Mechanisms of C3 effects in ARPKD pathogenesis

描述(申请人提供)：这项资助申请涉及补体成分3(C3)，补体途径的轴成分，在常染色体隐性遗传性多囊肾病(ARPKD)的发病机制中的作用，ARPKD是最严重的多囊肾病(PKD)形式。随着局部C3产生的新的基本作用的发现而导致的范式转变，我们的研究指出，肾内C3的产生是决定ARPKD进展速度的膀胱形成途径的调节因素。我们提出这一假设是基于我们对表现为ARPKD的Cys1cpk/CPK模型中的肾脏进行的快速与缓慢的囊变的全基因组表达分析。我们通过证明：(I)ARPKD及其同源模型的肾脏中生物活性的分裂C3片段的含量增加；(Ii)肾囊和囊周细胞中存在C3mRNA和蛋白；以及(Iii)我们在C3缺陷(C3-/-)小鼠与Cys1cpk/+小鼠的杂交中证实了肾脏C3表达与肾脏囊变速度之间的强烈关联，从而支持了这一假设。此外，将Pkhd1pck大鼠导入到低C3表达的品系中，其囊性发生被减弱。虽然C3可能通过不同的途径发挥作用，但我们发现加速囊变与补体受体CR3调节的途径有一致的关联。CR3(或Mac-1)是C3片段iC3b的主要受体，在囊性肾脏中含量丰富，在单核/巨噬细胞的分化、附着和存活中起着核心作用。虽然我们已经确定C3、巨噬细胞标记物CD14和C3诱导因子MCP-1是PKD预后的候选预测因子，但其他研究表明，巨噬细胞耗竭通过减少囊性小管的增殖来减少常染色体显性PKD直系模型的囊变。CR3还可诱导促囊性肿瘤坏死因子的释放，并直接激活肾小管上皮细胞的c-Src。与局部补体因子的产生、肾小管细胞产生和激活补体C3的能力以及由致囊性缺陷引起的这一过程的失调相一致，我们认为ARPKD中的C3效应随着囊性小管的扩张而增加，形成了一个恶性循环。具体地说，我们假设C3途径的激活通过CR3依赖的过程加速ARPKD的包囊形成。我们在三个相互关联的目标中提出这一假说：1)剖析C3致囊作用的潜在机制；2)确定表达Pkhd1的细胞产生C3是否调节ARPKD的发病；3)确定补体成分受体CR3(或MAC1)在肾囊变中的作用。这些目标的目标将通过整合：(I)询问肾脏囊变的新调控机制的高度创新的研究设计与(Ii)新技术试剂的产生(例如，用于条件C3靶向)来实现。实现这些目标将：(I)通过将已建立的和新的致囊途径连接到C3-CR3联系来整合现有知识，以及(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开发。

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.