Heme oxygenase‐1 regulates sirtuin‐1–autophagy pathway in liver transplantation: From mouse to human

Liver ischemia–reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase‐1 (HO‐1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin‐1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO‐1–mediated autophagy induction in human OLT and in a murine OLT model with extended (20 hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO‐1. Fifty‐one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO‐1 levels correlated with well‐preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO‐1 overexpression by genetically modified HO‐1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO‐1–mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO‐1 cytoprotection and identifies SIRT1‐mediated autophagy pathway as a new essential regulator of HO‐1 function in IR‐stressed OLT.     

Negative CD4 + TIM‐3 Signaling Confers Resistance Against Cold Preservation Damage in Mouse Liver Transplantation

Ischemia‐reperfusion injury (IRI), an innate immunity‐driven local inflammation, remains the major problem in clinical organ transplantation. T cell immunoglobulin and mucin domain (TIM‐3)–Galectin‐9 (Gal‐9) signaling regulates CD4+ Th1 immune responses. Here, we explored TIM‐3–Gal‐9 function in a clinically relevant murine model of hepatic cold storage and orthotopic liver transplantation (OLT). C57BL/6 livers, preserved for 20 h at 4°C in UW solution, were transplanted to syngeneic mouse recipients. Up‐regulation of TIM‐3 on OLT‐infiltrating activated CD4+ T cells was observed in the early IRI phase (1 h). By 6 h of reperfusion, OLTs in recipients treated with a blocking anti‐TIM‐3 Ab were characterized by: (1) enhanced hepatocellular damage (sALT levels, liver Suzuki's histological score); (2) polarized cell infiltrate towards Th1/Th17‐type phenotype; (3) depressed T cell exhaustion markers (PD‐1, LAG3); and (4) elevated neutrophil and macrophage infiltration/activation. In parallel studies, adoptive transfer of CD4+ T cells from naïve WT, but not from TIM‐3 Tg donors, readily recreated OLT damage in otherwise IR‐resistant RAG^?/? test recipients. Furthermore, pre‐treatment of mice with rGal‐9 promoted hepatoprotection against preservation‐association liver damage, accompanied by enhanced TIM‐3 expression in OLTs. Thus, CD4+ T cell‐dependent “negative” TIM‐3 costimulation is essential for hepatic homeostasis and resistance against IR stress in OLTs.

     

Supplementing preservation solution with mitochondria‐targeted H2S donor AP39 protects cardiac grafts from prolonged cold ischemia–reperfusion injury in heart transplantation

Heart transplant has been accepted as the standard treatment for end‐stage heart failure. Because of its susceptibility to ischemia–reperfusion injury, the heart can be preserved for only 4 to 6 hours in cold static preservation solutions. Prolonged ischemia time is adversely associated with primary graft function and long‐term survival. New strategies to preserve donor hearts are urgently needed. We demonstrate that AP39, a mitochondria‐targeting hydrogen sulfide donor, significantly increases cardiomyocyte viability and reduces cell apoptosis/death after cold hypoxia/reoxygenation in vitro. It also decreases gene expression of proinflammatory cytokines and preserves mitochondria function. Using an in vivo murine heart transplant model, we show that preserving donor hearts with AP39‐supplemented University of Wisconsin solution (n = 7) significantly protects heart graft function, measured by quantitative ultrasound scan, against prolonged cold ischemia–reperfusion injury (24 hours at 4°C), along with reducing tissue injury and fibrosis. Our study demonstrates that supplementing preservation solution with AP39 protects cardiac grafts from prolonged ischemia, highlighting its therapeutic potential in preventing ischemia–reperfusion injury in heart transplant.     

