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，ARPKD是多囊肾病（PKD）的最严重形式。与局部C3产生的新的重要作用的发现所导致的范式转变一致，我们的研究指出肾内C3的产生是决定ARPKD进展速度的囊性途径的调节剂。我们基于我们对Cys 1cpk/cpk模型（其表型模仿ARPKD）中具有快速与缓慢囊性形成的肾脏的全基因组表达分析，制定了这一假设。我们通过证明：（i）ARPKD及其正态模型肾脏中生物活性裂解C3片段含量增加;（ii）肾囊肿和囊周细胞中存在C3 mRNA和蛋白;（iii）我们在C3缺陷（C3-/-）小鼠与Cys 1cpk/+小鼠杂交中验证的肾脏C3表达与肾囊肿发生速度之间的强相关性来支持这一假设。此外，在Pkhd 1 pck大鼠中，C3表达低的品系的基因渗入减弱了囊肿形成。虽然C3可能通过不同的途径起作用，但我们发现加速的囊肿形成与补体受体CR 3调节的途径特异性相关。CR 3（或Mac-1）是C3片段iC 3b的主要受体，其在囊性肾中高度丰富，在单核细胞/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CD 14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，巨噬细胞耗竭通过减少囊性小管的增殖来减弱常染色体显性PKD的正位模型中的囊性生成。CR 3还可诱导促囊生成性TNF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的基本和新效应一致，肾小管细胞产生和激活C3的能力以及由囊性缺陷引起的该过程的失调，我们认为ARPKD中的C3效应随着囊性小管扩张而增加，形成恶性囊性循环。 具体来说，我们假设C3途径激活通过CR 3依赖性过程加速ARPKD囊肿形成。我们在三个相互关联的目标中解决了这一假设：1）剖析C3的囊性效应的潜在机制; 2）确定Pkhd 1表达细胞产生的C3是否调节ARPKD的发病机制; 3）确定补体成分受体CR 3（或Mac 1）在肾囊肿形成中的作用。这些目标的目的将通过整合以下各项来实现：（i）探究肾囊肿发生的新型调控机制的高度创新的研究设计，（ii）产生新型最先进的试剂（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性途径与C3-CR 3联系起来，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.