Preventative effect of TSPO ligands on mixed antibody-mediated rejection through a Mitochondria-mediated metabolic disorder.

BACKGROUND: Immune-mediated rejection was the major cause of graft dysfunction. Although the advances in immunosuppressive agents have markedly reduced the incidence of T-cell-mediated rejection after transplantation. However, the incidence of antibody-mediated rejection (AMR) remains high. Donor-specific antibodies (DSAs) were considered the major mediators of allograft loss. Previously, we showed that treatment with 18-kDa translocator protein (TSPO) ligands inhibited the differentiation and effector functions of T cells and reduced the rejection observed after allogeneic skin transplantation in mice. This study we further investigate the effect of TSPO ligands on B cells and DSAs production in the recipients of mixed-AMR model. METHODS: In vitro, we explored the effect of treatment with TSPO ligands on the activation, proliferation, and antibody production of B cells. Further, we established a heart-transplantation mixed-AMR model in rats. This model was treated with the TSPO ligands, FGIN1-27 or Ro5-4864, to investigate the role of ligands in preventing transplant rejection and DSAs production in vivo. As TSPO was the mitochondrial membrane transporters, we then investigated the TSPO ligands effect on mitochondrial-related metabolic ability of B cells as well as expression of downstream proteins. RESULTS: In vitro studies, treatment with TSPO ligands inhibited the differentiation of B cells into CD138+CD27+ plasma cells; reduced antibodies, IgG and IgM, secretion of B cells; and suppressed the B cell activation and proliferation. In the mixed-AMR rat model, treatment with FGIN1-27 or Ro5-4864 attenuated DSA-mediated cardiac-allograft injury, prolonged graft survival, and reduced the numbers of B cells, including IgG+ secreting B cells, T cells and macrophages infiltrating in grafts. For the further mechanism exploration, treatment with TSPO ligands inhibited the metabolic ability of B cells by downregulating expression of pyruvate dehydrogenase kinase 1 and proteins in complexes I, II, and IV of the electron transport chain. CONCLUSIONS: We clarified the mechanism of action of TSPO ligands on B-cell functions and provided new ideas and drug targets for the clinical treatment of postoperative AMR.

A prediction model of delayed graft function in deceased donor for renal transplant: a multi-center study from China.

BACKGROUND: Kidneys obtained from deceased donors increase the incidence of delayed graft function (DGF) after renal transplantation. Here we investigated the influence of the risk factors of donors with DGF, and developed a donor risk scoring system for DGF prediction. METHODS: This retrospective study was conducted in 1807 deceased kidney donors and 3599 recipients who received donor kidneys via transplants in 29 centers in China. We quantified DGF associations with donor clinical characteristics. A donor risk scoring system was developed and validated using an independent sample set. RESULTS: The incidence of DGF from donors was 19.0%. Six of the donor characteristics analyzed, i.e., age, cause of death, history of hypertension, terminal serum creatinine, persistence of hypotension, and cardiopulmonary resuscitation (CPR) time were risk factors for DGF. A 49-point scoring system of donor risk was established for DGF prediction and exhibited a superior degree of discrimination. External validation of DGF prediction revealed area under the receiver-operating characteristic (AUC) curves of 0.7552. CONCLUSIONS: Our study determined the deceased donor risk factors related to DGF after renal transplantation pertinent to the Chinese cohort. The scoring system developed here had superior diagnostic significance and consistency and can be used by clinicians to make evidence-based decisions on the quality of kidneys from deceased donors and guide renal transplantation therapy.

Noninvasive quantification of intrarenal allograft C4d deposition with targeted ultrasound imaging.

