Diagnostic Value Of Tamm-Horsfall Protein And Neutrophil Gelatinase-associated Lipocalin In Chronic Kidney Disease With Renal Tubular Damage

With the increasing as well as changing of aging population and living environment,the incidence of chronic kidney disease(CKD)including diabetic nephropathy,hypertensive nephropathy and primary nephrotic were gradually increased.The pathological development of chronic kidney disease is slowly and early symptoms of kidney damage is not obvious.A lot of research found that Tamm-Horsfall Protein(THP)and other new indicators of renal function had changes in the early kidney damage such as: neutrophil gelatinase-associated lipocalin(NGAL),cystatinC(Cys C),etc.Currently,the radioimmunoassay is used to detect THP,but it could cause medical pollution.In this study,ELISA was developed to detect THP.Collecting the patients with Chronic Kidney disease in our hospital and detecting urine THP.The urinary NGAL,RBP and NAG,serum CysC,?2-MG and RBP of these patients were detected by using Hitachi 7600 automatic biochemical analyzer.According to the Organization to Improve Global Outcomes of Kidney Disease(KDIGO)in 2009,the patients were staged according to eGFR values.The THP and various indicators in CKD patients at different stages were detected.The correlation of new indicators of kidney function as urinary NGAL,urinary THP with traditional indicators such as Scr and Urea was analyzed.The curve of receiver operating characteristic of each indicator was drawn to judge the accuracy of early diagnosis of CKD.Finally,the urinary NGAL and THP were joint detected to observe the accuracy of joint predictors in diagnose of renal tubular damage,which provides the basis for early diagnosis and staging of CKD.Research ObjectiveFirstly,the method of ELISA is simple as well as low-cost.We should understandthe principles of preparation of monoclonal antibodies,the main steps and methods.Secondly,the changes of urine THP in renal tubular injury of CKD have clinical diagnostic significance.The monoclonal antibodies were prepared by using the THP fragment after purified and reorganized,and then the ELISA to detect THP was constructed.Finally,urine NGAL is a sensitive indicator of acute kidney injury.This study will analyze the diagnostic value of urine NGAL in patients with renal tubular injury of CKD.The sensitivity and specificity of combined detection of urinary NGAL and THP in the diagnosis of early stage CKD was analyzed.Meanwhile,the other new biological indicators of changes in different stages of CKD patients was analyzed.To observe the relevance of these indicators with glomerular filtration rate,serum creatinine and serum urea.All of these indicators could provide clinical evidence for early diagnosis,early treatment and delay the pathological progression of CKD.Research methodsBefore these experiments,genetic engineering technology was used to prepare the THP antigen,and then the THP was immuned to mice.Splenocytes were harvested when the blood antibody titers meet the requirements.The splenocytes were fused with myeloma cells by using conventional method to obtain hybridoma cells secreting THP antibodies and stored in liquid nitrogen.After being recoveried and cultured,the cells were injected into mice.The method of Protein G.Labeled antibody horseradish peroxidase was used to purify ascitic fluid,and the method of ELISA detecting the THP in the urine of the patients was constructed.The levels of urine NGAL and NAG,serum Cys C and ?2-MG were detected by using Hitachi 7600 automatic biochemical analyzer.At the same time,49 healthy volunteers were collected as a control group.Research resultThe ascites titer was higher than 1:720000 after the preparation of ascites with the hybrid tumor cells.The monoclonal antibody of THP were purified with Protein G.The antibody titer was 4.1ng/ml.After conjugated with HRP,the 4E4-HRP antibody titer was high enough and it could be used to detect clinical specimens.Statisticsresults showed that the level of urine THP appears significant increasing from the CKD2.The level of urine NGAL was significantly different from the control group from the period of CKD3 a.The results indicates that the levels of urine NGAL and THP were changed in the early period of CKD.These indicators were more sensitive than traditional indicators like serum Scr and Urea.The other biological indicators such as urinary NAG,serum Cys C and ?2-MG also have diagnostic value in the early period of CKD,which were better indicators in reflecting the early damage of chronic kidney disease.ROC curves showed that the accuracy of urine NGAL was higher than that of urine THP.The joint predictors had higher sensitivity and specificity,and the area under the ROC curve was 0.953.The level of urine THP was negatively correlated with serum Scr and Urea.While the level of other indicators such as: Urine NGAL,urine NAG and serum CysC were positively correlated with serum Scr and Urea.ConclusionAfter being recoveried and cultured,the cell of hybridoma could stably secrete monoclonal antibody of THP.The results shows that the hybridoma had good stability.The monoclonal antibody of THP could specially recognize the native THP.Compared with the traditional method of radioimmunoassay,ELISA is more convenient and concise.The urine THP and NGAL,serum CysC and ?2-MG could be used as the sensitive indicator of early evaluation of GFR in patients with CKD.The urine NGAL and THP as joint predictors could improve the sensitivity and specificity in early diagnosis of renal damage,which could have important clinical significance for early detection of CKD patients with tubular damage and evaluating the damage degree.