The Role And Mechanism Of Soluble Urokinase Plasminogen Activator Receptor In ANCA-associated Vasculitis

Antineutrophil cytoplasmic autoantibody(ANCA)-associated vasculitis(AAV)is a kind of autoimmune disease characterized by pauci-immune necrotizing inflammation of the small blood vessels.AAV involves multiple organ systems all over the body and kidneys are the most susceptible organs.The soluble urokinase plasminogen activator receptor(suPAR),a biomarker of inflammation,on one hand involves in the activation of the immune system,on the other hand is associated with kidney diseases and might be a prognostic factor of renal function progression.At present,the role of suPAR in AAV is unclear.Therefore,this study aims to analyze the correlation between suPAR and AAV disease activity,explore whether suPAR has a potential physiological role in AAV,and provide new ideas for the treatment of AAV.First,in the clinical research part,we collected 90 AAV patients,tested the plasma suPAR levels with a commercial kit,and analyzed the correlations between the plasma suPAR levels and the clinical parameters,and prognosis of the patients.It was found that the plasma suPAR levels in AAV patients in active stage were significantly higher than those in remission phase or in healthy controls.Plasma suPAR levels in patients were correlated with many clinical parameters,and higher levels of suPAR were closely related to poor prognosis.In vitro experiment,we used human u PAR recombinant protein andMPO-ANCA-positive IgG to stimulate glomerular endothelial cells(GEn C).And the effect of u PAR in MPO-ANCA induced GEn C activation and injury was measured.The results showed that u PAR enhanced MPO-ANCA-positive IgG-induced activation and injury of endothelial cells.Toll-like receptor 4(TLR4)was up-regulated at the protein and m RNA levels after co-stimulation.Down-regulated expression of TLR4 or pre-incubation with TLR4 inhibitors or could effectively reverse activation of endothelial cells induced by u PAR and ANCA-positive IgG.In summary,plasma levels of suPAR in AAV patients were significantly related to disease activity and prognosis.Human u PAR protein could enhance ANCA-positive IgG-induced endothelial cell activation through the TLR4 pathway.suPAR might be a potential biomarker and might participant in the pathogenesis of AAV.Therefore,intervention of suPAR may have potential therapeutic effects,providing new insights into the treatment of AAV.Part Ⅰ The expression and significance of plasma soluble urokinase plasminogen activator receptor in ANCA–associated vasculitisObjective: To explore whether circulating suPAR was associated with AAV disease activity and prognosis of the patients.Methods: We evaluated 90 AAV patients with follow-up data;their plasma suPAR and C-reactive protein(CRP)levels were measured by using ELISA kits.Associations between plasma suPAR levels and clinical parameters,and prognosis were analyzed.Results: Plasma suPAR levels in AAV patients were significantly higher than in healthy controls(5,920.08±3,447.17 pg/m L vs.1,441.97±835.04 pg/m L,P<0.001).Furthermore,suPAR was significantly elevated in AAV patients in active stage compared to those in partial remission(6,492.19±3,689.48 pg/m L vs.5,031.86±2,489.01 pg/m L,P=0.039).Correlation analyses demonstrated that suPAR levels positively correlated with initial serum creatinine,BVAS,CRP,and procalcitonin concentrations,and negatively correlated with e GFR and C3 circulating levels.In a Kaplan-Meier survival analysis,patients with plasma suPAR levels >5683.3 pg/m L showed poorer survival than patients with lower levels(log-rank,P=0.001).Besides,multivariate analyses confirmed that plasma suPAR levels were an independent adverse prognostic factor for a composite outcome of end-stage renal disease(ESRD)or death,after adjusting for age and gender(HR 1.05,95%CI=1.01-1.11,P=0.043).Receiver operating characteristic curves showed a suPAR cut-off value > 6662.2 pg/m L for composite outcome with 68% sensitivity and 88% specificity,with an AUC = 0.82(95 % CI=0.68-0.96,P<0.001).Conclusion: Circulating suPAR levels might be a marker of activity correlated with disease activity in AAV patients,and,to some extent,could be a factor of poor prognosis.Part Ⅱ suPAR contributes to ANCA-positive IgG-mediated glomerular endothelial cells activation through the TLR4 pathwayObjective: Based on the results of our previous study that plasma suPAR levels were significantly associated with disease activity and prognosis,we further investigated whether u PAR protein induces glomerular endothelial cell activation and injury,which is one of the important aspects in the pathogenesis of AAV.Methods: We used human u PAR recombinant protein and MPO-ANCA-positive IgG extracted from the serum of MPO-AAV patients to stimulate the glomerular endothelial cells(GEn C).For evaluating the endothelial activation,q RT-PCR was used to detect the m RNA levels of ICAM-1 and VCAM-1,and appropriate kits were used to measure the levels of lactate dehydrogenase,s ICAM-1,and s VCAM-1.Permeability experiments were performed to detect monolayer activation quantitatively,and immunofluorescence was used to observe morphological alterations of cells.Western-blot and q RT-PCR were performed to detect the expression of TLR4.In addition,TLR4 si RNA and TLR4 inhibitor was employed to determine its role.Results: Sera from AAV patients or human uPAR protein stimulation could mediate endothelial injury with increased expression of ICAM-1 and VCAM-1 m RNA levels.Compared with single stimulation,u PAR combined with ANCA-positive IgG stimulation could further increase the expression of ICAM-1 and VCAM-1 m RNA levels,increase the endothelial monolayer permeability,and impair tight junctions,and the expression of TLR4 was significantly up-regulated.TLR4 si RNA significantly alleviated increased expression of ICAM-1 and VCAM-1 m RNA levels induced by u PAR and ANCA-positive IgG.And pretreatment of TLR4 inhibitor significantly down-regulated the elevated levels of ICAM-1m RNA,s ICAM-1,and LDH induced by u PAR plus ANCA-positive IgG.Conclusion: uPAR protein can enhance the glomerular endothelial activation induced by ANCA-positive IgG through TLR4 pathway,indicating that suPAR may have potential pathophysiological effects in AAV and suPAR might be regarded as potential therapeutic target.