Recipient HO‐1 inducibility is essential for posttransplant hepatic HO‐1 expression and graft protection: From bench‐to‐bedside

By documenting potent antioxidative and anti‐inflammatory functions, preclinical studies encourage heme oxygenase‐1 (HO‐1)‐inducing regimens in clinical orthotopic liver transplantation (OLT). We aimed to determine the importance of recipient‐derived HO‐1 in murine and human OLTs. Hepatic biopsies from 51 OLT patients were screened for HO‐1 expression (Western blots) prior to put‐in (basal) and post reperfusion (stressed) and correlated with the hepatocellular function. In parallel, livers from HO‐1 proficient mice (WT; C57/BL6), subjected to ex vivo cold storage (18 hour), were transplanted to syngeneic myeloid HO‐1 deficient (mHO‐1 KO) or FLOX (control) hosts, and sampled postreperfusion (6 hour). In human OLT, posttransplant but not pretransplant HO‐1 expression correlated negatively with ALT levels (P = .0178). High posttransplant but not pretransplant HO‐1 expression trended with improved OLT survival. Compared with controls, livers transplanted into mHO‐1 KO recipient mice had decreased HO‐1 levels, exacerbated hepatic damage/frequency of TUNEL+ cells, increased mRNA levels coding for TNFα/CXCL1/CXCL2/CXCL10, higher frequency of Ly6G+/4HN+ neutrophils; and enhanced MPO activity. Peritoneal neutrophils from mHO‐1 KO mice exhibited higher CellRox+ ratio and increased TNFα/CXCL1/CXCL2/CXCL10 expression. By demonstrating the importance of posttransplant recipient HO‐1 phenotype in hepatic macrophage/neutrophil regulation and function, this translational study identifies recipient HO‐1 inducibility as a novel biomarker of ischemic stress resistance in OLT.     

Thrombomodulin Attenuates Inflammatory Damage Due to Liver Ischemia and Reperfusion Injury in Mice in Toll‐Like Receptor 4–Dependent Manner

Liver ischemia reperfusion injury (IRI) is an important problem in liver transplantation. Thrombomodulin (TM), an effective drug for disseminated intravascular coagulation, is also known to exhibit an anti‐inflammatory effect through binding to the high‐mobility group box 1 protein (HMGB‐1) known as a proinflammatory mediator. We examined the effect of recombinant human TM (rTM) on a partial warm hepatic IRI model in wild‐type (WT) and toll‐like receptor 4 (TLR‐4) KO mice focusing on the HMGB‐1/TLR‐4 axis. As in vitro experiments, peritoneal macrophages were stimulated with recombinant HMGB‐1 protein. The rTM showed a protective effect on liver IRI. The rTM diminished the downstream signals of TLR‐4 and also HMGB‐1 expression in liver cells, as well as release of HMGB‐1 from the liver. Interestingly, neither rTM treatment in vivo nor HMGB‐1 treatment in vitro showed any effect on TLR‐4 KO mice. Parallel in vitro studies have confirmed that rTM interfered with the interaction between HMGB‐1 and TLR‐4. Furthermore, the recombinant N‐terminal lectin‐like domain 1 (D1) subunit of TM (rTMD1) also ameliorated liver IRI to the same extent as whole rTM. Not only rTM but also rTMD1 might be a novel and useful medicine for liver transplantation. This is the first report clarifying that rTM ameliorates inflammation such as IRI in a TLR‐4 pathway–dependent manner.     

Anti‐CD47 monoclonal antibody therapy reduces ischemia‐reperfusion injury of renal allografts in a porcine model of donation after cardiac death

We investigated whether blockade of the CD47 signaling pathway could reduce ischemia‐reperfusion injury (IRI) of renal allografts donated after cardiac death (DCD) in a porcine animal model of transplantation. Renal allografts were subjected to 30 minutes of warm ischemia, 3.5 hours of cold ischemia, and then perfused with a humanized anti‐CD47 monoclonal antibody (CD47mAb) in the treatment group or HTK solution in the control group (n = 4/group). The animals were euthanized five days after transplantation. At the time of reperfusion, indocyanine green‐based in vivo imaging showed that CD47mAb‐treated organs had greater and more uniform reperfusion. On post‐transplant days 3‐5, the treatment group had lower values compared to the control for creatinine and blood urea nitrogen. Histological examination of allograft tissues showed a significant decrease of acute tubular injury in the CD47mAb‐treated group compared to control. Compared to the control group, CD47mAb treatment significantly decreased genes expression related to oxidative stress (sod‐1, gpx‐1, and txn), the inflammatory response (il‐2, il‐6, inf‐g, and tgf‐b), as well as reduced protein levels of BAX, Caspase‐3, MMP2, and MMP9. These data demonstrate that CD47mAb blockade decreases IRI and subsequent tissue injury in DCD renal allografts in a large animal transplant model.     

