Role Of C-kit Receptor In The Progression Of Venous Neointimal Hyperplasia In Autologous Arteriovenous Fistulae Among Uremic Patients

BackgroundChronic kidney disease(CKD)is a major public health problem associated with a high risk of progression to end-stage renal disease(ESRD).Hemodialysis(HD)is one of the major modes of renal replacement therapies,with arteriovenous fistula(AVF)as the preferred vascular access.However,the clinical utility of AVF has been limited by the high incidence of non-maturation or failure.Venous outflow tract stenosis is the common cause of AVF malfunction,with the pathological basis being neointimal hyperplasia(NIH).The development of the NIH is related to intravascular pathological mechanisms,including inflammation,oxidative stress,and their following cascade reactions,as well as the vascular remodeling,which can lead to stenosis,thrombosis and finally AVF failure.C-kit is a tyrosine kinase receptor that specifically binds the stem cell factor(SCF),and is involved in the regulation of cell proliferation and differentiation through a series of signaling pathways.It has been demonstrated that c-kit plays a significant role in arterial restenosis after injury and atherosclerosis,while its role in NIH,namely the key mechanism of AVF malfunction,has not been fully elucidated.ObjectivesA retrospective cohort study was conducted to investigate the AVF patency rate after creation in ESRD patients,as well as the clinical predictors and relevant biological markers of AVF failure.A patient biospecimen bank was established to study the changes of intimal c-kit expression and vascular morphological indicators and their correlation in patients with NIH-induced AVF failure and those undergoing first-time AVF creation,as well as the changes in the expression of the phosphatidylinositol 3-kinase(PI3K)/Akt signaling pathway downstream.A dual injury model of aortocaval fistula(ACF)in rats with renal failure was established to observe the changes of intimal c-kit expression and vascular morphological indices of ACF and their correlation at different time points after the application of c-kit specific inhibitor(imatinib)intervention therapy,and to investigate the effect of c-kit inhibition on NIH after ACF creation.MethodsWe investigated the patency rate of the AVF at 12,24,36,48,and 60 months after initial AVF surgery in 290 ESRD patients.Cox proportional risk regression analysis was applied to investigate independent risk factors for AVF failure.Enzyme-linked immunosorbent assay(ELISA)was used to determine serum levels of SCF and endothelial cell activation markers E-selectin(ES)and glypican-1(GPC1),and the correlations between SCF,ES,and GPC1 were investigated.Patients with ESRD who underwent first-time AVF creation(preoperative group,n=14)or AVF failure due to NIH(failed group,n=16)were included in the study.Hematoxylin-Eosin(HE)and Verhoeff-Van Gieson(EVG)staining were used for histomorphometric analysis.Immunohistochemical methods were applied to examine the expression of c-kit in the intima,and Pearson’s correlation analysis was performed to examine its correlation with the intimal area and the area of stenosis.Double-label immunofluorescence method was used to detect the co-localization of c-kit with SMA and CD31.Protein immunoblotting was used to detect c-kit and the associated PI3K/Akt signaling axis(PI3K,P-PI3K,Akt,P-Akt473,P-Akt308 and mTOR).A rat model of renal failure was first established by feeding a diet containing 0.75%adenine,and the control group were normal diet rats.The ACF model was then established based on the renal failure model.Saline gavage(model group)and imatinib gavage(imatinib group)were administered 1 week after surgery,and vascular samples were taken at 4,6 and 8 weeks after ACF surgery.Immunohistochemical methods were used to detect changes in c-kit expression,and EVG staining was used to observe morphological indices.Pearson correlation was used to evaluate the correlations of intimal c-kit expression and intimal thickness and lumen cross-sectional area.ResultsAccording to the Kaplan-Meier survival analysis,the primary patency rate of forearm AVF in ESRD patients was 89.0%,84.1%,79.3%,77.6%,and 77.6%at 12,24,36,48,and 60 months,respectively.Crude Cox proportional risk regression analysis showed that cardiovascular disease(CVD),antiplatelet agents,statins,D-dimer(per 1 mg/dL increase),serum albumin(per 1 g/L increase),and calcium(per 1 g/L increase)were independently associated with AVF failure,with HRs of 1.63(95%CI,1.00-2.65),1.92(95%CI,1.08-3.41),1.97(95%CI,1.05-3.69),1.08(95%CI,1.02-1.15),0.96(95%CI,0.92-0.99),and 0.37(95%CI,0.16-0.89),respectively.Cox analysis was used to adjust for potential confounders,and only hypertension was associated with the loss of AVF function(HR=8.47,95%CI=1.12-63.84),and serum SCF was positively correlated with ES and GPC1(ES:R=0.42,P<0.001;GPC1:R=0.44,P<0.001).The internal elastic lamina area,the intimal area,the percentage of stenosis and the average optical density(AOD)of c-kit in the intima were significantly higher in the ESRD patients of the failed AVF group than in the preoperative group(P≤0.001).The expression of c-kit in the intima was positively correlated with both intimal area and luminal stenosis(intimal area:R=0.744,P<0.001;stenosis:R=0.923,P<0.001).Immunofluorescence staining demonstrated that c-kit co-localized with α-SMA but not with CD31.Western blot showed that the levels of c-kit,P-PI3K,P-Akt473 and mTOR in the PI3K/Akt axis were higher in the failed AVF group than in the preoperative group(P<0.05).Biochemical and pathological staining and ultrasound confirmed that a dual injury model of renal failure and ACF could be successfully established in rats.C-kit was positively expressed on the intima of the inferior vena cava(IVC)in rats with renal failure but negative in rats without renal failure,and the difference in intimal thickness between the two groups was not statistically significant(P=0.06).The use of imatinib in the dual injury model inhibited c-kit expression in ACF intima at 8 weeks postoperatively(P=0.04).Intimal thickness decreased in the imatinib group at 6 and 8 weeks postoperatively(P=0.03 at 6 weeks and P=0.001 at 8 weeks)and lumen area increased at 8 weeks postoperatively(P=0.01).C-kit expression in the ACF intima was positively correlated with intimal thickness(R=0.664,P=0.003)and negatively correlated with lumen area(R=-0.563,P=0.015).Conclusions1.This retrospective cohort study showed that the primary patency rates in ESRD patients decrease each year after forearm AVF creation to 77.6%at 60 months.Baseline plasma D-dimer levels failed to predict AVF malfunction.Serum SCF was associated with endothelial cell activation,suggesting that SCF and its specific receptor c-kit may play a role in AVF failure.2.Increased c-kit expression in patients’ vascular tissue significantly correlated with the degree of stenosis due to NIH,accompanied by increased expression of PI3K/Akt pathway-related proteins,implying the possible involvement of c-kit and the downstream PI3K/Akt pathway in the development of NIH in AVF.3.After successfully establishing a dual injury model of adenine-induced renal failure and ACF in rats,imatinib,a c-kit specific inhibitor,was used as the interventional treatment,which can inhibit the c-kit expression in the ACF intima,reduce intimal thickness and increase luminal area,suggesting that interventional treatment with imatinib can inhibit the NIH development.