Immunoassays distinguishing between HNL/NGAL released in urine from kidney epithelial cells and neutrophils
Highlights
► Two ELISAs measure monomeric and dimeric HNL/NGAL in urine. ► The ELISA-2 mainly detected dimeric HNL/NGAL from neutrophils. ► The ELISA-1/ELISA-2 ratio mainly detected monomeric HNL/NGAL from kidney cells. ► The monomer-specific ELISA-1/ELISA-2 ratio increased during AKI development. ► Patients with urinary tract infections had high dimer-specific ELISA-2 levels.
Introduction
Human neutrophil lipocalin (HNL), also known as neutrophil gelatinase-associated lipocalin (NGAL), is released by kidney tubular cells under stressful conditions [1] and is a promising biomarker for acute kidney injury (AKI) [2]. Additionally, HNL/NGAL is released by activated neutrophils [3], [4]. HNL/NGAL exists in different molecular forms in the neutrophil [5], in plasma [6] and in urine [1], [7]. The protein exists as a 25-kDa monomer, a 45-kDa homodimer or a 135-kDa heterodimer, covalently conjugated with gelatinase. Kidney epithelial cells in culture mainly secrete the monomeric form, whereas neutrophils mainly release the dimeric form [1]. Hence, elevated urinary HNL/NGAL levels in AKI may emanate from injured kidney epithelium as well as from infiltrating neutrophils involved in the AKI-pathophysiology [8]. Furthermore, activation of neutrophils and the subsequent release of their, mainly dimeric, HNL/NGAL in urine have been observed in patients with urinary tract infections (UTIs) [1], [9], [10]. The interpretation of elevated urinary HNL/NGAL is further complicated by the fact that increased levels have been found in non-acutely ill patients without evidence of kidney damage or UTIs [11].
The clinical performance of the HNL/NGAL assay to catch the different forms of HNL/NGAL depends on the antibody configuration [7]. This can explain the varying ability of urinary HNL/NGAL to predict AKI in previous studies [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25].
To distinguish between different causes of elevated HNL/NGAL in urine, precise diagnostic assays measuring different forms of HNL/NGAL are desirable since they may increase the diagnostic accuracy of conditions such as AKI. Also, identifying the different sources of HNL/NGAL will aid in understanding the timing of different pathophysiological events during the AKI continuum [26].
This study aims to examine the ability of different combinations of monoclonal antibodies against HNL/NGAL to distinguish between the molecular forms primarily secreted by kidney epithelial cells and neutrophils, respectively.
Section snippets
Materials and methods
The regional ethical review board in Stockholm approved this study. Written informed consent was obtained from patients or next of kin.
Selection and categorization of urine samples
782 urine samples from 83 ICU patients were analyzed by RIA. 138 samples (from 48 patients) with a RIA-measured HNL/NGAL concentration ≥ 50 ng/mL were subjected to Western blot. In three urine samples the monomeric and dimeric bands were barely seen by visual inspection of the Western blots. These samples were therefore not categorized. Still, scanning of the electropherograms generated Western blot ratios. In one sample only the heterodimeric band was seen and neither monomeric nor dimeric
Discussion
This study shows that different antibody configurations of the HNL/NGAL ELISA distinguish between the monomeric and dimeric forms of HNL/NGAL in urine. The various molecular forms were detected by Western blotting using polyclonal anti-HNL/NGAL antibodies, likely identifying all forms of the molecule [6]. When comparing results from the RIA and ELISAs with the Western blot patterns (Fig. 2) we made some interesting findings. First, the polyclonal RIA measured both forms of HNL/NGAL as we
Acknowledgments
This work was partly supported by grants from Gambro (Stockholm, Sweden) and Diagnostics Development (Uppsala, Sweden). The authors thank Lena Moberg for performing the Western blot analyses and Ann-Katrin Eriksson for performing the ELISA assays. We thank research nurses Ola Friman, Åsa Bengtsson and Jenny Svedlund for their assistance with the urine sampling. We are grateful for the expert statistical advice concerning quantile regression provided by Professor Matteo Bottai at the Unit of
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