The lectin-like domain of thrombomodulin is a drug candidate for both prophylaxis and treatment of liver ischemia and reperfusion injury in mice

Ischemia and reperfusion injury (IRI) can occur in any tissue or organ. With respect to liver transplantation, the liver grafts from donors by definition experience transient ischemia and subsequent blood reflow. IRI is a problem not only in organ transplantation but also in cases of thrombosis or circulatory disorders such as mesenteric ischemia, myocardial, or cerebral infarction. We have reported that recombinant human soluble thrombomodulin (rTM), which is currently used in Japan to treat disseminated intravascular coagulation (DIC), has a protective effect and suppresses liver IRI in mice. However, rTM may not be fully safe to use in humans because of its inherent anticoagulant activity. In the present study, we used a mouse liver IRI model to explore the possibility that the isolated lectin-like domain of rTM (rTMD1), which has no anticoagulant activity, could be effective as a therapeutic modality for IRI. Our results indicated that rTMD1 could suppress ischemia and reperfusion-induced liver damage in a dose-dependent manner without concern of associated hemorrhage. Surprisingly, rTMD1 suppressed the liver damage even after IR insult had occurred. Taken together, we conclude that rTMD1 may be a candidate drug for prevention of and therapy for human liver IRI without the possible risk of hemorrhage.     

Activation of Autophagy by Everolimus Confers Hepatoprotection Against Ischemia–Reperfusion Injury

As the criteria for liver donation have been extended to include marginal donors, liver grafts are becoming particularly vulnerable to hepatic ischemia–reperfusion injury (IRI). However, no specific measures have been validated to ameliorate hepatic IRI. In this article, we explored whether everolimus has protective effects against hepatic IRI in relation with autophagy. The effects of everolimus were investigated in both in vitro and in vivo hepatic IRI models. Mouse hepatocyte AML12 cells and BALB/c mice were utilized for the establishment of each model. In the IRI‐induced AML12 cells, everolimus treatment increased the expressions of autophagic markers (microtubule‐associated protein 1 light chain 3 and p62) and decreased pro‐apoptotic proteins (cleaved caspase 3 and cleaved poly‐ADP ribose polymerase). The blockage of autophagy, using either bafilomycin A1 or si‐autophagy‐related protein 5, abrogated these anti‐apoptosis effects of everolimus. Subsequently, everolimus administration to the hepatic IRI‐induced mice provided hepatoprotective effects in terms of (1) decreasing the expressions of pro‐apoptotic proteins, (2) inhibiting the release of pro‐inflammatory cytokines (IL‐6 and tumor necrosis factor‐α), (3) reducing elevated liver enzymes (aspartate transaminase, alanine transaminase, and ammonia), and (4) restoring liver histopathology. These findings suggest that everolimus protects the liver against hepatic IRI by way of activating autophagy, and thus could be a potential therapeutic agent for hepatic IRI.     