Antibody-mediated rejection (AMR) has emerged as a major cause of renal allograft dysfunction. C4d, a specific marker for AMR diagnosis, was strongly recommended for routine surveillance; however, currently, C4d detection is dependent upon tissue biopsy, which is invasive and provides only local semi-quantitative data. Targeted ultrasound imaging has been used extensively for noninvasive and real-time molecular detection with advantages of high specificity and sensitivity. In this study, we designed C4d-targeted microbubbles (MBC4d ) using a streptavidin-biotin conjugated method and detected C4d deposition in vivo in a rat model of AMR by enhanced ultrasound imaging. This noninvasive procedure allowed successful acquisition of the first qualitative image of C4d deposition in a wide renal allograft section, which reflected real-time C4d distribution in grafts. Moreover, we introduced normal intensity difference for quantitative analysis, which exhibited a nearly linear correlation with the grade of C4d deposition according to pathologic analysis. In addition, this approach showed no influence on survival rates and pathologic features in the microbubble injection groups, thereby demonstrating its safety. These findings demonstrated a simple, noninvasive, quantitative, and safe evaluation method for C4d, with the utility of this approach potentially preventing patients from having to undergo an invasive biopsy.

Triptolide inhibits donor-specific antibody production and attenuates mixed antibody-mediated renal allograft injury.

Donor-specific antibodies (DSAs) are major mediators of renal allograft injury, and strategies to inhibit DSAs are important in promoting long-term graft survival. Triptolide exhibits a wide spectrum of antiinflammatory and immunosuppressive activities, and in autoimmune diseases it inhibits autoantibody levels. In this study, we investigated the suppressive role of triptolide in the generation of DSAs in transplant recipients. We found that triptolide treatment of skin allograft recipients in mice significantly suppressed the development of circulating anti-donor-specific IgG and effectively alleviated DSA-mediated renal allograft injury, which led to prolonged allograft survival. In vitro studies revealed that triptolide inhibited the differentiation of B cells into CD138+ CD27++ plasma cells; reduced the levels of IgA, IgG, and IgM secreted by plasma cells; and repressed somatic hypermutation and class switch recombination of B cells. Moreover, triptolide-treated recipients showed reduced numbers of B cells, plasma cells, and memory B cells in spleens and decreased numbers of T, B, natural killer (NK) cells, and macrophages infiltrating grafts. These findings highlight the importance of triptolide in suppressing DSAs and establish triptolide as a novel therapeutic agent for antibody-mediated allograft rejection.

Allogeneic mesenchymal stem cells as induction therapy are safe and feasible in renal allografts: pilot results of a multicenter randomized controlled trial.

BACKGROUND: Kidneys from deceased donors are being used to meet the growing need for grafts. However, delayed graft function (DGF) and acute rejection incidences are high, leading to adverse effects on graft outcomes. Optimal induction intervention should include both renal structure injury repair and immune response suppression. Mesenchymal stem cells (MSCs) with potent anti-inflammatory, regenerative, and immune-modulatory properties are considered a candidate to prevent DGF and acute rejection in renal transplantation. Thus, this prospective multicenter paired study aimed to assess the clinical value of allogeneic MSCs as induction therapy to prevent both DGF and acute rejection in deceased donor renal transplantation. METHODS: Forty-two renal allograft recipients were recruited and divided into trial and control groups. The trial group (21 cases) received 2 × 106/kg human umbilical-cord-derived MSCs (UC-MSCs) via the peripheral vein before renal transplantation, and 5 × 106 cells via the renal artery during the surgical procedure. All recipients received standard induction therapy. Incidences of DGF and biopsy-proven acute rejection were recorded postoperatively and severe postoperative complications were assessed. Graft and recipient survivals were also evaluated. RESULTS: Treatment with UC-MSCs achieved comparable graft and recipient survivals with non-MSC treatment (P = 0.97 and 0.15, respectively). No increase in postoperative complications, including DGF and acute rejection, were observed (incidence of DGF: 9.5% in the MSC group versus 33.3% in the non-MSC group, P = 0.13; Incidence of acute rejection: 14.3% versus 4.8%, P = 0.61). Equal postoperative estimated glomerular filtration rates were found between the two groups (P = 0.88). All patients tolerated the MSCs infusion without adverse clinical effects. Additionally, a multiprobe fluorescence in situ hybridization assay revealed that UC-MSCs administered via the renal artery were absent from the recipient's biopsy sample. CONCLUSIONS: Umbilical-cord-derived MSCs can be used as clinically feasible and safe induction therapy. Adequate timing and frequency of UC-MSCs administration may have a significant effect on graft and recipient outcomes. Trial registration NCT02490020 . Registered on June 29 2015.

