【作者】 黄炜；

【导师】 易凡； 孙玉；

【作者基本信息】 山东大学 ， 药理学， 2022， 博士

【摘要】 研究背景急性肾损伤是由缺血再灌注、肾毒性药物、脓毒血症、失血性休克等多种原因导致肾功能迅速下降的常见临床综合症,具有极高的发病率与死亡率,并最终发展成为慢性肾病与终末期肾病。缺血再灌注损伤是器官在经历短暂的血流减少或者中断后再灌注时所触发的炎症过程,肾缺血再灌注引起的急性肾损伤极易导致临床患者死亡,并且在肾移植中也会导致不良后果。而由于肾毒性药物在肾脏中的代谢引起的急性肾损伤限制了多种药物在临床治疗中的实际应用,如作为多种癌症的标准化学治疗方案的顺铂类药物。肾小管细胞死亡与炎症反应是缺血再灌注与顺铂引起的急性肾损伤的主要病理学特征。目前除了肾移植与肾透析之外,急性肾损伤尚缺乏有效治疗手段。急性肾损伤同时也是巨噬细胞、中性粒细胞、T细胞、B细胞等固有或适应性免疫细胞以及肾小管上皮细胞、血管内皮细胞等实质细胞共同参与的病理过程。在多种髓样炎症细胞中,巨噬细胞以其在肾脏炎症与损伤修复中的关键作用毫无疑问地成为极受关注的治疗靶点。炎症状态的M1型巨噬细胞被募集到肾脏后能够放大炎症反应并促进组织实质性损伤。与之相反的是,M2型巨噬细胞在急性肾损伤后展现出抗炎效应并促进损伤修复,并且这一作用在很大程度上与M2型巨噬细胞的胞葬作用相关。近年来,连接粘附型分子JAMs(junctional adhesion molecules)由于它们在免疫细胞活化与炎症反应中的重要功能而日益受到关注。而连接粘附型分子样蛋白JAML(junctional adhesion molecule-like protein)是其中新发现的成员,表达在多种固有以及适应性免疫细胞中,近期也有文章报道其在实质细胞中也发挥着重要作用。大量研究表明JAML能够调节炎症反应与组织修复,课题组前期研究也发现JAML在AKI小鼠肾脏的基因芯片结果中表达发生明显改变,提示JAML可能在急性肾损伤病理过程中发挥重要作用。因此深入探究JAML能否调控急性肾损伤及其具体作用机制对于寻找新的AKI治疗靶点具有重要的临床意义。研究目的1.明确JAML在急性肾损伤肾脏中的表达变化情况。2.探究巨噬细胞与肾脏实质细胞表达的JAML在急性肾损伤中的作用。3.对JAML调控急性肾损伤的具体分子机制与相关细胞功能进行深入探究,验证以其相关信号通路作为AKI治疗新靶点的可行性。研究内容与结果一、JAML在AKI患者及IRI小鼠肾脏中的表达我们使用免疫组化及荧光多重免疫组化对44例急性肾小管坏死患者肾活检组织以及7例接受肾切除术患者被切除肾脏组织健康端进行染色,结果表明,JAML在急性肾小管坏死患者肾小管细胞与肾脏间质巨噬细胞的表达量均明显上升。在小鼠肾IRI模型中,我们通过RT-PCR、WB、IHC、ELISA对缺血30分钟后再灌注24 h、48 h与72h的小鼠肾脏与血清样品进行检测,结果显示三个时间点的JAML表达均明显高于sham组小鼠。同时流式细胞术检测结果表明缺血再灌注损伤24 h后的肾脏中浸润巨噬细胞(CD11bhigh/F4/80low)与定居巨噬细胞(CD11blow/F4/80high)的JAML表达均明显升高,而中性粒细胞JAML变化并不明显。最后我们通过WB证明氧糖剥夺实验、AA/2-DG阻断呼吸链后复氧与CoCl2持续消耗培养基中氧气三种方式模拟的缺血再灌注损伤均能明显诱导JAML在NRK-52E中的表达。二、巨噬细胞与肾脏实质细胞JAML在IRI中发挥的作用为探究不同细胞表达的JAML在AKI中所发挥的具体作用,我们在这一部分逐步构建了JAML全身敲除小鼠、骨髓嵌合小鼠、髓样细胞特异性敲除JAML小鼠以及肾小管细胞特异性敲除JAML小鼠并进一步构建IRI模型。我们通过RT-PCR、WB、IHC以及流式细胞术对JAML敲除效率进行验证,结果表明我们成功构建了上述三种JAML敲除小鼠,骨髓移植实验也能成功替换嵌合小鼠体内80%左右的骨髓细胞。通过检测血清中肌酐与尿素氮含量以评价肾功能损伤;通过H&E、TUNEL与IF分别检测各组小鼠肾脏形态学损伤、肾脏细胞凋亡与肾损伤分子KIM-1的表达;通过IHC与RT-PCR检测肾脏中炎症细胞浸润与炎症因子表达。结果表明,全身缺失JAML与骨髓来源细胞特异性缺失JAML均能显著改善缺血再灌注诱导的肾脏损伤,而肾脏实质细胞缺失JAML只能发挥轻度改善所用。在体外实验中,我们通过RT-PCR对炎症因子的检测与流式细胞术对细胞死亡的检测分别发现JAML缺失能明显降低LPS诱导的骨髓来源巨噬细胞的炎症反应,但并不能减少氧糖剥夺实验引起的肾小管上皮细胞的死亡。三、JAML调控巨噬细胞表型极化与胞葬的作用及机制研究首先,在对WT与Jaml-/-小鼠IRI肾脏基因芯片的结果分析中,我们发现部分C型凝集素受JAML调控较为明显。并通过RT-PCR、WB、流式细胞术以及IHC逐步确认了肾脏巨噬细胞缺失JAML对巨噬细胞诱导型C型凝集素Mincle在IRI肾脏中表达的抑制作用最为明显。体外实验中,我们在BMDMs中进一步证实了 JAML缺失抑制Mincle表达并通过Mincle抑制下游效应分子Syk的磷酸化;同时也通过转染siRNA沉默NRK-52E的JAML后进行OGD实验证明了JAML在上皮细胞中的缺失抑制Mincle内源性配体SAP130的产生与分泌。接下来我们通过RT-PCR检测表型转换实验后巨噬细胞M2型标志物(Arg1、Ccl8)以及M1型标志物(Il6、iNos)的表达,发现JAML在BMDMs中的缺失促进其向M2型转换,同时抑制其向M1型转换,上述作用均由于Mincle过表达而被逆转。同时,流式细胞术对IRI肾脏中巨噬细胞M1型标志物(CD80、CD86)与M2型标志物(CD206)的检测结果表明,JAML缺失明显降低IRI肾脏中M1型巨噬细胞比例且增加M2型巨噬细胞比例。在探究JAML对巨噬细胞胞葬作用的调控中,所有实验均基于巨噬细胞对凋亡细胞的吞噬而设计。使用流式细胞术与荧光显微镜拍摄分别检测腹腔巨噬细胞对凋亡中性粒细胞的吞噬以及BMDMs对凋亡Jurkat细胞的吞噬,结果表明JAML缺失通过抑制Mincle表达而增加巨噬细胞对凋亡细胞的吞噬。为验证上述作用是否在AKI肾脏中也能发生,我们特异性过表达了Jawl-/-小鼠体内巨噬细胞的Mincle,并通过CD68与TUNEL试剂共染检测巨噬细胞对凋亡细胞的吞噬。结果表明,JAML缺失同样能够在AKI肾脏中通过抑制Mincle表达促进巨噬细胞对凋亡细胞吞噬。四、JAML在顺铂诱导的AKI中的作用及机制研究通过给与小鼠腹腔单次注射顺铂(30 mg/kg)诱导急性肾损伤。Western blot结果表明JAML在顺铂诱导的AKI小鼠肾脏中表达量随时间而明显增加。对各组小鼠血清检测结果显示JAML缺失明显减少血清中肌酐与尿素氮含量。