Alloimmune Activation Enhances Innate Tissue Inflammation/Injury in a Mouse Model of Liver Ischemia/Reperfusion Injury

The deleterious sensitization to donor MHC Ags represents one of the most challenging problems in clinical organ transplantation. Although the role of effector/memory T cells in the rejection cascade has been extensively studied, it remains unknown whether and how these ‘Ag‐specific’ cells influence host innate immunity, such as tissue inflammation associated with ischemia and reperfusion injury (IRI). In this study, we analyzed how allogeneic skin transplant (Tx) affected the sequel of host's own liver damage induced by partial warm ischemia and reperfusion. Our data clearly showed that allo‐Tx recipients had increased inflammatory response against IR insult in their native livers, as evidenced by significantly more severe hepatocelluar damage, compared with syngeneic Tx recipient controls, and determined by serum ALT levels, liver histology (Suzuki's score) and intrahepatic proinflammatory gene inductions (TNF‐α, IL‐1β and CXCL10). The CD4 T cells, but neither CD8 nor NK cells, mediated the detrimental effect of allo‐Ag sensitization in liver IRI. Furthermore, CD154, but not IFN‐γ, was the key mechanism in allo‐Tx recipients to facilitate IR‐triggered liver damage. These results provide new evidence that alloreactive CD4 T cells are capable of enhancing innate tissue inflammation and organ injury via an Ag‐nonspecific CD154‐dependent but IFN‐γ independent mechanism.     

Opposite acute potassium and sodium shifts during transplantation of hypothermic machine perfused donor livers

Liver transplantation is frequently associated with hyperkalemia, especially after graft reperfusion. Dual hypothermic oxygenated machine perfusion (DHOPE) reduces ischemia/reperfusion injury and improves graft function, compared to conventional static cold storage (SCS). We examined the effect of DHOPE on ex situ and in vivo shifts of potassium and sodium. Potassium and sodium shifts were derived from balance measurements in a preclinical study of livers that underwent DHOPE (n = 6) or SCS alone (n = 9), followed by ex situ normothermic reperfusion. Similar measurements were performed in a clinical study of DHOPE‐preserved livers (n = 10) and control livers that were transplanted after SCS only (n = 9). During DHOPE, preclinical and clinical livers released a mean of 17 ± 2 and 34 ± 6 mmol potassium and took up 25 ± 9 and 24 ± 14 mmol sodium, respectively. After subsequent normothermic reperfusion, DHOPE‐preserved livers took up a mean of 19 ± 3 mmol potassium, while controls released 8 ± 5 mmol potassium. During liver transplantation, blood potassium levels decreased upon reperfusion of DHOPE‐preserved livers while levels increased after reperfusion of SCS‐preserved liver, delta potassium levels were ‐0.77 ± 0.20 vs. +0.64 ± 0.37 mmol/L, respectively (P = .002). While hyperkalemia is generally anticipated during transplantation of SCS‐preserved livers, reperfusion of hypothermic machine perfused livers can lead to decreased blood potassium or even hypokalemia in the recipient.     

Diannexin, a Novel Annexin V Homodimer, Protects Rat Liver Transplants Against Cold Ischemia-Reperfusion Injury

Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4 degrees C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 microg/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF-alpha, IL-1beta, IP-10 expression, diminished expression of P-selectin, endothelial ICAM-1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO-1/Bcl-2/Bcl-xl, and reduced Caspase-3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool.     

