草酸钙肾结石核心区域基质蛋白质丰度变化与结石发生的相关性研究

Up to now, there was the lack of effective prevention measures for current and recurrent renal stones radically because that it was unknown of incidence and stone formation mechanism in renal stones. In spite of minimally invasive treatment making the great progress in therapy of renal stones by Extracorporeal Shock Wave Lithotripsy (ESWL) and endoscopes, however, the rate of incidence(1-10%) and recurrent (1 year 10%, 5 years 33% and 10 years 50-60%)was not declined. Calcium oxalate (CaOx) was the commonest constituent, in about over 80% of all renal stones. The accumulated evidences demonstrated that the renal tubular epithelium injured was essential to the adhesion of CaOx crystals to the membrane of renal tubular epithelium, which triggered the renal CaOx stone formation. The interaction between CaOx crystals and renal tubular epithelium, that is crystal-membrane interaction, played an important role in regulating the adhesion of crystals to cell membrane. So far, the knowledge of crystal-membrane interaction is limit. In the present study, our strategy was to employ the bioinformatics tools to explore the association between the known important pathways involved in crystal-membrane interaction and the abundance changes of stone matrix protein (SMP) within inner core of CaOx renal stones indicated by iTRAQ quantitative proteomics technology. On the basis of the predictive results above, the hypothesis that SMP within inner core of CaOx renal stone taking participate in regulating crystal-membrane interaction was proposed, which was addressed in human HK-2 renal tubular cell model of crystal-membrane interaction. Our study was hope to discovery the important proteins taking participate in regulating crystal-membrane interaction in the early stage of stone formation and explore their function. In conclusion, our study will provide the new inspiration and targets for further study to inhibit or control of occurrence, development and recurrence of renal CaOx stones.

肾结石病因和形成机理至今尚不明确，从根本上缺乏有效预防结石发生和复发的措施。尽管微创治疗（ESWL和腔镜）已取得巨大进展，但肾结石发病率和复发率仍居高不下。CaOx结石是最常见（≥80%）的肾结石。大量研究表明：CaOx晶体粘附到肾小管上皮细胞是CaOx结石形成的始动因素，而肾小管上皮细胞损伤是晶体粘附的中心环节。CaOx晶体与肾小管细胞的相互作用，即晶-膜相互作用是调控晶体粘附的最重要因素。目前，晶-膜相互作用研究仍处于起步阶段，很多分子机制不清楚。本项目中，我们拟采用生物信息学工具探讨CaOx结石核心区域蛋白丰度变化（iTRAQ定量）与已知的晶-膜相互作用主要信号通路或分子的相关性，提出工作假说，并在人肾小管上皮HK-2细胞晶-膜相互作用模型中验证，以期发现在结石形成起始阶参与晶-膜相互作用调控的重要蛋白质，并揭示其作用机制，为控制CaOx肾结石发生、发展和复发提供新的启示和研究靶点。

肾结石病因和形成机理不明确，从根本上缺乏有效预防结石发生和复发的措施。尽管肾结石的微创处理（ESWL和腔镜）已取得巨大进展，但肾结石发病率和复发率仍居高不下。CaOx结石是最常见（≥80%）的肾结石。大量研究表明：CaOx晶体粘附到肾小管上皮细胞是CaOx结石形成的始动因素，而肾小管上皮细胞损伤是晶体粘附的中心环节。CaOx晶体与肾小管细胞的相互作用，即晶-膜相互作用是调控晶体粘附的最重要因素。目前，晶-膜相互作用研究仍处于起步阶段，很多分子机制不清楚。本项目联合应用“尿液晶体法”和“结石基质分区法”两种新的结石基质研究策略，利用定量蛋白组学技术筛选129个与CaOx肾结石核心区域（蕴含大量结石形成起始阶段信息）高度相关的差异表达蛋白(含OPN，OPN是目前公认的参与CaOx形成最重要的分子)。生物信息学分析显示：这些差异蛋白与已知的晶-膜相互作用主要信号通路（p38 MAPK、ROS/OS）无明显相关性，而与钙粘蛋白介导的细胞间粘附作用关系密切。结合GEO数据库中人肾乳头Randall斑块定量蛋白组学数据，从上述差异表达蛋白中筛选出筛选出JUP、VSP35、MMP9、ILK、GAS6、Apoil1、UGDH、HPGD和CAPN1共9个最有价值的靶点验证。与生物信息学数据基本一致，在人肾小管上皮HK-2细胞晶-膜相互作用模型中，这些靶点在蛋白水平存在时间依赖性变化。推测它们很可能在肾结石形成起始阶段占有重要地位。目前，这些靶点在肾结石研究中鲜有报道。本研究不仅为进一步研究CaOx肾结石发生、发展和复发提供了新的启示和研究靶点，而且首次证实肾结石基质蛋白分区研究策略在结石发生机制的研究中的价值，为CaOx肾结石研究提供了新的线索和思路。