IDO+DCs与Tregs相互作用对AKI后出现“适应不良性修复”影响机制的研究

The G2/M arrest of tubular epeithlial cells is a key point of “maladaptive repair”. Our previous studies revealed that the expression of indoleamine 2,3-dioxygenase (IDO) in DCs is consistence with the increase of Mφ infiltration and T cells proliferation，which benefits to tissue restoration after renal IRI. The upregulation of IDO could inhibit the G2/M arrest of skin tissue and induce reapir. An important feature of the maladaptive repair process after severe injury is the development of cell cycle arrest in G2/M, which leads to DNA repair response by releasing profibrotic factors. Furthermore, studies show that IDO acts as a bridge between dendritic cells (DCs) and regulatory T cells (Tregs ). We hypothesized that the insufficient production of IDO would result in tubular epithelial cell cycle arrest at G2/M and promote subsequent downstream signaling of profibrotic factors, which lead to tubular atrophy and tubularintersitum fibrosis, eventually. Because of IDO acts as a bridge between DCs and regulatory Tregs, the of loss IDO production and Tregs proliferation might attribute to the abnormal cross-talk between IDO+DCs and Tregs. .To test this hypothesis, monocytes Ly6C+, naïve T cells and tubular epithelial cells will be isolated and co-cultured in vitro and IDO+DCs adoptive transfer will be used in vivo in this study to elucidate the molecular mechanism of IDO and cross-talk between IDO+DCs and Tregs in kidney maladaptive repair after AKI, and further to provide a novel clue for preventing AKI-CKD transition

AKI后，TECs在G2/M期阻滞是触发“适应不良性修复”的关键之一。我们研究表明，在AKI修复期，DCs细胞内NFκB非经典途径可促进IDO的表达、Mφ浸润和Tregs增殖，有利于肾脏修复。相关研究表明，IDO上调可抑制皮肤组织细胞G2/M期阻滞，促进修复；IDO缺乏则引起细胞G2/M期阻滞，促进纤维化因子产生和疤痕的形成。由于表达IDO的IDO+DCs与Tregs之间相互作用影响着IDO的产生，而IDO在DCs与Tregs之间充当桥梁作用。因此，我们提出科学假说：适应不良性修复是由于肾脏中IDO+DCs与Tregs相互作用异常、IDO生成不足、导致TECs在G2/M期阻滞、上调促纤维化分子表达而引起的。本课题将使用KO小鼠模型，采用细胞共培养，研究IDO+DCs与Tregs间相互作用对TECs在G2/M期阻滞、以及促纤维化因子的影响，阐明肾脏纤维化的机制。

吲哚胺-2，3-双加氧酶（IDO）是色氨酸是代谢的限速酶，其被报道与慢性肾脏病（CKD）相关，然而具体机制并不明确。本研究通过对肾脏缺血再灌注损伤（IRI）后肾脏单细胞数据分析发现集合管主细胞IDO1表达量在2d时达到高峰，后逐渐回落；远曲小管、成纤维细胞及髓袢细胞IDO1表达量在损伤6w时达到高峰。在此基础上，本研究构建了IDO-/-小鼠及“适应不良性修复”模型观察IDO对“适应不良性修复”模型小鼠纤维化水平的影响。结果发现：缺氧损伤可导致TECs中α-SMA的高表达，IDO+DCs可明显促进缺氧损伤后TECs中α-SMA的表达，而IDO-DCs组与对照组相比则无促TECs中α-SMA表达及激活Wnt/β-catenin的作用。IDO小鼠有正常的肾功能和肾脏形态。与假手术组相比，野生小鼠急性肾损伤后IDO表达增加。与野生小鼠相比，IDO-/-小鼠在IRI后14天肾功能及组织病理损伤减轻，马松染色提示IDO-/-小鼠纤维化程度较野生小鼠减轻，Western Blot显示纤维化指标α-SMA、fibronectin及vimentin表达降低。Wnt/β-catenin通路在野生小鼠急性肾损伤后表达增加，在IDO-/-小鼠表达降低。与生理盐水腹腔注射的野生急性肾损伤小鼠相比，前列腺素E2腹腔注射14天后，野生急性肾损伤小鼠肌酐水平更低，Western Blot显示IDO、α-SMA、fibronectin及vimentin表达更低，Wnt/β-catenin通路中GSK-3β及β-catenin表达减低。研究结果提示：IRI后IDO表达增加，通过激活Wnt/β-catenin通路诱导肾脏纤维化的发生；特异性敲除IDO表达，可抑制Wnt/β-catenin通路活性，改善肾脏纤维化。进一步，本研究进行了临床转化研究：IDO水平随CKD分期的增加而逐渐增加，IDO与eGFR负相关，是CKD的独立影响因素；同时，IDO对CKD具有良好的预测价值，其ROC面积为0.825(95% CI: 0.732 – 0.919, p< 0.001)，IDO的切点值为0.0466时具有83.8%的敏感性和75%的特异性；此外，IDO与ACR正相关，是CKD患者蛋白尿的独立影响因素，其ROC曲线下面积为0.753（95%CI: 0.68-0.826，p<0.01），IDO的最佳切点值为0.075