Variation Of Von Willebrand Factor Cleaving Protease In Plasma Of Patients With Nephrotic Syndrome And Expression As Well As Influencing Factors In Human Renal Tubular Epithelial Cells

Objective:To detect the antigen and activity level of von willebrand factor cleaving protease (ADAMTS13) in plasma of patients with Nephrotic Syndrome(NS), analyze the variation of ADAMTS13 level in plasma of patients with NS and discuss its clinical significance; observe the effect of salvianolate on expression of ADAMTS13 in human renal tubular epithelial cells(human kidney-2, HK2), and preliminarily analyze the significance.Methods:1.Collect fasting plasma of 70 patients with NS and 48 normal controls from June 2009 to May 2010 in First Affiliated Hospital of Soochow University, to measure the antigen levels of ADAMTS13 and VWF; also test the activity level of ADAMTS13 of partial patients and normal controls.2.All the patients underwent kidney biopsy. According to pathological diagnosises, they are divided into membranous nephropathy group (MN group,n=30) and non-membranous nephropathy group (non-MN group,n=40). Compared the differences of plasma ADAMTS13 antigen, ADAMTS13 activity, VWF antigen, ratio of VWF antigen to ADAMTS13 antigen and ratio of VWF antigen to ADAMTS13 activity, and analyze the correlation relationship among ADAMTS13 antigen, ADAMTS13 activity, and other clinical indexes.3.Cultivation of HK2 in vitro, inoculate the cells in culture flasks, when the cells fuse to 80-90%, replacement the medium of salvianolate contents of 0, 0.1, 0.5, 1.2mg/L. After cultured for 24 hours, collect HK2 cells, extract mRNA, and convert to cDNA. Use RT-PCR to detect the expression of ADAMTS13 mRNA of HK2, gel image analysis to analyze in semi-quantitatively.Results:1.Detection of plasma ADAMTS13 antigen, VWF antigen of patients with NS:①Compared with normal control group, plasma ADAMTS13 antigens of both MN group and non-MN group are decreased (P<0.05), VWF antigens are increased (P<0.05), and the ratios of VWF antigen to ADAMTS13 antigen are increased (P<0.01).②Compared with non-MN group, plasma ADAMTS13 antigen of MN group is lower (P<0.05), while VWF antigen level is higher (P<0.05), the ratio of VWF antigen to ADAMTS13 antigen is higher too(P<0.05).③Correlation analysis shows that plasma ADAMTS13 antigen of patients with NS is positively correlated with serum albumin(ALB) (r=0.385,P<0.01), while negatively correlated with the quantitative measurement of 24 hours urinary protein(r=-0.242,P<0.05), total cholesterol(TC) (r=-0.317,P<0.01) and triglyceride(TG) (r=-0.243,P<0.05); plasma VWF antigen is negatively correlated with ALB(r=-0.287, P<0.05), and positively correlated with fibrinogen(FIB) (r=0.366,P<0.01) and D-Dimer(r=0.255, P<0.05); there is no significant correlation among plasma ADAMTS13 antigen and VWF antigen(P>0.05).2. Detection of plasma ADAMTS13 antigen , VWF antigen and ADAMTS13 activity of partial patients and normal controls:①Compared with normal control group, ADAMTS13 activities of both MN group and non-MN group are decreased (P<0.01), VWF antigens are increased (P<0.05), and the ratios of VWF antigen to ADAMTS13 activity are increased (P<0.01). In the MN group, ADAMTS13 antigen is the lowest (P<0.01), and the ratio of VWF antigen to ADAMTS13 antigen is the highest(P<0.01), while there are no differences of those two are observed between non-MN group and normal control group(P>0.05).②Compared with non-MN group, ADAMTS13 antigen is lower(P<0.05), while the ratio of VWF antigen to ADAMTS13 antigen is higher(P<0.05) in MN group. No differences are observed in ADAMTS13 activity, VWF antigen and the ratio of VWF antigen to ADAMTS13 activity among MN group and non-MN group(P>0.05).③Correlation analysis shows that plasma ADAMTS13 activity of patients with NS is negatively correlated with VWF antigen(r=-0.393,P<0.01) and FIB(r=-0.457,P<0.05), there are no significant correlations among ADAMTS13 antigen and VWF antigen, ADAMTS13 activity and ADAMTS13 antigen (P>0.05).3.Taken the ADAMTS13 mRNA of HK2 with no salvianolate co-cultured as control(the reference value is 1), the ADAMTS13 mRNA of HK2 co-cultured by the medium of salvianolate contents of 0.1, 0.5, 1 and 2mg/L are 2.39,3.05,4.65 and 6.65 respectively, which are the averages of two repetitive tests.Conclusions:1.The increased VWF antigen, decreased ADAMTS13 antigen and activity in different extent of NS patients may be one of the pathogenesis of thrombosis which is the common complication of NS. Further more, the higher VWF antigen and the lower ADAMTS13 antigen and activity of MN patients may be one of the reasons why MN is more prone to thrombosis.2.ADAMTS13 antigen level reflects the pathological degree of NS in a certain extent. And ADAMTS13 activity level can be used as one of indexes of endothelial function and hypercoagulability determination.3.HK2 could express ADAMTS13 mRNA, and salvianolate could excite ADAMTS13 expression in a dose-dependent manner. The regulation of salvianolate to ADAMTS13 expressed in renal tubular epithelial cells may affect coagulation of kidney local circulation, which could be one pharmaco–mechanisms of salvianolate.