H&E、IF与TUNEL染色结果分别表明腹腔注射顺铂能明显导致小鼠肾脏形态学损伤、KIM-1表达与肾脏细胞凋亡,但JAML缺失能明显抑制顺铂引起的肾脏损伤。通过RT-PCR与WB对WT与Jaml-/-小鼠肾脏中Mincle表达进行检测,结果表明,顺铂诱导WT小鼠肾脏中Mincle表达明显增加,而JAML缺失则能显著抑制其表达。IHC结果也表明注射顺铂的Jaml-/-小鼠肾脏中Mincle阳性细胞数量明显低于WT小鼠。研究结论1.本研究发现JAML在临床AKI样本与构建的小鼠AKI模型中表达量显著升高,为以JAML作为AKI临床诊断的生物学标志物提供了实验基础。2.本课题首次证明了 JAML全身缺失以及在巨噬细胞中特异性缺失均显著改善急性肾损伤,表明JAML具有作为新的急性肾损伤治疗靶点的可能性。3.本课题证明了 JAML缺失抑制了 Mincle表达并通过Mincle抑制下游效应分子Syk的磷酸化,进一步完善了急性肾损伤病理进程中的分子机制。4.本研究发现JAML缺失通过抑制Mincle表达促进巨噬细胞转化为M2型且抑制其转化为M1型,同时增强巨噬细胞胞葬作用,进而改善急性肾损伤。证明了以JAML为靶点干预巨噬细胞的功能进而作为AKI的生物学疗法的可行性。更多还原

【Abstract】 BackgroundAlthough macrophages are undoubtedly attractive therapeutic targets for acute kidney injury(AKI)because of their critical roles in renal inflammation and repair,the underlying mechanisms of macrophage phenotype switching and efferocytosis in the regulation of inflammatory responses during AKI are still largely unclear.The present study elucidated the role of junctional adhesion molecule-like protein(JAML)in the pathogenesis of AKI.We found that JAML was significantly upregulated in kidneys from 2 different murine AKI models including renal ischemia/reperfusion injury(IRI)and cisplatin-induced AKI.By generation of bone marrow chimeric mice,macrophage-specific and tubular cell-specific Jaml conditional knockout mice,we demonstrated JAML promoted AKI mainly via a macrophage-dependent mechanism and found that JAML-mediated macrophage phenotype polarization and efferocytosis is one of the critical signal transduction pathways linking inflammatory responses to AKI.Mechanistically,the effects of JAML on the regulation of macrophages were,at least in part,associated with a macrophage inducible C-type lectin-dependent mechanism.Collectively,our studies explore for the first time to our knowledge new biological functions of JAML in macrophages and conclude that JAML is an important mediator and biomarker of AKI.Pharmacological targeting of JAML-mediated signaling pathways at multiple levels may provide a novel therapeutic strategy for patients with AKI.Objectives1.Illustrate the expression pattern of JAML in kidneys of patients with ATN and mice with AKI.2.Explore the role of macrophage JAML and parenchymal JAML in the pathology process of AKI.3.Verify the underlying molecular mechanisms and related cellular functions of JAML regulating AKI,and the feasibility of related signaling pathways as a new target for AKI therapy.Methods and Results1.The expression of JAML in kidneys from patients with ATN and mice with IRIBy IHC and fluorescence multiplexed IHC analysis,we determined that JAML was upregulated in renal tubules and macrophages of kidneys from patients with biopsy-proven acute tubular necrosis(ATN).The results of RT-PCR,WB and IHC show that JAML expression was elevated in the kidney after 30 minutes of renal ischemia followed by different time points of reperfusion in mice.The serum