Establishment Of Urinary TF-PCA Analysis And Its Application In Diabetic Nephropathy

Background & ObjectiveWith the modernization of life style, the continuous improvement of social life quality, the prevalence of diabetes mellitus (DM) becomes higher and higher. According to the latest estimation, the overall prevalence rate has reached 9.7%, the national total has reached nearly 100 million diabetic patients people, which ranks the first in the world. The tendency of younger patients with diabetes is gradually increasing, indicating the highest prevalence rate of diabetes among 30-45 year-olds. Diabetic nephropathy (DN) is one of the most common microvascular complication of diabetes mellitus, with about 33% incidence, and also the leading cause of mortality and morbidity in diabetic patients.The pathogenesis of DN is complicated and remains unclear. It is generally agreed that the abnormal metabolism of glucose, protein and fat, and renal hemodynamic changes are the basic pathophysiologic mechanisms. Activation and effects of Growth factors and cytokines are related with the direct renal lesions. The earliest manifestations of DN is the enlargement of renal, thickness of glomerular basement membrane, proliferation of mesangial cell and the increase of mesangial matrix, then, further development can occur with the deposition of fibrinogen, to the typical glomerular sclerosis lesions at last. At present, the role of coagulative dysfunction and hypercoagulable state in the initiation and development of DN draw much attention. Higher level and activity of tissue factor (TF) are observed in some renal diseases and are related with the glomerular sclerosis lesions and the loss of renal units.TF, the coagulation factorâ…¢, is the strongest initiation factor of coagulation cascade, is also reported to be closely related with many diseases. High level of TF in endothelial cell and plasma is observed and is belived to be related with the complications in DM. TF procoagulant activity (TF-PCA) in peripheral mononuclear cells from DM and its roles in the pathogenesis of DN remains unknown. If so, high levels of TF might be detected in urine as well. Therefore, we set up to investigate the change of TF-PCA both in peripheral mononuclear cells and urine from patients with DM, explore the relationship between the TF-PCA and the renal damage, so as to ascertain the role of TF-PCA in the occurrence and development of DN. Here, a simple and effective method to detect the TF-PCA in urine was established. TF-PCA were measured both in peripheral mononuclear cells and in urine from 86 patietns with type 2 diabetic patients and 21 healthy controls. Other renal damage markers, such as N-acetyl-b-D-glucosaminidase (NAG), kidney injury molecule 1 (KIM-1), neutrophil gelatinaseassociated lipocalin (NGAL) and smadl were also analyzed. Investigation the relationships between TF-PCA and renal damage in DN, ascertain the clinical value of established urinary TF-PCA analysis, so as to provide basis for TF research in DN.Method1. The clinic singnificance of tissue factor in patients with diabetic nephropathy86 cases with T2DM and 21 healthy volunteers were enrolled in this study.86 patients with T2DM were divided into three groups according to urinary albumin creatinine ratio (UACR):42 cases in normal albuminuria group,26 cases in microalbuminuria group and 18 cases in macroalbuminuria group. The levels of fasting blood glucose, uric acid(UA), serum cystatin C (Cysc), glycohemoglobinAlc (HbA1c) and hyper-sensitive CRP (hs-CRP) were measured. Estimated glomerular filtration rate (eGFR) were determined by Macisaac formulae. The peripheral blood mononuclear cells (MNC) were islolated by Ficoll-Hypaque density gradient centrifugation and were lysed by freezing and thawing. The TF-PCA of MNC was analyzed by one-stage coagulation with STAGO auto-analyzer. TF concentration in unine and MNC were determined by ELISA. Other routine clinic indexes were also analyzed.2. The establishment and evaluation of urinary TF-PCA analysisOne-stage coagulation analysis detecting urinary TF-PCA was performed in STAGO auto-analyzer. The effects of the pooled plasma, the storage time of pooled plasma, urinary phosphate, sample storage time, different diluents on TF-PCA analysis were observed. Imprecision, accuracy, lower limit of detection and linear evaluation was conducted to evaluate the established method.3. Urinary TF-PCA in patients with diabetic nephropathyUrine samples from the cases of part 1 were analyzed by established one-stage coagulation with STAGO auto-analyzer. KIM-1, NGAL, smadl and urinary TF were measured by ELISA. The urinary TF-PCA and TF antigen in the three different groups were compared and the relationships with other routine clinic indexes and renal damage markers were also analyzed. The diagnostic value of urinary TF-PCA was determined by ROC curve.Results1. The clinic significance of tissue factor in patients with diabetic nephropathyCompared with control, TF-PCA and TF antigen in MNC, TF antigen in urine from patients with T2DM were significantly increased, peaked in macroalbuminuria group, in this group, TF-PCA and TF antigen in MNC were negatively correlated with GFR, positively correlated with UACR. Among DM, there was no statistical difference between normal albuminuria group and microalbuminuria group (P>0.05).TF-PCA and TF antigen in MNC of T2DM were positively correlated with parameters respectively:fasting blood glucose (r=0.263, r=0.393),Hs-CRP (r=0.498, r=0.367),HbAlc(r=0.225, r=0.298),UACR(r=0.370, r=0.272),UA(r=0.278, r=0.324). Urinary TF antiege were positively correlated with Hs-CRP (r=0.288) and UACR (r=0.332). 2. The establishment and evaluation of urinary TF-PCA analysisThe urinary TF-PCA was analyzed by one-stage coagulation with STAGO auto-analyzer. No difference was found when different pooled plasma was used. So if it come from 5 more different plasmas, the pooled plasma could satisfy the test. However, the pooled plasma must keep fresh, as the TF-PCA could be affected by the pooled plasma if it was kept above 8 hours. There was no apparent difference when pooled plasma was used within 4 hours.As high level of phosphate in urine can inhibit the urinary TF-PCA, so the urinary must be diluted 5 times at least to avoid the interference. Deionized water, normal saline and owren-koller were used as urine diluents. Compared with the other two diluents, owren-koller could effectively sustain urinary TF-PCA.Urinary TF-PCA which was measured with a total imprecison of 6.98% and 5.0%, recovery rate of 80.8% respectively, met the analytical goal. The lower limit of detection was 5mu/l, and good linear was observed when the concentration range from 15 to 2200mu/L.3. Urinary TF-PCA in patients with diabetic nephropathyCompared with control, TF-PCA in urine from patients with T2DM were significantly increased, and there was statistical difference among three DM groups (P<0.05), peaked in macroalbuminuria group. Among DM, urinary TF-PCA correlated with parameters respectively:BUN (r=0.242, P=0.025), Cr (r=0.392, P<001), CysC (R=0.240, P=0.026), hs-CRP(r=0.257, P=0.017), UACR(r=0.430, P<0.001),NGAL(r=0.280, P=0.009), smadl(r=0.284, P=0.008) and eGFR (r=-0.458, P<0.01). Urinary TF-PCA might be a potentional diagnostic indicator for renal damage in DM, as the area under the ROC curve of urinary TF-PCA was 0.847.Conclusions1. TF-PCA and TF antigen in MNC, TF antigen in urine from patients with T2DM were remarkbly elevated, which signify the hypercoagulable state in patients with T2DM. High level of TF might be involved in the pathogenesis of DN. 2. One-stage urinary TF-PCA analysis was successfully established, some interference factors were investigated, layed the foundation for further study on urinary TF.3. Urinary TF-PCA and TF antigen were increased in patients with DM, urinary TF-PCA positively correlated with UACR and negatively correlated with GFR, which makes urinary TF-PCA potentially clinical applicable.