Objective To investigate the role and potential mechanism of secreted phosphoprotein 1 (SPP1)⁺ macrophages in the formation of chronic renal allograft fibrosis. Methods The expression features of SPP1⁺ macrophages in renal allografts of chronic allograft dysfunction (CAD) patients were analyzed based on single-cell transcriptome data of renal tissues from patients with CAD. Transcription factor VIPER analysis and DoRothEA transcription factor activity analysis were performed on the single-cell transcriptome data. Renal tissue samples were collected from kidney transplant recipients, including the CAD group (n=5) and the non-renal allograft fibrosis group (CTL group, n=5). A mouse model of chronic allograft rejection was established and divided into the allogeneic kidney transplantation group (CAD group, n=3) and the syngeneic kidney transplantation group (SYN group, n=3). Hematoxylin-eosin staining was used to detect renal tissue injury in mice, and Masson staining was used to detect renal tissue fibrosis. Immunofluorescence staining was performed to detect SPP1 expression in renal tissues of transplant recipients and mouse renal allografts. Bone marrow-derived macrophages (BMDMs) were extracted from mice and subjected to hypoxia stimulation. The expression of hypoxia-inducible factor (HIF)-1α and SPP1 was detected by Western blot, and SPP1 expression was detected by flow cytometry. BMDMs were transfected with HIF-1α overexpression plasmid and HIF-1α small interfering RNA (siRNA) followed by hypoxia intervention, and the expression of HIF-1α and SPP1 was detected by Western blot. Mouse aortic endothelial cells (MAECs) were co-cultured with the supernatant of BMDMs, and the expression of endothelial-mesenchymal transition (EndMT)-related markers was detected by Western blot and immunofluorescence. Results Single-cell transcriptome analysis showed that the proportion of SPP1⁺ macrophages in renal allograft tissues was significantly higher in the CAD group than in the CTL group (P<0.05). The renal injury score and the percentage of interstitial fibrotic area in the CAD group were significantly higher than those in the SYN group (both P<0.05). Immunofluorescence staining showed that the proportion of SPP1⁺ macrophages was increased in the CAD group compared with the CTL group, and also increased in the CAD group compared with the SYN group (both P<0.05). VIPER analysis and DoRothEA transcription factor activity analysis revealed activation of the hypoxia pathway and upregulated expression of transcription factors such as HIF-1α in SPP1⁺ macrophages. SPP1 expression was elevated in BMDMs under hypoxic conditions. Knockdown of HIF-1α inhibited hypoxia-induced SPP1 protein expression, whereas overexpression of HIF-1α upregulated SPP1 protein levels. After co-culture of hypoxia-induced BMDMs with MAECs, the expression levels of EndMT-related markers were increased. Conclusions SPP1⁺ macrophages differentiated under hypoxia are significantly infiltrated in the formation of chronic renal allograft fibrosis, and may promote renal allograft fibrosis by inducing EndMT in renal vascular endothelial cells.

Kidney transplantation is the most effective treatment for end-stage renal failure, and antibody-mediated rejection remains the leading cause of late allograft loss. Macrophages, as central effectors of innate immunity, play a crucial role in the initiation, progression and tissue damage of antibody-mediated rejection. This article reviews the spatiotemporal dynamic evolution of macrophage polarization status in different stages of antibody-mediated rejection, the fine regulation of key signaling pathways for macrophage polarization, macrophage related molecules and the application prospects of targeted macrophage therapy. In depth analysis of the research progress of macrophages in antibody-mediated rejection, aiming to provide important theoretical basis for the development of precision diagnostic tools based on macrophages and novel immune intervention targets for antibody mediated rejection, ultimately promoting the improvement of long-term prognosis in kidney transplantation.

Objective:To explore the role of macrophage polarization on pericyte-to-myofibroblast transition and renal allograft fibrosis after kidney transplantation(KT).Methods:Allograft tissues were harvestedfrom recipients with chronic allograft dysfunction(CGD)and normal kidney tissues.The expression and distribution of M1/M2 macrophages in kidney tissues were detected by routine and immunofluorescent staining; mRNA of CD68, CD206 and iNOS detected by polymerase chain reaction(PCR); Murine vascular pericytes subjected to TGF-β1 in vitro and the expressions of α-SMA and PDGFR-β in perivascular cells detected by immunoblotting and cellular fluorescence; The co-culturing models of vascular pericytes and M1/M2 macrophages were constructed.The expressions of α-SMA and PDGFR-β in pericytes were detected by immunoblotting, cellular fluorescence and PCR.Results:A marked infiltration of CD68+ iNOS+ M1 macrophages was present in allograft tissues of recipients with CGD while no obvious infiltration of CD68 + CD206 + was observed.The mRNA levels of CD68, iNOS and CD206 were significantly higher in CGD group than those in control group( P<0.05); In CGD allograft tissues, protein expressions of α-SMA and PDGFR-β spiked markedly( P<0.05)while cells with double staining of α-SMA and PDGFR-β were markedly infiltrated in interstitial area of CGD allograft.TGF-β1 could induce a marked elevation of PMT-related markers in a time-dependent manner( P<0.05); Immunoblotting and cellukar fluorescence indicated that M1 macrophages could promote the elevations of α-SMA and PDGFR-β in pericytes in vitro while M2 macrophages showed no effect on pericyte-to-myofibroblast transition in pericytes. Conclusions:M1 macrophage polarization may promote the formation of renal allograft interstitial fibrosis through promoting PMT.