The origin of multiple molecular forms in urine of HNL/NGAL

Abstract

Background and objectives: Several molecular forms of human neutrophil lipocalin/neutrophil gelatinase-associated lipocalin (HNL/NGAL), a novel biomarker for acute kidney injury (AKI), have been found in urine. The origin of these different forms and the effect of antibody configuration on assay performances were investigated in this report.

Design, setting, participants, & measurements: The molecular forms of HNL/NGAL from human neutrophils and present in urine obtained from cardiac surgery patients and patients with urinary tract infection (UTI), as well as secreted from HK-2 cells, were studied by Western blotting. The levels of HNL/NGAL in urine were measured by ELISAs. Kidney injury was simulated by incubation of HK-2 cells under stressful conditions.

Results: The major molecular form of HNL/NGAL secreted by neutrophils is dimeric, whereas the major form secreted by HK-2 cells is monomeric. This was reflected by a predominance of the monomeric form in urine from patients with AKI and the dimeric form in patients with UTIs. The epitope specificities of the antibody used in the ELISAs had a profound effect on assay performance and paralleled differences of the antibodies to identify the different forms of urine HNL/NGAL.

Conclusions: The monomeric form is the predominant form secreted by tubular epithelial cells, and the dimeric form is the predominant form secreted by neutrophils. The development of molecular form-specific assays for HNL/NGAL may be a means to identify the origin of HNL/NGAL in urine and construct more specific tools for the diagnosis of AKI.

Figures

Figure 1.

Detection by Western blotting of different molecular forms of HNL/NGAL in urine samples and released from neutrophils. Samples were separated on SDS-PAGE under nonreducing conditions. U1 and U2 represent the urine samples obtained from two patients undergoing cardiac surgery.

Figure 2.

Detection by Western blotting of different molecular forms of HNL/NGAL in urine samples of five patients with UTIs.

Figure 3.

Measurements of HNL/NGAL in the fractions from Superdex-75 gel filtration of urine using different antibody-based assays. The major molecular forms of HNL/NGAL in peak 1 and peak 2 are dimer and monomer, respectively. The insert shows the amplification of the peak 1.