The Impact Of Histopathological Characteristics And Cytokine Levels On Allograft Outcomes In Kidney Transplant Recipients With Acute Rejection–A Clinical And Experimental Study

PartⅠ The impact of histopathological characteristics on allograft outcomes in kidney transplant recipients with acute rejectionBackground:Acute rejection,especially antibody mediated rejection（ABMR）,is one of the major risk factors on graft survival post kidney transplantation.Microvascular inflammation is the primary histopathological manifestation of ABMR.Literatures have called awareness of the associations between microvascular inflammation and long-term graft failure.This study is of the first time to evaluate the incidence of microvascular inflammation in different acute rejections and investigate the impact of microvascular inflammation on renal allograft function and survival.Methods:A total of 525 kidney transplant patients underwent renal biopsy in our hospital from 2012 January 1^stto 2020 June 30^thwere included.The patients were divided into 4 subgroups according to Banff 2017 criteria with 143 patients of normal or non-specific histopathological changes（Normal）,140 patients with borderline change,194 patients with T-cell mediated rejection（TCMR）and 48 patients with ABMR.Patient demographics,the incidence of microvascular inflammation,serum creatinine and estimated glomerular filtration rate within 6 months after biopsy,and long-term graft survival in four subgroups were compared.Additionally,the impact of microvascular inflammation on the short-term and long-term allograft outcomes was also investigated.Results:The incidence rate of microvascular inflammation was significantly increased in acute rejection,rising in Borderlines,TCMRs and ABMRs,which were 40.71%、57.22%and 81.25%,respectively.ABMR was associated with highest serum creatinine levels at biopsy and 6-month follow-up as well as worst long-term graft survival（Log rank p<0.001）.Increased Banff histopathological injuries and inflammatory cell infiltrations were observed in allograft with microvascular inflammation.Moderate to severe microvascular inflammation（mvi score≥2）was an independent risk factor for acute rejection（OR=2.454,95%CI:1.064-5.660）.Microvascular inflammation indicates poorer prognosis of renal transplantation.Moderate to severe microvascular inflammation was related to significantly higher serum creatinine levels and increased graft loss（Log rank p=0.003）both in overall and non-ABMR groups.Conclusions:Microvascular inflammation is a risk factor of graft failure,which affected short-term graft function and long-term graft survival of kidney transplantation.Therefore,this study recommended the use of anti-humeral therapies to prevent graft loss in patients with microvascular inflammation.Part Ⅱ Differences of serum cytokine levels and antibody subtypes among different rejection groupsBackground: The activation of both adaptive and specific immune cells was involved in the progress of acute rejection,in which cytokine-dependent T-B cell cross-talk plays a vital role.The predictive value of specific urinary proteins and cytokine levels in recipients to monitor acute rejection has been widely investigated.The serum antibody levels and various cytokines were also associated with acute rejection.However,the values of detecting serum cytokines important in T-B cell cross-talk in acute rejection have not been fully studied.Methods: From September 1st 2017 to September 30 th 2019,a total of 78 kidney transplant patients in our center were enrolled in this study,of which were 30 in non-rejection stable group（NR）,28 in TCMRs and 20 in ABMR groups according to Banff 2017 criteria.Luminex assay was used to detect serum cytokines levels including IL-2,IL-4,IL-6,IL-10,IL-12,IL-17 A,IL-21,IL-27,IFN-γ and TNF-α,as well as antibody levels including Ig M and Ig G1-4 subtypes in kidney transplant recipients.Linear regression was applied to evaluate the correlation between cytokine and serum creatinine levels.Receiver operating characteristic（ROC）curve was used to test the diagnostic capacity of cytokines in acute rejection.Results: Compared with NR group,the serum levels of IL-21 in acute rejection group significantly decreased（p <0.001）,which was accompanied by increased levels of IL-10（p = 0.005）.The levels of IL-21 between TCMR and ABMR groups were comparable.The serum IL-2 and IL-10 levels in ABMR group increased significantly,while IL-17 and IFN-γ levels in TCMR group were slightly lower than NR.No difference was found in the levels of Ig M,Ig G1,Ig G2,Ig G3 and Ig G4 between acute rejection group and NR group.Subgroup analysis showed that the levels of Ig G3 and Ig G4 increased significantly in ABMR group.The serum levels of IL-21（ρ =-0.0149,p =0.012）,IL-10（ρ = 0.0096,p <0.001）and TNF-α（ρ = 0.0062,p <0.001）were correlated with serum creatinine levels.ROC curve analysis showed a promising diagnostic value of IL-21 to identify acute rejection with a sensitivity and specificity of75% and 80%,respectively（AUC: 0.821,p <0.001）.Conclusion: The serum IL-21 levels were significantly decreased in patients with acute rejection,which suggests IL-21 might be a potential marker for the diagnosis of acute rejection.Part Ⅲ The role of IL-21 in acute rejection: a mouse model of kidney transplantationBackground: IL-21 is a key immune regulatory cytokine which is considered as a driving factor of germinal center formation and B cell differentiation.Researches have showed IL-21 associated with both immune tolerance and acute rejection post kidney transplantation.Whereas,the impact of IL-21 on transplant immunity remains unclear.Methods: In this study,we established a mouse model of kidney transplantation and treated with IL-21（n = 9）and PBS（n = 7）respectively.The histopathological injuries in allograft,IL-2,IL-4,IL-6,IL-10,IL-17,IL-21,IFN-γ and TNF levels,as well as the proportion of CD19 + B cells and CD4 + T cells in peripheral blood and spleen were further analyzed.Results: Exogenous administration of IL-21 accelerated acute rejection in kidney transplant mouse model,manifesting as significantly increased inflammatory cell infiltration,higher Banff histopathological injury scores and higher serum DSA levels.Reduced IL-21 levels in the peripheral blood were observed in kidney transplant mice after IL-21 injection.Further analysis revealed that increased IL-21 levels in the spleen induced proliferation of CD4+ T cells and CD19+ B cells after IL-21 treatment.Conclusion: Exogenous administration of IL-21 accelerated acute rejection in kidney transplant mice,highlighting the critical function of IL-21 in acute rejection,which could be a potential therapeutic target of acute rejection.