Study On The Regulation Of Autophagy In Mesangial Cells By High Phosphorus And Thrombolytic Therapy With Urokinase In Radial Artery Cephalic Venous Fistula Thrombosis

Background and ObjectiveChronic kidney disease(CKD)is a serious public health problem.Most patients with CKD eventually have to undergo renal replacement therapy and continue to use large amounts of medical resources.Delaying the progression of kidney disease has always been the focus of CKD clinical and scientific research.Mesangial cells(MCs)show proliferation hypertrophy,apoptosis,excessive secretion of mesangial matrix under different injury factors,aggravating glomerulosclerosis and renal fibrosis,involved in CKD progression.Hyperphosphatemia is one of the characteristics of advanced CKD.Recent studies have found that elevated plasma phosphate(Pi)accelerates the progression of CKD.However,it is not completely clear how high Pi can promote CKD progress so far.Autophagy is one of the most basic physiological processes in the body.Autophagy plays important roles in many biological processes and diseases,including apoptosis,proliferation and inflammation.However,few studies have reported on the relationship between autophagy and renal diseases,especially autophagy and MCs stress injury.Studies have shown that down-regulated autophagy can increase the production of type I collagen in MCs,thereby increasing the glomerular mesangial matrix and promoting the progression of renal fibrosis.TGF-? is the most important molecule causing renal fibrosis,inducting the change of autophagy activity in MCs,which affects the level of downstream apoptosis-related caspase-3 molecules.Hyperphosphate increases extracellular matrix(ECM)production through TGF-?1 signaling pathway in MCs.If high Pi can increase the secretion of TGF-?1 in MCs,it may affect the autophagic activity of cells and thus change the apoptosis of MCs and increase extracellular matrix(ECM)production,promote glomerular sclerosis,and aggravate glomerular damage.So far,the effect of high Pi on autophagy of glomerular mesangial cells has not been reported in the literature.This study is to clarify the effect of high Pi on the autophagy and wheather high Pi increases the apoptosis and the secretion of extracellular matrix in mesangial cells by regulating autophagy activity.Methods1.The concentration of total TGF-?1 in the culture medium of human glomerular MCs under different phosphorus concentrations and PFA culture conditions was measured by ELISA.2.CCK8 and flow cytometry to determine the effect of high Pi on mesangial cell proliferation and apoptosis.The expression of cleaved caspase3,a protein marker related to the apoptosis of MCs,was detected by western blot in the presence of high Pi and high Pi combined with Rap,3-MA and CQ.3.The expression of autophagy-related proteins LC3,P62 and Beclin-1 in mesangial cells was detected by western blot.4.The expression of autophagy-associated protein LC3 in mesangial cells was detected by western blot with high Pi in the presence of Rap,3-MA and CQ.5.The RGP-GFP-LC3 adenovirus was transfected into MCs,and the number of autophagy spots in MCs with different Pi concentrations was observed by laser confocal microscopy.6.The number of autophagosomes in MCs with different Pi concentrations was observed by electron microscopy.7.Western blot was used to detect the expression levels of mesangial matrix-associated proteins ?-SMA and Collagen I in presence of high Pi and with Rap,3-MA,and CQ,respectively.8.Real-time PCR was used to detect MCs m RNA levels of LC3,Type IV collagen,TGF-?1 and fibronectin in the culture media of different Pi concentrations.Results1.TGF-?1 secreted by HMCs cultured in hyperphosphate medium was higher than that in normal control group(p<0.05),and TGF-?1 was higher in the P5.0m M group than in the P2.5m M group(p<0.05);TGF-?1 in a culture medium of P2.5 m M or P 5.0 m M for 48 hours increased more than that of each culture 24 hours(p<0.05).TGF-?1 in the high Pi with PFA group was significantly lower than that in the high Pi group(p<0.05).2.Compared with P0.9m M and P2.5m M groups,P5.0m M obviously induced apoptosis(p<0.05);P2.5m M compared to P0.9m M group,induced the tendence of apoptosis,which did not reach statistical significance between the two groups.The expression of Caspase3 protein in P5.0m M group was more than that in P0.9m M group;the expression of Caspase3 protein in P5.0m M+Rap group was lower than that in P5.0m M group;the expression of Caspase3 protein in P5.0m M+MA,P5.0m M+CQ group was higher than that in P5.0m M group.The above comparisons were statistically significant(p<0.05).3.The ratio of autophagic marker LC3II/LC3 I in P0.9m M,P2.5m M and P5.0m M groups and the autophagosomes observed by fluorescence microscope gradually decreased;electron microscopy showed that the autophagosome in P2.5m M group was less than P0.9m M.P5.0m M increased LC3II/LC3 I ratio in the presence of Rap and CQ and reduced LC3II/LC3 I ratio in the presence of 3-MA.The expression of P62 in P0.9m M,P2.5m M and P5.0m M groups were increased step by step.The yellow dots were observed by fluorescence microscope after the autophagic spots merged with green and red.4.Compared with P0.9m M group,the levels of ?-SMA and Collagen I protein increased in P5.0m M,P5.0m M+3-MA and P5.0m M+CQ groups,P5.0m M+Rap group decreases.The expression of ?-SMA and Collagen I protein in P5.0m M+3-MA,P5.0m M+CQ groups were higher than that in P5.0m M group.5.The m RNA levels of TGF-?1,LC3 II,Collagen IV and Fibronectin in P2.5m M cells were increased compared to P0.9m M,while the m RNA level of which in P2.5m M+Rap group was lower than that in P2.5m M group.Conclusion1.High Pi inhibited the autophagic activity of HMCs in vitro.2.High Pi inducted TGF-?1 in HMCs by the concentration and time-dependent manner;PFA reduced TGF-?1 of HMCs in high phosphorus condition.3.High Pi inhibited autophagy,promoted apoptosis and secreted ECM in MCs.High Pi may regulate apoptosis in a concentration dependent manner.Objective: We prospectively assessed the safety and efficacy of high-dose bolus urokinase and a tourniquet for anticoagulation for a radiocephalic arteriovenous fistula(AVF)occlusion.Methods: We prospectively studied 52 radiocephalic AVF occlusion events in 38 patients who were treated with bolus 100,000-200,000 IU urokinase injected into the outflow vein near the occlusion after tourniquet compression of the outflow vein.Bleeding complications within one week were recorded.Salvage and patency were calculated and patients were followed up during regular hemodialysis sessions.Results: The therapy was successful for 47 cases and urokinase doses ranged from 150,000-450,000 IU(mean 27.8±9.2×104 IU).Successful thrombolysis was achieved 10-1200 min after urokinase treatment.No bleeding was observed.All patients were followed and 5 had an occlusion recurrence.The shortest time to recurrence was 1 month and the longest was 56 months(mean 17.5±15.2 months).Primary patency was 84.9% at 3 months(95% CI,68.1-94.9%),69.7% at 6 months(95% CI,51.3-84.4%),and 33.3% at 24 months(95% CI,18.0-51.3%).Assisted primary patency was 90.9%(95% CI,75.7-98.1%)at 3 months,75.8% at 6 months(95% CI,57.7-88.9%)and 42.4% at 24 months(95% CI,25.5-60.8%).Conclusion: Thrombolytic treatment with local urokinase and a tourniquet may be used to salvage occluded AVF and systemic complications of urokinase can be avoided using the tourniquet.