New Factors Predicting Delayed Graft Function: a Multi-Center Cohort Study of Kidney Donation After Brain Death Followed by Circulatory Death.

BACKGROUND/AIMS: Delayed graft function (DGF) is a common complication following kidney transplantation adversely affecting graft outcomes. Donation after brain death followed by circulatory death (DBCD), a novel donation pattern, is expected to correlate with high incidence of DGF. However, little information is available about factors associated with DGF in DBCD. METHODS: A total of 383 kidney transplants from DBCD donation in three institutions were enrolled. Associations of DGF with the clinical characteristics of recipients and donors were quantified. RESULTS: In this retrospective multi-center study, the incidence of DGF was 19.3%. Lower incidence of DGF was found in recipients for whom antithymocyte globulin was used for induction (p < 0.05), which was an independent protective factor against DGF (odds ratio [OR] = 0.48; 95% CI 0.27-0.86). Two novel explicative variables were recognized as independent risk factors, including use of vasoactive drugs (OR = 3.15; 95% CI 1.39-7.14) and cardiopulmonary resuscitation (OR = 2.51; 95% CI 1.05-6.00), which contributed significantly to increased risk of DGF (p < 0.05). Prolonged warm ischemia time (> 18 min; OR = 2.42; 95% CI 1.36-4.32), was also predictive of DGF in DBCD. A prediction model was developed and achieved an area under the curve of 0.89 in predicting DGF when combined with reported parameters. CONCLUSION: The novel factors, confirmed for the first time in our study, will help to improve risk prediction of DGF and to determine optimal interventions to prevent DGF in clinical practice.

Donation after brain death followed by circulatory death, a novel donation pattern, confers comparable renal allograft outcomes with donation after brain death.

BACKGROUND: Organ donation after brain death (DBD) is the standard strategy for organ transplantation; however, the concept of brain death is not universally accepted due to cultural beliefs and barriers amongst billions of people worldwide. Hence, a novel donation pattern has been established in China which outlines the concept of donation after brain death followed by circulatory death (DBCD). Differently from any current donation classification, this new concept is formulated based on combination of recognizing brain death and circulatory death. Should approval be gained for this definition and approach, DBCD will pave a novel donation option for billions of people who cannot accept DBD due to their cultural beliefs. METHODS: A multi-center, cohort study was conducted from February 2012 to December 2015. 523 kidney transplant recipients from four kidney transplant institutions were enrolled into the study, of which, 383 received kidneys from DBCD, and 140 from DBD. Graft and recipient survivals following transplantation were retrospectively analyzed. Postoperative complications including delayed graft function,, and acute rejection, were also analyzed for both groups. RESULTS: DBCD could achieve comparable graft and recipient survivals in comparison with DBD (Log-rank P = 0.32 and 0.86,respectively). One-year graft and recipient survivals were equal between DBCD and DBD groups (97.4% versus 97.9%, P = 0.10;98.4% versus 98.6%, P = 1.0, respectively). Furthermore, DBCD did not increase incidences of postoperative complications compared with DBD, including delayed graft function (19.3% versus 22.1%, P = 0.46) and acute rejection (9.1% versus 8.6%, P = 1.0). Additionally, antithymocyte globulin as induction therapy and shorter warm ischemia time decreased incidence of delayed graft function in DBCD group (16.8% on antithymocyte globulin versus 27.2% on basiliximab, P = 0.03; 16.7% on ≤18 min versus 26.7% on > 18 min group, P = 0.03). CONCLUSIONS: Kidney donation through DBCD achieves equally successful outcomes as DBD, and could provide a feasible path to graft availability for billions of people who face barriers to organ donation from DBD.

Late and chronic antibody-mediated rejection: main barrier to long term graft survival.

