HNL/NGAL Used For Early Diagnosis Of Acute Kidney Injury And Its Immunoassay Study

Acute kidney injury (AKI) is a common and tough clinical problem. Despite tremendous progress in our understanding of the pathogenesis of AKI and substantial technical improvements in preservation and therapeutics in the past decades, the mobility and morbidity associated with AKI remain unacceptably high. One reason for this situation may be the lack of early and powerful markers for AKI. Recently, the value of HNL/NGAL for early detection of AKI has been highlighted.In the present study, we isolated and purified dimmer HNL/NGAL (around 1mg) from about 6L buffy coat obtained from 100 healthy blood donors, which was used as standard.In order to evaluate the potential value of HNL/NGAL as an early biomarker of AKI, 59 adult patients undergoing cardiac surgery were enrolled in this study and the levels of urine, serum and plasma HNL/NGAL pre and 2h, 24h, 48h, 72h post operation were measured by radioimmunoassay (RIA). The results indicate the HNL/NGAL levels postoperative were significantly elevated when compared with pre-operation, and the 2h level postoperative reached to the peak. There were significant correlations between extracorporeal circulation (EEC) time and the levels of HNL/NGAL. Additionally, the levels of HNL/NGAL have significant correlations with glomerular filtration rate (GFR) and the levels of plasma creatinine. The 2h urine HNL/NGAL levels are significantly different between AKI and no-AKI subjects (p=0.0044), and the 48h and 72h serum and plasma HNL/NGAL levels are significantly different between the two groups, respectively. The AUC of 2h urine, 48h serum and plasma HNL/NGAL levels are 0.777 (p=0.0064), 0.875 (p=0.0243) and 0.973 (p=0.004), respectively. Based on the above data, we concluded that urine HNL/NGAL might be a useful early biomarker of postoperative AKI and the serum and plasma HNL/NGAL could indicate the kidney injury but couldn't be used for early diagnosis of AKI.In order to reach the demands from lab researches and clinical diagnosis in the future, we developed and characterized a novel and sensitive sandwich ELISA for HNL/NGAL measuring, and the sensitivity is 0.1μg/L. The overall correlation between HNL/NGAL levels measured by RIA and ELISA was r2=0.72, however, the correlations at different time points have a wide range (r2′s ranging between 0.59-0.90). The correlations between HNL/NGAL(RIA) and EEC time, GFR and plasma creatinine are higher than HNL/NGAL(ELISA). Additionally, although the same standard applied in the two immonoassays, the concentrations of HNL/NGAL were measured by ELISA are much lower than those measured by RIA, and the ratios varied at different time points (10%～20%). In the present study, we fist proposed that the antibody configuration might have impact on the HNL/NGAL assay based on different antibodies. We also concluded the molecular forms of HNL/NGAL might change pre and different time points post operation. In order to further confirm our viewpoint, we investigated the existing molecular forms of HNL/NGAL pre and post operation. We also studied the affinities of different antibodies to various HNL/NGAL in urine and neutrophil granule release products. Additionally, five novel sandwich ELISA were developed and characterized for measuring the gel filtration chromatography products and clinical urine samples. The sensitivity of the new ELISA is 0.039μg/L. We found all three major molecular forms of HNL/NGAL in urine, and the composition changed pre and post operation. We also found the different antibodies have various affinities to different molecular forms of HNL/NGAL in urine. However, they have the similar performance when detecting different molecular forms of HNL/NGAL in neutrophil granule released products. The results also show that the assays based on different antibodies have different ability when measuring momomer and dimmer HNL/NGAL in urine separated by gel filtration chromatography, and the clinical performances of the assays were various. Based on the above findings, we concluded that although the monomer and dimmer HNL/NGAL in urine and in neutrophil granule released products the molecular configurations might have discrepancy. These results confirm that antibody configuration have impact on the assay based on different antibodies again. We also concluded the urine HNL/NGAL might be secreted partly by renal tubular epithelial cells. In addition, we proposed a hypothesis that we could raise a specific antibody against a definite molecular form of HNL/NGAL and develop an immunoassay based on the antibody, which can be used for searching the origin of HNL/NGAL and enhance the specificity and sensitivity of disease diagnosis.In order to investigate the origin of urine HNL/NGAL post cardiac surgery, we have studied the character of the expression of HNL/NGAL in HK-2 a renal tubule epithelial cell line. In this part, we have cultured the cells under adversity conditions or stimulated the cells with IL-1β, TNF-αor LPS. To some extent, these conditioned cultures have simulated the disadvantages caused by cardiac surgery. We also invested the molecular forms of HNL/NGAL secreted by HK-2. The results show adversity, IL-1β, TNF-αand LPS can induce HK-2 cells up-regulation HNL/NGAL protein, and IL-1βcan induce the up-regulation of HNL/NGAL mRNA. The major molecular form of HNL/NGAL secreted by HK-2 is monomer. We concluded that the renal tubular epithelial cell could express and secrete HNL/NGAL, and the urine HNL/NGAL elevated post cardiac surgery might be partly come from kidney synthesis. According to the existing molecular forms of HNL/NGAL in urine, neutrophil granule released products and secreted by HK-2, we concluded that the urine HNL/NGAL might partly due to neutrophil activation and release. Besides, the high molecular HNL/NGAL may come from the kidney expression and the combination of monomer HNL/NGAL and MMP-9 in urine.In summary, for the first time the study investigated the potential value of urine, serum and plasma HNL/NGAL used for postoperative AKI early diagnosis, simultaneously. We have developed and characterized six sensitivity sandwich ELISA based on various antibodies for HNL/NGAL quantitation. We first proposed and verified that the antibody configuration had impact on the immunoassays'clinical performances. It is a useful experimental consideration and theory to develop a molecular form definitely immunoassay, and such immunoassay may be a powerful way to investigate the origin of HNL/NGAL and it can be used for enhancing the clinical diagnosi's sensitivity and specificity. In the present study, we reported for the first time the expression characterize of HNL/NGAL in renal tubular epithelial cell line HK-2 cultured under adversity conditions or stimulated with IL-1β, TNF-αor LPS. Additionally, we reported for the first time the molecular form of HNL/NGAL in urine secreted by HK-2. Together with the existing forms in neutrophil granule release product, we draw a view on the possible origin of urine HNL/NGAL post cardiac surgery.