level of JAML was also increased compared with controls.The flow cytometry results show that JAML was upregulated on both 2 subtypes macrophages(F4/80lowa nd F4/80high)in kidney with IRI.No appreciable changes of JAML expression were observed on neutrophils.Western blot results show that JAML was significantly upregulated in NRK-52E under mimical hypoxia conditions.2.The roles of macrophage JAML and parenchymal JAML in renal IRIIn this part,we gradually generated global Jaml-knockout mice(Jaml-/-),bone marrow chimeras,myeloid cell-specific Jaml-knockout mice(Lysm-Cre+/Jamlfl/fl)and tubular cell-specific Jaml-knockout mice(Ksp-Cre+Jamlfl/fl),which were confirmed by RT-PCR、WB、IHC and flow cytometry.The renal injury of these mice after IRI was assessed by the concentration of blood serum creatinine and blood urea nitrogen,the tubular damage score,the expression of KIM-1 and the death of renal cells.As the results show,the deficiency of JAML in myeloid cells significantly ameliorated renal IRI,whereas JAML in renal parenchymal cells only promotes AKI slightly.In vitro,JAML deficiency significantly reduced the inflammation of BMDMs treated with LPS but could not influence the death of NRK-52E induced by OGD,which were confirmed by RT-PCR and flow cytometry individually.3.The role and mechanism of JAML in regulating macrophage polarization and efferocytosisThe result of mcroarray analysis shows the expression of some C-type lectin receptors(CLRs)changed notablely in kidneys from Jaml-/-mice with renal IRI compared with WT mice.RT-PCR,Western blot,flow cytometry and IHC analyses gradually verified that the deficiency of JAML markedly attenuated IRI-induced macrophage Mincle expression in the kidney.In vitro,the mRNA and Western blot analyses verified that the deficiency of JAML in BMDMs significantly inhibited the expression of Mincle and the activation of Syk,which was reversed by Mincle overexpression.And we also demonstrated that the deficiency of JAML inhibited the secretion SAP130 from NRK-52E,which is one of endogenous ligands of Mincle.Next,we polarized BMDMs isolated from WT or Jaml-/-mice into M1 or M2 macrophages,before switching them back into M2 or M1 macrophages,respectively.In the absence of JAML,M1 macrophages that had been switched to M2 showed significantly enhanced expressions of M2-associated genes Argl and Ccl8,while Jaml-deficient M2 macrophages that had been switched to M1 showed lower expression of M1-related genes 116 and iNos.All these changes were counteracted by Mincle overexpression.We also detected the polarization status of 2 subtypes of macrophages(F4/80low and F4/80high)in the mouse kidneys by flow cytometry analysis.A significantly higher proportion of M2(CD206high)or lower proportion of M1(CD80high)macrophages was found in Jaml-/-IRI mice than in control mice.To evaluate the efferocytosis capacity of peritoneal