cystogenesis.

肾囊肿病蛋白在原纤毛定位和肾囊肿病可发生原纤毛缺陷的发现，提示原纤毛与肾囊肿病关系密切。在前期研究中我们首次在合并肾囊肿的植烷酸贮积病患者中发现原纤毛形成缺陷和棕榈酸缺乏。由此推测：植烷酸代谢异常可导致原纤毛形成缺陷和肾囊肿发生，其分子机制与植烷酸代谢障碍时肾囊肿病蛋白发生棕榈酰化修饰缺乏继发原纤毛定位障碍有关。为验证该推测，本项目拟运用酰基生物素置换法鉴定植烷酸贮积病患者皮肤成纤维细胞中棕榈酰化修饰缺乏的肾囊肿病蛋白，进而通过体外过表达实验证实该肾囊肿蛋白在植烷酸代谢异常时发生原纤毛定位障碍；并进一步证实PHYH基因体外回补可纠正植烷酸贮积病中原纤毛形成缺陷和肾囊肿病蛋白的榈酰化修饰缺乏以及原纤毛定位障碍。同时通过构建斑马鱼疾病模型观察有无纤毛病表型，从体内水平验证植烷酸代谢异常对原纤毛形成缺陷和肾囊肿发生的影响。本项目旨在揭示原纤毛形成缺陷和肾囊肿发生的机制，为肾囊肿病治疗提供新思路。

原纤毛是体内重要的亚细胞器，原纤毛形成或功能障碍可导致20余种已知单基因遗传肾脏纤毛病。积极探讨导致原纤毛形成或功能缺陷的分子机制，对肾脏纤毛病的发病机制和防治研究具有重要意义。本课题运用全基因组连锁分析联合外显子组测序、cre/loxp系统、siRNA干扰、免疫荧光/组化、质谱分析、扫描电镜等技术，在肾脏纤毛病患者、高植醇饮食诱导的PHYH 敲除（PHYH-/-）小鼠（植烷酸贮积病小鼠模型）、PHYH敲低肾小管上皮细胞系等多层次的研究模型中探讨了植烷酸氧化代谢通路异常在原纤毛缺陷中的作用，进一步将植烷酸代谢异常与原纤毛缺陷联系在一起，研究结果为原纤毛缺陷发病机制研究提供了新的思路和潜在的干预靶点。.1.本课题立足于前期发现的过氧化物酶体病植烷酸贮积病的致病基因植烷酸辅酶A羟化酶（PHYH）纯合突变可导致肾脏纤毛病，运用全基因组连锁分析联合外显子组测序技术在一个病因未名、疑似常染色体隐性遗传纤毛病（肾衰竭、肥胖、共济失调、眼底黄斑病）近亲家系的一对患病兄妹中首次发现，位于PHYH基因下游的2-羟基植烷酸辅酶A裂解酶（HACL1）纯合无义突变（c.1049G>A;W349Xp.W349X)为致病基因。经患者皮肤成纤维细胞原纤毛染色证实原纤毛在长度及密度上均较正常对照显著减少。PHYH基因与HACL1基因突变均可导致肾脏纤毛病这一重要结果表明，植烷酸氧化代谢通路在原纤毛发生中可能具有重要作用。.2.运用cre/loxp系统构建了PHYH-/-小鼠模型（国内首次），随后运用高植醇饮食成功诱导了植烷酸贮积病小鼠模型。我们发现PHYH-/-小鼠肾脏发育异常（较WT小鼠显著缩小）。经高植醇饮食诱导后 PHYH-/-小鼠出现了植烷酸贮积病表型（营养不良、肝内脂肪淤积、共济失调等）及脂肪酸代谢异常（高植烷酸及长链脂肪酸缺乏）。经免疫荧光及电镜证实肾脏原纤毛生长发生缺陷。进一步经免疫组化、realtime PCR、western blot等实验发现植烷酸贮积病小鼠肾内存在PPAR 活化及自噬激活。体外研究证实，植烷酸刺激可导致PHYH敲低肾小管上皮细胞系原纤毛生长缺血，且呈剂量依赖性。