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

描述（由申请方提供）：导致囊肿持续生长的异常囊肿衬里细胞增殖被认为是ADPKD肾功能进行性丧失的基础。我们实验室使用PKD 1和PKD 2的正位模型发表的工作表明，巨噬细胞以异常高的数量进入肾脏，并在囊肿开始之前包围肾小管，并在生长的囊肿周围逐渐积聚。全动物巨噬细胞耗竭抑制囊肿衬里细胞增殖，减缓囊肿生长并改善肾功能。我们来自PC 1和PC 2缺失肾的初步RNAseq和qPCR数据揭示了在囊肿形成之前单核细胞趋化因子Mcp 1的表达增加14-16倍，这在无纤毛的PC 1或PC 2缺失肾中是不存在的。这些结果使我们假设纤毛对Mcp 1表达具有刺激信号，而Mcp 1表达通常被PC 1表达抑制。因此，预测缺乏功能性PC 1的细胞表达高水平的Mcp 1，导致持续的巨噬细胞积聚。我们以前已经证明，交替激活的巨噬细胞刺激肾小管细胞增殖。从多囊肾分离的巨噬细胞的FACS、qPCR和蛋白质组学数据表明，它们主要是交替活化的，并表达高水平的胰岛素样生长因子1（Igf 1）。Igf 1是一种主要的小管细胞增殖因子，可与已知在人类和啮齿动物ADPKD中上调的Egf协同作用，以刺激上皮细胞增殖。我们的目标是确定PKD依赖性单核细胞归巢/巨噬细胞活化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肾小管功能。我们所选择的方法也被设计为定义那些通路中的关键调节步骤，这些通路可以被治疗性地靶向以防止这种病理反应。为了实现这一点，我们将首先确定ADPKD中Mcp 1过度表达的来源，并在体外和体内中断该信号以防止巨噬细胞归巢到囊性肾（SA 1）。然后，我们将定义PC 1无效纤毛诱导Mcp 1表达的细胞机制，并确定多囊蛋白通常如何抑制该信号（SA 2）。最后，我们将鉴定巨噬细胞分泌的Igf 1单独或与局部表达的Egf共同促进体外PC 1缺失肾小管细胞增殖的作用 并使用Igf 1信号传导的体内中断来确定靶向该途径用于减缓囊肿生长的治疗效用（SA 3）。

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).