Notch信号通路在电针预处理诱导大鼠脑缺血耐受中的作用

目的 探讨Noah信号通路在电针预处理诱导大鼠脑缺血耐受中的作用.方法 健康成年雄性SD大鼠52只,体重280～320 g,随机分为2组(n=26):正常对照组(C组)不做任何处理;电针预处理组(EA组)于百会穴进行电针刺激(刺激条件:疏密波2/15Hz,电流强度1 mA),30 min次,1次/d,连续5 d.最后一次电针刺激结束后24 h采用阻断单侧大脑中动脉120 min再灌注72 h的方法制备局灶性脑缺血再灌注模型.分别于缺血前即刻、再灌注24 h及再灌注72 h时采用Western blot法测定缺血侧大脑皮层Notch细胞内片段(NICD)蛋白的表达,采用实时定量PCR法测定缺血侧大脑皮层Notch通路信号分子的表达;于再灌注72 h时进行神经功能损伤评分,评分完毕后测定脑梗死体积百分比.结果 缺血前即刻两组大脑皮层Hesl mRNA及NICD蛋白表达差异无统计学意义(P>0.05);与缺血前即刻比较,两组再灌注24、72 h时NICD蛋白表达上调,C组再灌注72 h时Hesl mRNA 表达上调,EA组再灌注24 h时Hesl mRNA表达上调(P<0.05);与再灌注24 h时比较,再灌注72 h时C组Hesl mRNA及NICD蛋白表达均上调,EA组Hesl mRNA及NICD蛋白表达均下调(P<0.05);与C组比较,EA组缺血前即刻Notchl mRNA、Notch4 mRNA及Jagl mRNA的表达上调,再灌注24 h时Hesl mRNA及NICD蛋白表达上调,再灌注72 h时Hesl mRNA及NICD蛋白表达下调,脑梗死体积百分比降低,神经功能损伤评分升高(P<0.05).结论 Notch信号通路可能参与了电针预处理诱导的大鼠脑缺血耐受.     

Hyperglycemia and Liver Ischemia Reperfusion Injury: A Role for the Advanced Glycation Endproduct and Its Receptor Pathway

Although pretransplant diabetes is a risk factor for mortality post–liver transplant, the underlying mechanism has not been fully defined. In a murine liver partial warm ischemia model, we addressed the question of how diabetes/hyperglycemia impacted tissue inflammatory injuries against ischemia reperfusion (IR), focusing on the advanced glycation endproduct (AGE) and its receptor (RAGE) pathway. Our results showed that hepatocellular injury was exacerbated in streptozotocin‐induced diabetic mice against IR, in association with hyper‐inflammatory immune activation in livers. Serum levels of AGEs, but not HMGB1, were increased in diabetic mice in response to liver IR. Both RAGE antagonist peptides and small interfering RNA alleviated liver injuries and inhibited inflammatory immune activation against IR in diabetic, but not normal, mice. Kupffer cells (KCs)/macrophages, but not hepatocytes, from diabetic mice expressed significantly higher levels of RAGE, leading to their hyper‐inflammatory responsiveness to both TLR ligands and AGEs. In vitro, hyperglycemia increased macrophage RAGE expression and enhanced their TLR responses. Our results demonstrated that activation of the AGE–RAGE signaling pathway in KCs was responsible for hyper‐inflammatory immune responses and exacerbated hepatocellular injuries in diabetic/hyperglycemic hosts against liver IR.     

CD47 blockade reduces ischemia/reperfusion injury in donation after cardiac death rat kidney transplantation

Modulation of nitric oxide activity through blockade of CD47 signaling has been shown to reduce ischemia–reperfusion injury (IRI) in various models of tissue ischemia. Here, we evaluate the potential effect of an antibody‐mediated CD47 blockade in a syngeneic and an allogeneic DCD rat kidney transplant model. The donor organ was subjected to 1 hour of warm ischemia time after circulatory cessation, then flushed with a CD47 monoclonal antibody (CD47mAb) in the treatment group, or an isotype‐matched immunoglobulin in the control group. We found that CD47mAb treatment improved survival rates in both models. Serum markers of renal injury were significantly decreased in the CD47mAb‐treated group compared with the control group. Histologically the CD47mAb‐treated group had significantly reduced scores of acute tubular injury and acute tubular necrosis. The expression of biomarkers related to mitochondrial stress and apoptosis also were significantly lower in the CD47mAb‐treated groups. Overall, the protective effects of CD47 blockade were greater in the syngeneic model. Our data show that CD47mAb blockade decreased the IRI of DCD kidneys in rat transplant models. This therapy has the potential to improve DCD kidney transplant outcomes in the human setting.     

