Jaml Promotes Acute Kidney Injury Mainly Through A Macrophage-dependent Mechanism

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.