Antibody-mediated rejection (AMR) is an important cause of graft loss after organ transplantation. It is caused by anti-donor-specific antibodies especially anti-HLA antibodies. C4d had been regarded as a diagnosis marker for AMR. Although most early AMR episodes can be successfully controlled or reversed, late and chronic AMR remains the leading cause of late graft loss. The strategies which work in early AMR have limited effect on late/chronic episodes. Here, we reviewed the lines of evidence that late/chronic AMR is the leading cause of late graft loss, characteristics of late AMR, and current strategies in managing late/chronic AMR. More effort should be put on the management of late/chronic AMR to make a better long term graft survival.

The diagnostic value of transcription factors T-bet/GATA3 ratio in predicting antibody-mediated rejection.

BACKGROUND: Previous data showed that the predominance of intraglomerular T-bet or GATA3 is correlated with different mechanisms of rejection, suggesting that the ratio of T-bet/GATA3 might be used to distinguish antibody-mediated rejection (ABMR) and T-cell-mediated rejection (TCMR). METHODS: We compared the intraglomerular T-bet/GATA3 ratio in ABMR and TCMR. The intragraft expression of T-bet and GATA3 was studied via immunohistochemistry. The correlation of the diagnosis of AMR with the ratio of T-bet/GATA3 was examined. RESULTS: Both intraglomerular T-bet- and GATA3-expressing cells were increased during acute rejection. T-bet/GATA3>1 was strongly correlated with ABMR (93.3% versus 18.2%). The incidence of positive HLA-I/II antibodies and glomerulitis is significantly higher in T-bet/GATA3>1 group (P < 0.001, 0.013, resp.). The scores of peritubular capillary inflammation and glomerulitis were also higher in T-bet/GATA3>1 group (P = 0.052, P < 0.001, resp.). Nevertheless, T-bet/GATA3>1 is also correlated with C4d-negative ABMR and resistance to steroid treatment. Compared with C4d deposition, T-bet/GATA3>1 had a slight lower (90% versus 100%) specificity but a much higher (87.5% versus 68.8%) sensitivity. CONCLUSION: Our data suggested that intraglomerular predominance of T-bet over GATA3 might be used as diagnosis maker of ABMR in addition to C4d, especially in C4d-negative cases.

Immunophenotyping in BK virus allograft nephropathy distinct from acute rejection.

Differentiating BK virus nephropathy (BKVN) from acute rejection (AR) is crucial in clinical practice, as both of them have interstitial inflammation in the grafts. The purpose of the study is to describe the inflammatory cellular constituents of BKVN and to determine the clinical utility of immunophenotyping findings in distinguishing BKVN from AR. In addition, the expression of the HLA-DR was investigated. Sixty-five renal allograft recipients were included in this study, including 22 cases of BKVN, 31 cases of AR, and 12 cases of stable allograft. Immunostaining for infiltrating lymphocytes showed that the number of CD20 cells (P < 0.001) and the percentages of CD3 (P < 0.001), CD4 (P = 0.004), CD8 (P = 0.005), and CD20 (P = 0.002) cells were all significantly different between BKVN and AR. Moreover, there were no statistically significant differences in tubule cell HLA-DR expression (P = 0.156). This observation suggests that the number of CD20 cells and the percentages of CD3, CD4, CD8, and CD20 cells in renal biopsies would aid the distinction between BKVN and AR. On the other hand, the presence of HLA-DR upregulation may not only be specific for acute rejection but also be a response to BKVN.

Endothelial injury in transplant glomerulopathy is correlated with transcription factor T-bet expression.

Transplant glomerulopathy is an important cause of late graft loss. Inflammatory lesions including glomerulitis and peritubular capillaritis, suggestive of endothelial injury, are prominent in this condition but the mechanism underlying this inflammation remains unclear. Here we measured the expression of T-bet (a member of the T-box family of transcription factors regulating Th1 lineage commitment) and its relationship with inflammation in 70 patients with transplant glomerulopathy. Within this cohort, 32 patients were diagnosed with transplant glomerulopathy, 23 with interstitial fibrosis/tubular atrophy, and 15 with stable grafts. There was a significant increase in T-bet expression in both glomerular and peritubular capillaries of the transplant glomerulopathy group. This expression was strongly correlated with CD4(+), CD8(+), and CD68(+) cell infiltration within glomerular and peritubular capillaries. The expression of GATA3, a Th2 regulator, was rarely found in the transplant glomerulopathy group. Transplant glomerulopathy was associated with diffuse peritubular capillary dilation without reduced capillary density. Moreover, the degree of capillary dilation was significantly correlated with the number of infiltrating CD68(+) cells. Since endothelial injury is a typical lesion that follows alloantibody reactivity, our results suggest that T-bet is involved in the pathogenesis of this glomerulopathy.