The first case of ischemia‐free organ transplantation in humans: A proof of concept

Ischemia and reperfusion injury (IRI) is an inevitable event in conventional organ transplant procedure and is associated with significant mortality and morbidity post‐transplantation. We hypothesize that IRI is avoidable if the blood supply for the organ is not stopped, thus resulting in optimal transplant outcomes. Here we described the first case of a novel procedure called ischemia‐free organ transplantation (IFOT) for patients with end‐stage liver disease. The liver graft with severe macrovesicular steatosis was donated from a 25‐year‐old man. The recipient was a 51‐year‐old man with decompensated liver cirrhosis and hepatocellular carcinoma. The graft was procured, preserved, and implanted under continuous normothermic machine perfusion. The recipient did not suffer post‐reperfusion syndrome or vasoplegia after revascularization of the allograft. The liver function test and histological study revealed minimal hepatocyte, biliary epithelium and vascular endothelium injury during preservation and post‐transplantation. The inflammatory cytokine levels were much lower in IFOT than those in conventional procedure. Key pathways involved in IRI were not activated after allograft revascularization. No rejection, or vascular or biliary complications occurred. The patient was discharged on day 18 post‐transplantation. This marks the first case of IFOT in humans, offering opportunities to optimize transplant outcomes and maximize donor organ utilization.     

Sotrastaurin, a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthotopic liver transplantation

Sotraustaurin (STN), a small molecule, targeted protein kinase C (PKC) inhibitor that prevents T-lymphocyte activation via a calcineurin-independent pathway, is currently being tested in Phase II renal and liver transplantation clinical trials. We have documented the key role of activated T cells in the inflammation cascade leading to liver ischemia/reperfusion injury (IRI). This study explores putative cytoprotective functions of STN in a clinically relevant rat model of hepatic cold ischemia followed by orthotopic liver transplantation (OLT). Livers from Sprague-Dawley rats were stored for 30 h at 4°C in UW solution, and then transplanted to syngeneic recipients. STN treatment of liver donors/recipients or recipients only prolonged OLT survival to >90% (vs. 40% in controls), decreased hepatocellular damage and improved histological features of IRI. STN treatment decreased activation of T cells, and diminished macrophage/neutrophil accumulation in OLTs. These beneficial effects were accompanied by diminished apoptosis, NF-κB/ERK signaling, depressed proapoptotic cleaved caspase-3, yet upregulated antiapoptotic Bcl-2/Bcl-xl and hepatic cell proliferation. In vitro, STN decreased PKCθ/IκBα activation and IL-2/IFN-γ production in ConA-stimulated spleen T cells, and diminished TNF-α/IL-1β in macrophage-T cell cocultures. This study documents positive effects of STN on liver IRI in OLT rat model that may translate as an additional benefit of STN in clinical liver transplantation.     

Anti‐LG3 Antibodies Aggravate Renal Ischemia–Reperfusion Injury and Long‐Term Renal Allograft Dysfunction

Pretransplant autoantibodies to LG3 and angiotensin II type 1 receptors (AT1R) are associated with acute rejection in kidney transplant recipients, whereas antivimentin autoantibodies participate in heart transplant rejection. Ischemia–reperfusion injury (IRI) can modify self‐antigenic targets. We hypothesized that ischemia–reperfusion creates permissive conditions for autoantibodies to interact with their antigenic targets and leads to enhanced renal damage and dysfunction. In 172 kidney transplant recipients, we found that pretransplant anti‐LG3 antibodies were associated with an increased risk of delayed graft function (DGF). Pretransplant anti‐LG3 antibodies are inversely associated with graft function at 1 year after transplantation in patients who experienced DGF, independent of rejection. Pretransplant anti‐AT1R and antivimentin were not associated with DGF or its functional outcome. In a model of renal IRI in mice, passive transfer of anti‐LG3 IgG led to enhanced dysfunction and microvascular injury compared with passive transfer with control IgG. Passive transfer of anti‐LG3 antibodies also favored intrarenal microvascular complement activation, microvascular rarefaction and fibrosis after IRI. Our results suggest that anti‐LG3 antibodies are novel aggravating factors for renal IRI. These results provide novel insights into the pathways that modulate the severity of renal injury at the time of transplantation and their impact on long‐term outcomes.