高表达KIM-1的肾小管上皮细胞调控M2巨噬细胞分化：急性肾损伤后慢性纤维化的新机制

M2 macrophages promote regenerative repair at the early phase of acute kidney injury (AKI), whereas their roles during the late repair phase and the fibrotic phase as well as their induction mechanisms remain unclarified. Our previous study has shown that in those AKI patients who developed chronic kidney disease, there were sustained infiltration of M2 macrophage and renal tubular KIM-1 overexpression in the biopsied kidney tissues, which were both correlated with the degree of interstitial fibrosis. Sustained hypoxia induced KIM-1 overexpression in cultured proximal tubular cells (PTCs), and the conditioned medium from these cells powerfully induced M2 macrophage differentiation. We then hypothesize that the sustained renal tubular KIM-1 expression triggers the chronic differentiation and infiltration of M2 macrophages in the late repair phase of AKI, which promotes kidney fibrosis. In our preliminary experiments, HNF3α was identified to mediate KIM-1 overexpression in PTCs, and GM-CSF from the tubular cell conditioned medium was found to play a critical role in M2 macrophage activation through the STAT5/ATF3 pathway. Next, we will perform AKI in mice with tamoxifen-induced conditional knockout target genes in renal tubules/macrophages, and take advantage of co-culture/3-D culture systems, to illustrate the roles of KIM-1 overexpressed PTCs in promoting M2 macrophage activation and the key modulating factors. Through this study, we will provide evidence and technological approaches for targeting M2 macrophages as novel therapeutic strategies in preventing the fibrosis progression after AKI.

M2巨噬细胞在AKI急性期可促进再生性修复，但在修复期和纤维化期的作用及分化调控机制不明。我们前期研究证实慢性转归的AKI患者肾组织中存在持续M2巨噬细胞浸润及肾小管KIM-1表达，二者均与纤维化程度正相关；持续缺氧可促进肾小管细胞表达KIM-1，而高表达KIM-1的肾小管细胞条件培养基可诱导M2巨噬细胞分化。据此我们设想：AKI后肾小管KIM-1持续表达通过旁分泌诱导M2巨噬细胞慢性活化，促发肾纤维化。本课题预实验发现HNF3α介导肾小管细胞缺氧下的KIM-1高表达，而GM-CSF是其旁分泌促进M2巨噬细胞分化的效应因子并通过STAT5/ATF3发挥作用。本课题拟应用他莫昔芬诱导的肾小管上皮和巨噬细胞条件性基因敲除小鼠制备AKI模型，结合细胞共培养和3D肾小管培养，阐明过表达KIM-1的肾小管细胞对M2巨噬细胞分化的影响及关键调控因子，揭示M2巨噬细胞在AKI后纤维化中的作用及调控机制。

急性肾损伤（AKI）患者约50%发生肾脏纤维化导致慢性肾功能损害，严重影响远期预后。本课题针对AKI后肾脏纤维化的进展机制和治疗靶标开展研究。我们采用单细胞转录组测序技术，绘制单侧缺血再灌注AKI自发肾纤维化小鼠模型在损伤期-修复期-纤维化期全过程中肾脏单核巨噬细胞亚型图谱并进行细胞溯源和功能分析。鉴定了4个肾脏固有巨噬细胞的亚型并解析其在AKI后炎症招募、抗原递呈、抗炎症和促修复中的功能异质性。进一步发现，在AKI进入修复期，促修复型固有巨噬细胞在损伤肾小管细胞释放的多种因子作用下，分化为CD38high巨噬细胞亚型，后者具有诱导肾小管细胞发生早衰和促进肾脏纤维化的效应。应用CD38特异性小分子抑制剂可以显著减轻单侧缺血再灌注AKI以及单侧尿路梗阻AKI 小鼠模型的肾小管早衰和肾脏纤维化程度。我们在临床AKI队列中亦证实CD38阳性巨噬细胞在肾组织中浸润以及与肾纤维化程度相关。进一步建立包括尿液巨噬细胞亚型分析在内的无创性肾脏炎症评估体系，可有效预测AKI发生、病情进展，以及辅助肾脏急性炎症损伤的定位诊断和活动度判断。以上研究成果为靶向巨噬细胞治疗的AKI临床转化研究提供有力证据和可行性策略。.课题进展过程中，全球爆发新冠疫情，课题组投入到抗疫防疫一线医疗工作，与此同时开展了新冠肺炎患者合并肾损伤的临床队列研究，揭示了不同病情程度新冠肺炎患者的肾损伤发生情况以及临床表型特征，解析了新冠肺炎患者发生器官损伤和重症结局事件的时间序列，发现新冠肺炎相关肾损伤是患者疾病进展及死亡的独立危险因素；进一步通过回顾性病例分析以及前瞻性队列研究，报告了新冠疫苗注射后新发肾脏病的疾病谱、肾脏病理特征、血清免疫学表型，以及治疗反应情况；分析了肾病患者注射新冠疫苗的安全性、免疫应答能力，以及免疫应答表现特征。系列研究结果为新冠肺炎患者的肾损伤防治以及新冠疫苗在肾病患者的安全有效应用提供了重要依据。