多囊肾组织微环境中囊肿衬里上皮细胞与巨噬细胞相互作用及对囊肿长大的影响

Autosomal dominant polycystic kidney disease (ADPKD) is the most common single gene inherited kidney disease. Unfortunately, so far its pathogenesis has not been clarified. Only 5% of nephrons with the same genetic background in the polycystic kidney tissues develop cysts by the microdissection, which suggests that the microenvironment of polycystic kidney tissues may play an important role in the development and growth of the renal cysts. The previous study found complement factor B (CFB) and component 3 (C3) were significantly increased, alternative complement pathway was aberrantly activated and abundant macrophages infiltrated around the renal cysts in the kidney tissues of ADPKD patients and Pkd1 conditional knockout mice, which are positively associated with the progression of ADPKD. Our further researches confirmed that M2-like macrophages in vitro and in vivo could remarkably stimulate cyst-lining epithelial cell proliferation and cyst growth. Based on the previous findings, we proposed a hypothesis that the interaction between cyst-lining epithelial cells and macrophages may promote cysts growth. The role and mechanism of CFB and C3 up-regulated in cyst-lining epithelial cells and Pkd1 knock-out mice kidney tissues on the recruitment and differentiation of macrophages will be investigated by western blot, cell co-culture technique and Pkd1 knock-out mice; the effect and mechanism of M2-like macrophages on cyst-lining epithelial cell proliferation and cyst growth will be further explored. The significance of our research will reveal the pathogenesis of the disease progression and provide a potential target for ADPKD intervention.

常染色体显性多囊肾病（ADPKD）是一种最常见的遗传性肾病，迄今发病机制未明。显微解剖表明只有5％具有相同遗传背景的肾单位发生囊肿，提示肾组织微环境与肾囊肿的形成及发展关系密切。先前发现ADPKD患者和Pkd1条件敲除鼠肾组织表达补体B因子和C3等显著增加，补体替代途径异常激活，伴随大量巨噬细胞在肾囊肿周围浸润，并与疾病进展相关。进一步研究表明，肾囊肿组织中M2型巨噬细胞可刺激囊肿衬里上皮细胞增殖及囊肿长大。本项目拟采用Western blot、体外细胞共培养及基因敲除鼠等技术及模型，研究囊肿衬里上皮细胞及Pkd1敲除鼠肾组织上调的补体B因子、C3等对巨噬细胞在肾组织募集及分化作用和机制；研究M2型巨噬细胞刺激囊肿衬里上皮细胞增殖及囊肿长大的作用和机制；从而证实多囊肾组织中细胞间相互作用促进囊肿长大假说，以进一步阐明ADPKD的发病机制，为寻找干预ADPKD新靶标提供新颖的理论及实验依据。

研究背景：常染色体显性多囊肾病（ADPKD）是人类最常见的单基因遗传性肾脏疾病，多种机制参与了ADPKD囊肿产生及生长，但目前哪种机制在该病发病中占主导地位尚存在争议，微环境为其机制研究提供了全新思路。在较早期研究中研究者已经发现巨噬细胞浸润在多囊肾间质中，且与囊肿生长关系密切，但是具体作用机制目前尚无研究。.方法与结果：在小鼠出生后第10天敲除体内PKD1基因造速发型ADPKD小鼠模型，在出生后第12，14，16，18，20，22，24，26，28，30，32天处死小鼠留取肾组织标本。用KI67免疫组化染色分析不同时间点小鼠肾脏增殖率变化，发现与野生型小鼠肾组织单峰增殖型不同，多囊肾小鼠肾脏呈双峰增殖。第一峰在出生后14天出现，与肾脏发育有关，在第16天后会回落至低谷点。而在第22天囊肿指数曲线会再次出现增高，与肾脏发育无关，呈持续性增高至死亡。利用基因芯片比较出生后22天和30天多囊肾小鼠肾脏差异表达基因，发现204个差异表达，其中包括ARG1。KEGG信号通路分析也提示代精氨酸谢通路在这一阶段呈现高度活化。这些结果提示ARG1在多囊肾囊肿生长过程中发挥重要作用。激光共聚焦发现ARG1主要表达在肾间质中巨噬细胞内，且随囊肿生长呈现逐渐增高表达，至出生后30天肾组织中多数巨噬细胞均高表达ARG1而呈现出M2型。清除此阶段巨噬细胞可以延缓多囊肾的进展，而靶向抑制ARG1的活性也可以有效抑制囊肿的生长。.结论：巨噬细胞和囊肿衬里上皮细胞相互作用构成的微环境变化参与了多囊肾进展的关键过程，也提供多种可能靶标抑制疾病的进展。