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

描述(由申请人提供)：囊壁细胞的异常增殖导致囊腔持续生长，被认为是ADPKD肾功能进行性丧失的基础。我们实验室使用PKD1和PKD2的同源模型发表的工作表明，巨噬细胞以异常高的数量进入肾脏，并在囊开始之前包围小管，并在不断增长的囊周围逐渐积聚。全动物去除巨噬细胞可抑制囊壁细胞的增殖，减缓囊壁生长，改善肾功能。我们对PC1和PC2缺失肾脏的初步RNAseq和qPCR数据显示，在无纤毛的PC1或PC2缺失肾脏中，单核细胞趋化物质Mcp1的表达增加了14-16倍，在无纤毛的PC1或PC2缺失肾脏中没有。这些结果使我们假设纤毛转导了Mcp1表达的刺激信号，而通常情况下，Mcp1表达被PC1表达抑制。因此，缺乏功能性PC1的细胞将被预测表达高水平的Mcp1，导致持续的巨噬细胞聚集。我们先前已经证明，激活的巨噬细胞可以刺激肾小管细胞的增殖。从多囊肾分离的巨噬细胞的流式细胞术、定量聚合酶链式反应和蛋白质组学数据表明，它们主要是交替激活的，并表达高水平的胰岛素样生长因子1(IGF1)。IGF1是一种主要的肾小管细胞增殖因子，它能与EGF协同作用，刺激上皮细胞增殖。EGF在人和啮齿动物ADPKD中上调。我们的目标是确定依赖PKD的单核细胞归巢/巨噬细胞激活和随后的囊泡生长的特异性通路，以便选择性地阻断这些事件，在不妨碍正常的巨噬细胞或肾小管功能的情况下减缓囊泡的生长。我们选择的方法也是为了定义这些通路中的关键调控步骤，这些步骤可以通过治疗靶向来防止这种病理反应。为了实现这一点，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阻断(S)来防止巨噬细胞归巢到囊性肾脏(SA1)。然后，我们将确定PC1零纤毛诱导Mcp1表达的细胞机制，并确定多囊蛋白通常如何抑制该信号(S)(SA 2)。最后，我们将确定巨噬细胞分泌的Igf1单独或与局部表达的EGF一起在体外促进pc1缺失的肾小管细胞增殖的作用。 并使用体内阻断Igf1信号来确定靶向减缓囊性生长的途径的治疗效果(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).