Picking transplant glomerulopathy out of the CAN: evidence from a clinico-pathological evaluation.

BACKGROUND: Since the term chronic allograft nephropathy (CAN) was removed from the Banff scheme in 2005, transplant glomerulopathy (TG) has been regarded as a clinicopathological entity that is one of the major causes of graft loss. To assess the distinction between CAN and TG, we performed a comprehensive evaluation comparing TG with traditional CAN. METHODS: We compared the clinicopathological features of 43 cases of TG with 43 matched cases of non-TG CAN (non-TG group) after renal transplantation. TG was diagnosed by light microscopy based on the double contours of the glomerular basement membranes, and the Banff 97 classification system was used to score TG severity (cg0-3). RESULTS: Compared to the control group, we found a significantly higher incidence of positivity for human leukocyte antigen class-I and II antibodies, a higher incidence of hepatitis C virus (HCV) infection, and poorer graft survival in TG patients. Clinically, TG was associated with a higher prevalence of proteinuria, hematuria, anaemia and hypoalbuminemia. Histologically, TG strongly correlated with antibody related microcirculatory injuries, including glomerulitis, peritubular capillaritis and peritubular capillary (PTC) C4d deposition. Interestingly, the TG patients showed a significantly higher incidence of IgA deposition than the control patients. C4d-positive TG was correlated with higher TG and PTC scores, and PTC C4d deposition was correlated with a more rapid progression to graft dysfunction. TG accompanied by HCV infection was associated with heavier proteinuria, higher TG and C4d scores, and poorer graft survival. CONCLUSIONS: TG presents clinicopathological features that are distinct from non-TG cases and leads to poorer outcomes. PTC C4d deposition is related to a more rapid progression to graft loss, suggesting ongoing antibody reactivity. HCV-positive TG is a more severe sub-entity, that requires further investigation.

Predominance of intraglomerular T-bet or GATA3 may determine mechanism of transplant rejection.

The transcription factors T-bet and GATA3 determine the differentiation of helper T cells into Th1 or Th2 cells, respectively. An altered ratio of their relative expression promotes the pathogenesis of certain immunological diseases, but whether this may also contribute to the pathogenesis of antibody-mediated rejection (ABMR) versus T cell-mediated rejection (TCMR) is unknown. Here, we characterized the intragraft expression of T-bet and GATA3 and determined the correlation of their levels with the presence of typical lesions of ABMR and TCMR. We found a predominant intraglomerular expression of T-bet in patients with ABMR, which was distinct from that in patients with TCMR. In ABMR, interstitial T-bet expression was typically located in peritubular capillaries, although the overall quantity of interstitial T-bet was less than that observed in TCMR. The expression of intraglomerular T-bet correlated with infiltration of CD4+ and CD8+ lymphocytes, which express T-bet, as well as intraglomerular CD68+ monocyte/macrophages, which do not express T-bet. The predominance of intraglomerular T-bet expression relative to GATA3 expression associated with poor response to treatment with bolus steroid. In summary, predominance of intraglomerular T-bet expression correlates with antibody-mediated rejection and resistance to steroid treatment.

Ex vivo anchored PD-L1 functionally prevent in vivo renal allograft rejection.