macrophages in vivo,PKH26+apoptotic neutrophils were injected into the peritoneum of mice.45 minutes later,the peritoneal cells were collected and analyzed by flow cytometry.Uptake of the injected apoptotic neutrophils by F4/80+macrophages in Jaml-/-mice was higher than that in WT mice.In vitro,we administrated the PKH26-labeled apoptotic Jurkat cells to BMDMs and fluorescence microscopy was used to assess efferocytosis.Our data showed that macrophages from mice had significantly higher efferocytosis than those from WT mice,which was reversed by overexpression of Mincle.To further investigate JAML’s modulation of macrophage efferocytosis through Mincle during AKI,we double stained kidney sections in situ with TUNEL reagents and CD68 antibody.As expected,we found that the macrophage efferocytosis was markedly enhanced in the injured kidneys of JAML deficiency mice compared with control mice.These effects were significantly inhibited by adoptive transfer with Mincle overexpressed Jaml-deficient macrophages.4.The role and mechanism of JAML in AKI induced by cisplatinAKI in WT and Jaml-/-mice was induced by a single intraperitoneal injection of cisplatin at a dose of 30 mg/kg.At 3 days,5 days,and 10 days after injection,mice were sacrificed,serum and kidney samples were collected for various analyses.The results of Western blot shows that the expression of JAML was increased at different time points after injected cisplatin.Compared with controls,JAML deficiency ameliorated renal dysfunction,tubular injury,and cell death which were verified by the level detection of ceratinine and blood urea nitrogen in the serum,the results of H&E,IF and TUNEL to the kidney.RT-PCR,Western blot and IHC also verified Mincle was upregulated in the kidneys after injected cisplatin,and the deficiency of JAML significantly decreased expression of Mincle.Conclusion1.This study verified that JAML was significantly upregulated in kidneys of patients with ATN and mice with IRI or cisplatin injection.Which means that JAML could be used as a biomarker for the clinical diagnosis of AKI.2.We first demonstrated that global JAML deficiency or macrophage-specific JAML deficiency significantly ameliorated AKI.Which provided an experimental basis for JAML as a potential therapeutic target for AKI.3.The deficiency of JAML inhibited the expression of Mincle,and reduced the activation of Syk via a Mincle dependent mechanism during the progress of AKI.Which further illustrated the molecular mechanism in the pathological process of AKI.4.This study verified that the deficiency of JAML promoted macrophages switching to M2,inhibited switching to M1 and enhanced the efferocytosis of macrophages during the process of AKI via a Mincle dependent mechanism.The results demonstrated the feasibility for JAML as a target to intervene in the function of macrophages and thus treat AKI.更多还原