Targeting polycystin-dependent macrophage responses to slow cyst growth in ADPKD

描述（由申请人提供）：异常的囊肿内膜细胞增殖导致囊肿持续生长被认为是ADPKD患者肾功能进行性丧失的基础。我们实验室发表的使用PKD1和PKD2同源模型的研究表明，巨噬细胞以异常高的数量进入肾脏，并在囊肿形成之前包围小管，并逐渐积聚在正在生长的囊肿周围。全动物巨噬细胞耗竭可抑制囊壁细胞增殖，减缓囊肿生长，改善肾功能。我们来自PC1和PC2缺失肾脏的初步RNAseq和qPCR数据显示，在囊肿形成之前，单核细胞趋化剂Mcp1的表达增加了14-16倍，而非纤毛PC1或PC2缺失肾脏中没有这种表达。这些结果使我们假设纤毛介导Mcp1表达的刺激信号通常被PC1表达抑制。因此，预计缺乏功能性PC1的细胞会表达高水平的Mcp1，导致巨噬细胞持续积聚。我们之前已经证明，交替激活的巨噬细胞刺激小管细胞增殖。从多囊肾中分离的巨噬细胞的FACS、qPCR和蛋白质组学数据表明，它们主要是选择性激活的，并表达高水平的胰岛素样生长因子1 （Igf1）。Igf1是一种主要的小管细胞增殖因子，可以与Egf协同作用，在人和啮齿类动物的ADPKD中，Egf被上调，从而刺激上皮细胞增殖。我们的目标是确定PKD依赖性单核细胞归巢/巨噬细胞激活和随后的囊肿生长的特异性途径，以便选择性地中断这些事件并减缓囊肿生长，而不阻止正常的巨噬细胞或小管功能。我们选择的方法还旨在确定这些途径中的关键调控步骤，这些步骤可以靶向治疗以防止这种病理反应。为了实现这一目标，我们将首先确定ADPKD中Mcp1过度表达的来源，并在体外和体内使用该信号的中断来阻止巨噬细胞归巢到囊肾（SA 1）。然后，我们将定义PC1缺失纤毛诱导Mcp1表达的细胞机制，并确定多囊毒素通常如何抑制该信号（SA 2）。最后，我们将确定巨噬细胞分泌的Igf1在体外促进PC1无管细胞增殖中的作用，无论是单独的还是与局部表达的Egf协同作用

DESCRIPTION (provided by applicant): Abnormal cyst-lining cell proliferation causing relentless cyst growth is believed to underlie the progressive loss of renal function in ADPKD. Published work from our laboratory using orthologous models of PKD1 and PKD2 demonstrates that macrophages enter the kidney in abnormally high numbers and surround the tubules just prior to cyst initiation and progressively accumulate around growing cysts. Whole animal depletion of macrophages inhibits cyst-lining cell proliferation, slows cyst growth and improves renal function. Our preliminary RNAseq and qPCR data from both PC1 and PC2 null kidneys reveals 14-16 fold increased expression of the monocyte chemoattractant Mcp1 preceding cyst formation that is absent in non-ciliated PC1 or PC2 null kidneys. These results have led us to hypothesize that cilia transduce a stimulatory signal for Mcp1 expression that is normally suppressed by PC1 expression. Cells lacking functional PC1 would thus be predicted to express high levels of Mcp1 causing sustained macrophage accumulation. We have previously demonstrated that alternatively activated macrophages stimulate tubular cell proliferation. FACS, qPCR and proteomic data from macrophages isolated from polycystic kidneys demonstrates that they are predominately alternatively activated and express high levels of insulin-like growth factor 1 (Igf1). Igf1 is a major tubule cell proliferative factor that can act cooperatively with Egf, which is known to be upregulated in human and rodent ADPKD, to stimulate cell proliferation in epithelia. Our goal is to define the pathways that are specific to PKD- dependent monocyte homing/macrophage activation and subsequent cyst growth in order to selectively interrupt those events and slow cyst growth without preventing normal macrophage or tubular function. The approach that we have chosen is designed to also define key regulatory steps in those pathways that can be therapeutically targeted to prevent this pathologic response. To achieve this we will first identify the source of exaggerated Mcp1 expression in ADPKD and use in vitro and in vivo interruption of that signal(s) to prevent macrophage homing to cystic kidneys (SA 1). We will then define the cellular mechanism by which PC1 null cilia induce Mcp1 expression and determine how polycystins normally suppress that signal(s)(SA 2). Finally, we will identify the role of macrophage-secreted Igf1, either alone or in concert with locally expressed Egf, in promoting PC1 null tubular cell proliferation in vitro and use in vivo interruption of Igf1 signaling to determine the therapeutic utility of targeting ths pathway for slowing cyst growth (SA 3).