Organ transplantation is the optimal treatment for patients with end-stage diseases. T cell activation is a major contributing factor toward the trigger of rejection. Induction therapy with T cell depleting agent is a common option but increases the risk of severe systemic infections. The ideal therapy should precisely target the allograft. Here, we developed a membrane-anchored-protein PD-L1 (map-PD-L1), which effectively anchored onto the surface of rat glomerular endothelial cells (rgEC). The expression of PD-L1 increased directly with map-PD-L1 concentration and incubation time. Moreover, map-PD-L1 was even stably anchored to rgEC at low temperature. Map-PD-L1 could bind to PD-1 and significantly promote T cell apoptosis and inhibited T cell activation. Using kidney transplantation models, we found that ex vivo perfusion of donor kidneys with map-PD-L1 significantly protected grafts against acute injury without using any immunosuppressant. We found map-PD-L1 could reduce T cell graft infiltration and increase intragraft Treg infiltration, suggesting a long-term effect in allograft protection. More importantly, modifying donor organs in vitro was not only safe, but also significantly reduced the side effects of systemic application. Our results suggested that ex vivo perfusion of donor organ with map-PD-L1 might provide a viable clinical option for organ-targeted induction therapy in organ transplantation.

De novo development of circulating anti-endothelial cell antibodies rather than pre-existing antibodies is associated with post-transplant allograft rejection.

Anti-endothelial cell antibodies (AECAs) are thought to be involved in the development of renal allograft rejection. To explore this further, we determine whether AECAs play a role both in predicting the incidence of allograft rejection and long-term outcomes by analysis of serum samples from 226 renal allograft recipients for AECAs pre- and post-transplant. Surprisingly, the presence of pre-existing AECAs was not associated with either an increased risk of rejection or a detrimental impact on recipient/graft survival. Subsequent de novo AECAs, however, were associated with a significantly increased risk of early acute rejection. Moreover, these rejections tended to be more severe with a significantly increased incidence of both steroid-resistant and multiple episodes of acute rejection. The acute rejections associated with de novo AECAs did not correlate with C4d deposition at the time of renal biopsy, but did demonstrate an association with the presence of glomerulitis and peritubular capillary inflammation. Significantly more patients with de novo AECAs developed graft dysfunction. Thus, our prospective study suggests the emergence of de novo AECAs is associated with transplant rejection that may lead to allograft dysfunction.

Corrigendum: Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

[This corrects the article DOI: 10.3389/fimmu.2021.778359.].

Blockade of IL-6/IL-6R Signaling Attenuates Acute Antibody-Mediated Rejection in a Mouse Cardiac Transplantation Model.

Acute antibody-mediated rejection (AAMR) is an important cause of cardiac allograft dysfunction, and more effective strategies need to be explored to improve allograft prognosis. Interleukin (IL)-6/IL-6R signaling plays a key role in the activation of immune cells including B cells, T cells and macrophages, which participate in the progression of AAMR. In this study, we investigated the effect of IL-6/IL-6R signaling blockade on the prevention of AAMR in a mouse model. We established a mouse model of AAMR for cardiac transplantation via presensitization of skin grafts and addition of cyclosporin A, and sequentially analyzed its features. Tocilizumab, anti-IL-6R antibody, and recipient IL-6 knockout were used to block IL-6/IL-6R signaling. We demonstrated that blockade of IL-6/IL-6R signaling significantly attenuated allograft injury and improved survival. Further mechanistic research revealed that signaling blockade decreased B cells in circulation, spleens, and allografts, thus inhibiting donor-specific antibody production and complement activation. Moreover, macrophage, T cell, and pro-inflammatory cytokine infiltration in allografts was also reduced. Collectively, we provided a highly practical mouse model of AAMR and demonstrated that blockade of IL-6/IL-6R signaling markedly alleviated AAMR, which is expected to provide a superior option for the treatment of AAMR in clinic.

Hydroxychloroquine Inhibits Macrophage Activation and Attenuates Renal Fibrosis After Ischemia-Reperfusion Injury.

