Mechanisms of C3 effects in ARPKD pathogenesis

描述（由申请人提供）：本资助申请涉及补体成分3 (C3)，补体途径的轴向成分，在常染色体隐性遗传性多囊肾病（ARPKD）发病机制中的作用，这是多囊肾病（PKD）最严重的形式。由于局部C3生成的新重要作用的发现而导致的范式转变，我们的研究指出，肾内C3的生成是决定ARPKD进展速度的囊生成途径的调节剂。我们基于对表现ARPKD的Cys1cpk/cpk模型中膀胱形成速度快与慢的肾脏的全基因组表达分析，提出了这一假设。我们支持这一假设，证明：(i) ARPKD及其同源模型肾脏中生物活性分裂的C3片段含量增加；（ii）肾囊细胞和囊周细胞中存在C3 mRNA和蛋白；（iii）我们在C3缺陷（C3-/-）小鼠与Cys1cpk/+小鼠的杂交中验证了肾C3表达与肾囊形成速度之间的强相关性。此外，Pkhd1pck大鼠渗入到低C3表达的品系后，囊发生减弱。虽然C3可能通过不同的途径起作用，但我们发现加速膀胱形成与补体受体CR3调节的途径有一致的联系。CR3（或Mac-1）是C3片段iC3b的主要受体，在囊肾中含量丰富，在单核/巨噬细胞的分化、附着和存活中起核心作用。虽然我们已经确定了C3、巨噬细胞标志物CD14和C3诱导因子MCP-1作为PKD结果的候选预测因子，但其他人已经证明巨噬细胞消耗通过减少囊小管的增殖来减弱常染色体显性PKD的同源模型中的囊发生。CR3也可能诱导前囊性肿瘤坏死因子释放并直接激活肾小管细胞中的c-Src。与局部补体因子产生、肾小管细胞产生和激活C3的能力以及这一过程因囊性缺陷而失调的基本和新作用一致，我们认为C3在ARPKD中的作用随着囊性小管扩张而增加，形成一个恶性囊性循环。具体来说，我们假设C3通路激活通过CR3依赖过程加速ARPKD中囊肿的形成。我们在三个相互关联的目标中提出了这一假设：1)剖析C3致囊作用的机制；2)确定表达ppkd1的细胞产生C3是否调节ARPKD的发病机制；3)确定补体成分受体CR3（或Mac1）在肾膀胱发生中的作用。这些目标将通过整合：(i)高度创新的研究设计，探究肾囊形成的新调节机制；（ii）新一代最先进的试剂（例如，用于条件C3靶向）。实现这些目标将：(i)通过将已建立的和新的囊生成途径与C3-CR3联系起来，从而整合现有知识；（ii）为ARPKD研究提供新的方向，可能导致新的预后和治疗策略的发展。

DESCRIPTION (provided by applicant): This grant application addresses the role of complement component 3 (C3), an axial component of complement pathway, in the pathogenesis of autosomal recessive polycystic kidneys disease (ARPKD), the most severe form of polycystic kidney disease (PKD). In line with paradigm shifts resulting from discoveries of novel essential roles of local C3 production, our studies point to intra-renal production of C3 as a regulator of a cystogenic pathway that dictates the pace of ARPKD progression. We formulated this hypothesis based on our genome-wide expression analyses of kidneys with rapid verses slow pace of cystogenesis in Cys1cpk/cpk model which phenocopies ARPKD. We supported this hypothesis by demonstrating: (i) increased content of biologically active split C3 fragments in kidneys from ARPKD and its orthologous model; (ii) presence of C3 mRNA and protein in renal cystic and pericystic cells; and (iii) strong association between renal C3 expression and pace of renal cystogenesis that we validated in crosses of C3 deficient (C3-/-) mice with Cys1cpk/+ mice. In addition, cystogenesis is attenuated in Pkhd1pck rats' introgressed to a strain with low C3 expression. While C3 may act through different pathways, we have found consistent association of accelerated cystogenesis specifically with the pathway regulated by complement receptor CR3. CR3 (or Mac-1), a major receptor for C3 fragment iC3b, which is highly abundant in cystic kidneys, plays a central role in differentiation, attachment and survival of monocytes/macrophages. While we have identified C3, macrophage marker CD14 and C3-inducible factor MCP-1 as candidate predictors of PKD outcomes, others have demonstrated that macrophage depletion attenuates cystogenesis in orthologous models of autosomal dominant PKD by reducing the proliferation of cystic tubules. CR3 may also induce pro-cystogenic TNF release and directly activate c-Src in renal tubule cells. Consistent with essential and novel effects of local complement factor production, the capacity of renal tubule cells to produce and activate C3 and dysregulation of this process by a cystogenic defect, we suggest that the C3 effects in ARPKD increase as cystic tubules dilate, forming a vicious cystogenic cycle. Specifically, we hypothesize that C3 pathway activation accelerates cyst formation in ARPKD through a CR3 dependent process. We address this hypothesis in three inter-related aims: 1) Dissect mechanisms underlying cystogenic effects of C3; 2) Determine whether C3 production by Pkhd1-expressing cells modulates ARPKD pathogenesis; and 3) Determine the role of complement component receptor CR3 (or Mac1) in renal cystogenesis. Objectives of these Aims will be accomplished by integrating: (i) highly innovative study design of interrogating novel regulatory mechanisms of renal cystogenesis with (ii) generation of novel state of the art reagents (e.g., for conditional C3 targeting). Achieving these aims will: (i) allo integration of existing knowledge by linking established and novel cystogenic pathways to the C3-CR3 nexus, and (ii) provide a new direction in ARPKD research that may lead to development of novel prognostic and therapeutic strategies.