Chronic kidney disease (CKD), which is associated with high morbidity, remains a worldwide health concern, while effective therapies remain limited. Hydroxychloroquine (HCQ), which mainly targets toll-like receptor-7 (TLR-7) and TLR-9, is associated with a lower risk of incident CKD. Taking into account that TLR-9 is involved in the development of renal fibrosis and serves as a potential therapy target for CKD, we investigated whether HCQ could attenuate CKD via TLR-9 signal pathway. The effects of HCQ on renal tubulointerstitial fibrosis were further explored using a mouse model of renal tubulointerstitial fibrosis after ischemia/reperfusion injury. Bone marrow-derived macrophages were isolated to explore the effects of HCQ in vitro. Judicious use of HCQ efficiently inhibited the activation of macrophages and MAPK signaling pathways, thereby attenuating renal fibrosis in vivo. In an in vitro model, results showed that HCQ promoted apoptosis of macrophages and inhibited activation of macrophages, especially M2 macrophages, in a dose-dependent manner. Because TLR-7 is not involved in the development of CKD post-injury, a TLR-9 knockout mouse was used to explore the mechanisms of HCQ. The effects of HCQ on renal fibrosis and macrophages decreased after depletion of TLR-9 in vivo and in vitro. Taken together, this study indicated that proper use of HCQ could be a new strategy for anti-fibrotic therapy and that TLR-9 could be a potential therapeutic target for CKD following acute kidney injury.

Depletion of Toll-Like Receptor-9 Attenuates Renal Tubulointerstitial Fibrosis After Ischemia-Reperfusion Injury.

Toll-like receptor-9 (TLR-9) is a potent proinflammatory receptor that mediates renal injury. However, the reported effects of TLR-9 are contradictory. Here, using a traditional mouse AKI→CKD transition model, the roles of TLR-9 during the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) were further explored. Using a TLR-9-/- mouse, the effects and mechanisms of TLR-9 were investigated. Loss of TLR-9 elicited no obvious effects as regards renal function or histology during AKI in the early phases (24-48 h), while TLR-9 KO attenuated renal fibrosis (as shown using fibronectin and collagen III) and epithelial-to-mesenchymal transition (EMT) [E-cadherin (E-Cad) and α-smooth muscle actin (α-SMA)] on the long-term after AKI through the inhibition of macrophages infiltration, especially M2 macrophages. The roles of TLR-9 on macrophages were also explored using Raw264.7 macrophage cell line, and results indicated that the inhibition of TLR-9 on Raw 264.7 macrophages decreased the induction of M2 type macrophage in a dose-dependent manner. The roles of TLR-9 on renal tubular epithelial (RTE) cells were also explored. Conversely, TLR-9 depletion did not contribute to the improvement of fibrosis and EMT in vitro. Therefore, TLR-9 plays a critical role in the AKI→CKD transition. Attenuation of CKD post-AKI in the TLR-9 KO group mainly relies on the effects of TLR-9 on macrophages. These results also suggest that TLR-9 could be a therapeutic target for CKD.

Plasma Macrophage Migration Inhibitory Factor Predicts Graft Function Following Kidney Transplantation: A Prospective Cohort Study.

Background: Delayed graft function (DGF) is a common complication after kidney transplantation (KT) with a poor clinical outcome. There are no accurate biomarkers for the early prediction of DGF. Macrophage migration inhibitory factor (MIF) release during surgery plays a key role in protecting the kidney, and may be a potential biomarker for predicting post-transplant renal allograft recovery. Methods: Recipients who underwent KT between July 2020 and December 2020 were enrolled in the study. Plasma MIF levels were tested in recipients at different time points, and the correlation between plasma MIF and DGF in recipients was evaluated. This study was registered in the Chinese Clinical Trial Registry (ChiCTR2000035596). Results: Intraoperative MIF levels were different between immediate, slowed, and delayed graft function groups (7.26 vs. 6.49 and 5.59, P < 0.001). Plasma MIF was an independent protective factor of DGF (odds ratio = 0.447, 95% confidence interval [CI] 0.264-0.754, P = 0.003). Combining plasma MIF level and donor terminal serum creatinine provided the best predictive power for DGF (0.872; 95%CI 0.795-0.949). Furthermore, plasma MIF was significantly associated with allograft function at 1-month post-transplant (R 2 = 0.42, P < 0.001). Conclusion: Intraoperative MIF, as an independent protective factor for DGF, has excellent diagnostic performance for predicting DGF and is worthy of further exploration.